Patent Application
20130274124
The invention relates generally to devices that culture, differentiate, or isolate cells based upon the surface expression of cellular ligands that associate and respond to polypeptides immobilized to a surface of the device. In some embodiments, the polypeptides mimic the extracellular matrix microenvironment. The invention also generally relates to methods of diagnosing patients with particular disorders based upon the presence or absence of cultured or isolated cells or the presence of absence of the display of certain cellular phenotypes.
There are a wide variety of contexts in biology and medicine when it is important or useful to be able to distinguish cells of different types from one another, to separate cells of different types from one another, and/or to identify, characterize, or define particular cells as members of one cell type or another. Current methods of isolation, differentiation, and characterization can be improved. Identification and characterization of certain cellular properties or expression profiles can be used for diagnosis or prognosis for certain disease states.
The present invention encompasses the recognition that cells can be identified and/or characterized by “adhesion signatures” that embody a cell's affinity for extracellular matrix components. In some embodiments, an adhesion signature embodies, or displays, an affinity for one or more such extracellular matrix components and is sufficient to distinguish or characterize relevant cells as compared with at least one other reference cell.
For example, in some embodiments, an adhesion signature is sufficient to distinguish or characterize cells of a particular cell type (e.g., host or tissue type, state of development, etc) from cells of one or more other types. In some embodiments, adhesion signatures allow isolating and/or culturing cells of a particular type and/or under defined conditions. In some embodiments, a cell sample that contains metastatic cells may bind an adhesion set comprising fibronectin in combination with galectin-3, galectin-8 or laminin more consistently than a cell sample that does not contain metastatic cells.
In accordance with the present invention, adhesion signatures are defined for particular cells or cell types relative to appropriate reference cells or cell types. In some embodiments, the particular cells differ from reference cells in that they are progeny of a different source (e.g., different cell lineage, organism, tissue type, etc.) as compared with the reference cells, the cells are at a different developmental stage than the reference cells, the cells suffer from or are susceptible to a particular disease, disorder, or condition. In some embodiments, cells are identical to reference cells with the exception of a characteristic or characteristics that results in a difference identifiable by differing adhesion signatures.
Further in accordance with the present invention, adhesion signatures are used to identify and/or characterize cells. For example, in some embodiments adhesion signatures are used to distinguish cells suffering from or susceptible to a particular disease, disorder, or condition from those that are not. In further embodiments, adhesion signatures are used to identify and/or characterize cells of a particular developmental stage, cell lineage, or tissue type.
The invention provides an array of polypeptides, the array comprising: a solid support and a plurality of adhesion sets, wherein each adhesion set comprises two or more different polypeptides comprising a polypeptide sequence associated with the extracellular matrix or a functional fragment thereof, and wherein the adhesion sets are attached to the solid support at an addressable location of the array. In some embodiments, the solid support is a slide optionally coated with a polymer. In some embodiments, the solid support is coated with a polymer. In some embodiments, the polymer is polyacrylamide. In some embodiments, the solid support is a material chosen from: polystyrene (TCPS), glass, quarts, quartz glass, poly(ethylene terephthalate) (PET), polyethylene, polyvinyl difluoride (PVDF), polydimethylsiloxane (PDMS), polytetrafluoroethylene (PTFE), polymethylmethacrylate (PMMA), polycarbonate, polyolefin, ethylene vinyl acetate, polypropylene, polysulfone, polytetrafluoroethylene, silicones, poly(meth)acrylic acid, polyamides, polyvinyl chloride, polyvinylphenol, and copolymers and mixtures thereof. In some embodiments, the at least one adhesion set comprises two different polypeptides attached to a solid support.
The invention further relates to an array of polypeptides, the array comprising: a solid support and a plurality of adhesion sets, wherein each adhesion set comprises two or more different polypeptides comprising a polypeptide sequence associated with the extracellular matrix or a functional fragment thereof, and wherein the adhesion sets are attached to the solid support at an addressable location of the array; and wherein the two or more of the different polypeptides sequences are chosen from: collagen I, collagen II, collagen III, collagen IV, collagen V, collagen VI, fibronectin, laminin, merosin, tenascin-R, chondroitin sulfate, agreccan, elastin, keratin, mucin, superfibronectin, F-spondin, nidogen-2, heparin sulfate, biglycan, decorin, galectin 1, galectin 3, galectin 3c, galectin 4, galectin 8, thrombospondin-4, osteopontin, osteonectin, testican 1, testican 2, fibrin, tenascin-C, nidogen-1, vitronectin, rat agrin, hyaluronan, brevican, or functional fragments thereof. In some embodiments, the at least one adhesion set comprises at least two different polypeptide sequences chosen from: osteopontin, thrombospondin-4, fibronectin, laminin, galectin 3, galectin 8, or functional fragments thereof. In some embodiments, the at least one adhesion set comprises at least two different polypeptide sequences chosen from: fibronectin, laminins and functional fragments thereof. In some embodiments, the at least one adhesion set comprises at least two different polypeptide sequences chosen from: fibronectin, galectin 3, and functional fragments thereof. In some embodiments, the at least one adhesion set comprises at least two different polypeptide sequences chosen from: fibronectin, galectin 8, and functional fragments thereof.
In some embodiments, the at least one adhesion set comprises at least two different polypeptide sequences chosen from: thrombospondin-4, galectin 8, and functional fragments thereof.
In some embodiments, wherein an array or system disclosed herein comprises at least one adhesion set comprising two polypeptide sequences associated with the extracellular matrix chosen from: Collagen 1 and Agreccan, Collagen IV and Nidogen-1, or a functional fragment thereof. In some embodiments, the at least one adhesion set comprises at least one polypeptide sequence that is osteopontin or a functional fragment thereof. In some embodiments, each adhesion set consists of a pair of different polypeptides associated with the extracellular matrix. In some embodiments, the array comprises at least about 700, about 750, or about 800 different adhesion sets. In some embodiments, the array comprises at least about 700, about 750, or about 800 different adhesion sets positioned at different discrete locations on the array.
The invention further relates to an array of polypeptides, the array comprising: a solid support and a plurality of adhesion sets, wherein each adhesion set comprises two or more different polypeptides comprising a polypeptide sequence associated with the extracellular matrix or a functional fragment thereof, and wherein the adhesion sets are attached to the solid support at an addressable location of the array; and wherein the array is free of animal-derived ECM material, embryonic fibroblasts, material deposited from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, or any combination thereof. In some embodiments, the array is free of serum derived or sourced from any animal species.
The invention relates to an array of polypeptides, the array comprising: a solid support and a plurality of adhesion sets, wherein each adhesion set comprises two or more different polypeptides comprising a polypeptide sequence associated with the extracellular matrix or a functional fragment thereof, and wherein the array further comprises one or a plurality of mammalian cells. In some embodiments, the one or a plurality of mammalian cells contains at least one lung cell.
The invention further provides an array or kit comprising at least one cell or at least one cell sample. In some embodiments, the cell sample contains at least one cancer cell or one stem cell.
In some embodiments, the cancer cell is derived from the cancer of the adrenal gland, bladder, bone, bone marrow, brain, spine, breast, cervix, gall bladder, ganglia, gastrointestinal tract, stomach, colon, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, or uterus. In some embodiments, the array or kit comprises a stem cell that is an embryonic stem cell, an adipose-derived stem cell, a mesenchymal stem cell, an umbilical stem cell or a pluripotent stem cell.
The invention relates to an array of polypeptides, the array comprising: a solid support and a plurality of adhesion sets, wherein each adhesion set comprises two or more different polypeptides comprising a polypeptide sequence associated with the extracellular matrix or a functional fragment thereof, wherein the two or more different polypeptides are attached to the solid support via passive electrostatic non-covalent binding.
The invention provides a system comprising: an array of polypeptides, the array comprising: a solid support and a plurality of adhesion sets, wherein each adhesion set comprises two or more different polypeptides comprising a polypeptide sequence associated with the extracellular matrix or a functional fragment thereof; and a cell culture vessel. In some embodiments, the system further comprises at least one or a plurality of cells. In some embodiments, the system further comprises at least one or a plurality of cells derived from cancer cells chosen from: cancer of the adrenal gland, bladder, bone, bone marrow, brain, spine, breast, cervix, gall bladder, ganglia, gastrointestinal tract, stomach, colon, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, or uterus. In some embodiments, the system further comprises cell media free of at least one of: serum, animal-derived ECM material, embryonic fibroblasts, or material deposited from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cell. In some embodiments, the system further comprises cell media free of: serum, animal-derived ECM material, embryonic fibroblasts, and material deposited from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cell. In some embodiments, the system further comprises at least one or a plurality of cells is a stem cell chosen from: an embryonic stem cell, an adipose-derived stem cell, a mesenchymal stem cell, an umbilical stem cell or a pluripotent stem cell.
The invention also provides a kit comprising: an array of polypeptides, the array comprising: a solid support and a plurality of adhesion sets, wherein each adhesion set comprises two or more different polypeptides comprising a polypeptide sequence associated with the extracellular matrix or a functional fragment thereof; and optionally comprising a cell culture vessel. In some embodiments, the kit further comprises at least one of the following: cell media, a volume of fluorescent stain or dye, a cell sample, and a set of instructions, optionally accessible remotely through an electronic medium.
The invention further provides a method of identifying an adhesion signature of a cell sample comprising: contacting a cell sample to an array or system disclosed herein; and determining a quantity of cells bound to one or a plurality of adhesion sets. In some embodiments, the cell sample contains at least one cell from a biopsy. The invention also provides a method of inducing differentiation of a cell comprising contacting a cell sample to an array or a system disclosed herein. In some embodiments, the method includes inducing differentiation of a stem cell chosen from: an embryonic stem cell, an adipose-derived stem cell, a mesenchymal stem cell, an umbilical stem cell or a pluripotent stem cell. In some embodiments, the step of contacting a cell or cell sample comprises exposing the cell or cell sample to the array or the system for a sufficient period of time for differentiation of a cell to a hepatic or pancreatic lineage.
The invention also provides for a method of culturing a cell comprising contacting a cell or a cell sample to an array or a system disclosed herein in the presence of cell media. In some embodiments, the cell media is serum free. In some embodiments, the cell media is free of at least one or a combination of: serum, animal-derived ECM material, embryonic fibroblasts, or material deposited from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cell. In some embodiments, the cell or cell sample is derived from a primary lineage of a cancer cells or stem cells. In some embodiments, the invention relates to a method of culturing a cell or cell sample wherein the cell or the cell sample comprises one or a plurality of stem cells chosen from: an embryonic stem cell, an adipose-derived stem cell, a mesenchymal stem cell, an umbilical stem cell or a pluripotent stem cell is a pluripotent stem cell or embryonic stem cell. In some embodiments, the invention relates to a method of culturing a cell comprising contacting a cell or a cell sample to an array or a system disclosed herein in the presence of cell media comprises a contacting cell wherein the cell is passaged at least about 30 times, at least 40 times, or at least 50 times.
The invention further provides a method of culturing one or a plurality of primary hepatocytes, the method comprising contacting one or a plurality of primary hepatocytes with an array or system disclosed herein.
The invention further relates to a method of diagnosing a hyperproliferative disease comprising: (a) contacting a cell sample to an array or system disclosed herein; (b) quantifying one or more adhesion values; (c) determining one or more adhesion signatures of the cell sample based upon the adhesion values; and (d) comparing the adhesion signature of the cell sample to an adhesion signature of a control cell sample. In some embodiments, the hyperproliferative disease is metastatic lung cancer. In some embodiments, the hyperproliferative disease is metastatic breast cancer.
The invention relates to a method of prognosing a clinical outcome of a subject comprising: (a) contacting a cell or cell sample to an array or system disclosed herein; (b) quantifying one or more adhesion values; (c) determining one or more adhesion signatures of the cell sample based upon the adhesion values; and (d) correlating the adhesion signature to an adhesion signature of a cell sample associated with a clinical outcome.
The invention further provides a method of determining patient responsiveness to a therapy comprising: (a) contacting a cell or cell sample to an array or system disclosed herein; (b) quantifying one or more adhesion values; (c) determining one or more adhesion signatures of the cell sample based at least partially upon the adhesion values; and (d) comparing the one or more adhesion signatures to one or more adhesion signature of a control cell sample.
The invention further provides a method of determining patient responsiveness to a therapy comprising: (a) contacting a cell or cell sample to an array or system disclosed herein; (b) quantifying one or more adhesion values by detecting fluorescence of cells through a computer-program product disclosed herein; (c) determining one or more adhesion signatures of the cell sample based at least partially upon the adhesion values; and (d) comparing the one or more adhesion signatures to one or more adhesion signature of a control cell sample.
The invention provides a method of isolating a cell comprising: contacting a cell sample to an array or system disclosed herein. In some embodiments, the method of isolating a cell comprises contacting a cell sample to an array or system disclosed herein for a sufficient time period and under sufficient conditions for a cell to adhere to the array or the system more tightly than other components of the cell sample. In some embodiments, the method of isolating a cell further comprises rinsing the array or system with a buffer that that washes other components of the cell sample from the cell.
The invention also provides a method of adhering hepatocytes derived from a primary lineage of human liver cells comprising contacting the hepatocytes to an array or system disclosed herein. The invention also provides a method of maintaining a culture of hepatocytes derived from a primary lineage of human liver cells comprising contacting the hepatocytes to an array or system disclosed herein.
The invention provides a method of sorting a mixture of cell types comprising: contacting a mixture of cell types to an array or system disclosed herein. In some embodiment, the method of sorting a mixture of cell types further comprises the step of determining one or more adhesion signatures of the cell sample based upon a calculated adhesion value. In some embodiments, the method further comprises the step of comparing the one or more adhesion signatures to one or more adhesion signature of a control cell type, and sorting the cell types based upon their similarities or differences to a phenotype of a the control cell type.
In some embodiments, particular cells are isolated from a composition also comprising other cells based on an adhesion signature common to the isolated cells and different from the other cells. For example, in some embodiments, a composition comprising more than one type of cells is contacted with one or more ECM components, wherein the affinity of the particular cells to be isolated for the ECM components constitutes part of an adhesion signature for the cells. In further embodiments, cells are cultured in media containing the ECM component composition.
In some embodiments, the present disclosure provides methods comprising contacting a sample comprising cells with a collection of extracellular matrix (ECM) components and detecting presence or level of interactions between cells in the sample and ECM components in the collection. In some embodiments, provided methods comprise determining that a particular set of detected interactions defines an adhesion signature that is characteristic of particular cells in the sample in that it distinguishes them from other cells in the sample or from reference cells. In some embodiments, detecting comprises detecting presence or level of a set of interactions that is characteristic of particular cells in the sample in that it distinguishes them from other cells in the sample or from reference cells.
In some embodiments, the present disclosure provides methods comprising contacting a sample comprising cells with a collection of extracellular matrix (ECM) components under conditions and for a time sufficient for a set of interactions to occur between particular cells in the sample and ECM components in the collection sufficient to isolate the cells from other components of the sample. In some embodiments, the other components of the sample from which the particular cells are isolated include other cells.
In certain embodiments, the other cells are cells that make a different set of interactions with the ECM components than do the isolated cells. In certain embodiments, the step of contacting comprises contacting with ECM components attached to a solid phase, under conditions and for a time sufficient for the set of interactions to occur on the solid phase. In some embodiments, provided methods comprise a step of separating solid phase from sample, so that particular cells making interactions with the solid phase are separated from the sample.
In some embodiments, the present disclosure provides methods for determining the effects on cells of interacting with extracellular matrix components comprising exposing a first population of cells to a first set of conditions that include contacting with a collection of extracellular matrix components, exposing a second population of cells, which second population of cells is comparable to the first population of cells, to a second set of conditions, which second set of conditions is comparable to the first set of conditions except that some or all of the extracellular matrix components are absent from the contacting; and determining one or more cell population features that differs between the first and second populations of cells after the exposing has occurred.
In some embodiments, the present disclosure provides methods of culturing a cell type of interest comprising contacting a sample comprising cells of a cell type of interest with a collection of extracellular matrix (ECM) components appropriate to promote growth and/or replication of cells of the cell type of interest as compared with cells of one or more different cell types. In some embodiments, the collection of ECM components is suspended in media. In certain embodiments, the collection of ECM components is attached to a solid phase. In some embodiments, the method further comprises isolating cells of the cell type of interest from the solid phase.
In some embodiments, the present disclosure provides kits for cell isolation and growth comprising a substrate coated with a collection of ECM components characterized in that, when a sample containing cells of a plurality of different cell types, which plurality of different cell types includes at least one cell type of interest, is contacted with the substrate, cells of the cell type of interest form a set of interactions with ECM components in the collection sufficient to isolate the cells of the cell type of interest from other cells in the sample. In some embodiments, isolation comprises growth of the cells of the cell type of interest. In some embodiments, growth comprises proliferation. In some embodiments, growth is sufficient to overpopulate the sample with the cell type of interest as compared with other cell types. In certain embodiments, the kit further comprises medium. In some embodiments, the kit further comprises cells of the cell type of interest.
In some embodiments, the present disclosure provides systems for culturing cells comprising a substrate coated with a collection of ECM components characterized in that, when a sample containing cells of a plurality of different cell types, which plurality of different cell types includes at least one cell type of interest, is contacted with the substrate, cells of the cell type of interest form a set of interactions with ECM components in the collection sufficient to isolate the cells of the cell type of interest from other cells in the sample. In some embodiments, isolation comprises growth of the cells of the cell type of interest. In certain embodiments, growth comprises proliferation. In some embodiments, growth is sufficient to overpopulate the sample with the cell type of interest as compared with other cell types.
In some embodiments, the present disclosure provides kits for cancer diagnosis comprising a substrate coated with a collection of ECM components characterized in that, when a sample containing cells of a plurality of different cell types, which plurality of different cell types includes at least one cell type of interest is contacted with the substrate, cells of the cell type of interest form a set of interactions with ECM components in the collection sufficient to isolate the cells of the cell type of interest from other cells in the sample. In some embodiments, the cell type of interest is cancer cells of a particular stage of metastasis. In some embodiments, isolation comprises growth of the cancer cells of a particular stage. In some embodiments, growth comprises proliferation. In some embodiments, the growth is sufficient to overpopulate the sample with the cancer cells of a particular stage as compared with other cell types. In some embodiments, the kit further comprises medium. In some embodiments, the kit further comprises a means for assessing abundance of the cancer cells of a particular stage.
The invention provides a method comprising steps of: contacting a sample comprising cells with a collection of extracellular matrix (ECM) components; detecting presence or level of interactions between cells in the sample and ECM components in the collection. In some embodiments, the method further comprises determining that a particular set of detected interactions defines an adhesion signature that is characteristic of particular cells in the sample in that it distinguishes them from other cells in the sample or from reference cells. In some embodiments, the step of detecting comprises detecting presence or level of a set of interactions that is characteristic of particular cells in the sample in that it distinguishes them from other cells in the sample or from reference cells. In some embodiments, the collection of ECM components is attached to a solid phase. In some embodiments, the invention provides a method comprising steps of: contacting a sample comprising cells with a collection of extracellular matrix (ECM) components; and detecting presence or level of interactions between cells in the sample and ECM components in the collection, wherein the ECM components in the collection are separately attached in discrete locations to the solid phase. In some embodiments, the step of detecting comprises quantifying binding levels at one or more of the discrete locations. In some embodiments, the step of detecting comprises quantifying binding levels at all of the discrete locations.
The invention further provides a method comprising steps of: contacting a sample comprising cells with a collection of extracellular matrix (ECM) components; detecting presence or level of interactions between cells in the sample and ECM components in the collection; and determining that a particular set of detected interactions defines an adhesion signature that is characteristic of particular cells in the sample in that it distinguishes them from other cells in the sample or from reference cells. In some embodiments, the step of detecting comprises determining presence or level of a predetermined set of interactions between cells in the sample and ECM components in the collection. In some embodiments, the method further comprises comparing the determined presence or level with reference presence or level of the predetermined set, so that identity with, similarity to, or difference from the reference presence or level is determined. In some embodiments, the reference presence or level is or comprises an adhesion signature that is characteristic of a particular cell type in that it distinguishes cells of the particular cell type from cells of at least one other cell type. In some embodiments, the reference presence or level is or comprises an adhesion signature of cells in a particular stage of development in that it distinguishes them from otherwise comparable cells in a different stage of development.
The invention also provides for a method comprising steps of: contacting a sample comprising cells with a collection of extracellular matrix (ECM) components under conditions and for a time sufficient for a set of interactions to occur between particular cells in the sample and ECM components in the collection sufficient to isolate the cells from other components of the sample. In some embodiments, the other components of the sample from which the particular cells are isolated include other cells. In some embodiments, the other cells are cells that make a different set of interactions with the ECM components than do the isolated cells.
The invention also provides for a method comprising steps of: contacting a sample comprising cells with a collection of extracellular matrix (ECM) components under conditions and for a time sufficient for a set of interactions to occur between particular cells in the sample and ECM components in the collection sufficient to isolate the cells from other components of the sample, wherein the step of contacting comprises contacting with ECM components attached to a solid phase, under conditions and for a time sufficient for the set of interactions to occur on the solid phase. In some embodiments, the method further comprises a step of separating the solid phase from the sample, so that the particular cells are separated from the sample.
The invention provides for a method comprising steps of: contacting a sample comprising cells with a collection of extracellular matrix (ECM) components, wherein the collection of extracellular matrix components comprises one or more of aggrecan, agrin, biglycan, brevican, chondroitin sulfate, collagen I, collagen II, collagen III, collagen IV, collagen V, collagen VI, decorin, elastin, f-spondin, fibrin, fibronectin, galectin 1, galectin 3, galectin 3c, galectin 4, galectin 8, heparan sulfate, hyaluronic acid, keratin, laminin, merosin, mucin, nidogen-1, nidogen-2, osteopontin, SPARC/osteonectin, superfibronectin, tenascin-C, tenascin-R, testican 1/SPOCKI, testican 2/SPOCK2, thrombospondin-4, vitronectin, and functional fragments thereof. The invention provides for a method comprising steps of: contacting a sample comprising cells with a collection of extracellular matrix (ECM) components, wherein the collection of extracellular matrix components comprises at least two ECM components selected from: agrin and collagen IV, agrin and fibrin, biglycan and collagen II, biglycan and fibrin, collagen I and thrombospondin-4, collagen II and decorin, collagen II and tenascin-C, collagen II and testican 2, collagen III and collagen VI, collagen III and thrombospondin-4, collagen IV and galectin 4, collagen IV and SPARC, collagen IV and vitronectin, collagen V and galectin 1, collagen VI and galectin 3, fibrin and galectin 3c, fibrin and galectin 4, fibrin and keratin, fibrin and osteopontin, fibrin and SPARC, f-spondin and fibronectin, fibronectin and galectin 3, fibronectin and galectin 8, fibronectin and laminin, fibronectin and testican 1, and or functional fragments thereof. In some embodiments, the invention provides for a method comprising steps of: contacting a sample comprising cells with a collection of extracellular matrix (ECM) components, wherein the collection of extracellular matrix components comprises at least two ECM components selected from: agrin and collagen II, agrin and laminin, biglycan and collagen II, brevican and fibronectin, collagen I and testican 2, collagen II and collagen IV, collagen II and laminin, collagen II and nidogen-1, collagen II and testican 2, collagen III and galectin 8, collagen III and superfibronectin, collagen V and fibronectin, collagen V and galectin 1, collagen VI and fibronectin, collagen VI and nidogen-1, collagen VI and tenascin-C, decorin and fibronectin, decorin and galectin 8, decorin and laminin, elastin and galectin 4, fibrin and galectin 3, fibronectin and galectin 1, fibronectin and galectin 3, fibronectin and galectin 4, fibronectin and mucin, fibronectin and SPARC, fibronectin and testican 2, galectin 1 and galectin 3, galectin 1 and keratin, galectin 3 and heparan sulfate, galectin 3 and superfibronectin, galectin 4 and nidogen-1, galectin 8 and tenascin-C, keratin and laminin, laminin and merosin, laminin and thrombospondin-4, SPARC and superfibronectin, superfibronectin and testican 1, and/or functional fragments thereof.
In some embodiments, the invention provides for a method comprising steps of: contacting a sample comprising cells with a collection of extracellular matrix (ECM) components, wherein the collection of extracellular matrix components comprises at least two ECM components selected from: at least two ECM components selected from biglycan and collagen IV, biglycan and galectin 4, brevican and collagen I, brevican and collagen IV, brevican and galectin 3c, collagen I and galectin 1, collagen I and galectin 3, collagen I and galectin 3c, collagen I and galectin 8, collagen I and nidogen-2, collagen I and SPARC, collagen I and tenascin-C, collagen I and testican 1, collagen I and vitronectin, collagen II and galectin 3, collagen II and galectin 8, collagen II and nidogen-1, collagen II and nidogen-2, collagen IV and decorin, collagen IV and galectin 8, collagen IV and nidogen-1, collagen IV and nidogen-2, collagen IV and testican 1, collagen IV and testican 2, collagen VI and f-spondin, collagen VI and galectin 3, collagen VI and galectin 8, collagen VI and tenascin-C, collagen VI and testican 2, collagen VI and thrombospondin-4, f-spondin and vitronectin, fibrin and galectin 4, fibronectin and galectin 4, fibronectin and nidogen-1, fibronectin and tenascin-C, fibronectin and testican 1, fibronectin and testican 2, galectin 3 and vitronectin, galectin 3c and merosin, galectin 3c and superfibronectin, galectin 4 and superfibronectin, galectin 8 and superfibronectin, galectin 8 and vitronectin, laminin and vitronectin, SPARC and testican 1, and/or superfibronectin and vitronectin.
The invention further provides for a method comprising steps of: contacting a sample comprising cells with a collection of extracellular matrix (ECM) components under conditions and for a time sufficient for a set of interactions to occur between particular cells in the sample and ECM components in the collection sufficient to isolate the cells, wherein the particular cells are human embryonic stem cells, human induced pluripotent stem cells, hepatocytes, mesenchymal stem cells, or cancer cells. In some embodiments, the mesenchymal stem cells are derived from bone marrow, adipose tissue, umbilical cord blood or umbilical cord. In some embodiments, the cells are cells in a certain stage of development. In some embodiments, the cancer cells are from a primary tumor, lymph nodes, or metastases at organ sites. In some embodiments, the cancer cells are from a primary tumor, lymph nodes, metastases at organ sites, or metastatic tissue. In some embodiments, the cancer cells are non-small cell lung cancer cells. In some embodiments, the cancer cells are breast cancer cells.
The invention also provides for a method of determining the effects on cells of interacting with extracellular matrix components, the method comprising steps of: exposing a first population of cells to a first set of conditions that includes contacting with a collection of extracellular matrix components, exposing a second population of cells, which second population of cells is comparable to the first population of cells, to a second set of conditions, which second set of conditions is comparable to the first set of conditions except that some or all of the extracellular matrix components are absent from the contacting; and determining one or more cell population features that differs between the first and second populations of cells after the exposing has occurred.
The invention also provides for a method for culturing a cell type of interest comprising contacting a sample comprising cells of a cell type of interest with a collection of extracellular matrix (ECM) components appropriate to promote growth and/or replication of cells of the cell type of interest as compared with cells of one or more different cell types. In some embodiments, the collection of ECM components is suspended in media. In some embodiments, the collection of ECM components is attached to a solid phase. In some embodiments, the method of culturing a cell type of interest further comprises isolating cells of the cell type of interest from the solid phase. In some embodiments, the collection of ECM components comprises ECM components that participate in interactions defining an adhesion signature characteristic of the cell type of interest in that it distinguishes cells of the cell type of interest from otherwise comparable cells of a different cell type. In some embodiments, the cell type of interest is cells in a developmental stage of interest and the different cell type is otherwise comparable cells in a different developmental stage.
In some embodiments, the invention provides for any of the disclosed methods wherein the cell type of interest is human embryonic stem cells or human induced pluripotent stem cells, and wherein the collection of ECM components comprises at least two ECM components selected from collagen II and galectin 4, collagen IV and galectin 8, collagen I and Laminin, or functional fragments thereof.
In some embodiments, the invention provides for any of the disclosed methods wherein the cell type of interest is human mesenchymal stem cells. In some embodiments, the mesenchymal stem cells are derived from bone marrow, adipose tissue, umbilical cord blood or umbilical cord.
The invention further provides for a method for culturing a cell type of interest comprising contacting a sample comprising cells of a cell type of interest with a collection of extracellular matrix (ECM) components appropriate to promote growth and/or replication of cells of the cell type of interest as compared with cells of one or more different cell types, wherein the collection of ECM components comprises at least two ECM components selected from biglycan and collagen IV, biglycan and galectin 4, brevican and collagen I, brevican and collagen IV, brevican and galectin 3c, collagen I and galectin 1, collagen I and galectin 3, collagen I and galectin 3c, collagen I and galectin 8, collagen I and nidogen-2, collagen I and SPARC, collagen I and tenascin-C, collagen I and testican 1, collagen I and vitronectin, collagen II and galectin 3, collagen II and galectin 8, collagen II and nidogen-1, collagen II and nidogen-2, collagen IV and decorin, collagen IV and galectin 8, collagen IV and nidogen-1, collagen IV and nidogen-2, collagen IV and testican 1, collagen IV and testican 2, collagen VI and f-spondin, collagen VI and galectin 3, collagen VI and galectin 8, collagen VI and tenascin-C, collagen VI and testican 2, collagen VI and thrombospondin-4, f-spondin and vitronectin, fibrin and galectin 4, fibronectin and galectin 4, fibronectin and nidogen-1, fibronectin and tenascin-C, fibronectin and testican 1, fibronectin and testican 2, galectin 3 and vitronectin, galectin 3c and merosin, galectin 3c and superfibronectin, galectin 4 and superfibronectin, galectin 8 and superfibronectin, galectin 8 and vitronectin, laminin and vitronectin, SPARC and testican 1, superfibronectin and vitronectin and/or functional fragments thereof. In some embodiments, the cell type of interest is human embryonic stem cells, mouse embryonic stem cells and/or human induced pluripotent stem cells. In some embodiments, the collection of ECM components comprises fibronectin and merosin.
In some embodiments, the invention further provides for a method for culturing hepatocytes comprising contacting a sample comprising hepatocytes with a collection of extracellular matrix (ECM) components appropriate to promote growth and/or replication of the hepatocytes. In some embodiments, the collection of ECM components comprises at least two ECM components selected from agrin and collagen I, collagen I and laminin, collagen I and merosin, collagen II and galectin 8, collagen II and SPARC, and/or collagen IV and nidogen-1.
The invention further provides for any of the disclosed methods herein wherein each of the disclosed steps is performed in a serum-free environment. The invention also provides for any of the disclosed methods herein comprising cells of a cell type of interest that are isolated from serum-free media or fully defined synthetic media.
In some embodiments, the invention provides for a kit for cell isolation and growth comprising: a substrate coated with a collection of ECM components characterized in that, when a sample containing cells of a plurality of different cell types, which plurality of different cell types includes at least one cell type of interest, is contacted with the substrate, cells of the cell type of interest form a set of interactions with ECM components in the collection sufficient to isolate the cells of the cell type of interest from other cells in the sample. In some embodiments, the isolation comprises growth of the cells of the cell type of interest. In some embodiments, the growth of cells comprises proliferation of the cell type of interest. In some embodiments, the growth is sufficient to overpopulate the sample with the cell type of interest as compared with other cell types.
The invention further provides for a kit comprising: a substrate coated with a collection of ECM components characterized in that, when a sample containing cells of a plurality of different cell types, which plurality of different cell types includes at least one cell type of interest, is contacted with the substrate, cells of the cell type of interest form a set of interactions with ECM components in the collection sufficient to isolate the cells of the cell type of interest from other cells in the sample. In some embodiments, the kit further comprises cell media. In some embodiments, the kit further comprises serum-free media or fully defined synthetic cell media. In some embodiments, the kit further comprises cells of the cell type of interest. In some embodiments, the kit further comprises cells of the cell type of interest, wherein the cell type of interest is a stem cell, cancer cell, or hepatocyte. In some embodiments, the substrate is coated with an array of ECM components. In some embodiments, the substrate is coated with an array of any pair of ECM components disclosed herein.
The invention further provides for a system for culturing cells comprising: a substrate coated with a collection of ECM components characterized in that, when a sample containing cells of a plurality of different cell types, which plurality of different cell types includes at least one cell type of interest, is contacted with the substrate, cells of the cell type of interest form a set of interactions with ECM components in the collection sufficient to isolate the cells of the cell type of interest from other cells in the sample. In some embodiments, the isolation comprises growth of the cells of the cell type of interest. In some embodiments, the growth comprises proliferation. In some embodiments, the growth is sufficient to overpopulate the sample with the cell type of interest as compared with other cell types. In some embodiments, the system comprises a substrate comprised of polystyrene or polypropylene. In some embodiments, the cell type of interest is human mesenchymal stem cells. In some embodiments, the cell type of interest is mesenchymal stem cells are derived from bone marrow, adipose tissue, umbilical cord blood or umbilical cord. In some embodiments, the cell type of interest is human mesenchymal stem cells are derived from bone marrow, adipose tissue, umbilical cord blood or umbilical cord.
The invention further provides for a system for culturing cells comprising: a substrate coated with a collection of ECM components wherein the collection of ECM components comprises at least two ECM components selected from biglycan and collagen IV, biglycan and galectin 4, brevican and collagen I, brevican and collagen IV, brevican and galectin 3c, collagen I and galectin 1, collagen I and galectin 3, collagen I and galectin 3c, collagen I and galectin 8, collagen I and nidogen-2, collagen I and SPARC, collagen I and tenascin-C, collagen I and testican 1, collagen I and vitronectin, collagen II and galectin 3, collagen II and galectin 8, collagen II and nidogen-1, collagen II and nidogen-2, collagen IV and decorin, collagen IV and galectin 8, collagen IV and nidogen-1, collagen IV and nidogen-2, collagen IV and testican 1, collagen IV and testican 2, collagen VI and f-spondin, collagen VI and galectin 3, collagen VI and galectin 8, collagen VI and tenascin-C, collagen VI and testican 2, collagen VI and thrombospondin-4, f-spondin and vitronectin, fibrin and galectin 4, fibronectin and galectin 4, fibronectin and nidogen-1, fibronectin and tenascin-C, fibronectin and testican 1, fibronectin and testican 2, galectin 3 and vitronectin, galectin 3c and merosin, galectin 3c and superfibronectin, galectin 4 and superfibronectin, galectin 8 and superfibronectin, galectin 8 and vitronectin, laminin and vitronectin, SPARC and testican 1, and/or superfibronectin, vitronectin, or functional fragments thereof. In some embodiments, the cell type of interest is human embryonic stem cells or human induced pluripotent stem cells and the collection of ECM components comprises at least two ECM components selected from collagen II and galectin 4, collagen IV and galectin 8, or collagen I and Laminin. In some embodiments, the cell type of interest comprises hepatocytes.
The invention further provides for a system for culturing cells comprising: a substrate coated with a collection of ECM components wherein the collection of ECM components comprises at least two ECM components selected from agrin and collagen I, collagen I and laminin, collagen I and merosin, collagen II and galectin 8, collagen II and SPARC, and/or collagen IV and nidogen-1.
The invention provides for a kit for cancer stage diagnosis comprising: a substrate coated with a collection of ECM components characterized in that, when a sample containing cells of a plurality of different cell types, which plurality of different cell types includes at least one cell type of interest is contacted with the substrate, cells of the cell type of interest form a set of interactions with ECM components in the collection sufficient to isolate the cells of the cell type of interest from other cells in the sample. In some embodiments, the cell type of interest is cancer cells of a particular stage of metastasis. In some embodiments, the kit comprises a substrate coated with a collection of ECM components characterized in that, when a sample containing cells of a plurality of different cell types, which plurality of different cell types includes at least one cell type of interest is contacted with the substrate, cancer cells form a set of interactions with ECM components in the collection sufficient to isolate the growth of the cancer cells of a particular stage. In some embodiments, the growth comprises proliferation. In some embodiments, the growth is sufficient to overpopulate the sample with the cancer cells of a particular stage as compared with other cell types. In some embodiments, wherein the cancer cells at a particular stage of metastasis are from a primary tumor, lymph nodes, or metastases at organ sites or are non-small cell lung cancer cells. In some embodiments, the kit further comprises cell media.
In some embodiments, the kit further comprises a means for assessing abundance of the cancer cells of a particular stage. In some embodiments, the cancer cells at a particular stage of metastasis are breast cancer cells.
The invention further provides a kit for cancer stage diagnosis comprising: a substrate coated with a collection of ECM components characterized in that, when a sample containing cells of a plurality of different cell types, which plurality of different cell types includes at least one cell type of interest is contacted with the substrate, cells of the cell type of interest form a set of interactions with ECM components in the collection sufficient to isolate the cells of the cell type of interest from other cells in the sample, wherein the collection of extracellular matrix components comprises at least two ECM components selected from agrin and collagen IV, agrin and fibrin, biglycan and collagen II, biglycan and fibrin, collagen I and thrombospondin-4, collagen II and decorin, collagen II and tenascin-C, collagen II and testican 2, collagen III and collagen VI, collagen III and thrombospondin-4, collagen IV and galectin 4, collagen IV and SPARC, collagen IV and vitronectin, collagen V and galectin 1, collagen VI and galectin 3, fibrin and galectin 3c, fibrin and galectin 4, fibrin and keratin, fibrin and osteopontin, fibrin and SPARC, f-spondin and fibronectin, fibronectin and galectin 3, fibronectin and galectin 8, fibronectin and laminin, and/or fibronectin and testican 1.
In some embodiments, the collection of extracellular matrix components comprises at least two ECM components selected from agrin and collagen II, agrin and laminin, biglycan and collagen II, brevican and fibronectin, collagen I and testican 2, collagen II and collagen IV, collagen II and laminin, collagen II and nidogen-1, collagen II and testican 2, collagen III and galectin 8, collagen III and superfibronectin, collagen V and fibronectin, collagen V and galectin 1, collagen VI and fibronectin, collagen VI and nidogen-1, collagen VI and tenascin-C, decorin and fibronectin, decorin and galectin 8, decorin and laminin, elastin and galectin 4, fibrin and galectin 3, fibronectin and galectin 1, fibronectin and galectin 3, fibronectin and galectin 4, fibronectin and mucin, fibronectin and SPARC, fibronectin and testican 2, galectin 1 and galectin 3, galectin 1 and keratin, galectin 3 and heparan sulfate, galectin 3 and superfibronectin, galectin 4 and nidogen-1, galectin 8 and tenascin-C, keratin and laminin, laminin and merosin, laminin and thrombospondin-4, SPARC and superfibronectin, and/or superfibronectin and testican 1.
Cell isolation and expansion.
Various terms relating to the methods and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein.
As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise.
The term “about” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
The term “addressable location” as used herein means a discrete surface area or position on a solid support onto which one or a plurality of adhesion sets are immobilized or absorbed such that exposure of the one or plurality of adhesion sets to a sample comprising a biomaterial or cell for a sufficient time period results in contact between the cell or biomaterial and the adhesion set. In some embodiments, the invention relates to an array comprising one or a plurality of addressable locations of the array with a width or diameter of about 10 nanometers. In some embodiments, the invention relates to an array comprising one or a plurality of addressable locations of the array with a width or diameter of about 20 nanometers. In some embodiments, the invention relates to an array comprising one or a plurality of addressable locations of the array with a width or diameter of about 30 nanometers. In some embodiments, the invention relates to an array comprising one or a plurality of addressable locations of the array with a width or diameter of about 40 nanometers. In some embodiments, the invention relates to an array comprising one or a plurality of addressable locations of the array with a width or diameter of about 50 nanometers. In some embodiments, the invention relates to an array comprising one or a plurality of addressable locations of the array with a width or diameter of about 60 nanometers. In some embodiments, the invention relates to an array comprising one or a plurality of addressable locations of the array with a width or diameter of about 70 nanometers. In some embodiments, the invention relates to an array comprising one or a plurality of addressable locations of the array with a width or diameter of about 80 nanometers. In some embodiments, the invention relates to an array comprising one or a plurality of addressable locations of the array with a width or diameter of about 90 nanometers. In some embodiments, the invention relates to an array comprising one or a plurality of addressable locations of the array with a width or diameter of about 100 nanometers. In some embodiments, the one or a plurality of addressable locations of the array is no more than 250 nanometers in diameter. In some embodiments, the one or a plurality of addressable locations of the array is no more than 120 nanometers in diameter or width. In some embodiments, the one or a plurality of addressable locations of the array is no more than 80 nanometers in diameter or width. In some embodiments, the one or a plurality of addressable locations of the array is no more than 70 nanometers in diameter or width. In some embodiments, the one or plurality of addressable locations of the array is no more than 60 nanometers in diameter or width. In some embodiments, the one or plurality of addressable locations of the array is no more than 50 nanometers in diameter or width. In some embodiments, the one or plurality of addressable locations of the array is no more than 40 nanometers in diameter or width. In some embodiments, the one or plurality of addressable locations of the array is no more than 30 nanometers in diameter or width. In some embodiments, the one or plurality of addressable locations of the array is no more than 20 nanometers in diameter or width. In some embodiments, the one or plurality of addressable locations of the array is no more than 10 nanometers in diameter or width. In some embodiments, the one or plurality of addressable locations of the array is from about 10 nanometers in diameter or width to about 100 nanometers in diameter or width. In some embodiments, the one or plurality of addressable locations of the array is spotted manually by a pipet or automatically by a robotic device.
As used herein, the terms “attach,” “attachment,” “adhere,” “adhered,” “adherent,” or like terms generally refer to immobilizing or fixing, for example, a group, a compound or adhesion set, to a surface, such as by physical absorption, chemical bonding, and like processes, or combinations thereof.
The term “adhesion set” or “adhesion sets” as used herein means at least two polypeptides comprising a protein or functional fragment of a protein that are covalently or non-covalently immobilized to a surface at a discrete, addressable location. In some embodiments, the adhesion set comprises a pair of polypeptides or functional fragments thereof. In some embodiments, the adhesion set comprises a plurality of polypeptides or functional fragments thereof covalently or non-covalently bound to a surface at a discrete location. In some embodiments, the adhesion set comprises three or more of polypeptides or functional fragments thereof covalently or non-covalently bound to a surface at a discrete location.
As used herein, the terms “animal-derived ECM material” mean any macromolecule component of an extracellular matrix or biomaterial derived therefrom, including a protein, polysaccharide, polypeptide modified with a polysaccharide, or group of the same that is produced by, originated from, or sourced from an animal species, including a human.
The terms “adhesion value” as used herein means a single quantitative value that can be used as a criterion for whether a particular cell or cell sample expresses or does not express a particular quantity of protein such that, when normalized against a quantitative value calculated for a control tissue, the adhesion value can be used in a predictive model for the diagnosis, prognosis, or clinical treatment plan of a subject. In some embodiments, the adhesion value means a single quantitative value that can be used as a criterion for how tightly or how readily a particular cell or cell sample does or does not associate (or bind) to a particular quantity of protein such that, when normalized against a calculated quantitative value for a reference or control sample, the adhesion value can be used in a predictive model for the diagnosis, prognosis, or clinical treatment plan of a subject. In some embodiments, the quantitative value is calculated by combining quantitative data regarding the association of a cell or cell sample to one or a plurality of adhesion sets through an interpretation function or algorithm described herein. In some embodiments, the subject is suspected of having, is at risk of developing, or has been diagnosed with a metastatic cancer. In some embodiments, the subject is suspected of having, is at risk of developing, or has been diagnosed with a metastatic lung or metastatic breast cancer.
As used herein, the terms “biopsy” means a cell sample, collection of cells, or tissue removed from a subject or patient for analysis. In some embodiments, the biopsy is a bone marrow biopsy, punch biopsy, endoscopic biopsy, needle biopsy, shave biopsy, incisional biopsy, excisional biopsy, or surgical resection.
As used herein the terms “electronic medium” mean any physical storage employing electronic technology for access, including a hard disk, ROM, EEPROM, RAM, flash memory, nonvolatile memory, or any substantially and functionally equivalent medium. In some embodiments, the software storage may be co-located with the processor implementing an embodiment of the invention, or at least a portion of the software storage may be remotely located but accessible when needed.
As used herein, the term “hyperproliferative diseases” is meant to refer to those diseases and disorders characterized by hyperproliferation of cells. Examples of hyperproliferative diseases include all forms of cancer, psoriasis, neoplasia, and hyperplasia.
As used herein, “sequence identity” is determined by using the stand-alone executable BLAST engine program for blasting two sequences (bl2seq), which can be retrieved from the National Center for Biotechnology Information (NCBI) ftp site, using the default parameters (Tatusova and Madden, FEMS Microbiol Lett., 1999, 174, 247-250; which is incorporated herein by reference in its entirety).
The term “subject” is used throughout the specification to describe an animal from which a cell sample is taken. In some embodiment, the animal is a human. For diagnosis of those conditions which are specific for a specific subject, such as a human being, the term “patient” may be interchangeably used. In some instances in the description of the present invention, the term “patient” will refer to human patients suffering from a particular disease or disorder. In some embodiments, the subject may be a human suspected of having or being identified as at risk to develop a hyperproliferative disease. In some embodiments, the subject may be diagnosed as having malignant cancer and of having or being identified as at risk to develop a metastatic hyperproliferative disease. In some embodiments, the subject is suspected of having or has been diagnosed with breast cancer or lung cancer. In some embodiments, the subject may be a human suspected of having or being identified as at risk to develop lung cancer or breast cancer. In some embodiments, the subject may be a mammal which functions as a source of the isolated cell sample. In some embodiments, the subject may be a non-human animal from which a cell sample is isolated or provided. The term “mammal” encompasses both humans and non-humans and includes but is not limited to humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.
As used herein, “conservative” amino acid substitutions may be defined as set out in Tables A, B, or C below. Hyperactive transposases include those wherein conservative substitutions have been introduced by modification of polynucleotides encoding polypeptides of the invention. Amino acids can be classified according to physical properties and contribution to secondary and tertiary protein structure. A conservative substitution is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties. Exemplary conservative substitutions are set out in Table A.
Alternately, conservative amino acids can be grouped as described in Lehninger, (Biochemistry, Second Edition; Worth Publishers, Inc. NY, N.Y. (1975), pp. 71-77) as set forth in Table B.
Alternately, exemplary conservative substitutions are set out in Table C.
It should be understood that the polypeptides comprising polypeptide sequences associated with the extracellular matrix described herein are intended to include polypeptides bearing one or more insertions, deletions, or substitutions, or any combination thereof, of amino acid residues as well as modifications other than insertions, deletions, or substitutions of amino acid residues.
As used herein, the term “prognosing” means determining the probable course and outcome of a disease.
As used herein, the term “functional fragment” means any portion of a polypeptide that is of a sufficient length to retain at least partial biological function that is similar to or substantially similar to the wild-type polypeptide upon which the fragment is based. In some embodiments, a functional fragment of a polypeptide associated with the extracellular matrix is a polypeptide that comprises 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity of any polypeptide disclosed in Table 1 and has sufficient length to retain at least partial binding affinity to one or a plurality of ligands that bind to the polypeptide in Table 1. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 contiguous amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 50 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 100 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table I and has a length of at least about 150 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 200 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table I and has a length of at least about 250 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 300 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 350 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 400 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 450 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 500 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 550 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 600 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 650 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 700 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 750 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 800 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 850 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 900 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 950 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 1000 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 1050 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 1250 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 1500 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 1750 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 2000 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 2250 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 2500 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 2750 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed in Table 1 and has a length of at least about 3000 amino acids.
As used herein, the terms “polypeptide sequence associated with the extracellular matrix” means any polypeptide or fragment thereof, modified or unmodified by any macromolecule (such as a sugar molecule or macromolecule), that is produced naturally by cells in any multicellular organism and is an ECM component or whose structure is based upon an ECM component. In some embodiments, a polypeptide sequence associated with the extracellular matrix is any polypeptide that polypeptide sequence comprising any of the polypeptides disclosed in Table 1. In some embodiments, a polypeptide sequence associated with the extracellular matrix is any polypeptide sequence comprising any of the polypeptides disclosed in Table 1 or a sequence that shares 85, 90, 95, 96, 97, 98, or 99% sequence identity with the polypeptides disclosed in Table 1 or a functional fragment thereof. In some embodiments, a polypeptide sequence associated with the extracellular matrix consists of any of the polypeptides disclosed in Table 1 or a sequence that shares 85, 90, 95, 96, 97, 98, or 99% sequence identity with the polypeptides disclosed in Table 1.
As used herein, the terms “xeno-free” media mean cell culture media free of animal serum or animal-derived components or macromolecules, except those proteins or other macromolecules derived and/or isolated from human tissue or human samples. In some embodiments, the arrays, the systems, kits or the composition described herein comprise xeno-free media. In some embodiments, the methods described herein comprise a step of culturing or contacting cells (such as stem cells) in the presence of xeno-free media. In some embodiments, the array or system does not comprise animal-derived ECM material. In some embodiments, the array or system or kit comprises xeno-free media. In some embodiments, the array or system or kit comprises media free of animal-derived components. In some embodiments, the system or array is free of any macromolecule derived from an animal, except a human. In some embodiments, the system or array is free of any macromolecule derived from an animal.
As used herein, the terms “media free of animal-derived components” mean any cell media that is free of any macromolecule component of an extracellular matrix or biomaterial derived therefrom, including a protein, polysaccharide, polypeptide modified with a polysaccharide, or group of the same that is produced by, originated from, or sourced from an animal species, including a human. In some embodiments, media free of animal-derived components comprises vegetable-derived components. In some embodiments, the media free of animal-derived components comprises only synthetic ECM components. In some embodiments, media free of animal-derived components does not comprise vegetable-derived components or macromolecules. In some embodiments, media free of animal-derived components does not comprise any human-derived ECM material or components. In some embodiments, the arrays, the systems, kits or the composition described herein comprise media free of animal-derived components. In some embodiments, the methods described herein comprise a step of culturing or contacting cells (such as stem cells) in the presence of media free of animal-derived components.
Adhesion signature: An “adhesion signature”, as that term is used herein, refers to a set of ECM binding affinity values (or range(s) of values) sufficient to characterize or distinguish a particular cell or cell type of interest from one or more different cells or cell types. In some embodiments, an adhesion signature includes a binding affinity value or range for at least one ECM component; in some embodiments, an adhesion signature includes binding affinity values or ranges for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more different ECM components and/combinations thereof. In some embodiments, an adhesion signature is a collection of data collected by a user of an array or system disclosed herein related to the quantity, intensity, presence, absence of cellular binding of a cell in a cell sample to one or more adhesion sets relative to the quantity, intensity, presence, absence of binding of a reference, or control, cell or reference cell sample. In some embodiments, the adhesion signature is a collection of data collected by a user of an array or system disclosed herein related to the quantity or proportion of cells that bind one or more adhesion sets as compared to the quantity or proportion of reference cells or control cells that bind the same one or more adhesion sets. In some embodiments, adhesion values are quantified by measuring the number of cells bound to one or more adhesion sets through fluorescent microscopy after staining the cells in a cell sample with fluorescent dye or other fluorescent marker.
“Cell type” means the organism, organ, and/or tissue type from which the cell is derived or sourced, state of development, phenotype or any other categorization of a particular cell that appropriately forms the basis for defining it as “similar to” or “different from” another cell or cells.
Affinity: As is known in the art, “affinity” is a measure of the tightness with which a particular ligand binds to (e.g., associates non-covalently with) and/or the rate or frequency with which it dissociates from, its partner. As is known in the art, any of a variety of technologies can be utilized to determine affinity. In many embodiments, affinity represents a measure of specific binding. In some embodiments a binding affinity is a measure of binding between a cell and an ECM component or collection of ECM components. In some embodiments, a binding affinity of cells to ECM components is expressed relative to binding affinities of cells to other ECM components. In some embodiments, a relative binding affinity of cells to an ECM component or collection of ECM components is expressed as a fold change relative to an average of all binding affinities of cells to ECM components or collection of ECM components assayed. In some embodiments, a relative binding affinity is 0. In some embodiments, a relative binding affinity is between 0 and 1. In some embodiments, a relative binding affinity is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more fold. In some embodiments, a relative binding affinity is between 0 and −1. In some embodiments, a relative binding affinity is −1, −2, −3, −4, −5, −6, −7, −8, −9, −10 or more fold.
Aggrecan polypeptide: In accordance with the present invention, the term “aggrecan polypeptide” is used to refer to a polypeptide that 1) shares an overall level of sequence identity and/or 2) shares at least one characteristic sequence element with an aggrecan protein, for example as set forth in Table 1 of the Appendix.
Agrin polypeptide: In accordance with the present invention, the term “agrin polypeptide” is used to refer to a polypeptide that 1) shares an overall level of sequence identity and/or 2) shares at least one characteristic sequence element with an agrin protein, for example as set forth in Table 1 of the Appendix.
Antibody: As used herein, the term “antibody” refers to any immunoglobulin, whether natural or wholly or partially synthetically produced. In some embodiments, an antibody is a complex comprised of 4 full-length polypeptide chains, each of which includes a variable region and a constant region, e.g., substantially of the structure of an antibody produced in nature by a B cell. In some embodiments, an antibody is a single chain. In some embodiments, an antibody is cameloid. In some embodiments, an antibody is an antibody fragment. In some embodiments, an antibody is chimeric. In some embodiments, an antibody is bi-specific. In some embodiments, an antibody is multi-specific. In some embodiments, an antibody is monoclonal. In some embodiments, an antibody is polyclonal. In some embodiments, an antibody is conjugated (i.e., antibodies conjugated or fused to other proteins, radiolabels, cytotoxins). In some embodiments, an antibody is a human antibody. In some embodiments, an antibody is a mouse antibody. In some embodiments, an antibody is a rabbit antibody. In some embodiments, an antibody is a rat antibody. In some embodiments, an antibody is a donkey antibody.
Array: An “array”, as that term is used herein, typically refers to an arrangement of entities (e.g., ECM components) in spatially discrete locations with respect to one another, and usually in a format that permits simultaneous exposure of the arranged entities to potential interaction partners (e.g., cells) or other reagents, substrates, etc. In some embodiments, an array comprises entities arranged in spatially discrete locations on a solid support. In some embodiments, spatially discrete locations on an array are termed “spots” (regardless of their shape). In some embodiments, spatially discrete locations on an array are arranged in a regular pattern with respect to one another (e.g., in a grid).
Biglycan polypeptide: In accordance with the present invention, the term “biglycan polypeptide” is used to refer to a polypeptide that 1) shares an overall level of sequence identity and/or 2) shares at least one characteristic sequence element with a biglycan protein, for example as set forth in Table 1 of the Appendix.
Binding partners: In general, the term “binding partner” is used herein to refer to any two entities that specifically bind with each other in a given context. In some embodiments, binding is specific in that a binding agent has a greater affinity for its target binding partner than for other potential binding partners in its environment. Binding partners may be of any chemical type. In some embodiments, binding partners are polypeptides. In some embodiments, binding partners are integrins, syndecans, proteoglycans, glycosaminoglycans, and/or lectins. In some embodiments, binding partners are carbohydrates.
Brevican polypeptide: In accordance with the present invention, the term “brevican polypeptide” is used to refer to a polypeptide that 1) shares an overall level of sequence identity and/or 2) shares at least one characteristic sequence element with a brevican protein, for example as set forth in Table 1 of the Appendix.
Collagen I polypeptide: In accordance with the present invention, the term “collagen I polypeptide” is used to refer to a polypeptide that 1) shares an overall level of sequence identity and/or 2) shares at least one characteristic sequence element with a collagen I protein, for example as set forth in Table 1 of the Appendix.
Collagen II polypeptide: In accordance with the present invention, the term “collagen II polypeptide” is used to refer to a polypeptide that 1) shares an overall level of sequence identity and/or 2) shares at least one characteristic sequence element with a collagen II protein, for example as set forth in Table 1 of the Appendix.
Collagen III polypeptide: In accordance with the present invention, the term “collagen III polypeptide” is used to refer to a polypeptide that 1) shares an overall level of sequence identity and/or 2) shares at least one characteristic sequence element with a collagen III protein, for example as set forth in Table 1 of the Appendix.
Collagen IV polypeptide: In accordance with the present invention, the term “collagen IV polypeptide” is used to refer to a polypeptide that 1) shares an overall level of sequence identity and/or 2) shares at least one characteristic sequence element with a collagen IV protein, for example as set forth in Table 1 of the Appendix.
Collagen V polypeptide: In accordance with the present invention, the term “collagen V polypeptide” is used to refer to a polypeptide that 1) shares an overall level of sequence identity and/or 2) shares at least one characteristic sequence element with a collagen V protein, for example as set forth in Table 1 of the Appendix.
Collagen VI polypeptide: In accordance with the present invention, the term “collagen VI polypeptide” is used to refer to a polypeptide that 1) shares an overall level of sequence identity and/or 2) shares at least one characteristic sequence element with a collagen VI protein, for example as set forth in Table 1 of the Appendix.
Characteristic: As is used herein, the term “characteristic” refers to any detectable feature of a cell type that allows it to be distinguished from a comparable cell type. In some embodiments, a characteristic is an amount or sequence of a gene. In some embodiments, a characteristic is an amount or sequence of a gene transcript. In some embodiments, a characteristic is an amount, sequence of, or modification of a protein. In some embodiments a characteristic is an amount of a carbohydrate. In some embodiments, a characteristic is an amount of a small molecule. In some embodiments, a characteristic is an amount of an ECM component.
Comparable: As is used herein, the term “comparable” is used to refer to two entities that are sufficiently similar to permit comparison, but differing in at least one feature.
Decorin polypeptide: In accordance with the present invention, the term “decorin polypeptide” is used to refer to a polypeptide that 1) shares an overall level of sequence identity and/or 2) shares at least one characteristic sequence element with a decorin protein, for example as set forth in Table 1 of the Appendix.
ECM component: In accordance with the present invention, the term “ECM component” is used to refer to any molecule or molecular complex that is part of an ECM of a cell and that has contributes to one or more adhesion signatures for a cell. In some embodiments, an ECM component is or comprises a polypeptide. In some embodiments, an ECM component is or comprises a polysaccharide. In some embodiments, an ECM component is or comprises a glycosaminoglycan. In some embodiments, an ECM component is or comprises a proteoglycan. In some embodiments an ECM component comprises a carbohydrate. In some embodiments, the ECM component is any fragment of a polypeptide, glycosaminoglycan, proteoglycan, or carbohydrate disclosed herein. In some embodiments, the ECM component is a polypeptide that shares at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the polypeptides disclosed in Table 1.
Elastin polypeptide: In accordance with the present invention, the term “elastin polypeptide” is used to refer to a polypeptide that 1) shares an overall level of sequence identity and/or 2) shares at least one characteristic sequence element with an elastin protein, for example as set forth in Table 1 of the Appendix.
F-Spondin polypeptide: In accordance with the present invention, the term “F-spondin polypeptide” is used to refer to a polypeptide that 1) shares an overall level of sequence identity and/or 2) shares at least one characteristic sequence element with an F-spondin protein, for example as set forth in Table 1 of the Appendix.
Fibrin polypeptide: In accordance with the present invention, the term “fibrin polypeptide” is used to refer to a polypeptide that 1) shares an overall level of sequence identity and/or 2) shares at least one characteristic sequence element with a fibrin protein, for example as set forth in Table 1 of the Appendix.
Fibronectin polypeptide: In accordance with the present invention, the term “fibronectin polypeptide” is used to refer to a polypeptide that 1) shares an overall level of sequence identity and/or 2) shares at least one characteristic sequence element with a fibronectin protein, for example as set forth in Table 1 of the Appendix.
Galectin 1 polypeptide: In accordance with the present invention, the term “galectin 1 polypeptide” is used to refer to a polypeptide that 1) shares an overall level of sequence identity and/or 2) shares at least one characteristic sequence element with a galectin 1 protein, for example as set forth in Table 1 of the Appendix.
Galectin 3 polypeptide: In accordance with the present invention, the term “galectin 3 polypeptide” is used to refer to a polypeptide that 1) shares an overall level of sequence identity and/or 2) shares at least one characteristic sequence element with a galectin 3 protein, for example as set forth in Table 1 of the Appendix.
Galectin 3c polypeptide: In accordance with the present invention, the term “galectin 3c polypeptide” is used to refer to a polypeptide that 1) shares an overall level of sequence identity and/or 2) shares at least one characteristic sequence element with a galectin 3c protein, for example as set forth in Table 1 of the Appendix.
Galectin 4 polypeptide: In accordance with the present invention, the term “galectin 4 polypeptide” is used to refer to a polypeptide that 1) shares an overall level of sequence identity and/or 2) shares at least one characteristic sequence element with a galectin 4 protein, for example as set forth in Table 1 of the Appendix.
Galectin 8 polypeptide: In accordance with the present invention, the term “galectin 8 polypeptide” is used to refer to a polypeptide that 1) shares an overall level of sequence identity and/or 2) shares at least one characteristic sequence element with a galectin 8 protein, for example as set forth in Table 1 of the Appendix.
Glycosaminoglycan: In accordance with the present invention, the term “glycosaminoglycan” is used to refer to an unbranched polysaccharides consisting of a repeating disaccharide unit. The repeating unit consists of a hexose or a hexuronic acid, linked to a hexosamine.
Keratin polypeptide: In accordance with the present invention, the term “keratin polypeptide” is used to refer to a polypeptide that 1) shares an overall level of sequence identity and/or 2) shares at least one characteristic sequence element with a keratin protein, for example as set forth in Table 1 of the Appendix.
Kit: As used herein, the term “kit” refers to a set of components provided in the context of a delivery system for delivering materials. Such delivery systems may include, for example, systems that allow for storage, transport, or delivery of various diagnostic or therapeutic reagents (e.g., oligonucleotides, enzymes, extracellular matrix components etc. in appropriate containers) and/or supporting materials (e.g., buffers, media, cells, written instructions for performing the assay etc.) from one location to another. For example, in some embodiments, kits include one or more enclosures (e.g., boxes) containing relevant reaction reagents and/or supporting materials. As used herein, the term “fragmented kit” refers to delivery systems comprising two or more separate containers that each contain a subportion of total kit components. Containers may be delivered to an intended recipient together or separately. For example, a first container may contain a petri dish or polysterence plate for use in a cell culture assay, while a second container may contain cells. The term “fragmented kit” is intended to encompass kits containing Analyte Specific Reagents (ASR's) regulated under section 520(e) of the Federal Food, Drug, and Cosmetic Act, but are not limited thereto. Indeed, any delivery system comprising two or more separate containers that each contain a subportion of total kit components are included in the term “fragmented kit.” In contrast, a “combined kit” refers to a delivery system containing all components in a single container (e.g., in a single box housing each of the desired components). The term “kit” includes both fragmented and combined kits.
Laminin polypeptide: In accordance with the present invention, the term “laminin polypeptide” is used to refer to a polypeptide that 1) shares an overall level of sequence identity and/or 2) shares at least one characteristic sequence element with a laminin protein, for example as set forth in Table 1 of the Appendix.
Lineage: In accordance with the present invention, the term “lineage” encompasses cells at any point in a developmental process from undifferentiated cells to fully differentiated cells of a specific cell type.
Merosin polypeptide: In accordance with the present invention, the term “merosin polypeptide” is used to refer to a polypeptide that 1) shares an overall level of sequence identity and/or 2) shares at least one characteristic sequence element with a merosin protein, for example as set forth in Table 1 of the Appendix.
Mucin polypeptide: In accordance with the present invention, the term “mucin polypeptide” is used to refer to a polypeptide that 1) shares an overall level of sequence identity and/or 2) shares at least one characteristic sequence element with a mucin protein, for example as set forth in Table 1 of the Appendix.
Nidogen-1 polypeptide: In accordance with the present invention, the term “nidogen-1 polypeptide” is used to refer to a polypeptide that 1) shares an overall level of sequence identity and/or 2) shares at least one characteristic sequence element with a nidogen-1 protein, for example as set forth in Table 1 of the Appendix.
Nidogen-2 polypeptide: In accordance with the present invention, the term “nidogen-2 polypeptide” is used to refer to a polypeptide that 1) shares an overall level of sequence identity and/or 2) shares at least one characteristic sequence element with a nidogen-2 protein, for example as set forth in Table 1 of the Appendix.
Osteopontin polypeptide: In accordance with the present invention, the term “osteopontin polypeptide” is used to refer to a polypeptide that 1) shares an overall level of sequence identity and/or 2) shares at least one characteristic sequence element with an osteopontin protein, for example as set forth in Table 1 of the Appendix.
Polypeptide: The term “polypeptide”, as used herein, generally has its art-recognized meaning of a polymer of at least three amino acids. Those of ordinary skill in the art will appreciate that the term “polypeptide” is intended to be sufficiently general as to encompass not only polypeptides having the complete sequence recited herein, but also to encompass polypeptides that represent functional fragments (i.e., fragments retaining at least one activity) of such complete polypeptides. Moreover, those of ordinary skill in the art understand that protein sequences generally tolerate some substitution without destroying activity. Thus, any polypeptide that retains activity and shares at least about 30-40% overall sequence identity, often greater than about 50%, 60%, 70%, or 80%, and further usually including at least one region of much higher identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99% in one or more highly conserved regions, usually encompassing at least 3-4 and often up to 20 or more amino acids, with another polypeptide of the same class, is encompassed within the relevant term “polypeptide” as used herein.
Reference cell: As will be understood from context, a reference cell or cell type is one that is sufficiently similar to a particular cell or cell type of interest to permit a relevant comparison. In some embodiments, information about a reference cell or cell type is obtained simultaneously with information about the particular cell or cell type. In some embodiments, information about a reference cell or cell type is historical. In some embodiments, information about a reference cell or cell type is stored for example in a computer-readable medium. In some embodiments, comparison of a particular cell or cell type of interest with a reference cell or cell type establishes identity with, similarity to, or difference of the particular cell or cell type of interest relative to the reference.
Sample: As used herein, the term “sample” refers to a biological sample obtained or derived from a source of interest, as described herein. In some embodiments, a source of interest comprises an organism, such as an animal or human. In some embodiments, a biological sample comprises biological tissue or fluid. In some embodiments, a biological sample may be or comprise bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy samples; cell-containing body fluids; free floating nucleic acids; sputum; saliva; urine; cerebrospinal fluid, peritoneal fluid; pleural fluid; feces; lymph; gynecological fluids; skin swabs; vaginal swabs; oral swabs; nasal swabs; washings or lavages such as a ductal lavages or bronchioalveolar lavages; aspirates; scrapings; bone marrow specimens; tissue biopsy specimens; surgical specimens; feces, other body fluids, secretions, and/or excretions; and/or cells therefrom, etc. In some embodiments, a biological sample is or comprises cells obtained from an individual. In some embodiments, a sample is a “primary sample” obtained directly from a source of interest by any appropriate means. For example, in some embodiments, a primary biological sample is obtained by methods selected from the group consisting of biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, collection of body fluid (e.g., blood, lymph, feces etc.), etc. In some embodiments, as will be clear from context, the term “sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and/or by adding one or more agents to) a primary sample. For example, filtering using a semi-permeable membrane. Such a “processed sample” may comprise, for example nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to techniques such as amplification or reverse transcription of mRNA, isolation and/or purification of certain components, etc.
Superfibronectin polypeptide: In accordance with the present invention, the term “superfibronectin polypeptide” is used to refer to a polypeptide that 1) shares an overall level of sequence identity and/or 2) shares at least one characteristic sequence element with a superfibronectin protein, for example as set forth in Table 1 of the Appendix.
SPARC/Osteonectin polypeptide: In accordance with the present invention, the term “SPARC/osteonectin polypeptide” is used to refer to a polypeptide that 1) shares an overall level of sequence identity and/or 2) shares at least one characteristic sequence element with a SPARC/osteonectin protein, for example as set forth in Table 1 of the Appendix.
Tenascin-C polypeptide: In accordance with the present invention, the term “tenascin-C polypeptide” is used to refer to a polypeptide that 1) shares an overall level of sequence identity and/or 2) shares at least one characteristic sequence element with a tenascin-C protein, for example as set forth in Table 1 of the Appendix.
Tenascin-R polypeptide: In accordance with the present invention, the term “tenascin-R polypeptide” is used to refer to a polypeptide that 1) shares an overall level of sequence identity and/or 2) shares at least one characteristic sequence element with a tenascin-R protein, for example as set forth in Table 1 of the Appendix.
Testican 1/SPOCK1 polypeptide: In accordance with the present invention, the term “testican 1/SPOCK1 polypeptide” is used to refer to a polypeptide that 1) shares an overall level of sequence identity and/or 2) shares at least one characteristic sequence element with a testican 1/SPOCK1 protein, for example as set forth in Table 1 of the Appendix.
Testican 2/SPOCK2 polypeptide: In accordance with the present invention, the term “testican 2/SPOCK2 polypeptide” is used to refer to a polypeptide that 1) shares an overall level of sequence identity and/or 2) shares at least one characteristic sequence element with a testican 2/SPOCK2 protein, for example as set forth in Table 1 of the Appendix.
Thrombospondin-4 polypeptide: In accordance with the present invention, the term “thrombospondin-4 polypeptide” is used to refer to a polypeptide that 1) shares an overall level of sequence identity and/or 2) shares at least one characteristic sequence element with a thrombospondin-4 protein, for example as set forth in Table 1 of the Appendix.
Vitronectin polypeptide: In accordance with the present invention, the term “vitronectin polypeptide” is used to refer to a polypeptide that 1) shares an overall level of sequence identity and/or 2) shares at least one characteristic sequence element with a vitronectin protein, for example as set forth in Table 1 of the Appendix.
Many significant cellular components reside within the extracellular matrix (ECM), a gelatinous layer on the exterior surface of cells. The ECM plays a variety of important roles, including serving as scaffolding for cellular components and providing biochemical and mechanical cues involved in intracellular communication and tissue differentiation. The ECM includes proteoglycan and fibrous protein, typically produced within cells and then secreted to form the ECM.
ECMs of different cell types are highly variable. For example, differing ECM compositions of different types of fibroblasts determine properties of connective tissue. Chondrocytes secrete an ECM composed primarily of collagen II, which forms cartilage, whereas osteoplasts secrete an ECM composed primarily of osteoid, a progenitor of bone tissue. This variability is created during development by an interaction of cells with the microenvironments in which they are located.
In addition to providing structural support, another important role of the ECM is intracellular communication, in particular through integrins. Integrins are cell surface receptors that regulate attachment of a cell to the ECM, and also transduce intracellular signals from the ECM to the interior of a cell. In addition to having a unique ECM composition, each respective cell type also has an individualized profile of cell surface integrins and other receptors for best interacting with its specific ECM. Thus, the ECM composition and the affinity for ECM components of a cell represents a potentially useful way of distinguishing between genetically identical cell types.
The present invention relates generally to definition and/or use of adhesion signatures that embody or characterize a cell's affinity for of Extracellular Matrix (ECM) components.
In some embodiments, an ECM component is or comprises any polypeptide present in the ECM. In some embodiments, an ECM component is or comprises an aggrecan polypeptide, an agrin polypeptide, a biglycan polypeptide, a brevican polypeptide, a collagen I polypeptide, a collagen II polypeptide, a collagen III polypeptide, a collagen IV polypeptide, a collagen V polypeptide, a collagen VI polypeptide, a decorin polypeptide, an elastin polypeptide, an f-spondin polypeptide, a fibrin polypeptide, a fibronectin polypeptide, a galectin 1 polypeptide, a galectin 3 polypeptide, a galectin 3c polypeptide, a galectin 4 polypeptide, a galectin 8 polypeptide, a keratin polypeptide, a laminin polypeptide, a merosin polypeptide, a mucin polypeptide, nidogen-1 polypeptide, a nidogen-2 polypeptide, an osteopontin polypeptide, a SPARC/osteonectin superfibronectin polypeptide, a tenascin-C polypeptide, a tenascin-R polypeptide, a testican 1/SPOCK1 polypeptide, a testican 2/SPOCK2 polypeptide, a thrombospondin-4 polypeptide, a vitronectin polypeptide and/or combinations thereof.
In some embodiments, an ECM component is or comprises one or more carbohydrate moieties. In some embodiments, an ECM component is or comprises a carbohydrate moiety that is naturally found in ECM produced by cells (e.g., on an ECM polypeptide). Representative such carbohydrate moieties include, for example, ECM components chondroitin sulfate glycosaminoglycans, heparan sulfate glycosaminoglycans, hyaluronic acid glycosaminoglycans or other glycosaminoglycans, and/or combinations thereof.
In some embodiments, an ECM component is or comprises a protein, peptide, glycoprotein, proteoglycans, glycosaminoglycans, and/or carbohydrate that is secreted by cells into the extracellular environment. In some embodiments, the secreted protein, peptide, glycoprotein, proteoglycans, glycosaminoglycans, and/or carbohydrate, or structures composed thereof can be bound to by cells as a means of immobilizing the cell permanently or transiently (as in cases of providing a means for directional motility).
ECM components interact with cells, typically through non-covalent binding interactions with one or more entities on or near cell surfaces. In some embodiments, cell components that interact or bind with ECM components include entities selected from groups consisting of cell membranes, cell surface entities (e.g., proteins, proteoglycans, glycoproteins, etc.), secreted entities (e.g., cell signaling molecules), laminins, integrins, syndecans, and actin.
In various embodiments, the present invention is useful in the identification, characterization, detection, isolation, and/or culturing of cells. In general, teachings of the invention are relevant to any cell that has, produces, and/or interacts with an ECM or ECM component(s).
In some embodiments, cells utilized in accordance with the present invention are cells that retain viability, and optionally growth capabilities, when suspended in solution. In some embodiments, cells are eukaryotic cells. In certain embodiments, cells are human cells. In some embodiments, cells are mouse cells. In certain embodiments, cells are obtained from cell culture. In some embodiments, cells are obtained from a living organism. In some embodiments, cells are hepatic cells. In some embodiments, cells are immune cells. In certain embodiments, cells are blood cells. In some embodiments, cells are nerve cells. In certain embodiments, cells are epithelial cells. In certain embodiments, cells are reproductive cells. In some embodiments, cells are stem cells. In some embodiments, cells are cancer cells. In some embodiments, the cell sample comprises an individual cell. In some embodiments, the cell sample is a composition comprising a plurality of cells. In some embodiments, the cell sample is a tissue sample taken from a subject suspected of having cancer or being diagnosed as having cancer. In some embodiments, the cell sample is a tissue sample taken from a subject with lung cancer or breast cancer. In some embodiments, the cell sample comprises a plurality of cells from the adrenal gland, bladder, blood, bone, bone marrow, brain, spine, breast, cervix, gall bladder, ganglia, gastrointestinal tract, stomach, colon, heart, kidney, liver, lung, lymphnodes, muscle, overay, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, or uterus. In some embodiment, the cell sample comprises a plurality of cells derived from the lung. In some embodiments, the cell sample comprises a plurality of cells derived from the breast.
In various embodiments, the present invention is useful in identification, characterization, detection, isolation, and/or culturing of stem cells at particular states of differentiation. As is commonly understood in the art, stem cells are cells with a capacity to differentiate into diverse specialized cell types. Different types of stem cells are at different stages of differentiation, ranging from completely undifferentiated (totipotent) to mostly differentiated (multipotent). In some embodiments stem cells are totipotent stem cells (e.g., undifferentiated cells having an ability to differentiate into any mature cell type). Types of totipotent stem cells include, for example, embryonic stem cells. In some embodiments, stem cells are pluripotent stem cells (e.g., having an ability to differentiate into most mature cell types). Types of pluripotent stem cells include, for example, induced pluripotent stem cells. In some embodiments, stem cells are multipotent stem cells (e.g., having an ability to differentiate into several related types of cells). Types of multipotent stem cells include, for example, mesenchymal stem cells. In some embodiments, mesenchymal stem cells are derived from bone marrow, adipose tissue, umbilical cord blood and/or umbilical cord. In certain embodiments, cells utilized in accordance with the present invention are cells differentiated from stem cells.
In various embodiments, the present invention is useful in identification, characterization, detection, isolation, and/or culturing of cancer cells generally and specifically cancer cells at particular states of metastasis. As is commonly understood in the art, metastasis is a process of cancer spreading from an initial tumor site and is correlated with a poor prognosis for cancer patients. Metastatic cells are characterized by an altered gene expression profile that directly correlates with ability to metastasize (Ramaswamy S. et al. “A molecular signature of metastasis in primary solid tumors”. Nature Genetics 33 (1): 49-54, 2003). Types of cancer cells include but are not limited to lung adenocarcinoma cells, non-metastatic primary tumor cells, metastatic primary tumor cells, metastatic lymph node cells, metastatic liver cells, breast cancer cells, colon cancer cells, prostate cancer cells, ovarian cancer cells, testicular cancer cells and/or leukemia cells.
In accordance with certain embodiments of the present invention, cells are contacted with ECM components, under conditions and for a time sufficient to allow cells to bind to ECM components. In certain embodiments, contacted cells are suspended in a solution. In some embodiments, cells are suspended at a concentration ranging from 100 to 10,000,000 cells/ml, from 1,000 to 1,000,000 cells/ml, or from 10,000 to 100,000 cells/ml. In one exemplary embodiment, cells are suspended at a concentration of 80,000 cells/ml. In certain embodiments, cells and ECM components are contacted in the presence of culture media. Any of a variety of cell culture media, including complex media and/or serum-free culture media, that support survival and/or growth of the one or more cell types or cell lines may be used in accordance with the present disclosure. Typically, a cell culture medium contains a buffer, salts, energy source, amino acids, vitamins and/or trace elements. Cell culture media may optionally contain a variety of other ingredients, including but not limited to, carbon sources, cofactors, lipids, sugars, nucleosides, animal-derived components, hydrolysates, hormones/growth factors, surfactants, indicators, minerals, activators/inhibitors of specific enzymes, and organics, and/or small molecule metabolites.
In certain embodiments, cell culture media utilized in accordance with the present invention is or comprises serum-free cell culture media. In certain embodiments, utilized cell culture media is fully defined synthetic cell culture media. In certain embodiments, utilized cell culture media is Dulbecco's Modified Eagle Medium (DMEM). In certain embodiments, utilized cell culture media is RPMI, Ham's F-12, or Mammary Epithelial Cell Growth Media (MEGM). In some embodiments, the cell culture media comprises additional components including Fetal Bovine Serum (FBS), Bovine Serum (BS), and/or Glutamine or combinations thereof. In some embodiments, utilized media are supplemented with an antibiotic to prevent contamination. Useful antibiotics in such circumstances include, for example, penicillin, streptomycin, and/or gentamicin and combinations thereof. Those of skill in the art are familiar with parameters relevant to selection of appropriate cell culture media.
In many embodiments, an array comprises a solid support to whose surface(s) ECM components are affixed in spatially discrete locations. Such an array can be prepared using ECM components from any source (e.g., recombinantly produced, biochemically isolated, commercially purchased, etc). Moreover, identity and relative amounts of individual ECM components may be determined or adjusted in accordance with requirements of a particular project or interests of a particular researcher.
For example, in many embodiments, it will be desirable to design, prepare and/or utilize an ECM array that includes as many different ECM components as is feasible. Alternatively or additionally, in some embodiments, it may be desirable to design, prepare, and/or utilize an ECM array that includes only ECM components known to be associated with (or not associated with) a particular cell or cell type. To give a few particular examples, in some embodiments, an ECM array is utilized that contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more different “spots” (physically discrete locations) containing different ECM components. In some embodiments, an ECM array is utilized that contains between about 1 and about 100,000 spots, between about 100 and about 10,000, or between about 1,000 and about 5,000 spots.
In some embodiments, spots on an array show spatial organization. In some embodiments, spots on an array are arranged in a grid.
In some embodiments, a variety of ECM components and combinations thereof are represented in spots of an ECM array with each spot corresponding to both a known location on the ECM array and a known composition of ECM components. In certain embodiments, at least one ECM component is spotted upon the ECM array. In certain embodiments, the ECM components are spotted individually. In some embodiments, mixtures of several ECM components are contained within a single spot. In some embodiments, an ECM array for use in accordance with the present invention includes both spots of single ECM components and spots of combinations of ECM components. In some embodiments, ECM components are spotted multiple times in the same array, so that the array includes replicate spots. In some embodiments, an ECM array for use in accordance with the present invention contains spots that lack an ECM component, and therefore for example may be utilized as negative controls in addition to spots containing ECM components. In certain embodiments, rhodamine dextran is included in a negative control spot.
An ECM array for use in accordance with the present invention may be prepared on any suitable substrate material. In many embodiments, the material will support viability and/or growth of cells, e.g., mammalian cells. In some embodiments, an ECM arrays utilizes a substrate material selected from the group consisting of polyamides, polyesters, polystyrene, polypropylene, polyacrylates, polyvinyl compounds (e.g. polyvinylchloride), polycarbonate, polytetrafluoroethylene (PTFE), nitrocellulose, cotton, polyglycolic acid (PGA), cellulose, dextran, gelatin, glass, fluoropolymers, fluorinated ethylene propylene, polyvinylidene, polydimethylsiloxane, polystyrene, silicon substrates (such as fused silica, polysilicon, or single silicon crystals), and the like, or combinations thereof. Alternatively or additionally, metals (gold, silver, titanium films) can be used. In a some embodiments, acrylic slides coated with polyacrylamide are used.
In some embodiments, the present invention provides ECM arrays for use in culturing cells. In some embodiments the ECM arrays for use in culturing cells are provided with medium. In some embodiments the ECM arrays for use in culturing cells are provided with a sufficient volume of medium to support cell culture for 1, 2, 3, 4, 5 or more days.
In some embodiments, the present invention provides ECM arrays for use as diagnostic assays. In some embodiments the ECM arrays are provided as part of a diagnostic or detection kit. In some embodiments the ECM arrays are provided as part of a detection kit. In certain embodiments, kits for use in accordance with the present invention may include one or more reference samples; instructions (e.g., for processing samples, for performing tests, for interpreting results, etc.); media; and/or other reagents necessary for performing tests.
The invention provides an array of polypeptides, the array comprising: a solid support and a plurality of adhesion sets, wherein each adhesion set comprises two or more different polypeptides comprising a polypeptide sequence associated with the extracellular matrix or a functional fragment thereof, and wherein the adhesion sets are attached to the solid support at an addressable location of the array. In some embodiments, the solid support is a slide optionally coated with a polymer. In some embodiments, the solid support is coated with a polymer. In some embodiments, the polymer is polyacrylamide. In some embodiments, the solid support is a material chosen from: polystyrene (TCPS), glass, quarts, quartz glass, poly(ethylene terephthalate) (PET), polyethylene, polyvinyl difluoride (PVDF), polydimethylsiloxane (PDMS), polytetrafluoroethylene (PTFE), polymethylmethacrylate (PMMA), polycarbonate, polyolefin, ethylene vinyl acetate, polypropylene, polysulfone, polytetrafluoroethylene, silicones, poly(meth)acrylic acid, polyamides, polyvinyl chloride, polyvinylphenol, and copolymers and mixtures thereof. In some embodiments, the at least one adhesion set comprises two different polypeptides attached to a solid support.
The invention further relates to a system comprising one or a plurality of arrays, wherein the one or plurality of arrays comprises: a solid support and a plurality of adhesion sets, wherein each adhesion set comprises two or more different polypeptides comprising a polypeptide sequence associated with the extracellular matrix or a functional fragment thereof, and wherein the adhesion sets are attached to the solid support at an addressable location of the array. In some embodiments, the one or plurality of arrays comprises a solid support is a slide optionally coated with a polymer. In some embodiments, the solid support is coated with a polymer. In some embodiments, the one or plurality of arrays comprises a solid support coated with a polymer that is polyacrylamide. In some embodiments, the one or plurality of arrays comprises a solid support comprising a material chosen from: polystyrene (TCPS), glass, quarts, quartz glass, poly(ethylene terephthalate) (PET), polyethylene, polyvinyl difluoride (PVDF), polydimethylsiloxane (PDMS), polytetrafluoroethylene (PTFE), polymethylmethacrylate (PMMA), polycarbonate, polyolefin, ethylene vinyl acetate, polypropylene, polysulfone, polytetrafluoroethylene, silicones, poly(meth)acrylic acid, polyamides, polyvinyl chloride, polyvinylphenol, and copolymers and mixtures thereof. In some embodiments, the at least one adhesion set comprises two different polypeptides attached to a solid support. In some embodiments, the system comprises a horizontally positioned or substantially horizontally positioned divide comprising at least one receptacle within which one or a plurality of solid supports is mounted. In some embodiments, the system comprises a horizontally positioned or substantially horizontally positioned divide comprising at least one receptacle and at least one gasket, such that the gasket is mounted between the one or a plurality of arrays and the divide. In some embodiments, the system comprises a horizontally positioned or substantially horizontally positioned divide defining an upper portion and a lower portion of the system wherein the divide comprises at least one receptacle and at least one gasket within which one or a plurality of arrays are mounted such that the gasket is positioned between the array and the divide. In some embodiments, the system comprises: (i) a horizontally positioned or substantially horizontally positioned divide defining an upper portion and a lower portion of the system wherein the divide comprises at least one receptacle and at least one gasket within which one or a plurality of arrays are mounted such that the gasket is positioned between the array and the divide; and (ii) a pair of side walls positioned orthogonally to the divide; and (iii) a base comprising an air inlet positioned between the pair of side walls such that the divide, the pair of side walls, and the base define a cavity; wherein the air inlet is adapted to receive a connector through which a vacuum is drawn, the vacuum capable of drawing fluid from the upper portion of the system to the lower portion. In some embodiments, the system comprises: (i) a horizontally positioned or substantially horizontally positioned divide defining an upper portion and a lower portion of the system wherein the divide comprises at least one receptacle and at least one gasket within which one or a plurality of arrays are mounted such that the gasket is positioned between the array and the divide; and (ii) a pair of side walls positioned orthogonally to the divide; and (iii) a base comprising an air inlet positioned between the pair of side walls such that the divide, the pair of side walls, and the base define a cavity; wherein the air inlet is adapted to receive a connector through which a vacuum is drawn, the vacuum capable of drawing fluid from the upper portion of the system to the lower portion. In some embodiments, the system comprises a vacuum pump operably connected to the base via a tube adapted to fit the air inlet.
The invention relates to a system comprising at least one, two, three, or four arrays as described herein. The invention also relates to a system comprising at least one array comprising at least 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 710, 720, 730, 740, 750, 760, 770, or 780 adhesion sets positioned at separate addressable locations on the at least one array. In some embodiments, the system is free of animal-derived ECM material, embryonic fibroblasts, material deposited from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, or any combination thereof. In some embodiments, the array is free of serum derived or sourced from any animal species. In some embodiments, the system comprises at least one array wherein the at least one array comprises no less than 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, or more adhesion sets comprising at least one polypeptide sequence associated with the extracellular matrix chosen from the polypeptides of Table 1 or functional fragments thereof.
In some embodiments, the system comprises at least one array, prepared by the step comprising: affixing no fewer than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, or 825 adhesion sets to discrete addressable locations on a solid support.
In some embodiments, the system comprises at least one array, prepared by the steps comprising: affixing no fewer than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, or 825 adhesion sets to discrete addressable locations on a solid support; wherein the adhesion sets comprise at least two or more polypeptides each of which comprising a polypeptide sequence associated with the extracellular matrix or a functional fragment thereof chosen from the polypeptides of Table 1. In some embodiments, the system comprises at least one array for the diagnosis or prognosis of a disorder of a patient, prepared by the steps comprising: (i) affixing no fewer than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, or 825 adhesion sets to discrete addressable locations on a solid support. In some embodiments, the system comprises at least one array for the diagnosis or the prognosis of a disorder of a patient, prepared by the steps comprising: (i) affixing no fewer than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, or 825 adhesion sets to discrete addressable locations on a solid support; wherein the adhesion sets comprise at least two or more polypeptides and wherein each of the two or more polypeptides comprises a polypeptide sequence associated with the extracellular matrix or a functional fragment thereof chosen from the polypeptides of Table 1. In some embodiments, the system comprises at least one array comprising a solid support, prepared by the steps comprising: (i) coating a solid support with a polymer; (ii) affixing no fewer than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, or 825 adhesion sets to discrete, addressable locations on the polymer; wherein the adhesion sets comprise at least two or more polypeptides each of which comprising a polypeptide sequence associated with the extracellular matrix or a functional fragment thereof chosen from the polypeptides of Table 1.
In some embodiments, the system comprises at least one array comprising a solid support, prepared by the steps comprising: affixing at least one adhesion set to the solid support; wherein the adhesion set comprises at least two or more polypeptides each comprising a polypeptide sequence associated with the extracellular matrix or a functional fragment thereof chosen from the polypeptides of Table 1. In some embodiments, the system comprises at least one array comprising a solid support, prepared by the steps comprising: affixing at least one adhesion set to the solid support; wherein the adhesion set comprises at least two or more polypeptides each comprising a polypeptide sequence associated with the extracellular matrix or a functional fragment thereof chosen from the polypeptides of Table 1; wherein the solid support comprises a material chosen from: polystyrene (TCPS), glass, quarts, quartz glass, poly(ethylene terephthalate) (PET), polyethylene, polyvinyl difluoride (PVDF), polydimethylsiloxane (PDMS), polytetrafluoroethylene (PTFE), polymethylmethacrylate (PMMA), polycarbonate, polyolefin, ethylene vinyl acetate, polypropylene, polysulfone, polytetrafluoroethylene, silicones, poly(meth)acrylic acid, polyamides, polyvinyl chloride, polyvinylphenol, and copolymers mixtures thereof.
In some embodiments, the system comprises at least one array comprising a solid support, prepared by the steps comprising: (i) preparing a first and second solution, each first and second solution comprising a known concentration of a polypeptide comprising a polypeptide sequence associated with the extracellular matrix or a functional fragment thereof; (ii) contacting the first and second solution with the solid support for a sufficient time period to adsorb polypeptide comprising a polypeptide sequence associated with the extracellular matrix or a functional fragment thereof to the solid support; wherein the polypeptide sequence associated with the extracellular matrix or a functional fragment thereof is chosen from the polypeptides of Table 1. In some embodiments, the system comprises at least one array comprising a solid support, prepared by the steps comprising: (i) preparing a first and second solution, each first and second solution comprising a known concentration of a polypeptide comprising a polypeptide sequence associated with the extracellular matrix or a functional fragment thereof; (ii) contacting the first and second solution with the solid support for a sufficient time period to adsorb polypeptide comprising a polypeptide sequence associated with the extracellular matrix or a functional fragment thereof to the solid support; wherein the polypeptide sequence associated with the extracellular matrix or a functional fragment thereof is chosen from the polypeptides of Table 1; wherein the solid support comprises a material chosen from: polystyrene (TCPS), glass, quarts, quartz glass, poly(ethylene terephthalate) (PET), polyethylene, polyvinyl difluoride (PVDF), polydimethylsiloxane (PDMS), polytetrafluoroethylene (PTFE), polymethylmethacrylate (PMMA), polycarbonate, polyolefin, ethylene vinyl acetate, polypropylene, polysulfone, polytetrafluoroethylene, silicones, poly(meth)acrylic acid, polyamides, polyvinyl chloride, polyvinylphenol, and copolymers mixtures thereof.
In some embodiments, the system comprises at least one array comprising a solid support, prepared by the steps comprising: (i) preparing a first and second solution, each first and second solution comprising a known concentration of a polypeptide comprising a polypeptide sequence associated with the extracellular matrix or a functional fragment thereof; (ii) contacting the first and second solution with the solid support for a sufficient time period absorb polypeptide comprising a polypeptide sequence associated with the extracellular matrix or a functional fragment thereof to the solid support; wherein the polypeptide sequence associated with the extracellular matrix or a functional fragment thereof is chosen from the polypeptides of Table 1; and wherein the steps of preparing a solution and contacting the solution with the solid support is repeated at least 700 times corresponding to the number of adhesion sets present on the at least one array. In some embodiments, the one or more repeated steps of contacting the first and second solution with the solid support is performed by an automated device such that each polypeptide comprising a polypeptide sequence associated with the extracellular matrix or fragment thereof is absorbed at discrete addressable locations on the at least one array.
The present invention encompasses the recognition that cells can be identified and/or characterized by “adhesion signatures” that embody a cell's affinity for one or more Extracellular Matrix (ECM) components. In some embodiments, an adhesion signature includes binding information sufficient to compare a particular cell or cell type of interest with a reference cell or cell type and/or to identify, characterize, and/or distinguish a particular cell or cell type with respect to other cells or cell types.
In some embodiments, an adhesion signature comprises information respecting absence, presence and/or level of binding interactions with one or more ECM components selected from the group consisting of aggrecan, agrin, biglycan, brevican, chondroitin sulfate, collagen I, collagen II, collagen III, collagen IV, collagen V, collagen VI, decorin, elastin, f-spondin, fibrin, fibronectin, galectin 1, galectin 3, galectin 3c, galectin 4, galectin 8, heparan sulfate, hyaluronic acid, keratin, laminin, merosin, mucin, nidogen-1, nidogen-2, osteopontin, SPARC/osteonectin, superfibronectin, tenascin-C, tenascin-R, testican 1/SPOCKI, testican 2/SPOCK2, thrombospondin-4, vitronectin and combinations thereof.
In some embodiments, an adhesion signature distinguishes a cell or cell type from comparable cells or cell types of other tissue origin. In some embodiments, an adhesion signature distinguishes a cell or cell type from comparable cells or cell types of a different developmental stage (or point in development). In some embodiments, an adhesion signature distinguishes a cell or cell type from comparable cells or cell types that differ in presence of and/or susceptibility to one or more disease states, disorders, or conditions. In some embodiments, an adhesion signature distinguishes a cell or cell type from comparable cells or cell types that differ in physiologic state. In some embodiments, an adhesion signature distinguishes a cell or cell type from comparable cells or cell types that differ with respect to extent, degree, or type of exposure to one or more environmental factors (including drugs, toxins, etc).
In some embodiments, detection or determination of an adhesion signature reveals information about identity, extent, and or nature of one or more components of ECM produced by a cell, and/or of one or more factors present on (e.g., expressed or captured on) a cell surface. To give but one example, existence and/or level of particular binding interactions in an adhesion signature of a cell can reveal identity, extent, and or nature of a cell surface component such as, for example, an integrin that participates in binding interaction(s).
In some embodiments, adhesion signatures are determined by contacting a cell or cell sample with an array or system disclosed herein; quantifying one or more adhesion values; and compiling the one or more adhesion values to create or determine one or more adhesion signatures, or profiles. In some embodiments, the step of quantifying one or more adhesion values comprises detecting a quantitative signal or signals relative to the cell or cell sample binding to one or a plurality of adhesion sets, normalizing the quantitative signals as compared to a control or reference cell or cell sample, and applying an algorithm or interpretation function disclosed herein to the quantitative signal or signals such that the output of the algorithm or interpretation function disclosed herein is one or a plurality of adhesion values. In some embodiments, the step of applying the algorithm or interpretation function disclosed herein is performed by a non-transitory computer program product. In some embodiments, one or more steps of the methods disclosed herein are performed by a non-transitory computer implemented method. In some embodiments, the algorithm or interpretation function for quantifying one or more adhesion values is performed using CellProfiler software (Carpenter A E, J. T., Lamprecht M R, Clarke C, Kang I H, Friman O, Guertin D A, Chang J H, Lindquist R A, Moffat J, Golland P, Sabatini D M (2006). CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biology 7, R100; which is herein incorporated by reference in its entirety). Nuclei are identified using the “IdentifyPrimaryObjects” module of the CellProfiler software with the Otsu Global thresholding method. Clumped objects are distinguished using “Intensity”. In some embodiments, adhesion values for a given cell line are determined by computing the average of replicate slides run for that given cell line. In some embodiments, the step of normalizing the adhesion values as compared to a control or reference cell or cell sample is accomplished by hierarchical clustering using Spotfire software, a hierarchical agglomerative method. For row clustering, the cluster analysis begins with each row placed in a separate cluster. Then the distance between all possible combinations of two rows is calculated using the Euclidean distance measure. The two most similar clusters are then grouped together and form a new cluster. In subsequent steps, the distance between the new cluster and all remaining clusters is recalculated using the UPGMA (Unweighted Pair-Group Method with Arithmetic mean) method. The number of clusters is thereby reduced by one in each iteration step. Eventually, all rows are grouped into one large cluster. The order of the rows in a dendrogram are defined by the average value weight. In some embodiments, no column clustering was performed.
Once the one or plurality of adhesion values are calculated using the algorithm or interpretation function, one can create or determine an adhesion signature for the cell or cell sample which, in some embodiments, is a quantitative binding profile (collection of adhesion values) of a cell or cell sample relative to the one or plurality of adhesion sets to which a reference cell or reference cell sample has been contacted. A user of the array or system disclosed herein can subsequently compare the adhesion signature of the cell or cell sample to one or a plurality of adhesion control or reference cells. In some embodiments, the adhesion signatures of the one or plurality of control samples is predetermined and/or catalogued so that the user of the array or system disclosed herein can compare the signatures of the cell or cell sample to the predetermined and/or catalogued control signature to identify or characterize the phenotype of the cell or cell sample. In some embodiments, the adhesion signatures of the one or plurality of control is predetermined and/or catalogued so that the user of the array or system disclosed herein can compare the signatures of the cell or cell sample to the predetermined and/or catalogued control adhesion signature to qualitatively assess the cell or cell sample as having physical characteristics more or less similar to the control adhesion signature. In some embodiments, the user of the array or system disclosed herein and generate a profile related to similarities or dissimilarities as between the cell or cell sample adhesion signature and the control adhesion signature. In some embodiments, the control adhesion signature is adhesion signature that quantitatively describes a set of adhesion values from a metastatic tissue. In some embodiments, the control adhesion signature is an adhesion signature that quantitatively describes a set of adhesion values from cancerous tissue. In some embodiments, the control adhesion signature is adhesion signature that quantitatively describes a set of adhesion values from a pre-cancerous tissue. In some embodiments, the control adhesion signature is adhesion signature that quantitatively describes a set of adhesion values from a stem cell. In some embodiments, the control adhesion signature is adhesion signature that quantitatively describes a set of adhesion values from an embryonic stem cell. In some embodiments, the control adhesion signature is adhesion signature that quantitatively describes a set of adhesion values from a mesenchymal stem cell. In some embodiments, the control adhesion signature is adhesion signature that quantitatively describes a set of adhesion values from an induced pluripotent stem cell. In some embodiments, the control adhesion signature is adhesion signature that quantitatively describes a set of adhesion values from a primary lineage of hepatocytes. In some embodiments, the control adhesion signature is adhesion signature that quantitatively describes a set of adhesion values from a cellular stage of development in respect to any of the cells disclosed herein. In some embodiments, the control adhesion signature is adhesion signature that quantitatively describes a set of adhesion values from one or various stages of tumor growth. In some embodiments, the control adhesion signature is adhesion signature that quantitatively describes a set of adhesion values from one or more induced pluripotent stem cells. In some embodiments, the control adhesion signature is adhesion signature that quantitatively describes a set of adhesion values from one or more mesenchymal stem cells. In some embodiments, the control adhesion signature is adhesion signature that quantitatively describes a set of adhesion values from one or more bone-derived stem cells. In some embodiments, the control adhesion signature is adhesion signature that quantitatively describes a set of adhesion values from one or more embryonic stem cells. In some embodiments, the control adhesion signature is adhesion signature that quantitatively describes a set of adhesion values from one or more adipose-derived stem cells. In some embodiments, the control adhesion signature is adhesion signature that quantitatively describes a set of adhesion values from one or more stem cells.
According to some embodiments, the invention provides a software component or other non-transitory computer program product that is encoded on a computer-readable storage medium, and which optionally includes instructions (such as a programmed script or the like) that, when executed, cause operations related to the calculation of adhesion values and/or adhesion signatures. In some embodiments, the computer program product is encoded on a computer-readable storage medium that, when executed: quantifies one or more adhesion values; normalizes the one or more adhesion values over a control set of data; creates an adhesion profile or signature; and displays the adhesion profile or signature to a user of the computer program product. In some embodiments, the computer program product is encoded on a computer-readable storage medium that, when executed: calculates one or more adhesion values, normalizes the one or more adhesion values, and creates an adhesion signature, wherein the computer program product optionally displays the adhesion signature and/or adhesion values on a display operated by a user. In some embodiments, the invention relates to a non-transitory computer program product encoded on a computer-readable storage medium comprising instructions for: quantifying one or more adhesion values; and displaying the one or more adhesion values to a user of the computer program product. In some embodiments, the invention provides a non-transitory computer program product encoded on a computer-readable storage medium comprising instructions for: quantifying one or more adhesion values; normalizing the one or more adhesion values to a control set of data; creating an adhesion signature; and displaying the adhesion profile to a user of the computer program product. In some embodiments, the step of calculating one or more adhesion values comprises quantifying an average and standard deviation of counts on replicate spots. In some embodiments, the step of calculating one or more adhesion values comprises discarding the spots for which the count is greater or less than one standard deviation above or below the mean, respectively, and computing an average of the remaining counts (such average denoted as “x”). In some embodiments, the step of normalizing the one or more adhesion values over a control set of data is performed by first computing the average count across all ECM combinations on the slide for which the count is greater than zero (X). In some embodiments, the normalized adhesion value for each combination is then computed by dividing the average of the raw counts for the combination by the average of the non-zero counts for the slide (x/X).
In general, one challenge faced by researchers and medical professionals is a need to identify cell types, differentiation states, and phenotypes, and to adequately isolate and grow specific cell populations. For example, because of interplay between genetic and environmental factors, two sub-populations of cells may be genetically identical and differ detectably only in composition of or adhesion to ECM components. Thus, one advantage of determining adhesion signature of cells as provided herein is that it can permit researchers to distinguish between cell populations that have not previously been distinguishable. Alternatively or additionally, provided methods and compositions allow characterization and/or classification of cells in ways not previously available or appreciated. Provided methods and compositions also provide basis for isolation or separation of cells from one another and/or from other components, materials, or entities.
The arrays, compositions, kits and systems disclosed herein allow the performance of methods to isolate, expand, differentiate, and maintain culture of mesenchymal stem cells and/or induced pluripotent stem cells. In some embodiments, the invention relates to a method of expanding of mesenchymal stem cells and/or induced pluripotent stem cells comprising the step of contacting mesenchymal stem cells and/or induced pluripotent stem cells to an array, composition, kit and/or system disclosed herein comprising at least one adhesion set. In some embodiments, the adhesion set comprises a polypeptide comprising a polypeptide sequence associated with the extracellular matrix that is chosen from one or a combination of: collagen I, laminin, collagen II, collagen IV, galectins-4, galectin-8, and/or fibronectin. In some embodiments, the adhesion set consists of collagen I and laminin. In some embodiments, the adhesion set consists of collagen II and galectin-4. In some embodiments, the adhesion set consists of collagen IV and galectin-4. In some embodiments, the adhesion set consists of collagen IV and galectin-8. In some embodiments, the adhesion set consists of collagen I and laminins and fibronectin. In some embodiments, the adhesion set consists of collagen II and galectin-4 and fibronectin. In some embodiments, the adhesion set consists of collagen IV and galectin-4 and fibronectin. In some embodiments, the adhesion set consists of collagen IV and galectin-8 and fibronectin. In some embodiments, the arrays, compositions, kits and systems disclosed herein are free of any polypeptide sequence associated with the extracellular matrix except collagen I, laminin, collagen II, collagen IV, galectins-4, galectin-8, and/or fibronectin. In some embodiments, the arrays, compositions, kits and systems disclosed herein are free of any media comprising inhibitors or antagonists of integrins.
In some embodiments, the invention relates to a method of isolating, expanding, differentiating, and/or maintaining a culture of mesenchymal stem cells and/or induced pluripotent stem cells by contacting a cell sample with one or more adhesion sets described herein in the presence of xeno-free media. In some embodiments, the invention relates to a method of isolating, expanding, differentiating, and/or maintaining a culture of mesenchymal stem cells and/or induced pluripotent stem cells by contacting a cell sample with one or more adhesion sets described herein in the presence of media free of animal-derived components. In some embodiments, the invention relates to a method of isolating, expanding, differentiating, and/or maintaining a culture of mesenchymal stem cells and/or induced pluripotent stem cells by contacting a cell sample with one or more adhesion sets described herein in the presence of media free of any inhibitors of any integrins.
In some embodiments, the invention relates to a method of maintaining or culturing hepatocytes in culture derived from primary lineages of cells comprising the step of contacting any of the arrays or systems disclosed herein to a primary hepatocyte.
In some embodiments, the invention relates to a method of culturing mesenchymal stem cells comprising the step of contacting any of the arrays or systems disclosed herein to a MSC.
In some embodiments, the invention relates to a method of differentiating an MSC comprising the step of contacting any of the arrays or systems disclosed herein to a MSC.
In some embodiments, the invention relates to a method of differentiating an iPSC into a cardiac lineage, liver lineage, or neural lineage comprising the step of contacting any of the arrays or systems disclosed herein to iPSC.
In some embodiments, the invention relates to a method of culturing a iPSCs comprising the step of contacting any of the arrays or systems disclosed herein to a iPSC.
In some embodiments, the invention relates to a method of culturing normal mammary epithelial cells in culture comprising the step of contacting any of the arrays or systems disclosed herein to a cell sample comprising a mammary epithelial cell.
In some embodiments, the invention relates to a method of culturing metastatic mammary epithelial cells in culture comprising the step of contacting any of the arrays or systems disclosed herein to a cell sample comprising a metastatic mammary epithelial cell.
In some embodiments, the invention relates to a method of proliferating normal mammary epithelial cells in culture comprising the step of contacting any of the arrays or systems disclosed herein to a cell sample comprising a mammary epithelial cell.
In some embodiments, the invention relates to a method of proliferating metastatic mammary epithelial cells in culture comprising the step of contacting any of the arrays or systems disclosed herein to a cell sample comprising a metastatic mammary epithelial cell.
In some embodiments, the invention relates to a array or system, or kit consisting of any one or plurality of adhesion sets disclosed herein adsorbed to solid support comprising polystyrene.
In some embodiments, the invention relates to a pharmaceutical composition comprising: a therapeutically effective amount or prophylactically effective amount of a nucleic acid molecule that interferes with the expression of any of the cognate integrins disclosed herein; and a pharmaceutical acceptable carrier. In some embodiments, the invention relates to a pharmaceutical composition comprising: a therapeutically effective amount of a nucleic acid molecule that interferes with the expression of any of the cognate integrins disclosed herein; and a pharmaceutical acceptable carrier; wherein the therapeutically effective amount of a nucleic acid molecule that interferes with the expression of any of the cognate integrins disclosed herein inhibits migration of cancer cells from the lymph node to distant organs.
In some embodiments, the invention relates to a pharmaceutical composition comprising: a therapeutically effective amount or prophylactically effective amount of a polypeptide or functional fragment thereof that interferes with the expression or binding of any of the cognate integrins disclosed herein; and a pharmaceutical acceptable carrier. In some embodiments, the invention relates to a pharmaceutical composition comprising: a therapeutically effective or prophylactically effective amount of a polypeptide or functional fragment thereof that interferes with the expression or binding of any of the cognate integrins disclosed herein; and a pharmaceutical acceptable carrier; wherein the therapeutically effective amount of a polypeptide or functional fragment thereof that interferes with the expression of any of the cognate integrins disclosed herein inhibits migration of cancer cells from the lymph node to distant organs. In some embodiments, the invention relates to a pharmaceutical composition comprising: a therapeutically effective or prophylactically effective amount of a polypeptide or functional fragment thereof that interferes with the expression of any of the cognate integrins disclosed herein; and a pharmaceutical acceptable carrier; wherein the therapeutically effective amount of a polypeptide or functional fragment thereof that interferes with the expression of any of the cognate integrins disclosed herein inhibits migration of cancer cells from the lymph node to distant organs. In some embodiments, the polypeptide or functional fragment thereof that interferes with the expression or binding of any of the cognate integrins disclosed herein is an antibody or antibody fragment. In some embodiments, the composition comprises a polypeptide or nucleic acid sequence that inhibits migration of cancer cells from the tissue from which the cancer cell originates to a lymph node.
The present invention provides for the use of an antibody or binding composition which specifically binds to a specified cognate binding pair to an ECM components disclosed herein or to an integrin disclosed herein. In some embodiments the antibody specifically binds the integrin from a mammalian polypeptide, e.g., a polypeptide derived from a primate, human, cat, dog, rat, or mouse. Antibodies can be raised to various integrins, including individual, polymorphic, allelic, strain, or species variants, and fragments thereof, both in their naturally occurring (full-length) forms or in their synthetic forms. Additionally, antibodies can be raised to the analogs in their inactive state or active state. Anti-idiotypic antibodies may also be used.
A number of immunogens may be selected to produce antibodies specifically reactive with ligand or receptor proteins. Synthetic integrins disclosed herein may serve as an immunogen for the production of monoclonal or polyclonal antibodies. Such antibodies may be used as antagonists or agonists for their targets modulating the disease state associated with the naturally occurring integrins or cognate integrins disclosed herein. Synthetic polypeptides of the claimed invention may also be used either in pure or impure form. Synthetic peptides, made using the appropriate protein sequences, may also be used as an immunogen for the production of antibodies. Naturally folded or denatured material can be used, as appropriate, for producing antibodies. Either monoclonal or polyclonal antibodies may be generated, e.g., for subsequent use in immunoassays to measure the protein, or for immunopurification methods. Methods of producing polyclonal antibodies are well known to those of skill in the art.
Typically, an immunogen, such as a purified integrin disclosed herein of the invention, is mixed with an adjuvant and animals are immunized with the mixture. The animal's immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to the protein of interest. For example, when appropriately high titers of antibody to the immunogen are obtained, usually after repeated immunizations, blood is collected from the animal and antisera are prepared. Further fractionation of the antisera to enrich for antibodies reactive to the protein can be performed if desired. See, e.g., Harlow and Lane; or Coligan. Immunization can also be performed through other methods, e.g., DNA vector immunization. See, e.g., Wang, et al. (1997) Virology 228:278-284.
Monoclonal antibodies may be obtained by various techniques familiar to researchers skilled in the art. Typically, spleen cells from an animal immunized with a desired integrin disclosed herein are immortalized, commonly by fusion with a myeloma cell. See, Kohler and Milstein (1976) Eur. J. Immunol. 6:511-519. Alternative methods of immortalization include transformation with Epstein Barr Virus, oncogenes, or retroviruses, or other methods known in the art. See, e.g., Doyle, et al. (eds. 1994 and periodic supplements) Cell and Tissue Culture: Laboratory Procedures, John Wiley and Sons, New York, N.Y. Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cells may be enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate host. Alternatively, one may isolate DNA sequences which encode a monoclonal antibody or a binding fragment thereof by screening a DNA library from human B cells according, e.g., to the general protocol outlined by Huse, et al. (1989) Science 246:1275-1281.
Antibodies or binding compositions, including binding fragments, single chain antibodies, Fv, Fab, single domain VH, disulfide-bridged Fv, single-chain Fv or F(ab′)2 fragments of antibodies, diabodies, and triabodies against predetermined fragments of the integrins disclosed herein can be raised by immunization of animals with integrins disclosed herein or conjugates of integrins disclosed herein. Monoclonal antibodies are prepared from cells secreting the desired antibody. These antibodies can be screened for binding to integrins disclosed herein. These monoclonal antibodies will usually bind with at least a KD of about 1 mM, usually at least about 300 μM, typically at least about 10 μM, at least about 30 μM, at least about 10 μM, and at least about 3 μM or more. These antibodies can be screened for binding to the naturally occurring polypeptides upon which the antibodies bind.
In some instances, it is desirable to prepare monoclonal antibodies (mAbs) from various mammalian hosts, such as mice, rodents, primates, humans, etc. Description of techniques for preparing such monoclonal antibodies may be found in, e.g., Stites, et al. (eds.) Basic and Clinical Immunology, 4th ed., Lange Medical Publications, Los Altos, Calif., and references cited therein; Harlow and Lane (1988) Antibodies: A Laboratory Manual CSH Press; Goding (1986) Monoclonal Antibodies: Principles and Practice, 2nd ed., Academic Press, New York, N.Y.; and particularly in Kohler and Milstein (1975) Nature 256:495-497, which discusses one method of generating monoclonal antibodies. Summarized briefly, this method involves injecting an animal with an polypeptide that binds an integrin disclosed herein. The animal is then sacrificed and cells taken from its spleen, which are then fused with myeloma cells.
The elucidation of the role played by the integrins associated metastatic cancer and adhesion profiles described herein in adhesion to ECM of a subject facilitates the development of pharmaceutical compositions useful for treatment and diagnosis of metastatic cancer. In some embodiments, the elucidation of the role played by the integrins associated metastatic cancer and adhesion profiles described herein in adhesion to ECM of a subject facilitates the development of pharmaceutical compositions useful for treatment and diagnosis of metastatic breast or lung cancer. These compositions may comprise, in addition to one of the above substances, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes. Whether it is a polypeptide, antibody, peptide, nucleic acid molecule, small molecule or other pharmaceutically useful compound according to the present invention that is to be given to an individual, administration is preferably in a “prophylactically effective amount” or a “therapeutically effective amount” (as the case may be, although prophylaxis may be considered therapy), this being sufficient to show benefit to the individual.
The present invention encompasses the recognition that adhesion signatures characteristic of particular cells of interest are useful in a variety of contexts, for example to identify, characterize, detect, and/or isolate cells of interest.
The present invention provides systems for determining adhesion signatures characteristic of cells. In certain embodiments, the system comprises contacting a sample comprising cells with a collection of extracellular matrix (ECM) components and detecting presence or level of interactions between cells in the sample and ECM components in the collection.
In some embodiments, the system comprises contacting cells with ECM components to allow the cells to adhere to the ECM components. In many embodiments, the interaction between ECM components and particular cells will be higher for cells that interact with higher affinity to a given collection of ECM components. In some embodiments, higher overall affinity may be achieved through individual high affinity interactions. In some embodiments, higher overall affinity may be achieved through a larger number of interactions, whether or not all are particularly high affinity. In some embodiments, overall affinity is affected or determined by multiple interactions between a plurality of distinct pairs of interacting entities. Alternatively or additionally overall affinity is affected or determined by copy number of individual interacting entities; as is understood in the art, a higher concentration of interacting entities can result in a higher number of interactions, which can achieve a higher overall affinity even when individual interactions are relatively modest affinity.
In some embodiments, the systems described herein comprise contacting a sample comprising cells with a collection of extracellular matrix (ECM) components. In some embodiments, a collection of ECM components comprises a single ECM component. In some embodiments, a collection of ECM components comprises 2 ECM components. In some embodiments, a collection of ECM components comprises 3, 4, 5, 6, 7, 8, 9, 10 up to 4,000 or more ECM components.
In some embodiments collections of ECM components for identifying, characterizing, detecting, and/or isolating cancer cells including non-small cell lung cancer cells and cells from primary tumors, lymph nodes, or metastases at organ sites comprises at least two ECM components selected from agrin and collagen IV, agrin and fibrin, biglycan and collagen II, biglycan and fibrin, collagen I and thrombospondin-4, collagen II and decorin, collagen II and tenascin-C, collagen II and testican 2, collagen III and collagen VI, collagen III and thrombospondin-4, collagen IV and galectin 4, collagen IV and SPARC, collagen IV and vitronectin, collagen V and galectin 1, collagen VI and galectin 3, fibrin and galectin 3c, fibrin and galectin 4, fibrin and keratin, fibrin and osteopontin, fibrin and SPARC, f-spondin and fibronectin, fibronectin and galectin 3, fibronectin and galectin 8, fibronectin and laminin, and/or fibronectin and testican 1.
In some embodiments collections of ECM components for identifying, characterizing, detecting, and/or isolating breast cancer cells comprise at least two ECM components selected from agrin and collagen II, agrin and laminin, biglycan and collagen II, brevican and fibronectin, collagen I and testican 2, collagen II and collagen IV, collagen II and laminin, collagen II and nidogen-1, collagen II and testican 2, collagen III and galectin 8, collagen III and superfibronectin, collagen V and fibronectin, collagen V and galectin 1, collagen VI and fibronectin, collagen VI and nidogen-1, collagen VI and tenascin-C, decorin and fibronectin, decorin and galectin 8, decorin and laminin, elastin and galectin 4, fibrin and galectin 3, fibronectin and galectin 1, fibronectin and galectin 3, fibronectin and galectin 4, fibronectin and mucin, fibronectin and SPARC, fibronectin and testican 2, galectin 1 and galectin 3, galectin 1 and keratin, galectin 3 and heparan sulfate, galectin 3 and superfibronectin, galectin 4 and nidogen-1, galectin 8 and tenascin-C, keratin and laminin, laminin and merosin, laminin and thrombospondin-4, SPARC and superfibronectin, and/or superfibronectin and testican 1.
In some embodiments, ECM components are attached to a solid phase. In some embodiments, a solid phase comprises any solid or semi-solid surface. In some embodiments, a solid phase comprises any traditional laboratory material for growing or maintaining cells including petri dishes, beakers, flasks, test tubes, microtitre plates, and/or culture slides. In some embodiments, a solid phase comprises a glass slide.
In some embodiments, ECM components in the collection are attached to discrete sites on a solid phase. In some embodiments the collection of ECM components are attached to a plurality of discrete sites on the solid phase. In some embodiments, a plurality of discrete sites comprises individual site containing only one ECM component. In some embodiments, a plurality of discrete sites comprises individual site containing two or more different ECM components. In some embodiments, a plurality of discrete sites comprises individual sites containing only one ECM component and individual sites containing two or more different ECM components. In some embodiments, different sites within the plurality of sites contain same component(s). In some embodiments, different sites within the plurality of sites contain different component(s). In some embodiments, the plurality of sites comprises sites comprising the same component(s) as other sites within the plurality of sites and sites comprising different component(s) from other sites within the plurality of sites. In some embodiments, the ECM components in the collection attached to discrete sites on a solid phase comprises an array.
In some embodiments, the solid or semi-solid surface comprising a solid phase is comprised of any material on which ECM components can be attached. In some embodiments, a solid phase comprises polyamides, polyesters, polystyrene, polypropylene, polyacrylates, polyvinyl compounds (e.g. polyvinylchloride), polycarbonate, polytetrafluoroethylene (PTFE), nitrocellulose, cotton, polyglycolic acid (PGA), cellulose, dextran, gelatin, glass, fluoropolymers, fluorinated ethylene propylene, polyvinylidene, polydimethylsiloxane, polystyrene, silicon substrates (such as fused silica, polysilicon, or single silicon crystals) or combinations thereof.
In some embodiments, contacting cells with a collection of ECM components in accordance with systems of the present invention comprises mixing cells with a collection of ECM components. In some embodiments, contacting cells with a collection of ECM components comprises overlaying cells on a collection of ECM components on a solid support. In some embodiments, contacting cells with a collection of ECM components comprises submerging ECM components on a solid support in cells. In some embodiments, contacting cells with a collection of ECM components comprises seeding cells onto ECM components on a solid support. In some embodiments, contacting cells with a collection of ECM components comprises seeding from 0.1 to 100 ml or from 1 to 10 ml of cells onto ECM components on a solid support. Alternatively, cells can be brought into contact with ECM components using any other means of transporting liquid.
In some embodiments, cells are contacted with ECM components under conditions and for a time sufficient to allow cells to interact with ECM components. In some embodiments, cells are contacted with ECM components for from 10 minutes to 48 hours, from 30 minutes to 24 hours, or from 1 hour to 12 hours. In a specific exemplary embodiment, cells are contacted to ECM components for 2 hours.
In some embodiments, contacting is performed at a temperature within a range consistent with cell viability and/or metabolic function. In some embodiments, contacting is performed at a temperature of between 10 to 70, of between 20 to 60, or of between 25 to 40 degrees Celsius. In a specific exemplary embodiment, the temperature is 37 degrees Celsius.
In some embodiments, contacting cells with a collection of ECM components further comprises washing ECM components. In some embodiments, ECM components are washed to remove excess cells. In some embodiments, ECM components are washed to remove non-interacting cells. In some embodiments, ECM components are washed in any solution that will not damage the cells or ECM components. In certain embodiments, ECM components are washed in the same cell culture media that is used to contact the cells to the ECM components. Alternatively, ECM components can be washed with PBS.
In certain embodiments, ECM components are washed in any arrangement that allows the cells interacting with ECM components to maintain their interaction with the ECM components. In certain embodiments, ECM components are washed in a stationary arrangement. In certain embodiments, ECM components, are mechanically agitated during washing. Methods for agitating cells in culture are well known in the art and include but are not limited to use of nutators, rockers, rotators, and shakers.
In some embodiments, the level of interactions between cells in the sample and ECM components in the collection is detected. In some embodiments, interactions between cells and ECM components is detected using any technology that allows cells interacting with ECM components to be quantified. In some embodiments interactions between cells and ECM components is detected by microscopy. In some embodiments interactions between cells and ECM components is detected by confocal microscopy. In some embodiments interactions between cells and ECM components is detected by fluorescence microscopy. In some embodiments interactions between cells and ECM components is detected by microscopy on live cells. In some embodiments interactions between cells and ECM components is detected by microscopy on fixed cells. Appropriate fixatives are well known in the art and include but are not limited to formaldehyde, glutaraldehyde, and formalin. In some embodiments interactions between cells and ECM components is detected by microscopy on stained cells. Appropriate stains for counting cells via microscopy are well known in the art. Examples include but are not limited to Hoechst, 4′,6-diamidino-2-phenylindole (DAPI), and acridine orange. In some embodiments interactions between cells and ECM components is detected by immunocytochemistry.
In some embodiments, detecting interactions between cells and ECM components comprises quantifying the interactions. In some embodiments interactions between cells and ECM components is/are quantified by any means that allows quantification of cells interacting with ECM components. In some embodiments interactions between cells and ECM components detected by microscopy is/are quantified visually. In some embodiments interaction between cells and ECM components detected by microscopy is/are quantified with the aid of a computer program or other computational device. Computer programs for quantifying cell number from microscopic images are well known in the art. One exemplary mathematical programs for quantification of cells visualized by microscopy includes CellProfiler (Carpenter, A. E., et al. CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biology, 7: R100, 2006, which is incorporated by reference in its entirety).
In some embodiments cluster analysis is performed on quantified interactions between cells and ECM components. Analyzing array data is a technique that is well known in the art. Computer programs for analyzing array data include but are not limited to Spotfire (Tibco) and Genespring (Agilent).
In some embodiments, methods in accordance with the disclosure may be used as a diagnostic tool to distinguish between cell types by detecting adhesion signatures characteristic of particular cells that distinguish those cells from other cells in the sample or from reference cells. For example, Example 4 of the present application demonstrates two metastatic cancer cell lines that cluster more closely to each other than to parental tumor cells from which they are derived. When the same cells are characterized by microarray analysis, however, each metastatic cell line clusters with the parental line from which it is derived. These data suggests that adhesion signatures can be used to detect metastatic changes in cancer cells that are undetectable by microarrays.
In certain embodiments, cells differing in one or more characteristic are distinguished by adhesion signatures. In certain embodiments, cells are distinguished from reference cells by adhesion signatures. In certain embodiments, cells at different stages of disease progression are distinguished by adhesion signatures. In certain embodiments, cancer cells at different stages of metastasis are distinguished by adhesion signatures. In some embodiments, cells at different stages of development are distinguished by adhesion signatures. In some embodiments, stem cells at differing stages of differentiation are distinguished by adhesion signatures. In some embodiments, stem cells at differing stages of differentiation include mesenchymal stem cells at different stages of differentiation towards osteogenic, chondrogenic or adipogenic lineages. In some embodiments, stem cells at differing stages of differentiation include human induced pluripotent stem cells or embryonic stem cells at different stages of differentiation towards any cell lineage of circulatory, nervous, or immune systems.
The present invention also provides systems for determining effects on cells of interacting with extracellular matrix components comprising exposing a first population of cells to a first set of conditions that includes contacting with a collection of extracellular matrix components, exposing a second population of cells, which second population of cells is comparable to the first population of cells, to a second set of conditions, which second set of conditions is comparable to the first set of conditions except that some or all of the extracellular matrix components are absent from the contacting, and determining one or more cell population features that differs between the first and second populations of cells after the exposing has occurred. In certain embodiments, information about cells or cell types, including information that characterizes the particular cell or cell type as compared with a different cell type, may be obtained while cells are in contact with ECM components. In certain embodiments effects on cells that result from exposure to and/or interaction with one or more ECM components are determined in accordance with the present invention, for example by determining features that differ in cells that are exposed to different ECM components. In some embodiments, presence or degree of features is determined to correlate with presence or level of one or more ECM components and/or with one or more adhesion signatures. In certain embodiments, cells are probed.
In certain embodiments, a population of cells comprises any collection of cells. In certain embodiments, a population of cells comprises cells of a certain type, wherein the cell type is unknown. In certain embodiments, a population of cells comprises cells of a known cell type. In some embodiments, a population of cells comprises a mixture of known or unknown cell types. In some embodiments, a population of cells comprises cells of a biological sample.
In some embodiments, cells are probed with antibodies that allow cells with different characteristics to be distinguished. It will be appreciated that the use of antibodies as probes is well known to those in the art. Antibodies are available to detect cell lineages or disease states. For example, anti-AFP antibodies can be used to distinguish undifferentiated stem cells from those differentiated towards hepatic lineages and anti-Pdx1 antibodies can be used to distinguish undifferentiated stem cells from those differentiated towards pancreatic lineages. Degree of antibody staining can be detected by techniques well known in the art using fluorescently or chemiluminescently labeled antibodies or by probing with a fluorescently or chemiluminescently labeled secondary antibodies.
In some embodiments, cells are probed with any sort of DNA probe. In some embodiments, cells are probed with DNA probes that allow cells with different characteristics to be distinguished by genotype. In some embodiments, cells are probed with DNA probes that allow cells with different characteristics to be distinguished by RNA transcripts. In some embodiments, cells are probed with any sort of labeled substrate. In some embodiments, cells are probed with labeled substrate that allows cells with different characteristics to be distinguished by enzymatic activity. In some embodiments, cells probed with any sort of protein. In some embodiments, cells are probed with a protein that allow cells with different characteristics to be distinguished by affinity for proteins other than ECM components.
As described above, certain embodiments of the present invention may be used to distinguish between cells at different states of cancer progression, making it a promising tool for diagnosing disease. This system is potentially useful, for example, when testing cells of a patient to determine whether disease is present. Diagnosing a patient using adhesion signatures would include, for example, comparing an adhesion signature of a sample from a patient with and adhesion signature of reference cells.
In certain embodiments, adhesion signatures are used to diagnose and/or prognose a patient suspected of having any condition causing his or her cells to have a distinguishing characteristic from reference cells as a result of the condition. In certain embodiments, adhesion signatures are used to diagnose and/or prognose a patient suspected of having any disease that affects adhesion signatures of his or her cells. In certain embodiments, adhesion signatures are used to diagnose and/or prognose a patient suspected of having any form of cancer. In certain embodiments, adhesion signatures are used to diagnose and/or prognose a patient suspected of having lung cancer. In certain embodiments, adhesion signatures are used to diagnose and/or prognose a patient suspected of having metastatic cancer. In certain embodiments, adhesion signatures are used to diagnose and/or prognose a patient suspected of having breast cancer. In certain embodiments, adhesion signatures are used to diagnose and/or prognose a patient suspected of having colon cancer. In certain embodiments, adhesion signatures are used to diagnose and/or prognose a patient suspected of having prostate cancer. In certain embodiments, adhesion signatures are used to diagnose and/or prognose a patient suspected of having testicular cancer. In certain embodiments, adhesion signatures are used to diagnose and/or prognose a patient suspected of having brain cancer. In certain embodiments, adhesion signatures are used to diagnose and/or prognose a patient suspected of having leukemia.
In some embodiments, kits in accordance with the disclosure provide a means of diagnosing cancer stage. Providing tools for diagnosis and/or prognosis via adhesion signatures in kit form brings adhesion signature technology to clinical settings. In some embodiments, kits for cancer stage diagnosis comprise a substrate coated with a collection of ECM components characterized in that, when a sample containing cells of a plurality of different cell types, which plurality of different cell types include cancer cells of a particular stage of metastasis, is contacted with the substrate, cancer cells of a particular stage of metastasis form a set of interactions with ECM components in the collection sufficient to isolate the cells of the cell type of interest from other cells in the sample. In some embodiments, the kit further comprises medium. In some embodiments, kits in accordance with the disclosure provide a means of detecting non-small cell lung cancer cells and cells from primary tumors, lymph nodes, or metastases at organ sites and comprise at least two ECM components as disclosed herein.
In some embodiments, presence of cancer cells of a particular stage of metastasis is detected by growth of those cells. In some embodiments, the kit further comprises a means for assessing growth or abundance of the cells. Methods for detecting and/or assessing cell growth and/or abundance are well known in the art and include but are not limited to spectrophotometry, FACS, microscopy, and/or plating. In some embodiments, a means for assessing growth or abundance of the cells comprises a container for sending the kit to a facility where growth and/or abundance is assessed.
In some embodiments, methods in accordance with the disclosure may be used as a tool to isolate cells of interest. This system is useful, for example, when trying to isolate certain types of cells out of a mixed cell population. When given a complex mixture of cells, for example partially differentiated stem cells, a patient biopsy, or a bone marrow sample, deconvolving this mixture using traditional methods can be difficult. In general, it is thought that one of the easiest ways to achieve this result is by flow cytometry, but flow cytometry requires an initial prediction of what might be present in a sample to establish a panel of markers that would represent that population. In some embodiments of the present invention, the use of adhesion signatures simplifies this process. Example 7, for instance, demonstrates that mesenchymal stem cells, which are normally isolated out of bone marrow, have high affinity for a combination of galectin-8 and thrombospondin-4. Stem cells can be human or derived from any other type of animal.
In some embodiments, the steps of isolating a particular cell type comprises contacting a sample comprising cells with a collection of extracellular matrix (ECM) components under conditions and for a time sufficient for a set of interactions to occur between particular cells in the sample and ECM components in the collection sufficient to isolate the cells from other components of the sample. In some embodiments, ECM components are used to separate cells from other cells. In some embodiments, ECM components are used to separate cells from other cells that make a different set of interactions with the ECM components than do the isolated cells. In some embodiments, cells are isolated using ECM components attached to a solid phase. In some embodiments, cells are isolated using ECM components attached to a solid phase by separating the solid phase from the sample.
In some embodiments, methods in accordance with the disclosure may be used to identify suitable culture conditions for and/or to propagate cells or cell types of interest. Any type of cell grown in culture that originates from a tissue requires a solid surface on which to attach and proliferate. It is generally understood that ECM components facilitate attachment to surfaces. There exist many cell types for which ideal culturing conditions remain unknown and ECM arrays could potentially provide this information.
This system is particularly useful for culturing stem cells because current methods to grow induced pluripotent stem cells require mitotically inactivated feeder cells (MEFs) or undefined extracellular matrix (ECM) mixes (i.e. Matrigel) and thus introduce animal factors and lot variability. Use of defined ECM components, particularly combinations of collagen II and galectin 4, collagen IV and galectin 8, or collagen I and laminin in combination with a defined media offer the potential to generate and maintain pluripotent stem cells without contamination by animal products and may therefore have translational implications for treatment of human disease.
This system is also particularly useful for cell types that are difficult to culture because it allows testing a wide variety of conditions simultaneously. One example is culturing of hepatocytes—the main hepatic cell types. In general, it is thought that only around 10% of donor cells are plateable after isolation. As described in example 8, for all of 6 different lots of unplateable hepatocytes, several ECM matrix combinations were identified that promoted cell adhesion. Combinations of collagen I with aggrecan and of collagen IV with nidogen-1 seem to have a universal effect.
In certain embodiments, culturing a cell type of interest comprises contacting a sample comprising cells of a cell type of interest with a collection of extracellular matrix (ECM) components appropriate to promote growth and/or replication of cells of the cell type of interest as compared with cells of one or more different cell types.
In certain embodiments, a cell type of interest in accordance with the present disclosure comprises human embryonic stem cells or human induced pluripotent stem cells; in some such embodiments, the collection of ECM components comprises at least two ECM components selected from collagen II and galectin 4, collagen IV and galectin 8, or collagen I and Laminin.
In certain embodiments, a cell type of interest in accordance with the present disclosure comprises hepatocytes; in some such embodiments, the collection of ECM components comprises at least two ECM components selected from agrin and collagen I, collagen I and laminin, collagen I and merosin, collagen II and galectin 8, collagen II and SPARC, and/or collagen IV and nidogen-1.
In certain embodiments, a cell type of interest in accordance with the present disclosure comprises mesenchymal stem cells; in some such embodiments, the collection of ECM components comprises at least two ECM components selected from biglycan and collagen IV, biglycan and galectin 4, brevican and collagen I, brevican and collagen IV, brevican and galectin 3c, collagen I and galectin 1, collagen I and galectin 3, collagen I and galectin 3c, collagen I and galectin 8, collagen I and nidogen-2, collagen I and SPARC, collagen I and tenascin-C, collagen I and testican 1, collagen I and vitronectin, collagen II and galectin 3, collagen II and galectin 8, collagen II and nidogen-1, collagen II and nidogen-2, collagen IV and decorin, collagen IV and galectin 8, collagen IV and nidogen-1, collagen IV and nidogen-2, collagen IV and testican 1, collagen IV and testican 2, collagen VI and f-spondin, collagen VI and galectin 3, collagen VI and galectin 8, collagen VI and tenascin-C, collagen VI and testican 2, collagen VI and thrombospondin-4, f-spondin and vitronectin, fibrin and galectin 4, fibronectin and galectin 4, fibronectin and nidogen-1, fibronectin and tenascin-C, fibronectin and testican 1, fibronectin and testican 2, galectin 3 and vitronectin, galectin 3c and merosin, galectin 3c and superfibronectin, galectin 4 and superfibronectin, galectin 8 and superfibronectin, galectin 8 and vitronectin, laminin and vitronectin, SPARC and testican 1, and/or superfibronectin and vitronectin. In some embodiments, the mesenchymal stem cells are derived from bone marrow, adipose tissue, umbilical cord blood or umbilical cord.
In certain embodiments, culturing a cell type of interest comprises culturing cells in any type of media that is capable of supporting growth of the cell type of interest. In certain embodiments, media comprises cell culture media. In certain embodiments, media comprises complex media. In certain embodiments, media comprises serum-free media. The selection of appropriate cell culture media appropriate for various cell types is well known in the art.
In some embodiments, the cells are cultured at a temperature within a range consistent with cell viability and/or metabolic function. In some embodiments, the cells are cultured a temperature of between from 10 to 70, of between 20 to 60, or of between 25 to 40 degrees Celsius.
In some embodiments, systems in accordance with the present disclosure may be used to culture and/or to propagate cells or cell types of interest. In some embodiments, systems for culturing cells comprise a substrate coated with a collection of ECM components characterized in that, when a sample containing cells of a plurality of different cell types, which plurality of different cell types includes at least one cell type of interest is contacted with the substrate, cells of the cell type of interest form a set of interactions with ECM components in the collection sufficient to isolate the cells of the cell type of interest from other cells in the sample by promoting growth of the cell type of interest. In some embodiments, systems in accordance with the present disclosure may be used to culture and/or to propagate mesenchymal stem cells, hepatocytes, human induced pluripotent stem cells or embryonic stem cells and comprise at least two ECM components as described herein.
In some embodiments, kits in accordance with the present disclosure may be used to culture and/or to propagate cells or cell types of interest. In some embodiments, kits for culturing cells comprise the substrate described above and optionally further comprise medium and a cell type of interest. Any array, system, or component thereof disclosed may be arranged in a kit either individually or in combination with any other array, system, or component thereof. The invention provides a kit to perform any of the methods described herein. In some embodiments, the kit comprises at least one container comprising one or a plurality of polypeptides comprising a polypeptide sequence associated with the extracellular matrix or functional fragments thereof. In some embodiments, the kit comprises at least one container comprising any of the polypeptides or functional fragments described herein. In some embodiments, the polypeptides are in solution (such as a buffer with adequate pH and/or other necessary additive to minimize degradation of the polypeptides during prolonged storage). In some embodiments, the polypeptide are lyophilized for the purposes of resuspension after prolonged storage. In some embodiments, the kit comprises: at least one container comprising one or a plurality of polypeptides comprising a polypeptide sequence associated with the extracellular matrix (or functional fragments thereof); and a solid support upon which the polypeptides or fragments may be affixed. In some embodiments, the kit optionally comprises instructions to perform any or all steps of any method described herein. In some embodiments, the kit comprises an array or system described herein and instructions for implementing one or a plurality of steps using a computer program product disclosed herein. It is understood that one or a plurality of the steps from any of the methods described herein can be performed by accessing a computer program product encoded on computer storage medium directly through one or more computer processors or remotely through one or more computer processors via an internet connection or other virtual connection to the one or more computer processors. In some embodiments, the kit comprises a computer-program product described herein or requisite information to access a computer processor comprising the computer program product encoded on computer storage medium remotely. In some embodiments, the computer program product, when executed by a user, calculates one or more adhesion values, normalizes the one or more adhesion values, generates one or more adhesion signatures or one or more adhesion profiles, and/or displays any of the adhesion values, adhesion signatures, adhesion profiles to a user. In some embodiments, the kit comprises a computer program product encoded on a computer-readable storage medium that comprises instructions for performing any of the steps of the methods described herein. In some embodiments, the invention relates to a kit comprising instructions for providing one or more adhesion values, one or more normalized adhesion values, one or more adhesion profiles, one or more adhesion signatures, or any combination thereof. In some embodiments, the kit comprises a computer program product encoded on a computer storage medium that when, executed on one or a plurality of computer processors, quantifies an adhesion value, determines an adhesion signature or adhesion profile, and/or displays an adhesion signature, adhesion value, adhesion signature, and/or any combination thereof. In some embodiments, the kit comprises a computer program product encoded on a computer storage medium that, when executed by one or a plurality of computer processors, quantifies adhesion values of one or more cells samples and determines an adhesion signature based at least partially upon the adhesion values. In some embodiments, kit comprises instructions for accessing the computer storage medium, quantifying adhesion values, normalizing adhesion values, determining an adhesion signature of a cell type, and/or any combination of steps thereof. In some embodiments, the computer-readable storage medium comprises instructions for performing any of the methods described herein. In some embodiments, the kit comprises an array or system disclosed herein and a computer program product encoded on computer storage medium that, when executed, performs any of the method steps disclosed herein individually or in combination and provides instructions for performing any of the same steps. In some embodiments, the instructions comprise an instruction to adhere any one or plurality of polypeptides disclosed herein to a solid support.
The invention further provides for a kit comprising one or a plurality of containers that comprise one or a plurality of the polypeptides or fragments disclosed herein. In some embodiments, the kit comprises cell media free of serum, or any animal-based derivative of serum that enhances the culture or proliferation of cells. In some embodiments, the kit comprises: an array disclosed herein, any cell media disclosed herein, and a computer program product disclosed herein optionally comprising instructions to perform any one or more steps of any method disclosed herein. In some embodiments, the kit does not comprise cell media. In some embodiments, the kit comprises a solid support free of any one individual pair of polypeptides disclosed herein. In some embodiments, the kit comprises a device for affixing one or more adhesion sets to a solid support.
The kit may contain two or more containers, packs, or dispensers together with instructions for preparation of an array. In some embodiments, the kit comprises at least one container comprising the array or system described herein and a second container comprising a means for maintenance, use, and/or storage of the array such as storage buffer. In some embodiments, the kit comprises a composition comprising any polypeptide disclosed herein in solution or lyophilized or dried and accompanied by a rehydration mixture. In some embodiments, the polypeptides and rehydration mixture may be in one or more additional containers.
The compositions included in the kit may be supplied in containers of any sort such that the shelf-life of the different components are preserved, and are not adsorbed or altered by the materials of the container. For example, suitable containers include simple bottles that may be fabricated from glass, organic polymers, such as polycarbonate, polystyrene, polypropylene, polyethylene, ceramic, metal or any other material typically employed to hold reagents or food; envelopes, that may consist of foil-lined interiors, such as aluminum or an alloy. Other containers include test tubes, vials, flasks, and syringes. The containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components of the compositions to mix. Removable membranes may be glass, plastic, rubber, or other inert material.
Kits may also be supplied with instructional materials. Instructions may be printed on paper or other substrates, and/or may be supplied as an electronic-readable medium, such as a floppy disc, CD-ROM, DVD-ROM, zip disc, videotape, audio tape, or other readable memory storage device. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an internet web site specified by the manufacturer or distributor of the kit, or supplied as electronic mail.
The invention also provides a kit comprising: an array of polypeptides, the array comprising: a solid support and a plurality of adhesion sets, wherein each adhesion set comprises two or more different polypeptides comprising a polypeptide sequence associated with the extracellular matrix or a functional fragment thereof; and optionally comprising a cell culture vessel. In some embodiments, the kit further comprises at least one of the following: cell media, a volume of fluorescent stain or dye, a cell sample, and a set of instructions, optionally accessible remotely through an electronic medium.
Any and all journal articles, patent applications, issued patents, or other cited references disclosed herein are incorporated by reference in their respective entireties.
The following example describes an ECM array. Among other things, the present invention provides a collection of ECM components attached to a solid surface useful in accordance with the present invention to define, detect, or utilize one or more features of an adhesion signature of a cell or cell type. In some embodiments, this collection can be defined as an ECM array. In the following example, an expanded Extracellular Matrix (ECM) array is developed for the purpose of identifying different cell types via their adhesion signature. The array described in US Published Patent Application 2006/0160066 A1 was adapted to incorporate all single and pair-wise combinations of 38 different ECM components (Table 1) for a total of 768 combinations presented in quintuplicate in the ECM array resulting in an overall number of 4000 spots per microscope slide (
Briefly, vantage acrylic slides (CEL Associates VACR-25C) were coated with polyacrylamide as previously described (Flaim, C. J. et al. An extracellular matrix microarray for probing cellular differentiation. Nat Meth, 2(2): 119-125, 2005). Before deposition of the molecules, slides are coated with a polyacrylamide hydrogel that is allowed to dry after soaking to remove any unpolymerized monomer. The dehydrated hydrogel acts to entrap molecules without requiring their chemical modification. Slides were then spotted using a DNA Microarray spotter (Cartesian Technologies Pixsys Microarray Spotter and ArrayIt 946 Pins) from source plates prepared using a Tecan liquid handler. Molecules were prepared at a concentration of 200 μg/ml using a buffer described previously (Flaim et al.). 768 pairwise combinations were spotted in replicates of five (
In this example, a protocol for detection of adhesion signatures using ECM arrays is described. Extracellular matrix microarrays preparation. Vantage acrylic slides (CEL Associates VACR-25C) were coated with polyacrylamide by depositing prepolymer containing Irgacure 2959 photoinitiator (Ciba) between the slide and a glass coverslip22. Following polymerization, slides were soaked in ddH2O and the coverslips were removed. Slides were allowed to dry before molecule deposition. Slides were spotted using a DNA Microarray spotter (Cartesian Technologies Pixsys Microarray Spotter and ArrayIt 946 Pins). 768 combinations were spotted in replicates of five. Rhodamine dextran (Invitrogen) was spotted as negative controls and for use in image alignment. The following molecules were used: Collagen I (Millipore), Collagen II (Millipore), Collagen III (Millipore), Collagen IV (Millipore), Collagen V (BD Biosciences), Collagen VI (BD Biosciences), Fibronectin (Millipore), Laminin (Millipore), Merosin (Millipore), Tenascin-R(R&D Systems), Chondroitin Sulphate (Millipore), Aggrecan (Sigma), Elastin (Sigma), Keratin (Sigma), Mucin (Sigma), Superfibronectin (Sigma), F-Spondin (R&D Systems), Nidogen-2 (R&D Systems), Heparan Sulphate (Sigma), Biglycan (R&D Systems), Decorin (R&D Systems), Galectin 1 (R&D Systems), Galectin 3 (R&D Systems), Galectin 3c (EMD Biosciences), Galectin 4 (R&D Systems), Galectin 8 (R&D Systems), Thrombospondin-4 (R&D Systems), Osteopontin (R&D Systems), Osteonectin (R&D Systems), Testican 1 (R&D Systems), Testican 2 (R&D Systems), Fibrin (Sigma), Tenascin-C(R&D Systems), Nidogen-1 (R&D Systems), Vitronectin (R&D Systems), Rat Agrin (R&D Systems), Hyaluronan (R&D Systems), Brevican (R&D Systems). The laminin used is Millipore catalogue no. AG56P, and is a mixture of human laminins that contain the beta1 chain. Source plates used in the spotter were prepared using a Tecan liquid handler. Molecules were prepared at a concentration of 200 μg ml-1 using a buffer described previously22. Slides were stored in a humidity chamber at 4° C. before use. Extracellular matrix microarray seeding and analysis. Slides were washed in PBS and treated with UV before seeding cells. Slides were washed in PBS and treated with UV prior to seeding cells. To measure cell-ECM interactions, cells are seeded onto the arrays in serum-free media and allowed to adhere for 1.5 h at 37° C. To ensure uniform seeding, the slides are agitated every 15 minutes. Furthermore, the top surfaces of the slides are held flush with the bottom of the plate through the use of a custom-designed seeding device that employs a vacuum seal (
Adhesion signatures of mouse tumor cells were characterized. To determine whether metastatic progression is characterized by discrete changes in the ability of cancer cells to adhere to ECM components a panel of cell lines derived from a genetically-engineered model of lung adenocarcinoma was used. Cell lines have been described in Winslow, M. M. et al. Suppression of lung adenocarcinoma progression by Nkx2-1; Nature 473, 101-104 (2011). Lung adenocarcinoma cells in people often contain an activating mutation in the KRAS oncogene and an inactivating mutation in the p53-tumor suppressor pathway. In this mouse model, inhalation of lentiviral Cre-recombinase by genetically-engineered mice G12D containing a loxP-Stop-loxP Kras knock-in allele and both p53 alleles flanked by loxP sites (KrasLSL-G12D/+; p53flox/flox) initiates lung adenocarcinoma development (DuPage, M. et al. Conditional mouse lung cancer models using adenoviral or lentiviral delivery of Cre recombinase. Nat. Protocols, 4(8): 10641072, 2009, Jackson, E. L., et al., Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras. Genes & Development, 15(24): 3243-3248, 2001). Distal metastases form over months in lymph nodes as well as many secondary organs (kidneys, adrenal glands, liver, etc.). Tumors can be resected from the lung and metastatic sites and cultured in vitro as cell lines. Metastatic populations can be correlated to their primary tumor of origin through the use of linker-mediated polymerase chain reaction (LMPCR) and specific PCR for the integration site, since the lentiviruses integrate stably into the genome (Winslow, M. M., et al. Suppression of lung adenocarcinoma progression by Nkx2-1. Nature, 473(7345): 101-104, 2011). Thus, cell lines were generated from primary tumors that did not form detectable metastases (TnonMet), primaries that did form metastases (TMet), lymph node metastases (LN), and metastases to other sites (Met) (
They were placed in a seeding device that holds the top surface of the slides flush with bottom of the well (
To uncover changes in global adhesion signatures of cancer cells during progression and metastatic spread, a panel of murine lung adenocarcinoma cell lines derived from nonmetastatic primary tumors (TnonMet), metastatic primary tumors (TMet), and metastases from the lymph node (LN) and liver (Met) were analyzed. Technically, analysis of these cell lines showed very highly reproducible adhesion between replicate spots confirming the overall high quality of the ECM spotting and quantitative nature of the assay. Analysis of the adhesion signatures of these cell lines highlighted the diverse adhesion of each cell line to different ECM combinations (
Whether cells had greater or lesser adhesion to combinations of ECM components than to the molecules in isolation was assessed.
ECM arrays spotted and seeded similar to the above Example 2 were then used to analyze cell lines from each of the four classes of cell lines (
This result is particularly surprising since two of these metastatic lines (389N1 and 393M1) were from tumors that directly disseminated from two of the primary lines screened (389T2 and 393T5, respectively), and yet clustered more closely with the other metastases than to their parental lines. This finding suggests that there is a conserved phenotypic change in the ECM adhesion signature of cancer cells from a metastatic site versus those that remain in the primary tumor. Interestingly, this differential clustering was not evident from unsupervised hierarchical clustering of gene expression of these lines (Winslow, M. M. et al. Suppression of lung adenocarcinoma progression by Nkx2-1. Nature 473, 101-104 (2011)). The present disclosure therefore indicates that this phenotype, which may influence metastatic progression, is undetectable by examining specific mRNA or protein expression of specific genes.
To determine whether phenotype-based adhesion screening using an ECM array uncovered characteristics of tumor progression that could not have been detected by gene expression studies, whether changes in adhesion found could be explained by changes in expression of related molecules was assessed. Gene expression profiling was performed on cell-lysate harvested from cells at the time the ECM arrays were run.
Expression data for each of the ECM genes was compared to adhesion to those molecules for each of the eleven cell lines. While some expression data correlated well with adhesion (low adhesion/low expression, high adhesion/high expression), many molecules exhibited either high expression with little adhesion or high adhesion with little expression. There was no statistically significant difference in adhesion between ECM genes expressed at a low, medium, or high level (p>0.6). The present disclosure therefore indicates that there is likely a complex interplay with other parenchymal or stromal cells that either act to provide molecules necessary for adhesion of the tumor cells or react to ECM components produced by tumor cells, perhaps in a manner that promotes tumorigenesis.
In light of the hierarchical clustering results, we asked whether there were particular combinations of molecules that are favored by metastatic cells rather than by cells from primary tumours. Thus, we compared the average adhesion of the liver metastasis-derived cell lines (M) for each ECM combination to the average adhesion of the TMet lines (
Furthermore, the differential adhesion to the aforementioned ECM combinations was clear in both the group-wise comparison (
Overall, the present disclosure demonstrates that adhesion signatures allow one to determine a cellular state that is predictive of disease state and that is otherwise unpredictable using available techniques. This signature can act as a diagnostic test for metastatic disease, predicting the TNM stage of a clinical sample and potentially identifying which distal organs the disease will most readily metastasize to. This finding is of major significance to the diagnosis of cancer.
Next we sought to correlate our in vitro adhesion profiles with ECM expression in vivo. To investigate whether the identified ECM molecules may be important in natural tumorigenesis, organs containing primary autochthonous tumors and their metastases were resected from KrasLSL-G12D/+; p53flox/flox mice and stained. Trichrome staining of lungs with extensive tumor burden revealed a significant presence of ECM deposition in the tumor-bearing lung (
We next asked whether the lymph node and distant organ metastases contained the metastasis-associated ECM molecules. Again, trichrome staining revealed the presence of significant matrix deposition within the lymph nodes (Data not shown). As expected, the entirety of the lymph node tumors was histologically high-grade and was Hmga2pos. There was also clear expression of all four of the metastasis-associated molecules (fibronectin, laminin, galectin-3 and galectin-8) within the lymph node metastases (
We also examined common metastatic sites for the presence of the metastasis-associated molecules (
Integrin Surface Expression Correlates with ECM-Binding Profiles.
Additionally, whether adhesion to specific ECM components correlated with expression of their cognate integrins was assessed. As was the case with the ECM component expression, expression of integrins often did not correlate with adhesion to their known ligands. This finding suggests that small alterations in expression of many integrins may result large changes in adhesion to molecules that they interact with or that more complex mechanisms, such as ECM or integrin post-processing, contribute dramatically to adhesion.
Whether presentation of molecules of interest at the protein level correlated with adhesion to those molecules was assessed. Western blots of lysate from the characteristic primary line 393T5 were compared to that of metastatic line 393M1. The data presented herein demonstrate that expression of both galectins was unchanged between the lines despite an increase in adhesion to them in the 393M1s (
We noted that comparisons of adhesion trends on our ECM arrays did not necessarily correlate with transcriptional profiles of the cognate integrins (
Nonetheless, the surface expression trends were consistent for the other TMet and M lines as well (data not shown). Furthermore, within a given cell line, we observed relatively homogeneous surface expression of the metastasis-associated integrins as measured by flow cytometry (data not shown), suggesting that variations in adhesion between lines are due to global increases in surface receptor expression rather than binding patterns of select subpopulations. Immunohistochemistry revealed that these integrins were also present in the metastases of mice bearing autochthonous tumors, but not the adjacent tissue (
The finding that the transcriptional levels of the integrins do not agree with the adhesion trends suggests that post-transcriptional regulation, post-translational modifications such as altered glycosylation or alterations in activation state of the integrins are likely responsible for the changes in adhesion. Thus, by utilizing our platform that investigates specific ECM binding rather than receptor gene or protein expression, we are able to identify candidate ECM interactions that might otherwise have been overlooked.
Integrin α3β1 Mediates Adhesion and Seeding In Vitro and In Vivo.
To examine which candidate receptor/ECM interactions may participate in the observed binding patterns, we performed in silico network mapping of the metastasis-associated ECM molecules using GeneGO software (Metacore) of manually curated molecular interactions. We generated a network map that we termed the lung adenocarcinoma metastasis network that has a greatest disease association with ‘Neoplasm Metastasis’ (P=1.094×10−45, hypergeometric test,
We next assessed whether this integrin dimer has a role in metastatic seeding in vivo. Thus, we conducted experimental metastasis assays by intrasplenic injection of 393M1-shα3 or 393M1-shFF cells into wild-type mice, and monitoring for liver tumor formation. We found that mice injected with the 393M1-shα3 cells formed fewer tumor nodules than the controls (
Galectin-3/8 is Present in Human Lung Cancer Metastases.
Based on the in vitro adhesion data and in vivo mouse findings, we sought to explore the role of the metastasis-associated ECM molecules in human samples. Using Oncomine-32, a human genetic dataset analysis tool, we examined the correlation of ECM gene expression and disease severity (for example, clinical stage or the presence of metastases). Results of these queries demonstrate that increased gene expression or copy number of LGALS3 or LGALS8 (galectin-3 and galectin-8, respectively) correlate with increased clinical stage or the presence of metastases (
Protein analysis. Western blot analysis of ECM molecules was performed with the following antibodies: galectin-3 (Abcam, ab53082, 1:500), galectin-8 (Abcam, ab69631, 1:500), osteopontin (Abcam, ab8448, 1:2,000), fibronectin (Abcam, ab2413, 1:1,000), laminin (Abcam, b11575, 1:1,000), collagen I (Abcam, ab34710, 1:5,000) and α-tubulin (Cell Signaling, 2125, 1:1,000). Immunohistochemistry of ECM molecules was performed with the following antibodies: galectin-3, galectin-8 (1:75), osteopontin, laminin (Abcam, ab11575, 1:100), fibronectin (Millipore, AB2033, 1:80), Hmga2 (Biocheck, 59170AP, 1:1,000), collagen I (Abcam, ab34710, 1:500) and collagen VI (Abcam ab6588, 1:100). Integrin staining was performed using the following antibodies: integrin αv (Millipore AB1930, 1:200), integrin α5 (Chemicon AB1928, 1:200), integrin α3 antibody was prepared using known methods. Tissue microarrays were acquired from LifeSpan Biosciences (LS-SLUCA50), and were stained with the same galectin-3 antibody. Murine tissues were harvested from KrasLSL-G12D, p53flox/flox mice27-29. IHC was performed following resection from mice, fixation in formalin and embedding in paraffin. Flow cytometry analysis of integrin expression was performed using the following antibodies: integrin α5 (Abcam and BioLegend-clone 5H10-27, 1:100), integrin αv (BD-clone RMV-7, 1:100), integrin α6 (BD and BioLegend-clone GoH3, 1:100), integrin α3 (R&D, 1:100), integrin α1 (BD-clone Ha31/8 and BioLegend-clone HMα1, 1:100) and integrin α2 (BD-clone HMα2, 1:100).
RNA isolation and expression profiling. Cell lysates were harvested using Trizol (Sigma). Chloroform extraction was performed followed by RNA purification using Qiagen RNeasy spin columns. Lysates were analyzed for RNA integrity and prepared with Affymetrix GeneChip WT Sense Target Labelling and Control Reagents kit, followed by hybridization to Affymetrix Mouse 3′ Arrays (Mouse 430A 2.0) Lysates used for gene expression microarrays were harvested at the same time as the ECM microarrays were seeded to ensure minimal variability introduced by cell culture. R/Bioconductor software was used to process array images. Unsupervised hierarchical clustering analysis was performed in Spotfire (Tibco) for all probe sets with variance >0.5 and expression >3.0 using Euclidean distances. Data sets are publically available from NCBI under accession number GSE40222 Retroviral short hairpin RNA (shRNA) constructs. miR30-based shRNAs targeting integrins β1 (5′ TGCTGTTGACAGTGAGCGCGGCTCTCAAACTATAAAGAAATAGTGAAGCCACAGATGT ATTTCTTTATAGTTTGAGAGCCTTGCCTACTGCCTCGGA-3′), α3 (5′-TGCTGTTGACAGTGAGCGCCGGATGGACATTTCAGAG AAATAGTGAAGCCACAGATGTATTTCTCTGAAATGTCCATCCGTTGCCTACTGCCTCGG A-3′), or control firefly luciferase (5′-AAGGTATATTGCTGTTGACAGTGAGCGAGCTCCC GTGA ATTGGAATCCTAGTGAAGCCACAGATGTAGGATTCCAATTCAGCGGGAG CCTGCCTACTGCCTCG-3′) were designed using the shRNA retriever software available at the Katandin homepage (http://katandin.cshl.edu/homepage/siRNA/RNAi.cgi?type=shRNA), synthesized (IDT, Coralville, Iowa), and then cloned into the MSCV-ZSG-2A-Puro-miR30 vector. Packaging of retrovirus and transduction of cells was done as described previously.
All animal procedures were performed in accordance with the MIT Institutional Animal Care and Use Committee under protocol 0211-014-14. Cell injection studies were performed in B6129SF1/J mice (Jackson Laboratory, Stock Number 101043). Intrasplenic injections were performed using 5×105 cells resuspended in 100 μl of phosphate-buffered saline (PBS) and injected into the tip of the spleen following existing protocols29. Animals were anaesthetized with avertin before surgery. Fur was removed from the animals and they were sterilized with Betadine and 70% ethanol. The spleen was exteriorized following incisions in the skin and body wall. Cells were injected into the end of the spleen with a 27-gauge syringe and allowed to travel into circulation for 2 min. Spleens were then excised from the animals following cauterization of the splenic vessels. The muscle wall was closed using 5-0 dissolvable sutures, and the skin was closed using 7 mm wound clips (Roboz). Mice were killed 2.5-4 weeks following injection, and their livers were excised. Quantification of surface nodules and imaging of livers was performed using a dissection microscope. Tissues were embedded in paraffin following fixation in 4% paraformaldehyde and stained using hematoxylin and eosin.
Our ECM microarrays provide a high-throughput multiplexed platform capable of measuring a variety of cellular responses to ECM. Here, we show they are capable of identifying adhesion patterns that differentiate metastatic populations from primary tumors. We found that metastatic lung cancer cells preferentially bind to fibronectin in combination with laminin, galectin-3 or galectin-8 compared with cells derived from primary tumors. These changes in adhesion correlate with changes in surface presentation of various integrins. In particular, α3β1 mediates adhesion to these molecules in vitro and permits metastatic seeding in vivo. Furthermore, metastases derived from both a genetically engineered mouse lung cancer model and from human lung cancers express the metastasis-associated ECM molecules. It is worth noting that the combinations of these ECM components elicited the strongest effects, highlighting the importance of using a platform that is capable of measuring responses to more than individual molecules.
Galectins are a class of lectins that bind β-galactosides and can associate with other ECM molecules such as fibronectin. Galectin-3 is associated with metastasis in a variety of cancers and can bind to the oncofetal Thomsen-Friedenreich antigen, a carbohydrate antigen overexpressed by many carcinomas. Our platform confirmed its importance in lung adenocarcinoma, and also identified galectin-8 as having similar importance. Although galectin-8 is known to affect adhesion of cells to other matrix molecules, its role in cancer and metastasis has been less clear as it has been found to have both a positive and negative association with adhesion and tumorigenesis. Using the ECM microarrays, we showed that binding to galectin-8 in combination with fibronectin is strongly associated with metastatic progression in lung adenocarcinoma.
Furthermore, in addition to many collagens, we found that loss of adhesion to osteopontin accompanied metastatic progression. Osteopontin levels correlate with prognosis in patients with metastatic disease, and secretion of osteopontin by primary tumors results in mobilization of bone marrow-derived stromal precursors that help establish the metastatic niche. In addition to confirming the presence of the metastatic molecules at the sites of metastases, we found that the invasive portions of primary tumors and the invasive front of the metastases secrete osteopontin (
The value of the ECM microarray platform extends beyond the specific application of cancer metastasis. Although this study documents the ability to profile adhesion patterns, cells bound to the arrays can be kept in culture for multiple days to monitor longterm responses to ECM such as cell death, proliferation and alterations in gene or protein expression. Toward that end, one could use multiplexed antibody staining to probe the effects of ECM on stem cell differentiation or activation. Orthogonal screens can be performed to look at the effects of growth factors, small molecules or RNAinterference agents in the context of ECM. Reduction of requisite cell numbers can be achieved using miniaturized arrays to screen rare cell populations such as circulating tumor cells or cancer stem cells and to help expand those populations in vitro for further biological studies.
The findings and utility of the array to identify characterizing protein expression information of lung cancer metastases can be applied to other cancer types, such as breast cancer. The Epithelial-Mesenchymal Transition (EMT) describes a process by which epithelial cells that are typically tightly bound to each other and a basement membrane undergo a transition to a mesenchymal state in which they exhibit enhanced migratory capabilities. While this process occurs naturally during embryogenesis and wound healing, recent studies have implicated its role in a variety of pathologies. In particular, it is now appreciated that, in at least some instances, it is the driving force behind the acquisition of metastatic potential by neoplasms. Carcinomas that turn on this embryonic program known as EMT are capable of breaking free from the cells and extracellular matrix (ECM) around them and can invade through tissue, blood vessels, lymphatics, and eventually reach distant sites in the body. In order to form a secondary tumor at these distant sites, however, it is thought that the cells must undergo the reverse transition, known as mesenchymal to epithelial transition (MET), in order to colonize that distant site and grow into clinically detectable overt metastases. While a variety of extracellular signaling molecules such as TGFbeta are known to induce EMT, the factors driving MET are still poorly understood. Perhaps, the most well-studied tissue in the field of EMT as it relates to cancer, is the breast. Others have developed model systems to characterize breast cancer metastasis in the context of EMT (Genes Dev. Jan. 1, 2001 15: 50-65; Yang, J., et al. (2004); Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. Cell 117, 927-939). A variety of transcription factors are known to turn on this program. In particular, Twist, Snail, and Slug are potent inducers of the EMT phenotype. Thus, in this work we have used a pair of cell lines that represent the two states: the epithelial cells (wild-type) and those that have undergone EMT (mesenchymal) (See
The ECM array was created using the techniques described above resulting in an ECM array comprising more than 700 different pairs of ECM components to determine how this process affects the interactions of cells with the ECM. We ran both the Epithelial (wt) and Mesenchymal (Twist+) cells (HMLERs) on the array. Alterations in their interactions may likely be representative of changes that occur to confer greater metastatic potential and be representative of more advanced stages of malignancy. Algorithms used in determining adhesion values and adhesion signature of the particular cells were previously described herein. The results from arrays are shown in the
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To identify whether certain adhesion sets can stimulate proliferation and not simply adhesion of certain cell populations, experiments were performed on the arrays to measure cell doubling times with normal epithelial cells (labeled “wild-type” or “wt” in
To study differential or selective proliferation capabilities of the array or system in respect to the both wild type mammary epithelial cells or metastatic mammary epithelial cells, adhesion signatures were collected for both cell types at 48 hours after seeding.
A heatmap was generated to illustrate raw differential adhesion values (data not shown). Normalization is as follows: variations in cell numbers put on each slide due to error from pipetting, counting, or both; and global adhesive changes that are not representative in changes to particular ECM combinations (i.e. one cell type being generally more “sticky” than another) The latter normalization step is particularly relevant as metastatic cells that still exhibit the same relative adhesion to a particular combination as their primary tumor counterparts will not appear to have reduced adhesion to it simply because those cells tend to be globally less adhesive.
48 Hour E-Cadherin Expression on ECM Microarrays:
48 Hour TWIST+E-Cadherin Expression on ECM Microarrays: This graph is the same as the first but with the mesenchymal (twist+) cells instead of the epithelial cells. Here, black dots depict combinations containing galectin-3. It is worth noting that E-Cadherin intensity (even of the top combinations) is much lower than the epithelial cells. This is due to their mesenchymal state (which should be lacking E-Cadherin expression). We expect that galectin-3 would induce a potent upregulation of epithelial markers such as E-Cadherin in this case. Nonetheless, the strong evidence for increased E-Cadherin expression in the context of the epithelial cells is quite convincing for its role in inducing an epithelial phenotype and likely conferring the ability of metastatic tumors to colonize distant sites.
Taken together,
The following example demonstrates the use of ECM arrays to characterize differentiation states of stem cells. Stem cells are a promising approach to treatment of human disease due to their inherent ability to proliferate and differentiate to all cell types in the human body. These proprieties make stem cells an ideal cell source for cellular therapy, but so far there is no available method to access and identify if a differentiated stem cell indeed resembles a native cell and to track the differentiation status of the cell. To address this issue, adhesion signatures generated by ECM arrays of human mesenchymal stem cells during differentiation towards osteogenic and adipogenic lineages (
Adhesion signatures generated by ECM arrays are able to distinguish between differentiation states of stem cells from different sources and towards different lineages, enabling the clear identification of a differentiation status of a given cell sample and to compare it to the native tissue. Out of these signatures it is also possible, in accordance with the present disclosure, to select an appropriate ECM component to isolate and culture cells in specific states of differentiation out of a mixed culture. For instance during hepatic differentiation vitronectin in combination with galectin-3 restricts cells in the endoderm stage.
In addition to the ability to isolate and expand these cells, induction of differentiation is an area of active research. Definition of specific cell fates is still unclear for the majority of cellular fates, and the extra cellular signaling component has been mostly ignored in these efforts. The ability of ECM to support and induce differentiation of stem cells towards a specific lineage was investigated, using the liver-pancreatic fate switch as a model system (
In the present example, determining growth rate as a function of adhesion signature is described. Growth rate of the cells on different ECM components or combinations thereof can be determined. This system allows selection of the ECM composition that supports both the highest adhesion to the ECM array and the greatest growth rate of attached cells. In preferred embodiments, the steps of culturing a cell type of interest would additionally include determining or obtaining a second adhesion signature of cells that had been incubated with ECM components, and thus allowed to multiply, and comparing this adhesion signature to the adhesion signature of the cells without the added incubation step to determine the growth rate of the attached cells. In certain embodiments, both adhesion signatures are obtained using ECM arrays. In further embodiments, the second ECM array is incubated for between 30 minutes and 5 days. In preferred embodiments, the second ECM array is incubated for between 12 and 48 hours.
The following example describes the use of ECM arrays to identify growth conditions for mesenchymal stem cells. Adult stem cells, in particular mesenchymal stem cells (MSCs), are actively being explored in the clinic for their immune-modulatory proprieties. Currently there are around 200 clinical trials ongoing using these cells. A major bottleneck to the use of these cells in a clinical setting is their isolations and culture in xeno-free conditions. Currently, there are xeno-free medias available, but they all rely on complex, non-characterized animal derived matrices for isolation and culture of these cells. The FDA has mandated that clinical trials for efficacy require a xeno-free culture system for these cells. Thus, an alternative is needed to animal derived extracellular matrices that are currently being used.
Arrays were fabricated using vantage acrylic slides (CEL-1 Associates VACR-25C) coated with polyacrylamide gel pads (60×22 mm) as described previously 30. ECM arrays were spotted using a DNA Microarray spotter (Cartesian Technologies Pixsys Microarray Spotter and ArrayIt 946 Pins) from 384 well source plates containing the ECM combinations previously prepared using a Tecan EVO 150 liquid handler. Molecules were prepared to a final concentration of 200 g/ml in a buffer described previously. 741 combinations were spotted in replicates of five and rhodamine dextran (invitrogen) was spotted as negative controls and alignment reference for analysis. ECM arrays were stored in a humidified chamber at 4° C., until later use. The following ECM molecules were incorporated in the array: Collagen I, Collagen II, Collagen III, Collagen IV, Fibronectin, Laminin, Chondroitin Sulfate, Merosin (Millipore), Collagen V, Collagen VI (BD Biosciences), Aggrecan, Elastin, Keratin, Mucin, Heparan Sulfate, Superfibronectin, Fibrin, Hyaluronan (Sigma), Tenascin-R, F-Spondin, Nidogen-2, Biglycan, Decorin, Galectin 1, Galectin 3, Galectin 4, Galectin 8, Thrombospondin-4, Osteopontin, Osteonectin, Testican 1, Testican 2, Tenascin-C, Nidogen-1, Vitronectin, Rat, Agrin, Brevican (R&D Systems) and Galectin 3c (EMD Biosciences).
Before cell seeding slides were washed in PBS and sterilized with UV light. Cell seeding occurs in specially designed devices that hold the top surface of the slides flush with bottom of the well and secure the slides under vacuum. Cell were seeded on ECM arrays in serum free conditions and cultured in appropriate conditions. After seeding, slides were transferred to quadriperm plates (NUNC, 167063), and fresh media was added. Cells grew for different periods under these conditions and fed daily in longer studies. Slides were then stained for nuclei and marker expression. Briefly, slides were washed three times with PBS and fixed with 4% paraformaldehyde. At the same time nuclei were stained using Hoechst (Invitrogen) in combination with 0.1% Triton-X and PBS. Slides were then washed again and blocked using a blocking solution containing the anti-sera from the animal where secondary antibodies were raised for one hour. After blocking slides were incubated with primary overnight at 4° C. Secondary antibody (Invitrogen) incubation for 45 minutes followed after PBS washes. Slides were finally washed and mounted with Fluoromount-G (Southern Biotech) and stored at 4° C. until imaging. The entire slide was imaged using a Nikon Ti-Eclipse inverted fluorescence microscope and NIS Elements Software (Nikon). Image processing and analysis was performed in MATLAB (Mathworks) and nuclei and marker intensity quantification using CellProfiler (Carpenter, et al., “CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biology 7, R100 (2006)). Replicate spots on each slide were averaged and those whose values were greater than one standard deviation above or below the mean of the replicates were excluded. Data was then normalized to allow averaging independent experiment data points.
MSCs were isolated from the mononuclear fraction of bone marrow cells of healthy donors, via adhesion in DMEM supplemented with fetal bovine serum and pen/strep (Invitrogen). Differentiation of MSCs towards the osteogenic and adipogenic lineages was carried out using the Invitrogen adipogenic and osteogenic differentiation kits and following the manufacturer's recommendations.
Immunostaining of ECM arrays for the presence ECM molecules was done by a 2 step immunofluorescence protocol. First slides were blocked for 1 hour with BSA and incubated overnight at 4 C with primary antibodies. After three washing steps slides were incubated with secondary antibodies labeled with near IR dies and imaged using the Licor System. Obtained images were colored and merged to form the 38 antibody stain ECM array representation. Cells were labeled for nuclear content using Hoechst stain and for actin with Alexa488 conjugated phalloidin (Invitrogen) according to supplier's instructions.
To quantify cell-ECM interactions in the ECM array we developed and automated an image acquisition and analysis process that combines both publicly available and in house developed software. After nuclear and specific marker staining according to conventional fluorescent staining protocols slides are imaged and ECM effects quantified. First, slides are imaged using an automated inverted epifluorescent microscope with NIS Elements software. This generates a multi-channel image of the entire ECM array (20×40 mm) at 4× magnification. Large images are then imported to MATLAB, individual channels isolated, and individual spots are cropped and indexed. Individual images are then fed to CellProfiler where specific cell parameters can be quantified 497. Typically, parameters as nuclei number, nuclei occupied area, nuclei intensity, and specific marker intensity and area are calculated. CellProfiler output data is then imported back to MATLAB where array data is extracted. The first step is to transform the output data in a 40 by 100 matrix that represents each individual island in the ECM array. Replicate ECM combinations are then averaged to generate an 8 by 100 matrix comprising the average ECM score for each ECM combination in the array. Before further analysis, a statistic test is run to exclude outlier spots. Outliers are considered if the statistical distance between the spot score and the average of the five replicas present in the array is larger than the standard deviation in the five replicates. Analysis of multiple experiments revealed that less than 3% of the 4000 features per array are considered outlying points (data not shown). To allow comparison between independent experiments, the ECM array data is normalized. This step facilitates comparison of measurements between experimental batches and corrects for overall differences in the imaging process (mostly due to fluorescence lamp intensity fluctuations). The score, or adhesion value, for each ECM combination is normalized against the average and standard deviation of the array according to the following formula:
Obtained data is centered on 0 and individual ECM scores represent the distance in standard deviations from the mean of the slide creating a relative score for each ECM combination. ECM combinations close to the average value of the array have a score close to 0 whereas combinations with high or low scores have positive and negative values depending on the distance from the average of the slide.
Initial tests with MSCs focused on the multiple parameters that can be obtained during image quantification using CellProfiler for the selection of the best descriptors of cell-ECM interactions. In these experiments, MSCs were differentiated in vitro towards the adipogenic and osteogenic lineages and the cell-ECM interaction descriptors of both these differentiated cell types where compared with undifferentiated MSCs by analysis on ECM arrays. Specifically, arrays 12 hours post seeding were fixed and stained both for DNA content and actin (
The quantification of adherent nuclei was used to generate MSC adhesion profiles (
To address FDA requirements, ECM arrays were used to identify xeno-free extracellular matrices for isolation and expansion of MSCs (
Adhesion profiles can also be used to compare and distinguish different cell states. For instance, during adipogenic and osteogenic differentiation of MSCs adhesion profiles of differentiating cells change overtime (
Table 3 below is a list of the combination of adhesion sets useful for mesenchymal stem cell isolation, culture and differentiation:
In the following example, the use of ECM arrays to identify growth conditions for hepatocytes is described. Hepatocytes are the main cell type in the liver. They are responsible for metabolism of the majority of drugs in the human body. Hepatic disease affects around 20 million Americans. The availability of cells to study liver disease is limited, mainly because only around 10% of donor cells are plateable after isolation. The definition of plateability is adhesion to collagen I, an ECM component traditionally used to culture hepatocytes. The complexity of ECM in the human body is significantly greater than that of a single ECM component, so we sought to look for an appropriate ECM that would enable plating unplateable hepatocytes (
In the following example, the use of ECM arrays to identify conditions for maintaining stem cells is described. Human embryonic and induced pluripotent stem cells (hESC/hiPSC) have the ability to differentiate into all cell lineages and thus hold great promise for the treatment of human disease. hESC and hIPSC have the ability to differentiate into all cell lineages and thus hold great promise for the treatment of human disease. However, current methods to grow hIPSC require mitotically inactivated feeder cells (MEFs) or undefined ECM mixes (i.e. Matrigel) and thus introduce animal factors and lot-to-lot variability. To identify human native or recombinant ECM and understand the role of ECM in the maintenance of pluripotency, we employed the ECM platform characterized in the disclosure herein.
To identify human native or recombinant ECM components capable of maintaining pluripotency, we looked for adhesion signatures of hESC/hiPSC on our ECM array (
Follow-up showed dependence of pluripotency on specific ECM combinations: (i) single matrix molecules are unable to maintain the pluripotency phenotype and (ii) blocking ECM components and signaling induces loss of pluripotency. ECM combinations are also able to support iPSC in defined media. These ECM combinations also support differentiation of cells to specific lineages. Based on these results we were also able to study the relationships between ECM and pluripotency signal cascades. We show that different ECM combinations induce different SMAD activation profiles and that SMAD levels are related to AKT levels. Maintenance of pluripotency requires an initial activation of SMAD2/3 and this protein appears to interact with AKT. Overall, by employing an unbiased and high-throughput approach we were able to identify ECM combinations that support pluripotency and to translate these results to a simple tissue culture system that revealed aspects of the molecular mechanisms responsible for this maintenance.
Briefly, vantage acrylic slides (CEL-1 Associates VACR-25C) were coated with polyacrylamide gel pads (60×22 mm) as described previously 30. ECM arrays were spotted using a DNA Microarray spotter (Cartesian Technologies Pixsys Microarray Spotter and ArrayIt 946 Pins) from 384 well source plates containing the ECM combinations previously prepared using a Tecan EVO 150 liquid handler. Molecules were prepared to a final concentration of 200 μg/ml in a buffer described previously. 741 combinations were spotted in replicates of five and rhodamine dextran (invitrogen) was spotted as negative controls and alignment reference for analysis. ECM arrays were stored in a humidified chamber at 4° C., until later use. The following ECM molecules were incorporated in the array: Collagen I, Collagen II, Collagen III, Collagen IV, Fibronectin, Laminin, Chondroitin Sulfate, Merosin (Millipore), Collagen V, Collagen VI (BD Biosciences), Aggrecan, Elastin, Keratin, Mucin, Heparan Sulfate, Superfibronectin, Fibrin, Hyaluronan (Sigma), Tenascin-R, F-Spondin, Nidogen-2, Biglycan, Decorin, Galectin 1, Galectin 3, Galectin 4, Galectin 8, Thrombospondin-4, Osteopontin, Osteonectin, Testican 1, Testican 2, Tenascin-C, Nidogen-1, Vitronectin, Rat, Agrin, Brevican (R&D Systems) and Galectin 3c (EMD Biosciences).
Before cell seeding slides were washed in PBS and sterilized with UV light. Cell seeding occurs in specially designed devices that hold the top surface of the slides flush with bottom of the well and secure the slides under vacuum. One million cells were seeded on each slide in 5 mL of conditioned media 513(hESC media described elsewhere condition by mouse CF-1 embryonal fibroblasts) and seeded overnight at 37° C. After seeding, slides were transferred to quadriperm plates (NUNC, 167063), and fresh media was added. Cells grew for 48 hours under these conditions and fed daily. Slides were then stained for nuclei and marker expression. Briefly, slides were washed three times with PBS and fixed with 4% paraformaldehyde. At the same time nuclei were stained using Hoechst (Invitrogen) in combination with 0.1% Triton-X and PBS. Slides were then washed again and blocked using a blocking solution containing the anti-sera from the animal where secondary antibodies were raised for one hour. After blocking slides were incubated with primary antibodies oct3/4 (BD), tra1-60 and ssea4 (EBiosciences) overnight at 4° C. Secondary antibody (Invitrogen) incubation for 45 minutes followed after PBS washes. Slides were finally washed and mounted with Fluoromount-G (Southern Biotech) and stored at 4° C. until imaging. The entire slide was imaged using a Nikon Ti-Eclipse inverted fluorescence microscope and NIS Elements Software (Nikon). Image processing and analysis was performed in MATLAB (Mathworks) and nuclei and marker intensity quantification using CellProfiler as described herein. Replicate spots on each slide were averaged and those whose values were greater than one standard deviation above or below the mean of the replicates were excluded. Data was then normalized to allow averaging independent experiment data points.
hIPSC/hESC Culture
Undifferentiated iPSC and hESC were maintained as described 95. In short, Human H9 (WA09) ESC and iPSC (IPSC2A and RC2) were cultured in hESC cell media (DMEM F12 medium supplemented with 20% knockout serum replacement, non-essential amino acids, glutamine, penicillin/streptomycin and bFGF (4 ng/ml; Invitrogen)) on mitotically inactivated mouse embryonic fibroblasts (MEFs) or on Matrigel coated plates using MEF-conditioned medium. Alternatively mTESR1 media was used for studies with defined media compositions. hIPSC and hESC cultured on ECM combinations were dispersed as singe cells and seed on regular TCP plates with adsorbed ECM molecules. ECM molecules were adsorbed in diH2O at a concentration of 15 g/ml for at least six hours and then UV treated for sterilization. The regular culture conditions were otherwise maintained.
ImmunoFluorescence (IF) on cultured cells followed the protocol previously described for ECM array slides, with the adequate adaptations. For flow cytometry analysis cells were incubated for 10 min with Accutase (Millipore) at 37° C. Cells were then fixed, permeabilized and blocked with the Cytofix/Cytoperm solution (BD) for 15 min. Cells were incubated for 30 min at 4° C. with primary antibodies diluted in perm/wash buffer (BD), washed and kept on ice until analysis. Flow cytometry analysis was performed using the FACScalibur or LSRII system (BD). For phosphorylated proteins analyzed via flow cytometry, cells were incubated with for 10 min with Accutase (Millipore) at 37° C. and then fixed, permeabilized and blocked as previously reported. In brief, after accutase treatment cells were immediately fixed with 1.6% paraformaldehyde for 10 minutes at room temperature in phosphatase inhibitor containing solution (Roche). Cells were then pelleted, washed and then permeabilized with ice cold methanol for 10 minutes at 4° C. Cells were incubated for 30 min at 4° C. with primary antibodies diluted in blocking buffer (PBS with 1% BSA and PHOSSTOP), washed and kept on ice until analysis. Flow cytometry analysis was performed using the LSRII system (BD).
Adhesion Blocking Assay
Adhesion blocking experiments were done using the α and β integrin investigator kits from Millipore. Cells were incubated with integrin blocking antibodies (2 μg/ml) for 30 minutes on ice. Cells where then incubated for one hour at 37° C., fixed with 4% paraformaldehyde containing Hoechst nuclear counterstains. A scan image of the entire well was acquired using a Nikon Ti-Eclipse inverted fluorescence microscope and cell counts where performed on the NIS Elements Software (Nikon).
All animals were housed in the Koch Institute animal facility and the Committee for Animal Care in the Department of Comparative Medicine at Massachusetts Institute of Technology approved all animal procedures. To generate teratomas cells were retrieved using accutase followed by centrifugation and resuspension in 250 μl of Matrigel (2 mg/ml in DMEM-F12; BD Bioscience). Cells were injected into the dorsal flank of Nude male mice (Taconic) using a 27G needle, and teratomas were dissected 8 to 11 weeks after injection and processed for histology using hematoxylin and eosin. Sections were analyzed by a trained pathologist to determine the nature of the obtained tumors.
Karyotyping analysis was done by Cell Line Genetics (Madison, Wis.) using the high-resolution G-band standard protocols.
To generate hepatocytes, monolayers of pluripotent cells harvested using Accutase (Millipore) were plated on 6 well plates pre-coated with 2 mg/ml Matrigel (Growth Factor Reduced; BD Bioscience) at a density of 5.0×105 cells per well in hESC cell media and 10 μM Y27632 and washed the following day. Once cells reached confluency, differentiations were initiated by culture for 5 days with 100 ng/ml Activin A (R&D Systems) in RPMI/B27 medium (Invitrogen) under ambient oxygen/5% CO2, followed by 5 days with 20 ng/ml BMP4 (Peprotech)/10 ng/ml FGF-2 (Invitrogen) in RPMI/B27 under 4% O2/5% CO2, then 5 days with 20 ng/ml HGF (Peprotech) in RPMI/B27 supplement under 4% O2/5% CO2, and finally for 5 days with 20 ng/ml Oncostatin-M (R&D Systems) in Hepatocyte Culture Media (Lonza) supplemented with SingleQuots (without EGF) in ambient oxygen/5% CO2.
To generate cardiomyocytes, monolayers of pluripotent cells harvested using Accutase (Millipore) were plated on 6 well plates pre-coated with 2 mg/ml Matrigel (Growth Factor Reduced; BD Bioscience) at a density of 1.0×105 cells per well in hESC cell media and 10 μM Y27632 and washed the following day. Once cells reached confluency around day 10, differentiations were initiated by culture for 5 days with 50 ng/ml Activin A (R&D Systems) and 20 ng/mL BMP4 in RPMI/B27 medium (Invitrogen) under ambient oxygen/5% CO2, followed by 10 days with 20 ng/ml BMP4 (Peprotech)/10 ng/ml FGF-2 (Invitrogen) in RPMI/B27 under 4% O2/5% CO2, and then finally 5 days in RPMI/B27 supplement under 4% O2/5% CO2.
iPSC were differentiated according to previously established methods to the neuronal lineage. Briefly, iPSC were cultured on Matrigel-coated plates and with hES MEF conditioned media. Colonies were lifted off with dispase solution and cultured in suspension for 4 days with fresh hES media and 3 days with neural differentiation media (consisting of DMEM/F12, N2 supplement, and nonessential amino acid) (all from Invitrogen Corporation, Carlsbad, Calif.). Aggregates were then plated on a laminin coated surface (Sigma-Aldrich, St. Louis, Mo.). On day 10, 0.1 μM retinoic acid (Sigma-Aldrich) was added to the neural differentiation media. Neural tube-like rosettes formed at day 15 of differentiation and were then detached mechanically and cultured in suspension in neural induction medium containing B27, 0.1 μM retinoic acid, and 1 μM purmorphamine (Cayman Chemical). After 5 days, neurospheres were collected and split using accutase (Millipore) and passaged in suspension. A sample was collected and lyzed for PCR analysis. Neurospheres were cultured in neural induction medium with B27, FGF8b 50 ng/mL, SHH 100 ng/mL, and ascorbic acid (200 μM) for 7 days. Neurospheres were then treated with accutase/trypsin and seeded as single cells onto polyornithine/laminin coated tissue culture plates in neural differentiation medium with FGF8b 50 ng/mL, SHH 100 ng/mL, ascorbic acid (200 μM), cAMP (1.0 μM), TGFβ3 (1 ng/mL), BDNF (10 ng/mL), GDNF (10 ng/mL), IGF-1 (10 ng/mL), and WNT3A (10 ng/mL) for 21 days. All cytokines are from Peprotech except WNT3A (R&D systems) A sample was collected and lyzed for PCR analysis.
Western and Immune-Precipitation
Total protein was extracted with radioimmuneprecipitation assay lysis buffer with STOP protease inhibitors (Roche), and samples were separated by electrophoresis on 12% (wt/vol) polyacrylamide gels and electrophoretically transferred to a PVDF membrane (Bio-Rad Laboratories). Blots were probed with primary antibodies, followed by HRP-conjugated secondary antibodies, and were developed by SuperSignal West Pico substrate (Thermo Scientific).
RT-PCR
Total RNA was isolated with the RNeasy Plus Mini Kit (Qiagen). First-strand cDNA was synthesized using Moloney murine leukemia virus reverse transcriptase (Bio-Rad). Quantitative PCR was carried out with Taq polymerase and SYBR Green in the supplier's reaction buffer containing 1.5 mM MgCl2 (Bio-Rad). Oligonucleotide primer sequences are available by request. Amplicons were analyzed by both melt curve analysis on the Biorad MYIQ QRTPCR machine and confirmed by 2% (wt/vol) agarose gel electrophoresis (Sigma).
Albumin and α-1-Antitrypsin ELISA.
Spent medium was stored at −20° C. α-1-Antitrypsin and albumin media concentrations were measured using sandwich ELISA technique with HRP detection (Bethyl Laboratories) and 3,3,5,5-tetramethylbenzidine (Thermo Scientific) as a substrate.
To fully characterize these results we selected a set of ECM combinations to be validated in a system that would be easily translatable to a regular tissue culture strategy and that would enable studying the impact of ECM on pluripotency. We selected 8 ECM combinations from the four categories mentioned above (
In defined media conditions (mTESR1), all ECM combinations were able to maintain pluripotency but only one matrix combination (collagen I and laminin) could maintain the robust expansion of pluripotent stem cells. Cells on collagen II/galectin-4 and on collagen IV/galectin-8 maintained the expression of oct3/4, ssea4 and tra1-60, but had a tendency to detach from the dish forming spheroid colonies instead of spreading in the surface (
The present disclosure therefore indicates that ECM molecules, when presented in specific combinations, are a reliable and defined platform to support iPSC, a potential alternative to MEFs and Matrigel.
Pluriptient stem cells (PSCs) maintain their pluripotent potential. PSCs have been widely explored as sources for cellular modeling or as replacement therapies for human disease. The adoption of long term culture systems for PSC requires that expanded cells are still able to be directly differentiated towards functional somatic cells. We differentiated hIPSC expanded on ECM combinations towards hepatocytes (endodermal lineage), cardiomyocytes (mesodermal lineage) and neurons (ectodermal lineage) following established protocols. ECM expanded hIPSC robustly generated hepatocyte-like cells after the stimulation with activin A, BMP4/bFGF, HGF and OSM. Differentiated cells secrete albumin and al antitrypsin to levels compared to matrigel expanded cells (
In vivo tissues are formed through the combination of several ECM molecules and the specificity of ECM in each tissue can be used as a signature of the tissue. The differences in ECM composition account for the structural differences in tissues although their role in regulating cell fate is unclear. The ECM array data (
To promote adhesion in defined media conditions (such as mTESR1), fibronectin was added to our hit ECM combinations (
In the following example, growth of cells on a solid substrate on which ECM components have been absorbed is described. Human H9 (WA09) embryonic stem cells and iPSC (IPSC2A and RC2) were cultured in hESC cell media (DMEM F12 medium supplemented with 20% knockout serum replacement, non-essential amino acids, glutamine, penicillin/streptomycin and bFGF (4 ng/ml; Invitrogen)) on mitotically inactivated mouse embryonic fibroblasts (MEFs) or on Matrigel coated plates using MEF-conditioned medium. Alternatively mTESR1 media was used for studies with defined media compositions. hIPSC and hESC cultured on ECM combinations were dispersed as single cells and seed on regular TCP plates with adsorbed ECM molecules. ECM molecules were adsorbed in diH2O at a concentration of 15 μg/ml or 8 μg/ml for at least six hours and then UV treated for sterilization. The culture conditions described in the previous Example were otherwise maintained. Selected ECM combinations for validation in a regular tissue culture approach (
The example demonstrates that ECM component adsorption to TCP (polystyrene) can function in a similar fashion to multiplexed arrays of ECM components spotted to slides.
Illustrations, Figures, specification, and other diagrams may contain abbreviations for the various ECm components tested. Meaning of the abbreviations are set forth below in Table 2:
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims:
This application is an international application designating the United States of America and filed under 35 U.S.C. §120, which claims priority to U.S. Provisional Ser. No. 61/609,115, filed on Mar. 9, 2012, which is herein incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 61609115 | Mar 2012 | US |
