The invention generally relates to cell-derived extracellular matrices and uses thereof including isolation, maintenance, and proliferation of mammalian cells and differentiation of stem cells.
In vitro cell culture is perhaps the most ubiquitous, important, and poorly understood aspect of all cell biology as well as the developing fields of regenerative medicine and tissue engineering. Firstly, it allows for the observation of cell behavior so that various aspects of cell function may be studied in detail. Secondly, it allows for increase in numbers of specific cell groups. For basic research, as well as many clinical applications, it is necessary to achieve large quantities of relatively rare cells from small biological samples. In vitro cell culture permits small numbers of cells to be expanded outside the body to achieve more relevant numbers. Lastly, it permits the storage of cells for later use. By expanding cell numbers in vitro, and freezing viable cells for later use, relatively small biological samples can yield cells for multiple experiments over the span of days, months, or even years.
Despite the omnipresence of cell culture, the effects that in vitro culture has on the native characteristics of the cells it is still relatively poorly understood. Many of our current practices have arisen not from deliberate thought, planning, and experimentation, but instead from chance observations. Mammalian cell culture began in the early 1900s when, in 1911, Alexis Carrel and Montrose Burrows first published an academic paper on the cultivation of mammalian tissues in vitro. They were studying the physiology and anatomy of tissues by cutting sections of mammalian tissues and placing them on microscope slides. They then noticed that some cells migrated out of the tissue onto the slide. They went on to describe techniques for culturing cells in perpetuity. It now appears that some of their observations may not have been valid, but their work paved the way for modern cell culture.
After the discovery of hematopoietic stem cells (HSCs), groups all over the world were studying (HSCs). During their culture (in suspension), it was observed that a sub-population of bone marrow cells stuck to the bottom of the plastic flasks and began to proliferate. These cells were later recognized to be distinct from HSCs, and were eventually dubbed mesenchymal stem cells (MSCs). Because of this chance observation that lead to the discovery of MSCs, plastic adherence is still widely used as a defining attribute of MSCs and many other mammalian cell types.
The practice of culturing cells on plastic substrates is problematic because there is substantial evidence, that is now widely accepted in the literature, demonstrating the critical role of the microenvironment in regulating cell function. The microenvironment has been shown to help direct the differentiation of stem and progenitor cells, and regulate the behavior of mature cell types.
When cells are removed from their native environment to be expanded in vitro they lose important cues from their surrounding extracellular matrix or microenvironment which relay important information to the cells regarding the composition and state of their surroundings. Changes to a cell's microenvironment have a profound effect on the behavior of those cells. The current standard for isolation and expansion of most adherent cells in vitro is to place the cells in culture vessels composed of polystyrene (plastic). The polystyrene may have been treated in some manner to facilitate cell attachment and growth but the surface is, in most cases, completely foreign to the cell. In other cases, the surface may be coated with individual matrix proteins (e.g. fibronectin or collagen) or some combination of proteins. These simple substrates disregard the complexity of the native microenvironment as well as the critical role of the microenvironment in normal cell function. The cell will immediately begin to respond to this foreign environment in a manner that is much different than when the cell is in its native environment.
Five major approaches are currently employed to address this issue of culturing cells on plastic substrates:
1. Ignore the problem.—Instead of trying to achieve a desired function that matches what would be expected in vivo, a multitude of cell types can be tested in various media in order to find cells that will exhibit a specific desired function without the appropriate matrix substrate. This approach is unsophisticated and often fails to produce desired results because of the complex interplay of variables and the breadth of interactions between cells and the extracellular matrix.
2. Identify key components.—Many academic laboratories and several companies have taken the approach of considering the tissue from which cells are isolated and looking for unique elements of that tissue that may be important for cell function. Cells are then cultured on simple substrates consisting of only one or a few matrix components. This approach often fails because matrices are naturally very complex environments including over one-hundred different proteins in some cases. Cells respond just as strongly to signals they need and fail to receive, as to signals they do not need and do receive.
3. Shotgun approach—The use of protein gels like MATRIGEL™ employs a sort of shotgun approach. A gel is created that contains many different matrix proteins with the hopes that it will contain the necessary binding motifs for many different cell types. This approach may fail by providing cues that push cells in a particular direction or by failing to provide all the cues that cells are expecting.
4. Tissue-derived matrices—This is a biomimetic approach that typically involves isolating a tissue of interest from a genetically similar animal, physically disrupting or chemically digesting the tissue to obtain a solution or uniform suspension, and then coating culture vessels with the deconstructed tissue. For example, someone who wishes to culture satellite cells, might collect muscle, homogenize the tissue, and then coat a culture vessel in homogenized muscle prior to seeding the cells. This method often fails for a few key reasons. Firstly, even within a specific tissue type, the stem cell/progenitor cell niche, may be distinct from the rest of the tissue. Simply homogenizing muscle does not guarantee that an appropriate niche is being created. Secondly, the niche consists of structural and physical cues, in addition to biochemical cues. Even if many/most of the biochemical cues are present in a tissue homogenate, the structure has been destroyed, and cells may sense very different mechanical cues. Lastly, manufacturability of tissue derived matrices is dependent on availability of tissues. This affects the total amount of cell culture possible and contributes to lot-to-lot variability.
5. Cell-derived matrices—Cells in culture can be induced to secrete a matrix in their culture vessel. This matrix is the best approximation available of the in vivo niche, and can be manufactured in vitro. Cells can be induced in vitro to elaborate a matrix and then the cells can subsequently be eliminated from the matrix, for example by using non-ionizing detergent to retain structure and chemistry of the matrix. This approach has several key advantages. (1) The matrix structure can be recreated and left undisturbed. (2) The matrix can be customized based on tissue/cell type of interest. (3) The matrix can be specific to stem and progenitor cell niche. (4) The matrix can be manufactured in large quantities.
With respect to cell-derived matrices, not all cell types can be efficiently isolated and expanded on any given cell-derived matrix. In fact, pluripotent stem cells (PSCs) appear to have much different requirements for a supportive growth substrate than do other types of cells. It is known that the specific cell type used to produce a matrix will have an effect on the composition of the matrix, and therefore, the reaction of various cell types to that matrix (see Marinkovic, M. et al., One size does not fit all: developing a cell-specific niche for in vitro study of cell behavior. Matrix Biol. 54-55, 426-441 (2016)). Prior work disclosed in U.S. Pat. No. 8,084,023 has described the production and composition of an extracellular matrix produced by bone marrow stromal or mesenchymal stem cells (see also Chen, X. et al., Extracellular Matrix Made by Bone Marrow Cells Facilitates Expansion of Marrow-Derived Mesenchymal Progenitor Cells and Prevents Their Differentiation into Osteoblasts. Journal of Bone and Mineral Research 22, 1943-1956 (2007) and Lai, Y. et al., Reconstitution of marrow-derived extracellular matrix ex vivo: a robust culture system for expanding large-scale highly functional human mesenchymal stem cells. Stem cells and development 19, 1095-107 (2010)). This bone marrow cell derived matrix has been shown to support the expansion of other MSCs but has not been effective for the attachment and growth of other types of stem cells, specifically, induced pluripotent stem cells (iPSCs). iPSCs have exhibited an expanded potential to form cells and tissues from a much broader category than MSCs. This represents a particularly interesting challenge, because the difficulty of growing a confluent monolayer of iPSCs in standard culture conditions makes it impractical to produce a cell-derived matrix from iPSCs. A major limitation of previous cell-derived matrices, is that in order to make a tissue-specific matrix (e.g., bone marrow matrix from bone marrow MSCs, adipose matrix from adipose MSCs, or endothelial matrix from hUVECs), it is necessary that the target population of cells already be capable of adhering to the starting substrate. A difficulty with iPSCs, embryonic stem cells (ES), and many other cell types is that they do not readily adhere to simple substrates.
The present invention provides a solution to at least some of the aforementioned limitations and deficiencies in the art relating to cell-derived extracellular matrices (ECMs) to support the isolation, expansion and proliferation of pluripotent stem cells (PSCs), including but not limited to induced pluripotent stem cells (iPSCs) and embryonic stem cells (ES). The solution is premised on the use of an amniotic fluid cell-derived extracellular matrix. The use of uncommitted, readily adherent, and highly proliferative perinatal cells found in amniotic fluid allows for the creation of an extracellular matrix (ECM) that surprisingly, supports adhesion, isolation, expansion, and proliferation of these PSCs. This technical achievement was not possible with the cell-derived ECMs of the prior art. Additionally, the amniotic fluid cell-derived extracellular matrix (AFC-ECM) of the present invention is effective for the isolation, expansion, and proliferation of adherent cell types including but not limited to other stem cells such as mesenchymal stem cells, somatic cells, progenitor cells, mature cells, and cells from multiple germ layers. Surprisingly, the AFC-ECM of the invention supports increased proliferation of MSCs produced by bone marrow relative to a bone marrow cell-derived ECM. Furthermore, the AFC-ECM of the invention can support differentiation of stem cells into differentiated cell types.
In one aspect of the invention, disclosed is a cell-derived extracellular matrix (ECM) derived in vitro from cells isolated from amniotic fluid. In some embodiments, the ECM comprises laminin, collagen alpha-1 (XVIII), basement membrane-specific heparan sulfate proteoglycan core protein, agrin, vimentin, and collagen alpha-2 (IV), and/or isoforms thereof. In some embodiments, the isoform of collagen alpha-1 (XVIII) is isoform 2. In some embodiments, the isoform of agrin is isoform 6. In some embodiments, the cell-derived ECM further comprises fibronectin and/or an isoform thereof. In some embodiments, the cell-derived ECM does not contain any one of or all of decorin, perlecan, and collagen (III). In some embodiments, the cells isolated from amniotic fluid comprise stem cells. In some embodiments, the cell-derived ECM is decellularized. In some embodiments, the cell-derived ECM includes any one of, any combination of, or all of the components listed in Table 2 (see Example 2) and/or any variants, derivatives, or isoforms thereof listed in Table 2, preferably all of the components listed in Table 2 and/or any derivatives or isoforms thereof.
In another aspect of the invention, disclosed is method of proliferating adherent cells in culture, the method comprising culturing the adherent cells in the presence of a cell-derived extracellular matrix (ECM) in a culture media thereby proliferating the adherent cells, wherein the cell-derived ECM is derived in vitro from cells isolated from amniotic fluid. In some embodiments, the cell-derived ECM comprises laminin, collagen alpha-1 (XVIII), basement membrane-specific heparan sulfate proteoglycan core protein, agrin, vimentin, and collagen alpha-2 (IV), and/or isoforms thereof. In some embodiments, the isoform of collagen alpha-1 (XVIII) is isoform 2. In some embodiments, the isoform of agrin is isoform 6. In some embodiments, the cell-derived ECM further comprises fibronectin and/or an isoform thereof. In some embodiments, the cell-derived ECM does not contain decorin, perlecan, or collagen (III). In some embodiments, the cells isolated from amniotic fluid comprise stem cells. In some embodiments, the cell-derived ECM is decellularized. In some embodiments, the adherent cells comprise mammalian adherent cells. In various embodiments, the adherent cells comprise stem cells, somatic cells, progenitor cells, mature cells, or cells from multiple germ layers. In some embodiments, the adherent cells comprise stem cells. In some embodiments, the stem cells are maintained in an undifferentiated state. In some embodiments, the stem cells comprise pluripotent stem cells (PSCs). In some embodiments, the PSCs comprise induced PSCs (iPSC) and/or embryonic stem cells (ES). In some embodiments, the stem cells comprise mesenchymal stem cells (MSCs). In some embodiments, the MSCs are obtained from bone marrow. In some embodiments, the adherent cells comprise progenitor cells. In some embodiments, the progenitor cells comprise endothelial progenitor cells. In some embodiments, the adherent cells comprise mature cells. In some embodiments, the mature cells comprise chondrocytes. In some embodiments, the cell-derived ECM includes any one of, any combination of, or all of the components listed in Table 2 and/or any variants, derivatives, or isoforms thereof listed in Table 2, preferably all of the components listed in Table 2 and/or any derivatives or isoforms thereof.
In another aspect of the invention, disclosed is a method of inducing differentiation of stem cells into differentiated cell types, the method comprising culturing the stem cells in the presence of a cell-derived extracellular matrix (ECM) in a differentiation media, wherein the cell-derived ECM is derived in vitro from cells isolated from amniotic fluid. In some embodiments, the cell-derived ECM comprises laminin, collagen alpha-1 (XVIII), basement membrane-specific heparan sulfate proteoglycan core protein, agrin, vimentin, and collagen alpha-2 (IV), and/or isoforms thereof. In some embodiments, the isoform of collagen alpha-1 (XVIII) is isoform 2. In some embodiments, the isoform of agrin is isoform 6. In some embodiments, the cell-derived ECM further comprises fibronectin and/or an isoform thereof. In some embodiments, the cell-derived ECM does not contain decorin, perlecan, or collagen (III). In some embodiments, the cells isolated from amniotic fluid comprise stem cells. In some embodiments, the cell-derived ECM is decellularized. In some embodiments, the stem cells comprise pluripotent stem cells (PSCs). In some embodiments, the PSCs comprise induced PSCs (iPSC). In some embodiments, the PSCs comprise embryonic stem cells (ES). In some embodiments, the stem cells comprise mesenchymal stem cells (MSCs). In some embodiments, the MSCs are obtained from bone marrow. In some embodiments, the differentiated cell types comprise adipocytes, osteoblasts, chondrocytes, or myocytes. In some embodiments, the cell-derived ECM includes any one of, any combination of, or all of the components listed in Table 2 and/or any variants, derivatives, or isoforms thereof listed in Table 2, preferably all of the components listed in Table 2 and/or any derivatives or isoforms thereof.
In another aspect of the invention, disclosed is a method of producing a cell-derived extracellular matrix (ECM) in vitro, the method comprising:
In another aspect of the invention, disclosed is an amniotic fluid cell-derived extracellular matrix (AFC-ECM) produced in vitro by the method comprising:
In another aspect of the invention, disclosed is a cell-derived extracellular matrix (ECM) derived in vitro from cells isolated from an umbilical cord. In some embodiments, the cells isolated from the umbilical cord are from the cord blood and/or the Wharton's jelly.
In another aspect of the invention, disclosed is a cell-derived extracellular matrix (ECM) derived in vitro from cells isolated from placenta tissue. In some embodiments, the cells isolated from the placenta tissue are from the membrane sheets (amnion and/or chorion), the villi, and/or the blood.
Also disclosed in the context of the present invention are embodiments 1 to 53. Embodiment 1 is a cell-derived extracellular matrix (ECM) derived in vitro from cells isolated from amniotic fluid. Embodiment 2 is the cell-derived ECM of embodiment 1, wherein the ECM comprises laminin, collagen alpha-1 (XVIII), basement membrane-specific heparan sulfate proteoglycan core protein, agrin, vimentin, and collagen alpha-2 (IV), and/or isoforms thereof. Embodiment 3 is the cell-derived ECM of embodiment 2, wherein the isoform of collagen alpha-1 (XVIII) 15 isoform 2, and/or wherein the isoform of agrin is isoform 6. Embodiment 4 is the cell derived ECM of any one of embodiments 2 or 3, wherein the cell-derived ECM further comprises fibronectin and/or an isoform thereof. Embodiment 5 is the cell-derived ECM of any one of embodiments 1 to 4, wherein the cell-derived ECM does not contain decorin, perlecan, and/or collagen (III). Embodiment 6 is the cell-derived ECM of any one of embodiments 1 to 5, wherein the cells isolated from amniotic fluid comprise stem cells. Embodiment 7 is the cell-derived ECM of any one of embodiments 1 to 6, wherein the cell-derived ECM is decellularized.
Embodiment 8 is a method of proliferating adherent cells in culture, the method comprising culturing the adherent cells in the presence of a cell-derived extracellular matrix (ECM) in a culture media thereby proliferating the adherent cells, wherein the cell-derived ECM is derived in vitro from cells isolated from amniotic fluid. Embodiment 9 is the method of embodiment 8, wherein the cell-derived ECM comprises laminin, collagen alpha-1 (XVIII), basement membrane-specific heparan sulfate proteoglycan core protein, agrin, vimentin, and collagen alpha-2 (IV), and/or isoforms thereof. Embodiment 10 is the method of embodiment 9, wherein the isoform of collagen alpha-1 (XVIII) is isoform 2, and/or wherein the isoform of agrin is isoform 6. Embodiment 11 is the method of any one of embodiments 9 or 10, wherein the cell-derived ECM further comprises fibronectin and/or an isoform thereof. Embodiment 12 is the method of any one of embodiments 8 to 11, wherein the cell-derived ECM does not contain decorin, perlecan, and/or collagen (III). Embodiment 13 is the method of any one of embodiments 8 to 12, wherein the cells isolated from amniotic fluid comprise stem cells. Embodiment 14 is the method of any one of embodiments 8 to 13, wherein the cell-derived ECM is decellularized prior to contact with the adherent stem cells. Embodiment 15 is the method of any one of embodiments 8 to 14, wherein the adherent cells comprise mammalian adherent cells. Embodiment 16 is the method of any one of embodiments 8 to 15, wherein the adherent cells comprise stem cells, somatic cells, progenitor cells, mature cells, or cells from multiple germ layers. Embodiment 17 is the method of embodiment 16, wherein the adherent cells comprise stem cells. Embodiment 18 is the method of embodiment 17, wherein the stem cells are maintained in an undifferentiated state. Embodiment 19 is the method any one of embodiments 17 or 18, wherein the stem cells comprise pluripotent stem cells (PSCs). Embodiment 20 is the method of embodiment 19, wherein the PSCs comprise induced PSCs (iPSC). Embodiment 21 is the method of embodiment 19, wherein the PSCs comprise embryonic stem cells (ES). Embodiment 22 is the method of any one of embodiments 17 or 18, wherein the stem cells comprise mesenchymal stem cells (MSCs). Embodiment 23 is the method of embodiment 22, wherein the MSCs are obtained from bone marrow. Embodiment 24 is the method of embodiment 16, wherein the adherent cells comprise progenitor cells. Embodiment 25 is the method of embodiment 24, wherein the progenitor cells comprise endothelial progenitor cells. Embodiment 26 is the method of embodiment 16, wherein the adherent cells comprise mature cells. Embodiment 27 is the method of embodiment 26, wherein the mature cells comprise chondrocytes.
Embodiment 28 is a method of inducing differentiation of stem cells into differentiated cell types, the method comprising culturing the stem cells in the presence of a cell-derived extracellular matrix (ECM) in a differentiation media, wherein the cell-derived ECM is derived in vitro from cells isolated from amniotic fluid. Embodiment 29 is the method of embodiment 28, wherein the cell-derived ECM comprises laminin, collagen alpha-1 (XVIII), basement membrane-specific heparan sulfate proteoglycan core protein, agrin, vimentin, and collagen alpha-2 (IV), and/or isoforms thereof. Embodiment 30 is the method of embodiment 29, wherein the isoform of collagen alpha-1 (XVIII) is isoform 2, and/or wherein the isoform of agrin is isoform 6. Embodiment 31 is the method of any one of embodiments 29 or 30, wherein the cell-derived ECM further comprises fibronectin and/or an isoform thereof. Embodiment 32 is the method of any one of embodiments 28 to 31, wherein the cell-derived ECM does not contain decorin, perlecan, and/or collagen (III). Embodiment 33 is the method of any one of embodiments 28 to 32, wherein the cells isolated from amniotic fluid comprise stem cells. Embodiment 34 is the method of any one of embodiments 28 to 33, wherein the stem cells comprise pluripotent stem cells (PSCs). Embodiment 35 is the method of embodiment 34, wherein the PSCs comprise induced PSCs (iPSC). Embodiment 36 is the method of embodiment 34, wherein the PSCs comprise embryonic stem cells (ES). Embodiment 37 is the method of any one of embodiments 28 to 33, wherein the stem cells comprise mesenchymal stem cells (MSCs). Embodiment 38 is the method of embodiment 22, wherein the MSCs are obtained from bone marrow. Embodiment 39 is the method of any one of embodiments 37 or 38, wherein the differentiated cell types comprise adipocytes, osteoblasts, chondrocytes, or myocytes.
Embodiment 40 is a method of producing a cell-derived extracellular matrix (ECM) in vitro, the method comprising: (a) isolating cells from amniotic fluid; (b) seeding the isolated cells onto a cell culture container or onto a cell culture container coated with a substrate; (c) adding a culture media to the cell culture container; and (d) culturing the cells, thereby producing a cell-derived ECM; and (e) optionally decellularizing the cell-derived ECM. Embodiment 41 is the method of embodiment 40, wherein the isolated cells from the amniotic fluid comprise stem cells. Embodiment 42 is the method of embodiment 40 or 41, wherein the substrate is fibronectin. Embodiment 43 is the method of any one of embodiments 40 to 42, wherein the cell-derived ECM comprises laminin, collagen alpha-1 (XVIII), basement membrane-specific heparan sulfate proteoglycan core protein, agrin, vimentin, and collagen alpha-2 (IV), and/or isoforms thereof. Embodiment 44 is the method of embodiment 43, wherein the isoform of collagen alpha-1 (XVIII) is isoform 2, and/or wherein the isoform of agrin is isoform 6. Embodiment 45 is the method of any one of embodiments 43 or 44, wherein the cell-derived ECM further comprises fibronectin and/or an isoform thereof. Embodiment 46 is the method of any one of embodiments 40 to 45, wherein the cell-derived ECM does not contain decorin, perlecan, and/or collagen (III).
Embodiment 47 is an amniotic fluid cell-derived extracellular matrix (AFC-ECM) produced in vitro by the method comprising: (a) isolating cells from amniotic fluid, (b) seeding the isolated cells onto a cell culture container or onto a cell culture container coated with a substrate, (c) adding a culture media to the cell culture container, and (d) culturing the cells, thereby producing the AFC-ECM, and (e) optionally decellularizing the AFC-ECM. Embodiment 48 is the method of embodiment 47, wherein the isolated cells from the amniotic fluid comprise stem cells. Embodiment 49 is the method of embodiment 47 or 48, wherein the substrate is fibronectin. Embodiment 50 is the method of any one of embodiments 47 to 49, wherein the cell-derived ECM comprises laminin, collagen alpha-1 (XVIII), basement membrane-specific heparan sulfate proteoglycan core protein, agrin, vimentin, and collagen alpha-2 (IV), and/or isoforms thereof. Embodiment 51 is the method of any embodiment 50, wherein the isoform of collagen alpha-1 (XVIII) is isoform 2, and/or wherein the isoform of agrin is isoform 6. Embodiment 52 is the method of any one of embodiments 50 or 51, wherein the cell-derived ECM further comprises fibronectin and/or an isoform thereof. Embodiment 53 is the method of any one of embodiments 47 to 52, wherein the cell-derived ECM does not contain decorin, perlecan, and/or collagen (III).
The term “stem-like characteristic” or “stemness” refers a characteristic that can be observed in or associated with a stem cell. For example, one of the stem-like characteristics can be chosen from an ability of differentiating into different cell types, an ability of self-renewal, an ability of survival under certain conditions (including hypoxia conditions), an ability to resist chemotherapeutic agents, and an expression of a wide variety of markers.
The term “isoform” refers to different forms of a biomolecule or a protein. The different forms of the biomolecule or protein may be produced by a variety of processes or mechanisms. In embodiments in which the biomolecule is a protein, the isoforms may be proteins that differ in sequence by one or more amino acids. For example, the protein isoforms may be genetic alleles. Alternatively, the protein isoforms may be the products of alternate splicing, RNA editing, posttranslational processing, and the like.
The terms “about” or “approximately” are defined as being close to as understood by one of ordinary skill in the art, and in one non-limiting embodiment the terms are defined to be within 10%, preferably within 5%, more preferably within 1%, and most preferably within 0.5%.
The term “substantially” and its variations are defined to include ranges within 10%, within 5%, within 1%, or within 0.5%.
The terms “wt. %,” “vol. %,” or “mol. %” refers to a weight percentage of a component, a volume percentage of a component, or molar percentage of a component, respectively, based on the total weight, the total volume of material, or total moles, that includes the component. In a non-limiting example, 10 grams of component in 100 grams of the material is 10 wt. % of component.
The terms “inhibiting” or “reducing” or “preventing” or “avoiding” or any variation of these terms, when used in the claims and/or the specification includes any measurable decrease or complete inhibition to achieve a desired result.
The term “effective,” as that term is used in the specification and/or claims, means adequate to accomplish a desired, expected, or intended result.
The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
The use of the word “a” or “an” when used in conjunction with the terms “comprising,” “having,” “including,” or “containing” (or any variations of these words) may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”
The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of” any of the ingredients or steps disclosed throughout the specification. With respect to the transitional phrase “consisting essentially of,” in one non-limiting aspect, a basic and novel characteristic of the cell-derived extracellular matrix of the present invention is that it (1) is derived in vitro from cells isolated from amniotic fluid and (2) provides an environment that supports adhesion, isolation, expansion, and/or proliferation of PSCs.
It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
The present invention discloses a cell-derived extracellular matrix (ECM) derived in vitro from cells isolated from amniotic fluid and methods of making the ECM. Also disclosed are methods of using the ECM for the isolation, maintenance, and expansion/proliferation of adherent cells which can be mammalian cells.
The function of mammalian cells is determined, largely, by the environment, e.g., an extracellular matrix, in which they reside. They react to signals that are present in their environment (positive signals) and also to signals that are required but are not present (negative signals). It is likely that uncommitted stem cells can produce a matrix that contains niche motifs necessary to maintain stem cell viability and stemness, but lack many lineage specific signals that more mature cells may secrete which would push a stem cell toward a particular fate. Without being bound by theory, it is suggested that a less mature cell, e.g., a perinatal cell or perinatal stem cell, may produce an ECM that is different from ECMs disclosed previously in the art, such as bone marrow stromal cell-derived ECMs, and may allow for better isolation and expansion/proliferation of stem cells with higher potential than mesenchymal stem cells (MSCs), such as pluripotent stem cells (PSCs). Mass spectrometry demonstrated, that compared to previously known cell-derived ECMs, the amniotic fluid cell-derived ECM of the invention contains matrix proteins found in all 3 germ layers and lacked specific proteins strongly associated with osteogenic lineages. Moreover, the ECM of the invention contained specific motifs, such as laminin, that are known to facilitate pluripotent cell adhesion and expansion.
Perinatal cells can be divided into three groups: cells from amniotic fluid; cells from the placenta; and cells from the umbilical cord. Amniotic fluid has several sources of cells including cells derived from the developing fetus sloughed from the fetal amnion membrane, skin, and alimentary, respiratory, and urogenital tracts. Placenta also has several sources of cells including the membrane sheets (amnion and chorion), the villi, and the blood. Umbilical cord cells generally come from two sources, cord blood and Wharton's jelly. The cells from these three perinatal sources can include stem cells. The cells used to produce the amniotic fluid cell-derived ECM of the invention are obtained from the amniotic fluid of a mammal including but not limited to a human (Homo sapiens), murine, rabbit, cat, dog, pig, equine, or primate. In preferred embodiments, the cells are from the amniotic fluid of a human. The amniotic fluid can be sourced from humans at full-term births (greater than about 37 weeks gestational age) or pre-term births (less than about 37 weeks gestational age). Pre-term births include late pre-term births (about 33 to about 37 weeks gestational age) moderate pre-term births (about 29 to about 33 weeks gestational age), and extreme pre-term births (about 23 to about 29 weeks gestational age). The amniotic fluid can be sourced from humans prior to birth at any gestational age where amniotic fluid is present, and can be combined with sources of amniotic fluid from births. Generally, prior to birth, amniotic fluid is collected by an amniocentesis procedure. In some embodiments the amniotic fluid is sourced from humans at full-term births, at pre-term births, at late pre-term births, at moderate pre-term births, at extreme pre-term births, or prior to birth, or combinations thereof. In some embodiments, the amniotic fluid is sourced prior to birth and is collected from about 10 weeks gestational age up to birth, or from about 10 weeks to about 23 weeks gestational age, or from about 10 weeks to about 16 weeks gestational age, or from about 12 weeks gestational age up to birth, or from about 12 weeks to about 23 weeks gestational age, or from about 12 weeks to about 16 weeks gestational age. In some embodiments, the amniotic fluid sourced prior to birth is collected by an amniocentesis procedure. The cells can be obtained and isolated from amniotic fluid by techniques known in the art, such as those disclosed in Murphy et. al., Amniotic Fluid Stem Cells, Perinatal Stem Cells, Second Ed. 2013.
Amniotic fluid is comprised of cells having the ability to differentiate into cell types derived from all 3 embryonic germ layers (ectoderm, endoderm, mesoderm) spontaneously or as a result of treatment with specific growth factors or combinations of growth factors known to one of skill in the art. That is, a single cell has the capacity to be induced to express genes which are specific to any of the three germ layers. Amniotic fluid also contains a mixture of different cell types including cells derived from the developing fetus sloughed from the fetal amnion membrane, skin, and alimentary, respiratory, and urogenital tracts. Because of the origin of the amniotic fluid and placental membranes, these cells can maintain highly multipotent differentiation potential and comprise a cell population that contains cells of all three germ layers. The amniotic fluid cells can comprise stem cells. In some embodiments, the amniotic fluid cells are isolated stem cells. In some embodiments, the amniotic fluid cells comprise stem cells having the ability to differentiate into cell types derived from all 3 embryonic germ layers (ectoderm, endoderm, mesoderm) and/or multipotent stem cells, and/or pluripotent stem cells.
The amniotic fluid cell-derived ECM of the invention is comprised of various proteins. The proteins of the ECM can be identified by techniques known in the art and include mass spectroscopy and immunohistochemical staining. The ECM can include, but is not limited to the components listed in Table 2 (see Example 2 below) and any variants, derivatives, or isoforms thereof. The amniotic fluid-cell derived ECM can include any combination of any of the components and any variants, derivatives, or isoforms thereof from Table 2. In some embodiments, a combination can comprise, consist essentially of, or consist of: laminin, collagen alpha-1 (XVIII), basement membrane-specific heparan sulfate proteoglycan core protein, agrin, vimentin, and collagen alpha-2 (IV), and/or isoforms thereof. In some embodiments, the isoform of collagen alpha-1 (XVIII) is isoform 2. In some embodiments, the isoform of agrin is isoform 6. In some embodiments, the cell-derived ECM further comprises, consist essentially of, or consists of fibronectin and/or an isoform thereof. In some embodiments, the amniotic fluid cell-derived ECM does not contain any one of or all of decorin, perlecan, and collagen (III). The most abundant collagens in Table 2 are collagens I, IV, and XVIII. Some noteworthy differences in proteins between the amniotic fluid cell-derived ECM of the present inventions and a bone marrow cell-derived matrix are described in Table 1.
The amniotic fluid cell-derived ECM (AFC-ECM) of the invention can be produced in vitro by the following process:
Disclosed herein is a method of producing in vitro a cell-derived extracellular matrix (ECM), the method comprising:
Disclosed herein is an amniotic fluid cell-derived extracellular matrix (AFC-ECM) produced in vitro by the method comprising:
Any seeding density may be used which allows cells to form a confluent monolayer immediately or after a period of time in culture. In some embodiments, the seeding density is about 10 cells/cm2-about 100,000 cells/cm2, or about 100 cells/cm2-about 75,000 cells/cm2, or about 500 cells/cm2-about 50,000 cells/cm2, or about 500 cells/cm2-about 10,000 cells/cm2, or about 500 cells/cm2-about 5,000 cells/cm2, or about 500 cells/cm2-about 2,500 cells/cm2, or about 1,000 cells/cm2-about 25,000 cells/cm2, or about 2,000 cells/cm2-about 10,000 cells/cm2, or about 3,000 cells/cm2-about 5000 cells/cm2.
Any type of container suitable for cultivation of cells can be used for the present invention. Examples include, but are not limited to cell culture flasks, T-flasks, stirred flasks, spinner flasks, fermenters, and bioreactors. Rocking bottles, shaking flasks, tubes, and other containers are also suitable containers when placed on a rocking platform or shaker. The cell culture container can be coated with a substrate to allow for better cell adhesion. A non-limiting example of a suitable substrate for coating the cell container is fibronectin.
Various commercially available cell culture media, e.g., alpha Minimum Essential Media (α-MEM) culture media (Thermo Fisher Scientific, Grand Island, N.Y.), are suitable for culturing amniotic fluid cells. The commercially available culture media can be modified by adding various supplemental substances to the media, e.g. sodium bicarbonate, L-glutamine, penicillin, streptomycin, Amphotericin B and/or serum. The serum can be fetal bovine serum. The media can also be serum free. Additionally, substances such as L-ascorbic acid can be added to the media or modified media to induce cell production of an ECM.
The initial culture media can be changed and/or replaced with another media at various times during the culturing process. For example, the initial media can be a “Complete Media” and then be replaced by an “Inducing Media” during the culturing process. A non-limiting example of a “Complete Media” contains (α-MEM) plus 2 mM L-Glutamine plus antibiotic-antimycotic plus 15% Fetal Bovine Serum. A non-limiting example of an “Inducing Media” contains the “Complete Media” plus 50 mM L-Ascorbic Acid.
The culturing of the amniotic fluid cells can take place in an incubator at 37° C., 5% CO2, and 90% humidity. Culturing can take place under various environmental conditions including, but not limited to normoxic, i.e., 20-21% oxygen in the atmosphere, or hypoxic conditions.
Decellularizing the amniotic fluid cell-derived ECM of the amniotic fluid cells can include removing the viable amniotic fluid cells or rendering the amniotic fluid cells non-viable. The amniotic fluid cells can be decellularized from the ECM by using methods known in the art and can include, but are not limited to lysing the amniotic fluid cells and then removing the lysed amniotic fluid cells by washing. Various substances can be used to remove the amniotic fluid cells from the ECM. Non-limiting examples include an “Extraction Buffer” containing TRITON X-100 and ammonium hydroxide in PBS buffer. After the ECM has been decellularized of amniotic fluid cells, the resulting ECM is thereby essentially cell-free or free of viable amniotic fluid cells. If feeder cells are used, then the decellularizing methods also apply to any viable feeder cells present on the ECM, thereby resulting in the ECM being essentially free or free of viable feeder cells. The decellularizing methods also apply to any viable cells present on the ECM, thereby resulting in the ECM being essentially free or free of any viable cells. Thus, a decellularized ECM means that the ECM is acellular, meaning that the ECM is free of any viable cells.
In some embodiments, the amniotic fluid cell-derived ECM is a three-dimensional (3D) ECM.
The methods described supra also apply to producing cell-derived ECMs from other perinatal cells such as cells from the umbilical cord including the cord blood and Wharton's jelly; and cells from placenta tissue including the membrane sheets (amnion and chorion), the villi and the blood.
In one embodiment, a perinatal cell-derived ECM is produced in vitro by the following process:
In another embodiment, a perinatal cell-derived ECM is produced in vitro by the following process:
In one aspect of the invention, disclosed is a cell-derived extracellular matrix (ECM) derived in vitro from cells isolated from an umbilical cord. In some embodiments, the cells isolated from the umbilical cord are from the cord blood and/or the Wharton's jelly.
In another aspect of the invention, disclosed is a cell-derived extracellular matrix (ECM) derived in vitro from cells isolated from placenta tissue. In some embodiments, the cells isolated from the placenta tissue are from the membrane sheets (amnion and/or chorion), the villi, and/or the blood
Methods to expand/proliferate adherent cells include obtaining the adherent cells and culturing them in the presence of the amniotic fluid cell-derived ECM of the invention. In some embodiments, the adherent cells are mammalian adherent cells. Any seeding density may be used which allows cells to form a confluent monolayer immediately or after a period of time in culture. In some embodiments, the seeding density is about 10 cells/cm2-about 100,000 cells/cm2, or about 100 cells/cm2-about 75,000 cells/cm2, or about 500 cells/cm2-about 50,000 cells/cm2, or about 500 cells/cm2-about 10,000 cells/cm2, or about 500 cells/cm2-about 5,000 cells/cm2, or about 500 cells/cm2-about 2,500 cells/cm2, or about 1,000 cells/cm2-about 25,000 cells/cm2, or about 2,000 cells/cm2-about 10,000 cells/cm2, or about 3,000 cells/cm2-about 5000 cells/cm2. In various embodiments, the adherent cells are stem cells, somatic cells, progenitor cells, mature cells, and/or cells from multiple germ layers.
In some embodiments, the adherent cells are stem cells. In some embodiments, the stem cells are pluripotent stem cells (PSCs) or mesenchymal stem cells (MSCs). Pluripotent stem cells (PSCs) can self-renew and differentiate into any of the three germ layers: ectoderm, endoderm, and mesoderm, from which all tissues and organs develop. Embryonic stem cells (ES) are currently the only known natural pluripotent stem cells. Induced pluripotent stem (iPSCs) cells also are PSCs. iPSCs are derived from cells generally taken from adult tissue or adult cells, and reprogrammed to the level of embryonic stem cells. Methods for producing iPSCs are known in the art. Mesenchymal stem cells (MSCs) are multipotent stromal cells. MSCs can be obtained from the following non-limiting sources: bone marrow, umbilical cord tissue, e.g. Wharton's jelly, umbilical cord blood, adipose tissue, and amniotic fluid. In some embodiments the stem cells are maintained in an undifferentiated state and maintain their stemness.
In some embodiments, the adherent cells are somatic cells also known as vegetal cells, which are any biological cells in a multicellular organism other than reproductive cells, gametes, germ cells, gametocytes, or undifferentiated stem cells.
In some embodiments, the adherent cells are cells from multiple germ layers including cells from the ectoderm, endoderm, and/or mesoderm.
In some embodiments, the adherent cells are progenitor cells including but not limited to endothelial progenitor cells (EPCs), angioblasts, pancreatic progenitor cells, progenitor cells from the periosteum, and bone marrow stromal cells.
In some embodiments, the adherent cells are mature cells including but not limited to chondrocytes and osteoblasts.
Cell culture techniques suitable for proliferation of adherent cells in culture are known in the art and can be followed. Suitable commercially available culture media for cell proliferation includes, but is not limited to: StemMACS™ iPS-Brew XF available from Miltenyl Biotec; alpha minimum essential media (aMEM) which can be supplemented with fetal bovine serum, antibiotic-antimycotic (anti-anti), and/or GlutaMax™ available from Gibco; and Iscove's Modified Dulbecco's Medium (IMDM) which can be supplemented with fetal bovine serum, antibiotic-antimycotic (anti-anti), GlutaMax and/or growth factors such as EGF and FGF. In some embodiments, no Rock inhibitor is used. Once cells begin to approach confluence (e.g., as determined by brightfield microscopy), cells can be passaged manually, by cutting large colonies into smaller colonies and then re-plate those by physically lifting them off the dish and placing them on a fresh plate of amniotic fluid cell-derived ECM. This procedure can be repeated indefinitely. In some embodiments disclosed is a method of proliferating adherent cells in culture, the method comprising culturing the cells in the presence of a cell-derived extracellular matrix (ECM) in a culture media thereby proliferating the adherent cells, wherein the cell-derived ECM is derived in vitro from cells isolated from amniotic fluid.
The methods of expanding/proliferating adherent cells described supra also apply to the use of expanding/proliferating adherent cells in culture in the presence of other perinatal cell-derived ECMs. In some aspects, disclosed is a method of proliferating adherent cells in culture, the method comprising culturing the adherent cells in the presence of a cell-derived extracellular matrix (ECM) in a culture media thereby proliferating the adherent cells, wherein the cell-derived ECM is derived in vitro from cells isolated from an umbilical cord or placenta tissue. In some embodiments, the cells isolated from the umbilical cord are from the cord blood and/or the Wharton's jelly. In other embodiments, the cells isolated from the placenta tissue are from the membrane sheets (amnion and/or chorion), the villi, and/or the blood.
Disclosed herein are methods of inducing differentiation of stem cells into differentiated cell types, the method comprising culturing the stem cells in the presence of a cell-derived extracellular matrix (ECM) in a differentiation media, wherein the cell-derived ECM is derived in vitro from cells isolated from amniotic fluid, i.e. the amniotic fluid cell-derived ECM of the invention. In some embodiments, the cell-derived ECM comprises laminin, collagen alpha-1 (XVIII), basement membrane-specific heparan sulfate proteoglycan core protein, agrin, vimentin, and collagen alpha-2 (IV), and/or isoforms thereof. In some embodiments, the isoform of collagen alpha-1 (XVIII) is isoform 2. In some embodiments, the isoform of agrin is isoform 6. In some embodiments, the cell-derived ECM further comprises fibronectin and/or an isoform thereof. In some embodiments, the cell-derived ECM does not contain decorin, perlecan, or collagen (III). In some embodiments, the cells isolated from amniotic fluid comprise stem cells. In some embodiments, the cell-derived ECM is decellularized. The stem cells can be pluripotent stem cells (PSCs), such as induced PSCs (iPSC) or embryonic stem cells (ES). The stem cells can be mesenchymal stem cells (MSCs). MSCs can be obtained from the following non-limiting sources: bone marrow, umbilical cord tissue, e.g. Wharton's jelly, umbilical cord blood, adipose tissue, and amniotic fluid. In some embodiments, the MSCs are obtained from bone marrow. In some embodiments, the differentiated cell types can be adipocytes, osteoblasts, chondrocytes, or myocytes.
Cell culture techniques for differentiation of stem cells into differentiated cell types are known in the art and can be followed. Various commercially available differentiation media are suitable for use and can be specific for a particular desired differentiated cell type. For example, an adipogenic differentiation media can comprise DMEM containing FBS, IBMX, dexamethasone, insulin, and/or indomethacin. Another example is an osteoblast differentiation media which can be a growth media supplemented with dexamethasone and/or L-ascorbate-2-phosphate.
The methods of inducing differentiation of stem cells described supra also apply to the use of culturing stem cells in the presence of other perinatal cell-derived ECMs in differentiation media. In some aspects, disclosed is a method of inducing differentiation of stem cells into differentiated cell types, the method comprising culturing the stem cells in the presence of a cell-derived extracellular matrix (ECM) in a differentiation media, wherein the cell-derived ECM is derived in vitro from cells isolated from an umbilical cord or placenta tissue. In some embodiments, the cells isolated from the umbilical cord are from the cord blood and/or the Wharton's jelly. In other embodiments, the cells isolated from the placenta tissue are from the membrane sheets (amnion and/or chorion), the villi, and/or the blood.
The following examples are included to demonstrate certain non-limiting aspects of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the applicants to function well in the practice of the invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Four amniotic fluid cell-derived ECMs (Matrix A, Matrix B, Matrix C, and Matrix D) were made using the following procedure: cells aseptically isolated from amniotic fluid collected from full term birth (>37 weeks gestational age) from 4 donors were seeded onto fibronectin coated tissue-culture treated flasks and cultured in Complete Media at 37° C., 5% CO2 and 90% RH in an incubator. The Complete Media was alpha Minimum Essential Media (aMEM) plus 2 mM L-Glutamine plus antibiotic-antimycotic plus 15% Fetal Bovine Serum.
At day 3-4, one-half of the complete medium was aspirated from the flasks and replaced with one-half of new Complete Media. The flasks were placed back into the incubator at the same conditions as stated above.
At day 7-8, the Complete Media was aspirated from the culture flasks and was replenished with Inducing Media. The flasks were placed back into the incubator at the same conditions as stated above. The Inducing Media was Complete Media plus 50 mM L-Ascorbic Acid.
At day 10-11, the Inducing Media was aspirated from the culture flasks and the ECM which had formed inside the flasks was washed one time with phosphate buffered saline (PBS). Then the PBS was aspirated from the flasks. An Extraction Buffer was added to the flasks and incubated for 7-10 minutes at RT to decellularize each ECM, then the Extraction Buffer was aspirated from the flasks. The Extraction Buffer was PBS containing 0.5% (v/v) TRITON-X100 and 20 mM ammonium hydroxide (NH4OH).
Each of the decellularized ECMs in the flasks was washed three times with PBS followed by one wash with sterile water and then the sterile water was aspirated from the flasks. The four decellularized ECMs in the flasks were allowed to dry at RT and then stored at 4° C.
A photomicrograph of a Brightfield Image of an amniotic fluid cell-derived ECM (Matrix B) is shown in
The composition of the each of the amniotic fluid cell-derived ECMs produced in Example 1 was determined by mass spectrometry. The components with their spectral count and molecular weight are listed in Table 2.
sapiens OX = 9606 GN = SPTAN1 PE = 1 SV = 3
sapiens OX = 9606 GN = SPTBN1 PE = 1 SV = 2
sapiens OX = 9606 GN = GAPDH PE = 1 SV = 3
sapiens OX = 9606 GN = HSPA5 PE = 1 SV = 2
sapiens OX = 9606 GN = HSPD1 PE = 1 SV = 2
sapiens OX = 9606 GN = COL12A1
sapiens OX = 9606 GN = IQGAP1 PE = 1 SV = 1
sapiens OX = 9606 GN = COL18A1
sapiens OX = 9606 GN = H2AFY
sapiens OX = 9606 GN = ATP5F1B PE = 1 SV = 3
sapiens OX = 9606 GN = VCP PE = 1 SV = 4
sapiens OX = 9606 GN = HNRNPU PE = 1 SV = 6
sapiens OX = 9606 GN = ATP5F1A PE = 1 SV = 1
sapiens OX = 9606 GN = HNRNPM PE = 1 SV = 3
sapiens OX = 9606 GN = HNRNPK PE = 1 SV = 1
sapiens OX = 9606 GN = EPRS PE = 1 SV = 5
sapiens OX = 9606 GN = IMMT
sapiens OX = 9606 GN = YWHAB
sapiens OX = 9606 GN = HNRNPA1 PE = 1 SV = 5
sapiens OX = 9606 GN = XRCC6 PE = 1 SV = 2
sapiens OX = 9606 GN = AP2B1
sapiens OX = 9606 GN = HMGA1 PE = 1 SV = 3
sapiens OX = 9606 GN = PDIA6
sapiens OX = 9606 GN = KRT2 PE = 1 SV = 2
sapiens OX = 9606 GN = TINAGL1 PE = 1 SV = 1
sapiens OX = 9606 GN = ESYT1
sapiens OX = 9606 GN = TOP2B
sapiens OX = 9606 GN = RACK1 PE = 1 SV = 3
sapiens OX = 9606 GN = XRCC5 PE = 1 SV = 3
sapiens OX = 9606 GN = MYO1C
sapiens OX = 9606 GN = RHOC PE = 1 SV = 1
sapiens OX = 9606 GN = RSL1D1 PE = 1 SV = 3
sapiens OX = 9606 GN = PSMC3 PE = 1 SV = 1
sapiens OX = 9606 GN = ATP5PO PE = 1 SV = 1
sapiens OX = 9606 GN = HNRNPR PE = 1 SV = 1
sapiens OX = 9606 GN = TPM1
sapiens OX = 9606 GN = PTBP1 PE = 1 SV = 1
sapiens OX = 9606 GN = SFPQ PE = 1 SV = 2
sapiens OX = 9606 GN = ARPC2 PE = 1 SV = 1
sapiens OX = 9606 GN = NARS PE = 1 SV = 1
sapiens OX = 9606 GN = HNRNPA3 PE = 1 SV = 2
sapiens OX = 9606 GN = AP1B1
sapiens OX = 9606 GN = ALDH1B1 PE = 1 SV = 3
sapiens OX = 9606 GN = DDX3X PE = 1 SV = 3
sapiens OX = 9606 GN = CAPZA1 PE = 1 SV = 3
sapiens OX = 9606 GN = HP1BP3 PE = 1 SV = 1
sapiens OX = 9606 GN = TPM3
sapiens OX = 9606 GN = MARS PE = 1 SV = 2
sapiens OX = 9606 GN = MYADM PE = 1 SV = 2
sapiens OX = 9606 GN = WARS PE = 1 SV = 2
sapiens OX = 9606 GN = VPS35 PE = 1 SV = 2
sapiens OX = 9606 GN = STOM PE = 1 SV = 3
sapiens OX = 9606 GN = HNRNPL PE = 1 SV = 2
sapiens OX = 9606 GN = GLG1
sapiens OX = 9606 GN = PCBP2
sapiens OX = 9606 GN = SNRPD1 PE = 1 SV = 1
sapiens OX = 9606 GN = CDC42 PE = 1 SV = 2
sapiens OX = 9606 GN = CLIC1 PE = 1 SV = 4
sapiens OX = 9606 GN = HNRNPH1 PE = 1 SV = 1
sapiens OX = 9606 GN = DDX21
sapiens OX = 9606 GN = PDLIM7
sapiens OX = 9606 GN = RPL17
sapiens OX = 9606 GN = PRPF8 PE = 1 SV = 2
sapiens OX = 9606 GN = RBMX PE = 1 SV = 3
sapiens OX = 9606 GN = TRIM28 PE = 1 SV = 5
sapiens OX = 9606 GN = DDX18 PE = 1 SV = 2
sapiens OX = 9606 GN = DPYSL2 PE = 1 SV = 1
sapiens OX = 9606 GN = CAPZA2 PE = 1 SV = 3
sapiens OX = 9606 GN = LRRC59 PE = 1 SV = 1
sapiens OX = 9606 GN = HSD17B12 PE = 1 SV = 2
sapiens OX = 9606 GN = PSMC6 PE = 1 SV = 1
sapiens OX = 9606 GN = RPL23A PE = 1 SV = 1
sapiens OX = 9606 GN = ARPC3 PE = 1 SV = 3
sapiens OX = 9606 GN = ARPC4 PE = 1 SV = 3
sapiens OX = 9606 GN = APEX1 PE = 1 SV = 2
sapiens OX = 9606 GN = COL6A3
sapiens OX = 9606 GN = GPI
sapiens GN = AP3D1
sapiens GN = MOGS PE = 1 SV = 5
sapiens OX = 9606 GN = SRP72 PE = 1 SV = 3
sapiens OX = 9606 GN = SNRPD2 PE = 1 SV = 1
sapiens OX = 9606 GN = SMARCC1 PE = 1 SV = 3
sapiens OX = 9606 GN = SSR4 PE = 1 SV = 1
sapiens OX = 9606 GN = OGDH PE = 1 SV = 3
sapiens OX = 9606 GN = EIF6 PE = 1 SV = 1
sapiens OX = 9606 GN = RPS24
sapiens OX = 9606 GN = PRKCSH
sapiens OX = 9606 GN = PSMA3
sapiens OX = 9606 GN = MYO1B
sapiens OX = 9606 GN = FUS
sapiens OX = 9606 GN = MTCH1 PE = 1 SV = 1
sapiens OX = 9606 GN = YBX1 PE = 1 SV = 3
sapiens GN = HSD17B4 PE = 1 SV = 3
sapiens GN = PRMT1 PE = 1 SV = 2
sapiens OX = 9606 GN = SRP9 PE = 1 SV = 2
sapiens OX = 9606 GN = VASP PE = 1 SV = 3
sapiens OX = 9606 GN = MRPL28 PE = 1 SV = 4
sapiens OX = 9606 GN = ACAT1 PE = 1 SV = 1
sapiens OX = 9606 GN = ABCD3 PE = 1 SV = 1
sapiens OX = 9606 GN = CYFIP1 PE = 1 SV = 1
sapiens OX = 9606 GN = IMPDH2 PE = 1 SV = 2
sapiens OX = 9606 GN = COL5A1
sapiens GN = NME2
sapiens GN = SEC31A
sapiens OX = 9606 GN = NUP205 PE = 1 SV = 3
sapiens OX = 9606 GN = SEC61B PE = 1 SV = 2
sapiens OX = 9606 GN = SARNP PE = 1 SV = 3
sapiens OX = 9606 GN = MRPL41 PE = 1 SV = 1
sapiens OX = 9606 GN = ARPC5 PE = 1 SV = 3
sapiens OX = 9606 GN = ASPH PE = 1 SV = 3
sapiens OX = 9606 GN = ATP5MF PE = 1 SV = 1
sapiens GN = CCAR2 PE = 1 SV = 2
sapiens GN = EDC4 PE = 1 SV = 1
sapiens OX = 9606 GN = EIF5B PE = 1 SV = 4
sapiens OX = 9606 GN = KHSRP PE = 1 SV = 4
sapiens OX = 9606 GN = RPS20
sapiens OX = 9606 GN = AP2A2
sapiens OX = 9606 GN = COL22A1
sapiens OX = 9606 GN = RAD50
sapiens OX = 9606 GN = FERMT2
sapiens OX = 9606 GN = PABPN1
sapiens OX = 9606 GN = SYF2
sapiens OX = 9606 GN = SEC16A
sapiens OX = 9606 GN = ILK
sapiens OX = 9606 GN = WASHC2C
sapiens OX = 9606 GN = MGST3 PE = 1 SV = 1
sapiens OX = 9606 GN = NAMPT PE = 1 SV = 1
sapiens OX = 9606 GN = HMGN1 PE = 1 SV = 1
sapiens OX = 9606 GN = PAWR PE = 1 SV = 1
sapiens OX = 9606 GN = PSME2 PE = 1 SV = 4
sapiens OX = 9606 GN = TPT1 PE = 1 SV = 1
sapiens OX = 9606 GN = SSR1 PE = 1 SV = 3
sapiens GN = MRPS35 PE = 1 SV = 1
sapiens OX = 9606 GN = CYC1 PE = 1 SV = 3
sapiens OX = 9606 GN = DLD PE = 1 SV = 2
sapiens OX = 9606 GN = SAP18 PE = 1 SV = 1
sapiens OX = 9606 GN = HYDIN PE = 1 SV = 3
sapiens OX = 9606 GN = CTSA
sapiens OX = 9606 GN = SCARB2
sapiens GN = RRAS2
sapiens OX = 9606 GN = EWSR1
sapiens OX = 9606 GN = LRRC17 PE = 2 SV = 1
sapiens OX = 9606 GN = PEX14 PE = 1 SV = 1
sapiens GN = HSPE1 PE = 1 SV = 2
sapiens OX = 9606 GN = PIKFYVE PE = 1 SV = 3
sapiens GN = MRPS34 PE = 1 SV = 2
sapiens GN = MRPL11 PE = 1 SV = 1
sapiens GN = MRPL34 PE = 1 SV = 1
sapiens OX = 9606 GN = ACAA2 PE = 1 SV = 2
sapiens OX = 9606 GN = RPS4Y1 PE = 1 SV = 2
sapiens GN = RPL23A PE = 1 SV = 1
sapiens GN = RPL32 PE = 1 SV = 1
sapiens GN = ARPC1B PE = 1 SV = 3
sapiens GN = ATP5L PE = 1 SV = 3
sapiens GN = MTHFD1 PE = 1 SV = 3
sapiens GN = CLIC4 PE = 1 SV = 4
sapiens GN = CLIC6 PE = 2 SV = 3
sapiens GN = CBX1 PE = 1 SV = 1
sapiens GN = CCDC124 PE = 1 SV = 1
sapiens GN = CCDC30 PE = 2 SV = 1
sapiens OX = 9606 GN = DNAJB11 PE = 1 SV = 1
sapiens OX = 9606 GN = DCDC2 PE = 1 SV = 2
sapiens GN = EBNA1BP2 PE = 1 SV = 1
sapiens OX = 9606 GN = FUBP1 PE = 1 SV = 3
sapiens OX = 9606 GN = GLUD1 PE = 1 SV = 2
sapiens GN = NMT2 PE = 1 SV = 1
sapiens GN = HNRNPA0 PE = 1 SV = 1
sapiens GN = HNRNPH2 PE = 1 SV = 1
sapiens OX = 9606 GN = HAPLN1 PE = 2 SV = 2
sapiens GN = HAPLN3 PE = 2 SV = 1
sapiens OX = 9606 GN = AKAP13
sapiens OX = 9606 GN = AP2M1
sapiens GN = CDYL
sapiens OX = 9606 GN = COL7A1
sapiens OX = 9606 GN = FGF2
sapiens GN = GFRA1
sapiens OX = 9606 GN = GOLGA5
sapiens OX = 9606 GN = RBBP4
sapiens GN = KHDRBS3
sapiens GN = CAPG
sapiens OX = 9606 GN = ABCB1
sapiens OX = 9606 GN = NCOR1
sapiens OX = 9606 GN = RBP1
sapiens OX = 9606 GN = SPTB
sapiens OX = 9606 GN = TEX10
sapiens OX = 9606 GN = TFPI2
sapiens OX = 9606 GN = BAZ1B
sapiens OX = 9606 GN = ZFHX4
sapiens OX = 9606 GN = GLYR1
sapiens GN = SAFB
sapiens GN = TOR1AIP1
sapiens OX = 9606 GN = IQCN
sapiens OX = 9606 GN = KIF24
sapiens GN = DIAPH3
sapiens OX = 9606 GN = EP400
sapiens GN = LMO7
sapiens OX = 9606 GN = COL11A1
sapiens OX = 9606 GN = COL4A6
sapiens OX = 9606 GN = MECP2
sapiens OX = 9606 GN = CLTA
sapiens OX = 9606 GN = LAMC2
sapiens OX = 9606 GN = MGST1 PE = 1 SV = 1
sapiens GN = MARCKS PE = 1 SV = 4
sapiens GN = CMAS PE = 1 SV = 2
sapiens GN = NNT PE = 1 SV = 1
sapiens GN = DFNA5 PE = 1 SV = 2
sapiens GN = FKBP10 PE = 1 SV = 1
sapiens GN = FKBP3 PE = 1 SV = 1
sapiens OX = 9606 GN = PWP1 PE = 1 SV = 1
sapiens GN = PTRF PE = 1 SV = 1
sapiens GN = POU3F3 PE = 2 SV = 2
sapiens GN = IQGAP1 PE = 1 SV = 1
sapiens GN = RAB14 PE = 1 SV = 1
sapiens OX = 9606 GN = BRIX1 PE = 1 SV = 2
sapiens GN = RPF2 PE = 1 SV = 2
sapiens GN = FBL PE = 1 SV = 2
sapiens GN = SRP14 PE = 1 SV = 2
sapiens GN = SMARCA5 PE = 1 SV = 1
sapiens OX = 9606 GN = SMARCE1 PE = 1 SV = 2
sapiens GN = TBC1D1 PE = 1 SV = 3
sapiens GN = TPM1 PE = 1 SV = 1
sapiens GN = TPM1 PE = 1 SV = 1
sapiens GN = SART1 PE = 1 SV = 1
sapiens OX = 9606 GN = USP24 PE = 1 SV = 3
Induced pluripotent stem cells (iPSCs) were allowed to proliferate on an amniotic fluid cell-derived ECM (Matrix B) from Example 1 in culture using the following procedure: commercially available, cryopreserved iPSCs were thawed using a water bath at 37° C. Cell suspension was diluted into commercially available media for stem cell proliferation (Miltenyi Biotec MACS iPS Brew) and seeded onto the ECM at approximately 1,000 cells/cm2 in a 6-well-plate with 2 mL of media/well. No Rock inhibitor was used. At day 1, the full volume of media was aspirated gently from cells in culture and replaced with fresh media. Every 24 hours, full media was replaced with fresh media. Once cells began to approach confluence (as determined by brightfield microscopy), cells were passage manually, using a sterile needle to cut large colonies into approximately 100 smaller colonies and then re-plate those by physically lifting them off the dish with the sterile needle and placing them on a fresh plate of the ECM. This procedure can be repeated indefinitely.
A photomicrograph showing Day 0 and Day 2 culture of iPSCs on amniotic fluid cell-derived ECM and a bone marrow cell-derived ECM is shown in
A plot of iPSC colony growth curves of iPSCs cultured in the presence of the amniotic fluid cell-derived ECM and a bone marrow cell-derived ECM is shown in
As can be seen in
In another study, iPSCs were plated onto an amniotic fluid cell-derived ECM (AFC-ECM). Cells were passaged two times and then RNA was collected for gene expression analysis. Quantitative PCR was used to determine the gene expression. The iPSCs grown on the AFC-ECM maintained pluripotency during the course of the experiment and maintained expression of the core pluripotent transcription factors POU5F1 (OCT4), SOX2 and NANOG as shown in
Mesenchymal stem cells (MSCs) obtained from bone marrow, endothelial progenitor cells (EPCs), and chondrocytes were seeded onto amniotic fluid cell-derived ECM (AF-ECM), bone marrow cell-derived ECM (BM-ECM), and tissue culture plastic (TCP) at equal seeding density in standard commercially available media. For MSCs and chondrocytes the media was alpha minimum essential media (aMEM) modified without phenol red, supplemented with 15% by volume fetal bovine serum, 1% antibiotic-antimycotic (anti-anti), and 1% GlutaMax. For EPCs, the media was Iscove's Modified Dulbecco's Medium (IMDM) Supplemented with 20% FBS, 1% anti-anti, 1% GlutaMax, and growth factors (EGF and FGF). Photomicrographs showing increased proliferation on the two ECMs relative to TCP are shown in
Mesenchymal stem cells (MSCs) obtained from human bone marrow were seeded on amniotic fluid cell-derived ECM (AFC-ECM) at 6×103 cells/cm2 and cultured for 14 days in growth media.
To assess the adipogenic differentiation efficiency of the cells, cultures were transferred at Day 7 to adipogenic media (DMEM containing 10% FBS, 0.5 mM IBMX, 10−6 M dexamethasone, 10 μM insulin, and 200 μM indomethacin), maintained for an additional 10 days, fixed with 10% formalin for 1 hour at room temperature, and then stained with Oil Red O. Adipogenesis is verified by staining the differentiated cells with the Oil Red O. The Oil Red O stains the lipid droplets (red staining) formed within the cells following the differentiation of the MSCs to adipocytes in culture. Photomicrographs showing adipogenic differentiation of the MSCs on the AFC-ECM is shown in
To assess osteogenic differentiation efficiency, the cultures were transferred to osteoblast differentiation media (growth media supplemented with 10−7 M dexamethasone (Sigma-Aldrich, St. Louis, Mo.) and 10−4 M L-ascorbate-2-phosphate (Wako Chemicals, Richmond, Va.), maintained an additional 25 days, fixed with 10% formalin for 1 hour at room temperature, and then stained with 1% Silver Nitrate (Von Kossa Staining). Osteogenesis is verified by staining the differentiated cells with 1% Silver Nitrate. The Silver Nitrate stains the minerals (dark staining) deposited following the differentiation of the MSCs to osteoblasts in culture. Photomicrographs showing osteogenic differentiation of the MSCs on the AFC-ECM is shown in
This application claims the benefit of U.S. Provisional Patent Application No. 62/740,817, filed Oct. 3, 2018, which is incorporated by reference herein in its entirety.
Number | Date | Country | |
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62740817 | Oct 2018 | US |