Patent Grant
11220671
The disclosure generally relates to the use of cellular constructs of cardiomyocytes derived from human stem cells on cell-derived extracellular matrices, methods of making the constructs, and methods for cardiotoxicity and proarrhythmic screening assays of drug compounds using the constructs.
Cardiotoxicity, or the perceived potential for cardiotoxicity, is a leading cause of toxicity related drug attrition during the investigation and selection of new drugs. Cardiac safety testing of new chemical entities that become lead drug candidates is a critical aspect of the drug discovery and development pipeline. A large number of cardiac side effects of cardiac and non-cardiac drugs are caused by drug interaction with one or more cardiac ion channels. Cardiac ion channels regulate cellular excitability, contractility and overall cardiac performance, and alteration of cardiac ion channel function can lead to sudden cardiac death. This contributed to the release of drug candidate testing guidelines from The International Conference on Harmonization (ICH).
The current preclinical drug candidate testing guidelines from the IHC (ICH S7A and S7B-Pharmacology Studies) rely on genetically modified heterologous cells and in vivo animal models. It has become increasingly recognized that these studies, such as hERG assay and QT prolongation studies, do not accurately predict cardiotoxicity and proarrhythmia risk for humans. Since 2005, cardiac safety of a drug compound has been determined almost exclusively by its effect or potential effect on the QT interval of the electrocardiogram (ECG) or the action potential duration (APD), and the potential to lead to a life-threatening arrhythmia called Torsades de Pointes (TdP). However, QT prolongation is not the ideal indicator for TdP as drugs that prolong the QT interval do not always cause TdP. It is now recognized that the QT prolongation parameter is only a surrogate marker for proarrhythmia. Data from pre-clinical and clinical trials have shown that there is no fixed relationship between the magnitude of QT prolongation and the risk for development of fatal arrhythmias such as TdP.
Therefore, the US Food and Drug Administration (FDA) and other stakeholders in drug discovery have called for an evolution in pre-clinical cardiotoxicity testing. The proposed new paradigm is called the Comprehensive In-Vitro Proarrhythmia Assay (CiPA). An integral part of the CiPA Initiative (cipaproject.org/) includes incorporation of data collected from human stem cell derived cardiomyocytes for cardiotoxicity and proarrhythmia assays. The overall goal of these new proposed guidelines is to provide a more accurate and comprehensive mechanistic based assessment of proarrhythmic potential that would more accurately assess the risk of new drugs. In review of the proposed CiPA Initiative's guidelines, the FDA has defined two advances that must be made before human cardiomyocytes can be incorporated in the new initiative. First, the growth and maturation state of human stem cell derived cardiomyocytes needs to be advanced to more closely resemble the structure and function of adult human cardiomyocytes. Second, a reliable high throughput screening platform using these cells must be developed.
There are generally three types of systems currently utilized to evaluate the electrophysiology of cardiomyocytes in vitro: 1) Patch clamping systems; 2) micro-electrode array (MEA) systems; and 3) voltage sensitive dye (VSD) visualization methods. Manual patch clamping systems are most commonly used in very early research studies to evaluate the electrophysiology of individual cells. These devices, while providing accurate and sensitive ionic current measurements, cannot be used for in vitro cell systems that more closely mimic cell-cell interaction in cardiac tissue. MEA systems include electrodes that are incorporated into cell culture wells allowing for the measurement of current across the well. While allowing for high throughput analysis and ability to measure impedance in in vitro cell systems, these systems have low spatial resolution, do not provide data on action potential shape, and hinder direct visualization of the cells and the ability to assess 3-D culture systems that more closely mimic cardiac tissue architecture. VSD systems are gaining interest as they address many of the shortcomings of the current technologies described above with features that include: 1) allowing for high throughput analysis; 2) providing high spatial resolution; 3) allowing visualization of impulse propagation across a culture dish; and 4) allowing for modification of the culture conditions including addition of extracellular matrix leading to a more natural testing environment.
The use of cardiomyocytes derived from human stem cells, such as induced pluripotent stem cells, have had limited success to date in cardiotoxicity and proarrhythmic assay screening. Cardiomyocytes derived from induced human pluripotent stem cells (hiPSC-CMs) are commercially available and can be purchased from several companies in cryopreserved vials that can be thawed and plated as monolayers. These hiPSC-CMs can be made in large scale in vitro. Also, hiPSC-CMs can be obtained from patient specific hiPSCs for a specific individual. However, there are hurdles that still need to be overcome to make the hiPSC-CMs a meaningful part of the new CiPA Initiative paradigm. 1) The maturation of hiPSC-CM structure and function must be advanced. Notably, the Kir2.1 potassium channel is absent in currently available hiPSC-CMs and sodium channel expression is low. Currently available hiPSC-CMs are very immature functionally and structurally. The vast majority of currently used hiPSC-CM-based proarrhythmia screening assays rely on immature, fetal-like cells that do not resemble the structure of function of the adult cardiomyocyte. 2) Electrical pacing of hiPSC-CM monolayers in a high throughput electrophysiological screening platform is needed. The vast majority of current hiPSC-CM-based proarrhythmia screens rely solely on field potential duration (MEA technology) or action potential duration prolongation (VSD technology) as surrogate marker for TdP induction. This is a limitation because not all drugs that prolong the action potential (QT interval) will cause fatal TdP arrhythmias. Although certain maturation states of immature hiPSC-CMs have been achieved using Matrigel™ ECM and bone-marrow cell-derived ECM in culture, use of these hiPSC-CMs in proarrhythmia screening assays did not produce consistent results in the observation of arrhythmia activation patters consistent with what is known to occur in cases of TdP in humans, especially between low risk and high risk drugs.
Thus, advanced materials and new methods are needed for the precise assessment and screening of the cardiac safety liability of drug compounds for accurate, reliable and efficient development of new drug candidates.
The present disclosure provides a solution to at least some of the aforementioned limitations and deficiencies in the art relating to the assessment and screening of the cardiac safety liability of drug candidates. The solution is premised on the discovery of an extracellular matrix derived from cells isolated from amniotic fluid (AFC-ECM), which can be used for the maturation of human stem cell derived cardiomyocytes. These mature cardiomyocytes can then be used in a cellular construct with the AFC-ECM for cardiotoxicity and proarrhythmic testing of drugs.
In one aspect, disclosed is a method for the maturation of immature cardiomyocytes derived from human induced pluripotent stem cells, the method comprising: (a) providing immature cardiomyocytes derived from human induced pluripotent stem cells (immature hiPSC-CMs); (b) providing an extracellular matrix derived in vitro from cells isolated from amniotic fluid (AFC-ECM); (c) contacting the immature hiPSC-CMs with the AFC-ECM; and (d) culturing the immature hiPSC-CMs with the AFC-ECM in a culture media to induce maturation of the immature hiPSC-CMs, thereby forming mature cardiomyocytes; wherein the mature cardiomyocytes are characterized by rod shaped cells with distinct sarcomere structure resembling adult human cardiac tissue. In some embodiments the mature cardiomyocytes have a similar or the same morphology as illustrated in any one of
In another aspect, disclosed is a cellular construct comprising a monolayer of mature cardiomyocytes on an extracellular matrix derived from cells isolated in vitro from amniotic fluid (AFC-ECM), wherein the mature cardiomyocytes are AFC-ECM cultured cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs), and wherein the mature cardiomyocytes are characterized by rod shaped cells with distinct sarcomere structure resembling adult human cardiac tissue. In some embodiments the mature cardiomyocytes have a similar or the same morphology as illustrated in any one of
In another aspect, disclosed is a method for making a cellular construct of mature cardiomyocytes on an extracellular matrix derived in vitro from cells isolated from amniotic fluid (AFC-ECM), the method comprising: (a) providing immature cardiomyocytes derived from human induced pluripotent stem cells (immature hiPSC-CMs); (b) providing an extracellular matrix derived in vitro from cells isolated from amniotic fluid (AFC-ECM); (c) plating the immature hiPSC-CMs on the AFC-ECM; and (d) culturing the plated immature hiPSC-CMs on the AFC-ECM in a culture media to induce maturation of the immature hiPSC-CMs into mature cardiomyocytes and to form a monolayer of the mature cardiomyocytes on the AFC-ECM, thereby forming the cellular construct, wherein the mature cardiomyocytes are characterized by rod shaped cells with distinct sarcomere structure resembling adult human cardiac tissue. In some embodiments the mature cardiomyocytes have a similar or the same morphology as illustrated in any one of
In another aspect, disclosed is a method for determining the cardiotoxicity and/or proarrhythmic effect of a drug compound in vitro, the method comprising contacting the drug compound with the mature cardiomyocytes of any one of the cellular constructs disclosed throughout the specification, and observing for a change in the electrophysiology of the mature cardiomyocytes to confirm whether the drug compound has a cardiotoxic and/or proarrhythmic effect on the mature cardiomyocytes. A change in the electrophysiology of the mature cardiomyocytes confirms that the drug compound has a cardiotoxic and/or proarrhythmic effect on the mature cardiomyocytes. The changes in the electrophysiology of the mature cardiomyocytes can include, but are not limited to, APD prolongation, APD prolongation plus rotors, and/or various types of arrhythmias, such as tachyarrhythmia (TA), quiescence (Q), delayed after depolarization (DAD), and/or early after depolarization (EAD). Observations can also include the cell viability, cell density, and/or morphology of the cells. In some embodiments, the change in the electrophysiology of the mature cardiomyocytes is prolongation of action potential duration (APD). In some embodiments, the change in the electrophysiology of the mature cardiomyocytes is early after depolarization (EAD). In some embodiments, the change in the electrophysiology of the mature cardiomyocytes is delayed after depolarization (DAD). In some embodiments, the change in the electrophysiology of the mature cardiomyocytes is action potential duration prolongation (APD prolongation) plus rotors. In some embodiments, the change in the electrophysiology of the mature cardiomyocytes is an arrhythmia. In some aspects, the cellular construct can be prepared by a process comprising: (a) providing immature cardiomyocytes derived from human induced pluripotent stem cells (immature hiPSC-CMs); (b) providing an extracellular matrix derived in vitro from cells isolated from amniotic fluid (AFC-ECM); (c) plating the immature hiPSC-CMs on the AFC-ECM; and (d) culturing the plated immature hiPSC-CMs on the AFC-ECM in a culture media to induce maturation of the immature hiPSC-CMs into mature cardiomyocytes and to form a monolayer of the mature cardiomyocytes on the AFC-ECM, thereby forming a cellular construct, wherein the mature cardiomyocytes are characterized by rod shaped cells with distinct sarcomere structure resembling adult human cardiac tissue. In some embodiments the mature cardiomyocytes have a similar or the same morphology as illustrated in any one of
Also, disclosed in the context of the present invention are the following embodiments 1 to 20:
Embodiment 1 is a method for the maturation of immature cardiomyocytes derived from human induced pluripotent stem cells, the method comprising:
(a) providing immature cardiomyocytes derived from human induced pluripotent stem cells (immature hiPSC-CMs);
(b) providing an extracellular matrix derived in vitro from cells isolated from amniotic fluid (AFC-ECM);
(c) contacting the immature hiPSC-CMs with the AFC-ECM; and
(d) culturing the immature hiPSC-CMs with the AFC-ECM in a culture media to induce maturation of the immature hiPSC-CMs, thereby forming mature cardiomyocytes;
wherein the mature cardiomyocytes are characterized by rod shaped cells with distinct sarcomere structure resembling adult human cardiac tissue.
Embodiment 2 is the method of embodiment 1, wherein the immature hiPSC-CMs are plated on the AFC-ECM.
Embodiment 3 is the method of any one of embodiments 1 or 2, wherein the mature cardiomyocytes form as a monolayer on the AFC-ECM, thereby forming a cellular construct comprising a monolayer of mature cardiomyocytes on the AFC-ECM, wherein the mature cardiomyocytes are aligned on the AFC-ECM.
Embodiment 4 is the method of any one of embodiments 1 to 3, wherein the immature hiPSC-CMs do not express inward-rectifier potassium channel Kir2.1.
Embodiment 5 is a cellular construct comprising a monolayer of mature cardiomyocytes on an extracellular matrix derived from cells isolated in vitro from amniotic fluid (AFC-ECM), wherein the mature cardiomyocytes are AFC-ECM cultured cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs), and wherein the mature cardiomyocytes are characterized by rod shaped cells with distinct sarcomere structure resembling adult human cardiac tissue.
Embodiments 6 is the cellular construct of embodiment 5, wherein the monolayer of mature cardiomyocytes is aligned on the AFC-ECM.
Embodiment 7 is the cellular construct of any one of embodiments 5 or 6, wherein fiber tracks are present on the construct.
Embodiment 8 is the cellular construct of any one of embodiments 5 to 7, wherein the AFC-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 9 is the cellular construct of embodiment 8, wherein the isoform of collagen alpha-1 (XVIII) is isoform 2, and/or wherein the isoform of agrin is isoform 6.
Embodiment 10 is the cellular construct of any one of embodiments 8 or 9, wherein the AFC-ECM further comprises fibronectin and/or an isoform thereof.
Embodiment 11 is the cellular construct of any one of embodiments 5 to 10, wherein the AFC-ECM does not contain decorin, perlecan, and/or collagen (III).
Embodiment 12 is a method for making a cellular construct of mature cardiomyocytes on an extracellular matrix derived in vitro from cells isolated from amniotic fluid (AFC-ECM), the method comprising:
(a) providing immature cardiomyocytes derived from human induced pluripotent stem cells (immature hiPSC-CMs),
(b) providing an extracellular matrix derived in vitro from cells isolated from amniotic fluid (AFC-ECM);
(c) plating the immature hiPSC-CMs on the AFC-ECM;
(d) culturing the plated immature hiPSC-CMs on the AFC-ECM in a culture media to induce maturation of the immature hiPSC-CMs into mature cardiomyocytes and to form a monolayer of the mature cardiomyocytes on the AFC-ECM, thereby forming the cellular construct; wherein the mature cardiomyocytes are characterized by rod shaped cells with distinct sarcomere structure resembling adult human cardiac tissue.
Embodiment 13 is the method of embodiment 12, wherein the monolayer of mature cardiomyocytes is aligned on the AFC-ECM.
Embodiment 14 is the method of any one of embodiments 12 or 13, wherein fiber tracks are present on the cellular construct.
Embodiment 15 is a method for determining the cardiotoxicity and/or proarrhythmic effect of a drug compound in vitro, the method comprising contacting the drug compound with the mature cardiomyocytes of any one of the cellular constructs of embodiments 5 to 11, and observing for a change in the electrophysiology of the mature cardiomyocytes to confirm whether the drug compound has a cardiotoxic and/or proarrhythmic effect on the mature cardiomyocytes.
Embodiment 16 is the method of embodiment 15, wherein the change in the electrophysiology of the mature cardiomyocytes is prolongation of action potential duration (APD), and wherein prolongation of APD confirms that the drug compound has a cardiotoxic and/or proarrhythmic effect on the mature cardiomyocytes.
Embodiment 17 is the method of embodiment 15, wherein the change in the electrophysiology of the mature cardiomyocytes is early after depolarization, and wherein early after depolarization (EAD) confirms that the drug compound has a cardiotoxic and/or proarrhythmic effect on the mature cardiomyocytes.
Embodiment 18 is the method of embodiment 15, wherein the change in the electrophysiology of the mature cardiomyocytes is delayed after depolarization, and wherein delayed after depolarization (DAD) confirms that the drug compound has a cardiotoxic and/or proarrhythmic effect on the mature cardiomyocytes.
Embodiment 19 is the method of embodiment 15, wherein the change in the electrophysiology of the mature cardiomyocytes is action potential duration (APD) plus rotors, and wherein prolongation of APD plus rotors confirms that the drug compound has a cardiotoxic and/or proarrhythmic effect on the mature cardiomyocytes.
Embodiment 20 is the method of embodiment 15, wherein the change in the electrophysiology of the mature cardiomyocytes is an arrhythmia, and wherein the arrhythmia confirms that the drug compound has a cardiotoxic and/or proarrhythmic effect on the mature cardiomyocytes.
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%.
As used herein, the terms “% w/w” or “wt. %” refers to a weight percentage of a component based on the total weight of material (e.g. a composition) that includes the component. In a non-limiting example, 10 grams of a component in 100 grams of a composition is 10% w/w of the component in the total weight of composition. As used herein, the terms “% v/v” or “vol. %” refers to a volume percentage of a component based on the total volume of material (e.g. a composition) that includes the component. In a non-limiting example, 10 mL of a component in 100 mL of a composition is 10% v/v of the component in the total volume of composition. As used herein, the term “% w/v” refers to a weight percentage of a component based on the total volume of material (e.g. a composition) that includes the component. In a non-limiting example, 10 grams of a component in 100 mL of a composition is 10% w/v of the component in the total volume of composition. As used herein, the term “% v/w” refers to a volume percentage of a component based on the total weight of material (e.g. a composition) that includes the component. In a non-limiting example, 10 mL of a component in 100 grams of a composition is 10% v/w of the component in the total weight of the composition.
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 cellular constructs disclosed herein is their use in cardiotoxicity and/or proarrhythmic screening testing of drug compounds due to their ability to mature cardiomyocytes derived from stem cells to a maturation state resembling that of mature native adult cardiomyocytes and native heart tissue.
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.
Human stem cell derived cardiomyocytes, such as those derived from human induced pluripotent stem cells (hiPSC-CMs), that have been matured in culture on an extracellular matrix derived in vitro from cells isolated from amniotic fluid (AFC-ECM) have demonstrated better, more consistent cardiotoxicity and proarrhythmia assay results than immature hiPSC-CMs or hiPSC-CMs matured on other ECMs, such as Matrigel™ ECM and bone marrow cell-derived ECM. The hiPSC-CMs matured on an AFC-ECM surprisingly have demonstrated a higher state of maturation than hiPSC-CMs matured on other ECMs, such as Matrigel™ ECM and even on other natural cell derived ECMs such as bone marrow cell-derived ECM, as shown by their cellular morphology and sarcomere structure that are found in cardiomyocytes of normal adult human cardiac tissue. Furthermore, a more robust expression of cTnI (cardiac troponin I) protein, as demonstrated by western blotting techniques, has been seen for hiPSC-CMs cultured on AFC-ECM than for hiPSC-CMs cultured on Matrigel™ ECM. Also, hiPSC-CMs cultured on AFC-ECM have more mitochondria and mitochondria with more polarized inner membrane potential than hiPSC-CMs cultured on Matrigel™ ECM. Thus, the AFC-EMC can stimulate mitochondrial biogenesis, maturation, and function in the hiPSC-CMs. Normal adult or mature human cardiac human tissue is characterized by rod shaped cells with sarcomere structure (striped appearance). The morphology and sarcomere structures of the cardiomyocytes can be identified visually by microscopy (transmitted light or by immunofluorescent staining). As a non-limiting example, the morphology and sarcomere structure of the cardiomyocytes matured on AFC-ECM can be distinctly seen by the presence of rod shaped cells with a striped appearance identified by arrows in the photomicrograph of
Disclosed herein are methods of using a cell-derived extracellular matrix derived in-vitro from cells isolated from amniotic fluid (AFC-ECM) for the maturation of immature cardiomyocytes derived from human induced pluripotent stem cells (immature hiPSC-CMs) in culture forming mature cardiomyocytes (mature hiPSC-CMs). Also disclosed herein is a cell construct comprising a monolayer of these mature cardiomyocytes on an AFC-ECM useful for cardiotoxicity and/or proarrhythmic screening assays of drug compounds. Also disclosed herein are methods for determining the cardiotoxicity and/or proarrhythmic effect of a drug compound in vitro using such cell constructs.
A. Amniotic Fluid Cell-Derived Extracellular Matrix (AFC-ECM)
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 disclosed herein can comprise 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 1 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). 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 can be produced by the following process:
Any cell 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 (AFC-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 by the following process:
In some embodiments, the cells isolated from the umbilical cord are from the cord blood and/or the Wharton's jelly.
In another embodiment, a perinatal cell-derived ECM is produced by the following process:
In one aspect, 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, 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.
B. Cellular Constructs and Methods for the Maturation of Immature hiPSC-CMs
Disclosed herein is a method for the maturation of immature cardiomyocytes derived from human induced pluripotent stem cells, the method comprising: (a) providing immature cardiomyocytes derived from human induced pluripotent stem cells (immature hiPSC-CMs); (b) providing an extracellular matrix derived in vitro from cells isolated from amniotic fluid (AFC-ECM); (c) contacting the immature hiPSC-CMs with the AFC-ECM; and (d) culturing the immature hiPSC-CMs with the AFC-ECM in a culture media to induce maturation of the immature hiPSC-CMs, thereby forming mature cardiomyocytes (mature hiPSC-CMs); wherein the mature cardiomyocytes are characterized by rod shaped cells with distinct sarcomere structure resembling adult human cardiac tissue. Normal adult human cardiac human tissue is characterized by rod shaped cells with sarcomere structure (striped appearance). The morphology and sarcomere structures of the cardiomyocytes can be identified visually by microscopy. As a non-limiting example, the morphology and sarcomere structure of the cardiomyocytes matured on AFC-ECM can be distinctly seen by the presence of rod shaped cells with a striped appearance identified by arrows in the photomicrograph of
Also disclosed herein is a cellular construct comprising a monolayer of mature cardiomyocytes on an extracellular matrix derived from cells isolated in vitro from amniotic fluid (AFC-ECM), wherein the mature cardiomyocytes are AFC-ECM cultured cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs), and wherein the mature cardiomyocytes are characterized by rod shaped cells with distinct sarcomere structure resembling adult human cardiac tissue. In certain aspects, the mature cardiomyocytes can be matured from immature cardiomyocytes derived from human induced pluripotent stem cells (immature hiPSC-CMs) in culture on the AFC-ECM, wherein the mature cardiomyocytes are characterized by rod shaped cells with distinct sarcomere structure resembling adult human cardiac tissue. In some aspects, the mature cardiomyocytes include the inward-rectifier Kir2.1 potassium channel and/or express inward-rectifier potassium channel Kir2.1. In some embodiments, the monolayer of mature cardiomyocytes is aligned on the AFC-ECM. In some embodiments, fiber tracks are present on the construct. In some embodiments, the AFC-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, and/or wherein the isoform of agrin is isoform 6. In some embodiments, the AFC-ECM further comprises fibronectin and/or an isoform thereof. In some embodiments, the AFC-ECM does not contain decorin, perlecan, and/or collagen (III). In some embodiments, the immature hiPSC-CMs do not include rod shaped cells with distinct sarcomere structure. In some embodiments, the immature hiPSC-CMs can be characterized by having less mitochondria than mature cardiomyocytes, by having disorganized myofilaments, by having a circular shape, and/or by having a single nucleus. In some embodiments, the mature cardiomyocytes can be further characterized by having greater amounts of mitochondria than immature hiPSC-CMs, by having organized, compact myofilaments, and/or by having two nuclei (bi-nucleated).
Also disclosed herein is a method for the preparation of a cellular construct comprising mature cardiomyocytes on an extracellular matrix derived in vitro from cells isolated from amniotic fluid (AFC-ECM), the method comprising (a) providing immature cardiomyocytes derived from human induced pluripotent stem cells (immature hiPSC-CMs), (b) providing an extracellular matrix derived in vitro from cells isolated from amniotic fluid (AFC-ECM); (c) plating the immature hiPSC-CMs on the AFC-ECM; and (d) culturing the plated immature hiPSC-CMs on the AFC-ECM in a culture media to induce maturation of the immature hiPSC-CMs into mature cardiomyocytes and to form a monolayer of the mature cardiomyocytes on the AFC-ECM, thereby forming the cellular construct, wherein the mature cardiomyocytes are characterized by rod shaped cells with distinct sarcomere structure resembling adult human cardiac tissue. In some embodiments, the monolayer of mature cardiomyocytes is aligned on the AFC-ECM. In some embodiments, fiber tracks are present on the construct.
Immature hiPSC-CMs can be obtained from commercial sources such as Cellular Dynamics International-FUJI under the trade name iCell®, and Takara Bio under the trade name Cellartis®. iCell® Cardiomyocytes, iCell® Cardiomyocytes2, and Cellartis® Cardiomyocytes are cryopreserved viable cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) available in vials. Immature hiPSC-CMs can also be generated from patient specific hiPSCs in a laboratory setting for a specific individual. In this case, differentiation of the hiPSCs can be accomplished using the small molecule protocol to obtain beating cardiomyocytes by day 8-10 using GSK3 inhibitor, RPMI/B27 minus insulin, Wnt Inhibitor and RPMI/B27 plus insulin at various days during the 7-day differentiation period. Suitable non-limiting examples of methods to generate immature hiPSC-CMs are disclosed in US publication 2015/0329825 herein incorporated by reference. In some embodiments, the immature hiPSC-CMs do not express inward-rectifier potassium channel Kir2.1. In some embodiments, the immature hiPSC-CMs do not include rod shaped cells with distinct sarcomere structure. In some embodiments, the immature hiPSC-CMs can be characterized by having less mitochondria than mature cardiomyocytes, by having disorganized myofilaments, by having a circular shape, and/or by having a single nucleus.
The AFC-ECM can be obtained using the production methods disclosed herein in this disclosure and can have the characteristics as described in this disclosure. In some embodiments, the AFC-ECM is decellularized prior to contact with the immature hiPSC-CMs.
The immature hiPSC-CMs can be in suspension when in contact with the AFC-ECM or the cells can be plated directly on the AFC-ECM which is in or on suitable cell culture containers or in multi-well plates. Non-limiting examples of suitable multi-well plates include 6-, 12-, 24-, 48, 96- and 384-well plates. In some embodiments, the contact surfaces of the cell culture containers or multi-well plates are coated with polydimethylsiloxane (PDMS) prior to the formation of the AFC-ECM. In some embodiments, the contact surfaces of the cell culture containers or multi-well plates are not coated with PDMS prior to the formation of the AFC-ECM. Any cell seeding density of immature hiPSC-CMs can be used. In some embodiments, a cell seeding density of immature hiPSC-CMs which allows the cells to form a confluent monolayer immediately or after a period of time in culture is used. Cell density can be modified as desired to improve monolayer formation. In multi-well plates, the immature hiPSC-CM cells are placed in the center of each well. Non-limiting examples of cell seeding densities of immature hiPSC-CMs in various multi-well plates are as follows: in 6-well plates, about 200,000 cells can be plated per well; in 12-well plates, about 150,000 cells can be plated per well; in 24-well plates about 175,000 cells can be plated per well; in 48-well plates about 100,000 cells can be plated per well; in 96-well plates, about 50,000 cells can be plated per well; in 384-well plates, about 15,000 cells can be plated per well. In some embodiments, the cell seeding density of immature hiPSC-CMs is about 50,000 cells per well in a 96-well plate. In some embodiments, the cell seeding density is about 200,000 cells per well in a 6-well plate. To induce maturation of the immature hiPSC-CMs, a suitable culture media such as RPMI media or Media 199 media is added to the immature hiPSC-CMs in contact with the AFC-ECM, and the cells are cultured with the AFC-ECM using standard cell culture techniques for a period of time, generally 7 days, until the cells have matured into mature cardiomyocytes having similar morphology as native adult cardiomyocytes. During the first 3 to 4 days, the immature hiPCS-CMs are adhering, forming a continuous monolayer, and beginning the maturation process. Surprisingly, the period of time for maturation of the cardiomyocytes can take 7 days or less, whereas generally a much longer period of time is typical with other substrates, e.g., up to 100 days. The morphology of these mature cardiomyocytes can be characterized by rod shaped cells with distinct sarcomere structure (striped appearance), the fundamental contractile unit of muscle, that can be visualized using conventional light microscopy techniques. In some embodiments, the mature cardiomyocytes can be further characterized by having greater amounts of mitochondria than immature hiPSC-CMs, by having organized, compact myofilaments, and/or by having two nuclei (bi-nucleated). Also, the AFC-ECM can naturally produce fiber tracks that the mature cardiomyocytes follow. This produces a degree of anisotropy to the monolayer that more closely mimics the native heart. Thus, the cardiomyocytes can be naturally aligned on the AFC-ECM following the alignment of the AFC-ECM that has been laid down in a natural anisotropic configuration by the amniotic fluid cells during formation of the AFC-ECM. In various embodiments, the period of time for the immature hiPSC-CMs to mature in culture can be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120 days. Preferably, the period of time for the maturation of the cardiomyocytes is 14 days, or more preferably, 10 days, or even more preferably 7 days. In some embodiments the period of time for the immature hiPSC-CMs to mature in culture is 7 days. In some embodiments, the immature hiPSC-CMs are plated on the AFC-ECM. In some embodiments, the mature cardiomyocytes form as a confluent monolayer on the AFC-ECM during culture, thereby forming a cellular construct comprising a monolayer of mature cardiomyocytes on the AFC-ECM. In some embodiments, the mature cardiomyocytes are aligned on the AFC-ECM. In some embodiments, the immature hiPSC-CMs are plated on the AFC-ECM in multi-well plates. In some embodiments, the multi-well plates have inserts of polydimethylsiloxane (PDMS). In some embodiment, the multi-well plates do not have inserts of PDMS.
C. Methods for Determining the Cardiotoxicity and/or Proarrhythmic Effect of a Drug Compound
Disclosed herein is a method for determining the cardiotoxicity and/or proarrhythmic effect of a drug compound in vitro, the method comprising contacting the drug compound with the mature cardiomyocytes of any one of the cellular constructs disclosed throughout the specification, and observing for one or more changes in the electrophysiology of the mature cardiomyocytes to confirm whether the drug compound has a cardiotoxic and/or proarrhythmic effect on the mature cardiomyocytes. One or more changes in the electrophysiology of the mature cardiomyocytes indicates and confirms that the drug compound has a cardiotoxic and/or proarrhythmic effect on the mature cardiomyocytes. The one or more changes of the electrophysiology of the mature cardiomyocytes can include, but are not limited to, APD prolongation, APD prolongation plus rotors, and/or various types of arrhythmias, such as tachyarrhythmia (TA), quiescence (Q), delayed after depolarization (DAD), and/or early after depolarization (EAD). In some embodiments, the change in the electrophysiology of the mature cardiomyocytes is prolongation of action potential duration (APD). In some embodiments, the change in the electrophysiology of the mature cardiomyocytes is early after depolarization (EAD). In some embodiments, the change in the electrophysiology of the mature cardiomyocytes is delayed after depolarization (DAD). In some embodiments, the change in the electrophysiology of the mature cardiomyocytes is action potential duration (APD) plus rotors. In some embodiments, the change in the electrophysiology of the mature cardiomyocytes is an arrhythmia. In some embodiments, the change in the electrophysiology of the mature cardiomyocytes is tachyarrhythmia (TA). In some embodiments, the change in the electrophysiology of the mature cardiomyocytes is quiescence (Q). Observations can also include the cell viability, cell density, and/or morphology of the cells. In some aspects, the cellular construct can be prepared by a process comprising: (a) providing immature cardiomyocytes derived from human induced pluripotent stem cells (immature hiPSC-CMs); (b) providing an extracellular matrix derived in vitro from cells isolated from amniotic fluid (AFC-ECM); (c) plating the immature hiPSC-CMs on the AFC-ECM; and (d) culturing the plated immature hiPSC-CMs on the AFC-ECM in a culture media to induce maturation of the immature hiPSC-CMs into mature cardiomyocytes and to form a monolayer of the mature cardiomyocytes on the AFC-ECM, thereby forming a cellular construct, wherein the mature cardiomyocytes are characterized by rod shaped cells with distinct sarcomere structure resembling adult human cardiac tissue. In other aspects, the method can comprise contacting the drug compound with any one of the cellular constructs disclosed throughout the specification and observing for cardiotoxic and/or proarrhythmic events. In one particular instance, the method comprises: (a) providing immature cardiomyocytes derived from human induced pluripotent stem cells (immature hiPSC-CMs); (b) providing an extracellular matrix derived in vitro from cells isolated from amniotic fluid (AFC-ECM); (c) plating the immature hiPSC-CMs on the AFC-ECM; (d) culturing the plated immature hiPSC-CMs on the AFC-ECM in a culture media to induce maturation of the immature hiPSC-CMs into mature cardiomyocytes and to form a monolayer of the mature cardiomyocytes on the AFC-ECM, thereby forming a cellular construct; (e) contacting the drug compound with the monolayer of the mature cardiomyocytes of the construct; and (f) observing for a change in the electrophysiology of the mature cardiomyocytes to confirm whether the drug compound has a cardiotoxic and/or proarrhythmic effect on the mature cardiomyocytes; wherein the mature cardiomyocytes are characterized by rod shaped cells with distinct sarcomere structure resembling adult human cardiac tissue. In some embodiments, the immature hiPSC-CMs are plated on the AFC-ECM in a multi-well plate. In some embodiments, the multi-well plates have inserts of polydimethylsiloxane (PDMS). In some embodiment, the multi-well plates do not have inserts of PDMS. In some embodiments, the immature hiPSC-CMs do not express inward-rectifier potassium channel Kir2.1. In some embodiments, the monolayer of mature cardiomyocytes is aligned on the AFC-ECM. In some embodiments, fiber tracks are present on the cellular construct.
The cardiotoxicity and/or proarrhythmia testing can be conducted using any type of equipment suitable for measuring such activity. In some embodiments, the cellular constructs of monolayers of mature cardiomyocytes on AFC-ECM are prepared as described supra. After the maturation process (generally 7 days or less), the electrophysiology of each well is observed using a plate reader. A suitable voltage sensitive or calcium sensitive fluorescent dye is loaded into each well. Non-limiting voltage sensitive dyes include FluoVolt™ dye commercially available from ThermoFisher. In some embodiments, the plate reader can rely on a suitable high spatiotemporal CCD camera combined with suitable lighting, such as light emitting diodes (LEDs), of the appropriate wavelength to excite each dye. Such high spatiotemporal CCD cameras are commercially available from SciMeasure. In some embodiments, the camera image acquisition rate is greater than or equal to 150 frames per second. In some embodiments, the camera and lens combination are designed such that it allows visualization of all the wells of the multi-well plate simultaneously with sufficient resolution to observe action potential and calcium wave propagation. Each plate is centered under the camera system, lighting is switched on and camera acquisition is initiated and electrophysiological activity is recorded. Experiments are performed at about 37° C. After baseline readings are made, the drug to be tested is added to the wells and the effects are recorded. Spontaneous activity is recorded for a sufficient period of time to obtain images, e.g. at least 10 seconds. The images can be stored on a computer. Images are analyzed and action potential duration, conduction velocity, beat rate and activation patterns can be quantified using image analysis software. Visualization of electrical wave patterns is important to determine a drug compound's effect to cause potentially fatal arrhythmias, e.g., Torsades de Pointes (TdP). Thus, in addition to providing information on a compound's effect on spontaneous action potential duration, the methods disclosed herein can also provide information on impulse conduction velocity and activation pattern depending on the type of equipment used.
D. Methods to Expand/Proliferate Mammalian Stem Cells
Also disclosed herein are methods of using a cell-derived extracellular matrix derived in vitro from cells isolated from amniotic fluid (AFC-ECM) for the isolation, maintenance, and expansion/proliferation of mammalian 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 is still relatively poorly understood. Many of the 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 can be 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 can 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 can 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 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 disclosure 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.
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 disclosure contain specific motifs, such as laminin, that are known to facilitate pluripotent cell adhesion and expansion.
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.
Methods to expand/proliferate pluripotent stem cells (PSCs) include obtaining PSCs and culturing them in the presence of the amniotic fluid cell-derived ECM of the invention. The PSCs can be iPSCs or ES. 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 some embodiments the PSCs are maintained in an undifferentiated state and maintain their stemness. Cell culture techniques suitable for proliferation of PSCs in culture are known in the art. Suitable commercially available culture media for stem cell proliferation includes, but is not limited to StemMACS™ iPS-Brew XF, available from Miltenyl Biotec. 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 pluripotent stem cells (PSCs) in culture, the method comprising culturing the PSCs in the presence of a cell-derived extracellular matrix (ECM) in a culture media thereby proliferating the PSCs, wherein the cell-derived ECM is derived in-vitro from cells isolated from amniotic fluid.
The methods of expanding/proliferating PSCs described supra also apply to the use of expanding/proliferating PSCs in culture in the presence of other perinatal cell-derived ECMs. In some embodiments, disclosed is a method of proliferating pluripotent stem cells (PSCs) in culture, the method comprising culturing the PSCs in the presence of a cell-derived extracellular matrix (ECM) in a culture media thereby proliferating the PSCs, 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 = COL11Al
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
Cellular constructs comprising monolayers of mature cardiomyocytes on extracellular matrices derived from cells derived in-vitro from amniotic fluid (AFC-ECMs) were prepared using the following method.
Using the methodology as outlined in Example 1, AFC-ECMs were prepared in 96-well plates (no silicone inserts were used). Using standard cell culture techniques, commercially available immature hiPSC-CMs from Cellular Dynamics International-FUJI (iCell® Cardiomyocytes) were plated on the AFC-ECMs. The immature hiPSC-CMs were plated at a density of 50,000 cells per well (96 well plate) or 200,000 cells per well (6 well plate), and were cultured for 7 days in RPMI media forming confluent monolayers of mature cardiomyocytes on the AFC-ECMs, thereby forming cellular constructs of mature cardiomyocyte monolayers on the AFC-ECMs.
Over the 7-day period, the immature hiPSC-CMs were observed to mature into the morphology and alignment of mature native adult cardiomyocytes as characterized by rod shaped cells with distinct sarcomere structure. For comparison purposes, iCell® immature hiPSC-CMs were also plated (no silicone inserts were used) in 96-well plates and cultured in a similar fashion on standard Matrigel™ ECM, and on a bone marrow cell-derived ECM (BM-ECM) as prepared by methods as disclosed in U.S. Pat. No. 8,084,023, herein incorporated by reference. Results of the studies are shown in photomicrographs of the cardiomyocytes on the different ECMs in
As can be seen in the
Thus, the immature hiPSC-CMs cultured on the AFC-ECM achieved a higher state of maturation than did the immature hiPSC-CMs cultured on the standard Matrigel™ ECM or the natural cell-derived ECM from bone marrow cells. It is evident that the hiPSC-CM morphology was affected differently by different ECM.
Cellular constructs of monolayers of mature cardiomyocytes on AFC-ECM were prepared as described in Example 4 using 96-well plates (no silicone inserts were used). After the 7-day maturation process the electrophysiology of each well was observed using a plate reader using the following high throughput screen method. FluoVolt™ dye in Hanks Balanced Salt Solution was loaded into each well. A high spatiotemporal CCD camera (SciMeasure DaVinci camera) combined with light emitting diodes (LEDs) as shown in the schematic in
In additional studies, the drugs domperidone (3 μM), diisopyramide (100 μM), azimilide (10 μM), D,1 Sotalol (100 μM), ibutilide (0.10 μM), and Bepridil (10 μM) were tested with mature cardiomyocytes on AFC-ECM as prepared in Example 4 (no silicone inserts) in 96-well plates using the plate reader method described above. The results of the testing are shown in
Various drugs shown in Table 3 and 4 below were also tested with mature cardiomyocytes on AFC-ECM as prepared in Example 4 (no silicone inserts) in 96-well plates using the plate reader method described above and observations for any arrhythmia detected and APD90 prolongation at 10 times the effective therapeutic plasma concentration (ETPC) were notated. The drugs were selected from the CiPA Initiative's list of compounds for validation and testing of CiPA and are classified as high risk, intermediate risk or low risk for causing fatal arrhythmias (TdPs) in patients. The complete list of CiPA compounds can be found at cipaproject.org/wp-content/uploads/sites/24/2016/05/CiPA-Compounds .pdf.
Further analyses of the data from the drugs tested in Tables 3 and 4 are shown in
Cellular constructs of mature cardiomyocytes on an AFC-ECM were prepared following the procedures as described in Example 4 above with the following modifications as noted below:
AFC-ECM was deposited onto Thermanox coverslips to enable immunostaining of cells and imaging using laser scanning confocal microscopy (Nikon A1R).
Matrigel™ ECM was applied to a separate subset of Thermanox coverslips for comparison.
Cells from Cellular Dynamics International-FUJI (iCell® Cardiomyocytes) were plated as monolayers on these Thermanox coverslips coated with each ECM at a density of 200,000 cells per well in 6 well plates.
After 7 days of incubation in cell culture media, cells were fixed in 3% paraformaldehyde and processed for immunocytochemistry with application of commercially available primary antibodies to determine cellular expression and localization in hiPSC-CMs. The following primary antibodies for key cardiac myofilament proteins were used: Troponin I, α-actinin, cardiac troponin T (cTnT), cardiac troponin I (cTnI) and N-cadherin. Commercially available fluorescently labelled secondary antibodies were used for detection. DAPI diamidino-2-phenylindole) fluorescent stain was used to mark the nuclei.
All procedures for cell labeling and visualization are as described in the following references incorporated by reference herein. Herron, T. J. et al. Extracellular Matrix-Mediated Maturation of Human Pluripotent Stem Cell-Derived Cardiac Monolayer Structure and Electrophysiological Function. Circulation: Arrhythmia and Electrophysiology 9 (2016). da Rocha, M. A. et al. Deficient cMyBP-C protein expression during cardiomyocyte differentiation underlies human hypertrophic cardiomyopathy cellular phenotypes in disease specific human ES cell derived cardiomyocytes. J. Mol. Cell. Cardiol. 99, 197-206 (2016). da Rocha, A. M. et al. hiPSC-CM Monolayer Maturation State Determines Drug Responsiveness in High Throughput Pro-Arrhythmia Screen. Sci. Rep. 7, 13834 (2017).
Cell shape was quantified using fluorescent images analyzed in NIS Elements Software. Using cell area and perimeter, cellular circularity was quantified using the established mathematical equation; Circularity Index=4π*Area/Perimeter2.
In some cases, mitochondria were stained using MitoTracker™ Red CMXRos (Thermo Fisher).
Results: Consistent with the results as seen in Example 4 above, data using fluorescent labeling and imaging demonstrates that the AFC-ECM promotes rapid (7-day) maturation of hiPSC-CMs, and shows that hiPSC-CMs cultured on Matrigel™ ECM are circular in shape and have disorganization of sarcomeres, whereas the same batch of hiPSC-CMs (isogenic coparator) cultured on AFC-ECM are rod shaped and have tight compaction/organization of sarcomeres and myofilaments.
The photomicrophraphs in
The photomicrographs in
The photomicrographs in
The photomicrographs (transmitted light) in
This application claims the benefit of U.S. Provisional Patent Application No. 62/808,690, filed Feb. 21, 2019, which is incorporated by reference herein in its entirety
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| Number | Date | Country | |
|---|---|---|---|
| 20200270577 A1 | Aug 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62808690 | Feb 2019 | US |
