Patent Application
20230035127
The present disclosure relates to cell differentiation including preparing stem cells that maintain pluripotency in two and three-dimensional cultures.
Heart diseases are the leading cause of death in the United States. A major reason for this is that the human heart has very limited capacity to regenerate cardiomyocytes once they are damaged by some malfunction (e.g., ischemia), leading to myocardial infarction. Stem cell therapy has been considered as a promising strategy for treating heart diseases. It is well accepted now that functional/beating human cardiomyocytes can be differentiated only from human pluripotent stem cells (PSCs) including human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), although mesenchymal stem cells may be used for cardiac regeneration via their cytokine effect and differentiation into some cardiac stromal cells. Human PSC-derived cardiomyocytes can be used as not only a therapeutic agent for treating heart diseases, but also a valuable tool for elucidating the etiology and pathogenesis of heart diseases and developing engineered heart tissues to test the cardiotoxicity of pharmaceutical drugs. There is an urgent need to develop a stable system to upscale the generation of human PSC-derived cardiomyocytes for both basic and translational applications.
Applicants have discovered that Rock inhibitor (RI), which has been ubiquitously used as a medium supplement to prevent apoptosis during handling and culture of human PSCs, including both ESCs and iPSCs, compromises the quality of PSCs in three-dimensional (3D) culture and their capacity of directed cardiac differentiation in 3D. By reducing the RI concentration for handling and culturing PSCs, cardiac differentiation is improved with the outset beating time (OBT) synchronized within 24 hours (versus 7 or more days for all contemporary protocols) and the time required for cardiac differentiation is shortened by at least 7 days.
Methods of maintaining pluripotency of stem cells in 3D culture are provided. The methods comprise culturing pluripotent stem cells in suspension to form an aggregate in a medium comprising 5 μM or less of a Rock inhibitor. In certain embodiments, the medium comprises from about 0.01 μM to about 5 μM of the Rock inhibitor. In certain embodiments, the medium comprises about 1 μM of the Rock inhibitor. In certain embodiments, the methods further compromise inducing differentiation of the pluripotent stem cells.
Methods of producing cardiomyocytes or cardiac organoids are also provided. The methods comprise culturing pluripotent stem cells in suspension to form an aggregate in a medium comprising 5 μM or less of a Rock inhibitor; culturing the aggregate of pluripotent stem cells in suspension in a medium without Rock inhibitor; and inducing cardiac differentiation of the aggregate of pluripotent stem cells, thereby producing cardiomyocytes or cardiac organoids.
Cardiomyocytes and cardiac organoids produced by the methods are provided. Therapeutic agents comprising the cardiomyocytes or cardiac organoids are also provided.
Further provided are methods for screening an agent for improving or diminishing cardiac function as well as methods for treating a disorder of a cardiac tissue.
While multiple embodiments are disclosed, still other embodiments will become apparent to those skilled in the art from the following detailed description, which shows and describes illustrative embodiments. Accordingly, the figures and detailed description are to be regarded as illustrative in nature and not restrictive.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
The following drawings form part of the specification and are included to further demonstrate certain embodiments. In some instances, embodiments can be best understood by referring to the accompanying figures in combination with the detailed description presented herein. The description and accompanying figures may highlight a certain specific example, or a certain embodiment. However, one skilled in the art will understand that portions of the example or embodiment may be used in combination with other examples or embodiments.
The present disclosure relates to the surprising discovery that Rock inhibitor (RI), used ubiquitously to improve the survival and yield of PSCs, induces early gastrulation-like change to PSCs in 3D culture and causes their heterogeneous differentiation into all the three germ layers (i.e., ectoderm, mesoderm, and endoderm) at the commonly used concentration (10 μM). This greatly compromises the quality of PSCs for homogeneous 3D cardiac differentiation. By reducing the RI to 1 μM for 3D culture, the PSCs retain high pluripotency and high quality in inner cell mass-like solid 3D spheroids. Consequently, the beating efficiency of 3D cardiac differentiation can be improved to more than 95% in about 7 days (compared to less than about 50% in 14 days for the 10 μM RI condition). The outset beating time (OBT) of all resultant cardiac organoids is synchronized within only 1 day and they form a synchronously beating 3D construct after 5-day culture, showing high homogeneity (in terms of the OBT) in functional maturity of the cardiac organoids. The resultant cardiomyocytes are of high quality with key functional ultrastructures and highly responsive to cardiac drugs.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which embodiments of the disclosure pertain. Many methods and materials similar, modified, or equivalent to those described herein can be used in the practice of the embodiments of the present disclosure without undue experimentation, the preferred materials and methods are described herein.
It is to be understood that all terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting in any manner or scope. For example, as used in this specification and the appended claims, the singular forms “a”, “an”, and “the” can include plural referents unless the content clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. The word “or” means any one member of a particular list and also includes any combination of members of that list. Further, all units, prefixes, and symbols may be denoted in their SI accepted form.
Numeric ranges recited within the specification are inclusive of the numbers defining the range and include each integer within the defined range. Throughout this disclosure, various aspects are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present disclosure or the associated claims. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges, fractions, and individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6, and decimals and fractions, for example, 1.2, 3.8, 1½, and 4¾. This applies regardless of the breadth of the range.
The term “about”, as used herein, refers to variation in the numerical quantity that can occur, for example, through typical measuring and liquid handling procedures in the real world; through inadvertent error in these procedures; through differences in the manufacture, measurement quantifications (e.g., weight, volume, temperature, and time), source, or purity of the ingredients used to make the compositions or carry out the methods; and the like. Whether or not modified by the term “about”, the claims include equivalents to the quantities.
The term “stem cell” is used herein to refer to a mammalian cell that has the ability both to self-renew and to generate a differentiated cell type (see Morrison et al. (1997) Cell 88:287-298). In the context of cell ontogeny, the adjective “differentiated”, or “differentiating” is a relative term. A “differentiated cell” is a cell that has progressed further down the developmental pathway than the cell it is being compared with. Thus, pluripotent stem cells (described below) can differentiate into germ layer-restricted stem cells (e.g., endodermal, mesodermal, ectodermal stem cells), which in turn can differentiate into cells that are further restricted (e.g., cardiac progenitors), which can differentiate into end-stage cells (i.e., terminally differentiated cells, e.g., cardiomyocytes), which play a characteristic role or function in a certain tissue type, and may or may not retain the capacity to proliferate further. Stem cells may be characterized by both the presence of specific markers (e.g., proteins, RNAs, etc.) and the absence of specific markers. Stem cells may also be identified by functional assays both in vitro and in vivo, particularly assays relating to the ability of stem cells to give rise to multiple differentiated progenies.
The stem cells of interest are mammalian, where the term refers to cells isolated from any animal classified as a mammal, including humans, domestic and farm animals, and zoo, laboratory, sports, or pet animals, such as dogs, horses, cats, cows, mice, rats, rabbits, etc. In some embodiments, the mammal is a human and the mammalian cells are therefore human cells.
A “progenitor cell” is a type of stem cell that typically does not have extensive self-renewal capacity (i.e., the number of self-renewing divisions is limited), and often can only generate a limited number of differentiated cell types (e.g., a specific subset of cells found in the tissue from which they derive). Thus, a progenitor cell is differentiated relative to its mother stem cell, but can also give rise to cells that are further differentiated (e.g., terminally differentiated cells). For the purposes of the present disclosure, progenitor cells are those cells that are committed to a lineage of interest (e.g., a cardiac progenitor), but have not yet differentiated into a mature cell (e.g., a cardiomyocyte).
When a stem cell divides symmetrically, both resulting daughter cells are equivalent. For example, a stem cell may undergo a self-renewing symmetric division in which both resulting daughter cells are stem cells with an equal amount of differentiation potential as the mother cell. However, a symmetric division is not necessarily a self-renewing division because both resulting daughter cells may instead be differentiated relative to the mother cell. When a stem cell divides asymmetrically, the resulting daughter cells are different from one another. For example, if a stem cell undergoes a self-renewing asymmetric division, then one of the resulting daughter cells is a stem cell with the same amount of differentiation potential as the mother cell while the other daughter cell is differentiated relative to the mother cell (e.g., a more lineage restricted progenitor cell, a terminally differentiated cell, etc.). A stem cell may directly differentiate, or may instead produce a differentiated cell type through an asymmetric or symmetric cell division.
Stem cells of interest include pluripotent stem cells (PSCs). The term “pluripotent stem cell” or “PSC” is used herein to mean a stem cell capable of producing all cell types of the organism. Therefore, a PSC can give rise to cells of all germ layers of the organism (e.g., the endoderm, mesoderm, and ectoderm of a mammal). Pluripotent cells are capable of forming teratomas and of contributing to ectoderm, mesoderm, or endoderm tissues in a living organism.
PSCs can be derived in a number of different ways. For example, embryonic stem cells (ESCs) are derived from the inner cell mass of an embryo (Thomson et al, Science. 1998 Nov. 6; 282(5391):1145-7) whereas induced pluripotent stem cells (iPSCs) are derived from somatic cells (Takahashi et. al, Cell. 2007 Nov. 30; 131(5):861-72; Takahashi et. al, Nat Protoc. 2007; 2(12):3081-9; Yu et. al, Science. 2007 Dec. 21:318(5858):1917-20. Epub 2007 Nov. 20). Because the term PSC refers to pluripotent stem cells regardless of their derivation, the term PSC encompasses the terms ESC and iPSC, as well as the term embryonic germ stem cells (EGSC), which are another example of a PSC. A human PSC can be referred to as an “hPSC”, an “hESC”, an “hEGSC”, and/or an “hiPSC”, depending on the context and the derivation of the PSC. PSCs may be in the form of an established cell line, they may be obtained directly from primary embryonic tissue, or they may be derived from a somatic cell. The methods described herein can be used to produce cardiomyocytes from any mammalian PSC population, including but not limited to an ESC population, an iPSC population, and/or an EGSC population.
By “embryonic stem cell” (ESC) is meant a PSC that was isolated from an embryo, typically from the inner cell mass of the blastocyst. ESC lines are listed in the NIH Human Embryonic Stem Cell Registry, e.g. hESBGN-01, hESBGN-02, hESBGN-03, hESBGN-04 (BresaGen, Inc.); HES-1, HES-2, HES-3, HES-4, HES-5, HES-6 (ES Cell International); Miz-hES1 (MizMedi Hospital-Seoul National University); HSF-1, HSF-6 (University of California at San Francisco); and H1, H7, H9, H13, H14 (Wisconsin Alumni Research Foundation (WiCell Research Institute)). Stem cells of interest also include embryonic stem cells from other primates, such as Rhesus stem cells and marmoset stem cells. The stem cells may be obtained from any mammalian species, e.g. human, equine, bovine, porcine, canine, feline, rodent (e.g. mice, rats, hamster), primate, etc. (Thomson et al. (1998) Science 282:1145; Thomson et al. (1995) Proc. Natl. Acad. Sci USA 92:7844; Thomson et al. (1996) Biol. Reprod. 55:254; Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998). In culture, ESCs typically grow as flat colonies with large nucleo-cytoplasmic ratios, defined borders and prominent nucleoli. In addition, ESCs express SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and Alkaline Phosphatase, but not SSEA-1. Examples of methods of generating and characterizing ESCs may be found in, for example, U.S. Pat. Nos. 7,029,913, 5,843,780, and 6,200,806, the disclosures of which are incorporated herein by reference. Methods for proliferating hESCs in the undifferentiated form are described in WO 99/20741, WO 01/51616, and WO 03/020920.
By “embryonic germ stem cell” (EGSC) or “embryonic germ cell” or “EG cell” is meant a PSC that is derived from germ cells and/or germ cell progenitors, e.g. primordial germ cells, i.e. those that would become sperm and eggs. Embryonic germ cells (EG cells) are thought to have properties similar to embryonic stem cells as described above. Examples of methods of generating and characterizing EG cells may be found in, for example, U.S. Pat. No. 7,153,684; Matsui, Y., et al. (1992) Cell 70:841; Shamblott, M., et al. (2001) Proc. Natl. Acad. Sci. USA 98: 113; Shamblott, M., et al. (1998) Proc. Natl. Acad. Sci. USA, 95:13726; and Koshimizu, U., et al. (1996) Development, 122:1235. the disclosures of which are incorporated herein by reference.
By “induced pluripotent stem cell” or “iPSC” it is meant a PSC that is derived from a cell that is not a PSC (i.e., from a cell that is differentiated relative to a PSC). iPSCs can be derived from multiple different cell types, including terminally differentiated cells. iPSCs have an ES cell-like morphology, growing as flat colonies with large nucleo-cytoplasmic ratios, defined borders and prominent nuclei on a 2D substrate. In addition, iPSCs express one or more key pluripotency markers known by one of ordinary skill in the art, including but not limited to Alkaline Phosphatase, SSEA3, SSEA4, Sox2, Oct3/4, Nanog, TRA160, TRA181, TDGF 1, Dnmt3b, FoxD3, GDF3, Cyp26a1, TERT, and zfp42. Examples of methods of generating and characterizing iPSCs may be found in, for example, U.S. Patent Publication Nos. US20090047263, US20090068742, US20090191159, US20090227032, US20090246875, and US20090304646, the disclosures of which are incorporated herein by reference. Generally, to generate iPSCs, somatic cells are provided with reprogramming factors (e.g., Oct4, SOX2. KLF4, MYC, Nanog, Lin28, etc.) known in the art to reprogram the somatic cells to become pluripotent stem cells.
The term “episomal induced pluripotent stem cells” or “eiPSCs” refers to somatic cells that are reprogrammed into induced pluripotent stem cells (iPSCs) using non-integrative episomal vector methods. eiPSCs are virus-free, which further enhances their translational value.
Compared to human ESCs, human iPSCs have no ethical concerns and are more easily accepted by society because they are not from human embryos. Of note, human iPSC banks may be established for generating human leukocyte antigen (HLA)-matched cell transplantation for known HLA types of donor and recipient, and the human iPSCs have great potential for allogeneic cell therapy besides autologous cell therapy.
By “somatic cell” it is meant that a cell of an organism that is not a germ cell. Thus, in the absence of experimental manipulation, a mammalian somatic cell does not ordinarily give rise to all types of cells in the body, although adult somatic stem cells do exist (e.g., lineage restricted progenitor cells)
Rho-kinase (Rock) inhibitor (RI) has been ubiquitously used to improve the survival and yield of iPSCs and ESCs at a concentration of 10 μM, which is the optimal concentration for preventing apoptosis of the PSCs under both 2D and 3D cultures. With all other conditions being kept consistent, using 10 μM RI in the medium for culturing the PSCs in 3D significantly improves the cell yield (i.e., number of viable cells) by about 3 and 2 times, compared to 0 or 1 μM RI, respectively. The use of 1 μM RI significantly increases the cell survival and yield compared to a 0 μM RI control. Unfortunately, RI at 10 μM induces uncontrolled spontaneous differentiation of PSCs into heterogeneous lineages including the ectoderm (e.g., neural and eye development), endoderm (e.g., lung and gut development), and non-cardiac mesoderm (e.g., urogenital and cartilage development) both before (i.e., on days −4 to 0) and after (i.e., after day 0) cardiac differentiation. The heterogeneous differentiation of PSCs cultured with 10 μM RI before cardiac differentiation is also evidenced by an archenteron-like cavity of the PSC spheroids, similar to the epiblast and hypoblast cells in the early gastrula. This uncontrolled spontaneous differentiation into heterogeneous lineages greatly compromises the efficiency and functional homogeneity (in terms of OBT) of directed or guided cardiac differentiation. In contrast, the use of 1 μM RI results in more than 95% beating cardiac organoids for cardiac differentiation of PSCs with the OBT being synchronized within 24 hours (compared to more than 7 days for the 10 μM RI group), indicating a highly efficient and homogeneous cardiac differentiation. This is attributed to the high pluripotency and high quality of the PSCs cultured with 1 μM RI in the solid inner cell mass-like spheroids.
Thus, in certain embodiments, the methods of the disclosure comprise the step of: (a) culturing pluripotent stem cells (e.g., single cells or clusters) in suspension to form an aggregate in a medium comprising 5 μM or less of a Rock inhibitor. In certain embodiments, the methods further comprise the step of: (b) culturing the aggregate of pluripotent stem cells in suspension in a medium without Rock inhibitor. Various media formulations are available and known in the art, and can be used to culture the PSCs. In certain embodiments, the medium is mTeSR1 or StemFlex.
As used herein, an “aggregate” refers to a three-dimensional association of cells in which the association is caused by cell-cell interaction rather than adherence to a substrate.
In one embodiment, the medium is supplemented with from about 0.01 μM to about 5 μM of the Rock inhibitor, from about 0.1 μM to about 4 μM of the Rock inhibitor, from about 0.5 μM to about 2 μM of the Rock inhibitor, or from about 0.75 μM to about 1.25 μM of the Rock inhibitor. In one embodiment, the medium is supplemented with about 0.5 μM of the Rock inhibitor, about 0.75 μM of the Rock inhibitor, about 1 μM of the Rock inhibitor, about 1.25 μM of the Rock inhibitor, about 1.5 μM of the Rock inhibitor, about 1.75 μM of the Rock inhibitor, or about 2 μM of the Rock inhibitor.
Examples of Rock inhibitors include Y27632 ((+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclo-hexanecarboxamide dihydrochloride), Fasudil (1-(5-Isoquinolinesulfonyl)homopiperazine), and Thiazovivin (N-Benzyl-2-(pyrimidin-4-ylamino)thiazole-4-carboxamide). In certain embodiments, the Rock inhibitor is Y27632.
The medium can be supplemented with a viscosity enhancer. The term “viscosity enhancer” refers to any substance that can increase the viscosity of a liquid such as a medium. The presence of a viscosity enhancer reduces the fusion of the PSC aggregates during the suspension culture. Viscosity enhancers include, for example, methylcellulose, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, hydroxypropylmethyl cellulose acetate stearate, hydroxypropylmethyl cellulose phthalate, polyacrylic acid, polyvinyl alcohol, polyethylene glycol, alginate, xanthan gum, acacia, chitosan, and combinations thereof. In certain embodiments, the viscosity enhancer is methylcellulose.
In a specific embodiment, the 3D culture is prepared by the following protocol: PSC colonies under 2D culture at about 80% confluence are treated with Versene for 2 minutes, rinsed with isotonic (by default) phosphate-buffered saline (PBS), and further detached from the substrate by gentle pipetting. The detached PSCs are re-suspended in mTeSR1 with 5 μM or less Rock inhibitor. Afterward, the medium is supplemented with 0.35% (v/v) methylcellulose. The cell suspension is pushed through a cell strainer with 100 μm mesh size. Later, the suspension of PSC clumps or clusters is transferred into a petri dish for culture in a humidified incubator at 37° C. and 5% CO2 for 2 days. Then, the medium is changed to mTeSR1 (supplemented with 0.35% methylcellulose) with no Rock inhibitor, to further culture for 2 days before differentiation.
In some embodiments, prior to 3D culture, the PSCs are cultured in a maintenance media on a substrate (i.e., 2D culture). In a specific embodiment, PSCs are cultured in a maintenance media by the following protocol: the PSCs are cultured in Matrigel-coated plates in a maintenance medium made of DMEM/F12 supplemented with bFGF (120 ng/ml), TGF-β (1 ng/ml), γ-aminobutyric acid (100 μg/ml), LiCl 30 (μg/ml), L-glutamine (100 μg/ml, Gibco), MEM non-essential amino acid (NEAA) solution (0.5%), NaHCO3 (500 μg/ml), chemically defined lipid concentrate (1%), sodium selenite (50 ng/ml), bovine serum albumin (20 mg/ml), and β-mercaptoethanol (4 μl per 500 ml medium). The cells are passaged twice a week at a ratio between 1:4 and 1:5 with Versene consisting of 0.48 nM ethylenediaminetetraacetic acid (EDTA) in 1× (by default) phosphate buffered saline (PBS).
As used herein, a “cardiomyocyte” or “myocardial cell” is a terminally differentiated heart muscle cell. A “cardiomyocyte progenitor” is defined as a cell that is capable (without dedifferentiation or reprogramming) of giving rise to progeny that include cardiomyocytes. Such progenitors may express various cytoplasmic and nuclear markers typical of the lineage, including, without limitation, cardiac troponin I (cTnI), cardiac troponin T (cTnT), sarcomeric myosin heavy chain (MHC), GATA-4, Nkx2.5, N-cadherin, β1-adrenoceptor (β1-AR), ANF, the MEF-2 family of transcription factors, creatine kinase MB (CK-MB), myoglobin, or atrial natriuretic factor (ANF).
The pluripotent stem cells of the disclosure can be differentiated into cardiomyocytes or cardiac organoids. The terms “cardiac organoid” and “cardiac spheroid” are used interchangeably herein to refer to a 3D multicellular in vitro cardiac tissue that recapitulates aspects of the in vivo organ.
In certain embodiments, the method for inducing cardiac differentiation of a pluripotent stem cell comprises the steps of (1) culturing a pluripotent stem cell in a medium comprising a Wnt signaling activator and (2) culturing a pluripotent stem cell produced in step (1) in a medium comprising a Wnt signaling inhibitor.
The term “Wnt signaling activator” or “Wnt agonist” as used herein refers to a substance which activates the Wnt signaling pathway. Examples of the Wnt signaling activator include a glycogen synthase kinase 3β (Gsk-3β) inhibitor such as 6-bromoindirubin-3′-oxime (BIO) or CHIR99021. More than one Wnt signaling activator, for example, 2, 3, or 4 Wnt signaling activators may be used in combination.
The term “Wnt signaling inhibitor” or “Wnt antagonist” as used herein refers to a substance which inhibits the Wnt signaling pathway. Examples of the Wnt signaling inhibitor include compounds such as KY02111, IWP2, XAV939, and IWR1, and proteins such as IGFBP4 and Dkk1. More than one Wnt signaling inhibitor, for example, 2, 3, or 4 Wnt signaling inhibitors may be used in combination.
The medium used in the step (1), the step of culturing a pluripotent stem cell in a medium comprising a Wnt signaling activator, and the medium used in the step (2), the step of culturing a pluripotent stem cell produced in step (1) in a medium comprising a Wnt signaling inhibitor, may be any conventional medium used for cardiac differentiation of a pluripotent stem cell and the composition of the differentiation medium is not specifically limited. Examples of the medium include DMEM/F12-based medium, RPMI1640-based medium, or α-MEM-based medium for cardiac differentiation.
In the methods of the disclosure, the period from the start of culture in a medium for cardiac differentiation (i.e., culture for cardiac differentiation) to the start of step (1) or (2) and the periods of steps (1) and (2) of may be appropriately determined. Step (2) may be started just after the end of step (1), or after a certain period from the end of step (1). The Wnt signaling activator and the Wnt signaling inhibitor may be added at early and middle phases of cardiac differentiation of a pluripotent stem cell, respectively. The early phase of cardiac differentiation of a pluripotent stem cell means a stage at which differentiation of a pluripotent stem cell into mesoderm is induced and the expression of a mesoderm marker gene is increased. The middle phase of cardiac differentiation of a pluripotent stem cell means a stage at which differentiation of mesoderm into cardiac muscle is induced. Examples of the mesoderm marker includes T, MIXL1, and NODAL. For example, step (1) may be conducted at day 0 to day 2 or day 0 to day 3 of culture for cardiac differentiation, in other words, for 2 or 3 days from the start of culture for cardiac differentiation, and step (2) may be conducted, up until day 28 of culture for cardiac differentiation, for 2 days or more (specifically, for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days), preferably for 3 to 10 days, more preferably for 4 to 10 days, still more preferably for 4 to 8 days, even more preferably for 4 to 6 days. Preferably, step (2) is conducted for 4 to 6 days up until day 10 of culture for cardiac differentiation, for example day 3 to day 9, day 3 to day 8, day 3 to day 7, day 4 to day 10, day 4 to day 9, or day 4 to day 8 of culture for cardiac differentiation.
Concentrations of the Wnt signaling activator and the Wnt signaling inhibitor are not particularly limited. When the Wnt signaling activator is BIO or CHIR99021, the Wnt signaling activator may be used at a final concentration of 100 nM to 100 μM, preferably 1 μM to 10 μM. When the Wnt signaling inhibitor is KY02111 or XAV939, the Wnt signaling inhibitor may be used, for example, at a final concentration of 0.5 to 20 μM, preferably 1 to 10 μM.
In a specific embodiment, PSCs are differentiated into cardiomyocytes by the following protocol: on day 0 of differentiation, the PSCs are induced into mesoderm by up-regulation of the Wnt signaling pathway using 8 μM CHIR99021 and 2 μM BIO in mesoderm induction medium for 1 day. The mesoderm induction medium is a mixture of DMEM/F12 and α-MEM (v/v, 1:1), containing 2% Knockout Serum Replacement (KOSR), 1 mM L-glutamine, 1% MEM non-essential amino acids (NEAA), and 0.1 mM β-mercaptoethanol. After 1 day, the cells are induced for cardiac commitment by down-regulating the Wnt signaling pathway using 10 μM KY02111 and 10 μM XAV939 in cardiac maintenance medium for 6 days with the medium being changed every other day. The cardiac maintenance medium is a mixture of RPMI1640 and α-MEM (v/v, 1:1), containing 5% fetal bovine serum (FBS). Starting from day 8, the cardiac maintenance medium without KY02111 and XAV939 is used, and it is changed every other day for cardiac maturation. Xenogeneic KOSR and FBS used in the media for cardiac differentiation and maintenance, respectively, may be replaced with materials of human origin to further improve translational value.
Differentiation into a cardiomyocyte may be detected from, for example, the number of beating cardiac organoids, expression of a cardiac marker, expression of an ion channel, a response to an electrophysiological stimulus, or the like. Examples of a cardiac marker include α-MHC, β-MHC, cTnT, α-actinin, and NKX2.5. Examples of the ion channel include HCN4, Nav1.5, Cav1.2, Cav3.2 HERG1b, and KCNQ1.
Therapeutic agents comprising the cardiomyocytes or cardiac organoids of the disclosure are provided. A “therapeutic agent” as used herein refers to any, composition useful for therapeutic or diagnostic purposes. The term as used herein is understood to mean any composition that is administered to a subject for the diagnosis, cure, mitigation, treatment, or prevention of a condition.
In vitro cardiomyocytes produced by the methods of the disclosure provide a source of donor cardiomyocytes for cell replacement in damaged hearts. Many forms of heart disease, including congenital defects and acquired injuries, are irreversible because they are associated with the loss of non-regenerative, terminally differentiated cardiomyocytes. Current therapeutic regimes are palliative, and in the case of end-stage heart failure, transplantation remains the last resort. However, transplantation is limited by a severe shortage of both donor cells and organs. In cases of myocardial infarction, 1 billion cells would potentially need to be replaced, highlighting the need for high-throughput and reproducible methodologies for de novo cardiomyocyte production.
As such, the cardiomyocytes may be used for tissue reconstitution or regeneration in a human patient or other subject in need of such treatment. The cells are administered in a manner that permits them to graft or migrate to the intended tissue site and reconstitute or regenerate the functionally deficient area. Special devices are available that are adapted for administering cells capable of reconstituting cardiac function directly to the chambers of the heart, the pericardium, or the interior of the cardiac muscle at the desired location. The cells may be administered to a recipient heart by intracoronary injection, e.g., into the coronary circulation. The cells may also be administered by intramuscular injection into the wall of the heart.
Medical indications for such treatment include treatment of acute and chronic heart conditions of various kinds, such as coronary heart disease, cardiomyopathy, endocarditis, congenital cardiovascular defects, and congestive heart failure. Efficacy of treatment can be monitored by clinically accepted criteria, such as reduction in area occupied by scar tissue or revascularization of scar tissue, and in the frequency and severity of angina; or an improvement in developed pressure, systolic pressure, end diastolic pressure, patient mobility, and quality of life.
The differentiating cells may be administered in any physiologically acceptable excipient, where the cells may find an appropriate site for regeneration and differentiation. The cells may be introduced by injection, catheter, or the like.
The cells of the disclosure can be supplied in the form of a pharmaceutical composition, comprising an isotonic excipient prepared under sufficiently sterile conditions for human administration. For general principles in medicinal formulation, the reader is referred to Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy, by G. Morstyn & W. Sheridan eds, Cambridge University Press, 1996; and Hematopoietic Stem Cell Therapy, E. D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000. Choice of the cellular excipient and any accompanying elements of the composition will be adapted in accordance with the route and device used for administration. The composition may also comprise or be accompanied with one or more other ingredients that facilitate the engraftment or functional mobilization of the cells. Suitable ingredients include matrix proteins that support or promote adhesion of the cells, or complementary cell types.
Cells of the disclosure may be genetically altered in order to introduce genes useful in the differentiated cardiomyocyte, e.g., repair of a genetic defect in an individual, selectable marker, etc. Cells may also be genetically modified to enhance survival, control proliferation, and the like. Cells may be genetically altering by transfection or transduction with a suitable vector, homologous recombination, or other appropriate technique, so that they express a gene of interest. The cells of the disclosure can also be genetically altered in order to enhance their ability to be involved in tissue regeneration, or to deliver a therapeutic gene to a site of administration. A vector is designed using the known encoding sequence for the desired gene, operatively linked to a promoter that is either pan-specific or specifically active in cardiomyocytes. Many vectors useful for transferring exogenous genes into target mammalian cells are available. The vectors may be episomal, e.g. plasmids, virus derived vectors such cytomegalovirus. adenovirus, etc., or may be integrated into the target cell genome, through homologous recombination or random integration, e.g., retrovirus derived vectors such MMLV, HIV-1, ALV, etc. For modification of stem cells, lentiviral vectors are preferred. Lentiviral vectors such as those based on HIV or FIV gag sequences can be used to transfect non-dividing cells, such as the resting phase of human stem cells (see Uchida et al. (1998) P.N.A.S 95(20):11939-44).
In vitro cardiomyocytes and cardiac organoids produced by the methods of the disclosure also provide a source cells for novel cardiac drug discovery, development, and safety testing. Among the drugs that ultimately make it to market, many are later withdrawn due to side effects associated with electrophysiological alterations of the heart (Braam et al., 2010). The use of in vitro cardiomyocytes and cardiac organoids produced by the methods of the disclosure offers the pharmaceutical industry an invaluable tool for preclinical screening of candidate drugs to treat cardiomyopathy, arrhythmia, and heart failure, as well as therapeutics to combat secondary cardiac toxicities. The development of new screens using in vitro cardiomyocytes produced by the methods of the disclosure should reduce the time and cost of bringing new drugs to market.
In screening assays for biologically active agents (e.g., small molecule compounds, peptides, viruses, etc.) of the cardiomyocytes, usually a culture comprising the cardiomyocytes, is contacted with the agent of interest, and the effect of the agent assessed by monitoring output parameters, such as expression of markers, cell viability, electrophysiology, and the like.
Agents of interest for screening include known and unknown compounds that encompass numerous chemical classes, primarily organic molecules, which may include organometallic molecules, inorganic molecules, genetic sequences, etc. An important aspect of the disclosure is to evaluate candidate drugs, including toxicity testing; and the like.
In addition to complex biological agents, such as viruses, candidate agents include organic molecules comprising functional groups necessary for structural interactions, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, frequently at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules, including peptides, polynucleotides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.
Included are pharmacologically active drugs, genetically active molecules, etc. Compounds of interest include chemotherapeutic agents, hormones or hormone antagonists, etc. Exemplary of pharmaceutical agents suitable for this disclosure are those described in, “The Pharmacological Basis of Therapeutics,” Goodman and Gilman, McGraw-Hill, New York, N.Y., (1996), Ninth edition, under the sections: Water, Salts and Ions; Drugs Affecting Renal Function and Electrolyte Metabolism; Drugs Affecting Gastrointestinal Function; Chemotherapy of Microbial Diseases; Chemotherapy of Neoplastic Diseases; Drugs Acting on Blood-Forming organs; Hormones and Hormone Antagonists; Vitamins, Dermatology; and Toxicology, all incorporated herein by reference.
Test compounds include all of the classes of molecules described above, and may further comprise samples of unknown content. Of interest are complex mixtures of naturally occurring compounds derived from natural sources such as plants. While many samples will comprise compounds in solution, solid samples that can be dissolved in a suitable solvent may also be assayed. Samples of interest include manufacturing samples, pharmaceuticals, libraries of compounds prepared for analysis, and the like. Samples of interest include compounds being assessed for potential therapeutic value, i.e. drug candidates.
Compounds, including candidate agents, are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds, including biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.
Agents are screened for biological activity by adding the agent to at least one and usually a plurality of cell samples, usually in conjunction with cells lacking the agent. The change in parameters in response to the agent is measured, and the result evaluated by comparison to reference cultures, e.g., in the presence and absence of the agent, obtained with other agents, etc.
The agents are conveniently added in solution, or readily soluble form, to the medium of cells in culture. The agents may be added in a flow-through system, as a stream, intermittent or continuous, or alternatively, adding a bolus of the compound, singly or incrementally, to an otherwise static solution. In a flow-through system, two fluids are used, where one is a physiologically neutral solution, and the other is the same solution with the test compound added. The first fluid is passed over the cells, followed by the second. In a single solution method, a bolus of the test compound is added to the volume of medium surrounding the cells. The overall concentrations of the components of the culture medium should not change significantly with the addition of the bolus, or between the two solutions in a flow through method.
Preferred agent formulations do not include additional components, such as preservatives, that may have a significant effect on the overall formulation. Thus, preferred formulations consist essentially of a biologically active compound and a physiologically acceptable carrier, e.g., water, ethanol, DMSO, etc. However, if a compound is liquid without a solvent, the formulation may consist essentially of the compound itself.
A plurality of assays may be run in parallel with different agent concentrations to obtain a differential response to the various concentrations. As known in the art, determining the effective concentration of an agent typically uses a range of concentrations resulting from 1:10, or other log scale, dilutions. The concentrations may be further refined with a second series of dilutions, if necessary. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration or below the level of detection of the agent or at or below the concentration of agent that does not give a detectable change in the phenotype.
The cells may be freshly isolated, cultured, genetically altered as described above, or the like. The cells may be environmentally induced variants of clonal cultures: e.g., split into independent cultures and grown under distinct conditions, for example with or without virus; in the presence or absence of other biological agents. The manner in which cells respond to an agent, particularly a pharmacologic agent, including the timing of responses, is an important reflection of the physiologic state of the cell.
Parameters are quantifiable components of cells, particularly components that can be accurately measured, desirably in a high throughput system. A parameter can be any cell component or cell product including cell surface determinant, receptor, protein or conformational or posttranslational modification thereof, lipid, carbohydrate, organic or inorganic molecule, nucleic acid, e.g., mRNA, DNA, etc. or a portion derived from such a cell component or combinations thereof. While most parameters will provide a quantitative readout, in some instances a semi-quantitative or qualitative result will be acceptable. Readouts may include a single determined value, or may include mean, median value or the variance, etc. Characteristically a range of parameter readout values will be obtained for each parameter from a multiplicity of the same assays. Variability is expected and a range of values for each of the set of test parameters will be obtained using standard statistical methods with a common statistical method used to provide single values.
In vitro cardiomyocytes and cardiac organoids produced by the methods of the disclosure can also be used in developmental biology, disease modeling, and post-genomic personalized medicine. Deriving hiPSCs from patients with specific cardiac diseases, differentiating them to cardiomyocytes using the methods of the disclosure, and then performing electrophysiological and molecular analyses will provide a powerful tool for deciphering the molecular mechanisms of disease (Josowitz et al., 2011). Studies to date have largely concentrated on recapitulating genetic disease phenotypes in vitro, such as long QT syndromes (Itzhaki et al., 2011a; Matsa et al., 2011; Moretti et al., 2010), Timothy syndrome (Yazawa et al., 2011), and LEOPARD syndrome (Carvajal-Vergara et al., 2010). The possibility of modeling cardiac diseases without a known genetic element is another exciting prospect. The combination of novel drug discovery and efficacy testing with cardiomyocytes derived from patient-specific hiPSCs is a potentially groundbreaking option for personalized medicine.
The following numbered embodiments also form part of the present disclosure:
1. A method of maintaining pluripotency of stem cells in three-dimensional (3D) culture, the method comprising: culturing pluripotent stem cells in suspension to form an aggregate in a medium comprising 5 μM or less of a Rock inhibitor.
2. The method of embodiment 1, wherein the medium comprises from about 0.01 μM to about 5 μM of the Rock inhibitor.
3. The method of embodiment 1 or embodiment 2, wherein the medium comprises from about 0.5 μM to about 2 μM of the Rock inhibitor.
4. The method of any one of embodiments 1-3, wherein the medium comprises about 1 μM of the Rock inhibitor.
5. The method of any one of embodiments 1-4, wherein the pluripotent stem cells are induced pluripotent stem cells.
6. The method of any one of embodiments 1-5, wherein the medium comprises a viscosity enhancer, optionally wherein the viscosity enhancer comprises methylcellulose.
7. The method of any one of embodiments 1-6, wherein the medium is mTeSR1 or StemFlex supplemented with the Rock inhibitor and methylcellulose.
8. The method of any one of embodiments 1-7, wherein prior to the culturing step, the pluripotent stem cells are cultured in a maintenance media on a substrate.
9. The method of embodiment 8, wherein the maintenance medium comprises DMEM/F12 supplemented with bFGF, TGF-β, γ-aminobutyric acid, LiCl, L-glutamine, MEM non-essential amino acids, NaHCO3, chemically defined lipid concentrate, sodium selenite, bovine serum albumin, and β-mercaptoethanol.
10. The method of any one of embodiments 1-9, further comprising culturing the aggregate of pluripotent stem cells in suspension in a medium without Rock inhibitor.
11. The method of any one of embodiments 1-10, wherein a 3D pluripotent stem cell spheroid is produced.
12. The method of any one of embodiments 1-11, further comprising inducing differentiation of the pluripotent stem cells.
13. The method of embodiment 12, wherein the pluripotent stem cells are differentiated into ectoderm, mesoderm, or endoderm.
14. The method of embodiment 12 or embodiment 13, wherein the pluripotent stem cells are differentiated into cardiomyocytes.
15. A 3D pluripotent stem cell spheroid produced by the method of any one of embodiments 1-11.
16. A method of producing cardiomyocytes or a cardiac organoid, the method comprising: (a) culturing pluripotent stem cells in suspension to form an aggregate in a medium comprising 5 μM or less of a Rock inhibitor; (b) culturing the aggregate of pluripotent stem cells in suspension in a medium without Rock inhibitor; and (c) inducing cardiac differentiation of the aggregate of pluripotent stem cells, thereby producing the cardiomyocytes or a cardiac organoid.
17. The method of embodiment 16, wherein the medium of step (a) comprises from about 0.01 μM to about 5 μM of the Rock inhibitor.
18. The method of embodiment 16 or embodiment 17, wherein the medium of step (a) comprises from about 0.5 μM to about 2 μM of the Rock inhibitor.
19. The method of any one of embodiments 16-18, wherein the medium of step (a) comprises about 1 μM of the Rock inhibitor.
20. The method of any one of embodiments 16-19, wherein the pluripotent stem cells are induced pluripotent stem cells.
21. The method of any one of embodiments 16-20, wherein the medium of step (a) or the medium of step (b) comprises a viscosity enhancer, optionally wherein the viscosity enhancer comprises methylcellulose.
22. The method of any one of embodiments 16-21, wherein the medium of step (a) is mTeSR1 or StemFlex supplemented with the Rock inhibitor and methylcellulose.
23. The method of any one of embodiments 16-22, wherein prior to the culturing step, the pluripotent stem cells are cultured in a maintenance media on a substrate.
24. The method of embodiment 23, wherein the maintenance medium comprises DMEM/F12 supplemented with bFGF, TGF-β, γ-aminobutyric acid, LiCl, L-glutamine, MEM non-essential amino acids, NaHCO3, chemically defined lipid concentrate, sodium selenite, bovine serum albumin, and β-mercaptoethanol.
25. The method of any one of embodiments 16-24, wherein inducing cardiac differentiation comprises culturing the aggregate of pluripotent stem cells in a medium comprising a Wnt signaling activator; and culturing the aggregate of pluripotent stem cells in a medium comprising a Wnt signaling inhibitor.
26. The method of embodiment 25, wherein the Wnt signaling activator comprises CHIR99021 and 6-bromoindirubin-3′-oxime (BIO).
27. The method of embodiment 25 or embodiment 26, wherein the Wnt signaling inhibitor comprises XAV939 and KY02111.
28. The method of any one of embodiments 16-27, wherein the outset beating time (OBT) is synchronized within about 24 hours.
29. The cardiomyocytes or cardiac organoid produced by the method of any one of embodiments 16-28.
30. A method for screening an agent for improving or diminishing cardiac function, the method comprising: contacting the cardiomyocytes or cardiac organoid of embodiment 29 with the agent; and measuring at least one response of the cardiomyocytes or cardiac organoid.
31. A therapeutic agent comprising the cardiomyocytes or cardiac organoid of embodiment 29.
32. A method for treating a disorder of a cardiac tissue, the method comprising: transplanting the cardiomyocytes or cardiac organoid of embodiment 29 into a heart of a subject in need of treatment.
All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this disclosure pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.
The following examples are offered by way of illustration and not by way of limitation.
The timeline for the 4-day (from day −4 to 0) 3D culture and the subsequent 3D cardiac differentiation of iPSCs is illustrated in
Cardiac differentiation of the 3D eiPSC spheroids was conducted by modulating the canonical Wnt signaling pathway with agonists and antagonists sequentially, which has also been used for differentiation of human PSCs into cardiomyocytes under 2D culture. Two Wnt agonists, CHIR99021 (8 μM) and 6-bromoindirubin-3′-oxime (BIO, 2 μM), were supplemented into the culture medium on day 0, to up-regulate the Wnt signaling and induce iPSC differentiation into mesoderm for 1 day. On day 1, two Wnt antagonists, XAV939 (10 μM) and KY02111 (10 μM), were used to suppress the Wnt signaling for inducing cardiac commitment of the mesoderm cells in the following 6 days to obtain cardiac spheroids. Afterward, the cardiac spheroids were cultured in pure cardiac maintenance medium for maturation until day 13.5. Spontaneous beating was observed for ˜48% of the cardiac spheroids in the 1 μM RI group on as early as day 6.5, and the percentage of beating cardiac spheroids peaked at ˜97% at ˜24 h later on day 7.5 (
To further investigate the difference of cells in cardiac spheroids between the 1 and 10 μM RI groups, the cardiac spheroids on day 13.5 from both groups were re-plated in regular cell culture petri dish. After 3 days, ˜61% of the cardiac spheroids in the 10 μM RI group attached to the 2D surface, and some of the cells migrated out of the spheroids appeared to be neuron-like with neurite-like processes (
To investigate the mechanisms for the different outcomes when using different RI concentrations for culturing the eiPSC spheroids in 3D for cardiac differentiation, samples at various stages of the 3D culture and differentiation shown in
To understand the number of genes that are uniquely and co-expressed within the cells of the 1 versus 10 μM RI group at different stages of 3D culture and cardiac differentiation as compared to the 2D control, the Venn diagrams were generated first and shown in
The differentially expressed genes were further examined through an enrichment analysis with the Gene Ontology (GO) database as the reference, to determine the biological functions or pathways significantly enriched with the genes. Notably, the top 18 significantly upregulated differentially expressed gene groups of MO with regard to MT (MO vs. MT) were enriched to heart development, muscle system process, and the development of Z-disc and I band (
The heterogeneous protein expression induced by high RI was first indicated by the morphology of the eiPSC spheroids (
To further confirm the aforementioned difference in morphology and protein expression between the 1 and 10 μM RI groups, the 3D iPSC spheroids obtained with 0, 1, and 10 μM RI concentrations collected on day 0 (i.e., after 4-day culture in 3D) were plated on the 2D surface coated with Matrigel in Petri dish (i.e., under conventional 2D culture) and cultured for 2 days to observe the cell morphology. Although eiPSC colonies were observable for all the three groups, the ones in the 0 and 1 μM RI groups were typical with tightly packed cells consisting mainly of nuclei inside them and a largely smooth outer boundary (
Lastly and importantly, protein markers for urogenital system development (WNT4) and cartilage development (CD44) are also observable in the eiPSC-derived cardiac spheroids of the MT group but not the MO group (
Probably due to the high and homogeneous pluripotency of the eiPSC spheroids in the low RI group according to the aforementioned transcriptomic and protein analyses, the cardiac differentiation procedure results in high purity of cells at each of the three stages of modulating the Wnt signal pathway (
The quality of the cardiac spheroids in the low RI group on day 12.5 was further analyzed with transmission electron microscopy (TEM) to identify the key cardiomyocyte functional ultrastructures. The cardiomyocytes in the cardiac spheroids developed plenty of myofibrils (MF, up to 18 μm) and sarcoplasmic reticula (SR), which were typical functional components of cardiac muscle (
A calcium spike assay was also conducted with the cardiac spheroids on day 12.5 from the low HR group to examine their beating function, for which the fluo-4 staining was used to visualize the calcium transient in the cardiac spheroids. Furthermore, cardiac drugs isoproterenol (ISO) that speeds up beating and propranolol (PRO) that slows down beating were used to test the drug response of the cardiac spheroids (
It is worth noting that the human eiPSC-derived homogeneous cardiac spheroids were compatible with GelMA that has been widely used for 3D bioprinting. The cardiac spheroids collected on day 5 post-cardiac differentiation (before the initiation of spontaneous beating) and homogeneously suspended/cultured in 7% GelMA, fuse together within 2 days of culture in the GelMA (
Another commonly utilized iPSC line (IMR90-1) was tested to confirm that the improved cardiac differentiation of iPSCs with low RI for 3D culture before differentiation is not because of the eiPSCs used. The beating of the resultant IMR90-1 cardiac spheroids peaked at ˜98% on day 7.5 with the OBT being synchronized within ˜1 day for the 1 μM RI group (
The human eiPSCs (DF19-9-11T.H) and IMR90-1 human iPSCs were cultured in Matrigel-coated 6-well plate in an iPSC maintenance medium made of DMEM/F12 supplemented with bFGF (120 ng/ml), TGF-β (1 ng/ml), γ-aminobutyric acid (100 μg/ml), LiCl 30 (μg/ml), L-glutamine (100 μg/ml), MEM non-essential amino acid (NEAA) solution (0.5%), NaHCO3 (500 μg/ml), chemically defined lipid concentrate (1%, Invitrogen), sodium selenite (50 ng/ml), bovine serum albumin (20 mg/ml), β-mercaptoethanol (4 μl per 500 ml medium). The cells were passaged twice a week at a ratio between 1:4 and 1:5 with Versene consisting of 0.48 nM ethylenediamineetraacetic acid (EDTA) in 1× (by default) phosphate buffered saline (PBS).
To obtain 3D iPSC spheroids, the iPSC colonies under 2D culture at ˜80% confluence were treated with Versene for 2 min, rinsed with PBS, and further detached from the substrate by gentle pipetting. The detached iPSCs were re-suspended in mTeSR1 with Rock inhibitor (Y27632) at 0, 1, 5, or 10 μM. Afterward, the medium was supplemented with 0.35% (v/v) methylcellulose. The cell suspension (12 ml) containing ˜4×106 cells was pushed through a cell strainer with 100 μm mesh size. Later, the suspension of iPSC clumps was transferred into a petri dish (diameter: 10 cm) for culture in a humidified incubator at 37° C. and 5% CO2 for 2 days, during which the 2D iPSC clumps grew into 3D iPSC spheroids. Then, the medium was changed to mTeSR1 (supplemented with 0.35% methylcellulose) with no rock inhibitor, to further culture for 2 days before cardiac differentiation. To determine the number of cells per eiPSC spheroid, roughly equal numbers of the spheroids from either the 1 or 10 μM RI group were pooled together and trypsinized to dissociate the cells. The dissociated cells were then resuspended in 1.4 ml of the aforementioned iPSC maintenance medium and the cell number was counted using a hemocytometer. Three independent runs with 282, 260, and 256 spheroids for the 1 μM RI group and 273, 298, and 286 spheroids for the 10 μM RI group were conducted.
To test the pluripotency of the cells in the 3D iPSC spheroids in vivo with the teratoma assay, the eiPSC spheroids were directly injected subcutaneously (s.c.) into the dorsal rear flank of severe combined immunodeficient mice (NOD.CB17-scid). A total of 2×106 cells in 300 μL of PBS was injected into each mouse (age: 5 weeks, and 5 mice per group). After 5 weeks, the mice were sacrificed, and the resulting teratoma were collected and fixed in 4% paraformaldehyde (PFA) for 3 days. Afterward, the samples were cut into small pieces of ˜0.5 cm3, embedded in paraffin, and sectioned into slices of 5 μm thick. The slices were stained with hematoxylin and eosin (H&E) and imaged with a Zeiss LSM 710 microscope. All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC, #R-MAY-18-24) at the University of Maryland, College Park.
The eiPSC spheroids obtained after 4 days of culture on day 0 were induced into mesoderm by up-regulation of the Wnt signaling pathway using 8 μM CHIR99021 and 2 μM GSK inhibitor 6-bromoindirubin-3′-oxime (BIO) in the mesoderm induction medium for 1 day. The mesoderm induction medium was a mixture of DMEM/F12 and α-MEM (v/v, 1:1), containing 2% Knockout Serum Replacement (KOSR), 1 mM L-glutamine, 1% MEM non-essential amino acids (NEAA), and 0.1 mM β-mercaptoethanol. After 1 day, the spheroids were induced for cardiac commitment by down-regulating the Wnt signaling pathway using 10 μM KY02111 and 10 μM XAV939 in the cardiac maintenance medium for 6 days with the medium being changed every other day. The cardiac maintenance medium was a mixture of RPMI1640 and α-MEM (v/v, 1:1) containing 5% fetal bovine serum (FBS). Starting from day 8, the cardiac maintenance medium without KY02111 and XAV939 was used, and it was changed every other day for cardiac maturation. Images and videos of the resultant cardiac spheroids were taken using the Zeiss LSM 710 microscope before medium change.
The iPSC spheroids and the iPSC-derived Cardiac spheroids were fixed in 4% PFA in 1×PBS at 4° C. overnight. The fixed spheroids were incubated sequentially in 10% and 15% sucrose solutions in saline for 4 h, respectively. Afterward, the spheroids were put in a plastic box and embedded in OCT for cryosectioning. Slices of the spheroids of 10 μm thick were obtained by cutting the frozen sample on a Leica cryostat platform and then immediately attached onto the Leica Apex high adhesive glass slides.
For immunostaining, the slides were gently rinsed twice with 1×PBS to remove the OCT and then incubated with 0.1% Tween-20 and 5% normal goat serum in 1×PBS for 1 h at room temperature (RT) to block non-specific bindings. Later, the samples were incubated with primary antibodies at 4° C. overnight. The dilution and product information of the primary antibodies were as follows: OCT-4 (1:500 dilution, Cell Signaling Technologies), NANOG (1:500 dilution, Cell Signaling Technologies), SSEA-4 (1:500 dilution, Cell Signaling Technologies), cTnT (1:500 dilution, Cell Signaling Technologies), CONNEXIN-43 (CX-43, 1:400 dilution, Santa Cruz Biotechnology), DESMIN (1:500 dilution, Santa Cruz Biotechnology), α-ACTININ (1:500 dilution; Santa Cruz Biotechnology), BRACHYURY (1:500; Santa Cruz Biotechnology), NKX2.5 (1:500; Santa Cruz Biotechnology), NESTIN (1:500; R & D Systems), MUSASHI-1 (1:500; R & D Systems), and TUJ-1 (1:500; R & D Systems). Afterward, the samples were rinsed with 1×PBS thrice and then incubated with the associated secondary antibodies (goat anti-rabbit IgG FITC and goat-anti-mouse IgG PE, Invitrogen) in 1×PBS for 1.5 h at RT. Lastly, the samples were rinsed with 1 ml of 1×PBS for 3 min and the nuclei were stained with 1 μg/ml DAPI solution for 5 min at RT. The samples were imaged with the Zeiss LSM 710 microscope.
For scanning electron microscopy (SEM), the eiPSC spheroids collected on day −3 were fixed by 4% PFA in 1×PBS at 4° C. overnight. Then, the spheroids were incubated in 15% sucrose solution in saline for 4 h. Afterward, the spheroids were suspended in 100% ethanol and loaded on the SEM sample carrier and dried at RT overnight. The samples were then sputter-coated with gold at 15 mA for 2 min using the Ted Pella Cressington-108 sputter coater. SEM images of the spheroids were obtained with a Hitachi SU-70 FEG scanning electron microscope.
For flow cytometry, the eiPSC spheroids were collected on day 0 and the cardiac spheroids were collected on days 1.5, 2.5, 12.5, 15. The spheroids were dissociated to single cells by 0.25% trypsin for 5 min at 37° C. and then fixed with 75% ethanol at 4° C. overnight. The cells were permeabilized with 0.05% Triton X-100 for 3 min and then rinsed with 1×PBS twice. The cell numbers were adjusted to 1×106 cells/tube in 700 μL of saline for each marker. These cells were incubated with primary antibodies including OCT-4 (1:500 dilution, Cell Signaling Technologies), NANOG (1:500 dilution, Cell Signaling Technologies), SSEA-4 (1:500 dilution, Cell Signaling Technologies), BRACHYURY (1:500; Santa Cruz Biotechnology), NKX2.5 (1:500; Santa Cruz Biotechnology), cTnT (1:500 dilution, Cell Signaling Technologies), cTnI (1:500 dilution, Cell Signaling Technologies), α-ACTININ (1:500 dilution; Santa Cruz Biotechnology), MUSASHI-1 (1:500; R&D Systems), and TUJ-1 (1:500; R&D Systems) at 4° C. overnight. Subsequently, the samples were rinsed with 1×PBS thrice before incubation with corresponding secondary antibodies: goat anti-mouse IgG FITC and goat anti-rabbit IgG PE, respectively (1:1000; Invitrogen) for 1 h at RT. The samples were then rinsed with 1×PBS thrice before flow cytometry. The negative controls were the cells incubated with respective primary antibodies as aforementioned (no incubation with a secondary antibody).
For RNA sequencing (RNA-Seq), total RNA was extracted from the eiPSC clumps from 2D culture on day −4 (termed as 2D) and spheroids collected on day −2.5 after 1.5 days in the 3D culture (termed as UO and UT for 1 μM and 10 μM RI conditions, respectively), day 2.5 (termed as CO and CT), and day 13.5 (termed as MO and MT). The DNase I from bovine pancreas was used to remove DNA in samples. The RNA concentration in the samples was measured with Nanodrop and the integrity and quality of RNAs in the samples were confirmed with the Nano RNA Bioanalyzer. Samples with RNA integrity number greater than 9 were used for the preparation of the next-generation sequencing library using the NEB Ultra Directional RNA library preparation kit. Pair-end sequencing was performed by the Illumina HiSeq2500 platform. RNA-seq sequencing data quality was verified by Novogene. Raw reads were mapped to the human reference genome version (hg38) and the gene expression level based on reads per kilobase of exon per million reads mapped for annotated genes was measured and normalized using the Cufflink program. Samples were compared in different combinations and genes with an expression change of more than 1.5 in |log2 fold change| were defined as differentially expressed genes (DEG). Different classes of genes were subjected to functional and pathway analysis with the Gene Ontology (GO) database.
For transmission electron microscopy (TEM) studies, the cardiac spheroids collected on day 12.5 were fixed with 2.5% glutaraldehyde, 2% PFA, and 0.1 M PIPES buffer at pH 7.4 for 2 h at 4° C., rinsed with 1×PBS, and subsequently incubated with 1% osmium tetroxide for 2 h at 4° C. Afterward, the samples were dehydrated by a series of ethanol solutions (75%, 85%, 95%, and 100%) and acetone sequentially. Then, the samples were embedded in the resin EMbed 812 by following the manufacturer's instructions. Slices (70 nm-thick) of the embedded samples were cut with a Leica UC6 ultramicrotome and subsequently stained with 1% (w/v) uranyl acetate for 10 min at RT. The slices were examined and imaged with an FEI Tecnai T12 transmission electron microscope. The lengths of myofibrils and sarcomeres were measured with the NIH ImageJ (v1.52a).
To quantify the calcium spike, the cardiac spheroids collected on day 12.5 either without or with drug treatment were incubated with 2 nM Fluo-4 probes in cardiac maintenance medium for 30 min. Then, the medium was replaced with a fresh cardiac maintenance medium for 20 min. Afterward, the cardiac spheroids were transferred into a 1 cm diameter glass-bottom dish containing 500 μL of cardiac maintenance medium and incubated in the stage incubator of the Zeiss LSM 710 microscope at 37° C. and 5% CO2 for imaging and video recording. Cardiac drugs Isoproterenol (ISO) and propranolol (PRO) that increase and decrease the heart beating rate, respectively, were used to test the drug response of the cardiac spheroids. To do this, a cardiac maintenance medium containing 10 NM ISO was incubated with the cardiac spheroids for 10 min. After acquiring the calcium spike activity with the ISO treatment, the medium with ISO was removed and the cardiac spheroids were rinsed with fresh cardiac maintenance medium for 3 min. Then, the cardiac spheroids were incubated with a cardiac maintenance medium containing 10 μM PRO for 10 min and the calcium spike activity was recorded. All the video-recording was done at 5 frames per second for 1 min using the Zeiss LSM 710 microscope and analyzed with the Zeiss Zen Blue software. Calcium spike activities were collected from three independent experiments to quantify the beating frequency/rate of the cardiac spheroids. The data of the calcium spikes of the cardiac spheroids were presented as ΔF/F0, where F represents fluorescence intensity, F0 is the fluorescence intensity at the resting state of the cardiac spheroids, and ΔF (=F−F0) is the change of fluorescence intensity.
To synthesize gelatin methacryloyl (GelMA), type A porcine skin gelatin (300 bloom) was dissolved at 10% (w/v) into 1×PBS at 50° C. for 20 min. Methacrylic anhydride (MA) was added dropwise into the gelatin solution under vigorous stirring for 1 h (0.6 g of MA per gram of gelatin). The mixture was diluted with 1×PBS to stop the reaction and centrifuged at 2000 g for 2 min. To remove excess acid, the supernatant containing dissolved GelMA was collected and dialyzed (10 kDa molecular weight cutoff) against water. The dialyzed GelMA was then frozen, lyophilized, and stored at −80° C. To make the GelMA solution suspended with cardiac spheroids, the lyophilized GelMA was dissolved at 7% (w/v) in the cardiac maintenance medium at 50° C. for 20 mins. Irgacure 2959 (0.1% (w/v)) was added into the GelMA solution at 50° C. and stirred for 15 min. The resultant GelMA solution was slowly cooled to 37° C. before mixing it with the cardiac spheroids collected on day 5 post-cardiac differentiation (before beating). The GelMA solution suspended with cardiac spheroids was transferred into a well of 12-well plate at 4×106 cells in 0.5 ml of the GelMA solution. The GelMA solution was crosslinked into GelMA hydrogel by exposing it to ultraviolet light at 5 mW cm−2 for 1 min. Lastly, 1 ml of the cardiac maintenance medium was added into the well and the medium was changed every other day.
All quantitative data were collected from at least three independent experiments. The data were presented as mean±standard deviation. Student's t-test (two-tails, unpaired, and assuming equal variance) was performed for comparisons between two groups. A p value less than 0.05 was considered to be statistically significant.
This application claims priority to provisional application U.S. Ser. No. 63/224,283, filed Jul. 21, 2021, which hereby is incorporated herein by reference in its entirety.
This invention was made with government support under R01EB023632 awarded by the National Institutes for Health and CBET1831019 awarded by the National Science Foundation. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63224283 | Jul 2021 | US |
