Methods of making cardiomyocytes

Abstract

This disclosure describes methods and materials for producing cardiomyocytes from pluripotent stem cells.

Description

[0001] F&R RefNo.: 48514-0013W01

[0002] METHODS OF MAKING CARDIOMYOCYTES

[0003] CLAIM OF PRIORITY

[0004] This application claims priority to U.S. Provisional Patent Application No. 63 / 716,480 filed on November 5, 2024, the entire contents of which are hereby incorporated by reference.

[0005] TECHNICAL FIELD

[0006] This disclosure generally relates to methods of making and using cardiomyocytes.

[0007] BACKGROUND

[0008] Cardiomyocytes derived from pluripotent stem cells (PSCs) have the potential to be used in cell-based therapies for cardiac repair, however, improved methods of reproducibly generating large numbers of cardiomyocytes are necessary before any type of cell therapy can be brought to the clinic. This disclosure provides such methods.

[0009] SUMMARY

[0010] In one aspect, methods of differentiating pluripotent stem cells (PSCs) into cardiomyocytes are provided. Such methods typically include the steps of: providing PSCs in a low shear strength 3D bioreactor; introducing about 0.5 ng / ml to about 2 ng / ml of an activator of BMP4 receptor and about 25 nM to about 100 nM of a ROCK1 / 2 inhibitor to reduce aggregation of cells into the bioreactor for a first period of time; introducing about 2 ng / ml to about 10 ng / ml of an activator of BMP4 receptor, about 1 ng / ml to about 8 ng / ml of an activator of the activin signaling pathway, about 2 ng / ml to about 5 ng / ml of a fibroblast growth factor, and about 0 to about 3 pM of a Wnt pathway agonist into the bioreactor for a second period of time; introducing about 5 ng / ml to about 15 ng / ml of vascular endothelial growth factor (VEGF) and about 1 pM to about 5 pM of a Wnt signaling antagonist into the bioreactor for a third period of time; introducing about 2.5 ng / ml to about 10 ng / ml of VEGF into the bioreactor for a fourth period of time; thereby differentiating the PSCs into cardiomyocytes. F&R RefNo.: 48514-0013W01

[0011] In some embodiments, the low shear strength 3D bioreactor is a vertical wheel bioreactor (VWBR). In some embodiments, about 60 million to about 100 million PSCs are provided to the bioreactor per 100 ml and about 400 million to about 500 million PSCs are provided to the bioreactor per 500 ml.

[0012] In some embodiments, the activator of BMP4 receptor is BMP4. In some embodiments, the ROCK1 / 2 inhibitor is Chroman-1. In some embodiments, the activator of the Nodal / activin signaling pathway is Activin A. In some embodiments, the fibroblast growth factor is basic FGF. In some embodiments, the Wnt pathway agonist is CHIR99021 . In some embodiments, the Wnt signaling antagonist is IWP2.

[0013] In some embodiments, the first period of time is about one day. In some embodiments, the second period of time is about two days. In some embodiments, the third period of time is about three days. In some embodiments, the fourth period of time is at least about 2 weeks.

[0014] In some embodiments, greater than about 3 x 10A9 cardiomyocytes are produced.

[0015] In some embodiments, the methods described herein further include expanding the PSCs in the low shear strength 3D bioreactor prior to the method of differentiating. In some embodiments, the PSCs are expanded from about 40 million PSCs to about 500 million PSCs per 500 ml.

[0016] In another aspect, methods of producing left ventricular cardiomyocytes are provided. Such methods typically include providing cardiomyocytes in a low shear strength 3D bioreactor; and introducing a suitable amount of BMP4 and Activin A under conditions that result in the production of left ventricular cardiomyocytes. In some embodiments, the suitable amount of BMP4 and Activin A is about 5 ng / ml to about 10 ng / ml BMP4 and about 3 ng / ml to about 10 ng / ml of Activin A.

[0017] In still another aspect, methods of producing atrial cardiomyocytes are provided. Such methods typically include providing cardiomyocytes in a low shear strength 3D bioreactor; and introducing a suitable amount of BMP4, Activin A, and all-trans-retinoic acid under conditions that result in the production of atrial cardiomyocytes. In some embodiments, the suitable amount of BMP4, Activin A, and all-trans-retinoic acid is about 2 ng / ml to about 5 ng / ml of BMP4, about 1 ng / ml to about 3 ng / ml of Activin A, and about 250 nM to about 750 nM of all-trans-retinoic acid. F&R RefNo.: 48514-0013W01

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods and compositions of matter belong. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the methods and compositions of matter, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0019] DESCRIPTION OF DRAWINGS

[0020] FIG. 1 shows an overview of one embodiment of the methods described herein.

[0021] FIGs. 2A - 2G show data in which the inoculation density was optimized for PSC expansion.

[0022] FIGs. 3 A - 3G show data in which the vertical wheel bioreactor (VWBR) agitation rate was optimized for PSC expansion.

[0023] FIGs. 4 A - 4E show the adaptation of the standard PSC-CM differentiation protocol to the VWBR. Differentiation of 3D expanded cells using this protocol produced sub-optimal PSC-CM yields.

[0024] FIGs. 5A - 5D show the modulation of Wnt signaling and aggregation improves PSC-CM yield by limiting the emergence of unwanted subpopulations.

[0025] FIGs. 6A - 6G show growth factor-based patterning of mesoderm and cardiomyocyte subtype in the VWBR enables left ventricular and atrial cell production.

[0026] FIGs. 7A - 7G show the expansion and differentiation of PSCs in the 500 ml VWBR.

[0027] DETAILED DESCRIPTION

[0028] While several groups have reported the large-scale generation of hPSC-derived cardiomyocytes (hPSC-CMs) in bioreactors using small molecules, no comparable bioreactor-based protocol has been described using growth factors. This is despite evidence that differentiation with optimized concentrations of growth factors (e.g., BMP4, Activin A, and bFGF) offers greater control of ultimate cardiomyocyte phenotype, including patterning to become specialized cardiac subtypes (e.g., atrial F&R RefNo.: 48514-0013W01 versus mature left ventricular cardiomyocytes). See, e.g., Dark et al., 2023, Cell Reports Methods, 3(4): 100456. To overcome this gap, we established a system for expanding and differentiating hPSCs into cardiomyocytes in suspension using a low shear strength 3D bioreactor. Examples of low shear strength 3D bioreactors include, without limitation, a vertical wheel bioreactor (VWBR) or a wave (bag) bioreactor. Using this protocol, we also show that BMP4 and Activin A concentrations can be modified in the VWBR to control the cardiomyocyte subtype, enabling both ventricular and atrial cardiomyocyte production. Together, this protocol allows large- scale, economic production of cardiomyocytes from hPSCs.

[0029] Described herein is a protocol for growth factor-based large-scale manufacturing of hPSC-CMs in a low shear strength 3D bioreactor system. This first involves the expansion of hPSCs in an undifferentiated state using commercial media such as mTesRl or StemScale. Ideal growth curves and aggregate sizes were determined by optimizing culture inoculation density and agitation rate. Next, aggregates were dissociated into single cells and subjected to our cardiomyocyte differentiation protocol in the VWBR system. Cells were aggregated in StemPro-34 media with media components present to promote differentiation and cell aggregation. Following aggregation, cells were subjected to our guided cardiac differentiation protocol optimized to limit non-cardiac forming cell contamination while maximizing cardiomyocyte cell yield. To establish this cardiac differentiation protocol, we optimized parameters including initial hPSC seeding density, aggregate size, agitation speeds (e.g., rpm), growth factor concentrations, and timing of hPSC harvesting. Additional minor modifications were required when we transitioned between different bioreactor vessel sizes.

[0030] Undifferentiated hPSC expansion requires about 3-4 days for the non- bioreactor-based methods (i.e., much smaller volumes) and about 5 days for the current bioreactor-based method. The protocol length is very similar between our nonbioreactor and bioreactor methods for hPSC-CM differentiation (e.g., about 20 days). The labor required each day, however, is substantially less for the bioreactor-based method compared to the non-bioreactor, again approximately one order of magnitude less hands-on time.

[0031] Our lab has successfully produced 500 million - 1 billion hPSC-CMs using “conventional” static culture (non-bioreactor) methods, but this approach is very F&R RefNo.: 48514-0013W01 technically challenging, is inconsistent, and is impractical for most academic and industrial applications. The amount of labor required to complete this production by this approach is substantial, and it is an open system and much more prone to contamination. The protocol described here is done in a closed system and involves significantly reduced labor requirements; one person with the same time and incubator space should be able to produce 2-3 billion hPSC-CMs.

[0032] The protocol described herein is the first growth-factor-based, bioreactor-based differentiation protocol that employs optimized concentrations of BMP4, Activin A, bFGF, and CHIR99021 (CHIR) to produce a cardiac mesoderm and IWP2 (Wnt inhibitor) to induce cardiogenesis. In contrast, other bioreactor-based protocols solely use CHIR99021 and a Wnt inhibitor to produce cardiomyocytes, which may impact the ability to pattern towards the intended mature left ventricular cardiomyocyte phenotype. hPSC-CM purity is often measured by the percentage of cells expressing the cardiomyocyte protein cardiac troponin T (cTnT). Based on purity and yield alone, our protocol has at least comparable results to CHIR-based protocols. For example, using the protocol described herein, hPSC-CM purity reached about 93%, based on cTnT, with an average yield of 1.24 x 10A6 cells / mL in a 500 mb vessel.

[0033] The benefits of the protocol described herein are numerous. For example, our protocol can be used for PSC expansion and PSC-CM differentiation, and modulation of growth factor concentrations enables production of cardiomyocyte subtypes in the same bioreactor. In addition, we demonstrated that improved cell aggregation and Wnt activation increases PSC-CM yields in the 3D bioreactor and that, despite accelerated kinetics, earlier induction does not improve cell yields and reduces cardiomyocyte purity. The protocol described herein provided comparable yields and high MLC2v potential compared to previously published protocols (see, for example, Kriedemann et al., 2024, Nat. Protoc., 19(7): 1911-39; Burridge et al., 2014, Nat. Methods, 11(8):855- 60), and can be scaled to produce about 600 million cardiomyocytes per 500 ml volume run. This yield can be scaled to even higher numbers simply by increasing the incubator capacity.

[0034] Differentiated hPSC-CM production using non-bioreactor and bioreactor-based methods would be expected to have similar costs when the same media compositions are used. However, if we compare the methods described herein to previously employed differentiation protocol that utilized undiluted Stem-Pro-34 and RPMI-B27 F&R RefNo.: 48514-0013W01 media, the current bioreactor-based method would be considerably less expensive. Moreover, even at comparable prices, the current bioreactor method has a substantially lower labor requirement at larger cell numbers when compared to an equivalent conventional non-bioreactor method.

[0035] Simply by way of example, consumable costs to produce 500 million hPSC- CMs using a conventional suspension method involving 15 cm plates are estimated to cost approximately $4500, whereas consumable costs to produce 500 million hPSC- CMs in a single 500 ml VWBR are estimated to cost approximately $3500. Similarly, consumable expenses to produce approximately 500 million undifferentiated hPSCs cultured using conventional 2D methods (e.g., in T175 flasks) is estimated to cost approximately $2067 CAD, whereas using the VWBR protocol described herein is estimated to cost approximately $1526 CAD. In addition, labor requirements (e.g., hrs per hPSC) are reduced by approximately an order of magnitude using the VWBR protocol described herein. Significantly, the VWBR expansion methods described herein are predicted to cost 25% less than comparable 2D culture methods.

[0036] Specifically, methods of differentiating pluripotent stem cells (PSCs) into cardiomyocytes are described herein. Briefly, PSCs are provided that have been cultured in a low shear-strength 3D bioreactor. A low shear-strength 3D bioreactor refers to, without limitation, a vertical wheel bioreactor (VWBR). For example, about 60 million to about 100 million (e.g., about 75-80 million) PSCs can be provided to a 100 ml bioreactor or about 400 million to about 500 million (e.g., about 450 million) PSCs can be provided to a 500 ml bioreactor.

[0037] About 0.5 ng / ml to about 2 ng / ml (e.g., about 0.75 ng / ml to about 1.5 ng / ml) of an activator of BMP4 receptor (e.g., BMP4) and about 25 nM to about 100 nM (e.g., about 50 nM to about 75 mN) of a ROCK1 / 2 inhibitor (e.g., Chroman-1) are introduced into the bioreactor for a first period of time to reduce aggregation of cells. A first period of time can refer to, without limitation, about one day (e.g., about 18 hours, about 24 hours, about 30 hours).

[0038] After the first period of time has elapsed, about 2 ng / ml to about 10 ng / ml (e.g., about 4 ng / ml to about 8 ng / ml) of an activator of BMP4 receptor (e.g., BMP4), about 1 ng / ml to about 8 ng / ml (e.g., about 2 ng / ml to about 6 ng / ml; about 4 nm / ml to about 5 ng / ml) of an activator of the activin signaling pathway (e.g., Activin A), about 2 ng / ml to about 5 ng / ml (e.g., about 3 ng / ml to about 4 ng / ml) of a fibroblast growth F&R RefNo.: 48514-0013W01 factor (e.g., basic FGF (bFGF)), and about 0 to about 3 pM (e.g., about 1 pM to about 2 pM) of a Wnt pathway agonist (e.g., CHIR99021) are introduced into the bioreactor for a second period of time. A second period of time can refer to, without limitation, about two days (e.g., about 36 hours, about 48 hours, about 60 hours).

[0039] After the second period of time has elapsed, about 5 ng / ml to about 15 ng / ml (e.g., about 7.5 ng / ml to about 12.5 ng / ml; about 10 ng / ml) of vascular endothelial growth factor (VEGF) and about 1 pM to about 5 pM (e.g., about 2 pM to about 4 pM) of a Wnt signaling antagonist (e.g., IWP2) are introduced into the bioreactor for a third period of time. A third period of time refers to, without limitation, about three days (e.g., about 48 hours, about 72 hours, about 96 hours).

[0040] After the third period of time has elapsed, about 2.5 ng / ml to about 10 ng / ml (e.g., about 4 ng / ml to about 7.5 ng / ml; about 6 ng / ml) of VEGF are introduced into the bioreactor for a fourth period of time. A fourth period of time refers to, without limitation, at least about 2 weeks (e.g., at least about 3 weeks, at least about 4 weeks). After the fourth period of time has elapsed, cardiomyocytes are present in significant numbers (e.g., greater than about 3 x 10A9 cardiomyocytes).

[0041] Methods of producing left ventricular cardiomyocytes also are described herein. Briefly, cardiomyocytes are provided that have been cultured in a low shearstrength 3D bioreactor (e.g., a vertical wheel bioreactor (VWBR)). A suitable amount (e.g., about 5 ng / ml to about 10 ng / ml; about 7.5 ng / ml) of BMP4 and a suitable amount (e.g., about 3 ng / ml to about 10 ng / ml; about 6 ng / ml) of Activin A are introduced under conditions that result in the production of left ventricular cardiomyocytes.

[0042] Methods of producing atrial cardiomyocytes also are described herein. Briefly, cardiomyocytes are provided that have been cultured in a low shear-strength 3D bioreactor (e.g., a vertical wheel bioreactor (VWBR)). A suitable amount (e.g., about 2 ng / ml to about 5 ng / ml; about 3.5 ng / ml) of BMP4, a suitable amount (e.g., about 1 ng / ml to about 3 ng / ml; about 2 ng / ml) of Activin A, and a suitable amount (e.g., about 250 nM to about 750 nM; about 500 nM) of all-trans-retinoic acid are introduced under conditions that result in the production of atrial cardiomyocytes.

[0043] In some instances, it may be desirable to expand the PSCs in the low shear strength 3D bioreactor prior to differentiating. Simply by way of example, PSCs can F&R RefNo.: 48514-0013W01 be expanded from about 40 million PSCs to about 500 million PSCs in a 500 ml bioreactor.

[0044] In some instances, it may be desirable to measure cardiac troponin T (cTnT) amounts produced by the cells as an indicator of the presence of cardiomyocytes. Methods of measuring cTnT are known in the art and suitable for use herein.

[0045] Embodiments

[0046] Embodiment 1 is a method of differentiating pluripotent stem cells (PSCs) into cardiomyocytes, comprising the steps of: providing PSCs in a low shear strength 3D bioreactor; introducing about 0.5 ng / ml to about 2 ng / ml of an activator of BMP4 receptor and about 25 nM to about 100 nM of a ROCK1 / 2 inhibitor to reduce aggregation of cells into the bioreactor for a first period of time; introducing about 2 ng / ml to about 10 ng / ml of an activator of BMP4 receptor, about 1 ng / ml to about 8 ng / ml of an activator of the activin signaling pathway, about 2 ng / ml to about 5 ng / ml of a fibroblast growth factor, and about 0 to about 3 pM of a Wnt pathway agonist into the bioreactor for a second period of time; introducing about 5 ng / ml to about 15 ng / ml of vascular endothelial growth factor (VEGF) and about 1 pM to about 5 pM of a Wnt signaling antagonist into the bioreactor for a third period of time; introducing about 2.5 ng / ml to about 10 ng / ml of VEGF into the bioreactor for a fourth period of time; thereby differentiating the PSCs into cardiomyocytes.

[0047] Embodiment 2 is the method of Embodiment 1, wherein about 60 million to about 100 million PSCs are provided to the bioreactor per 100 ml and wherein about 400 million to about 500 million PSCs are provided to the bioreactor per 500 ml.

[0048] Embodiment 3 is the method of any of the preceding Embodiments, wherein the low shear strength 3D bioreactor is a vertical wheel bioreactor (VWBR).

[0049] Embodiment 4 is the method of any of the preceding Embodiments, wherein the activator of BMP4 receptor is BMP4.

[0050] Embodiment 5 is the method of any of the preceding Embodiments, wherein the ROCK1 / 2 inhibitor is Chroman-1.

[0051] Embodiment 6 is the method of any of the preceding Embodiments, wherein the activator of the Nodal / activin signaling pathway is Activin A.

[0052] Embodiment 7 is the method of any of the preceding Embodiments, wherein the fibroblast growth factor is basic FGF (bFGF). F&R RefNo.: 48514-0013W01

[0053] Embodiment 8 is the method of any of the preceding Embodiments, wherein the Wnt pathway agonist is CHIR99021.

[0054] Embodiment 9 is the method of any of the preceding Embodiments, wherein the Wnt signaling antagonist is IWP2.

[0055] Embodiment 10 is the method of any of the preceding Embodiments, wherein the first period of time is about one day.

[0056] Embodiment 11 is the method of any of the preceding Embodiments, wherein the second period of time is about two days.

[0057] Embodiment 12 is the method of any of the preceding Embodiments, wherein the third period of time is about three days.

[0058] Embodiment 13 is the method of any of the preceding Embodiments, wherein the fourth period of time is at least about 2 weeks.

[0059] Embodiment 14 is the method of any of the preceding Embodiments, wherein greater than about 3 x 10A9 cardiomyocytes are produced.

[0060] Embodiment 15 is the method of any of the preceding Embodiments, further comprising expanding the PSCs in the low shear strength 3D bioreactor prior to the method of differentiating.

[0061] Embodiment 16 is the method of any of the preceding Embodiments, wherein the PSCs are expanded from about 40 million PSCs to about 500 million PSCs per 500 ml.

[0062] Embodiment 17 is a method of producing left ventricular cardiomyocytes, comprising: providing cardiomyocytes in a low shear strength 3D bioreactor; and introducing a suitable amount of BMP4 and Activin A under conditions that result in the production of left ventricular cardiomyocytes.

[0063] Embodiment 18 is the method of Embodiment 17, wherein the suitable amount of BMP4 and Activin A is about 5 ng / ml to about 10 ng / ml BMP4 and about 3 ng / ml to about 10 ng / ml of Activin A.

[0064] Embodiment 19 is a method of producing atrial cardiomyocytes, comprising: providing cardiomyocytes in a low shear strength 3D bioreactor; and introducing a suitable amount of BMP4, Activin A, and all-trans-retinoic acid under conditions that result in the production of atrial cardiomyocytes.

[0065] Embodiment 20 is the method of Embodiment 19, wherein the suitable amount of BMP4, Activin A, and all-trans-retinoic acid is about 2 ng / ml to about 5 ng / ml of F&R RefNo.: 48514-0013W01

[0066] BMP4, about 1 ng / ml to about 3 ng / ml of Activin A, and about 250 nM to about 750 nM of all-trans-retinoic acid.

[0067] In accordance with the present invention, there may be employed molecular biology, microbiology, biochemical, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. The invention will be further described in the following examples, which do not limit the scope of the methods and compositions of matter described in the claims.

[0068] EXAMPLES

[0069] Example 1 — Methods and Compositions for Producing Cardiomyocytes

[0070] The schematic in FIG. 1 provides an overview of the protocol for hPSC expansion and cardiac differentiation in the vertical -wheel bioreactor (VWBR) system. Undifferentiated human pluripotent stem cells (hPSC) were first expanded in the VWBR system. Optimal inoculation density (FIGs. 2A-2G) and culture agitation rate (FIGs. 3A-3G) varied slightly from cell line to cell line but was optimized as shown in each data set. FIG. 4A shows the adaptation of the standard differentiation protocol to the VWBR using cells grown in the conventional 2D method and the current 3D method. While cells grown in 2D or 3D at 30 rpm had a greater than 1 hPSC : IhPSC- CM yield, cells grown in 3D at 40 rpm or 60 rpm had a lower yield (FIGs. 4B-4E), demonstrating the benefits of optimizing differentiation of the 3D-grown cells in the VWBR system.

[0071] Examination of 3D-grown cells at day 3 post-differentiation revealed increased emergence of unwanted subpopulations (ckit+ / Cxr4+), and modification of Wnt signaling through the addition of CHIR99021 on day 1 and aggregation through the addition of chroman-1 on day 0 reduced the unwanted subpopulations (FIGs. 5A-5D). These steps significantly improved hPSC-CM yield without significantly affecting cardiomyocyte purity.

[0072] Using this technology, we also demonstrated that BMP4 and Activin A concentrations can be modified in the VWBR system to enable the production of atrial-like cardiomyocytes (FIGs. 6A-6B). While 10 ng / mL BMP4 and 6 ng / mL Activin A produced a mesoderm that enabled left ventricular cardiomyocyte production, 4 ng / mL BMP4 and 2 ng / mL Activin A enabled the production of atrial F&R RefNo.: 48514-0013W01 cardiomyocytes, which was confirmed by flow cytometry and analysis of gene expression at day 20 (FIGs. 6C-6G). For atrial cell production, 500 nM all-trans- retinoic acid also was added from day 3-5 of differentiation.

[0073] Example 2 — Scaling the Methods and Compositions for Producing Cardiomyocytes The protocol described herein was adapted to the 500 m VWBRto increase the cardiomyocyte production capacity. In brief, 40 million hPSCs were expanded in the 500 m VWBR in mTeSRl medium as per the media exchange schedule shown in FIG. 7A. On day 5 of hPSC expansion, aggregates were collected and enzymatically dissociated into single cells using Accutase. Following dissociation, cells were counted and inoculated into a 500 mb VWBR vessel at IxlO6cells / mb in Stem Pro-34 media supplemented with E-glutamine (2 mM), E-ascorbic acid (50 pg / mb), monothioglycerol (MTG) 0.004%, transferrin (150 pg / mb). bone morphogenetic protein-4 (rhBMP4, 1 ng / mb) and 50 nM Chroman-1. Aggregation speed was determined empirically to achieve an average aggregate size of 80-100 pm on day 1. See FIGs. 7B-7D.

[0074] On day 1, media was exchanged, and cells were resuspended in Stem Pro-34 media supplemented with the supplements above (excluding chroman-1), as well as 1 pM CHIR99021, BMP4, activin A, and basic fibroblast growth factor (bFGF, 5 ng / mb). BMP4 and activin A concentrations were titrated for optimal concentrations. On day 3, cells were completely removed from the VWBR vessel, and aggregates were centrifuged at 400 rpm for 3 minutes. Aggregates and culture vessels were washed once with Iscove's Modified Dulbecco's Medium (IMDM) media after which aggregates were cultured in the “cardiomyocyte specification media” that includes our base StemPro-34 media (including E-glutamine, ascorbic acid, MTG and transferrin) supplemented with the Wnt inhibitor IWP2 (2 pM) and vascular endothelial growth factor (VEGF, 10 ng / mb) for an additional 3 days.

[0075] From day 6 to 12, cells were cultured in media consisting of 25% (v / v) StemPro-34 and 75% (v / v) IMDM supplemented with E-glutamine, ascorbic acid, MTG, and transferrin at the concentrations indicated above, as well as 5 ng / mb VEGF. At day 12, post-induction cells were maintained in 25% (v / v) StemPro-34 and 75% (v / v) IMDM supplemented with E-glutamine, ascorbic acid, and MTG as listed above. At day 20, hPSC-CM aggregates were harvested by enzymatic digestion with 300 F&R RefNo.: 48514-0013W01

[0076] U / mL collagenase II (Worthington) in Hanks Balanced Salt Solution (Wisent) overnight at room temperature. The following day, cells were washed using Stem Pro / IMDM media with DNase (10 U / mL) and filtered through a 37 pm reversible strainer. The remaining aggregates were further dissociated using TrypLE for 3-5 minutes at 37°C and washed with base Stem Pro / IMDM media. Single cells were counted and cryopreserved by freezing cells in Cryostore CS10 freezing media using a controlled rate freezer at l°C / min.

[0077] FIGs. 7E-7F show the flow cytometry and analysis of gene expression of cells grown in the conventional 2D VWBR method and the current 3D VWBR method with or without Wnt signaling modification (via the addition of CHIR99021). While cells grown in 2D or 3D without Wnt signaling modification resulted in a comparable yield, cells grown in the current 3D VWBR method with Wnt signaling modification resulted in a significantly increased yield (FIG. 7G), almost two-fold as much as the yield resulting from the conventional 2D VWBR method and the current 3D VWBR method without Wnt signaling modification.

[0078] It is to be understood that, while the methods and compositions of matter have been described herein in conjunction with a number of different aspects, the foregoing description of the various aspects is intended to illustrate and not limit the scope of the methods and compositions of matter. Other aspects, advantages, and modifications are within the scope of the following claims.

[0079] Disclosed are methods and compositions that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that combinations, subsets, interactions, groups, etc. of these methods and compositions are disclosed. That is, while specific reference to each various individual and collective combinations and permutations of these compositions and methods may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular composition of matter or a particular method is disclosed and discussed and a number of compositions or methods are discussed, each and every combination and permutation of the compositions and the methods are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed.

Claims

F&R RefNo.: 48514-0013W01WHAT IS CLAIMED IS:

1. A method of differentiating pluripotent stem cells (PSCs) into cardiomyocytes, comprising the steps of: providing PSCs in a low shear strength 3D bioreactor; introducing about 0.5 ng / ml to about 2 ng / ml of an activator of BMP4 receptor and about 25 nM to about 100 nM of a ROCK1 / 2 inhibitor to reduce aggregation of cells into the bioreactor for a first period of time; introducing about 2 ng / ml to about 10 ng / ml of an activator of BMP4 receptor, about 1 ng / ml to about 8 ng / ml of an activator of the activin signaling pathway, about 2 ng / ml to about 5 ng / ml of a fibroblast growth factor, and about 0 to about 3 pM of a Wnt pathway agonist into the bio reactor for a second period of time; introducing about 5 ng / ml to about 15 ng / ml of vascular endothelial growth factor (VEGF) and about 1 pM to about 5 pM of a Wnt signaling antagonist into the bioreactor for a third period of time; introducing about 2.5 ng / ml to about 10 ng / ml of VEGF into the bioreactor for a fourth period of time; thereby differentiating the PSCs into cardiomyocytes.

2. The method of claim 1, wherein about 60 million to about 100 million PSCs are provided to the bioreactor per 100 ml and wherein about 400 million to about 500 million PSCs are provided to the bioreactor per 500 ml.

3. The method of claims 1, wherein the low shear strength 3D bioreactor is a vertical wheel bioreactor (VWBR).

4. The method of claim 1, wherein the activator of BMP4 receptor is BMP4.F&R RefNo.: 48514-0013W016. The method of claim 1, wherein the activator of the Nodal / activin signaling pathway is Activin A.

7. The method of claim 1, wherein the fibroblast growth factor is basic FGF (bFGF).

8. The method of claim 1, wherein the Wnt pathway agonist is CHIR99021.

9. The method of claim 1, wherein the Wnt signaling antagonist is IWP2.

10. The method of claim 1, wherein the first period of time is about one day.

11. The method of claim 1, wherein the second period of time is about two days.

12. The method of claim 1, wherein the third period of time is about three days.

13. The method of claim 1, wherein the fourth period of time is at least about 2 weeks.

14. The method of claim 1, wherein greater than about 3 x 10A9 cardiomyocytes are produced.

15. The method of claim 1, further comprising expanding the PSCs in the low shear strength 3D bioreactor prior to the method of differentiating.

16. The method of claim 15, wherein the PSCs are expanded from about 40 million PSCs to about 500 million PSCs per 500 ml.

17. A method of producing left ventricular cardiomyocytes, comprising:F&R RefNo.: 48514-0013W01 providing cardiomyocytes in a low shear strength 3D bioreactor; and introducing a suitable amount of BMP4 and Activin A under conditions that result in the production of left ventricular cardiomyocytes.

18. The method of claim 17, wherein the suitable amount of BMP4 andActivin A is about 5 ng / ml to about 10 ng / ml BMP4 and about 3 ng / ml to about 10 ng / ml of Activin A.

19. A method of producing atrial cardiomyocytes, comprising: providing cardiomyocytes in a low shear strength 3D bioreactor; and introducing a suitable amount of BMP4, Activin A, and all-trans- retinoic acid under conditions that result in the production of atrial cardiomyocytes.

20. The method of claim 19, wherein the suitable amount of BMP4, Activin A, and all-trans -retinoic acid is about 2 ng / ml to about 5 ng / ml of BMP4, about 1 ng / ml to about 3 ng / ml of Activin A, and about 250 nM to about 750 nM of all -transretinoic acid.

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