METHODS AND COMPOSITIONS FOR MAKING AND USING ENDOCARDIAL CELLS
20230265391 · 2023-08-24
Inventors
- Gordon M. Keller (Toronto, Ontario, CA)
- Alexander Mikryukov (Toronto, Ontario, CA)
- Amine Mazine (Toronto, Ontario, CA)
- Ian Fernandes (Toronto, Ontario, CA)
Cpc classification
A61K35/44
HUMAN NECESSITIES
C12N2501/165
CHEMISTRY; METALLURGY
A61P9/10
HUMAN NECESSITIES
C12N2501/115
CHEMISTRY; METALLURGY
A61K35/34
HUMAN NECESSITIES
C12N2501/155
CHEMISTRY; METALLURGY
International classification
Abstract
Methods and compositions for making endocardial cells from pluripotent stem cells are described, as are methods and compositions for using such cells.
Claims
1. A method of producing a population of endocardial cells, comprising: providing cardiovascular progenitor cells; and contacting the cardiovascular progenitor cells with FGF and BMP under appropriate culture conditions, thereby producing a population of endocardial cells, wherein the population of endocardial cells is phenotypically NKX2-5+ and CD31+.
2. The method of claim 1, wherein the cardiovascular progenitor cells are pluripotent stem cell-derived cardiovascular progenitor cells.
3. The method of claim 1, wherein the cardiovascular progenitor cells are cardiovascular mesoderm cells.
4. (canceled)
5. The method of claim 1, wherein the BMP is selected from BMP10 and BMP4.
6. (canceled)
7. The method of claim 1, wherein the appropriate culture conditions comprises the absence of VEGF or the absence of a Wnt inhibitor.
8. (canceled)
9. The method of claim 1, wherein the population of endocardial cells is phenotypically GATA4+, GATA5+, NFATC +, NPR3+ and NRG1+ or wherein the population of endocardial cells is phenotypically ENG(CD105)+.
10. (canceled)
11. Endocardial cells made by the method of claim 1.
12. The method of claim 1, further comprising: culturing PDGFRb+ cells from the population of endocardial cells in the presence of bFGF, BMP2 and TGFbeta2, thereby producing valvular interstitial-like cells (VICs), wherein VICs are phenotypically SOX9+, MSX2+, VIM+, VCAN+, COL1A1+, COL3A1+, POSTN+, CDH11+, NR4A2+, PRRX2+, TIMP3+, RGS5+ and ITGA2+.
13. Valvular interstitial-like cells (VICs) made by the method of claim 12.
14. A method of producing coronary endothelial-like cells, comprising: contacting the endocardial cells of claim 11 with a) VEGFA followed by b) VEGFB, thereby producing coronary endothelial-like cells.
15. The method of claim 1, further comprising: culturing the population of endocardial cells in the presence of ventricular cardiomyocytes, wherein the ventricular cardiomyoctyes are phenotypically MYL2(MLC2V)+ and CTNT+, thereby generating trabecular myocardial cells, wherein the trabecular myocardial cells are phenotypically BMP10+, NPPA+, NPPB+, IRX3+, GJA5+, MYL2+, and CTNT+.
16. The method of claim 15, wherein the culturing is in the presence of VEGFA.
17. Trabecular ventricular cardiomyocytes made by the method of claim 15, wherein the trabecular ventricular cardiomyocytes are phenotypically BMP10+, NPPA+, NPPB+, IRX3+, GJA5+, MYL2+, and CTNT+.
18. A method of replenishing coronary vasculature in myocardium, comprising: delivering the endocardial cells of claim 11 to heart tissue, thereby replenishing the coronary vasculature.
19. (canceled)
20. A method of improving cardiomyocyte grafts by replenishing the myocardium, comprising: delivering ventricular cardiomyocytes in conjunction with the endocardial cells of claim 11, thereby replenishing the myocardium.
21-23. (canceled)
24. A method of producing valvular interstitial-like cells, comprising: contacting the endocardial cells of claim 11 with BMP2/4 and TGFbeta2 under appropriate culture conditions, or contacting the endocardial cells of claim 11 with a) CHIR99021, SB-431542, bFGF and BMP2/4 followed by b) bFGF, BMP2/4 and TGFbeta2, thereby producing valvular interstitial-like cells.
25. The method of claim 24, wherein the valvular interstitial-like cells (VICs) are phenotypically SOX9+, MSX2+, VIM+, VCAN+, COL1A1+, COL3A1+, POSTN+, CDH11+, NR4A2+, PRRX2+, TIMP3+, RGS5+ and ITGA2+.
26. A method of producing trabecular ventricular cardiomyocytes, comprising: contacting ventricular cardiomyocytes with the endocardial cells of claim 11 or exogenous neuregulin (NRG1) under appropriate culture conditions, thereby producing trabecular ventricular cardiomyocytes.
27. (canceled)
28. A method of inducing the production of neuregulin in a cell, comprising: contacting the endocardial cells of claim 11 with ventricular cardiomyocytes under appropriate conditions.
29. A biological valve comprising the endocardial cells of claim 11.
30. A method of replenishing coronary vasculature in myocardium, comprising: delivering the coronary endothelial cells made by the method of claim 14 to heart thereby replenishing the coronary vasculature.
31. A method of improving cardiomyocyte grafts by replenishing the myocardium, comprising: delivering ventricular cardiomyocytes in conjunction with the coronary endothelial cells made by the method of claim 14, thereby replenishing the myocardium.
32. A biological valve comprising the valvular interstitial-like cells of claim 13.
Description
DESCRIPTION OF DRAWINGS
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DETAILED DESCRIPTION
[0041] The embryonic endocardium is essential for early heart development. The embryonic endocardium is, together with the myocardium, one of the first heart tissues to be formed. It is the source of the cells that make up the valves and a portion of the coronary vasculature, and it functions to induce trabecular myocardium. Given such potential, endocardial cells provide unique therapeutic opportunities that include engineering biological valves and cell-based therapy strategies to replace heart valves and coronary vasculature in damaged hearts. This disclosure describes methods of making a population of endocardial cells, as well as a number of methods for using such cells.
Endocardial Cells
[0042] To produce a population of endocardial cells, cardiovascular progenitor cells can be cultured as described herein to generate a population of cells that displays characteristics of endocardium including expression of a cohort of genes that identifies the endocardial lineage in vivo (e.g., NKX2-5, GATA4, GATA5,NFATC1, NPR3, NRG1 and ENG(CD105)). In addition, the population of endocardial cells produced using the methods described herein have the capacity to induce a trabecular fate in immature cardiomyocytes in vitro and, surprisingly, also have the potential to produce mesenchymal cells that express markers of valve interstitial cells (VICs).
[0043] As used herein, cardiovascular progenitor cells can refer to pluripotent stem cell (PSC)-derived cells, which, in turn, can refer to embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). In some instances, the cardiovascular progenitor cells are human cells.
[0044] This disclosure describes methods of producing endocardial cells from cardiovascular progenitor cells (or, e.g., from pluripotent stem cells if the presence of BMP4, bFGF and ActivinA agonists are included). Endocardial cells can be identified by the expression of NKX2-5 and CD31, and can be produced by contacting pluripotent stem cell-derived cardiovascular progenitor cells with an FGF agonist and a BMP agonist under appropriate culture conditions. For example, endocardial cells can be generated by first differentiating pluripotent stem cells into cardiovascular mesoderm using a bFGF agonist, a BMP4 agonist, and an ActivinA agonist. Cardiovascular mesoderm then can be cultured in the presence of a bFGF agonist to generate cardiovascular progenitor cells, which then can be cultured in the presence of a bFGF agonist and a BMP10 agonist to specify an NKX2-5+ CD31+ endocardial population. In addition to NKX2-5+ CD31+, the population of endocardial cells described herein also expresses GATA4, GATA5, NFATC1, NPR3, NRG1, and ENG(CD105).
[0045] Representative FGF agonists are FGF2 (also known as basic FGF or bFGF), FGF1, FGF4, FGF8, FGF9, FGF10, FGF16 and FGF20, and representative BMP agonists include a BMP10, BMP9, BMP4, or BMP2 polypeptide. In some instances, the culture conditions appropriate to produce endocardial cells includes the absence of, or low levels (0-30 ng/mL) of, a VEGFA agonist. In some instances, the culture conditions appropriate to produce endocardial cells includes the absence of a Wnt inhibitor.
[0046] As the endocardial cells mature, NKX2-5 can be downregulated, giving rise to a NKX2-5− CD31+ population. It would be understood that NKX2-5− CD31+ cells derived from the NKX2-5+ CD31+ population are endocardial cells, but it would be appreciated that NKX2-5− CD31+ cells not derived from the NKX2-5+ CD31+ population (e.g., cultured in the presence of a FGF agonist and in the absence of a BMP10 agonist; or cultured in the presence of a FGF agonist and a VEGFA agonist) are endothelial cells and not endocardial cells.
[0047] Endocardial cells, or a population of endocardial cells, made by the methods described herein can be maintained by culturing them in serum free-media (e.g., StemPro) supplemented with a VEGFA agonist and a BMP10 agonist. The endocardial cells described herein also can be used to produce neuregulin (i.e., from the NRG1 gene), which is a cytokine.
Valvular Interstitial-Like Cells (VICs)
[0048] Methods of making valvular interstitial-like cells (VICs) also are described herein. As described herein, the population of endocardial cells described herein includes valve progenitor cells having a PDGFRb+ phenotype. Such valve progenitor cells can be cultured in the presence of an FGF agonist, a BMP agonist, and a TGFbeta agonist. The resulting VICs have a phenotype of SOX9+, MSX2+, VIM+, VCAN+, COL1A1+, COL3A1+, POSTN+, CDH11+, NR4A2+, PRRX2+, TIMP3+, RGS5+ and ITGA2+.
[0049] When generating VICs, a representative FGF agonist is bFGF, representative BMP agonists include, without limitation, BMP2 or BMP4, and representative TGFbeta agonists include, for example, TGFbeta1, TGFbeta2 or TGFbeta3.
[0050] VICs produced as described herein can be used to model valvular heart disease and can be seeded on a biomimetic construct to generate a living tissue engineered heart valve for therapeutic applications.
Cells Having a Trabecular Cardiomyocyte Fate
[0051] Methods of producing cells having a trabecular cardiomyocyte fate also are described herein. As described herein, immature ventricular cardiomyocytes can be cultured with the population of endocardial cells described herein, or with neuregulin produced from such a population of endocardial cells, such that the immature ventricular cardiomyocytes adopt a trabecular fate. The immature ventricular cardiomyocytes that can be used in this method typically are CTNT+ and can be derived, for example, from ventricular mesoderm. A trabecular phenotype, or cells having a trabecular fate, generally exhibit an upregulation of BMP10, NPPA, NPPB, IRX3, GJA5 and MYL2, and also can exhibit a downregulation of HEY2.
[0052] To produce cells having a trabecular fate, the combination of the population of endocardial cells, or neuregulin produced from such cells, and the immature ventricular cardiomyocytes can be cultured in the presence of a VEGFA agonist for about 4 to 5 days, after which trabecular marker expression in the cardiomyocytes can be maintained using neuregulin. Representative VEGFA agonists include, without limitation, VEGFA.
[0053] Cells having a trabecular fate as described herein can be maintained by culturing them in media containing neuregulin or with endocardial cells producing neuregulin in the presence of VEGFA to maintain endocardial cell identity, and can be used to generate cardiac Purkinje fibers in vitro. Purkinje fibers are the end point to which electrical signal arrives from the heart pacemakers (sinoatrial node and AV-node) and stimulates ventricular contraction. Purkinje fibers have been implicated in both the maintenance and the initiation of arrhythmias in the heart ventricles, which makes them an important cell type for modelling disease and for drug screening. In addition, the presence of cells exhibiting a trabecular fate can be used as an indicator of endocardial function.
Biological Valves and Replenishing or Repairing Coronary Vasculature in Myocardium
[0054] A population of endocardial cells as described herein and/or the VICs described herein can be used in the construction of a biological valve, and such biological valves (e.g., made using the endocardial cells and/or the VICs described herein, or including cells differentiated from the endocardial cells and/or the VICs described herein) are provided. In addition, methods of replenishing or repairing coronary vasculature in myocardium (e.g., damaged myocardium) are provided, which include delivering the endocardial cells described herein to myocardium.
[0055] The endocardial cells described herein can be used to replenish coronary vasculature in myocardium. For example, the endocardial cells described herein and/or cells produced from such endocardial cells (e.g., coronary endothelial cells) can be delivered to heart tissue (e.g., via injection). Upon engraftment, the cells then can revascularize the myocardium. Such therapeutic applications of the cells described herein can be beneficial when the heart tissue is damaged.
[0056] Methods are being developed to transplant cardiomyocytes in order to remuscularize damaged myocardium. Current strategies are focused on transplanting cardiomyocytes alone, however, this approach is inefficient, in part, because many of the transplanted cardiomyocytes do not survive. However, the transplantation of coronary endothelial cells generated from the endocardial cells described herein together with cardiomyocytes can improve, in some cases significantly, the efficiency of engraftment (e.g., using fewer cells, with larger grafts). It would be appreciated that each of types of cells (i.e., the endocardial cells and the cardiomyocytes) can be delivered (e.g., transplanted) separately or together, and can be delivered using, for example, injection (e.g., co-injection).
Screening Methods
[0057] The endocardial cells described herein, the VICs described herein, or the cells having a trabecular fate described herein can be used to screen test compounds to identify those that exhibit toxicity. The endocardial cells described herein, the VICs described herein, or the cells having a trabecular fate described herein can be contacted with test compounds from classes such as proteins, small molecules, or nucleic acids, and the viability of the cells determined. Those compounds that reduce the viability of the cells can be deemed to have some toxicity against such cells.
[0058] Methods of determining the viability of cells are known in the art and include, for example, assays for apoptosis (e.g., Annexin V, TUNEL, or caspase) or for cell proliferation (e.g., methyl violet, neutral red uptake, trypan blue, BrdU).
[0059] The endocardial cells described herein can be used in vitro to study valve diseases. For example, endocardial cells can be produced using the methods described herein from patients having a valve disease (e.g., hypoplastic left heart syndrome (HLHS)) in order to study the etiology of the disease and/or the effects of one or more test compounds.
[0060] In accordance with the present invention, there may be employed conventional 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.
EXAMPLES
Example 1—Generation and Maintenance of Human ESC/iPSC Lines
[0061] The generation of the HES3-NKX2-5eGFP/w (karyotype: 46, XX) and HES2:RFP hESC (karyotype: 46, XX) reporter cell lines was described previously (Irion et al., 2007, Nat. Biotechnol., 25(12):1477-82; Elliott et al., 2011, Nat. Methods, 8(12):1037-40). The HES2:GFP hESC line was generated from the HES2:RFP hESC line by exchanging the tdRFP cassette with a EGFP expressing cassette. The hPSC lines were maintained on irradiated mouse embryonic fibroblasts in hPSC culture media consisting of DMEM/F12 (Cellgro), penicillin/streptomycin (1%, ThermoFisher), L-glutamine (2 mM, ThermoFisher), non-essential amino acids (1×, ThermoFisher), beta-mercaptoethanol (55 μM, ThermoFisher) and KnockOut™ serum replacement (20%, ThermoFisher) as described previously (Kennedy et al., 2007, Blood, 109(7):2679-87).
Example 2—Directed Differentiation of Human PSC Lines Into Cardiomyocytes
[0062] For cardiac differentiation, a previously described protocol was used for generating ventricular cardiomyocytes from mesoderm induced with 10 ng/ml BMP4 and 6 ng/ml activin A (Lee et al., 2017, Cell Stem Cell., 21(2):179-94 e174). To initiate differentiation, hPSCs at 80%-90% confluence were dissociated into single cells (TrypLE, ThermoFisher) and re-aggregated to form EBs at a cell density of 5×10e5 cells/ml in StemPro-34 media (ThermoFisher) supplemented with penicillin/streptomycin (1%, ThermoFisher), L-glutamine (2 mM, ThermoFisher), ascorbic acid (50 μg/ml, Sigma), monothioglycerol (50 μg/ml, Sigma), transferrin (150 μg/ml, ROCHE), ROCK inhibitor Y-27632 (10 μM, TOCRIS) and rhBMP4 (1 ng/ml, R&D). For EB generation, the cultures were rotated for 18 h on an orbital shaker (MaxQ 2000 shaker, Thermofisher) in 6 cm petri dishes (VWR) at 60 RPM. Following the rotation step, the EBs were transferred to fresh 6 cm petri dishes (VWR) in mesoderm induction media consisting of StemPro-34 supplemented with penicillin/streptomycin (1%), L-glutamine (2 mM), ascorbic acid (50 μg/ml), monothioglycerol (50 μg/ml), transferrin (150 μg/ml), rhBMP4 (10 ng/ml, R&D), rhActivinA (6 ng/ml, R&D) and rhbFGF (5 ng/ml, R&D). At day 3, the EBs were harvested, washed with IMDM and transferred to cardiac specification media consisting of StemPro-34 supplemented with penicillin/streptomycin (1%), L-glutamine (2 mM), ascorbic acid (50 μg/ml), monothioglycerol (50 μg/ml), transferrin (150 μg/ml), the Wnt inhibitor IWP2 (2 μM, TOCRIS) and rhVEGFA (5 ng/mL, R&D). At day 5, the EBs were harvested, washed with IMDM and cultured in StemPro-34 supplemented with penicillin/streptomycin (1%), L-glutamine (2 mM), ascorbic acid (50 μg/ml), monothioglycerol (50 μg/ml), transferrin (150 μg/ml) and rhVEGFA (5 ng/ml) for another 4 days. EBs cultures for periods longer than 9 days were maintained in StemPro-34 supplemented with penicillin/streptomycin (1%), L-glutamine (2 mM), ascorbic acid (50 μg/ml) and monothioglycerol (50 μg/ml). The media was changed every 2 days. At days 9, 16 or 23 HES2:RFP− or HES2:GFP-derived cardiomyocytes were isolated based on the expression of SIRPA (Dubois et al., 2011, Nat. Biotechnol., 29(11):1011-8). To generate cardiomyocyte aggregates, sorted cells were plated at a density 5×10e4 cells per well in 96-well flat bottom ultra-low attachment microplates (Corning) for 8 days in StemPro-34 supplemented with penicillin/streptomycin (1%), L-glutamine (2 mM), ascorbic acid (50 μg/ml), monothioglycerol (50 μg/ml) with or without NRG1 (50 ng/ml). The media was changed every 2 days. The cultures were incubated in a low oxygen environment (5% CO2, 5%O2, 90% N2) for first 9 days and a normoxic environment (5% CO2) after day 9.
Example 3—Generation of Endocardial/Endothelial Cells
[0063] To generate endocardial/endothelial cells, hPSCs were differentiated to cardiogenic mesoderm using the protocol described above. At day 3, the EBs were dissociated with TrypLE for 3 min at 37° C. and the cells plated in 96-well flat bottom microplates (Falcon) coated with Matrigel (25% v/v, Corning) at a density of 10e5 cells per well. The monolayers were cultured in StemPro-34 supplemented with penicillin/streptomycin (1%, ThermoFisher), L-glutamine (2 mM, ThermoFisher), ascorbic acid (50 μg/ml, Sigma), monothioglycerol (50 μg/ml, Sigma), transferrin (150 μg/ml, ROCHE) and rhbFGF (50 ng/ml, R&D). For endocardial induction, rhBMP10 (10 ng/ml) was added to the media from day 5 to day 9. For generation of control endothelial cells, VEGFA (100 ng/mL, R&D) was added to the differentiation media from day 3 to day 9. The cultures were incubated in a low oxygen environment (5% CO2, 5%O2, 90% N2) and the media was changed every 2 days. At day 9, HES3-NKX2-5eGFP/w-derived endocardial/endothelial cells were analyzed and isolated based on the expression of NKX2-5:GFP and CD31. The endocardial cells generated from the non-transgenic hPSC lines were analyzed and isolated as CD31+ populations. For dissociation, the monolayers were incubated in Collagenase type 2 (1 mg/ml, Worthington) in HANKs buffer at 37° C. for one hour.
Example 4—Co-Culture of Endocardial/Endothelial With Cardiomyocytes
[0064] For culture in the absence of cardiomyocytes, the endocardial or control endothelial cells (HES3-NKX2-5eGFP/w line) isolated at day 9 of differentiation were plated in a 96-well flat bottom microplate (Falcon) coated with Matrigel (25% v/v, Corning) at a density 2.5×10e4 cells per well in StemPro-34 supplemented with penicillin/streptomycin (1%, ThermoFisher), L-glutamine (2 mM, ThermoFisher), ascorbic acid (50 μg/ml, Sigma), monothioglycerol (50 μg/ml, Sigma), rhVEGFA (100 ng/ml, R&D) with or without rhBMP10 (10 ng/ml) or rhBMP4 (10 ng/ml). For co-culture of the two cell types, 5×10e4 hPSC-derived SIRPA+ cardiomyocytes (day 9) were mixed together with either 2.5×10e4 HES3-NKX2-5eGFP/w- or 5×10e4 HES2:RFP-derived endocardial or endothelial cells and cultured for 8 days in 96-well flat bottom microplates (Falcon) coated with Matrigel (25% v/v, Corning) in StemPro-34 supplemented with penicillin/streptomycin (1%, ThermoFisher), L-glutamine (2 mM, ThermoFisher), ascorbic acid (50 μg/ml, Sigma), monothioglycerol (50 μg/ml, Sigma) and rhVEGFA (100 ng/ml, R&D). For inhibition of the NRG-ERBB pathway lapatinib (10 μM, LC labs) was added to the media. Control cardiomyocytes were cultured as aggregates as described above in the same media with or without NRG1 (50 ng/ml). The cultures were incubated in a normoxic environment (5% CO2) and the media was changed every 2 days.
Example 5—Generation of Mesenchymal/Valvular Interstitial-Like Cells From Endocardial/Endothelial Cells
[0065] Day 9 endocardial/endothelial bulk cultures (HES3-NKX2-5eGFP/w line) were dissociated with Collagenase type 2 as described above. Single cell suspensions were enriched for CD31+ CD34+ cells by magnetic activated cell sorting (MACS, Miltenyi, 130-146-702) to a purity of over 95%. Cells were incubated with anti-CD34 microbeads for 30 minutes at 4° C. in base media supplemented with DNAse (1 U/ml, Millipore)(10 μl microbeads/5×10e6 cells in 100 μl of media) and purified by MS or LS columns. After MACS cells from endocardium were further sub-fractionated by FACS into CD31+ CD34+ PDGFRb+ and CD31+ CD34+ PDGFRb− populations. After the sort, total CD31+ CD34+, CD31+ CD34+ PDGFRb+ and CD31+ CD34+ PDGFRb− endocardial cells as well as total CD31+ CD34+ endothelial cells were plated in a 12-well flat bottom microplate (Falcon) coated with Matrigel (25% v/v, Corning) at a density 8×10e5 cells per well and cultured for 8 days in StemPro-34 supplemented with penicillin/streptomycin (1%, ThermoFisher), L-glutamine (2 mM, ThermoFisher), ascorbic acid (50 μg/ml, Sigma), monothioglycerol (50 μg/ml, Sigma), rhbFGF (10 ng/ml, R&D), rhBMP2 (100 ng/ml), TGFbeta2 (0.3 ng/ml, R&D).
[0066] For enrichment of CD31− PDGFRB+ VIC-like cells, MACS-sorted CD31+ CD34+ endocardial-like cells were plated in a 12-well flat bottom microplate (Falcon) coated with Matrigel (25% v/v, Corning) at a density 2×10e5 cells per well and cultured for 12 days in StemPro-34 supplemented with penicillin/streptomycin (1%, ThermoFisher), L-glutamine (2 mM, ThermoFisher), ascorbic acid (50 μg/ml, Sigma), monothioglycerol (50 μg/ml, Sigma) in the presence of rhbFGF (10 ng/ml, R&D), rhBMP2/4 (100 ng/ml), CHIR99021 (1 μM), SB-431542 (5.4 μM) for 4 days, followed by rhbFGF (10 ng/ml, R&D), rhBMP2/4 (100 ng/ml), TGFbeta2 (0.3 ng/ml, R&D) for 8 days.
Example 6—Generation of Coronary Endothelial-Like Cells From Endocardial Cells
[0067] Day 9 endocardial/endothelial bulk cultures (HES3-NKX2-5eGFP/w line) were dissociated with Collagenase type 2 as described above. Single cell suspensions were enriched for CD31+ CD34+ cells by magnetic activated cell sorting (MACS, Miltenyi, 130-146-702) to a purity of over 95% as for VIC protocol. Cells were plated in a 12-well flat bottom microplate (Falcon) coated with Matrigel (25% v/v, Corning) at a density 4×10e5 cells per well and cultured for 8 days in StemPro-34 supplemented with penicillin/streptomycin (1%, ThermoFisher), L-glutamine (2 mM, ThermoFisher), ascorbic acid (50 μg/ml, Sigma), monothioglycerol (50 μg/ml, Sigma) in the presence of rhVEGFA (50 ng/ml, R&D) for 4 days, followed by rhVEGFB (50 ng/ml, R&D) and PPARa agonist GW7647 (1 μM) for additional 4 days.
Example 7—Flow Cytometry and Cell Sorting
[0068] Day 3 EBs were dissociated with TrypLE for 3 min at 37° C. Day 9-23 EBs or mixed populations of cardiomyocytes and endocardial/endothelial cells were dissociated by incubation in Collagenase type 2 (1 mg/ml, Worthington) in HANKS buffer at 37° C. for one hour followed by TrypLE treatment (5 min at 37° C.). The following antibodies were used for staining: anti-SIRPA-PeCy7 (Biolegend, 1:1000), anti-CD31-PE (BD Pharmingen, 1:100), anti-CD31-FITC (BD Pharmingen, 1:100), anti-CD31-AF647 (BD Pharmingen, 1:100), anti-CD90-APC (BD Pharmingen, 1:1000), anti-PDGFRB-BV421 (BD Pharmingen, 1:100), anti-CD105-APC (eBioscience, 1:200), anti-CD56-APC (BD Pharmingen, 1:100), anti-PDGFRA-PE (BD Pharmingen, 10:100), anti-CD36-APC (Biolegend, 1:100), anti-LDLR-BV421 (Biolegend, 1:100), anti-cardiac isoform of CTNT (ThermoFisher Scientific, 1:2000), or anti-myosin light chain 2 (Abcam, 1:1000), anti-ANP (Abcam, 1:500). For unconjugated primary antibodies, the following secondary antibodies were used for detection: goat anti-mouse IgG-APC (BD Pharmigen, 1:250), or donkey anti-rabbit IgG-AF488 (ThermoFisher Scientific, 1:1000). Detailed antibody information is described in the Key Resources Table. For cell-surface marker analyses, cells were stained for 30 min at 4° C. in FACS buffer consisting of PBS with 5% fetal calf serum (FCS) (Wisent) and 0.02% sodium azide. For intracellular staining, cells were fixed for 15 min at 4° C. with 4% PFA in PBS followed by permeabilization using 90% methanol for 20 min at 4° C. Cells were washed with PBS containing 0.5% BSA (Sigma) and stained with unconjugated primary antibodies in FACS buffer overnight at 4° C. Stained cells were washed with PBS with 0.5% BSA and stained with secondary antibodies in FACS buffer for 1 h at 4° C. Stained cells were analyzed using an LSR II Flow cytometer (BD). For cell sorting, cells were stained, washed and kept in IMDM with 0.2% KnockOut™ serum replacement and sorted using either Influx (BD), FACSAriaII (BD), MoFlo-XDP (BD) of FACSAria Fusion (BD) cells sorter at the Sickids/UHN flow cytometry facility. Data were analyzed using FlowJo software (Tree Star).
Example 8—Immunohistochemistry
[0069] Day 3 EBs generated from HES3-NKX2-5eGFP/w line under 10B/6A mesoderm induction conditions were dissociated as described above and the cells plated onto 12 mm cover glasses (VWR) pre-coated with Matrigel (25% v/v, BD) in 24-well plates (Falcon) at a density 2×10e5 cells per well. The cells were cultured for 6 days as monolayers under endocardial, control endothelial or cardiomyocyte differentiation conditions. Following culture, the cells were fixed with 4% PFA in PBS for 10 min at room temperature and permeabilized with PBS containing 0.2% TritonX for 20 min at RT. The fixed cells were blocked with PBS containing 10% FCS and 2% BSA. The following antibodies were used for staining: rabbit anti-NKX2-5 (Cell Signaling, 1:100), rabbit anti-GATA4 (Abcam, 1:100), rabbit anti-NFAT2 (Abcam, 1:100), mouse anti human CD31 (Dako, 1:100), mouse anti-cardiac isoform of CTNT (ThermoFisher Scientific, 1:100), goat anti-GFP (Rockland, 1:500). For detecting unconjugated primary antibodies, the following secondary antibodies were used: donkey anti-mouse IgG-A647 (ThermoFisher, 1:1000), donkey anti-rabbit IgG-A555 (ThermoFisher, 1:1000) and donkey anti-goat IgG-A488 (ThermoFisher, 1:1000). Detailed antibody information is described in the Key Resources Table. The cells were stained with primary antibodies in staining buffer consisting of PBS with 0.05% TritonX and 2% BSA overnight at 4° C. The stained cells were washed with PBS containing 0.1% BSA 3× for 10 min each wash at room temperature. The cells were then stained with secondary antibodies in staining buffer for 1 h at room temperature followed by a wash step as described above. The cell nuclei were stained with DAPI (Biotium, 0.3 μg/ml) in wash buffer for 5 min at room temperature. Following staining, the samples were mounted using ProLong Diamond Antifade Mountant (ThermoFisher). The stained cells were analyzed using an Olympus FluoView 1000 Laser Scanning Confocal Microscope. FV10-ASW software was used for image acquisition.
Example 9—Quantitative Real-Time PCR
[0070] Total RNA from hPSC-derived populations was isolated using RNAqueous-micro Kit including RNase-free DNase treatment (Ambion). Between 100 ng and 1 mg of isolated RNA was reverse transcribed into cDNA using oligo (dT) primers and random hexamers and Superscript III Reverse Transcriptase (ThermoFisher). RT-qPCR was performed on an EP Real-Plex MasterCycler (Eppendorf) using QuantiFast SYBR Green PCR kit (QIAGEN). All experiments were prepared in duplicates and included a 10-fold dilution series of sonicated human genomic DNA standards ranging from 25 ng/ml to 2.5 pg/ml for evaluating the efficiency of the PCR reaction and the copy number of each gene relative to the house keeping gene TBP. Heat maps of gene expression data were generated using the MultiExperiment Viewer (MeV) open source software. Primer sequences are listed in the Table below.
Example 10—Quantification and Statistical Analysis
[0071] All data are represented as mean±standard error of mean (SEM). Sample sizes (n) represent biological replicates of differentiation experiments. No statistical method was used to predetermine the samples size. Due to the nature of the experiments, randomization was not performed and the investigators were not blinded. Statistical significance was determined by using Student's t test (paired, two-tailed) and one-way or two-way ANOVA analysis with Bonferroni post-hoc test in GraphPad Prism 7 software. Results were considered to be significant at p<0.05 (*), p<0.005 (**), p<0.0005 (***). All statistical parameters are reported in the respective figures and figure legends.
Example 11—FGF and BMP Induce NKX2-5 Expression in Cardiogenic Mesoderm
[0072] Initial experiments were set up to identify the signaling pathways that would promote the generation of a NKX2-5+ CD31+ cell that we propose represents the earliest stage of human endocardial development. The transgenic HES3-NKX2-5eGFP/w reporter line (Elliott et al., 2011, Nat. Methods, 8(12):1037-40) was used to allow for the identification of NKX2-5+ cells by flow cytometry. As previous studies have shown that both FGF and BMP signaling are involved in the specification of NKX2-5+ cardiac progenitors in vivo, the role of these pathways in the generation of the target NKX2-5+ CD31+ population in the hPSC differentiation cultures was evaluated. For these studies, either BMP4 or BMP10 was used, as both are expressed in the early cardiomyocytes that develop adjacent to the endocardial cells. These pathways were manipulated in cardiogenic mesoderm induced with the concentrations of activin and BMP4 (6 ng/ml activin, 10 ng/ml BMP4) used in previous studies to generate ventricular cardiomyocytes (
[0073] In the first set of experiments, the effects of varying the concentration of bFGF between days 3 and 9 of differentiation were evaluated in the presence of either BMP4 or BMP10 that were added in the following time intervals: days 3-9, 5-9 or 7-9. These times were chosen to match the emergence of cardiomyocytes (between days 6 and 9) in the cultures, in an effort to recapitulate the coordinated development of the endocardial and cardiomyocyte lineages in the early embryo. For the initial analyses, a single concentration of BMP4 or BMP10 was used (10 ng/ml). At day 9, the populations were harvested and analyzed for the presence of NKX2-5+ and CD31+ cells. None of the populations induced with BMP4 or BMP10 alone in the absence of bFGF contained CD31+ cells, indicating that exogenous FGF signaling enhances endothelial development under these conditions (
[0074] The effects of different concentrations of BMP on the induction of the NKX2-5+ CD31+ population at a defined bFGF dose was determined. As shown in
[0075] The protocol for cardiomyocytes specification from hPSC-derived cardiovascular mesoderm included a WNT inhibition step from day 3 to day 5. As the same mesoderm was used to generate the NKX2-5+ CD31+ cells in the above studies, we were next interested in determining if WNT signaling plays a role in development of these cells. To address this, the WNT pathway was either activated through the addition of the small molecule GSK-3 inhibitor, CHIR99021, or inhibited by the addition of IWP2 for 2, 4 or 6 days, beginning at day 3 of differentiation (
[0076] The kinetics of NKX2-5+ CD31+ development was analyzed by comparing BMP4 to BMP10 induction using the concentrations of factors and timing of their addition determined in the above experiments (
[0077] Exploiting the above observation that high concentrations of VEGFA promotes the development of a NKX2-5− CD31+ population, the cells were treated with VEGFA and bFGF without BMP, in an effort to generate NKX2-5− CD31+ non-endocardial endothelial cells. As shown in
[0078] Given the findings from these kinetic analyses and the fact that the protocol was optimized in the previous experiments for a day 9 population, the NKX2-5+ CD31+ cells from day 9 cultures were isolated for the following studies.
Example 12—Characterization of NKX2-5+ CD31+ Cells
[0079] Endocardial cells can be distinguished from other endothelial cells by the levels of expression of specific transcription factors including GATA4, GATA5 and NFATC1 as well as by the expression of atrial natriuretic peptide receptor, NPR3, and neuregulin (NRG1), the ligand to the ERBB family receptors (de la Pompa et al, 1998, Nature, 392(6672):182-6; Ranger et al., 1998, Nature, 392(6672):186-90; Charron and Nemer, 1999, Semin. Cell Dev. Biol., 10(1):85-91; Nemer and Nemer, 2002, Development, 129(17):4045-55; Rivera-Feliciano et al., 2006, Development, 133(18):3607-18; Wu et al., 2013, Trends Cardiovasc. Med., 23(8):294-300; Zhang et al., 2016, Circ. Res., 118(12):1880-93). To further characterize the NKX2-5+ and CD31+ populations generated with the optimized protocol, the different populations were isolated by Fluorescence-Activated Cell Sorting (FACS) and analyzed for the expression of these genes (
[0080] To determine if the method of induction impacted the molecular profile of the NKX2-5+ CD31+ population, the expression patterns in cells induced with either BMP4 or BMP10 was compared. Although the levels of expression of GATA4, GATA5 and NFATC1 were similar in both, the BMP10-induced population expressed higher levels of NRG1 than the cells induced with BMP4 (
[0081] Collectively, the findings from the above comparative studies demonstrate that BMP signaling plays a pivotal role in the generation of NKX2-5+ CD31+ cells that express markers indicative of endocardial cells. Additionally, they suggest that BMP10 is more effective than BMP4 in specifying this fate. To further investigate differences between these pathway agonists, BMP10-induced NKX2-5+ CD31+ cells were cultured in the presence of different concentrations of either BMP4 or BMP10 for 8 days. VEGFA was added to maintain the endothelial phenotype of the cells during this culture period. Analyses of the cultured populations revealed that BMP10 was more effective than BMP4 in maintaining the NKX2-5+ CD31+ cell phenotype at all concentrations of agonist tested (
[0082] To further characterize the BMP10-induced NKX2-5+ CD31+ endocardial-like cells, the cells were analyzed for the presence of NKX2-5, GATA4 and NFATC1 proteins by immunostaining analyses. As shown in
Example 13—NRG1 Induces Markers of Trabecular Myocardium in hPSC-Derived Cardiomyocytes
[0083] If the NKX2-5+ CD31+ cells represent the equivalent of endocardium, they should be able to induce a trabecular fate in target cardiomyocytes, thereby replicating the interaction that takes place in the early heart tube. As specification of the trabecular fate is mediated by NRG-ERBB signaling in the developing heart tube, the cardiac cells generated with the protocol described herein were tested to determine if they were responsive to this pathway. For this analysis, SIRPA+ cardiomyocyte populations (Dubois et al., 2011, Nat. Biotechnol., 29(11):1011-8) isolated at days 9, 16 and 23 of differentiation were treated with NRG1 (
[0084] The treated populations were evaluated by flow cytometric analyses to determine the proportion of MLCV+ cells that co-express the trabecular marker, atrial natriuretic peptide (ANP; encoded by NPPA). These analyses showed that the day 9-derived population contained the highest proportion of ANP+ cells, with an average of 90% of the cells expressing this marker (
Example 14—NKX2-5+ CD31+ Endocardial-Like Cells Induce Markers of Trabecular Myocardium in hPSC-Derived Cardiomyocytes
[0085] To determine if the NKX2-5+ CD31+ endocardial-like cells have the potential to induce a trabecular fate in the cardiomyocyte target population, day 9 SIRPA+ cardiomyocytes generated from a HES2:RFP hPSC line were mixed with day 9 NKX2-5+ CD31+ cells produced from the HES3:NKX2-5:GFP line and cultured them together in a monolayer format in the presence of VEGFA (
[0086] RT-qPCR analyses revealed that expression of the markers of trabecular myocardium (BMP10, NPPA, NPPB, IRX3) were significantly upregulated in the RFP+ SIRPA+ cardiomyocytes co-cultured with the NKX2-5+ CD31+ endocardial-like cells in a pattern similar to that observed in the aggregates treated with NRG1. By contrast, expression of these genes was not upregulated in the cardiomyocytes cultured with the control endothelial cells or in the cardiomyocyte aggregates cultured in the absence of NRG1. The expression pattern of the compact marker HEY2 was opposite to that of the trabecular markers, as it was upregulated in the cardiomyocytes cultured with the control endothelial cells, but not in the cells cultured with the endocardial-like cells. All populations expressed comparable levels of CTNT. MYL2 expression was upregulated in the cardiomyocytes co-cultured with the NKX2-5+ CD31+ endocardial-like cells and in the cardiomyocyte aggregates induced with NRG1, indicative of ventricular maturation. As observed in the above analyses, expression of GJA5 did not segregate with the patterns of the other trabecular markers.
[0087] Induction of the trabecular fate by the NKX2-5+ CD31+ endocardial-like cells was inhibited by the addition of the ERBB2 inhibitor lapatinib (lap), indicating that the effect is mediated by NRG-ERBB signaling. This interpretation is further supported by the finding that the NKX2-5+ CD31+ cells isolated from the co-culture population express NRG1 (
[0088] The majority of the NKX2-5+ CD31+ endocardial-like cells from the co-culture population maintained expression of NKX2-5 similar to those cultured with exogenous BMP10 (
[0089] Taken together, the findings from these co-culture experiments demonstrate that the NKX2-5+ CD31+ endocardial-like cells can induce a trabecular fate in the cardiomyocyte target population and that this induction is mediated through NRG-ERBB signaling. The ability to induce a trabecular fate appears to be specific to the endocardial like cells, as the control endothelial cells do not display this activity. Given this specificity, the upregulation of trabecular genes can be used as an in vitro developmental assay to assess endocardial potential of any endothelial population. To test this, the potential of BMP4 induced NKX2-5+ CD31+ cells to those induced with BMP10 was compared. As shown in
Example 15—Generation of NKX2-5+ CD31+ Endocardial Cells From Different Mesoderm Populations
[0090] Ventricular and atrial cardiomyocytes develop from distinct mesoderm subpopulations that are induced with different concentrations of BMP4 and Activin A. The NKX2-5+ CD31+ endocardial cells characterized in the above studies were generated from mesoderm induced with a ventricular protocol (10 ng/ml of BMP4, 5 ng/ml of bFGF and 6 ng/ml of ActA, referred to as “10B/6A”). To determine if the endocardial fate is restricted to this mesoderm and to establish the overall importance of the mesoderm induction step in the generation of these cells, mesoderm were induced with a range of concentrations of BMP4 (3-20 ng/ml) and Activin A (1-12 ng/ml) and then the cells were cultured in either endocardial or control endothelial conditions (
[0091] Put another way, different subsets of mesoderm were produced by culturing hESCs in the presence of different amounts of BMP4 and Activin A. All the mesodermal populations that were generated with the range of concentrations of Activin A and BMP4 that were tested were capable of producing control endothelial cells upon culturing them in the presence of VEGFA and bFGF, but only a small subset of amounts of BMP4 and Activin A from the range that were tested produced mesodermal populations that were capable of generating endocardial cells upon culturing them in the presence of bFGF and BMP10.
[0092] To further characterize cells induced under different conditions, NKX2-5+ CD31+ cells generated from 3 different inductions (“3B/6A”, “5B/9A”, or “10B/12A”) were analyzed and compared to cells generated under our standard conditions (10B/6A). The different NKX2-5+ CD31+ cells were evaluated for their ability to induce a trabecular fate in day 9 target cardiomyocytes and for levels of NRG1 expression. All 3 NKX2-5+ CD31+ populations were able to induce a trabecular profile similar to that induced by the 10B/6A cells (
Example 16—Generation of Endocardial-Like Cells From HES2-RFP hESCs
[0093] As the development of NKX2-5+ CD31+ cells correlated with the development of total CD31+ cells under endocardial conditions, this readout was used to optimize induction strategies for the generation of endocardial-like cells from a second hPSC line that did not contain a NKX2-5 reporter. These cells were engineered to constitutively expresses RFP (HES2-RFP) (
[0094] To measure endocardial potential (induction of trabecular fate), both populations were isolated from day 9 EBs and co-cultured with SIRPA+ cardiomyocytes generated from a hPSC line that expresses GFP (HES2-GFP) (
[0095] Collectively, the findings from this set of experiments clearly demonstrate that it is possible to generate endocardial-like cells from non-targeted (NKX2-5) hPSCs and that expression of CD31 can be used to monitor the development of this population and to isolate it for functional analyses.
Example 17—Generation of VIC-Like Cells From hPSC-Derived Endocardial-Like Cells
[0096] To assess the potential of hPSC-derived endocardial-like cells to undergo endothelial-to-mesenchymal transition, the endothelial fractions of hPSC-derived endocardial-like cells and control endothelial cells were isolated at day 9 using magnetic-activated cell sorting (MACS) (
[0097] In order to enrich for CD31− PDGFRb+ VIC-like cells, activation of the Wnt signaling pathway with 1 μM CHIR99021 was tested prior to specification of the VIC fate with BMP2/4, bFGF and TGFb2. Day 9 CD31+ MACS-sorted endocardial-like cells were cultured in the presence of 1 μM CHIR99021, 10 ng/mL of bFGF and 100 ng/mL of BMP2/4 as well as 5.4 μM of the TGFbeta inhibitor SB-431542 to stimulate cell proliferation for four days (
[0098] Collectively, the findings from this set of experiments demonstrate that a subpopulation of hPSC-derived endocardial-like cells, marked by the expression of PDGFRb, can undergo an endothelial-to-mesenchymal transition, and do so more efficiently than control endothelial cells. Furthermore, when compared with mesenchymal cells derived from control endothelial cells, these endocardial-derived mesenchymal cells looked more like VICs, based on signature gene expression, than the mesenchymal cells derived from control endothelial cells.
TABLE-US-00001 Key resources table Reagent or Resource Source Identifier Antibodies Mouse monoclonal to Biolegend Cat. # 323808; RRID: SIRPα (clone SE5A5), PeCy7 AB_1236443 conjugated Mouse monoclonal to CD90 BD Pharmingen Cat. # 559869; RRID: (clone 5E10), APC conjugated AB_398677 Mouse monoclonal to CD31 BD Pharmingen Cat. # 555446; RRID: (clone WM59), PE conjugated AB_395839 Mouse monoclonal to CD31 BD Pharmingen Cat. # 555445; RRID: (clone WM59), FITC conjugated AB_395838 Mouse monoclonal to CD31 BD Pharmingen Cat. # 561654; RRID: (clone WM59), Alexa Fluor 647 AB_10896969 conjugated Mouse monoclonal to CD34 ThermoFisher, Cat. # 25-0349-42; (clone 4H11), PeCy7 conjugated eBioscience RRID: AB_1963576 Mouse monoclonal to CD36 Biolegend Cat. # 336208 RRID; (clone 5-271), APC conjugated AB_1279224 Mouse monoclonal to CD140b BD Horizon Cat. # 564124; RRID: (PDGFRβ) (clone 28D4), BV421 AB_2738609 conjugated Mouse monoclonal to CD105 ThermoFisher, Cat. # 17-1057-42; (Endoglin) (clone SN6), APC eBioscience RRID: AB_1582211 conjugated Mouse monoclonal to PDGFRα BD Pharmingen Cat. # 556002; RRID: (clone αR1), PE conjugated AB_396286 Mouse monoclonal to CD56 BD Pharmingen Cat. # 555518; RRID: (clone B159), APC conjugated AB_398601 Mouse monoclonal anti human Dako Cat. # M0823; RRID: CD31 (clone JC70A), non- AB_2114471 conjugated Mouse monoclonal to CTNT ThermoFisher Cat. # MS-295-P; RRID: (clone 13-11), non-conjugated AB_11000742 Rabbit polyclonal to MLC2V Abcam Cat. # ab79935; RRID: (clone 13-11), non-conjugated AB_1952220 Mouse monoclonal to ANP Abcam Cat. # ab2093; RRID: (M622709), non-conjugated AB_302831 Rabbit monoclonal to NKX2-5 Cell Signaling Cat. # 8792S; RRID: (E1Y8H), non-conjugated AB_2797667 Rabbit polyclonal to NFAT2, Abcam Cat. # ab25916; RRID: non-conjugated AB_448901 Rabbit polyclonal to GATA4, Abcam Cat. # ab84593; RRID: non-conjugated AB_10670538 Mouse monoclonal anti-human Biolegend Cat. # 744847; RRID LDLR (clone c7), BrilliantViolet421 conjugated Goat anti-mouse IgG (H + L), BD Pharmingen Cat. # 550826; RRID: APC conjugated AB_398465 Donkey anti-rabbit IgG (H + L), ThermoFisher Cat. # A21206; RRID: AlexaFluor488 conjugated AB_2535792 Goat anti-GFP, non-conjugated Rockland Cat. # 600-101-215; RRID: AB_218182 Donkey anti-goat IgG (H + L), ThermoFisher Cat. # A11055; RRID: AlexaFluor488 conjugated AB_2534102 Donkey anti-rabbit IgG (H + L), ThermoFisher Cat. # A31572; RRID: AlexaFluor555 conjugated AB_162543 Donkey anti-mouse IgG (H + L), ThermoFisher Cat. # A31571; RRID: AlexaFluor647 conjugated AB_162542 Chemicals, Peptides, and Recombinant Proteins Penicillin/streptomycin ThermoFisher Cat. # 15070063 L-glutamine ThermoFisher Cat. # 25030081 Non-essential amino acids ThermoFisher Cat. # 11140-050 Transferrin ROCHE Cat. # 10652202001 Ascorbic acid Sigma Cat. # A4544-100G Monothioglycerol Sigma Cat. # M-6145-25 ml β-Mercaptoethanol ThermoFisher Cat. # 21985-023 ROCK inhibitor Y-27632 Tocris Cat. # 1254 Recombinant human BMP2 R&D Cat. # 355-BM Recombinant human BMP4 R&D Cat. # 314-BP Recombinant human ActivinA R&D Cat. # 338-AC Recombinant human TGFβ2 R&D Cat. # 302-B2 Recombinant human bFGF R&D Cat. # 233-FB Recombinant human BMP10 R&D Cat. # 2926-BP Recombinant human VEGFA R&D Cat. # 293-VE Recombinant human VEGFB R&D Cat. # 751-VEB-025 Recombinant human NRG1β1 R&D Cat. # 396-HP IWP2 (Wnt inhibitor) Tocris Cat. # 3533 CHIR 99021 (GSK-3 inhibitor) Tocris Cat. # 4423 SB-431542 (TGFbeta inhibitor, Sigma-Aldrich Cat. # S4317 SB) Lapatinib LC labs Cat. # L-4804 LDN 193189 (BMP inhibitor) Tocris Cat. # 6053 GW7647 (PPARa agonist) Sigma Cat. # G6793 Collagenase type2 Worthington Cat. # LS004-176 DAPI Biotium Cat. # 40043 Fetal calf serum (FCS) Wisent Cat. # 080-150 Bovine serum albumin (BSA) Sigma Cat. # A1470 Matrigel, growth factor reduced Coming Cat. # 356230 Critical Commercial Assays RNAqueous-micro kit with Ambion Cat. # AM1931 RNase-free Dnase treatment Superscript III Reverse ThermoFisher Cat. # 18080044 Transcriptase kit QuantiFast SYBR Green PCR kit QIAGEN Cat. # 204057 CD34 MicroBead Kit, human Miltenyi Biotec Cat. # 130-046-702 MACS cell separation, LS Miltenyi Biotec Cat. # 130-042-401 columns Experimental models: Cell Lines Human ESC: HES3-NKX2- Gift from Drs. E. NA 5.sup.eGFP/w line Stanley and A. Elefanty, Monash University, AU (Elliott, Braam et al. 2011) Human ESC: HES2:RFP line Generated in Keller lab NA from HES2 (ESI International Singapore) (Irion, Luche et al. 2007) Human ESC: HES2:GFP line Generated in Keller lab NA from HES2 (ESI International Singapore) Oligonucleotides See Table below for PCR primer This paper Table below sequences Software and Algorithms FlowJo Tree Star https://www.flowjo.com FV10-ASW Olympus https://www.olympus- lifescience.com GraphPad Prism 6 GraphPad Software http://www.graphpad.co m/scientific- software/prism Other StemPro-34 media (kit) ThermoFisher Cat. # 10639011 DMEM/F12 Cellgro Cat. # 10-092-CV KnockOut serum replacement ThermoFisher Cat. # 10828028 TrypLE ThermoFisher Cat. # 12605010 96-well clear flat bottom TC- Falcon Cat. # 353072 treated culture microplate 24-well clear flat bottom TC- Falcon Cat. # 353047 treated culture microplate 96-well clear flat bottom ultra- Coming Cat. # 3474 low attachment microplate Petri dishes 60 × 15 mm VWR Cat. # 25384-090 Micro cover glasses 12 mm VWR Cat. # 89015-725 ProLong diamond antifade ThermoFisher Cat. # P36965 mountant
TABLE-US-00002 SEQ SEQ ID ID Gene Forward (5′-3′) NO Reverse (5′-3′) NO: APOD TGG CAA GAA ACC CTA ATC TCC 1 CTG GTC TGT GAC CGT CAT TT 2 BMP10 GCC CAT CTC CAT CCT CTA TTT 3 AAG CCA TAG GAC TCT TCT TCT ATC 4 COLIA1 CAA TGC TGC CCT TTC TGC TCC TTT 5 CAC TTG GGT GTT TGA GCA TTG CCT 6 COL3A1 GAA TTT GGT GTG GAC GTT GG 7 CTT GCA CTG GTT GAC AAG ATT AG 8 CDH11 ACC ACT CAT TGT CTT TGA GGA A 9 ATT CTG GAG GGT GGC AAT ATC 10 CD36 GGT GAT GAG AAG GCA AAC A 11 GGTGATGAGAAGGCAAACA 12 CTNT TTC ACC AAA GAT CTG CTC CTC GCT 13 TTA TTA CTG GTG TGG AGT GGG TGT GG 14 DLL4 CCT GCA TTG TGA ACA CAG CAC CTT 15 ACC TGT CCA CTT TCT TCT CGC AGT 16 ETV2 CCG GGC ATG AAT TAC GAG AA 17 CGA AGC GGT ACG TGT ACT TT 18 FABP4 CGT CAC TTC CAC GAG AGT TTA T 19 TCC CAC AGA ATG TTG TAG AGT TC 20 GATA4 CGA ATG ACG GCA TCT GTT TGC CAT 21 ATT TGG TAT TAG GGA TGC AGG GCG 22 GATA5 ACC AAG ATT CCC AGT GAA GCA CCT 23 TCC GTC TAT CCA TGT GGG CAA TGA 24 GJA4 CTA CCT CGT GGA CTG CTT TG 25 GTT AAG CAC CAG GGA GAT GAG 26 GJA5 AAT CTT CCT GAC CAC CCT GCA TGT 27 CAG CCA CAG CCA GCA TAA AGA CAA 28 HEY2 GAG TGA GAG AGT CGT GTT TC 29 ACT TCT GTC CCT TTC CTT TC 30 IRX3 TCT GCC TTT GTG TGT GTG 31 GTG GCA GCA GCT CAT TTA 32 ISL1 GAA GGT GGA GCT GCA TTG GTT TGA 33 TAA ACC AGC TAC AGG ACA GGC CAA 34 ITGA2 GAT GAG ATT GAT GAG ACC ACA GA 35 TTC ACG CAA ACA GCA AAC C 36 JAG1 TGC TAC AAC CGT GCC AGT GAC TAT 37 AGT GGT CTT TCA GGT GTG AGC AGT 38 MSX2 CAC GCC CTT TAC CAC ATC C 39 AGG TTC AGA GAG CTG GAG AA 40 MYL2 TGT CCC TAC CTT GTC TGT TAG CCA 41 ATT GGA ACA TGG CCT CTG GAT GGA 42 NFATC1 CCT GCT GCC TTA CAC AGT GCA TTT 43 TGC GAC TAT GAA CAA GCC TAC GGT 44 NKX2-5 TTT GCA TTC ACT CCT GCG GAG ACC TA 45 ACT CAT TGC ACG CTG CAT AAT CGC 46 NOTCH1 CGG GTC CAC CAG TTT GAA TG 47 GTT GTA TTG GTT CGG CAC CAT 48 NOTCH2 TTT GGC AAC TAA CGT AGA AAC TCA AC 49 TGC CAA GAG CAT GAA TAC AGA GA 50 NPPA GGG TCT CTG CTG CAT TTG TGT CAT 51 AGA GGC GAG GAA GTC ACC ATC AAA 52 NPPB TCC TGC TCT TCT TGC ATC TGG CTT 53 TGT AAC CCG GAC GTT TCC AAG T 54 NPR3 CGG GAA GAT TCC ATC AGA TCC 55 GTC TTT CAA CCC GTC TAT CTC C 56 NR4A2 CCC AGA ACT TCG TAC CCT TTG 57 TGC TTG GGA GGA GGT CTT A 58 NRG1 CCG AAA GCC ACT CTG TAA TC 59 CCT GAG GAA GCT GTT ACA TTC 60 POSTN CCG AGC CTT GTA TGT ATG TTA TG 61 TTT GTA CAC CAA GCA CCT ATT T 62 PRRX2 TCG CTG CTC AAG TCC TAC A 63 CCA GGA GAG ATA ATC TGG ACT CA 64 RGS5 TGG ATT GCC TGT GAG GAT TAC 65 TTA GGA GCC TCC GTT TGA ATG 66 SOX9 GAA AGA GAG GAC CAA CCA GAA T 67 TTG GGT ACG AGT TGC CTT TAG 68 TBP TGA GTT GCT CAT ACC GTG CTG CTA 69 CCC TCA AAC CAA CTT GTC AAC AGC 70 TIMP3 GCG TCT ATG ATG GCA AGA TGT A 71 ACC CAG GTG ATA CCG ATA GT 72 VCAN TGG AAA GAT GAA ACC TCG TTA T 73 TCC TAA GCA CCG GAT AGT T 74 VIM AGCATCACGATGACCTTGAATA 75 GGCACTTGAAAGCTGTTTCTT 76
Example 18—Generation of Coronary Endothelial-Like Cells From PSC-Derived Endocardial-Like Cells
[0099] Several lineage tracing studies have demonstrated the contribution of endocardial cells to the coronary endothelium of the adult, with these cells being preferentially localized to the septal arteries and inner myocardial walls (see, e.g., Zhang et al., 2016, Circ. Res., 118:1880-93; Sharma et al., 2017, Develop. Cell, 42:655-66; Wu et al., 2021, Cell, 151:1083-96). Upon ablation of the other predominant progenitor pool, the sinus venosus endothelium, the endocardial cells will give rise to most of the coronary endothelial cells in the adult (see, e.g., Sharma et al., 2017, Develop. Cell, 42:655-66; Chen et al., 2014, Develop., 141:4500-12). These observations demonstrate that endocardial cells represent a source of coronary endothelium that can function across the vascular bed.
[0100] One of the defining characteristics of the coronary arterial endothelium is its capacity to transport fatty acids (FA) to the adjacent cardiomyocytes (see, e.g., Su et al., 2018, Nature, 559:356-62; Hagberg et al., 2010, Nature, 464:917-21). To achieve this, the coronary endothelial cells upregulate expression of genes that encode proteins required for this process, including CD36 and LDLR, proteins involved in the trafficking of FA from the blood stream into the cell, FABP4, a cytosolic FA binding protein, and APOD, a secreted regulator of FA metabolism.
[0101] To generate coronary cells in vitro, important signals known to regulate their development from endocardial cells in the developing embryo were recapitulated, using a two-step protocol that involves both specification to immature coronary endothelium and their maturation to functional cells (
[0102] In the embryo, endocardial cells expand from the subluminal surface of the heart into the developing myocardium through a VEGFA dependent process (see, for example, Wu et al., 2021, Cell, 151:1083-96). This is considered to be the first step in the development of the coronary lineage. To mimic this step, the hPSC-derived endocardial cells were cultured in the presence of VEGFA for 4 days. Treatment with VEGFA promoted the development of a CD34+ CD31+ endothelial cell population that downregulated expression of the endocardial markers NKX2-5 and CD140b and initiated upregulation of LDLR, suggesting specification to the coronary lineage (
[0103] In the heart, CD36 expression is regulated by VEGFB secreted from the adjacent cardiomyocytes (Hagberg et al., 2010, Nature, 464:917-21). To recapitulate this stage, the cells were cultured in the presence of VEGFB together with a PPARa agonist, GW7647, a known regulator of fatty acid transport and metabolism (Chanda et al., 2016, FEBS Letters, 590:2364-74). As a control, cells were cultured in VEGFA for the entire culture period. Flow cytometric analyses showed that treatment with the combination of VEGFB and PPARa agonist induced the upregulation of CD36 on a subset of cells expressing LDLR (
[0104] 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.
[0105] 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.