BMP10 Signaling Promotes the Development of Endocardial Cells from Human Pluripotent Stem Cell-Derived Cardiovascular Progenitors

•Endocardial cells can be generated from hPSC-derived cardiovascular mesoderm•BMP10 is required for specification and maintenance of human endocardial cells•Human induce a trabecular fate in cardiomyocytes•hPSC-derived generate valvular interstitial-like The embryonic endocardium essential early heart development as it functions to myocardium, the first tissue form, source that make up valves portion coronary vasculature. With this potential, could provide unique therapeutic opportunities include engineering biological cell-based therapy strategies replace vasculature damaged hearts. To access cells, we pluripotent stem cell (hPSC)-derived endothelial population displays many characteristics endocardium, including expression cohort genes identifies lineage vivo, capacity immature cardiomyocytes vitro, ability undergo an endothelial-to-mesenchymal transition. Analyses signaling pathways identified novel role BMP10 mesoderm. One earliest stages formation primitive tube consists inner layer specialized known surrounded by outer (Buijtendijk et al., 2020Buijtendijk M.F.J. Barnett P. van den Hoff M.J.B. Development heart.Am. J. Med. Genet. C. Semin. 2020; 184: 7-22Crossref PubMed Scopus (12) Google Scholar; Sedmera 2000Sedmera D. Pexieder T. Vuillemin M. Thompson R.P. Anderson R.H. Developmental patterning myocardium.Anat. Rec. 2000; 258: 319-337Crossref (416) Sylva 2014Sylva M.J. Moorman A.F. A. 2014; 164A: 1347-1371Crossref (72) Scholar). These play pivotal development, they are responsible inducing functional cardiomyocytes, namely, form muscle myocardium (Del Monte-Nieto 2018Del G. Ramialison Adam A.A.S. Wu B. Aharonov D’Uva Bourke L.M. Pitulescu M.E. Chen H. de la Pompa J.L. al.Control cardiac jelly dynamics NOTCH1 NRG1 defines building plan trabeculation.Nature. 2018; 557: 439-445Crossref (67) Tian Morrisey, 2012Tian Y. Morrisey E.E. Importance myocyte-nonmyocyte interactions disease.Circ. Res. 2012; 110: 1023-1034Crossref (89) Induction mediated through neuregulin/ERBB2 via neuregulin secreted cells. In addition promoting serves progenitors several other types (Nakano 2016Nakano Nakano Smith K.A. Palpant N.J. developmental origins contributions endothelium.Biochim. Biophys. Acta. 2016; 1863: 1937-1947Crossref (13) Zhang 2018Zhang Lui K.O. Zhou Endocardial Cell Plasticity Cardiac Development, Diseases Regeneration.Circ. 122: 774-789Crossref (27) Scholar) endothelium makes part (Chen 2014Chen H.I. Sharma Akerberg B.N. Numi H.J. Kivelä R. Saharinen Aghajanian McKay A.S. Bogard P.E. Chang A.H. al.The sinus venosus contributes VEGFC-stimulated angiogenesis.Development. 141: 4500-4512Crossref (109) He Zhou, 2018He L. Regeneration Coronary Arteries.Curr. Cardiol. Rep. 20: 54Crossref (7) 2017Sharma Ho Ford G.H. Goldstone A.B. Woo Y.J. Quertermous Reversade Red-Horse K. Alternative Progenitor Cells Compensate Rebuild Vasculature Elabela- Apj-Deficient Hearts.Dev. Cell. 2017; 42: 655-666Abstract Full Text PDF (39) 2014Tian X. Hu Huang Liu Q. Yu W. Yang Z. Yan al.Vessel formation. De novo distinct vascular neonatal heart.Science. 345: 90-94Crossref (113) 2012Wu Wang Chamberlain A.A. Moreno-Rodriguez R.A. Markwald R.R. O’Rourke B.P. Sharp D.J. al.Endocardial arteries angiogenesis myocardial-endocardial VEGF signaling.Cell. 151: 1083-1096Abstract (229) (VECs) that, transition (EndoMT), give rise interstitial (VICs) seed (Combs Yutzey, 2009Combs M.D. Yutzey K.E. Heart valve development: regulatory networks 2009; 105: 408-421Crossref (292) Hinton 2011Hinton R.B. structure function disease.Annu. Rev. Physiol. 2011; 73: 29-46Crossref (259) MacGrogan 2018MacGrogan Münch Notch interacting signalling disease, regeneration.Nat. 15: 685-704Crossref (53) From perspective, distinguished populations fact specified progenitor expresses NKX2-5, key transcription factor, ISL1, regulator secondary field (Cai 2003Cai C.L. Liang Shi Chu P.H. Pfaff S.L. Evans S. Isl1 proliferates prior differentiation majority heart.Dev. 2003; 5: 877-889Abstract (1171) Ferdous 2009Ferdous Caprioli Iacovino Martin C.M. Morris Richardson J.A. Latif Hammer R.E. Harvey Olson E.N. al.Nkx2-5 transactivates Ets-related protein 71 gene specifies endothelial/endocardial developing embryo.Proc. Natl. Acad. Sci. USA. 106: 814-819Crossref (140) Harris Black, 2010Harris I.S. Black B.L. endocardium.Pediatr. 2010; 31: 391-399Crossref (60) Ma 2008Ma Pu W.T. Reassessment Nkx2-5 maps using Gata4-based reporter Cre activity.Dev. Biol. 2008; 323: 98-104Crossref (156) Milgrom-Hoffman 2011Milgrom-Hoffman Harrelson Ferrara N. Zelzer E. S.M. Tzahor derived progenitors.Development. 138: 4777-4787Crossref (74) Moretti 2006Moretti Caron Lam J.T. Bernshausen Qyang Bu Sasaki Martin-Puig al.Multipotent isl1+ lead cardiac, smooth muscle, diversification.Cell. 2006; 127: 1151-1165Abstract (766) Stanley 2002Stanley E.G. Biben Elefanty Koentgen F. Robb Efficient Cre-mediated deletion conferred 3’UTR-ires-Cre allele homeobox Nkx2-5.Int. Dev. 2002; 46: 431-439PubMed During NKX2-5 directly activates ETV2, factor lineages (Ferdous Within pool NKX2-5+ progenitors, ETV2 upregulate NFATC1 expression, which turn promotes at expense cardiomyocyte (Palencia-Desai 2011Palencia-Desai Kohli V. Kang Chi N.C. Sumanas Vascular differentiate absence Etsrp/Etv2 function.Development. 4721-4732Crossref (63) regulate these factors within embryo not well defined. However, develop (Stanley Scholar), BMP, FGF, Wnt, likely candidates some level (Alsan Schultheiss, 2002Alsan B.H. Schultheiss T.M. Regulation avian cardiogenesis Fgf8 signaling.Development. 129: 1935-1943PubMed Barron 2000Barron Gao Lough Requirement BMP FGF during cardiogenic induction non-precardiac mesoderm specific, transient, cooperative.Dev. Dyn. 218: 383-393Crossref (134) Wijk 2007van Role bone morphogenetic proteins differentiation.Cardiovasc. 2007; 74: 244-255Crossref (143) Given central (hPSCs) would unlimited studying their origin, tissues disease modeling drug screening, generating applications. Several previous studies have shown possible express markers (Bao 2017Bao Bhute V.J. Han Qian Lian Palecek S.P. Human cell-derived epicardial endocardial-like cells.Bioeng. Transl. 2: 191-201Crossref Neri 2019Neri Hiriart Vliet P.P. Faure Norris Farhat Jagla Lefrancois Sugi Moore-Morris al.Human pre-valvular recapitulate pathophysiological valvulogenesis.Nat. Commun. 2019; 10: 1929Crossref (19) 2017Palpant Pabon Friedman C.E. Roberts Hadland Zaunbrecher R.J. Bernstein I. Zheng Murry Generating high-purity derivatives patterned cells.Nat. Protoc. 12: 15-31Crossref (77) 2015Palpant Jones Moon R.T. Ruzzo W.L. Inhibition ?-catenin respecifies anterior-like into beating cardiomyocytes.Development. 2015; 142: 3198-3209Crossref (36) report also demonstrated was able EndoMT mesenchymal vitro (Neri While findings encouraging, none showed marker evaluated full potential study, used NKX2-5:GFP hPSC line (Elliott 2011Elliott D.A. Braam S.R. Koutsis Ng E.S. Jenny Lagerqvist E.L. Hatzistavrou Hirst Q.C. al.NKX2-5(eGFP/w) hESCs isolation cardiomyocytes.Nat. Methods. 8: 1037-1040Crossref (279) identify characterize promote NKX2-5+CD31+ population. We show plays generation collection define vivo one shows high transcriptional similarity primary fetal endocardium. display following co-culture with target VIC lineage. As both involved transgenic HES3-NKX2-5eGFP/w evaluate population, propose represents stage development. either BMP4 or expressed adjacent (Neuhaus 1999Neuhaus Rosen Thies R.S. specific mouse BMP-10 member TGF-beta superfamily.Mech. 1999; 80: 181-184Crossref (102) Somi 2004Somi Buffing Van Den Expression protein-10 mRNA chicken development.Anat. A Discov. Mol. Evol. 2004; 279: 579-582Crossref (15) were manipulated induced concentrations activin (6 ng/mL Activin A, 10 BMP4) our (Lee 2017Lee J.H. Protze S.I. Laksman Backx Keller G.M. Pluripotent Stem Cell-Derived Atrial Ventricular Cardiomyocytes Develop Distinct Mesoderm Populations.Cell 21: 179-194Abstract ventricular (Figure 1A). effects varying basic fibroblast growth (bFGF) presence (10 ng/mL) added time intervals: days 3–9, 5–9, 7–9. At day 9, harvested analyzed CD31+ None alone bFGF contained indicating exogenous under conditions 1B). did large populations, regardless concentration used. When paired signaling, significantly increased proportion generated. 5–9 higher than extended agonist 3 9 (Figures 1C 1D). frame, highest frequency combination 50 100 1C). Addition 7–9 promoted comparable frequencies but larger NKX2-5–CD31+ subpopulations BMP10/FGF50- BMP10/FGF100-treated groups 1E). Based on observations number 1F), chose use together experiments. next determined optimal amount found 1G–1I). above carried out modified protocol, normally includes WNT inhibition step between 5. investigate Wnt activated pathway GSK-3 inhibitor CHIR99021 inhibited IWP2 2, 4, 6 days, beginning S1A–S1E). each different intervals resulted significant reduction CHIR impact total produced cultures, led decrease frequency. Together, indicate endogenously differentiating sufficient Next, investigated VEGF/KDR derivative low levels KDR (data shown). outlined Figure S1F increase S1G S1J). Rather, when 3–9 window, corresponding S1G–S1J). observations, included protocol described below. Kinetic analyses optimized culture revealed specification, emerged 8 persisted 12 2B). Although developed differ two 8, BMP10-induced BMP4-treated 2E). Exploiting observation treated without effort non-endocardial 2B, VEGF/bFGF detected differentiation. upregulated any conditions, reasoned should represent them “control” studies. kinetic experiments isolate cultures further isolated fluorescence-activated sorting (FACS) distinguish neuregulin, GATA4, GATA5, NFATC1, atrial natriuretic peptide receptor NPR3 (Charron Nemer, 1999Charron Nemer GATA development.Semin. 85-91Crossref (200) 1998de Timmerman L.A. Takimoto Yoshida Elia A.J. Samper Potter Wakeham Marengere Langille al.Role NF-ATc morphogenesis septum.Nature. 1998; 392: 182-186Crossref (523) 2002Nemer Cooperative interaction GATA5 regulates endothelial-endocardial cells.Development. 4045-4055PubMed Ranger 1998Ranger A.M. Grusby Hodge M.R. Gravallese E.M. Brousse F.C. Hoey Mickanin Baldwin H.S. Glimcher L.H. formation.Nature. 186-190Crossref (498) Rivera-Feliciano 2006Rivera-Feliciano Lee K.H. Kong S.W. Rajagopal Springer Izumo Tabin C.J. requires Gata4 endothelial-derived 133: 3607-3618Crossref (137) 2013Wu Nfatc1 directs primordium.Trends Cardiovasc. 2013; 23: 294-300Crossref (20) 2016Zhang Li Sucov H.M. Endocardium Minimally Contributes Endothelium Embryonic Free Walls.Circ. 118: 1880-1893Crossref (71) 3A). progenitors. 3B, all defining compared control NRG1, similar whereas NKX2-5+CD31+cells. suggest contain differences may reflect maturation. NKX2-5+CD31– markers, ISL1 suggesting contains Taken together, molecular consistent interpretation determine whether method impacted profile patterns BMP10. both, 3C). Collectively, comparative demonstrate more effective specifying agonists, cultured days. maintain phenotype period. maintaining tested 3D). sustained those maintained 3E). culture. Notably, period upregulation does Immunostaining analysis GATA4 protein, qRT-PCR profiles S2A S2B). contrast, no expression. nuclei S2C). If equivalent establishing assay, SIRPA+ (Dubois 2011Dubois Craft Elliott A.G. Gramolini SIRPA cell-surface isolating Biotechnol. 29: 1011-1018Crossref (362) 16, 23 BMP10, NPPA, NPPB, IRX3 2004Chen Acosta Lu Bao Schneider Chien K.R. al.BMP10 murine cardiogenesis.Development. 131: 2219-2231Crossref (336) Christoffels 2000Christoffels V.M. Keijser Houweling A.C. Clout D.E. Patterning heart: identification five Iroquois 224: 263-274Crossref (117) Kim 2012Kim Bruneau B.G. Hui C.C. homeodomain function.Circ. 1513-1524Crossref (31) Sergeeva Christoffels, 2013Sergeeva I.A. brain peptide, biomarkers disease.Biochim. 1832: 2403-2413Crossref (99) activation S3A). S3B, 9-treated later populations. most striking observed 9-derived only untreated Culture reduced compact HEY2 (Koibuchi Chin, 2007Koibuchi Chin M.T. CHF1/Hey2 left maturation suppression ectopic expression.Circ. 100: 850-855Crossref PubM