Stepwise Generation of Human Induced Pluripotent Stem Cell-Derived Cardiac Pericytes to Model Coronary Microvascular Dysfunction

HomeCirculationVol. 147, No. 6Stepwise Generation of Human Induced Pluripotent Stem Cell–Derived Cardiac Pericytes to Model Coronary Microvascular Dysfunction Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessLetterPDF/EPUBStepwise Generation of Human Induced Pluripotent Stem Cell–Derived Cardiac Pericytes to Model Coronary Microvascular Dysfunction Mengcheng Shen, Chun Liu, Shane Rui Zhao, Amit Manhas, Laksshman Sundaram, Mohamed Ameen and Joseph C. Wu Mengcheng ShenMengcheng Shen https://orcid.org/0000-0001-7037-6159 From Stanford Cardiovascular Institute (M.S., C.L., S.R.Z., A.M., M.A., J.C.W.), Stanford University, CA. Institute for Stem Cell Biology and Regenerative Medicine (M.S., C.L., S.R.Z., A.M., J.C.W.), Stanford University, CA. the Departments of Medicine (Division of Cardiology) (M.S., C.L., S.R.Z., A.M., J.C.W.), Stanford University, CA. *M. Shen and C. Liu contributed equally. Search for more papers by this author , Chun LiuChun Liu https://orcid.org/0000-0002-8242-3896 From Stanford Cardiovascular Institute (M.S., C.L., S.R.Z., A.M., M.A., J.C.W.), Stanford University, CA. Institute for Stem Cell Biology and Regenerative Medicine (M.S., C.L., S.R.Z., A.M., J.C.W.), Stanford University, CA. the Departments of Medicine (Division of Cardiology) (M.S., C.L., S.R.Z., A.M., J.C.W.), Stanford University, CA. *M. Shen and C. Liu contributed equally. Search for more papers by this author , Shane Rui ZhaoShane Rui Zhao https://orcid.org/0000-0002-2137-5536 From Stanford Cardiovascular Institute (M.S., C.L., S.R.Z., A.M., M.A., J.C.W.), Stanford University, CA. Institute for Stem Cell Biology and Regenerative Medicine (M.S., C.L., S.R.Z., A.M., J.C.W.), Stanford University, CA. the Departments of Medicine (Division of Cardiology) (M.S., C.L., S.R.Z., A.M., J.C.W.), Stanford University, CA. Search for more papers by this author , Amit ManhasAmit Manhas https://orcid.org/0000-0001-6044-4576 From Stanford Cardiovascular Institute (M.S., C.L., S.R.Z., A.M., M.A., J.C.W.), Stanford University, CA. Institute for Stem Cell Biology and Regenerative Medicine (M.S., C.L., S.R.Z., A.M., J.C.W.), Stanford University, CA. the Departments of Medicine (Division of Cardiology) (M.S., C.L., S.R.Z., A.M., J.C.W.), Stanford University, CA. Illumina Artificial Intelligence Laboratory, Illumina Inc., Foster City, CA (A.M., L.S.). Search for more papers by this author , Laksshman SundaramLaksshman Sundaram Computer Science (L.S.), Stanford University, CA. Illumina Artificial Intelligence Laboratory, Illumina Inc., Foster City, CA (A.M., L.S.). Search for more papers by this author , Mohamed AmeenMohamed Ameen From Stanford Cardiovascular Institute (M.S., C.L., S.R.Z., A.M., M.A., J.C.W.), Stanford University, CA. Cancer Biology (M.A.), Stanford University, CA. Search for more papers by this author and Joseph C. WuJoseph C. Wu Correspondence to: Joseph C. Wu, MD, PhD, 265 Campus Drive, Room G1120B, Stanford, CA 94305. Email E-mail Address: [email protected] https://orcid.org/0000-0002-6068-8041 From Stanford Cardiovascular Institute (M.S., C.L., S.R.Z., A.M., M.A., J.C.W.), Stanford University, CA. Institute for Stem Cell Biology and Regenerative Medicine (M.S., C.L., S.R.Z., A.M., J.C.W.), Stanford University, CA. the Departments of Medicine (Division of Cardiology) (M.S., C.L., S.R.Z., A.M., J.C.W.), Stanford University, CA. Search for more papers by this author Originally published6 Feb 2023https://doi.org/10.1161/CIRCULATIONAHA.122.061770Circulation. 2023;147:515–518Cardiac pericytes (CPs), a major mural cell type maintaining homeostasis, integrity, and perfusion of the coronary microvasculature, remain the most enigmatic and underappreciated cell population in the heart. Accumulating evidence suggests that CPs play a key role in cardiovascular complications such as coronary vasospasm, no reflow after myocardial infarction, and cancer drug–induced cardiotoxicity.1 However, the lack of unequivocal cell markers and specific tools for characterization, lineage tracing, and conditional targeting of CPs has precluded a comprehensive understanding of their pathogenic role in coronary microvascular dysfunction. Here, we report a stepwise approach to generate first-of-its-kind CPs from human induced pluripotent stem cells (iPSCs), which were shown to transcriptionally and functionally resemble their primary counterparts.To derive pure iPSC-CPs, we first generated epicardial cells (EPIs), the predominant progenitor cells giving rise to CPs, in a stepwise fashion (Figure [A]). We found that temporal activation or inhibition of key morphogens at different differentiation stages enabled the generation of pure EPIs (Figure [B, i]).2 In contrast, using an extant protocol that only manipulated Wnt signaling throughout the entire differentiation process (referred to as the GiWiGi protocol),3 we observed a large variation of EPI induction efficiency (ranging from 3% to 87%) even among iterative differentiations (Figure [B, ii]). Moreover, our stepwise protocol generated more mature EPIs (ALDH1A2 [aldehyde dehydrogenase 1 family member A2], UPK3B [uroplakin 3B],and ANXA8 [annexin A8]) than did the GiWiGi protocol (Figure [C]). Finally, we performed single-cell ATAC (assay for transposase-accessible chromatin) sequencing on stepwise and GiWiGi EPIs and projected them onto human fetal heart single-cell ATAC sequencing data (Figure [D, i]) based on their chromatin landscape similarities.4 Our data showed that >30% of GiWiGi EPIs acquired a fibroblast fate (Figure [D, ii]), precluding the generation of pure CPs for cell type–specific studies. In contrast, stepwise EPIs overlapped with primary EPIs and endocardial cells, 2 progenitors that can give rise to CPs (Figure [D, iii]).5Download figureDownload PowerPointFigure. Phenotypic characterization and functional assessment of stepwise differentiated iPSC-cardiac pericytes. A, Schematic showing stage-specific inhibition and activation of morphogens to generate pure EPIs from iPSCs. Representative bright-field images for each stage of cell differentiation are demonstrated. B, Immunofluorescent images (ZO1 and WT1), flow cytometry (WT1) graphs (iPSC lines, n=10; 5 males, 5 females), and quantitative data showing EPI induction efficiency by stepwise (B, i) and GiWiGi (B, ii) protocols. C, Quantitative reverse transcription polymerase chain reaction results showing expression levels of canonical (WT1, TBX18, and TCF21) and mature (ALDH1A2, UPK3B, and ANXA8) markers of EPIs derived by both protocols. D, scATAC-seq of EPIs generated by both GiWiGi (D, ii) and stepwise (D, iii) protocols are projected to that of human fetal heart cell clusters (D, i). Dotted frames indicate the EPI cluster in the human fetal heart scATAC-seq UMAP. E, Immunoblots showing time-dependent changes in CP and SMC markers during differentiation. iPSC-SMCs were used as a control. F, Flow cytometry and quantitative data (n=5; 2 males, 3 females) showing iPSC-CP yields with and without exogenous PDGF-BB. G, Heatmap showing transcriptomic similarities of pericyte markers between primary and iPSC-CPs by quantitative reverse transcription polymerase chain reaction. H, Bright-field and immunofluorescent images of iPSC-derived SMCs, CFs, and ECs. I, Calcium imaging using Fura-2 AM to quantitatively compare carbachol-induced intracellular calcium increases in iPSC-SMCs and iPSC-CPs. J, Tubular networks formed by iPSC-CPs, iPSC-ECs, and in combination in vitro (n=2; 1 male, 2 females). iPSC-CPs were fluorescently labeled with Calcein Red-Orange AM. K, Vasculatures formed by iPSC-ECs alone (1.2×106 cells/Matrigel plug) or in combination with iPSC-CPs (1.0×106 iPSC-ECs and 0.2×106 iPSC-CPs/Matrigel plug) after being implanted in male immunodeficiency NOD SCID mice (n=3 per group) for 7 days. Antibodies targeting iPSC-ECs and iPSC-CPs are human-specific. L, Dose–response curves for (L, i) iPSC-CMs, iPSC-CPs, and iPSC-ECs (n=3; 1 male, 2 females per group) and (L, ii) primary and iPSC-CPs (n=2; 1 male, 1 female per group) after 72 h of sunitinib (0–100 µM) treatment using a PrestoBlue viability assay. M, Bright-field images showing the cell morphology of primary and iPSC-CPs after 72 h of vehicle, sunitinib (5 µM), and sunitinib (5 µM)/thalidomide (1 µM) treatment. N, BrdU incorporation assays showing suppressed iPSC-CP proliferation by sunitinib (n=5; 2 males, 3 females per group) in a dose-dependent manner. O, Heatmap showing gene clustering patterns of iPSC-CPs treated with vehicle, sunitinib (5 µM), and sunitinib (5 µM)/thalidomide (1 µM) for 72 h. Key changes of hallmarks in each cluster are highlighted. P, Gene ontology pathway analysis of iPSC-CPs treated with sunitinib vs other conditions. GraphPad Prism 9 was used for statistical analysis. All data are presented as mean±SEM. Statistical significance was assessed using a nonparametric statistical procedure (Wilcoxon signed-rank test for 2 groups and Kruskal–Wallis test for >2 groups). *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. α-SMA indicates alpha smooth muscle actin; ABCC9, ATP binding cassette subfamily C member 9; ACTA2, actin alpha 2, smooth muscle; ALDH1A2, aldehyde dehydrogenase 1 family member A2; ANPEP, alanyl aminopeptidase; ANXA8, annexin A8; BMP4, bone morphogenetic protein 4; CD13, cluster of differentiation 13; CD146, cluster of differentiation 146; CF, cardiac fibroblast; CM, cardiomyocyte; CP, cardiac pericyte; CSPG4, chondroitin sulfate proteoglycan 4; EC, endothelial cell; EPI, epicardial cell; FC, fold change; FDR, false discovery rate; FGF2, fibroblast growth factor 2; iPSC, human induced pluripotent stem cell; KCNJ8, potassium Inwardly rectifying channel subfamily J member 8; LPM, lateral plate mesoderm; MCAM, melanoma cell adhesion molecule; MPS, mid-primitive streak; NG2, neural/glial antigen 2; ns, not significant; NOD SCID, nonobese diabetic and severe combined immunodeficiency; PEO, pre-epicardial organ; PDGFRB, platelet-derived growth factor receptor beta; PDGF-BB, platelet-derived growth factor BB; RGS5, regulator of G protein signaling 5; scATAC-seq, single-cell assay for transposase-accessible chromatin sequencing; SM, splanchnic mesoderm; SMC, smooth muscle cell; SM-MHC, smooth muscle-myosin heavy chain; ST, septum transversum; TBX18, T-box transcription factor 18; TCF21, transcription factor 21; UMAP, uniform manifold approximation and projection; UPK3B, uroplakin 3B; WT1, wilms tumor 1; and ZO1, zonula occludens 1.Next, we differentiated stepwise EPIs in a commercial pericyte medium and observed that it took at least 12 days to generate iPSC-CPs (Figure [E]) with low efficiency (Figure [F]). Since pericyte–endothelial cell crosstalk and PDGFR (platelet-derived growth factor receptor) signaling are critical for pericyte development, we successfully generated pure iPSC-CPs (PDGFRβ+/CD146+/NG2+/CD13+) with exogenous PDGF-BB(platelet-derived growth factor BB), a ligand predominantly secreted by endothelial cells (Figure [F]). Next, we confirmed comparable expression levels of cell markers between primary and iPSC-CPs using quantitative reverse transcription polymerase chain reaction (Figure [G]). Finally, we showed that iPSC-CPs are negative for smooth muscle cell markers and not reactive to antifibroblast antibody TE-7 (Figure [H]), further confirming their cell-type specificity.Next, we examined the relative cell contraction capacities of iPSC-CPs versus iPSC smooth muscle cells by measuring intracellular calcium transients after carbachol treatment. While both cell types showed a stereotypic contraction pattern to carbachol, iPSC-CPs had a much smaller calcium amplitude, indicating weaker contraction because of lower intracellular calcium increase and lower expression levels of contractile proteins (Figure [I]). We also tested the proangiogenic potentials of iPSC-CPs. We observed that iPSC-CPs not only spontaneously formed a tubular network in vitro, but also improved tube formation in vitro and neovessel maturation in vivo when they were mixed with iPSC endothelial cells (Figure [J and K]).A previous study reported that CPs were the primary targets of sunitinib-induced coronary microvascular abnormalities and cardiac dysfunction in mice.1 To validate whether our iPSC-CPs can recapitulate this phenotype, we treated iPSC-CPs, iPSC-ECs, and iPSC-cardiomyocytes with a wide dose range of sunitinib. In line with the in vivo findings,1 we observed that iPSC-CPs were more sensitive than iPSC cardiomyocytes to sunitinib-induced cell death (Figure [L, i]). In contrast, primary and iPSC-CPs showed a similar cytotoxicity profile to sunitinib treatment (Figure [L, ii and M]). Sunitinib significantly suppressed iPSC-CP proliferation in a dose-dependent manner (Figure [N]). Notably, thalidomide, a drug that can rescue mice from sunitinib-induced CP loss and cardiac dysfunction,1 profoundly rescued CP death (Figure [M]) and restored genes associated with cell cycle, pericyte function, and DNA damage to a baseline level (Figure [O and P]). Because the precise mechanisms by which sunitinib-induced CP cytotoxicity remain elusive, our iPSC model, if combined with high throughput screening tools, may help identify novel therapeutics to prevent cardiotoxicity induced by sunitinib or other agents that primarily inhibit PDGFR signaling.In summary, our human iPSC-CPs represent a novel model system essential to understanding coronary microvascular dysfunction. The high resemblance between iPSC-CPs and their in vivo counterparts will help researchers understand genetic- or environmental factor–induced coronary microvasculature malformation, spur the development of more effective proangiogenic cell therapies for patients experiencing myocardial infarction, and spark novel approaches for drug toxicity evaluation and discovery.Human heart tissue samples, animals, and iPSCs used in this study were approved by the Stanford Institutional Review Board (no. 42056), Administrative Panel on Laboratory Animal Care (no. 34262), and Stem Cell Research Oversight Committee (no. 563), respectively. All single-cell ATAC sequencing data can be accessed at GSE181346 and RNA sequencing data at GSE210652. Raw data can be made available upon request from the corresponding author.Article InformationAcknowledgmentsThe authors thank Blake Wu for proofreading this article and Dr Lingfeng Luo for technical support in OCT sample preparation.Sources of FundingThis study was supported by research grants from the Tobacco-Related Disease Research Program (no. 30FT0852 to M.S.); American Heart Association Career Development Award (no. 19CDA34760019 to C.L.); American Heart Association–Allen Initiative (no. 19PABHI34580007 to J.C.W); and the National Institutes of Health (nos. R01 HL126527, R01 HL130020, R01 HL146690, R01 HL141851, and P01 HL141084 to J.C.W).Nonstandard Abbreviations and AcronymsCPcardiac pericyteiPSChuman induced pluripotent stem cellECendothelial cellEPIepicardial cellDisclosures J.C.W. is a cofounder of Greenstone Biosciences but has no competing interests, as the work presented was performed independently. The other authors declare no competing interests.Footnotes*M. Shen and C. Liu contributed equally.For Sources of Funding and Disclosures, see page 518.Circulation is available at www.ahajournals.org/journal/circCorrespondence to: Joseph C. Wu, MD, PhD, 265 Campus Drive, Room G1120B, Stanford, CA 94305. Email joewu@stanford.eduReferences1. Chintalgattu V, Rees ML, Culver JC, Goel A, Jiffar T, Zhang J, Dunner K, Pati S, Bankson JA, Pasqualini R, et al. Coronary microvascular pericytes are the cellular target of sunitinib malate-induced cardiotoxicity.Sci Transl Med. 2013; 5:187ra–18169. doi: 10.1126/scitranslmed.3005066CrossrefGoogle Scholar2. Shen M, Liu C, Wu JC. Generation of embryonic origin-specific vascular smooth muscle cells from human induced pluripotent stem cells.Methods Mol Biol (Clifton, NJ). 2022; 2429:233–246. doi: 10.1007/978-1-0716-1979-7_15CrossrefMedlineGoogle Scholar3. Bao X, Lian X, Hacker TA, Schmuck EG, Qian T, Bhute VJ, Han T, Shi M, Drowley L, Plowright A, et al. Long-term self-renewing human epicardial cells generated from pluripotent stem cells under defined xeno-free conditions.Nat Biomed Eng. 2016; 1: doi: 10.1038/s41551-016-0003CrossrefMedlineGoogle Scholar4. Ameen M, Sundaram L, Shen M, Banerjee A, Kundu S, Nair S, Shcherbina A, Gu M, Wilson KD, Varadarajan A, et al. Integrative single cell analysis of human cardiogenesis.Cell. doi: 10.1016/j.cell.2022.11.028CrossrefGoogle Scholar5. Chen Q, Zhang H, Liu Y, Adams S, Eilken H, Stehling M, Corada M, Dejana E, Zhou B, Adams RH. Endothelial cells are progenitors of cardiac pericytes and vascular smooth muscle cells.Nat Commu. 2016; 7:12422. doi: 10.1038/ncomms12422CrossrefMedlineGoogle Scholar eLetters(0)eLetters should relate to an article recently published in the journal and are not a forum for providing unpublished data. Comments are reviewed for appropriate use of tone and language. Comments are not peer-reviewed. Acceptable comments are posted to the journal website only. 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