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• W2238713044 abstract "•CD13 and ROR2 separate hESC-derived MIXL1+ mesoderm from MIXL1+ endoderm•CD13 and ROR2 select for a population of highly enriched pre-cardiac mesoderm•CD13+/ROR2+ cells derived from hESCs engraft into porcine, but not murine hearts•CD13+/ROR2+ cells differentiate to all major cardiac lineages in the pig heart The generation of tissue-specific cell types from human embryonic stem cells (hESCs) is critical for the development of future stem cell-based regenerative therapies. Here, we identify CD13 and ROR2 as cell-surface markers capable of selecting early cardiac mesoderm emerging during hESC differentiation. We demonstrate that the CD13+/ROR2+ population encompasses pre-cardiac mesoderm, which efficiently differentiates to all major cardiovascular lineages. We determined the engraftment potential of CD13+/ROR2+ in small (murine) and large (porcine) animal models, and demonstrated that CD13+/ROR2+ progenitors have the capacity to differentiate toward cardiomyocytes, fibroblasts, smooth muscle, and endothelial cells in vivo. Collectively, our data show that CD13 and ROR2 identify a cardiac lineage precursor pool that is capable of successful engraftment into the porcine heart. These markers represent valuable tools for further dissection of early human cardiac differentiation, and will enable a detailed assessment of human pluripotent stem cell-derived cardiac lineage cells for potential clinical applications. The generation of tissue-specific cell types from human embryonic stem cells (hESCs) is critical for the development of future stem cell-based regenerative therapies. Here, we identify CD13 and ROR2 as cell-surface markers capable of selecting early cardiac mesoderm emerging during hESC differentiation. We demonstrate that the CD13+/ROR2+ population encompasses pre-cardiac mesoderm, which efficiently differentiates to all major cardiovascular lineages. We determined the engraftment potential of CD13+/ROR2+ in small (murine) and large (porcine) animal models, and demonstrated that CD13+/ROR2+ progenitors have the capacity to differentiate toward cardiomyocytes, fibroblasts, smooth muscle, and endothelial cells in vivo. Collectively, our data show that CD13 and ROR2 identify a cardiac lineage precursor pool that is capable of successful engraftment into the porcine heart. These markers represent valuable tools for further dissection of early human cardiac differentiation, and will enable a detailed assessment of human pluripotent stem cell-derived cardiac lineage cells for potential clinical applications. The mammalian heart has been reported to possess a limited regenerative capacity; however, this is not sufficient to effectively remuscularize the heart after a myocardial infarction (MI) (Ali et al., 2014Ali S.R. Hippenmeyer S. Saadat L.V. Luo L. Weissman I.L. Ardehali R. Existing cardiomyocytes generate cardiomyocytes at a low rate after birth in mice.Proc. Natl. Acad. Sci. USA. 2014; 111: 8850-8855Crossref PubMed Scopus (166) Google Scholar). In the case of severe MI the human heart experiences dramatic loss of cardiomyocytes, the basic functional unit of the heart, with estimates placing that loss upward of a billion cells (Bergmann et al., 2009Bergmann O. Bhardwaj R.D. Bernard S. Zdunek S. Barnabe-Heider F. Walsh S. Zupicich J. Alkass K. Buchholz B.A. Druid H. et al.Evidence for cardiomyocyte renewal in humans.Science. 2009; 324: 98-102Crossref PubMed Scopus (2252) Google Scholar, Laflamme and Murry, 2005Laflamme M.A. Murry C.E. Regenerating the heart.Nat. Biotechnol. 2005; 23: 845-856Crossref PubMed Scopus (807) Google Scholar). As heart disease continues to be a leading cause of mortality worldwide, the use of human pluripotent stem cells (hPSCs) for cardiac regeneration is a compelling approach and has become a major focus of stem cell research (Cibelli et al., 2013Cibelli J. Emborg M.E. Prockop D.J. Roberts M. Schatten G. Rao M. Harding J. Mirochnitchenko O. Strategies for improving animal models for regenerative medicine.Cell Stem Cell. 2013; 12: 271-274Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, Matsa et al., 2014Matsa E. Burridge P.W. Wu J.C. Human stem cells for modeling heart disease and for drug discovery.Sci. Transl. Med. 2014; 6: 239ps236Crossref Scopus (148) Google Scholar). Indeed, the first human subject receiving hPSC-derived cardiovascular progenitors as a therapeutic for heart failure has recently been reported (Menasche et al., 2015Menasche P. Vanneaux V. Hagege A. Bel A. Cholley B. Cacciapuoti I. Parouchev A. Benhamouda N. Tachdjian G. Tosca L. et al.Human embryonic stem cell-derived cardiac progenitors for severe heart failure treatment: first clinical case report.Eur. Heart J. 2015; 36: 2011-2017Crossref PubMed Scopus (309) Google Scholar). The progression of in vitro-derived cardiac cells toward therapeutic applications will be greatly assisted by an increasingly detailed understanding of cardiac lineage commitment. Moreover, it is still unclear whether committed progenitors or fully differentiated cells will be most efficacious for any particular therapeutic use. Indeed, homogeneous populations of cardiovascular progenitor cells that have the capacity to form multiple cardiac cell types (e.g., cardiomyocytes, fibroblasts, and vascular cells) may have a role to play in future stem cell-based therapies. In this context, further research is required to elaborate the cardiac lineage tree and to devise methods for isolating key cell types and their progenitors. Generation of a pure hPSC-derived cardiac population through an intermediate mesodermal germ layer (from which the cardiac tissue arises) may be of therapeutic importance. Previous studies have identified SSEA1, PDGFRα, and KDR as surface markers on PSC-derived mesodermal progenitors with capacity to generate cardiovascular lineages (Blin et al., 2010Blin G. Nury D. Stefanovic S. Neri T. Guillevic O. Brinon B. Bellamy V. Rucker-Martin C. Barbry P. Bel A. et al.A purified population of multipotent cardiovascular progenitors derived from primate pluripotent stem cells engrafts in postmyocardial infarcted nonhuman primates.J. Clin. Invest. 2010; 120: 1125-1139Crossref PubMed Scopus (261) Google Scholar, Kattman et al., 2011Kattman S.J. Witty A.D. Gagliardi M. Dubois N.C. Niapour M. Hotta A. Ellis J. Keller G. Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines.Cell Stem Cell. 2011; 8: 228-240Abstract Full Text Full Text PDF PubMed Scopus (860) Google Scholar, Yang et al., 2008Yang L. Soonpaa M.H. Adler E.D. Roepke T.K. Kattman S.J. Kennedy M. Henckaerts E. Bonham K. Abbott G.W. Linden R.M. et al.Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population.Nature. 2008; 453: 524-528Crossref PubMed Scopus (1152) Google Scholar). Subsequently, SIRPA and VCAM1 were identified as novel markers of cardiomyogenic lineages (Dubois et al., 2011Dubois N.C. Craft A.M. Sharma P. Elliott D.A. Stanley E.G. Elefanty A.G. Gramolini A. Keller G. SIRPA is a specific cell-surface marker for isolating cardiomyocytes derived from human pluripotent stem cells.Nat. Biotechnol. 2011; 29: 1011-1018Crossref PubMed Scopus (405) Google Scholar, Elliott et al., 2011Elliott D.A. Braam S.R. Koutsis K. Ng E.S. Jenny R. Lagerqvist E.L. Biben C. Hatzistavrou T. Hirst C.E. Yu Q.C. et al.NKX2-5(eGFP/w) hESCs for isolation of human cardiac progenitors and cardiomyocytes.Nat. Methods. 2011; 8: 1037-1040Crossref PubMed Scopus (316) Google Scholar, Skelton et al., 2014Skelton R.J. Costa M. Anderson D.J. Bruveris F. Finnin B.W. Koutsis K. Arasaratnam D. White A.J. Rafii A. Ng E.S. et al.SIRPA, VCAM1 and CD34 identify discrete lineages during early human cardiovascular development.Stem Cell Res. 2014; 13: 172-179Crossref PubMed Scopus (48) Google Scholar, Uosaki et al., 2011Uosaki H. Fukushima H. Takeuchi A. Matsuoka S. Nakatsuji N. Yamanaka S. Yamashita J.K. Efficient and scalable purification of cardiomyocytes from human embryonic and induced pluripotent stem cells by VCAM1 surface expression.PLoS One. 2011; 6: e23657Crossref PubMed Scopus (230) Google Scholar). These studies provide a foundation upon which to construct a human cardiovascular cell lineage tree based on cell-surface markers, analogous to that of the hematopoietic system. Other surface markers, such as CD13 and ROR2, have been used in combination with PDGFRα and KDR to isolate progenitors capable of giving rise to enriched cardiac cell populations (Ardehali et al., 2013Ardehali R. Ali S.R. Inlay M.A. Abilez O.J. Chen M.Q. Blauwkamp T.A. Yazawa M. Gong Y. Nusse R. Drukker M. et al.Prospective isolation of human embryonic stem cell-derived cardiovascular progenitors that integrate into human fetal heart tissue.Proc. Natl. Acad. Sci. USA. 2013; 110: 3405-3410Crossref PubMed Scopus (53) Google Scholar). The combination of these four markers led to isolation of committed cardiovascular cells as shown by in vitro and in vivo analyses. However, the utility of CD13 and ROR2 as stand-alone markers of cardiac intermediates remains unclear. Here, we define CD13 and ROR2 as markers of mesodermal progenitors of cardiac cell lineages. Furthermore, in vivo cardiac differentiation and engraftment efficiency of CD13+/ROR2+ cells was compared in large (porcine) and small (murine) animal models. Our data demonstrate that human embryonic stem cell-derived cardiovascular progenitor cells (hESC-CPCs) engraft and differentiate into all cardiovascular lineages more efficiently in the porcine heart than in the mouse heart. Consistent with previous reports, these data suggest that the murine heart may be an inappropriate xenotransplantation model (Cibelli et al., 2013Cibelli J. Emborg M.E. Prockop D.J. Roberts M. Schatten G. Rao M. Harding J. Mirochnitchenko O. Strategies for improving animal models for regenerative medicine.Cell Stem Cell. 2013; 12: 271-274Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, van Laake et al., 2008van Laake L.W. Passier R. Doevendans P.A. Mummery C.L. Human embryonic stem cell-derived cardiomyocytes and cardiac repair in rodents.Circ. Res. 2008; 102: 1008-1010Crossref PubMed Scopus (196) Google Scholar, van Laake et al., 2009van Laake L.W. Passier R. den Ouden K. Schreurs C. Monshouwer-Kloots J. Ward-van Oostwaard D. van Echteld C.J. Doevendans P.A. Mummery C.L. Improvement of mouse cardiac function by hESC-derived cardiomyocytes correlates with vascularity but not graft size.Stem Cell Res. 2009; 3: 106-112Crossref PubMed Scopus (65) Google Scholar). The pig heart, however, may provide a useful pre-clinical platform upon which to test the regenerative potential of hESC-CPCs (Ye et al., 2014Ye L. Chang Y.H. Xiong Q. Zhang P. Zhang L. Somasundaram P. Lepley M. Swingen C. Su L. Wendel J.S. et al.Cardiac repair in a porcine model of acute myocardial infarction with human induced pluripotent stem cell-derived cardiovascular cells.Cell Stem Cell. 2014; 15: 750-761Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). Collectively, these findings enhance our understanding of cardiac mesoderm lineage formation, provide well-defined tools for the enrichment of cardiac-committed mesoderm, and demonstrate engraftment and differentiation of transplanted hESC-CPCs in porcine hearts. Initially, a stencil differentiation protocol (Myers et al., 2013Myers F.B. Silver J.S. Zhuge Y. Beygui R.E. Zarins C.K. Lee L.P. Abilez O.J. Robust pluripotent stem cell expansion and cardiomyocyte differentiation via geometric patterning.Integr. Biol. (Camb). 2013; 5: 1495-1506Crossref Scopus (20) Google Scholar) was used to isolate mesodermal cells based on GFP expression from the MIXL1 locus (Davis et al., 2008Davis R.P. Ng E.S. Costa M. Mossman A.K. Sourris K. Elefanty A.G. Stanley E.G. Targeting a GFP reporter gene to the MIXL1 locus of human embryonic stem cells identifies human primitive streak-like cells and enables isolation of primitive hematopoietic precursors.Blood. 2008; 111: 1876-1884Crossref PubMed Scopus (190) Google Scholar) (Figure S1). Microarray analysis of isolated cells from day 3 of differentiation was used to identify differences between MIXL1eGFP+ and MIXL1eGFP− transcriptomes. We identified 6,757 differentially regulated genes, of which 2,520 were upregulated ≥2-fold in the eGFP+ (MIXL1+) mesoderm population (Figure 1A ). These included known mesodermal markers, such as T, PDGFRα, MESP1, and EOMES, as well as two genes encoding for cell-surface proteins, CD13 (an aminopeptidase) and ROR2 (a Wnt receptor) (Figure 1A). To further investigate the expression profile of CD13 and ROR2, we differentiated MIXL1eGFP/w hESCs toward mesoderm and conducted flow cytometry analysis. On day 3 of differentiation, approximately 30% of cells co-expressed CD13 and ROR2 in several hPSC lines that were tested (Figure 1B). More efficient differentiation schemes using the H3 hESC line produced populations consisting upward of 80% CD13+/ROR2+ cells (Figures 1C, S2A, and S2B). Later in differentiation, the majority of cells downregulated CD13 while maintaining ROR2+ expression out to day 10 (∼92%) (Figure 1C). Flow cytometry analysis also showed that approximately 70% of the eGFP+ (MIXL1+) population expressed CD13 and ROR2 on day 3 of differentiation (Figure 1D). Comparatively, very few CD13+/ROR2+ (13R2+) cells were detected in the eGFP− (MIXL1−) fraction (∼4%) (Figure 1D), suggesting that CD13 and ROR2 are predominantly restricted to a mesoderm population marked by MIXL1 expression. In addition, qPCR analysis of 13R2+ cells confirmed the high expression levels of cardiac precursor markers such as PDGFRα (1.1 × 104-fold), HAND1 (5.4 × 103-fold), MESP1 (530-fold), and EOMES (1.4 × 104-fold) relative to GAPDH (Figure 1E). Subsequently, we performed expression profiling of triple-positive (MIXL1+/CD13+/ROR2+) and triple-negative (MIXL1−/CD13−/ROR2−) populations. Gene Ontology (GO) analysis revealed that the transcripts enriched in the MIXL1+/CD13+/ROR2+ population correlated with the processes of heart development (p = 6.79 × 10−10), germ layer formation (p = 1.78 × 10−6), gastrulation (p = 2.77 × 10−6), mesoderm development (p = 5.52 × 10−6), and heart morphogenesis (p = 2.18 × 10−5) (Figures 1F and 1G). We next sought to determine whether CD13 and ROR2 are expressed on MIXL1+ endoderm-derived cells. Flow cytometric analysis of MIXL1GFP/w cells differentiated under endodermal conditions (D'Amour et al., 2005D'Amour K.A. Agulnick A.D. Eliazer S. Kelly O.G. Kroon E. Baetge E.E. Efficient differentiation of human embryonic stem cells to definitive endoderm.Nat. Biotechnol. 2005; 23: 1534-1541Crossref PubMed Scopus (1344) Google Scholar) revealed no substantial expression of CD13 or ROR2 on MIXL1-eGFP+ endoderm cells (Figure 2A ). This observation was confirmed by qPCR, demonstrating a 26- and a 2-fold decrease in the expression of CD13 and ROR2, respectively, relative to mesodermal MIXL1-eGFP+ cells (Figure 2B). Furthermore, throughout differentiation 13R2+ cells expressed low levels of endodermal markers, including SOX17, SOX7, FOXA2, and HFN4A, relative to the CD13−/ROR2− fraction (Figure 2C). To confirm the restriction of CD13 and ROR2 expression to mesoderm-derived cells, we tested for the presence of the definitive endoderm cell-surface marker, CXCR4 (McGrath et al., 1999McGrath K.E. Koniski A.D. Maltby K.M. McGann J.K. Palis J. Embryonic expression and function of the chemokine SDF-1 and its receptor, CXCR4.Dev. Biol. 1999; 213: 442-456Crossref PubMed Scopus (397) Google Scholar, Yusuf et al., 2005Yusuf F. Rehimi R. Dai F. Brand-Saberi B. Expression of chemokine receptor CXCR4 during chick embryo development.Anat. Embryol. (Berl). 2005; 210: 35-41Crossref PubMed Scopus (39) Google Scholar). Flow cytometric analysis of mesoderm cells derived from unmodified H9 hESCs revealed that expression of CXCR4 and CD13/ROR2 is mutually exclusive (Figure 2D). This differential expression pattern was further confirmed by immunofluorescence staining (Figures 3A and S2C). 13R2+ cells also downregulated pluripotency markers, and expressed the cardiac mesoderm markers MESP1 and MIXL1 (88% ± 2.3% SEM, n = 3) (Figures 1E, 1F, 2C, 3A, S2D, and S2E). Together, these results indicate that CD13 and ROR2 can be used to preferentially select for mesoderm from a mixed population of differentiating hESCs.Figure 3CD13 and ROR2 Mark a Distinct, Transitory Pre-cardiac Mesoderm PopulationShow full caption(A) Day-4 ICC analysis of sorted 13R2+ and 13R2− cells from hESC mesoderm differentiations, showing expression of MIXL1, MESP1, CXCR4, and GATA4. Scale bars, 25 μm. See also Figures S2C and S2D.(B) Day-5 flow cytometric analysis of unsorted and sorted 13R2+ and 13R2− cells for expression of CD326 and CD56. See also Figure S2F.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Day-4 ICC analysis of sorted 13R2+ and 13R2− cells from hESC mesoderm differentiations, showing expression of MIXL1, MESP1, CXCR4, and GATA4. Scale bars, 25 μm. See also Figures S2C and S2D. (B) Day-5 flow cytometric analysis of unsorted and sorted 13R2+ and 13R2− cells for expression of CD326 and CD56. See also Figure S2F. In accordance with previous reports that an epithelial-to-mesenchymal transition (EMT) occurs at an early stage of mesoderm commitment, we analyzed the expression of EpCAM/CD326 and NCAM/CD56 in 13R2+ fractions (Evseenko et al., 2010Evseenko D. Zhu Y. Schenke-Layland K. Kuo J. Latour B. Ge S. Scholes J. Dravid G. Li X. MacLellan W.R. et al.Mapping the first stages of mesoderm commitment during differentiation of human embryonic stem cells.Proc. Natl. Acad. Sci. USA. 2010; 107: 13742-13747Crossref PubMed Scopus (182) Google Scholar). We observed that approximately 90% of sorted 13R2+ cells expressed CD56 and downregulated CD326 after 2 days of reculture, suggestive of an EMT process and mesoderm specification (Figure 3B). Comparatively, the CD13−/ROR2− (13R2−) fraction was largely CD56−/CD326+ (∼87%), consistent with an epithelial phenotype (Figures 3B and S2F). A proportion of the day-3 CD13+ and 13R2+ fraction also expressed PDGFRα (45.7% ± 2.1%, n = 3) and C-KIT (1.3% ± 0.8%, n = 3), respectively (Figures S3A and S3B). Furthermore, 18.8% ± 6.4% (n = 3) of the ROR2+ fraction expressed KDR (Figure S3C). Nonetheless, a large majority of day-3 13R2+ cells were negative for SSEA1 (Figure S3E). Surface markers associated with later stages of cardiac differentiation were also absent from the 13R2+ population, including VCAM1, SIRPA, and CD34 (Figures S3F and S3G). Collectively, these data suggest that CD13 and ROR2 mark a distinct, transitory state of EMT committed cells and can be used to prospectively enrich for pre-cardiac mesoderm, depleting both endodermal and residual pluripotent cells. Next, we sought to determine the efficiency at which purified 13R2+ cells differentiate toward definitive cardiovascular lineages. To assist downstream characterization, we generated a double reporter hESC line in which eGFP is expressed upon activation of endogenous NKX2-5 (Elliott et al., 2011Elliott D.A. Braam S.R. Koutsis K. Ng E.S. Jenny R. Lagerqvist E.L. Biben C. Hatzistavrou T. Hirst C.E. Yu Q.C. et al.NKX2-5(eGFP/w) hESCs for isolation of human cardiac progenitors and cardiomyocytes.Nat. Methods. 2011; 8: 1037-1040Crossref PubMed Scopus (316) Google Scholar) and mCherry expression is controlled by the αMHC promoter (Kita-Matsuo et al., 2009Kita-Matsuo H. Barcova M. Prigozhina N. Salomonis N. Wei K. Jacot J.G. Nelson B. Spiering S. Haverslag R. Kim C. et al.Lentiviral vectors and protocols for creation of stable hESC lines for fluorescent tracking and drug resistance selection of cardiomyocytes.PLoS One. 2009; 4: e5046Crossref PubMed Scopus (188) Google Scholar). This dual-color hESC line facilitates identification and quantification of cardiac progenitors and cardiomyocytes based upon expression of NKX2-5 and αMHC, respectively. Sorted 13R2− cells did not survive in our monolayer differentiation protocols for the extended time period required for analysis, so unsorted cells served as a control. Isolated 13R2+ cells were recultured under conditions promoting cardiomyocyte differentiation to characterize their developmental potential. Seven days post sort, 86% ± 3.2% (n = 3) of 13R2+ cells proceeded to express NKX2-5eGFP, compared with 22% ± 4.9% (n = 3) in the unsorted population (Figure 4A ). Furthermore, 63% ± 4.1% (n = 3) of 13R2+ cells differentiated to express mCherry in addition to eGFP (indicating progression to cardiomyocytes), whereas only 16% ± 4.0% (n = 3) of the unsorted population was observed to be eGFP+/mCherry+ (Figures 4A and S4A). Although differentiation efficiency varied significantly in unsorted cells, we observed a persistently high rate of cardiomyocyte generation when selecting for CD13/ROR2. Gene expression analysis supported these findings, and demonstrated that differentiating 13R2+ cells temporally expressed high levels of cardiac mesoderm genes followed by cardiovascular progenitor and definitive cardiomyocyte genes (Figures 4B and S4B). This was illustrated by elevated expression levels of HAND1 and MIXL1 at day 4, followed by ISL-1, MEF2C, TBX5, and NKX2-5 by days 7–10, and cTnT, MYL2, IRX4, and NPPA at later stages (Figures 4B and S4B). In addition, when we analyzed expression of eGFP and mCherry on days 7 and 14 as markers for NKX2-5 and αMHC, respectively, we noted higher expression levels in 13R2+ cells when compared with either day-3 sorted single positive CD13 or ROR2, day-4 sorted SSEA1+, and day-5 sorted PDGFR+/KDR+ populations (Figures S4C–S4H). Furthermore, when maintained in a monolayer culture, 13R2+ cells displayed cardiac troponin T (cTnT)-positive sarcomeric structures, and formed contractile 3D layers (Figures 4C and Movie S1). Together, these results suggest that 13R2+ cells give rise to a highly enriched population of cardiomyocytes. We next sought to determine whether 13R2+ cells can give rise to other cardiovascular lineages. Under cardiomyocyte culture conditions, a proportion of the day-3 sorted 13R2+ fraction progressed toward an αSMA+ phenotype (Figure 4C). Furthermore, the expression level of smooth muscle transcripts, ACTA2 (αSMA) and CNN1, were significantly higher in 13R2+ cells at various time points during days 7–14 of differentiation (p < 0.05) (Figure 4D). To further investigate the lineage potential of the 13R2+ fraction, we cultured day-3 sorted 13R2+ cells under conditions to promote smooth muscle differentiation (transforming growth factor β 2 ng/ml, platelet-derived growth factor β 10 ng/ml) (Cheung et al., 2014Cheung C. Bernardo A.S. Pedersen R.A. Sinha S. Directed differentiation of embryonic origin-specific vascular smooth muscle subtypes from human pluripotent stem cells.Nat. Protoc. 2014; 9: 929-938Crossref PubMed Scopus (55) Google Scholar). After 11 days in culture, 13R2+ cells expressed high levels of ACTA2 (αSMA) and CNN1 transcripts, and low levels of VE-cadherin, consistent with a smooth muscle phenotype (Figure 4E). Enrichment for smooth muscle cells in the differentiated 13R2+ population was confirmed by protein-level expression of αSMA and CNN1, as determined by immunocytochemistry (ICC) (Figure S4I). A fraction of 13R2+ cells also differentiated toward a VE-cadherin+ phenotype in standard cardiomyocyte differentiations (Figure 4C). To further characterize endothelial differentiation, we cultured day-3 sorted 13R2+ cells under endothelial conditions (50 ng/ml vascular endothelial growth factor, 20 ng/ml stem cell factor, 10 ng/ml basic fibroblast growth factor). After 11 days of culture under these conditions, 13R2+ cells expressed high levels of the endothelial markers VE-cadherin, TAL1, TEK, KDR, and vWF (Figure 4E). Furthermore, after 14 days flow cytometric analysis revealed that a subset of sorted 13R2+ cells proceeded to co-express CD31/CD34 and CD31/KDR (32% and 37%, respectively), consistent with an endothelial phenotype (Figures 4F and S4J). Taken together, these results suggest that 13R2+ cells on day 3 of differentiation represent cardiovascular mesoderm capable of giving rise to cardiomyocytes, smooth muscle, and endothelial cells. To determine the gene expression profile at different stages of cardiac differentiation from hESCs, we performed transcriptome (RNA-seq) analysis on undifferentiated hESCs, 13R2+ and 13R2− populations from day 3, 13R2+/NKX2-5+, and 13R2+/NKX2-5− from day 7, and 13R2+/NKX2-5+/αMHC+ and 13R2+/NKX2-5+/αMHC− from day 14 (Figure S5). These data supported previous findings showing an enrichment of pre-cardiac mesodermal genes and concomitant downregulation of pluripotency genes in the 13R2+ population on day 3 (Figure 5A ). Day-7 13R2+/NKX2-5+ cells expressed cardiac transcription factors such as MEF2-C, NKX2-5, TBX20, and TBX5, suggestive of commitment to a cardiac progenitor cell type (Figure 5A). Day-7 13R2+/NKX2-5+ cells were also enriched for cardiomyocyte markers TNNT2, KCNIP2, KCNH7, and MYL4 (Figure 5A). The day-14 13R2+/NKX2-5+/αMHC+ fraction maintained expression of these cardiomyocyte markers, in addition to upregulating other cardiomyocyte genes such as NPPA, NPPB, MYH7, and MYL7, suggestive of a progression toward a more differentiated cardiomyocyte phenotype (Figure 5A). In addition, both day-14 13R2+/NKX2-5+/αMHC+ and 13R2+/NKX2-5+/αMHC− populations were enriched for smooth muscle genes, such as MYH11, CNN1, and ACTA1, suggesting that these populations may also contain vascular smooth muscle cells (Figure 5A). GO analysis of the upregulated transcripts in day-7 13R2+/NKX2-5+ and day-14 13R2+/NKX2-5+/αMHC+ fractions generated a list of 100 GO terms with p < 0.0005. These included voltage-gated calcium channels (p = 4.9 × 10−26), heart morphogenesis (p = 1.56 × 10−12), muscle contraction (p = 2.15 × 10−9), myofibril assembly (p = 2.46 × 10−8) and calcium ion transport (p = 1.04 × 10−6), further confirming the progression of 13R2+ progenitors toward cardiac cell types, and in particular cardiomyocytes (Figure 5B). The transcriptional profile of CD13/ROR2 fractions and their progeny maps out the developmental hierarchy of a putative pre-cardiac mesodermal cell population that differentiates to cardiac progenitors with subsequent specification to mature cardiomyocytes. To determine whether 13R2+ cells retain an in vivo latent potential to differentiate to a cardiovascular lineage, we transplanted these cells into the mouse kidney capsule and heart. Day-3 13R2+ cells were isolated from a differentiating NKX2-5GFP/w hESC reporter line (Elliott et al., 2011Elliott D.A. Braam S.R. Koutsis K. Ng E.S. Jenny R. Lagerqvist E.L. Biben C. Hatzistavrou T. Hirst C.E. Yu Q.C. et al.NKX2-5(eGFP/w) hESCs for isolation of human cardiac progenitors and cardiomyocytes.Nat. Methods. 2011; 8: 1037-1040Crossref PubMed Scopus (316) Google Scholar), recovered for 24 hr in culture, and approximately 5 × 105 cells were implanted under the kidney capsule of non-obese diabetic/SCID mice with common γ-chain knockout (NSG) (Figure 5C). Six weeks later, 13R2+ grafts had eGFP+ patches demonstrating NKX2-5 expression. Transplanted 13R2+ progeny also contained cells expressing cardiac progenitor (TBX5), smooth muscle (CNN1), and cardiomyocyte (cTnT) proteins (Figure 5D). However, these grafts were not contractile and did not form organized sarcomeric structures. Furthermore, transplanted 13R2+ cells did not express the endothelial markers CD31 and APJ. These data indicate that the mouse kidney capsule may not provide a supportive environment for human myocardial or endothelial differentiation. Differentiation potential of day-3 13R2+ cells was also tested in the mouse heart. Approximately 5 × 105 13R2+ (or 13R2−) cells were sorted and recultured for 24 hr before transplantation by direct injection into the left ventricle of healthy NSG mouse hearts, or into the peri-infarct area following occlusion of the left anterior coronary descending artery (n = 6 in each group). Control experiments included equivalent volumes of conditioned media administered in similar locations of healthy and injured mouse hearts (n = 6). Engraftment was examined 8 weeks after transplantation by screening for human mitochondria staining in sectioned hearts (Figure 6). We detected very limited survival and engraftment of transplanted 13R2+ cells in the healthy NSG mouse hearts, and no substantial human cells in injured hearts. Engrafted 13R2+ cells did not express markers of definitive cardiac cell lineages, and no teratomas were observed (Figure 6A). Conversely, transplanted 13R2− cells formed teratomas, with mesoderm, endoderm, and ectoderm derivatives, in healthy (two of six) and injured (one of six) mouse hearts, suggesting the presence of residual undifferentiated hESCs (Figures 6B and S6A). Cardiac function, assessed by echocardiography at baseline and 8 weeks after intervention, revealed no changes in the ejection fraction or fractional shortening between groups (sham, conditioned media, 13R2+, or 13R2− transplants; n = 6 in each group) (Figure S6B). While several studies have examined hESC-derived cardiomyocytes in murine hearts, less is known of the capacity of human smooth muscle and endothelial cells to improve heart function, possibly by neovascularization of the damaged tissue (Li et al., 2009Li Z. Wilson K.D. Smith B. Kraft D.L. Jia F. Huang M. Xie X. Robbins R.C. Gambhir S.S. Weissman I.L. et al.Functional and transcriptional characterization of human embryonic stem cell-derived endothelial cells for treatment of myocardial infarction.PLoS One. 2009; 4: e8443Crossref PubMed Scopus (98) Google Scholar, Xiong et" @default.
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• W2238713044 title "CD13 and ROR2 Permit Isolation of Highly Enriched Cardiac Mesoderm from Differentiating Human Embryonic Stem Cells" @default.
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