Selection of a common multipotent cardiovascular stem cell using the 3.4-kb MesP1 promoter fragment
- 532 Downloads
Common cardiovascular progenitor cells are characterized and induced by expression of the transcription factor MesP1. To characterize this population we used a 3.4-kb promoter fragment previously described by our group. This served to isolate MesP1-positive cells from differentiating ES stem cells via magnetic cell sorting based on a truncated CD4 surface marker. As this proximal promoter fragment omits a distal non-cardiovasculogenic enhancer region, we were able to achieve a synchronized fraction of highly enriched cardiovascular progenitors. These led to about 90 % of cells representing the three cardiovascular lineages: cardiomyocytes, endothelial cells and smooth muscle cells as evident from protein and mRNA analyses. In addition, electrophysiological and pharmacological parameters of the cardiomyocytic fraction show that almost all correspond to the multipotent early/intermediate cardiomyocyte subtype at day 18 of differentiation. Further differentiation of these cells was not impaired as evident from strong and synchronous beating at later stages. Our work contributes to the understanding of the earliest cardiovasculogenic events and may become an important prerequisite for cell therapy, tissue engineering and pharmacological testing in the culture dish using pluripotent stem cell-derived as well as directly reprogrammed cardiovascular cell types. Likewise, these cells provide an ideal source for large-scale transcriptome and proteome analyses.
KeywordsES cells Magnetic cell sorting MesP1 Cardiovascular stem cell Proximal MesP1 promoter
We thank Christiane Gross for technical assistance. R.D. and W.-M.F. are supported by the BMBF (01GN0960) and the DFG (DA 1296/2-1 and FR 705/14-2). F.S. is funded by the FöFoLe program of the LMU Munich. Additional funding was granted by the Dr. Helmut Legerlotz-Stiftung and the Fritz-Bender-Stiftung. W.-M.F. is the PI of the Munich Heart Alliance.
Conflict of interest
The authors declare that they have no conflict of interest.
Suppl. Movie 1 Increased spontaneous beating activity in EBs derived from cells reaggregated after MesP1-∆CD4 based MACS purification at day 24 of differentiation show vigorous and highly synchronized beating (WMV 300 kb)
Suppl. Movie 2 Increased spontaneous beating activity in EBs derived from cells overexpressing MesP1 at day 24 of differentiation contain high numbers of independently active foci due to a high content of spontaneously beating early/intermediate cardiomyocytes (WMV 7841 kb)
- 2.Bondue A, Tannler S, Chiapparo G, Chabab S, Ramialison M, Paulissen C, Beck B, Harvey R, Blanpain C (2011) Defining the earliest step of cardiovascular progenitor specification during embryonic stem cell differentiation. J Cell Biol 192:751–765. doi: 10.1083/jcb.201007063 PubMedCrossRefGoogle Scholar
- 6.David R, Brenner C, Stieber J, Schwarz F, Brunner S, Vollmer M, Mentele E, Muller-Hocker J, Kitajima S, Lickert H, Rupp R, Franz WM (2008) MesP1 drives vertebrate cardiovascular differentiation through Dkk-1-mediated blockade of Wnt-signalling. Nat Cell Biol 10:338–345. doi: 10.1038/ncb1696 PubMedCrossRefGoogle Scholar
- 15.Graichen R, Xu X, Braam SR, Balakrishnan T, Norfiza S, Sieh S, Soo SY, Tham SC, Mummery C, Colman A, Zweigerdt R, Davidson BP (2008) Enhanced cardiomyogenesis of human embryonic stem cells by a small molecular inhibitor of p38 MAPK. Differentiation. 76:357–370. doi: 10.1111/j.1432-0436.2007.00236.x PubMedCrossRefGoogle Scholar
- 17.Hattori F, Chen H, Yamashita H, Tohyama S, Satoh YS, Yuasa S, Li W, Yamakawa H, Tanaka T, Onitsuka T, Shimoji K, Ohno Y, Egashira T, Kaneda R, Murata M, Hidaka K, Morisaki T, Sasaki E, Suzuki T, Sano M, Makino S, Oikawa S, Fukuda K (2010) Nongenetic method for purifying stem cell-derived cardiomyocytes. Nat Methods 7:61–66. doi: 10.1038/nmeth.1403 PubMedCrossRefGoogle Scholar
- 23.Kita-Matsuo H, Barcova M, Prigozhina N, Salomonis N, Wei K, Jacot JG, Nelson B, Spiering S, Haverslag R, Kim C, Talantova M, Bajpai R, Calzolari D, Terskikh A, McCulloch AD, Price JH, Conklin BR, Chen HS, Mercola M (2009) Lentiviral vectors and protocols for creation of stable hESC lines for fluorescent tracking and drug resistance selection of cardiomyocytes. PLoS ONE 4:e5046. doi: 10.1371/journal.pone.0005046 PubMedCrossRefGoogle Scholar
- 26.Lindsley RC, Gill JG, Murphy TL, Langer EM, Cai M, Mashayekhi M, Wang W, Niwa N, Nerbonne JM, Kyba M, Murphy KM (2008) Mesp1 coordinately regulates cardiovascular fate restriction and epithelial–mesenchymal transition in differentiating ESCs. Cell Stem Cell 3:55–68. doi: 10.1016/j.stem.2008.04.004 PubMedCrossRefGoogle Scholar
- 29.Mauritz C, Schwanke K, Reppel M, Neef S, Katsirntaki K, Maier LS, Nguemo F, Menke S, Haustein M, Hescheler J, Hasenfuss G, Martin U (2008) Generation of functional murine cardiac myocytes from induced pluripotent stem cells. Circulation 118:507–517. doi: 10.1161/CIRCULATIONAHA.108.778795 PubMedCrossRefGoogle Scholar
- 31.Mummery CL, Ward D, Passier R (2007) Differentiation of human embryonic stem cells to cardiomyocytes by coculture with endoderm in serum-free medium. Curr Protoc Stem Cell Biol. Chapter 1:Unit 1F.2. doi: 10.1002/9780470151808.sc01f02s2
- 39.Soh BS, Zheng D, Li Yeo JS, Yang HH, Ng SY, Wong LH, Zhang W, Li P, Nichane M, Asmat A, Wong PS, Wong PC, Su LL, Mantalaris SA, Lu J, Xian W, McKeon F, Chen J, Lim EH, Lim B (2012) CD166(pos) subpopulation from differentiated human ES and iPS cells support repair of acute lung injury. Mol Ther 11:182Google Scholar
- 42.van Laake LW, Qian L, Cheng P, Huang Y, Hsiao EC, Conklin BR, Srivastava D (2010) Reporter-based isolation of induced pluripotent stem cell- and embryonic stem cell-derived cardiac progenitors reveals limited gene expression variance. Circ Res 107:340–347. doi: 10.1161/CIRCRESAHA.109.215434 PubMedCrossRefGoogle Scholar
- 44.Wobus AM, Kaomei G, Shan J, Wellner MC, Rohwedel J, Ji G, Fleischmann B, Katus HA, Hescheler J, Franz WM (1997) Retinoic acid accelerates embryonic stem cell-derived cardiac differentiation and enhances development of ventricular cardiomyocytes. J Mol Cell Cardiol 29:1525–1539. doi: 10.1006/jmcc.1997.0433 PubMedCrossRefGoogle Scholar
- 50.Zimmermann WH, Melnychenko I, Wasmeier G, Didie M, Naito H, Nixdorff U, Hess A, Budinsky L, Brune K, Michaelis B, Dhein S, Schwoerer A, Ehmke H, Eschenhagen T (2006) Engineered heart tissue grafts improve systolic and diastolic function in infarcted rat hearts. Nat Med 12:452–458. doi: 10.1038/nm1394 PubMedCrossRefGoogle Scholar