Advertisement

Selection of a common multipotent cardiovascular stem cell using the 3.4-kb MesP1 promoter fragment

Abstract

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.

This is a preview of subscription content, log in to check access.

Access options

Buy single article

Instant unlimited access to the full article PDF.

US$ 39.95

Price includes VAT for USA

Subscribe to journal

Immediate online access to all issues from 2019. Subscription will auto renew annually.

US$ 199

This is the net price. Taxes to be calculated in checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

References

  1. 1.

    Bondue A, Lapouge G, Paulissen C, Semeraro C, Iacovino M, Kyba M, Blanpain C (2008) Mesp1 acts as a master regulator of multipotent cardiovascular progenitor specification. Cell Stem Cell 3:69–84. doi:10.1016/j.stem.2008.06.009

  2. 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

  3. 3.

    Buckingham M, Meilhac S, Zaffran S (2005) Building the mammalian heart from two sources of myocardial cells. Nat Rev Genet 6:826–835. doi:10.1038/nrg1710

  4. 4.

    Christiaen L, Davidson B, Kawashima T, Powell W, Nolla H, Vranizan K, Levine M (2008) The transcription/migration interface in heart precursors of Ciona intestinalis. Science 320:1349–1352. doi:10.1126/science.1158170

  5. 5.

    Christiaen L, Stolfi A, Davidson B, Levine M (2009) Spatio-temporal intersection of Lhx3 and Tbx6 defines the cardiac field through synergistic activation of Mesp. Dev Biol 328:552–560. doi:10.1016/j.ydbio.2009.01.033

  6. 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

  7. 7.

    David R, Franz WM (2012) From pluripotency to distinct cardiomyocyte subtypes. Physiology 27:119–129. doi:10.1152/physiol.00044.2011

  8. 8.

    David R, Groebner M, Franz WM (2005) Magnetic cell sorting purification of differentiated embryonic stem cells stably expressing truncated human CD4 as surface marker. Stem Cells 23:477–482. doi:10.1634/stemcells.2004-0177

  9. 9.

    David R, Jarsch VB, Schwarz F, Nathan P, Gegg M, Lickert H, Franz WM (2011) Induction of MesP1 by Brachyury(T) generates the common multipotent cardiovascular stem cell. Cardiovasc Res 23:23. doi:10.1093/cvr/cvr158

  10. 10.

    David R, Stieber J, Fischer E, Brunner S, Brenner C, Pfeiler S, Schwarz F, Franz WM (2009) Forward programming of pluripotent stem cells towards distinct cardiovascular cell types. Cardiovasc Res 84:263–272. doi:10.1093/cvr/cvp211

  11. 11.

    Davidson B, Shi W, Levine M (2005) Uncoupling heart cell specification and migration in the simple chordate Ciona intestinalis. Development. 132:4811–4818. doi:10.1242/dev.02051

  12. 12.

    Dubois NC, Craft AM, Sharma P, Elliott DA, Stanley EG, Elefanty AG, Gramolini A, Keller G (2011) SIRPA is a specific cell-surface marker for isolating cardiomyocytes derived from human pluripotent stem cells. Nat Biotechnol 29:1011–1018. doi:10.1038/nbt.2005

  13. 13.

    Eschenhagen T, Zimmermann WH, Kleber AG (2006) Electrical coupling of cardiac myocyte cell sheets to the heart. Circ Res 98:573–575. doi:10.1161/01.RES.0000215627.13049.5d

  14. 14.

    Gassanov N, Er F, Zagidullin N, Hoppe UC (2004) Endothelin induces differentiation of ANP-EGFP expressing embryonic stem cells towards a pacemaker phenotype. Faseb J. 18:1710–1712. doi:10.1096/fj.04-1619fje

  15. 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

  16. 16.

    Haraguchi S, Kitajima S, Takagi A, Takeda H, Inoue T, Saga Y (2001) Transcriptional regulation of Mesp1 and Mesp2 genes: differential usage of enhancers during development. Mech Dev 108:59–69. doi:10.1016/S0925-4773(01)00478-6

  17. 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

  18. 18.

    Hirata H, Murakami Y, Miyamoto Y, Tosaka M, Inoue K, Nagahashi A, Jakt LM, Asahara T, Iwata H, Sawa Y, Kawamata S (2006) ALCAM (CD166) is a surface marker for early murine cardiomyocytes. Cells Tissues Organs 184:172–180. doi:10.1159/000099624

  19. 19.

    Ieda M, Fu JD, Delgado-Olguin P, Vedantham V, Hayashi Y, Bruneau BG, Srivastava D (2010) Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors. Cell 142:375–386. doi:10.1016/j.cell.2010.07.002

  20. 20.

    Ishiguro F, Murakami H, Mizuno T, Fujii M, Kondo Y, Usami N, Yokoi K, Osada H, Sekido Y (2012) Activated leukocyte cell-adhesion molecule (ALCAM) promotes malignant phenotypes of malignant mesothelioma. J Thorac Oncol 7:890–899. doi:10.1097/JTO.0b013e31824af2db

  21. 21.

    Joos TO, David R, Dreyer C (1996) xGCNF, a nuclear orphan receptor is expressed during neurulation in Xenopus laevis. Mech Dev 60:45–57. doi:10.1016/S0925-4773(96)00599-0

  22. 22.

    Kanno S, Kim PK, Sallam K, Lei J, Billiar TR, Shears LL 2nd (2004) Nitric oxide facilitates cardiomyogenesis in mouse embryonic stem cells. Proc Natl Acad Sci USA 101:12277–12281. doi:10.1073/pnas.0401557101

  23. 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

  24. 24.

    Kitajima S, Takagi A, Inoue T, Saga Y (2000) MesP1 and MesP2 are essential for the development of cardiac mesoderm. Development 127:3215–3226

  25. 25.

    Klug MG, Soonpaa MH, Koh GY, Field LJ (1996) Genetically selected cardiomyocytes from differentiating embryonic stem cells form stable intracardiac grafts. J Clin Invest 98:216–224. doi:10.1172/JCI118769

  26. 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

  27. 27.

    Maltsev VA, Rohwedel J, Hescheler J, Wobus AM (1993) Embryonic stem cells differentiate in vitro into cardiomyocytes representing sinusnodal, atrial and ventricular cell types. Mech Dev 44:41–50. doi:10.1016/0925-4773(93)90015-P

  28. 28.

    Maltsev VA, Wobus AM, Rohwedel J, Bader M, Hescheler J (1994) Cardiomyocytes differentiated in vitro from embryonic stem cells developmentally express cardiac-specific genes and ionic currents. Circ Res 75:233–244. doi:10.1161/01.RES.75.2.233

  29. 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

  30. 30.

    Muller M, Fleischmann BK, Selbert S, Ji GJ, Endl E, Middeler G, Muller OJ, Schlenke P, Frese S, Wobus AM, Hescheler J, Katus HA, Franz WM (2000) Selection of ventricular-like cardiomyocytes from ES cells in vitro. Faseb J 14:2540–2548. doi:10.1096/fj.00-0002com

  31. 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

  32. 32.

    Murakami Y, Hirata H, Miyamoto Y, Nagahashi A, Sawa Y, Jakt M, Asahara T, Kawamata S (2007) Isolation of cardiac cells from E8.5 yolk sac by ALCAM (CD166) expression. Mech Dev 124:830–839. doi:10.1016/j.mod.2007.09.004

  33. 33.

    Oerlemans MI, Goumans MJ, van Middelaar B, Clevers H, Doevendans PA, Sluijter JP (2010) Active Wnt signaling in response to cardiac injury. Basic Res Cardiol 105:631–641. doi:10.1007/s00395-010-0100-9

  34. 34.

    Oginuma M, Hirata T, Saga Y (2008) Identification of presomitic mesoderm (PSM)-specific Mesp1 enhancer and generation of a PSM-specific Mesp1/Mesp2-null mouse using BAC-based rescue technology. Mech Dev 125:432–440. doi:10.1016/j.mod.2008.01.010

  35. 35.

    Ooi J, Liu P (2012) Delineating nuclear reprogramming. Protein Cell 3:329–345. doi:10.1007/s13238-012-2920-x

  36. 36.

    Rust W, Balakrishnan T, Zweigerdt R (2009) Cardiomyocyte enrichment from human embryonic stem cell cultures by selection of ALCAM surface expression. Regen Med 4:225–237. doi:10.2217/17460751.4.2.225

  37. 37.

    Saga Y, Kitajima S, Miyagawa-Tomita S (2000) Mesp1 expression is the earliest sign of cardiovascular development. Trends Cardiovasc Med 10:345–352. doi:10.1016/S1050-1738(01)00069-X

  38. 38.

    Saga Y, Miyagawa-Tomita S, Takagi A, Kitajima S, Miyazaki J, Inoue T (1999) MesP1 is expressed in the heart precursor cells and required for the formation of a single heart tube. Development 126:3437–3447

  39. 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 11:182

  40. 40.

    Takahashi T, Lord B, Schulze PC, Fryer RM, Sarang SS, Gullans SR, Lee RT (2003) Ascorbic acid enhances differentiation of embryonic stem cells into cardiac myocytes. Circulation 107:1912–1916. doi:10.1161/01.CIR.0000064899.53876.A3

  41. 41.

    Uosaki H, Fukushima H, Takeuchi A, Matsuoka S, Nakatsuji N, Yamanaka S, Yamashita JK (2011) Efficient and scalable purification of cardiomyocytes from human embryonic and induced pluripotent stem cells by VCAM1 surface expression. PLoS ONE 6:e23657

  42. 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

  43. 43.

    Ventura C, Maioli M, Asara Y, Santoni D, Mesirca P, Remondini D, Bersani F (2005) Turning on stem cell cardiogenesis with extremely low frequency magnetic fields. FASEB J 19:155–157. doi:10.1096/fj.04-2695fje

  44. 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

  45. 45.

    Wobus AM, Wallukat G, Hescheler J (1991) Pluripotent mouse embryonic stem cells are able to differentiate into cardiomyocytes expressing chronotropic responses to adrenergic and cholinergic agents and Ca2+ channel blockers. Differentiation 48:173–182

  46. 46.

    Wu J, Li J, Zhang N, Zhang C (2011) Stem cell-based therapies in ischemic heart diseases: a focus on aspects of microcirculation and inflammation. Basic Res Cardiol 106:317–324. doi:10.1007/s00395-011-0168-x

  47. 47.

    Xu C, Police S, Rao N, Carpenter MK (2002) Characterization and enrichment of cardiomyocytes derived from human embryonic stem cells. Circ Res 91:501–508. doi:10.1161/01.RES.0000035254.80718.91

  48. 48.

    Xu XQ, Graichen R, Soo SY, Balakrishnan T, Rahmat SN, Sieh S, Tham SC, Freund C, Moore J, Mummery C, Colman A, Zweigerdt R, Davidson BP (2008) Chemically defined medium supporting cardiomyocyte differentiation of human embryonic stem cells. Differentiation 76:958–970

  49. 49.

    Yamanaka S (2007) Strategies and new developments in the generation of patient-specific pluripotent stem cells. Cell Stem Cell 1:39–49. doi:10.1016/j.stem.2007.05.012

  50. 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

Download references

Acknowledgments

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.

Author information

Correspondence to Robert David or Wolfgang-Michael Franz.

Electronic supplementary material

Below is the link to the electronic supplementary material.

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)

Suppl. Figure 1 Clustal-W2-Alignment analysing the 5 kb-upstream regions of Mesp1-ORFs comparing Human (H), Chimpanzee (P), Rhesus Monkey (M), Mouse (Mo), Rat (R) and Dog (C). Blue marks demarcate homologous regions at two different threshold levels; lower panel: 50 %-threshold; upper panel: 37 %-threshold. Black and green frames: enhancer regions as described above (PPT 541 kb)

Suppl. Figure 2 (A) Representative time course of ∆CD4 expression after induction of differentiation in ES cell clone 2 as analysed in FACS. (B) Corresponding time course for clone 3 and (C) for clone 4 (PPT 1076 kb)

Suppl. Figure 3 (A) Purification of the ∆CD4-positive fraction from ~ 2 % to over 99 % after two subsequent MACS steps is reflected in high enrichment of endogenous MesP1 mRNA within the MACS-positive fraction. (B) Spontaneous reaggregation of purified cells in hanging drops: reformed EBs after 24 h (left panel) and 48 h (right panel) (PPT 851 kb)

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)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

David, R., Schwarz, F., Rimmbach, C. et al. Selection of a common multipotent cardiovascular stem cell using the 3.4-kb MesP1 promoter fragment. Basic Res Cardiol 108, 312 (2013) doi:10.1007/s00395-012-0312-2

Download citation

Keywords

  • ES cells
  • Magnetic cell sorting
  • MesP1
  • Cardiovascular stem cell
  • Proximal MesP1 promoter