Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Expansion and patterning of cardiovascular progenitors derived from human pluripotent stem cells

Abstract

The inability of multipotent cardiovascular progenitor cells (CPCs) to undergo multiple divisions in culture has precluded stable expansion of precursors of cardiomyocytes and vascular cells. This contrasts with neural progenitors, which can be expanded robustly and are a renewable source of their derivatives. Here we use human pluripotent stem cells bearing a cardiac lineage reporter to show that regulated MYC expression enables robust expansion of CPCs with insulin-like growth factor-1 (IGF-1) and a hedgehog pathway agonist. The CPCs can be patterned with morphogens, recreating features of heart field assignment, and controllably differentiated to relatively pure populations of pacemaker-like or ventricular-like cardiomyocytes. The cells are clonogenic and can be expanded for >40 population doublings while retaining the ability to differentiate into cardiomyocytes and vascular cells. Access to CPCs will allow precise recreation of elements of heart development in vitro and facilitate investigation of the molecular basis of cardiac fate determination. This technology is applicable for cardiac disease modeling, toxicology studies and tissue engineering.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: The expansion of early hPSC-derived cardiac cells.
Figure 2: NKX2-5–eGFP expression is activated in spheres by concerted FGF and BMP signaling.
Figure 3: Characterization of sphere heterogeneity and differentiation potential.
Figure 4: Recapitulation of ventricular-like and pacemaker-like cardiomyocyte functions.
Figure 5: Long-term expansion and differentiation analysis of CPCs.
Figure 6: Working model of hPSC-derived cardiovascular progenitor self-renewal and differentiation in the context of heart development.

References

  1. 1

    Drawnel, F.M. et al. Disease modeling and phenotypic drug screening for diabetic cardiomyopathy using human induced pluripotent stem cells. Cell Reports 9, 810–820 (2014).

    PubMed  Article  CAS  Google Scholar 

  2. 2

    Matsa, E., Burridge, P.W. & Wu, J.C. Human stem cells for modeling heart disease and for drug discovery. Sci. Transl. Med. 6, 239ps6 (2014).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  3. 3

    Lundy, S.D., Gantz, J.A., Pagan, C.M., Filice, D. & Laflamme, M.A. Pluripotent stem cell derived cardiomyocytes for cardiac repair. Curr. Treat. Options Cardiovasc. Med. 16, 319 (2014).

    PubMed  PubMed Central  Article  Google Scholar 

  4. 4

    Bhave, M., Akhter, N. & Rosen, S.T. Cardiovascular toxicity of biologic agents for cancer therapy. Oncology 28, 482–490 (2014).

    PubMed  Google Scholar 

  5. 5

    Birket, M.J. & Mummery, C.L. Pluripotent stem cell derived cardiovascular progenitors—a developmental perspective. Dev. Biol. 400, 169–179 (2015).

    PubMed  Article  CAS  Google Scholar 

  6. 6

    Vincent, S.D. & Buckingham, M.E. How to make a heart: the origin and regulation of cardiac progenitor cells. Curr. Top. Dev. Biol. 90, 1–41 (2010).

    PubMed  Article  Google Scholar 

  7. 7

    Cai, C.-L. et al. Isl1 identifies a cardiac progenitor population that proliferates prior to differentiation and contributes a majority of cells to the heart. Dev. Cell 5, 877–889 (2003).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  8. 8

    Lints, T.J., Parsons, L.M., Hartley, L., Lyons, I. & Harvey, R.P. Nkx-2.5: a novel murine homeobox gene expressed in early heart progenitor cells and their myogenic descendants. Development 119, 419–431 (1993).

    PubMed  CAS  Google Scholar 

  9. 9

    Ma, Q., Zhou, B. & Pu, W.T. Reassessment of Isl1 and Nkx2–5 cardiac fate maps using a Gata4-based reporter of Cre activity. Dev. Biol. 323, 98–104 (2008).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  10. 10

    Laugwitz, K.-L. et al. Postnatal isl1+ cardioblasts enter fully differentiated cardiomyocyte lineages. Nature 433, 647–653 (2005).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  11. 11

    Moretti, A. et al. Multipotent embryonic isl1+ progenitor cells lead to cardiac, smooth muscle, and endothelial cell diversification. Cell 127, 1151–1165 (2006).

    PubMed  Article  CAS  Google Scholar 

  12. 12

    Wu, S.M. et al. Developmental origin of a bipotential myocardial and smooth muscle cell precursor in the mammalian heart. Cell 127, 1137–1150 (2006).

    PubMed  Article  CAS  Google Scholar 

  13. 13

    Mommersteeg, M.T.M. et al. Molecular pathway for the localized formation of the sinoatrial node. Circ. Res. 100, 354–362 (2007).

    PubMed  Article  CAS  Google Scholar 

  14. 14

    Wiese, C. et al. Circ. Res. 104, 388–397 (2009).

    PubMed  Article  CAS  Google Scholar 

  15. 15

    Liang, X. et al. HCN4 dynamically marks the first heart field and conduction system precursors. Circ. Res. 113, 399–407 (2013).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  16. 16

    Später, D. et al. A HCN4+ cardiomyogenic progenitor derived from the first heart field and human pluripotent stem cells. Nat. Cell Biol. 15, 1098–1106 (2013).

    PubMed  Article  CAS  Google Scholar 

  17. 17

    Kattman, S.J. et al. Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines. Cell Stem Cell 8, 228–240 (2011).

    PubMed  Article  CAS  Google Scholar 

  18. 18

    Wierstra, I. & Alves, J. The c-myc promoter: still mysterY and challenge. Adv. Cancer Res. 99, 113–333 (2008).

    PubMed  Article  CAS  Google Scholar 

  19. 19

    Gittenberger-De Groot, A.C. et al. Nkx2.5 negative myocardium of the posterior heart field and its correlation with podoplanin expression in cells from the developing cardiac pacemaking and conduction system. Anat. Rec. 290, 115–122 (2007).

    Article  CAS  Google Scholar 

  20. 20

    Dyer, L.A. et al. BMP signaling modulates hedgehog-induced secondary heart field proliferation. Dev. Biol. 348, 167–176 (2010).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  21. 21

    Gude, N. et al. Akt promotes increased cardiomyocyte cycling and expansion of the cardiac progenitor cell population. Circ. Res. 99, 381–388 (2006).

    PubMed  Article  CAS  Google Scholar 

  22. 22

    Keren-Politansky, A., Keren, A. & Bengal, E. Neural ectoderm-secreted FGF initiates the expression of Nkx2.5 in cardiac progenitors via a p38 MAPK/CREB pathway. Dev. Biol. 335, 374–384 (2009).

    PubMed  Article  CAS  Google Scholar 

  23. 23

    Liberatore, C.M., Searcy-Schrick, R.D., Vincent, E.B. & Yutzey, K.E. Nkx-2.5 gene induction in mice is mediated by a Smad consensus regulatory region. Dev. Biol. 244, 243–256 (2002).

    PubMed  Article  CAS  Google Scholar 

  24. 24

    Reifers, F., Walsh, E.C., Leger, S., Stainier, D.Y. & Brand, M. Induction and differentiation of the zebrafish heart requires fibroblast growth factor 8 (fgf8/acerebellar). Development 127, 225–235 (2000).

    PubMed  CAS  Google Scholar 

  25. 25

    Schultheiss, T.M., Burch, J.B. & Lassar, A.B. A role for bone morphogenetic proteins in the induction of cardiac myogenesis. Genes Dev. 11, 451–462 (1997).

    PubMed  Article  CAS  Google Scholar 

  26. 26

    Barron, M., Gao, M. & Lough, J. Requirement for BMP and FGF signaling during cardiogenic induction in non-precardiac mesoderm is specific, transient, and cooperative. Dev. Dyn. 218, 383–393 (2000).

    PubMed  Article  CAS  Google Scholar 

  27. 27

    Inman, G.J. et al. SB-431542 is a potent and specific inhibitor of transforming growth factor-β superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7. Mol. Pharmacol. 62, 65–74 (2002).

    PubMed  Article  CAS  Google Scholar 

  28. 28

    Den Hartogh, S.C. et al. Dual reporter MESP1mCherry/w-NKX2–5eGFP/w hESCs enable studying early human cardiac differentiation. Stem Cells 33, 56–67 (2015).

    PubMed  Article  CAS  Google Scholar 

  29. 29

    Yang, L. et al. Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population. Nature 453, 524–528 (2008).

    PubMed  Article  CAS  Google Scholar 

  30. 30

    Ardehali, R. et al. Prospective isolation of human embryonic stem cell-derived cardiovascular progenitors that integrate into human fetal heart tissue. Proc. Natl. Acad. Sci. USA 110, 3405–3410 (2013).

    PubMed  Article  Google Scholar 

  31. 31

    van Berlo, J.H. et al. c-kit+ cells minimally contribute cardiomyocytes to the heart. Nature 509, 337–341 (2014).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  32. 32

    James, D. et al. Expansion and maintenance of human embryonic stem cell-derived endothelial cells by TGFβ inhibition is Id1 dependent. Nat. Biotechnol. 28, 161–166 (2010).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  33. 33

    Marques, S.R., Lee, Y., Poss, K.D. & Yelon, D. Reiterative roles for FGF signaling in the establishment of size and proportion of the zebrafish heart. Dev. Biol. 321, 397–406 (2008).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  34. 34

    de Pater, E. et al. Bmp signaling exerts opposite effects on cardiac differentiation. Circ. Res. 110, 578–587 (2012).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  35. 35

    Ueno, S. et al. Biphasic role for Wnt/β-catenin signaling in cardiac specification in zebrafish and embryonic stem cells. Proc. Natl. Acad. Sci. USA 104, 9685–9690 (2007).

    PubMed  Article  CAS  Google Scholar 

  36. 36

    Goumans, M.-J. et al. TGF-β1 induces efficient differentiation of human cardiomyocyte progenitor cells into functional cardiomyocytes in vitro. Stem Cell Res. 1, 138–149 (2007).

    PubMed  Article  CAS  Google Scholar 

  37. 37

    Verkerk, A.O., van Ginneken, A.C.G. & Wilders, R. Pacemaker activity of the human sinoatrial node: role of the hyperpolarization-activated current, I(f). Int. J. Cardiol. 132, 318–336 (2009).

    PubMed  Article  Google Scholar 

  38. 38

    Xi, J. et al. Comparison of contractile behavior of native murine ventricular tissue and cardiomyocytes derived from embryonic or induced pluripotent stem cells. FASEB J. 24, 2739–2751 (2010).

    PubMed  Article  CAS  Google Scholar 

  39. 39

    Jackson, T. et al. The c-myc proto-oncogene regulates cardiac development in transgenic mice. Mol. Cell. Biol. 10, 3709–3716 (1990).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  40. 40

    Noseda, M., Peterkin, T., Simões, F.C., Patient, R. & Schneider, M.D. Cardiopoietic factors extracellular signals for cardiac lineage commitment. Circ. Res. 108, 129–152 (2011).

    PubMed  Article  CAS  Google Scholar 

  41. 41

    Cai, W. et al. Coordinate Nodal and BMP inhibition directs Baf60c-dependent cardiomyocyte commitment. Genes Dev. 27, 2332–2344 (2013).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  42. 42

    Waldo, K.L. et al. Conotruncal myocardium arises from a secondary heart field. Development 128, 3179–3188 (2001).

    PubMed  CAS  Google Scholar 

  43. 43

    Milgrom-Hoffman, M. et al. The heart endocardium is derived from vascular endothelial progenitors. Development 138, 4777–4787 (2011).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  44. 44

    Zhang, Q. et al. Direct differentiation of atrial and ventricular myocytes from human embryonic stem cells by alternating retinoid signals. Cell Res. 21, 579–587 (2011).

    PubMed  Article  CAS  Google Scholar 

  45. 45

    Devalla, H.D. et al. Atrial-like cardiomyocytes from human pluripotent stem cells are a robust preclinical model for assessing atrial-selective pharmacology. EMBO Mol. Med. 7, 394–410 (2015).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  46. 46

    Kattman, S.J., Huber, T.L. & Keller, G.M. Multipotent Flk-1+ cardiovascular progenitor cells give rise to the cardiomyocyte, endothelial, and vascular smooth muscle lineages. Dev. Cell 11, 723–732 (2006).

    PubMed  Article  CAS  Google Scholar 

  47. 47

    Qyang, Y. et al. The renewal and differentiation of Isl1+ cardiovascular progenitors are controlled by a WNT/β-catenin pathway. Cell Stem Cell 1, 165–179 (2007).

    PubMed  Article  CAS  Google Scholar 

  48. 48

    Bu, L. et al. Human ISL1 heart progenitors generate diverse multipotent cardiovascular cell lineages. Nature 460, 113–117 (2009).

    PubMed  Article  CAS  Google Scholar 

  49. 49

    Domian, I.J. et al. Generation of functional ventricular heart muscle from mouse ventricular progenitor cells. Science 326, 426–429 (2009).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  50. 50

    Moretti, A. et al. Mouse and human induced pluripotent stem cells as a source for multipotent Isl1+ cardiovascular progenitors. FASEB J. 24, 700–711 (2010).

    PubMed  Article  CAS  Google Scholar 

  51. 51

    Elliott, D.A. et al. NKX2–5eGFP/w hESCs for isolation of human cardiac progenitors and cardiomyocytes. Nat. Methods 8, 1037–1040 (2011).

    PubMed  Article  CAS  Google Scholar 

  52. 52

    Miano, J.M., Cserjesi, P., Ligon, K.L., Periasamy, M. & Olson, E.N. Smooth muscle myosin heavy chain exclusively marks the smooth muscle lineage during mouse embryogenesis. Circ. Res. 75, 803–812 (1994).

    PubMed  Article  CAS  Google Scholar 

  53. 53

    Bellin, M. et al. Isogenic human pluripotent stem cell pairs reveal the role of a KCNH2 mutation in long-QT syndrome. EMBO J. 32, 3161–3175 (2013).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  54. 54

    Maherali, N. et al. A high-efficiency system for the generation and study of human induced pluripotent stem cells. Cell Stem Cell 3, 340–345 (2008).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  55. 55

    Birket, M.J. et al. PGC-1-α and reactive oxygen species regulate human embryonic stem cell-derived cardiomyocyte function. Stem Cell Rep. 1, 560–574 (2013).

    Article  CAS  Google Scholar 

  56. 56

    Hao, J. et al. In vivo structure activity relationship study of dorsomorphin analogs identifies selective VEGF and BMP inhibitors. ACS Chem. Biol. 5, 245–253 (2010).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  57. 57

    Rape, A.D., Guo, W.-H. & Wang, Y.-L. The regulation of traction force in relation to cell shape and focal adhesions. Biomaterials 32, 2043–2051 (2011).

    PubMed  Article  CAS  Google Scholar 

Download references

Acknowledgements

We acknowledge the contributions of I. Marcuccio and S. Casini. Work in C.L.M.'s lab is supported by Cardiovascular Research Netherlands (CVON HUSTCARE), The Netherlands Institute of Regenerative Medicine (NIRM), the European Research Council (ERCAdG 323182 STEMCARDIOVASC) and The Netherlands Organization for Scientific Research (NWO-FOM FOM 09MMC02). Work in R.P.'s lab is supported by the Netherlands Organization for Scientific Research (ZonMW-TOP 40-00812-98-12086 and ZonMW-MKMD 40-42600-98-036) and the Rembrandt Institute of Cardiovascular Science. V.V.O. was supported by the European Community's Seventh Framework Programme (FP7/2007-2013) grant agreement 602423.

Author information

Affiliations

Authors

Contributions

M.J.B. and C.L.M. designed the study. M.J.B., M.C.R., A.O.V., A.R.L. and V.S. performed experiments and analyzed data. H.D.D. analyzed data. M.J.B., C.L.M. and A.O.V. wrote the manuscript. D.W. maintained and differentiated the PSCs. S.C.d.H. generated and provided the MESP1+ RNA. M.B. generated and provided the original hPSC lines. V.V.O. helped with experimental design. R.P. and C.L.M. supervised the study. All authors edited the manuscript.

Corresponding author

Correspondence to Christine L Mummery.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–14 and Supplementary Tables 1 and 2 (PDF 14182 kb)

Data Set 1

Microarray Gene Lists (XLSX 52 kb)

Source Data 1

Source Data Figure 1 (XLSX 14 kb)

Source Data 2

Source Data Figure 2 (XLSX 10 kb)

Source Data 3

Source Data Figure 3 (XLSX 14 kb)

Source Data 4

Source Data Figure 4 (XLSX 19 kb)

Source Data 5

Source Data Figure 5 (XLSX 13 kb)

Time-lapse of cardiac EB-derived cells expanding as clonal spheres. (MP4 446 kb)

Sorted-CPC populations differentiated to cardiomyocytes. (MP4 3013 kb)

Examples of single contracting NKX2-5eGFP+ or NKX2-5eGFP- cardiomyocytes. (MP4 1321 kb)

iPSC-derived CPC populations differentiated to cardiomyocytes. (MP4 2385 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Birket, M., Ribeiro, M., Verkerk, A. et al. Expansion and patterning of cardiovascular progenitors derived from human pluripotent stem cells. Nat Biotechnol 33, 970–979 (2015). https://doi.org/10.1038/nbt.3271

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing