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Generation of human vascular smooth muscle subtypes provides insight into embryological origin–dependent disease susceptibility

Abstract

Heterogeneity of embryological origins is a hallmark of vascular smooth muscle cells (SMCs) and may influence the development of vascular disease. Differentiation of human pluripotent stem cells (hPSCs) into developmental origin–specific SMC subtypes remains elusive. Here we describe a chemically defined protocol in which hPSCs were initially induced to form neuroectoderm, lateral plate mesoderm or paraxial mesoderm. These intermediate populations were further differentiated toward SMCs (>80% MYH11+ and ACTA2+), which displayed contractile ability in response to vasoconstrictors and invested perivascular regions in vivo. Derived SMC subtypes recapitulated the unique proliferative and secretory responses to cytokines previously documented in studies using aortic SMCs of distinct origins. Notably, this system predicted increased extracellular matrix degradation by SMCs derived from lateral plate mesoderm, which was confirmed using rat aortic SMCs from corresponding origins. This differentiation approach will have broad applications in modeling origin-dependent disease susceptibility and in developing bioengineered vascular grafts for regenerative medicine.

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Figure 1: Induction of mesoderm subtypes from hPSCs.
Figure 2: Efficient differentiation of intermediate lineages into vascular SMCs.
Figure 3: Functional characterization of hPSC-derived SMCs.
Figure 4: MKL2 knockdown and cytokine treatments validate the origin-specific characteristics of hPSC-derived SMC subtypes.
Figure 5: HPSC-derived SMC subtypes predict MMP and TIMP expression and activity in rat aortic SMCs of corresponding origins.
Figure 6: The different embryological origins of aortic SMCs may contribute to the site of aortic dissection.

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References

  1. Carmeliet, P. Mechanisms of angiogenesis and arteriogenesis. Nat. Med. 6, 389–395 (2000).

    Article  CAS  Google Scholar 

  2. Majesky, M.W. Developmental basis of vascular smooth muscle diversity. Arterioscler. Thromb. Vasc. Biol. 27, 1248–1258 (2007).

    Article  CAS  Google Scholar 

  3. Waldo, K.L. et al. Secondary heart field contributes myocardium and smooth muscle to the arterial pole of the developing heart. Dev. Biol. 281, 78–90 (2005).

    Article  CAS  Google Scholar 

  4. Jiang, X., Rowitch, D.H., Soriano, P., McMahon, A.P. & Sucov, H.M. Fate of the mammalian cardiac neural crest. Development 127, 1607–1616 (2000).

    CAS  PubMed  Google Scholar 

  5. Wasteson, P. et al. Developmental origin of smooth muscle cells in the descending aorta in mice. Development 135, 1823–1832 (2008).

    Article  CAS  Google Scholar 

  6. Mikawa, T. & Gourdie, R.G. Pericardial mesoderm generates a population of coronary smooth muscle cells migrating into the heart along with ingrowth of the epicardial organ. Dev. Biol. 174, 221–232 (1996).

    Article  CAS  Google Scholar 

  7. Etchevers, H.C., Vincent, C., Le Douarin, N.M. & Couly, G.F. The cephalic neural crest provides pericytes and smooth muscle cells to all blood vessels of the face and forebrain. Development 128, 1059–1068 (2001).

    CAS  PubMed  Google Scholar 

  8. Vrancken Peeters, M.P., Gittenberger-de Groot, A.C., Mentink, M.M. & Poelmann, R.E. Smooth muscle cells and fibroblasts of the coronary arteries derive from epithelial-mesenchymal transformation of the epicardium. Anat. Embryol. (Berl.) 199, 367–378 (1999).

    Article  CAS  Google Scholar 

  9. Pouget, C., Pottin, K. & Jaffredo, T. Sclerotomal origin of vascular smooth muscle cells and pericytes in the embryo. Dev. Biol. 315, 437–447 (2008).

    Article  CAS  Google Scholar 

  10. Armulik, A., Genove, G. & Betsholtz, C. Pericytes: developmental, physiological, and pathological perspectives, problems, and promises. Dev. Cell 21, 193–215 (2011).

    Article  CAS  Google Scholar 

  11. Haimovici, H. & Maier, N. Fate of aortic homografts in canine atherosclerosis. 3. Study of fresh abdominal and thoracic aortic implants into thoracic aorta: role of tissue susceptibility in atherogenesis. Arch. Surg. 89, 961–969 (1964).

    Article  CAS  Google Scholar 

  12. DeBakey, M.E., Lawrie, G.M. & Glaeser, D.H. Patterns of atherosclerosis and their surgical significance. Ann. Surg. 201, 115–131 (1985).

    Article  CAS  Google Scholar 

  13. Leroux-Berger, M. et al. Pathologic calcification of adult vascular smooth muscle cells differs on their crest or mesodermal embryonic origin. J. Bone Miner. Res. 26, 1543–1553 (2011).

    Article  CAS  Google Scholar 

  14. Ruddy, J.M., Jones, J.A., Spinale, F.G. & Ikonomidis, J.S. Regional heterogeneity within the aorta: relevance to aneurysm disease. J. Thorac. Cardiovasc. Surg. 136, 1123–1130 (2008).

    Article  Google Scholar 

  15. Cheung, C. & Sinha, S. Human embryonic stem cell-derived vascular smooth muscle cells in therapeutic neovascularisation. J. Mol. Cell. Cardiol. 51, 651–664 (2011).

    Article  CAS  Google Scholar 

  16. Vallier, L. et al. Early cell fate decisions of human embryonic stem cells and mouse epiblast stem cells are controlled by the same signalling pathways. PLoS ONE 4, e6082 (2009).

    Article  Google Scholar 

  17. Bernardo, A.S. et al. BRACHYURY and CDX2 mediate BMP-induced differentiation of human and mouse pluripotent stem cells into embryonic and extraembryonic lineages. Cell Stem Cell 9, 144–155 (2011).

    Article  CAS  Google Scholar 

  18. Dosch, R., Gawantka, V., Delius, H., Blumenstock, C. & Niehrs, C. Bmp-4 acts as a morphogen in dorsoventral mesoderm patterning in Xenopus. Development 124, 2325–2334 (1997).

    CAS  PubMed  Google Scholar 

  19. Schneider, M.D., Gaussin, V. & Lyons, K.M. Tempting fate: BMP signals for cardiac morphogenesis. Cytokine Growth Factor Rev. 14, 1–4 (2003).

    Article  CAS  Google Scholar 

  20. Goldman, D.C. et al. BMP4 regulates the hematopoietic stem cell niche. Blood 114, 4393–4401 (2009).

    Article  CAS  Google Scholar 

  21. Zhang, P. et al. Short-term BMP-4 treatment initiates mesoderm induction in human embryonic stem cells. Blood 111, 1933–1941 (2008).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  23. McLean, A.B. et al. Activin A efficiently specifies definitive endoderm from human embryonic stem cells only when phosphatidylinositol 3-kinase signaling is suppressed. Stem Cells 25, 29–38 (2007).

    Article  CAS  Google Scholar 

  24. Jain, R.K. Molecular regulation of vessel maturation. Nat. Med. 9, 685–693 (2003).

    Article  CAS  Google Scholar 

  25. Kramer, J., Quensel, C., Meding, J., Cardoso, M.C. & Leonhardt, H. Identification and characterization of novel smoothelin isoforms in vascular smooth muscle. J. Vasc. Res. 38, 120–132 (2001).

    Article  CAS  Google Scholar 

  26. Huang, X. & Saint-Jeannet, J.P. Induction of the neural crest and the opportunities of life on the edge. Dev. Biol. 275, 1–11 (2004).

    Article  CAS  Google Scholar 

  27. Wang, D.Z. et al. Potentiation of serum response factor activity by a family of myocardin-related transcription factors. Proc. Natl. Acad. Sci. USA 99, 14855–14860 (2002).

    Article  CAS  Google Scholar 

  28. Li, J. et al. Myocardin-related transcription factor B is required in cardiac neural crest for smooth muscle differentiation and cardiovascular development. Proc. Natl. Acad. Sci. USA 102, 8916–8921 (2005).

    Article  CAS  Google Scholar 

  29. Oh, J., Richardson, J.A. & Olson, E.N. Requirement of myocardin-related transcription factor-B for remodeling of branchial arch arteries and smooth muscle differentiation. Proc. Natl. Acad. Sci. USA 102, 15122–15127 (2005).

    Article  CAS  Google Scholar 

  30. Owens, A.P. III et al. Angiotensin II induces a region-specific hyperplasia of the ascending aorta through regulation of inhibitor of differentiation 3. Circ. Res. 106, 611–619 (2010).

    Article  CAS  Google Scholar 

  31. Topouzis, S. & Majesky, M.W. Smooth muscle lineage diversity in the chick embryo. Two types of aortic smooth muscle cell differ in growth and receptor-mediated transcriptional responses to transforming growth factor-beta. Dev. Biol. 178, 430–445 (1996).

    Article  CAS  Google Scholar 

  32. Gadson, P.F. Jr. et al. Differential response of mesoderm- and neural crest-derived smooth muscle to TGF-beta1: regulation of c-myb and alpha1 (I) procollagen genes. Exp. Cell Res. 230, 169–180 (1997).

    Article  CAS  Google Scholar 

  33. Isoda, K. et al. Deficiency of interleukin-1 receptor antagonist promotes neointimal formation after injury. Circulation 108, 516–518 (2003).

    Article  CAS  Google Scholar 

  34. Galis, Z.S. & Khatri, J.J. Matrix metalloproteinases in vascular remodeling and atherogenesis: the good, the bad, and the ugly. Circ. Res. 90, 251–262 (2002).

    Article  CAS  Google Scholar 

  35. Owens, G.K., Kumar, M.S. & Wamhoff, B.R. Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol. Rev. 84, 767–801 (2004).

    Article  CAS  Google Scholar 

  36. Ailawadi, G. et al. Smooth muscle phenotypic modulation is an early event in aortic aneurysms. J. Thorac. Cardiovasc. Surg. 138, 1392–1399 (2009).

    Article  Google Scholar 

  37. Libby, P., Ridker, P.M. & Hansson, G.K. Progress and challenges in translating the biology of atherosclerosis. Nature 473, 317–325 (2011).

    Article  CAS  Google Scholar 

  38. Milewicz, D.M., Dietz, H.C. & Miller, D.C. Treatment of aortic disease in patients with Marfan syndrome. Circulation 111, e150–e157 (2005).

    Article  Google Scholar 

  39. Loeys, B.L. et al. A syndrome of altered cardiovascular, craniofacial, neurocognitive and skeletal development caused by mutations in TGFBR1 or TGFBR2. Nat. Genet. 37, 275–281 (2005).

    Article  CAS  Google Scholar 

  40. Zhu, L. et al. Mutations in myosin heavy chain 11 cause a syndrome associating thoracic aortic aneurysm/aortic dissection and patent ductus arteriosus. Nat. Genet. 38, 343–349 (2006).

    Article  CAS  Google Scholar 

  41. Guo, D.C. et al. Mutations in smooth muscle alpha-actin (ACTA2) lead to thoracic aortic aneurysms and dissections. Nat. Genet. 39, 1488–1493 (2007).

    Article  CAS  Google Scholar 

  42. van de Laar, I.M. et al. Mutations in SMAD3 cause a syndromic form of aortic aneurysms and dissections with early-onset osteoarthritis. Nat. Genet. 43, 121–126 (2011).

    Article  CAS  Google Scholar 

  43. Lindsay, M.E. & Dietz, H.C. Lessons on the pathogenesis of aneurysm from heritable conditions. Nature 473, 308–316 (2011).

    Article  CAS  Google Scholar 

  44. Gittenberger-De Groot, A.C., Bartelings, M.M., Deruiter, M.C. & Poelmann, R.E. Basics of cardiac development for the understanding of congenital heart malformations. Pediatr. Res. 57, 169–176 (2005).

    Article  Google Scholar 

  45. Kalimo, H., Ruchoux, M.M., Viitanen, M. & Kalaria, R.N. CADASIL: a common form of hereditary arteriopathy causing brain infarcts and dementia. Brain Pathol. 12, 371–384 (2002).

    Article  CAS  Google Scholar 

  46. Brons, I.G. et al. Derivation of pluripotent epiblast stem cells from mammalian embryos. Nature 448, 191–195 (2007).

    Article  CAS  Google Scholar 

  47. Vallier, L. et al. Signaling pathways controlling pluripotency and early cell fate decisions of human induced pluripotent stem cells. Stem Cells 27, 2655–2666 (2009).

    Article  CAS  Google Scholar 

  48. Irizarry, R.A. et al. Nucleic Acids Res. 31, e15 (2003).

    Article  Google Scholar 

  49. King, J.Y. et al. Pathway analysis of coronary atherosclerosis. Physiol. Genomics 23, 103–118 (2005).

    Article  CAS  Google Scholar 

  50. Dennis, G. Jr. et al. DAVID: database for annotation, visualization, and integrated discovery. Genome Biol. 4, 3 (2003).

    Article  Google Scholar 

  51. Storey, J.D. & Tibshirani, R. Proc. Natl. Acad. Sci. USA 100, 9440–9445 (2003).

    Article  CAS  Google Scholar 

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Acknowledgements

The authors thank L. Vallier and S.T. Rashid for supplying us the wild-type human iPSCs; K. Jensen for the human fetal gut RNA; N. Figg and M. Ackers-Johnson for help rendered in Matrigel sectioning and the harvesting of rat aortic SMCs, respectively. We also thank T. Faial and D. Ortmann for validation of the mesoderm protocols. This work was supported by a Wellcome Trust Intermediate Clinical Fellowship for S.S. and the Cambridge National Institute for Health Research Comprehensive Biomedical Research Centre. C. Cheung was sponsored by a National Science Scholarship (PhD) from the Agency for Science, Technology and Research (Singapore). R.A.P. and M.W.B.T. were supported by a Medical Research Council centre grant and A.S.B. was supported by a Leukemia and Lymphoma Society grant.

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Authors

Contributions

C.C. and S.S. developed the concept of generating origin-specific SMCs and designed the experiments. A.S.B. and R.A.P. developed the mesoderm specification protocols. C.C. performed experiments, analyzed data, and wrote and prepared the manuscript. A.S.B. performed part of the mesoderm validation experiments. M.W.B.T. gave advice regarding design of the microarray experiment, processed the resulting data and contributed to further analysis. S.S. supervised the project. All authors edited the manuscript.

Corresponding author

Correspondence to Sanjay Sinha.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Tables 1–3 and Supplementary Figs. 1–6 (PDF 1511 kb)

Supplementary Video 1

HeLa cells (WMV 343 kb)

Supplementary Video 2

NE-SMC (WMV 142 kb)

Supplementary Video 3

LM-SMC (WMV 230 kb)

Supplementary Video 4

PM-SMC (WMV 198 kb)

Supplementary Video 5

ASMC (WMV 150 kb)

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Cheung, C., Bernardo, A., Trotter, M. et al. Generation of human vascular smooth muscle subtypes provides insight into embryological origin–dependent disease susceptibility. Nat Biotechnol 30, 165–173 (2012). https://doi.org/10.1038/nbt.2107

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