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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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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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.

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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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