Abstract
Human pluripotent stem cells (hPSCs) provide a valuable model for the study of human development and a means to generate a scalable source of cells for therapeutic applications. This protocol specifies cell fate efficiently into cardiac and endothelial lineages from hPSCs. The protocol takes 2 weeks to complete and requires experience in hPSC culture and differentiation techniques. Building on lessons taken from early development, this monolayer-directed differentiation protocol uses different concentrations of activin A and bone morphogenetic protein 4 (BMP4) to polarize cells into mesodermal subtypes that reflect mid-primitive-streak cardiogenic mesoderm and posterior-primitive-streak hemogenic mesoderm. This differentiation platform provides a basis for generating distinct cardiovascular progenitor populations that enable the derivation of cardiomyocytes and functionally distinct endothelial cell (EC) subtypes from cardiogenic versus hemogenic mesoderm with high efficiency without cell sorting. ECs derived from cardiogenic and hemogenic mesoderm can be matured into >90% CD31+/VE-cadherin+ definitive ECs. To test the functionality of ECs at different stages of differentiation, we provide methods for assaying the blood-forming potential and de novo lumen-forming activity of ECs. To our knowledge, this is the first protocol that provides a common platform for directed differentiation of cardiomyocytes and endothelial subtypes from hPSCs. This protocol yields endothelial differentiation efficiencies exceeding those of previously published protocols. Derivation of these cell types is a critical step toward understanding the basis of disease and generating cells with therapeutic potential.
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Acknowledgements
This work was supported by the following grants: PO1 GM081619, PO1 HL094374, RO1 HL084642, and UO1 HL100405 (C.E.M.); NIH DP2DK102258-01 (Y.Z.); T32 EB001650 (R.J.Z.); NIH T32EB001650 and NIH T32HL007312 (M.R.); NIH UO1 HL100395 and U01 HL099997 (I.B.); and Alex's Lemonade Stand Young Investigator Award and the Hyundai Hope Scholar Grant (B.H.).
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N.J.P. conceptually developed protocols, designed and performed experiments, analyzed data, and wrote the manuscript. L.P. conceptually developed protocols, designed experiments, and assisted in preparation of the manuscript. C.E.F., M.R., B.H., and R.J.Z. performed experiments, analyzed data, and helped prepare protocol details. Y.Z. and I.B. designed experiments and assisted in preparation of the manuscript. C.E.M. supervised the project, obtained funding for the research, and wrote and approved the manuscript.
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Integrated supplementary information
Supplementary Figure 1 Protocol development for directed differentiation of endothelium from human embryonic stem cells.
(a) Schematic diagram of time course of directed differentiation and days (a-f) in which changes were made as outlined in panels b-j to optimize conditions for differentiation into endothelium. (b-j) Flow cytometry analysis for the cardiac progenitor marker KDR+/PDGFRα+ and the endothelial marker KDR+/CD31+ (b-h) at day 5 of differentiation. Manipulation of conditions at time points a-f are described to the right of each panel.
Supplementary Figure 2 Time dependency of VEGF treatment during endothelial differentiation.
(a) Schematic of differentiation timeline. (b) Exposure to VEGF continuously starting on day 1 of differentiation vs. subsequent days as it impacts efficiency of endothelial cell production on day 5 by FACS analysis for KDR and CD34. (c) Exposure to VEGF for 24 hour increments starting on day 1 vs. subsequent days as it impacts efficiency of endothelial cell production on day 5 by FACS analysis for KDR and CD34.
Supplementary Figure 3 Examples of FACS profiles for analysis
(a-d) FSC/SSC plot (left) isotype gating (middle) and experimental gating (right) for all analyses reported in the protocol including cTnT (a), CD31 (b), CD43/CD235a (c) and KDR/CD34 (d).
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Supplementary Figures 1–3 and Supplementary Methods (PDF 929 kb)
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Palpant, N., Pabon, L., Friedman, C. et al. Generating high-purity cardiac and endothelial derivatives from patterned mesoderm using human pluripotent stem cells. Nat Protoc 12, 15–31 (2017). https://doi.org/10.1038/nprot.2016.153
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DOI: https://doi.org/10.1038/nprot.2016.153
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