Differentiation of human pluripotent stem cells to cells similar to cord-blood endothelial colony–forming cells

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Abstract

The ability to differentiate human pluripotent stem cells into endothelial cells with properties of cord-blood endothelial colony–forming cells (CB-ECFCs) may enable the derivation of clinically relevant numbers of highly proliferative blood vessel–forming cells to restore endothelial function in patients with vascular disease. We describe a protocol to convert human induced pluripotent stem cells (hiPSCs) or embryonic stem cells (hESCs) into cells similar to CB-ECFCs at an efficiency of >108 ECFCs produced from each starting pluripotent stem cell. The CB-ECFC-like cells display a stable endothelial phenotype with high clonal proliferative potential and the capacity to form human vessels in mice and to repair the ischemic mouse retina and limb, and they lack teratoma formation potential. We identify Neuropilin-1 (NRP-1)-mediated activation of KDR signaling through VEGF165 as a critical mechanism for the emergence and maintenance of CB-ECFC-like cells.

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Figure 1: Simple one-step, 2D, serum-free, endothelial lineage differentiation protocol does not require embryoid body formation or TGF-β inhibition and yields ECFCs similar to those found in cord blood.
Figure 2: Human iPSC-ECFCs contribute to vascular repair of both ischemic retina and limb.
Figure 3: NRP-1 is critical for the emergence of ECFCs from hPSCs.
Figure 4: NRP-1 is critical for the maintenance of ECFC proliferative potential.

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Acknowledgements

These studies were supported by funds provided by the Riley Children's Foundation (M.C.Y.), by an AHA postdoctoral fellowship (N.P.), and Public Health Service Grants R01 HL109602 (M.C.Y. and S.L.V.-H.), RO1 HL056416 (H.E.B.), RO1 HL067384 (H.E.B.), PO1 DK090948 (H.E.B. and M.C.Y.), and by the Bio & Medical Technology Development Program of the National Research Foundation (NRF), which is funded by the South Korean government (MEST; No. 2011-0019487). The authors thank H.P. Poudel (Indiana University Purdue University Indianapolis) for critical input in biostatistical analysis. The authors also thank H.J. Lee (CHA Stem Cell Institute, CHA University, South Korea) for HLI experiments.

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N.P., M.R.L., H.E.B., S.R., M.P.M., A.W.S., S.L.V.-H. and M.C.Y. all participated in the design of the experiments. N.P., M.R.L., S.V., J.L.M., M.Y., M.J.F., A.F., M.G., B.M.R., M.R.S., M.G., O.E., Y.L., H.M.C., K.S.H., E.R., C.L.O. and R.J.M. performed the experiments. N.P. and M.C.Y. wrote the manuscript, and M.R.L., H.E.B., A.W.S., M.G., M.Y., A.F., C.L.O., H.M.C. and K.S.H. provided manuscript edits.

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Correspondence to Mervin C Yoder.

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The authors declare no competing financial interests.

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Supplementary Figures 1–9 and Supplementary Tables 1 and 2 (PDF 3760 kb)

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Prasain, N., Lee, M., Vemula, S. et al. Differentiation of human pluripotent stem cells to cells similar to cord-blood endothelial colony–forming cells. Nat Biotechnol 32, 1151–1157 (2014). https://doi.org/10.1038/nbt.3048

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