Human pluripotent stem cells (hPSCs) are a promising source of autologous endothelial progenitor cells (EPCs) that can be used for the treatment of vascular diseases. However, this kind of treatment requires a large amount of EPCs. Therefore, a highly efficient, robust, and easily reproducible differentiation protocol is necessary. We present a novel serum-free differentiation protocol that exploits the synergy of multiple powerful differentiation effectors. Our protocol follows the proper physiological pathway by differentiating EPCs from hPSCs in three phases that mimic in vivo embryonic vascular development. Specifically, hPSCs are differentiated into (i) primitive streak, which is subsequently turned into (ii) mesoderm, which finally differentiates into (iii) EPCs. This differentiation process yields up to 15 differentiated cells per seeded hPSC in 5 days. Endothelial progenitor cells constitute up to 97% of these derived cells. The experiments were performed on the human embryonic stem cell line H9 and six human induced pluripotent stem cell lines generated in our laboratory. Therefore, robustness was verified using many hPSC lines. Two previously established protocols were also adapted and compared to our synergistic three-phase protocol. Increased efficiency and decreased variability were observed for our differentiation protocol in comparison to the other tested protocols. Furthermore, EPCs derived from hPSCs by our protocol expressed the high-proliferative-potential EPC marker CD157 on their surface in addition to the standard EPC surface markers CD31, CD144, CD34, KDR, and CXCR4. Our protocol enables efficient fully defined production of autologous endothelial progenitors for research and clinical applications.

1 Surgery Department St Anne's University Hospital Brno Brno Czechia

Department of Histology and Embryology Theoretical Departments Faculty of Medicine Masaryk University Brno Czechia

International Clinical Research Center St Anne's University Hospital Brno Brno Czechia

See more in PubMed

Adams W. J., Zhang Y., Cloutier J., Kuchimanchi P., Newton G., Sehrawat S., et al. (2013). Functional vascular endothelium derived from human induced pluripotent stem cells. Stem Cell Rep. 1 105–113. 10.1016/j.stemcr.2013.06.007 PubMed DOI PMC

Bao X., Lian X., Dunn K. K., Shi M., Han T., Qian T., et al. (2015). Chemically-defined albumin-free differentiation of human pluripotent stem cells to endothelial progenitor cells. Stem Cell Res. 15 122–129. 10.1016/j.scr.2015.05.004 PubMed DOI PMC

Carmeliet P. (2000). Mechanisms of angiogenesis and arteriogenesis. Nat. Med. 6 389–395. 10.1038/74651 PubMed DOI

Cheng C.-C., Chang S.-J., Chueh Y.-N., Huang T.-S., Huang P.-H., Cheng S.-M., et al. (2013). Distinct angiogenesis roles and surface markers of early and late endothelial progenitor cells revealed by functional group analyses. BMC Genomics 14:182. 10.1186/1471-2164-14-182 PubMed DOI PMC

Choi K.-D., Yu J., Smuga-Otto K., Salvagiotto G., Rehrauer W., Vodyanik M., et al. (2009). Hematopoietic and endothelial differentiation of human induced pluripotent stem cells. Stem Cells 27 559–567. 10.1634/stemcells.2008-0922 PubMed DOI PMC

Chong M. S. K., Ng W. K., Chan J. K. Y. (2016). Concise review: endothelial progenitor cells in regenerative medicine: applications and challenges: endothelial progenitors in regenerative medicine. Stem Cells Transl. Med. 5 530–538. 10.5966/sctm.2015-0227 PubMed DOI PMC

Harding A., Cortez-Toledo E., Magner N. L., Beegle J. R., Coleal-Bergum D. P., Hao D., et al. (2017). Highly efficient differentiation of endothelial cells from pluripotent stem cells requires the MAPK and the PI3K pathways: the MAPK and PI3K pathways in endothelial fate. Stem Cells 35 909–919. 10.1002/stem.2577 PubMed DOI

Joo H. J., Kim H., Park S.-W., Cho H.-J., Kim H.-S., Lim D.-S., et al. (2011). Angiopoietin-1 promotes endothelial differentiation from embryonic stem cells and induced pluripotent stem cells. Blood 118 2094–2104. 10.1182/blood-2010-12-323907 PubMed DOI

Joo H. J., Song S., Seo H.-R., Shin J. H., Choi S.-C., Park J. H., et al. (2015). Human endothelial colony forming cells from adult peripheral blood have enhanced sprouting angiogenic potential through up-regulating VEGFR2 signaling. Int. J. Cardiol. 197 33–43. 10.1016/j.ijcard.2015.06.013 PubMed DOI

Kang S. N., Park C., Kim S. M., Park K. W., Park B. J., Han D. K., et al. (2015). Effect of stromal cell derived factor-1α release from heparin-coated Co-Cr stent substrate on the recruitment of endothelial progenitor cells. Macromol. Res. 23 1159–1167. 10.1007/s13233-015-4002-z DOI

Kempf H., Olmer R., Haase A., Franke A., Bolesani E., Schwanke K., et al. (2016). Bulk cell density and Wnt/TGFbeta signalling regulate mesendodermal patterning of human pluripotent stem cells. Nat. Commun. 7:13602. 10.1038/ncomms13602 PubMed DOI PMC

Kitajima K., Nakajima M., Kanokoda M., Kyba M., Dandapat A., Tolar J., et al. (2016). GSK3β inhibition activates the CDX/HOX pathway and promotes hemogenic endothelial progenitor differentiation from human pluripotent stem cells. Exp. Hematol. 44 68.e10–74.e10. 10.1016/j.exphem.2015.09.007 PubMed DOI PMC

Lee J. H., Lee S. H., Choi S. H., Asahara T., Kwon S.-M. (2015). The sulfated polysaccharide fucoidan rescues senescence of endothelial colony-forming cells for ischemic repair. Stem Cells 33 1939–1951. 10.1002/stem.1973 PubMed DOI

Li Z., Hu S., Ghosh Z., Han Z., Wu J. C. (2011). Functional characterization and expression profiling of human induced pluripotent stem cell- and embryonic stem cell-derived endothelial cells. Stem Cells Dev. 20 1701–1710. 10.1089/scd.2010.0426 PubMed DOI PMC

Olmer R., Engels L., Usman A., Menke S., Malik M. N. H., Pessler F., et al. (2018). Differentiation of human pluripotent stem cells into functional endothelial cells in scalable suspension culture. Stem Cell Rep. 10 1657–1672. 10.1016/j.stemcr.2018.03.017 PubMed DOI PMC

Orlova V. V., van den Hil F. E., Petrus-Reurer S., Drabsch Y., ten Dijke P., Mummery C. L. (2014). Generation, expansion and functional analysis of endothelial cells and pericytes derived from human pluripotent stem cells. Nat. Protoc. 9 1514–1531. 10.1038/nprot.2014.102 PubMed DOI

Park S.-W., Koh Y. J., Jeon J., Cho Y.-H., Jang M.-J., Kang Y., et al. (2010). Efficient differentiation of human pluripotent stem cells into functional CD34+ progenitor cells by combined modulation of the MEK/ERK and BMP4 signaling pathways. Blood 116 5762–5772. 10.1182/blood-2010-04-280719 PubMed DOI

Patel J., Seppanen E., Chong M. S. K., Yeo J. S. L., Teo E. Y. L., Chan J. K. Y., et al. (2013). Prospective surface marker-based isolation and expansion of fetal endothelial colony-forming cells from human term placenta. Stem Cells Transl. Med. 2 839–847. 10.5966/sctm.2013-0092 PubMed DOI PMC

Patel J., Seppanen E. J., Rodero M. P., Wong H. Y., Donovan P., Neufeld Z., et al. (2017). Functional definition of progenitors versus mature endothelial cells reveals key SoxF-dependent differentiation process. Circulation 135 786–805. 10.1161/CIRCULATIONAHA.116.024754 PubMed DOI

Patsch C., Challet-Meylan L., Thoma E. C., Urich E., Heckel T., O’Sullivan J. F., et al. (2015). Generation of vascular endothelial and smooth muscle cells from human pluripotent stem cells. Nat. Cell Biol. 17 994–1003. 10.1038/ncb3205 PubMed DOI PMC

Prasain N., Lee M. R., Vemula S., Meador J. L., Yoshimoto M., Ferkowicz M. J., et al. (2014). Differentiation of human pluripotent stem cells to cells similar to cord-blood endothelial colony–forming cells. Nat. Biotechnol. 32 1151–1157. 10.1038/nbt.3048 PubMed DOI PMC

Sahara M., Hansson E. M., Wernet O., Lui K. O., Später D., Chien K. R. (2014). Manipulation of a VEGF-Notch signaling circuit drives formation of functional vascular endothelial progenitors from human pluripotent stem cells. Cell Res. 24 820–841. 10.1038/cr.2014.59 PubMed DOI PMC

Shafiee A., Patel J., Hutmacher D. W., Fisk N. M., Khosrotehrani K. (2018). Meso-endothelial bipotent progenitors from human placenta display distinct molecular and cellular identity. Stem Cell Rep. 10 890–904. 10.1016/j.stemcr.2018.01.011 PubMed DOI PMC

Simara P., Tesarova L., Padourova S., Koutna I. (2014). Generation of human induced pluripotent stem cells using genome integrating or non-integrating methods. Folia Biol. 60:6. PubMed

Sriram G., Tan J. Y., Islam I., Rufaihah A. J., Cao T. (2015). Efficient differentiation of human embryonic stem cells to arterial and venous endothelial cells under feeder- and serum-free conditions. Stem Cell Res. Ther. 6:261. 10.1186/s13287-015-0260-5 PubMed DOI PMC

Suknuntha K., Tao L., Brok-Volchanskaya V., D’Souza S. S., Kumar A., Slukvin I. (2018). Optimization of synthetic mRNA for highly efficient translation and its application in the generation of endothelial and hematopoietic cells from human and primate pluripotent stem cells. Stem Cell Rev. Rep. 14 525–534. 10.1007/s12015-018-9805-1 PubMed DOI PMC

Tan J. Y., Sriram G., Rufaihah A. J., Neoh K. G., Cao T. (2013). Efficient derivation of lateral plate and paraxial mesoderm subtypes from human embryonic stem cells through GSKi-mediated differentiation. Stem Cells Dev. 22 1893–1906. 10.1089/scd.2012.0590 PubMed DOI PMC

Tatsumi R., Suzuki Y., Sumi T., Sone M., Suemori H., Nakatsuji N. (2011). Simple and eighly efficient method for production of endothelial cells from human embryonic stem cells. Cell Transplant 20 1423–1430. 10.3727/096368910X547444 PubMed DOI

Tesarova L., Simara P., Stejskal S., Koutna I. (2016). The aberrant DNA methylation profile of human induced pluripotent stem cells is connected to the reprogramming process and is normalized during in vitro culture. PLoS One 11:e0157974. 10.1371/journal.pone.0157974 PubMed DOI PMC

Vodyanik M. A., Yu J., Zhang X., Tian S., Stewart R., Thomson J. A., et al. (2010). A mesoderm-derived precursor for mesenchymal stem and endothelial cells. Cell Stem Cell 7 718–729. 10.1016/j.stem.2010.11.011 PubMed DOI PMC

Wakabayashi T., Naito H., Suehiro J., Lin Y., Kawaji H., Iba T., et al. (2018). CD157 marks tissue-resident endothelial stem cells with homeostatic and regenerative properties. Cell Stem Cell 22 384.e6–397.e6. 10.1016/j.stem.2018.01.010 PubMed DOI

Yamamizu K., Kawasaki K., Katayama S., Watabe T., Yamashita J. K. (2009). Enhancement of vascular progenitor potential by protein kinase A through dual induction of Flk-1 and Neuropilin-1. Blood 114 3707–3716. 10.1182/blood-2008-12-195750 PubMed DOI

Ye L., Tan S.-H., Su L.-P., Cook S. (2016). Three-dimensional scaffolds for efficient arterial endothelial cell differentiation from human induced pluripotent stem cells. J. Am. Coll. Cardiol. 67:2291 10.1016/S0735-1097(16)32292-6 DOI

Zhang S., Dutton J. R., Su L., Zhang J., Ye L. (2014). The influence of a spatiotemporal 3D environment on endothelial cell differentiation of human induced pluripotent stem cells. Biomaterials 35 3786–3793. 10.1016/j.biomaterials.2014.01.037 PubMed DOI PMC

Zhao H., Zhao Y., Li Z., Ouyang Q., Sun Y., Zhou D., et al. (2018). FLI1 and PKC co-activation promote highly efficient differentiation of human embryonic stem cells into endothelial-like cells. Cell Death Dis. 9:131. 10.1038/s41419-017-0162-9 PubMed DOI PMC