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Efficient differentiation and purification of human induced pluripotent stem cell-derived endothelial progenitor cells and expansion with the use of inhibitors of ROCK, TGF-?, and GSK3?.

ABSTRACT: Endothelial cells (ECs) and endothelial progenitor cells (EPCs) play crucial roles in maintaining vascular health and homeostasis. Both cell types have been used in regenerative therapy as well as in various in vitro models; however, the properties of primary human ECs and EPCs are dissimilar owing to differences in genetic backgrounds and sampling techniques. Human induced pluripotent stem cells (hiPSCs) are an alternative cell source of ECs and EPCs. However, owing to the low purity of differentiated cells from hiPSCs, purification via an antigen-antibody reaction, which damages the cells, is indispensable. Besides, owing to limited expandability, it is difficult to produce these cells in large numbers. Here we report the development of relatively simple differentiation and purification methods for hiPSC-derived EPCs (iEPCs). Furthermore, we discovered that a combination of three small molecules, that is, Y-27632 (a selective inhibitor of Rho-associated, coiled-coil containing protein kinase [ROCK]), A 83-01 (a receptor-like kinase inhibitor of transforming growth factor beta [TGF-?]), and CHIR-99021 (a selective inhibitor of glycogen synthase kinase-3? [GSK3?] that also activates Wnt), dramatically stimulated protein synthesis-related pathways and enhanced the proliferative capacity of iEPCs. These findings will help to establish a supply system of EPCs at an industrial scale.

SUBMITTER: Aoki H

PROVIDER: S-EPMC7056658 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Efficient differentiation and purification of human induced pluripotent stem cell-derived endothelial progenitor cells and expansion with the use of inhibitors of ROCK, TGF-β, and GSK3β.

Aoki Hiromasa H Yamashita Misaki M Hashita Tadahiro T Ogami Koichi K Hoshino Shinichi S Iwao Takahiro T Matsunaga Tamihide T

Heliyon 20200303 3

Endothelial cells (ECs) and endothelial progenitor cells (EPCs) play crucial roles in maintaining vascular health and homeostasis. Both cell types have been used in regenerative therapy as well as in various <i>in vitro</i> models; however, the properties of primary human ECs and EPCs are dissimilar owing to differences in genetic backgrounds and sampling techniques. Human induced pluripotent stem cells (hiPSCs) are an alternative cell source of ECs and EPCs. However, owing to the low purity of ...[more]

PMID: 32154424

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Project description:BackgroundGeneration of large quantities of endothelial cells is highly desirable for vascular research, for the treatment of ischemia diseases, and for tissue regeneration. To achieve this goal, we developed a simple, chemically defined culture system to efficiently and rapidly differentiate endothelial cells from human pluripotent stem cells by going through an MESP1 mesoderm progenitor stage.MethodsMesp1 is a key transcription factor that regulates the development of early cardiovascular tissue. Using an MESP1-mTomato knock-in reporter human embryonic stem cell line, we compared the gene expression profiles of MESP1+ and MESP1- cells and identified new signaling pathways that may promote endothelial differentiation. We also used a 3D scaffold to mimic the in vivo microenvironment to further improve the efficiency of endothelial cell generation. Finally, we performed cell transplantation into a critical limb ischemia mouse model to test the repairing potential of endothelial-primed MESP1+ cells.ResultsMESP1+ mesoderm progenitors, but not MESP1- cells, have strong endothelial differentiation potential. Global gene expression analysis revealed that transcription factors essential for early endothelial differentiation were enriched in MESP1+ cells. Interestingly, MESP1 cells highly expressed Sphingosine-1-phosphate (S1P) receptor and the addition of S1P significantly increased the endothelial differentiation efficiency. Upon seeding in a novel 3D microniche and priming with VEGF and bFGF, MESP1+ cells markedly upregulated genes related to vessel development and regeneration. 3D microniches also enabled long-term endothelial differentiation and proliferation from MESP1+ cells with minimal medium supplements. Finally, we showed that transplanting a small number of endothelial-primed MESP1+ cells in 3D microniches was sufficient to mediate rapid repair of a mouse model of critical limb ischemia.ConclusionsOur study demonstrates that combining MESP1+ mesoderm progenitor cells with tissue-engineered 3D microniche and a chemically defined endothelial induction medium is a promising route to maximizing the production of endothelial cells in vitro and augment their regenerative power in vivo.

Project description:IntroductionPluripotent stem cells have been used by various researchers to differentiate and characterize endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) for the clinical treatment of vascular injuries. Studies continue to differentiate and characterize the cells with higher vascularization potential and low risk of malignant transformation to the recipient. Unlike previous studies, this research aimed to differentiate induced pluripotent stem (iPS) cells into endothelial progenitor cells (EPCs) and VSMCs using a step-wise technique. This was achieved by elucidating the spatio-temporal expressions of the stage-specific genes and proteins during the differentiation process. The presence of highly expressed oncogenes in iPS cells was also investigated during the differentiation period.MethodsInduced PS cells were differentiated into lateral mesoderm cells (Flk1+). The Flk1+ populations were isolated on day 5.5 of the mesodermal differentiation period. Flk1+ cells were further differentiated into EPCs and VSMCs using VEGF165 and platelet-derived growth factor-BB (PDGF-BB), respectively, and then characterized using gene expression levels, immunocytochemistry (ICC), and western blot (WB) methods. During the differentiation steps, the expression levels of the marker genes and proto-oncogenic Myc and Klf4 genes were simultaneously studied.ResultsThe optimal time for the isolation of Flk1+ cells was on day 5.5. EPCs and VSMCs were differentiated from Flk1+ cells and characterized with EPC-specific markers, including Kdr, Pecam1, CD133, Cdh5, Efnb2, Vcam1; and VSMC-specific markers, including Acta2, Cnn1, Des, and Myh11. Differentiated cells were validated based on their temporal gene expressions, protein synthesis, and localization at certain time points. Significant decreases in Myc and Klf4 gene expression levels were observed during the EPCs and VSMC differentiation period.ConclusionEPCs and VSMCs were successfully differentiated from iPS cells and characterized by gene expression levels, ICC, and WB. We observed significant decreases in oncogene expression levels in the differentiated EPCs and VSMCs. In terms of safety, the described methodology provided a better safety margin. EPCs and VSMC obtained using this method may be good candidates for transplantation and vascular regeneration.

Dataset Information

Efficient differentiation and purification of human induced pluripotent stem cell-derived endothelial progenitor cells and expansion with the use of inhibitors of ROCK, TGF-?, and GSK3?.

Publications

Efficient differentiation and purification of human induced pluripotent stem cell-derived endothelial progenitor cells and expansion with the use of inhibitors of ROCK, TGF-β, and GSK3β.

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