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Sex-dependent VEGF expression underlies variations in human pluripotent stem cell to endothelial progenitor differentiation.

ABSTRACT: Human pluripotent stem cells (hPSCs) offer tremendous promise in tissue engineering and cell-based therapies because of their unique combination of two properties: pluripotency and a high proliferative capacity. To realize this potential, development of efficient hPSC differentiation protocols is required. In this work, sex-based differences are identified in a GSK3 inhibitor based endothelial progenitor differentiation protocol. While male hPSCs efficiently differentiate into CD34?+?CD31+ endothelial progenitors upon GSK3 inhibition, female hPSCs showed limited differentiation capacity using this protocol. Using VE-cadherin-GFP knockin reporter cells, female cells showed significantly increased differentiation efficiency when treated with VEGF during the second stage of endothelial progenitor differentiation. Interestingly, male cells showed no significant change in differentiation efficiency with VEGF treatment, but did show augmented early activation of VE-cadherin expression. A sex-based difference in endogenous expression of VEGF was identified that is likely the underlying cause of discrepancies in sex-dependent differentiation efficiency. These findings highlight the importance of sex differences in progenitor biology and the development of new stem cell differentiation protocols.

SUBMITTER: Randolph LN

PROVIDER: S-EPMC6853961 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Sex-dependent VEGF expression underlies variations in human pluripotent stem cell to endothelial progenitor differentiation.

Randolph Lauren N LN Bao Xiaoping X Oddo Michael M Lian Xiaojun Lance XL

Scientific reports 20191113 1

Human pluripotent stem cells (hPSCs) offer tremendous promise in tissue engineering and cell-based therapies because of their unique combination of two properties: pluripotency and a high proliferative capacity. To realize this potential, development of efficient hPSC differentiation protocols is required. In this work, sex-based differences are identified in a GSK3 inhibitor based endothelial progenitor differentiation protocol. While male hPSCs efficiently differentiate into CD34 <sup>+ </sup> ...[more]

PMID: 31723192

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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

Sex-dependent VEGF expression underlies variations in human pluripotent stem cell to endothelial progenitor differentiation.

Publications

Sex-dependent VEGF expression underlies variations in human pluripotent stem cell to endothelial progenitor differentiation.

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