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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

ABSTRACT: Endothelial cells (ECs) and endothelial progenitor cells (EPCs) play crucial roles in maintaining vascular health and hemostasis. 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. Although human induced pluripotent stem cells (hiPSCs) are an alternative cell source of ECs and EPCs, the differentiation and purification processes have not been optimized. 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-β]), and CHIR-99021 (a selective inhibitor of glycogen synthase kinase-3β [GSK3β] 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE138225 | GEO | 2020/03/03

REPOSITORIES: GEO

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Project description:Exploiting an Asp-Glu “switch” in glycogen synthase kinase 3 to design paralog selective inhibitors for use in acute myeloid leukemia: Genome-wide transcriptional profile for the GSK3β selective inhibitor BRD3731. Glycogen synthase kinase 3 (GSK3), a key regulatory kinase in the WNT pathway, remains a therapeutic target of interest in many diseases. While dual GSK3α/β inhibitors have entered clinical trials, none has successfully translated to clinical application. Mechanism-based toxicities, driven in part by the inhibition of both GSK3 paralogs and subsequent β-catenin stabilization, are a concern in the translation of this target class to cancer therapy, particularly for the treatment of acute myeloid leukemia (AML). Knockdown of GSK3α or GSK3β individually does not increase β-catenin in certain cellular subtypes and offers a conceptual resolution to targeting GSK3: paralog-selective inhibition. However, only inadequate chemical tools exist. The design of selective ATP competitive inhibitors poses a drug discovery challenge due to the high homology (95% identity, 100% similarity) in their ATP binding domains. Taking advantage of an Asp133®Glu196 “switch” in their hinge binding domains, we present a rational design strategy towards the discovery of a paralog selective set of GSK3 inhibitors. These first-in-class GSK3α and GSK3β selective inhibitors provided insights into GSK3 targeting in AML where GSK3α has been identified as a therapeutic target using genetic approaches. Our GSK3α selective compound (BRD0705) inhibits kinase function and does not stabilize β-catenin, mitigating potential neoplastic concerns. BRD0705 induces myeloid differentiation and impairs colony formation in AML cells while no effect is observed on normal hematopoietic cells. Moreover, BRD0705 impairs leukemia initiation and prolongs survival in AML mouse models. These studies validate feasibility of paralog selective GSK3α inhibition offering a promising therapeutic approach in AML.

Project description:Background: In recent times a subset of bone marrow (BM) derived cells; endothelial progenitor cells (EPCs), have generated tremendous interest, as these cells are suggested to home to sites of neovascularization and neoendothelialization and differentiate into mature endothelial cells (ECs), a process referred to as postnatal vasculogenesis. EPCs are being considered as a potential regenerative tool for treating various pathophysiological disease states including cardiovascular disorder and as a possible target to restrict vessel growth in tumour pathology. However, conflicting results have been reported in the field due to lack of EPC specific biomarkers, and the identification, characterization, and the precise role of EPCs in vascular biology still remains a subject of great debate. Therefore, the objective of this study was to use a bioinformatics approach to identify putative novel EPC specific biomarkers. Methods: This study reports a detailed gene expression profile of human umbilical cord blood (UCB) derived non-adherent CD133 + cells at different time points during in vitro differentiation (day 4 and day 7) which were compared with differentiated donor matched human umbilical vein endothelial cells (HUVEC). Results: EPC gene expression was profiled at both days 4 and 7, using affymetrix human 1.0ST exon arrays. Affymetrix gene expression profiling revealed significant expression changes in genes associated with stem/progenitor cell properties such as adhesion, signalling, molecular transport, cell structure organisation and growth. Conclusions: This is the first study utilizing gene expression profiling to examine non adherent CD133+ progenitor cells. Alterations in the gene expression reported in this study may be involved in the cellular processes characteristic of EPC development and differentiation. Gene expression profiling was performed on EPCs cultured for D4 and D7, on Affymetrix human 1.0ST exon array chips. Gene Spring (version GX11) was used to perform the gene expression analysis on three experimental groups; (a) D4 EPCs versus HUVEC; (b) D7 EPCs versus HUVEC and (c) D4 versus D7 EPCs.

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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

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