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; Yamashita, Misaki; Hashita, Tadahiro; Ogami, Koichi; Hoshino, Shin‐ichi; Iwao, Takahiro; Matsunaga, Tamihide

doi:10.1016/j.heliyon.2020.e03493

Cited by 12 publications

(23 citation statements)

References 49 publications

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“…ARHGDIA overexpression did not seem to adversely affect self-renewal or differentiation, as ARHGDIA-transduced cultures were maintained for several months and differentiated into EBs comprising all three germ layers. This is consistent with the routine use of ROCKi for hPSC culture; however, some laboratories have begun to report using ROCKi in differentiation protocols [38][39][40][41].…”

Section: Discussionsupporting

confidence: 70%

ARHGDIAConfers Selective Advantage to Dissociated Human Pluripotent Stem Cells

Riggs

Sheridan

Rao

2021

Stem Cells and Development

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Human pluripotent stem cells (hPSCs) have generated significant interest in the scientific community based on their potential applications in regenerative medicine. However, numerous research groups have reported a propensity for genomic alterations during hPSC culture that poses concerns for basic research and clinical applications. Work from our laboratory and others has demonstrated that amplification of chromosomal regions is correlated with increased gene expression. To date, the phenotypic association of common genomic alterations remains unclear and is a cause for concern during clinical use. In this study, we focus on trisomy 17 and a list of candidate genes with increased gene expression to hypothesize that overexpressing 17q25 located ARHGDIA will confer selective advantage to hPSCs. HPSC lines overexpressing ARHGDIA exhibited culture dominance in cocultures of overexpression lines with nonoverexpression lines. Furthermore, during low-density seeding, we demonstrate increased clonality of our ARHGDIA lines against matched controls. A striking observation is that we could reduce this selective advantage by varying the hPSC culture conditions with the addition of ROCK inhibitor (ROCKi). This work is unique in (1) demonstrating a novel gene that confers selective advantage to hPSCs when overexpressed and may help explain a common trisomy dominance, (2) providing a selection model for studying culture conditions that reduce the appearance of genomically altered hPSCs, and (3) aiding in elucidation of a mechanism that may act as a molecular switch during culture adaptation.

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Section: Discussionsupporting

confidence: 70%

ARHGDIAConfers Selective Advantage to Dissociated Human Pluripotent Stem Cells

Riggs

Sheridan

Rao

2021

Stem Cells and Development

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“…In 2013, Bai et al, found that during the derivation of vascular endothelial cells from human ESCs, the expression level of TGF-β increased from the fourth day of differentiation, and the addition of TGF-β type I receptor inhibitor SB431542 reduced the expression levels of mesodermal markers and the number of endothelial progenitors upon mesoderm induction [37]. In 2020, when Aoki et al, derived endothelial cells from human iPSCs, they found that the combination of three small molecules, namely, Y-27632 (a selective inhibitor of ROCK), A83-01 (a receptor-like kinase inhibitor of TGF-β) and CHIR-99021 (a selective inhibitor of GSK3β), improved the expansion of endothelial cells [36]. In our study, during the induction of porcine iPSCs into vascular endothelial cells, the addition of SB431542 at the early stage of differentiation inhibited the expression of mesoderm-specific genes, while the addition of SB431542 at the later stage of differentiation increased the expression of endothelial-specific genes, suggesting that TGF-β signaling plays a positive role in mesoderm formation and a negative role in its further endothelialization.…”

Section: Discussionmentioning

confidence: 99%

“…Considering the above results from bioinformatic analysis and the roles of TGF-β signaling in the development of human and mouse endothelial cells [34][35][36][37], we firstly…”

Section: Effects Of Tgf-β On the Differentiation Of Porcine Ipsc-ecsmentioning

confidence: 99%

“…Considering the above results from bioinformatic analysis and the roles of TGF-β signaling in the development of human and mouse endothelial cells [34][35][36][37], we firstly detected the secretion level of TGF-β1 during the derivation of porcine iPSCs-ECs by ELISA. The results showed that at the early stage of differentiation, the secretion level of TGF-β1 was low, while along with the prolongation of culture time, the level gradually increased and reached the peak on day 5, and then it decreased and maintained a low level (Figure 5A).…”

Section: Effects Of Tgf-β On the Differentiation Of Porcine Ipsc-ecsmentioning

confidence: 99%

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Derivation and Characterization of Endothelial Cells from Porcine Induced Pluripotent Stem Cells

et al. 2022

IJMS

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Although the study on the regulatory mechanism of endothelial differentiation from the perspective of development provides references for endothelial cell (EC) derivation from pluripotent stem cells, incomplete reprogramming and donor-specific epigenetic memory are still thought to be the obstacles of iPSCs for clinical application. Thus, it is necessary to establish a stable iPSC-EC induction system and investigate the regulatory mechanism of endothelial differentiation. Based on a single-layer culture system, we successfully obtained ECs from porcine iPSCs (piPSCs). In vitro, the derived piPSC-ECs formed microvessel-like structures along 3D gelatin scaffolds. Under pathological conditions, the piPSC-ECs functioned on hindlimb ischemia repair by promoting blood vessel formation. To elucidate the molecular events essential for endothelial differentiation in our model, genome-wide transcriptional profile analysis was conducted, and we found that during piPSC-EC derivation, the synthesis and secretion level of TGF-β as well as the phosphorylation level of Smad2/3 changed dynamically. TGF-β-Smad2/3 signaling activation promoted mesoderm formation and prevented endothelial differentiation. Understanding the regulatory mechanism of iPSC-EC derivation not only paves the way for further optimization, but also provides reference for establishing a cardiovascular drug screening platform and revealing the molecular mechanism of endothelial dysfunction.

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“…A83-01, an effective inhibitor of TGF-β type I receptor (ALK5-TD), promotes cell proliferation 52 and colonosphere formation 53 . Y-27632, A83-01, and CHIR99021 are often used together to improve cell survival and cell proliferation 51 , 54 .…”

Section: Construction Of Pdosmentioning

confidence: 99%

Living biobank-based cancer organoids: prospects and challenges in cancer research

Liu

Chen

2022

Cancer Biol Med

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Biobanks bridge the gap between basic and translational research. Traditional cancer biobanks typically contain normal and tumor tissues, and matched blood. However, biospecimens in traditional biobanks are usually nonrenewable. In recent years, increased interest has focused on establishing living biobanks, including organoid biobanks, for the collection and storage of viable and functional tissues for long periods of time. The organoid model is based on a 3D in vitro cell culture system, is highly similar to primary tissues and organs in vivo, and can recapitulate the phenotypic and genetic characteristics of target organs. Publications on cancer organoids have recently increased, and many types of cancer organoids have been used for modeling cancer processes, as well as for drug discovery and screening. On the basis of the current research status, more exploration of cancer organoids through technical advancements is required to improve reproducibility and scalability. Moreover, given the natural characteristics of organoids, greater attention must be paid to ethical considerations. Here, we summarize recent advances in cancer organoid biobanking research, encompassing rectal, gastric, pancreatic, breast, and glioblastoma cancers. Living cancer biobanks that contain cancerous tissues and matched organoids with different genetic backgrounds, subtypes, and individualized characteristics will eventually contribute to the understanding of cancer and ultimately facilitate the development of innovative treatments.

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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

Cited by 12 publications

References 49 publications

ARHGDIAConfers Selective Advantage to Dissociated Human Pluripotent Stem Cells

ARHGDIAConfers Selective Advantage to Dissociated Human Pluripotent Stem Cells

Derivation and Characterization of Endothelial Cells from Porcine Induced Pluripotent Stem Cells

Living biobank-based cancer organoids: prospects and challenges in cancer research

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