Maintenance of an undifferentiated state of human induced pluripotent stem cells through migration-dependent regulation of the balance between cell–cell and cell–substrate interactions

TET1 is implicated in maintaining the pluripotency of embryonic stem cells. However, its precise effects on induced pluripotent stem cells (iPSCs), and particularly on porcine iPSCs (piPSCs), are not well defined. To investigate the role of TET1 in the pluripotency and differentiation of piPSCs, piPSCs were induced from porcine embryonic fibroblasts by overexpression ofPOU5F1(OCT4),SOX2,KLF4, andMYC(C-MYC). siRNAs targeting toTET1were used to transiently knockdown the expression ofTET1in piPSCs. Morphological abnormalities and loss of the undifferentiated state of piPSCs were observed in the piPSCs after the downregulation ofTET1. The effects ofTET1knockdown on the expression of key stem cell factors and differentiation markers were analyzed to gain insights into the molecular mechanisms underlying the phenomenon. The results revealed that knockdown ofTET1resulted in the downregulated expression of pluripotency-related genes, such asLEFTY2,KLF2, andSOX2, and the upregulated expression of differentiation-related genes includingPITX2,HAND1,GATA6, andLEF1. However,POU5F1,MYC,KLF4, andNANOGwere actually not downregulated. Further analysis showed that the methylation levels of the promoters forPOU5F1andMYCincreased significantly afterTET1downregulation, whereas there were no obvious changes in the promoters ofSOX2,KLF4, andNANOG. The methylation of the whole genome increased, while hydroxymethylation slightly declined. Taken together, these results suggest thatTET1may play important roles in the self-renewal of piPSCs and the maintenance of their characteristics by regulating the expression of genes and the DNA methylation.

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An effective detachment system for human induced pluripotent stem cells cultured on multilayered cultivation substrates using resonance vibrations

Scientific Reports ◽

10.1038/s41598-019-51944-w ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Yusuke Terao ◽

Yuta Kurashina ◽

Shugo Tohyama ◽

Yuki Fukuma ◽

Keiichi Fukuda ◽

...

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Driving Frequency ◽

Enzymatic Method ◽

Cell Recovery ◽

Undifferentiated State ◽

Resonance Vibrations ◽

Induced Pluripotent

Abstract Clinical application of human induced pluripotent stem cells (hiPSCs) has been hampered by the lack of a practical, scalable culture system. Stacked culture plates (SCPs) have recently attracted attention. However, final cell yields depend on the efficiency of cell detachment, and inefficient cell recovery from SCPs presents a major challenge to their use. We have developed an effective detachment method using resonance vibrations (RVs) of substrates with sweeping driving frequency. By exciting RVs that have 1–3 antinodes with ultra-low-density enzyme spread on each substrate of SCPs, 87.8% of hiPSCs were successfully detached from a 5-layer SCP compared to 30.8% detached by the conventional enzymatic method. hiPSC viability was similar after either method. Moreover, hiPSCs detached by the RV method maintained their undifferentiated state. Additionally, hiPSCs after long-term culture (10 passages) kept excellent detachment efficiency, had the normal karyotypes, and maintained the undifferentiated state and pluripotency. These results indicated that the RV method has definite advantages over the conventional enzymatic method in the scalable culture of hiPSCs using SCPs.

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Maintenance of hemiround colonies and undifferentiated state of mouse induced pluripotent stem cells on carbon nanotube-coated dishes

Carbon ◽

10.1016/j.carbon.2011.01.061 ◽

2011 ◽

Vol 49 (7) ◽

pp. 2287-2299 ◽

Cited By ~ 10

Author(s):

Tsukasa Akasaka ◽

Atsuro Yokoyama ◽

Makoto Matsuoka ◽

Takeshi Hashimoto ◽

Fumio Watari

Keyword(s):

Stem Cells ◽

Carbon Nanotube ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Undifferentiated State ◽

Induced Pluripotent

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Induced Pluripotent Stem Cells of Microtus levis x Microtus arvalis Vole Hybrids: Conditions Necessary for Their Generation and Self-Renewal

Acta Naturae ◽

10.32607/20758251-2015-7-4-56-69 ◽

2015 ◽

Vol 7 (4) ◽

pp. 56-69 ◽

Cited By ~ 2

Author(s):

E. V. Grigor’eva ◽

A. I. Shevchenko ◽

S. P. Medvedev ◽

N. A. Mazurok ◽

A. И. Zhelezova ◽

...

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Mammalian Species ◽

Molecular Genetic ◽

Experimental Studies ◽

Microtus Arvalis ◽

Undifferentiated State ◽

Induced Pluripotent ◽

Microtus Levis

Every year, the list of mammalian species for which cultures of pluripotent stem cells (PSCs) are generated increases. PSCs are a unique tool for extending the limits of experimental studies and modeling different biological processes. In this work, induced pluripotent stem cells (iPSCs) from the hybrids of common voles Microtus levis and Microtus arvalis, which are used as model objects to study genome organization on the molecular-genetic level and the mechanisms of X-chromosome inactivation, have been generated. Vole iPSCs were isolated and cultured in a medium containing cytokine LIF, basic fibroblast growth factor (bFGF), ascorbic acid, and fetal bovine serum. Undifferentiated state of vole iPSCs is maintained by activation of their endogenous pluripotency genes - Nanog, Oct4, Sox2, Sall4, and Esrrb. The cells were able to maintain undifferentiated state for at least 28 passages without change in their morphology and give rise to three germ layers (ectoderm, mesoderm and endoderm) upon differentiation.

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