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Enhanced self-renewal of human pluripotent stem cells by simulated microgravity

ABSTRACT: We developed a fast-rotating clinostat to simulate micrigravity (µg) and investigated various effects (including proliferation, self-renewal, and cell cycle regulation) of simulated microgravity (sµg) on human pluripotent stem cells (hPSC). Cells were cultured in sµg and control condition in normal gravity (1g). We observed significant upregulation of protein translation of human pluripotency transcription factors in hPSC cultured in sµg condition compared to 1g. We also noted a significant increase in the expression levels of genes involved in telomere elongation. Our induced differentiation experiments showed that hPSC cultured in sµg condition were less susceptible towards differentiation compared to cells cultured in 1g condition as indicated by the significant delayed in the process of differentiation of the cell in sµg condition. These results suggest that sµg conditions enhance the self-renewal of hPSC. Our study further revealed that sµg enhanced the cell proliferation of hPSC by regulating the expression of cell cycle associated kinases. Moreover, RNAseq analysis indicated that in sµg condition the expression of pathways related to differentiation and development and down-regulated, while multiple components of the ubiquitin proteasome system are up-regulated, thus further contributing to an enhanced self-renewal of hPSC. These effects of sµg were not replicated in human fibroblasts. Taken together, these results highlight pathways and mechanisms in hPSC vulnerable to µg that impose significant impacts on human health and performance, physiology, and cellular and molecular processes.

ORGANISM(S): Homo sapiens

PROVIDER: GSE205559 | GEO | 2022/06/11

REPOSITORIES: GEO

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Project description:We recently revealed that myeloid master regulator PU.1 directly represses metallothionein (MT)-1G through its epigenetic activity, but the functions of MT-1G in myeloid differentiation remain unknown. To clarify this, we established MT-1G-overexpressing acute promyelocytic leukemia NB4 (NB4MTOE) cells, and investigated whether MT-1G functionally contributes to all-trans retinoic acid (ATRA)-induced NB4 cell differentiation. Real-time PCR analyses demonstrated that the inductions of CD11b and CD11c and reductions in myeloperoxidase and c-myc by ATRA were attenuated in NB4MTOE cells. Since G1 arrest is a hallmark of ATRA-induced NB4 cell differentiation, we observed a decrease in G1 accumulation, as well as decreases in p21WAF1/CIP1 and cyclin D1 inductions, by ATRA in NB4MTOE cells. Nitroblue tetrazolium (NBT) reduction assays revealed that the proportions of NBT-positive cells were decreased in NB4MTOE cells in the presence of ATRA. Microarray analyses showed that the changes in expression of several myeloid differentiation-related genes (GATA2, azurocidin 1, pyrroline-5-carboxylate reductase 1, defensin-4, C-X3-C motif receptor 3, matrix metallopeptidase -8, S100 calcium-binding protein A12, neutrophil cytosolic factor 2 and oncostatin M) induced by ATRA were disturbed in NB4MTOE cells. Collectively, overexpression of MT-1G disturbs the proper differentiation of myeloid cells. The present study provides evidence that expression analysis of MT-1G in acute promyelocytic leukemia patients may be a good prediction marker to estimate the efficacy of ATRA. Cell culture and generation of MT-1G-overexpressing cells: To generate MT-1G-overexpressing cells and their control cells, the MT-1G expression vector and its parental pcDNA 3.1/myc-His(-) version A vector (Invitrogen) were transfected using a CLB-Transfection device (Lonza, Basel, Switzerland). NB4 clones stably transfected with the vectors were isolated by limiting dilution and selection with 400 µg/ml of neomycin in RPMI (Gibco BRL, Rockville, MD) containing 10% heat-inactivated fetal bovine serum (HIFBS). Cells were cultured under 5% CO2 at 37°C in a humidified atmosphere. Microarray analyses: MT-1G-overexpressing (NB4MTOE) cells and their control cells were seeded at a density of 1×105 cells/ml and treated with 1 µM all-trans retinoic acid (ATRA). The cells were harvested after 72 h and total cellular RNA was isolated from control (NB4pcDNA4, 6, 7) cells and NB4MTOE (NB4MT22, 23, 25) cells using an RNA Mini Purification Kit (Qiagen, Miami, FL) according to the manufacturer’s protocol. Aliquots containing 10 µg of RNA from each sample of control cells were mixed and used as controls. Similarly, 10 µg of RNA from each sample of NB4MTOE cells were mixed and used as NB4MTOE cells. The samples were subjected to microarray analyses using a CodeLink Human 54K Whole Genome Bioarray (Filgen, Nagoya, Japan).

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Enhanced self-renewal of human pluripotent stem cells by simulated microgravity

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