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. 2022 Jul 4;8(1):22.
doi: 10.1038/s41526-022-00209-4.

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

S Timilsina  1 , T Kirsch-Mangu  1 , S Werth  1 , B Shepard  1 , T Ma  2 , L G Villa-Diaz  3   4
Affiliations

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

S Timilsina et al. NPJ Microgravity. .

Abstract

A systematic study on the biological effects of simulated microgravity (sµg) on human pluripotent stem cells (hPSC) is still lacking. Here, we used a fast-rotating 2-D clinostat to investigate the sµg effect on proliferation, self-renewal, and cell cycle regulation of hPSCs. We observed significant upregulation of protein translation of pluripotent transcription factors in hPSC cultured in sµg compared to cells cultured in 1g conditions. In addition to a significant increase in expression of telomere elongation genes. Differentiation experiments showed that hPSC cultured in sµg condition were less susceptible to differentiation compared to cells in 1g conditions. These results suggest that sµg enhances hPSC self-renewal. Our study revealed that sµg enhanced the cell proliferation of hPSCs by regulating the expression of cell cycle-associated kinases. RNA-seq analysis indicated that in sµg condition the expression of differentiation and development pathways are downregulated, while multiple components of the ubiquitin proteasome system are upregulated, contributing to an enhanced self-renewal of hPSCs. These effects of sµg were not replicated in human fibroblasts. Taken together, our results highlight pathways and mechanisms in hPSCs vulnerable to microgravity that imposes significant impacts on human health and performance, physiology, and cellular and molecular processes.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Experimental setup to simulate microgravity.
A Clipmax chamber-slide flask with a small cell culture channel created (represented by a red double-sided arrow) after filling the two sides (represented by two blue double-sided arrows) and the top of the chamber-slide flask (not shown) with PDMS. B Distribution of microgravity forces in relation to the rotation axis at the area where cells are cultured. Oval circles represent cells. C Illustration of the developed device to simulate microgravity: (A) indicates 3-D printed adapter connecting two cell culture flasks (B) to the spinning bolt of a sample rotator instrument. C Indicates the bottom surfaces of the flasks where cells are attached (illustrated in red), which are positioned back-to-back and located in the axis of rotation. D Our developed rotary cell culture system (RCCS) with 2 flasks affixed to the system and located inside of the cell culture incubator, ready to generate simulated microgravity. E Experimental design used in this project.
Fig. 2
Fig. 2. Simulated microgravity (sμg) enhances the self-renewal of hPSCs.
Culture under sμg maintained self-renewal of hPSCs and enhanced the expression of the core set of pluripotent transcription factors (TF), transmembrane glycoproteins ITGA6 and ITGB1, and telomere genes. A Representative immunofluorescent micrographs of colonies from sμg and 1g conditions stained for the core set of pluripotent TFs. Scale bars, 200 µm. Graphs show the mean fluorescence intensity analysis from micrographs. B Representative immunoblots and densitometry analysis from protein lysates of cells cultured at sμg and 1g conditions. RT-qPCR analysis indicates relative mRNA levels of (C) pluripotent associated and (D) telomere elongation genes present in cells cultured at sμg and 1g conditions. *p < 0.05, **p < 0.005 (n = 3; unpaired t test). Error bars in graphs represent the SEM of the group.
Fig. 3
Fig. 3. Differentiation of hPSCs is reduced under simulated microgravity (sμg) conditions.
hPSCs were cultured under sμg or 1g for 48 h with a medium that sustains their self-renewal, followed by a further 48 h of culture with a differentiation medium to induce (A) trophectoderm and (B) neuroectoderm differentiation. RT-qPCR analysis of representative genes related to pluripotency, trophectoderm, and neuroectoderm indicated that differentiation under sμg condition is reduced compared to 1g condition. *p < 0.05, **p < 0.005 (n = 3; unpaired t test). Error bars in graphs represent the SEM of the group.
Fig. 4
Fig. 4. Simulated microgravity (sµg) enhances the proliferation of hPSCs.
A Representative micrographs of hPSC colonies cultured under sμg and 1g conditions. Scale bars, 1000 µm. B Graphs indicating significant fold change difference in hPSC colonies area after 96 h of culture under smg and 1g conditions. C Graph comparing the total cell number of hPSCs after 96 h of culture under sμg and 1g conditions. D Representative gel band images of rt-PCR analysis for the cell proliferation gene Ki67 in hPSCs after 96 h of culture under sμg and 1g conditions. The mRNA expression of TBP was used to show equal loading and to quantify the relative differences in expression, as indicated above each band. E Heat map showing the relative mRNA levels of 44 human cell cycle-associated genes in hPSCs culture under sμg for 96 h in relation to cells cultured in 1g condition. F RT-qPCR analysis was used to verify the expression of selected genes tested in the gene array assay. *p < 0.05, **p < 0.005 (n = 3; unpaired t test). Error bars in graphs represent the SEM of the group.
Fig. 5
Fig. 5. Volcano plot of differential gene expression between hPSCs cultured in simulated microgravity (sµg) and 1g condition.
The color intensity is proportional to Log2 (Fold Change). Genes are overly expressed under sµg condition if Log2 (Fold Change) > 0 (colored red in the figure). The vertical dashed lines correspond to Log2 (Fold Change) = ±1, and the horizontal line corresponds to the adjusted p-value = 0.01. There are a few genes that are highly expressed under 1g conditions such as HAS2, CYP26A1, SERPINB9, etc.
Fig. 6
Fig. 6. Simulated microgravity (sµg) enhances PSMD11 expression in hPSCs.
A Representative immunofluorescent micrographs of hPSCs cultured in sμg and 1g conditions and stained for PSMD11 and SOX2. DAPI was used to stain the nuclei of all cells. Scale bars, 200 µm. Graphs showing the mean fluorescence intensity analysis from micrographs. B Representative Immunoblots and densitometry analysis of PSMD11 from protein lysates of cells cultured at sμg and 1g conditions. GAPDH was used as a loading control. Graphs on the right show the densitometry analysis of the immunoblots. C RT-qPCR analysis indicating the relative mRNA levels of PSMD11 present in cells cultured at sμg compared to 1g condition. *p < 0.05 (n = 3; unpaired t test). Error bars in graphs represent the SEM of the group.
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