Simulated microgravity reduces proliferation and reorganizes the cytoskeleton of human umbilical cord mesenchymal stem cells

Abstract

The cytoskeleton plays a key role in cellular proliferation, cell-shape maintenance and internal cellular organization. Cells are highly sensitive to changes in microgravity, which can induce alterations in the distribution of the cytoskeletal and cell proliferation. This study aimed to assess the effects of simulated microgravity (SMG) on the proliferation and expression of major cell cycle-related regulators and cytoskeletal proteins in human umbilical cord mesenchymal stem cells (hucMSCs). A WST-1 assay showed that the proliferation of SMG-exposed hucMSCs was lower than a control group. Furthermore, flow cytometry analysis demonstrated that the percentage of SMG-exposed hucMSCs in the G0/G1 phase was higher than the control group. A western blot analysis revealed there was a downregulation of cyclin A1 and A2 expression in SMG-exposed hucMSCs as well. The expression of cyclin-dependent kinase 4 (cdk4) and 6 (cdk6) were also observed to be reduced in the SMG-exposed hucMSCs. The total nuclear intensity of SMG-exposed hucMSCs was also lower than the control group. However, there were no differences in the nuclear area or nuclear-shape value of hucMSCs from the SMG and control groups. A western blot and quantitative RT-PCR analysis showed that SMG-exposed hucMSCs experienced a downregulation of bata-actin and alpha-tubulin compared to the control group. SMG generated the reorganization of microtubules and microfilaments in hucMSCs. Our study supports the idea that the downregulation of major cell cycle-related proteins and cytoskeletal proteins results in the remodeling of the cytoskeleton and the proliferation of hucMSCs.

Fig. 1

The proliferation of hucMSCs from control and simulated microgravity (SMG) groups. (A) hucMSC proliferation assessed by a WST-1 kit. In the control group, the OD value of hucMSCs was higher than the SMG group. (B) Flow cytometry analysis of hucMSCs. In the SMG group, hucMSCs showed a higher G0/G1 ratio than the control group, resulting in the induction of cell cycle arrest in the hucMSCs. (C) The expression of major regulators was estimated by western blot. In the SMG group, the expression of cell cycle-related proteins in hucMSCs was downregulated. *** indicates a significant difference compared with the control group (P<0.001). ** indicates a significant difference compared with the control group (P<0.01). * indicates a significant difference compared with the control group (P<0.05).

Fig. 2

The evaluation of nuclear morphology in human umbilical cord mesenchymal stem cells (hucMSCs). (A), total nuclear intensity, (B) nuclear area, and (C) nuclear shape of hucMSCs generated by the cell cycle app. In the control group, the nuclei of the hucMSCs showed a higher intensity than those from the simulated microgravity (SMG) group. There was no difference in the nuclear area or nuclear-shape value between the control and SMG groups. (D) The distribution of nuclear-shape values in relation to total nuclear intensity. (E) The distribution of the nuclear area in the relation to total nuclear intensity. Blue color indicates the G0/G1 phase, red indicates the S phase, green indicates the G2/M phase, grey indicates <2n, and yellow indicates >4n. *** indicates a significant difference compared with the control group (P<0.001).

Fig. 3

The expression of β-actin and α-tubulin in human umbilical cord mesenchymal stem cells (hucMSCs). (A) A western blot analysis shows the expression β-actin and α-tubulin protein. In the simulated microgravity (SMG) group, hucMSCs demonstrated a lower expression of cytoskeletal proteins than the control group. (B) A qRT-PCR showing the transcript expression of β-actin and α-tubulin 3. The β-actin and α-tubulin 3 transcripts were attenuated in hucMSCs under SMG. *** indicates a significant difference compared with the control group (P<0.001).

Fig. 4

Cytoskeleton staining of human umbilical cord mesenchymal stem cells. Microtubules were stained with SiR-tubulin (red). Microfilaments were stained with phalloidin (green). The nucleus was counterstained with H33342 (blue). (Magnification ×100), Scale bar = 200 μm.