Human Wharton's jelly stem cells (hWJSCs), derived from the umbilical cord's Wharton's jelly, are multipotent mesenchymal stem cells (MSCs) with potential for various biotechnological applications. Their abundance and ease of access make them an attractive cell source for regenerative medicine and tissue engineering. hWJSCs possess several characteristics that distinguish them from other MSCs, including an immature phenotype, high plasticity, maintained morphology during prolonged in vitro passages, and sustained immunoprivilege. They've shown the ability to differentiate into various lineages, including endodermal, mesodermal, and ectodermal lineages, highlighting their therapeutic potential. However, the impact of simulated microgravity (sµG), specifically simulated lunar microgravity (0.16G), on hWJSC growth, differentiation, and viability remains incompletely understood. Previous studies have hinted at altered gene expression and reduced growth in hWJSCs exposed to sµG, but a comprehensive analysis correlating growth, cell marker expression, viability, and transcriptional changes was lacking. This study aimed to fill this gap by investigating the effects of acute (72-hour) sµG exposure on hWJSCs and whether these changes were transient or persistent upon return to normal gravity.

Several studies have explored the differentiation potential of hWJSCs in various lineages. Mitchell et al. reported neuronal lineage differentiation using specific growth factors. Conconi et al. demonstrated myogenic, osteogenic, and adipogenic differentiation in vitro. Zhang et al. reported hepatocyte lineage differentiation using hepatocyte growth factor and fibroblast growth factor. Other studies have examined the influence of altered oxygen tension, shear stress, and low-serum conditions on hWJSC differentiation and growth. However, research on the effects of simulated microgravity on hWJSCs is limited. Pala et al. observed reductions in NANOG and SOX2 expression after 6 hours of sµG exposure, along with downregulation of cell cycle proteins after 72 hours, suggesting impacts on growth and stemness. A study using limbal fibroblasts showed reduced cell growth after 48 hours of rotary cell culture with increased MSC marker expression. These studies suggested that sµG might induce rapid transcriptional changes in hWJSCs, affecting their differentiation and growth.

Seven primary hWJSC lines were cultured under standard 1.0 G conditions and then passaged and cultured under simulated lunar microgravity (0.16 G) using a random positioning machine (RPM) for 72 hours. Control and experimental control groups were maintained at 1.0 G. After 72 hours, a post-sµG group was established by returning the sµG-exposed cells to 1.0 G for another 72 hours. Various assays were conducted to assess cell morphology (phase contrast microscopy), cell numbers (trypan blue counts – manual and automated), CD marker profile (FACS analysis – CD34, CD45, CD73, CD90, CD105), apoptosis (Annexin V-FITC assay), and gene expression (bulk RNA sequencing and qPCR). Differentiation potential was evaluated by culturing sµG-exposed and control hWJSCs in osteogenic and chondrogenic media for 14 days followed by Von Kossa and Alcian blue staining and qRT-PCR analysis for osteocyte and chondrocyte-related genes. RNA sequencing data were analyzed using DESeq2 for differential gene expression (DEG) analysis and clusterProfiler for Gene Set Enrichment Analysis (GSEA).

Simulated lunar microgravity (sµG) for 72 hours significantly reduced hWJSC cell numbers compared to controls, although cell viability (as assessed by trypan blue exclusion) remained largely unaffected. FACS analysis revealed a significant reduction in the expression of stemness markers (CD73, CD90, and CD105) in the sµG group at day 3, with a minimal increase in apoptosis (Annexin V positive cells). RNA sequencing identified 308 upregulated and 328 downregulated genes in the sµG group compared to the control. MKI67 (cell proliferation marker) was significantly downregulated. Interestingly, several genes associated with osteocyte-chondrocyte lineage differentiation (SERPINI1, MSX2, TFPI2, BMP6, COMP, TMEM119, LUM, HGF, CHI3L1, and SPP1) were significantly upregulated in the sµG group. Genes involved in cell fate regulation, such as DNMT1 and EZH2, were downregulated. After returning the cells to 1.0 G for 72 hours (post-sµG), cell numbers increased significantly, and the expression of stemness markers approached normal levels. However, several osteocyte-chondrocyte lineage-associated genes remained upregulated. Functional pathway analysis revealed enrichment of pathways related to osteogenesis and chondrogenesis (glycosaminoglycan biosynthesis, sphingolipid metabolism) in the sµG group. Von Kossa and Alcian blue staining confirmed enhanced osteogenic and chondrogenic differentiation, respectively, in the sµG-exposed cells when cultured in appropriate differentiation media for 14 days.

This study demonstrates that acute sµG exposure transiently inhibits hWJSC growth and alters their differentiation potential. The observed reduction in cell proliferation and stemness markers is likely due to changes in gene expression and pathway activation, as evidenced by the downregulation of MKI67 and the upregulation of osteocyte-chondrocyte markers. The reversibility of growth inhibition and stemness marker expression upon return to 1.0 G suggests a temporary adaptation rather than irreversible damage. The persistent upregulation of certain osteocyte-chondrocyte lineage markers after sµG exposure points towards a potentially significant long-term impact on cell fate. The activation of pathways related to osteogenesis and chondrogenesis strongly suggests that sµG promotes these differentiation pathways. These results are intriguing, considering that previous research using other types of MSCs (such as bone marrow-derived MSCs) indicated inhibition of osteogenic differentiation under microgravity conditions. This discrepancy may be due to inherent differences between hWJSCs and other MSC subtypes. Further research is needed to fully elucidate the mechanisms underlying these effects and explore the clinical and biotechnological implications.

This study provides the first comprehensive analysis of the impact of simulated lunar microgravity on hWJSCs. Acute sµG exposure transiently arrests growth and induces a shift towards osteocyte-chondrocyte differentiation, although the growth and stemness characteristics are largely reversible upon return to normal gravity. These findings have implications for understanding the effects of microgravity on stem cells and may inform strategies for using hWJSCs in regenerative medicine applications in space or for simulating space-like conditions on Earth.

The study focused on acute (72-hour) sµG exposure. Longer-term exposure studies are necessary to determine the long-term effects of sµG on hWJSCs. While the study used seven different hWJSC lines, the sample size could be increased for greater statistical power. The mechanisms by which sµG induces osteocyte-chondrocyte lineage differentiation remain to be fully elucidated. Further investigation into specific signaling pathways and molecular mechanisms is warranted.