The effect of simulated microgravity on human mesenchymal stem cells cultured in an osteogenic differentiation system: a bioinformatics study

Abstract

One proposed strategy for bone regeneration involves ex vivo tissue engineering, accomplished using bone-forming cells, biodegradable scaffolds, and dynamic culture systems, with the goal of three-dimensional tissue formation. Rotating wall vessel bioreactors generate simulated microgravity conditions ex vivo, which lead to cell aggregation. Human mesenchymal stem cells (hMSCs) have been extensively investigated and shown to possess the potential to differentiate into several cell lineages. The goal of the present study was to evaluate the effect of simulated microgravity on all genes expressed in hMSCs, with the underlying hypothesis that many important pathways are affected during culture within a rotating wall vessel system. Gene expression was analyzed using a whole genome microarray and clustering with the aid of the National Institutes of Health's Database for Annotation, Visualization and Integrated Discovery database and gene ontology analysis. Our analysis showed 882 genes that were downregulated and 505 genes that were upregulated after exposure to simulated microgravity. Gene ontology clustering revealed a wide variety of affected genes with respect to cell compartment, biological process, and signaling pathway clusters. The data sets showed significant decreases in osteogenic and chondrogenic gene expression and an increase in adipogenic gene expression, indicating that ex vivo adipose tissue engineering may benefit from simulated microgravity. This finding was supported by an adipogenic differentiation assay. These data are essential for further understanding of ex vivo tissue engineering using hMSCs.

FIG. 1.

Experimental design scheme. Color images available online at www.liebertonline.com/ten.

FIG. 2.

Cell viability of hMSCs cultured in rotary bioreactor (μGr) versus static culture after 7 days in culture. Cell viability was validated by MTT staining and the microbead-cell constructs were imaged using light microscopy (A). Cell viability was quantified using CellTiter-Glo bioluminescent assay. (B) No significant change in viability was found between the two culture conditions. Arrows indicate stained cells; bars indicate SE; n = 5. hMSCs, human mesenchymal stem cells. Color images available online at www.liebertonline.com/ten.

FIG. 3.

Osteogenic differentiation of hMSCs cultured in rotary bioreactor versus static culture. Osteogenic differentiation of hMSCs in rotating bioreactor was validated using ALP staining in vitro after 7 days in presence of osteogenic supplements. The microbead-cell constructs were imaged using light microscopy (A). Colorimetric assay quantitatively assessed the activity of ALP comparing the two. (B) No significant change in ALP activity was detected between the two culture conditions. Arrows indicate stained cells; bars indicate SE; n = 5. ALP, alkaline phosphatase. Color images available online at www.liebertonline.com/ten.

FIG. 4.

Adipogenic differentiation of hMSCs cultured in rotary bioreactor versus static culture. Adipogenic differentiation of hMSCs in rotating bioreactor (A, B) and static culture (C) was validated using Oil Red O staining in vitro after 7 days in presence of adipogenic supplements. Afterward, the cellular constructs were distained with isopropanol and the accumulated Oil Red O was evaluated using spectrophotometer (D). The cells cultured in microgravity conditions were presented significantly higher stain for Oil Red O. Arrows indicate stained cells; bars indicate SE; *p < 0.05; n = 5. OD, optical density. Color images available online at www.liebertonline.com/ten.

FIG. 5.

Overview of down- and upregulated genes after thresholding of nonexpressed genes and genes that were not found to be statistically significant (p > 0.001). Color images available online at www.liebertonline.com/ten.

FIG. 6.

Diagram that summarizes the findings in the tissue-specific gene expression affected by microgravity (as listed in supplemental tables 5–10). Color images available online at www.liebertonline.com/ten.