Effect of microgravity on the mesenchymal stem cell characteristics of limbal fibroblasts

Abstract

Background: Mesenchymal stem cells (MSCs) are important for regenerative medicine. Limbal fibroblasts (LFs), present in the corneal limbus, have been shown to possess MSC characteristics, and can differentiate into other cell types. The current study sought to investigate the effect of microgravity on the proliferation and differentiation of LFs, and identify culture conditions to obtain a high proportion of LFs possessing MSC characteristics.

Methods: A rotary cell culture system was used to generate microgravity. Cellular proliferation and MSC marker (CD14, CD45, CD90, CD105, and SSEA4) expression were evaluated by WST-1 test and flow cytometry, respectively. Differentiation of LFs into adipocytes, osteocytes, and chondrocytes was examined. The effects of LF-conditioned medium on limbal stem cell differentiation were assessed.

Results: The cellular proliferation rates under microgravity were significantly lower than those under normal gravity (0.44 vs. 0.18 at 24 h, and 0.70 vs. 0.44 at 48 h, both P ≤ 0.004). Higher proportions of cells expressed CD90 (95.33% vs 81.69%), CD105 (95.32% vs 87.96%), and SSEA4 (68.26% vs 26.13%) under microgravity than under normal gravity. The differentiation potential of LFs was more prominent under microgravity. The LF-conditioned medium attenuated the differentiation of limbal corneal epithelial stem cells.

Conclusion: Under microgravity, LFs showed a higher proportion of MSC characteristics and were easily induced into different linage cells. Culture in a microgravity environment may allow harvesting a greater number of MSC-like LFs for stem cell therapy in ocular surface reconstruction.

Keywords: Differentiation; Limbal fibroblasts; Mesenchymal stem cells; Microgravity.