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Oss-Ronen L , Redden RA , Lelkes PI
Enhanced Induction of Definitive Endoderm Differentiation of Mouse Embryonic Stem Cells in Simulated Microgravity
Stem Cells Dev. 2020 Aug 26;29(19) :1275-1284
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Abstract
Directed in vitro differentiation of pluripotent stem cells towards definitive endoderm (DE) offers great research and therapeutic potential since these cells can further differentiate into cells of the respiratory and gastrointestinal tracts, as well as associated organs such as pancreas, liver, and thyroid. We hypothesized that culturing mouse embryonic stem cells (mESCs) under simulated microgravity (SMG) conditions in rotary bioreactors (BRs) will enhance the induction of directed DE differentiation. To test our hypothesis, we cultured the cells for 6 days in two dimensional (2D) monolayer colony cultures, or as embryoid bodies (EBs) in either static conditions or, dynamically, in the rotary BRs. We used flow cytometry and qPCR to analyze the expression of marker proteins and genes, respectively, for pluripotency (Oct3/4) and mesendodermal (Brachyury T), endodermal (FoxA2, Sox17, CxCr4), and mesodermal (Vimentin, Meox1) lineages. Culture in the form of EBs in maintenance media (MM) in the presence of LIF, in static or SMG conditions, induced expression of some of the differentiation markers, suggesting heterogeneity of the cells. This is in line with previous studies showing that differentiation is initiated as cells are aggregated into EBs even without supplementing differentiation factors to the media. Culturing EBs in static conditions in differentiation media (DM) in the presence of activin A, reduced Oct3/4 expression, and significantly increased Brachyury T and CxCr4 expression, but downregulated FoxA2 and Sox17. However, culturing in SMG BRs in DM upregulated Brachyury T and all of the DE markers, and reduced Oct3/4 expression, indicating the advantage of dynamic cultures in BRs to specifically enhance directed DE differentiation. Given the potential discrepancies between the SMG conditions on earth and actual microgravity conditions, as observed in other studies, future experiments in space flight are required to validate the effects of reduced gravity on mESCs differentiation.
Notes
1557-8534 Ronen, Liat Redden, Robert Lelkes, Peter I Journal Article United States Stem Cells Dev. 2020 Jul 31. doi: 10.1089/scd.2020.0097.