Current Featured Research Article

Liat Oss-Ronen, Robert A. Redden, and Peter I. Lelkes
Published Online: 26 Aug 2020 https://doi.org/10.1089/scd.2020.0097

Abstract

Directed in vitro differentiation of pluripotent stem cells toward 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 monolayer colony cultures or as embryoid bodies (EBs) in either static conditions or, dynamically, in the rotary BRs. We used flow cytometry and quantitative polymerase chain reaction to analyze the expression of marker proteins and genes, respectively, for pluripotency (Oct3/4) and mesendodermal (Brachyury T), endodermal (FoxA2Sox17CxCr4), and mesodermal (VimentinMeox1) lineages. Culture in the form of EBs in maintenance media in the presence of leukemia inhibitory factor, 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 mESC differentiation.

Figure 1: mESC embryoid bodies in culture

FIG. 1. mESCs cultured in 2D in MM expanded in small colonies, as observed on day 3 (A), while cells in DM started migrating out of the colonies on day 3 (B) and showed an epithelial-like morphology when confluent, on day 6 (B*). We observed static EBs in MM (C) and DM (D), and EBs in SMG BRs in MM (G) and DM (H), on day 6 using phase contrast microscopy. Histology and hematoxylin and eosin staining were performed for EBs in static conditions in MM (E) and DM (F) and in BRs in MM (I) and DM (J). Scale bars: 50 μm in (AB); 200 μm in (CJ). 2D, two-dimensional; mESC, mouse embryonic stem cell; MM, maintenance media; DM, differentiation media; EBs, embryoid bodies; SMG, simulated microgravity; BR, bioreactor. Color images are available online.

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