Stress forces first lineage differentiation of mouse Embryonic Stem Cells, validation of a high throughput screen for toxicant stress.

Mouse Embryonic Stem Cells (mESC) are unique in their self-renewal and pluripotency. Hypothetically, mESCs model gestational stress effects or stresses of in vitro fertilization/assisted reproductive technologies or drug/environmental exposures that endanger embryos. Testing mESCs stress responses should diminish and expedite in vivo embryo screening. Transgenic mESCs for green fluorescent protein (GFP) reporters of differ-entiation use the promoter for platelet-derived growth factor receptor (Pdgfr)a driving GFP expression to moni-tor hyperosmotic stress-forced mESC proliferation decrease (stunting), and differentiation increase that further stunts mESC population growth. In differentiating mESCs Pdgfra marks the first lineage extraembryonic primi-tive endoderm (ExEndo), Hyperosmotic stress forces mESC differentiation gain (Pdgfra-GFP) in monolayer or 3D embryoid bodies. Despite culture with potency-maintaining LIF, stress forces ExEndo as assayed using mi-croplate readers and validated by co-expression of Pdgfra-GFP, Disabled 2 (Dab2) and laminin by immunofluo-rescence and GFP protein and Dab2 by immunoblot. In agreement with previous reports, Rex1 and Oct4 loss was inversely proportional to increased Pdgfra-GFP mESC after treatment with high hyperosmotic sorbitol de-spite LIF. The increase in subpopulations of Pdgfra-GFP+ cells>background at ~23% was similar to the previ-ously reported ~25% increase in Rex1-RFP negative subpopulation at matched high sorbitol doses. By micro-plate reader there is a ~7-11fold increase in GFP at the a high submorbid and a morbid dose despite LIF, com-pared with LIF alone. By flow cytometry the subpopulation of Pdgfra-GFP+ cells>background increases ~8-16fold at these doses. Taken together the microplate, flow cytometry, immunoblot, and immunofluorescence data suggest that RA or hyperosmotic stress force dose-dependent differentiation whether LIF is present or not and this is negatively correlated with and possibly compensates for stress-forced diminished ESC population expansion and potency loss.