Stress Forces First Lineage Differentiation of Mouse Embryonic Stem Cells; Validation of a High-Throughput Screen for Toxicant Stress

Abstract

Mouse Embryonic Stem Cells (mESCs) 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 differentiation use the promoter for platelet-derived growth factor receptor (Pdgfr)a driving GFP expression to monitor 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 primitive endoderm (ExEndo). Hyperosmotic stress forces mESC differentiation gain (Pdgfra-GFP) in monolayer or three-dimensional embryoid bodies. Despite culture with potency-maintaining leukemia inhibitory factor (LIF), stress forces ExEndo as assayed using microplate readers and validated by coexpression of Pdgfra-GFP, Disabled 2 (Dab2), and laminin by immunofluorescence 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 despite LIF. The increase in subpopulations of Pdgfra-GFP + cells>background at *23% was similar to the previously reported *25% increase in Rex1-red fluorescent protein (RFP)-negative subpopulation at matched high sorbitol doses. By microplate reader, there is a *7-11-fold increase in GFP at a high nonmorbid and a morbid dose despite LIF, compared with LIF alone. By flow cytometry (FACS), the subpopulation of Pdgfra-GFP + cells>background increases *8-16-fold at these doses. Taken together, the microplate, FACS, immunoblot, and immunofluorescence data suggest that retinoic acid or hyperosmotic stress forces 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.

Blastocyst-Derived Stem Cell Populations under Stress: Impact of Nutrition and Metabolism on Stem Cell Potency Loss and Miscarriage

“…For example, introducing potency and/or first lineage differentiation reporters into ESCs and TSCs will provide a rapid way to screen for drugs, diet supplements, cosmetics, maternal stress hormones, and inflammatory and environmental stimuli [41,42,96] that may be potential pregnancy toxicants, since decreased ESC and TSC proliferation and forced-differentiation due to stress appear to apply for multiple types of stress tested. The cell types used for building HTSs should be extended to a variety of human racial and sex subtypes.…”

Stress Decreases Host Viral Resistance and Increases Covid Susceptibility in Embryonic Stem Cells

“…The experimental protocol used a 72hr stimulus of ESC to normal stemness (NS) with stemness and proliferation-maintaining Leukemia inhibitory factor (LIF), normal differentiation by LIF removal (ND), and three levels of hyperosmotic stress as a positive control of stress-forced differentiation (SFD) used in previous studies (Figure 1 [ 18 , 21 , 28 ]). The 72hr stimulus was previously established for use with Rex1-RFP [ 17 , 18 ] and Pdgfra-GFP [ 28 ] transgenic ESCs as it enables some differentiation and a 2-3fold change in stemness and 1st differentiated lineage, respectively. Doses of 200, 250 and 300mM sorbitol creates reduced growth, near zero growth, and negative growth, respectively.…”

“…Only minor increases were observed in intermediate bright Pdgfra+ cells at 200-250mM sorbitol. Interestingly, 300mM sorbitol despite LIF produced almost equal increases in Pdgfra+ intermediate and bright cells as retinoic acid at 1uM with LIF removal an established inducer of XEN cells [ 28 , 40 ] . Since retinoic acid at this level is a normal acid at 1uM with LIF removal an established inducer of XEN cells [ 28 , 40 ].…”

“…Interestingly, 300mM sorbitol despite LIF produced almost equal increases in Pdgfra+ intermediate and bright cells as retinoic acid at 1uM with LIF removal an established inducer of XEN cells [ 28 , 40 ] . Since retinoic acid at this level is a normal acid at 1uM with LIF removal an established inducer of XEN cells [ 28 , 40 ]. Since retinoic acid at this level is a normal morphogen for XEN cells [ 40 ], this suggests that 300mM overrides LIF and forces differentiation of a subpopulation of ESC.…”

“…4 First differentiated lineage XEN cells increase with 300mM sorbitol stress as much as positive control XEN morphogen retinoic acid, and mouse and human ESC differentiated to first lineage by culture conditions of Sox17 transgenic overexpression, cause induction of ACE2 and Tmprss2 Covid19 susceptibility genes. ( A ) Pdgfra-GFP ESC were exposed to 72hr LIF+ (NS), LIF- (ND), 1uM retinoic acid a normal morphogen for 1st lineage XEN induction from ESC or 200-400mM sorbitol in the presence of LIF to test for a stress dose that causes similar % of bright and intermediate bright 1st lineage Pdgfra+ cells as assayed by flow cytometry [ 28 ], ( B ) Three mouse ESC lines were culture under conditions to maintain stemness (far right) or XEN differentiation (3 histogram bars on left) and tested for ACE2, Tmprss2 Covid19 susceptibility genes or Gata6 XEN determining transcription factor, from NCBI GEO database; GSM40110/22/23/24 [ 52 ]. ( C ) Two human ESC lines were culture under conditions to maintain stemness (2 histogram bars on far right) or two human ESC lines transdifferentiated to XEN differentiation using first lineage driver Sox17 (2 histogram bars on left) and tested for ACE2, Tmprss2 C ovid19 susceptibility genes or Gata6 XEN determining transcription factor, from NCBI GEO database; GSM2722918/19/20/21 [ 53 ] …”