Stress-Induced Enzyme Activation Primes Murine Embryonic Stem Cells to Differentiate Toward the First Extraembryonic Lineage

Abstract

Extracellular stresses influence transcription factor (TF) expression and therefore lineage identity in the periimplantation mouse embryo and its stem cells. This potentially affects pregnancy outcome. To understand the effects of stress signaling during this critical period of pregnancy, we exposed cultured murine embryonic stem cells (mESCs) to hyperosmotic stress. We then measured stress-enzyme-dependent regulation of key pluripotency and lineage TFs. Hyperosmotic stress slowed mESC accumulation due to slowing of the cell cycle over 72 h, after a small apoptotic response within 12 h. Phosphoinositide 3-kinase (PI3K) enzymatic signaling was responsible for stem cell survival under stressed conditions. Stress initially triggered mESC differentiation after 4 h through MEK1, c-Jun N-terminal kinase ( JNK), and PI3K enzymatic signaling, which led to proteasomal degradation of Oct4, Nanog, Sox2, and Rex1 TF proteins. Concurrent with this post-transcriptional effect was the decreased accumulation of potency TF mRNA transcripts. After 12-24 h of stress, cells adapted, cell cycle resumed, and Oct4 and Nanog mRNA and protein expression returned to approximately normal levels. The TF protein recovery was mediated by p38MAPK and PI3K signaling, as well as by MEK2 and/or MEK1. However, due to JNK signaling, Rex1 expression did not recover. Probing for downstream lineages revealed that although mESCs did not differentiate morphologically during 24 h of stress, they were primed to differentiate by upregulating markers of the first lineage differentiating from mESCs, extraembryonic endoderm. Thus, although two to three TFs that mark pluripotency recover expression by 24 h of stress, there is nonetheless sustained Rex1 suppression and a priming of mESCs for differentiation to the earliest lineage.

Commonly used fertility drugs, a diet supplement, and stress force AMPK-dependent block of stemness and development in cultured mammalian embryos

“…Similar to past reports for two-cell embryos [45], blastocysts [41,45], TSCs [41,45,46,63,104,105], ESCs [41,64,65], and induced pluripotent stem cells (iPSCs) [43], AMPK is the master regulatory enzyme for stress-induced potency loss of Oct4, Rex1, Cdx1, Id2, [41,[43][44][45][46]106], and Nanog [42]. Oct4 and Rex1 and other potency factor loss in two-cellstage embryos, blastocysts, TSCs, and ESCs occur within 0.5-2 h, suggesting that the stemness program change may be an important strategy requiring rapid, molecular action.…”

“…Thus, transient high molecular responses may not lead to biological changes. In addition, three of four ESC potency factors that were decreased from 1 to 4 h of 200 mM sorbitol rebounded to normal levels at 24 h despite continuing hyperosmotic stress [65]. These data suggest that in vitro effects may be higher due to lack of clearance and that ESCs in the early embryo may regain potency after prolonged exposures.…”

“…We used hyperosmotic sorbitol at 200 mM because this dose is not toxic to embryos, TSCs, or ESCs [41,55,56,65,79] but slows their proliferation and has significant effects in causing AMPK-dependent Cdx2 and Id2 loss [41,45], PL1 increase in TSCs [80], Oct4 and Rex1 loss and first lineage Dab2 and LRP2 markers in ESCs [65,81,82], and significant AMPK-dependent Id2 loss in blastocysts [41]. We had previously shown that 200 mM sorbitol causes Cdx2 and Id2 loss in two-cell embryos in an AMPK-dependent manner [45], and this report adds AMPK-dependent Rex1 and Oct4 loss to make up a group of four AMPK-regulated potency factors expressed in both ESC and TSC lineages by the blastocyst stage.…”

“…Immunofluoresence Two-cell-stage embryos were fixed, quenched, permeabilized, and stained for Oct4 and Rex1 and counterstained for 4′,6-diamidino-2-phenylindole (DAPI) by modifying previous protocols used in mouse ESCs and validated by immunoblot [64,65]. Briefly, embryos were fixed in 2 % paraformaldehyde in phosphate-buffered saline (PBS; pH 7.4) for 30 min at room temperature, quenched in 0.5 M glycine, and rinsed three times in PBS containing 0.5 % bovine serum albumin (PBS/BSA).…”

Development and Validation of a Rex1-RFP Potency Activity Reporter Assay That Quantifies Stress-Forced Potency Loss in Mouse Embryonic Stem Cells

“…This decreases embryonic developmental rates and stem cell growth rates and causes potency loss affecting fetal growth, function, and viability [1,2]. Embryonic stem cells (ESCs) respond to hyperosmotic stress with a transient loss of Oct4 and long-term-loss of Rex1 proteins in a proteasome-dependent manner [3]. This correlates with stress-induced first cell lineage differentiation and suppression of later lineages.…”