Hypoxic stress induces, but cannot sustain trophoblast stem cell differentiation to labyrinthine placenta due to mitochondrial insufficiency

Abstract

Dysfunctional stem cell differentiation into placental lineages is associated with gestational diseases. Of the differentiated lineages available to trophoblast stem cells (TSC), elevated O2 and mitochondrial function are necessary to placental lineages at the maternal-placental surface and important in the etiology of preeclampsia. TSC lineage imbalance leads to embryonic failure during uterine implantation. Stress at implantation exacerbates stem cell depletion by decreasing proliferation and increasing differentiation. Implantation site O2 is normally ~2%. In culture, exposure to 2% O2 and fibroblast growth factor (FGF)4 enabled highest mouse TSC multipotency and proliferation. In contrast, hypoxic stress (0.5% O2) initiated the most TSC differentiation after 24 hr despite FGF4. However, hypoxic stress supported differentiation poorly after 4–7 days, despite FGF4 removal. At all tested O2 levels, FGF4 maintained Warburg metabolism; mitochondrial inactivity and aerobic glycolysis. However, hypoxic stress suppressed mitochondrial membrane potential, maintained low mitochondrial cytochrome c oxidase (oxidative phosphorylation/OxPhos), and high pyruvate kinase M2 (glycolysis) despite FGF4 removal. Inhibiting OxPhos inhibited differentiation at the differentiation optimum at 20% O2. Moreover, adding differentiation-inducing hyperosmolar stress failed to induce differentiation during hypoxia. Thus, differentiation depended on OxPhos at 20% O2; hypoxic and hyperosmolar stresses did not induce differentiation at 0.5% O2. Hypoxia-limited differentiation and mitochondrial inhibition and activation suggest that differentiation into two lineages of the labyrinthine placenta requires O2>0.5–2% and mitochondrial function. Stress-activated protein kinase increases an early lineage and suppresses later lineages in proportion to the deviation from optimal O2 for multipotency, thus it is the first enzyme reported to prioritize differentiation.

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.…”

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

“…Thus, induction of both Lrp2 and Dab2 by stress suggests that a primary function of stress-induced differentiation is to provide function that becomes essential rapidly after blastocyst implantation. Hyperosmotic stress begins to induce this absorptive capability in mESCs in monolayers, similar to the induction by several stressors of the placental hormone PL1 from TSCs [5,7,8,71] that increases nutritional supply to the fetal-maternal interface in vivo. PI3K and p38MAPK protect potency after adaptation to stress, leading to recovery of Nanog/Rex1 and Nanog/Oct4 between 4 and 24 h, respectively (Table 3).…”

“…Finally, the stress-induced differentiation of TSCs occurred even in the presence of FGF4 signaling that sustains their multipotent state [5][6][7][8]94]. Stress signaling was dominant over FGF4 signaling at doses where stem cell populations expanded, but at diminished rates.…”

“…Interestingly, JNK prioritizes differentiation of TSCs stressed by hyperosmotic sorbitol, benzopyrene, or hypoxic O 2 below 2%, by inducing first lineage [5,7,64] and suppressing later lineages [5]. JNK appears to mediate stress-induced endoderm, the first lineage arising from ESCs, but it remains to be determined whether JNK suppresses later lineages.…”

“…TGCs maintain early pregnancy by producing the hormones that stimulate uterine changes necessary to support an embryo. When placental trophoblast stem cells (TSCs), precursors to TGCs, were confronted with hyperosmotic stress in vitro, the stress enzymes that were activated modulated lineage TF expression [2,[5][6][7]. Nearly all surviving TSCs terminally differentiated to first-lineage TGCs [5,7,8] and later lineages were suppressed [5,8].…”