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Date of Award
Daniel A. Rappolee
Low level of oxygen (O2) occurs physiologically during in vivo embryo development. As developing embryos moving from fallopian tube to uterus, oxygen level gradually decreases to ≤ 5% at the time of blastocyst implantation. Blastocysts are made of two major cell populations, trophoblast cells and inner cell mass, from which trophoblast stem cells (TSCs) and embryonic stem cells (ESCs) are derived respectively. TSCs serve as placental stem cells that later on proliferate and differentiate into placenta. Previous study has shown that 2% O2 is the optimal O2 level for mTSC in vitro growth and potency maintenance, which agrees with their low O2 niche in vivo. Pathological hypoxia can happen to embryos in pregnancy complicated by certain medical conditions such as sleep apnea, anemia, hypertension or suboptimal living conditions such as high altitude or with carbon monoxide pollution.
Here we study the effect of hypoxia at 0.5% O2 on mTSC proliferation and differentiation. We found that 0.5% O2 reduces the growth of mTSC without high levels of apoptosis and forces differentiation despite the potency maintaining conditions. Hypoxic stress induced differentiation has a preference toward trophoblast giant cells (TGCs) lineage. As a matter of fact, at the end of 6 days’ 0.5% O2 culture, approximately 50% of cells became TGCs. One essential function of TGCs is to secrete placental lactogen 1 (PL1), the hormone that rescues corpus luteum function and maintains pregnancy. Increased PL1 expression was found in 0.5% O2 induced differentation. Compared with normal differentiaiton with fibroblast growth factor 4 (FGF4) removal, hypoxic stress induced differentiation has a longer reversible period, which eventually becomes irreversible with prolong hypoxic exposure. We think that stress induced differentiation initially serves to increase the chance of organimal survival by providing essential parenchymal function; but with prolonged stress, reduced growth and irreversible differentiaiton caused stem cell depletion would lead to miscarriage.
The study on the effect of 0.5% O2 on mTSC with FGF4 present is to model how pathological hypoxia might affect embryo development during the peri-implantation period. We also studied what can potentially be the optimal O2 for in vitro human blastocyst culture before implantation. 2%, 5% and 20% O2 were compared. 2% is the O2 level in human uterus at the time of implantation. 5% is the current standard O2 for human in vitro embryo culture and 20% is the tradition O2 level that has been used for 30 years since the start of in vitro fertilization (IVF) practice. We found that 20% O2 is most detrimental to post-thaw day 3 human embryo culture to blastocyst stage and 5% O2 is most beneficial. 2% and 5% O2 are remarkably similar to each other in terms of blastocyst cell number and stress related gene expression. However, 2% O2 slightly increased the level of apoptosis compared with 5% O2. Potential confounding factors from 20% O2 used at the first three days of culture before cryopreservation and insufficient nutrient supply associated with static culture may contribute to the result seen here.
We next investigated the effect of O2 on the dynamic of AMP-activated protein kinase (AMPK). AMPK is a central regulator of enegry metabolism and there are increasing evidences showing it is also related to stem cell potency regulation. We found that departing from optimal 2% O2 for mTSC in vitro culture induced fastest activation of AMPK, regardless which new O2 level cells were switched into. The speed of AMPK activation is similar to stress activated protein kinase (SAPK) when departing from 2% O2. The highest magnitude of AMPK and SAPK activation was observed in hypoxic O2 at 0.5% and anoxia. We think the speed of stress kinase activation reflects the starting cellular state while the magnitude of stress kinase activation reflects the final cellular state. Both speed and magnitude of stress kinase activation are important indicators of the environment cells are subjected to. Interestingly, we found that at 2% O2 AMPK was activated at 6 - 8h of mTSC culture, which probably reflects the need to change medium frequently in order to supply sufficient nutrition for the rapid cell proliferation at 2% O2.
Yang, Yu, "Identification Of Oxygen Optima For Mouse Trophoblast Stem Cells And Human Embryos And The Stress Responses Upon Departing Optima" (2017). Wayne State University Dissertations. 1758.