Ian O. Shum

During early embryonic development, cells transition from naive to primed pluripotent state. Various culture conditions have been established to revert primed cells back to naive state, to increase differentiation potential and to reset epigenetic abnormalities. In this study, we modified culture conditions to allow primed-to-naive conversion under feeder-independent and normoxic conditions (FINO medium), which exemplified the need for a quantitative measure of pluripotent states. DNA methylation (DNAm) profiling revealed extensive hypomethylation at naive state, but also significant gains of methylation at specific sites in the genome. We demonstrate that DNAm patterns can be used to benchmark culture protocols. Furthermore, we developed a naive-score based on DNAm at two genomic sites, which can be analyzed by digital PCR to monitor transition between pluripotent states. Our study describes a simplified culture protocol for primed-to-naive conversion, offers insights into the specific DNAm changes, and introduces a robust DNAm-based biomarker to track this process effectively.

Abnormalities at any stage of trophoblast development may result in pregnancy-related complications. Many of these adverse outcomes are discovered later in pregnancy, but the underlying pathomechanisms are constituted during the first trimester. Acquiring developmentally relevant material to elucidate the disease mechanisms is difficult. Human pluripotent stem cell (hPSC) technology can provide a renewable source of relevant cells. BMP4, A83-01, and PD173074 (BAP) treatment drives trophoblast commitment of hPSCs toward syncytiotrophoblast (STB), but lacks extravillous trophoblast (EVT) cells. EVTs mediate key functions during placentation, remodeling of uterine spiral arteries, and maintenance of immunological tolerance. We optimized the protocol for a more efficient generation of HLA-G^pos EVT-like trophoblasts from primed hiPSCs. Increasing the concentrations of A83-01 and PD173074, while decreasing bulk cell density resulted in an increase in HLA-G of up to 71%. Gene expression profiling supports the advancements of our treatment regarding the generation of trophoblast cells. The reported differentiation protocol will allow for an on-demand access to human trophoblast cells enriched for HLA-G^pos EVT-like cells, allowing for the elucidation of placenta-related disorders and investigating the immunological tolerance toward the fetus, overcoming the difficulties in obtaining primary EVTs without the need for a complex differentiation pathway via naïve pluripotent or trophoblast stem cells.