Establishment of high-throughput screening platform for the identification of pharmacological drugs in modulating early trophoblast differentiation from human expanded potential stem cells

Abstract

Development of trophectoderm of a blastocyst, and its subsequent differentiation to trophoblast and interaction with endometrium are critical to implantation success. Dysregulated trophoblast development can lead to miscarriages, preeclampsia and intrauterine growth restriction, but no effective drug is available for the treatments. To solve the ethical issue in using human embryos, a human embryonic stem cells (hESC) derived trophoblastic spheroids (BAP-EB) having selective interaction with receptive endometrium was established in our laboratory. Recently, a new stem cell named Expanded Potential Stem Cell (EPSC) that possesses totipotency features of cleavage stage embryos was also established. It was hypothesized that human EPSC (hEPSC) represented a new cell model for identifying critical pathways for early human trophectoderm/trophoblast development. This study aimed at developing a high throughput screening system to identify compounds that modulated such development. By using a hEPSC line derived from iPSC with GATA2 reporter, a cost-effective large screening platform was established. Trophoblast differentiation with the first 24h of BAP treatment and the inclusion of XTT cell viability assay were adopted for screening the LOPAC1280 pharmacologically active compounds library. Gene ontology analysis demonstrated that the target gene enriched pathways were related to trophoblast development. After dose dependent validation, two pairs of compounds that had agonistic or antagonistic effects on PPARγ and histamine H1-receptor 1 (HRH1), and two compounds (Ro 90-7501, K145 hydrochloride) with unknown function on early trophoblast differentiation were selected for further studies. Transcriptomic analysis from the published datasets indicated that the 4 target genes of the selected compounds had differentially higher expression in trophectoderm or early trophoblastic cells. Further molecular and functional studies demonstrated that PPARγ antagonist reduced syncytiotrophoblast (STB) but accelerated extravillous trophoblast (EVT) differentiation from hEPSC. It also reduced the BAP-EB attachment rates. PPARγ antagonist also reduced STB and EVT differentiation from TSC, but PPARγ agonist had no effect. The follow-up study of a feedback loop involving compounds targeting PPARγ/NO/TGFβ has identified a new regime depriving of BAP for early trophoblast differentiation from hEPSC. The study on HRH1 activator and inhibitor suggested that HRH1 inhibitor suppressed the gene expression of early trophoblast makers, reduced BAP-EB cystic structure formation and the attachment rates. However, stimulatory effect of HRH1 activator was found to be minimal. The effects of K145 hydrochloride and Ro 90-7501, and their target genes (SPHK2, APP) were followed. Treatments with the two compounds suppressed the trophoblast marker gene expressions. Ro 90-7501 also reduced the cystic structures formation and attachment rates of the treated BAP-EB. While K145 hydrochloride decreased EVT differentiation, Ro 90-7501 had no effect on both STB and EVT differentiation from TSC. Gene knockout (KO) by CRISPR/Cas9 approach indicated SPHK2-KO and APP-KO had similar impairment effects as the drug treatments. Mouse studies demonstrated that both compounds impaired preimplantation embryo developments. K145 hydrochloride, but not Ro 90-7501 also significantly decreased the implantation rate. In conclusion, hEPSC trophoblast differentiation provided a unique cellular platform to identify key molecules and signals in the trophoblasts differentiation which may strengthen our understanding on the pregnancy-associated disease during the early embryo implantation processes.