Establishment of trophoblastic spheroids from human embryonic stem cells for in vitro implanatation study

Abstract

A successful pregnancy requires a good quality embryo and a receptive endometrium. Implantation failure is one of the contributing factors to the low success rate of in vitro fertilization, however, the mechanism is not known mainly due to the lack of a proper in vitro human implantation model. With the use of BAP (BMP4, A83-01 and PD173074), a human embryonic stem cell (hESC) derived trophoblastic spheroid (BAP-EB) was established in our laboratory. It was hypothesized that BAP-EB could be used as embryo surrogate for the study of early embryo implantation and trophoblast differentiation. The objectives of this study were to investigate the molecular and functional characterization of BAP-EB and explore the application of this model. The BAP-EB differentiation protocol was confirmed in H9. H9-BAP-EB, like the reported VAL3-BAP-EB derived from another hESC, possessed blastocyst-like structure, molecular characteristics of trophoblast cells and specific attachment potential to receptive endometrial epithelial cells. In an attempt to optimize the BAP-EB differentiation, it was found that mouse embryonic fibroblast conditioned medium (MEF-CM) was indispensable during BAP-EB differentiation. Furthermore, BAP treatment that covered the first 24 hours was sufficient to induce the trophoblastic differentiation from hESC. BAP-EB was also found to have higher expression levels of trophectoderm (TE) signature genes when compared to BAP differentiated cells in 2-dimensional format. Target approach functional studies on BAP-EB have shown that the knock down of Cx43 in hESC favoured the trophoblast differentiation of BAP-EB as demonstrated by higher human Chorionic Gonadotropin (hCG) production and attachment rates. Besides, antibody blocking approach also suggested E-cadherin on endometrial epithelial cells might be a critical molecule involved in BAP-EB attachment. Next generation sequencing was used to obtain the transcriptome profile during BAP-EB differentiation. Transcriptomic analysis indicated BAP-EB differentiation protocol was highly reproducible. While epiblast signature genes were reduced upon BAP differentiation, trophoblast signature genes were induced progressively at later time-points. Specifically, TE signature genes were induced in BAP-EB at 48h and 72h, during which the blastocyst-like structure and attachment competency were observed. KEGG pathway analysis indicated Hippo signaling pathway was enriched for the genes induced in all the time point of BAP-EB differentiation. The role of Hippo signaling pathway was studied by using a YAP inhibitor and an inhibitory effect was observed during BAP-EB differentiation. The data suggested BAP-EB was the best available embryo surrogate for trophoblast development and endometrial receptivity study. A coculture model of BAP-EB with primary endometrial epithelial cells (EEC) isolated from the endometria of sub-fertile women was established. The EEC isolation protocol was first optimized using different approaches. With the optimized method, the cultured EEC was highly pure and responsive to hormone treatment. The attachment rate of BAP-EB on EEC isolated from patients at their receptive phases in natural cycles (LH+7) or stimulated cycles (HCG+7) were significantly higher than the rates at their corresponding pre-receptive phases (LH+2 or HCG+2). In summary, the data suggested the potential use of BAP-EB for studying early trophoblast differentiation and predicting the endometrial receptivity.