Generation and characterisation of a human embryonic stem cell- derived developmental model of the human female reproductive tract epithelium

Following the discovery of human embryonic stem cells (hESC), much effort has focused on their in vitro differentiation to therapeutically relevant cell types. Tissue recombination technology enabled recombination of embryonic stem cells with organ specific mouse mesenchyme for organogenesis in vivo. These differentiation strategies have also enabled researchers to gain an insight into human organ development previously not possible. The aim of this study was to establish a model of human female reproductive tract (FRT) epithelium to gain a better understanding of its development. A two-stage method was developed that recombined hESC with neonatal mouse uterine mesenchyme (nMUM) for development in vitro and then in vivo. In vitro recombination experiments showed that hESC-derived embryoid bodies (EBs) from two separate human embryonic stem cell lines (ENVY and MEL-1) fused together with nMUM as a recombinant structure in vitro. Following transplantation of the recombinant grafts in vivo into NOD.SCID mice, nMUM was able to direct hESCs to differentiate into epithelial structures (simple columnar ciliated epithelium) within small teratoma-like growths. The established model required further modifications to improve success rate. More importantly, a detailed characterisation was required to confirm the identity of the hESC derived epithelium. To increase the reproducibility of the established model, exogenous growth factors (BMP4 and Activin A) were added in vitro to enhance hESC survival. A new strain of severely immunodeficient mice was used for hosting recombinant grafts to increase viability of hESC in vivo. A comprehensive characterisation of the established model was carried out from the earliest stage of hESC differentiation (day 3 in vitro) to 8 weeks after transplantation by immunohistochemical and molecular methods. The expression of a panel of morphological (cilia, columnar/cuboidal morphology, CK18) and functional markers (Estrogen receptor, Ki-67 and Glycodelin A) confirmed that by week 8 of in vivo incubation, hESC derived FRT-like epithelium (surrounded by mouse uterine stroma) had characteristics similar to human adult upper FRT epithelium of the oviduct or endometrium. At early in vivo stages (week 2 and 4), temporally regulated expression of developmental homeodomain transcription factors (HOXA10, PAX2) recapitulated key events during FRT organogenesis. Moreover, nMUM was demonstrated for the first time to induce formation of primitive streak-like cells and upregulate mesoderm/endoderm gene expression in differentiating hESC-derived embryoid bodies (EBs) in vitro. Using the established hESC-derived FRT model, the ontogeny of a particular transcription factor, LIM1 was examined. The expression of the LIM1 protein in the hESC-derived FRT model instigated a novel investigation into the expression of LIM1 into human adult uterine tissue as well as human endometrial cancer cell lines. The findings of this investigation contradicts a previous notion that Lim1 is not expressed in adult FRT. Interestingly, the evolutionarily conserved expression of Lim1 was also detected in neonatal and adult mouse uterus using immunofluorescence and real time polymerase chain reaction (PCR) methods. Based on evidence of the inductive capabilities of the nMUM mentioned above, a microarray investigation was conducted to identify genes coding for mesenchyme-derived growth factors involved in hESC differentiation. Connective tissue growth factor (CTGF) was identified and confirmed by immunofluorescence and real time PCR in the nMUM. The presence of endogenous CTGF in the recombinant EBs, however, further complicated the investigation. Functional analysis into endogenous CTGF in the EB and the action of exogenous CTGF revealed that the production of the growth factor is dependent on initial formation of primitive streak-like cells in the EBs. Furthermore, CTGF may be involved in epithelial-mesenchymal transition (EMT) as well as mesoderm differentiation in EBs. The model established in this study provides a platform for future studies to explore various aspects of human FRT development, or as an alternative strategy for hESC differentiation using organ specific animal mesenchyme. The model may one day be used to study the detrimental effects of endocrine disruptors on human FRT development. Furthermore, the presence of the Lim1/LIM1 in adult reproductive tract shows the persistent expression of yet another developmental homeodomain transcription factor in the adult mouse and human uterus, further highlighting the complex transcriptional factor network regulating adult endometrial remodelling