Establishment of a novel human embryonic stem cell-derived trophoblastic spheroid implantation model.
OBJECTIVE
Can human embryonic stem cell-derived trophoblastic spheroids be used to study the early stages of implantation?


CONCLUSIONS
We generated a novel human embryonic stem cell-derived trophoblastic spheroid model mimicking human blastocysts in the early stages of implantation.


BACKGROUND
Both human embryos and choriocarcinoma cell line derived spheroids can attach onto endometrial cells and are used as models to study the early stages of implantation. However, human embryos are limited and the use of cancer cell lines for spheroid generation remains sub-optimal for research.


METHODS
Experimental induced differentiation of human embryonic stem cells into trophoblast and characterization of the trophoblast.


METHODS
Trophoblastic spheroids (BAP-EB) were generated by inducing differentiation of a human embryonic stem cell line, VAL3 cells with bone morphogenic factor-4, A83-01 (a TGF-β inhibitor), and PD173074 (a FGF receptor-3 inhibitor) after embryoid body formation. The expressions of trophoblastic markers and hCG levels were studied by real-time PCR and immunohistochemistry. BAP-EB attachment and invasion assays were performed on different cell lines and primary endometrial cells.


RESULTS
After 48 h of induced differentiation, the BAP-EB resembled early implanting human embryos in terms of size and morphology. The spheroids derived from embryonic stem cells (VAL3), but not from several other cell lines studied, possessed a blastocoel-like cavity. BAP-EB expressed several markers of trophectoderm of human blastocysts on Day 2 of induced differentiation. In the subsequent days of differentiation, the cells of the spheroids differentiated into trophoblast-like cells expressing trophoblastic markers, though at levels lower than that in the primary trophoblasts or in a choriocarcinoma cell line. On Day 3 of induced differentiation, BAP-EB selectively attached onto endometrial epithelial cells, but not other non-endometrial cell lines or an endometrial cell line that had lost its epithelial character. The attachment rates of BAP-EB was significantly higher on primary endometrial epithelial cells (EEC) taken from 7 days after hCG induction of ovulation (hCG+7 day) when compared with that from hCG+2 day. The spheroids also invaded through Ishikawa cells and the primary endometrial stromal cells in the co-culture.


CONCLUSIONS
The attachment rates of BAP-EB were compared between EEC obtained from Day 2 and Day 7 of the gonadotrophin stimulated cycle, but not the natural cycles.


CONCLUSIONS
BAP-EB have the potential to be used as a test for predicting endometrial receptivity in IVF cycles and provide a novel approach to study early human implantation, trophoblastic cell differentiation and trophoblastic invasion into human endometrial cells.
Department of Obstetrics and Gynaecology, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, China Shenzhen Key Laboratory of Fertility Regulation, The University of Hong Kong Shenzhen Hospital, The University of Hong Kong, Shenzhen, China Center for Reproduction, Development and Growth, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China.
Yin-Lau
YL
Lee
Department of Obstetrics and Gynaecology, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, China.
Sze-Wan
SW
Fong
Department of Obstetrics and Gynaecology, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, China.
Andy C H
AC
Chen
Department of Obstetrics and Gynaecology, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, China.
Tiantian
T
Li
Department of Obstetrics and Gynaecology, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, China.
Chaomin
C
Yue
Department of Obstetrics and Gynaecology, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, China Shenzhen Key Laboratory of Fertility Regulation, The University of Hong Kong Shenzhen Hospital, The University of Hong Kong, Shenzhen, China Center for Reproduction, Development and Growth, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China.
Cheuk-Lun
CL
Lee
Department of Obstetrics and Gynaecology, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, China Shenzhen Key Laboratory of Fertility Regulation, The University of Hong Kong Shenzhen Hospital, The University of Hong Kong, Shenzhen, China Center for Reproduction, Development and Growth, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China.
Ernest H Y
EH
Ng
Department of Obstetrics and Gynaecology, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, China Shenzhen Key Laboratory of Fertility Regulation, The University of Hong Kong Shenzhen Hospital, The University of Hong Kong, Shenzhen, China Center for Reproduction, Development and Growth, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China.
William S B
WS
Yeung
Department of Obstetrics and Gynaecology, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, China Shenzhen Key Laboratory of Fertility Regulation, The University of Hong Kong Shenzhen Hospital, The University of Hong Kong, Shenzhen, China Center for Reproduction, Development and Growth, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China ckflee@hku.hk.
Kai-Fai
KF
Lee
D002460:Cell Line; D010064:Embryo Implantation; D000066449:Human Embryonic Stem Cells; D006801:Humans; D008954:Models, Biological; D018874:Spheroids, Cellular; D014327:Trophoblasts
embryo development; human endometrium; implantation; in vitro implantation model; stem cells; trophoblasts