Establishment of human trophoblast stem cells from term smooth chorion.

INTRODUCTION

Human trophoblast stem (TS) cells derived from first-trimester placental villi (TSCT) and blastocyst (TSblast) are valuable for studying placental development and pathobiology. However, most placenta-mediated pregnancy complications are diagnosed in the third-trimester, and there are limited reports on TS cells from the third-trimester placental tissues. In this study, we report the successful derivation of TS cells from the term smooth chorion.

METHODS

Smooth chorion isolation was carried out from term placenta, followed by the preparation of single cells through enzymatic dissociation. ITGA6-positive cells were isolated from smooth chorion and cultured in TS medium. The chorion-derived TS (Ch-TS) cells were characterized through flow cytometry, immunocytochemical analysis, DNA methylation analyses via bisulfite sequencing, miRNA analysis using quantitative PCR, and RNA sequencing. To assess their differentiation potential, Ch-TS cells were induced to differentiate into extravillous trophoblast (EVT) cells and syncytiotrophoblast (ST) cells.

RESULTS

Human TS cells were successfully derived from the term smooth chorion. Immunohistochemistry confirmed trophoblast marker expression in Ch-TS cells. Bisulfite sequencing revealed that the ELF5 promoter region was hypomethylated in Ch-TS cells, consistent with trophoblastic DNA methylation status. Ch-TS cells expressed miRNAs from the chromosome 19 miRNA cluster (C19MC). Flow cytometryanalysis revealed that the expression patterns of HLA class I molecules were comparable between Ch-TS and TSCT/blast. Ch-TS cells successfully differentiated into EVT and ST cells. RNA sequencing showed transcriptomic similarities between Ch-TS cells and TSCT/blast cells.

DISCUSSION: Ch-TS cells may serve as a valuable in vitro model for studying trophoblast biology and placenta-mediated pregnancy complications, similar to TSCT/blast cells.