A comparative study of key physiological stem cell parameters between three human trophoblast cell lines

Highlights

• IPSs induced trophoblast stem cells are similar to primary trophoblast stem cells.

• IPSs induced trophoblast stem cells are useful to study trophoblast fusion.

• IPSs induced trophoblast stem cells exhibited placenta barrier function.

Abstract

Human trophoblast stem cells (TSCs) play a key role in the placenta. These cells are proliferative, undifferentiated, and can differentiate into mature trophoblast cell types. However, primary human TSCs are difficult to obtain. In our previous study, we established TSCs from human induced pluripotent stem cells (TS^hiPSC). Here, we aimed to characterize the identity of these TS^hiPSC cells by comparing them with BeWo choriocarcinoma cells and primary TSCs (CT cells). Compared with BeWo cells, CT and TS^hiPSC cells showed high secretion of human chorionic gonadotrophin (hCG) and syncytiotrophoblast differentiation ability. Global gene microarray analysis results showed that CT and TS^hiPSC cells, unlike BeWo cells, could be classified in the same group. Compared with BeWo cells, CT and TS^hiPSC cells showed high expression levels of TSC-specific genes and low expression of cancer adhesion and invasion genes. Analysis of placental barrier integrity showed that TS^hiPSC cells could form a good barrier. Prospective studies using TS^hiPSC cells hold great promise for elucidating the pathogenesis of infertility due to trophoblast defects.

Introduction

Trophoblast stem cells (TSCs), also known as mononuclear cytotrophoblast cells, are the precursors of differentiated trophoblasts in the placenta. As pregnancy progresses, TSCs differentiate into either syncytiotrophoblasts (STBs) or extravillous trophoblasts (EVTs). Aberrancy in trophoblasts (TBs) is associated with abnormal placental function, which can potentially lead to pregnancy-related complications, including the early onset form of preeclampsia, intrauterine growth restriction, preterm labor, and low-birth weight [[1], [2], [3]]. An appropriate TSC model for in vitro studies of TB and placenta function remains to be established. The models that have been used so far include choriocarcinoma cells, primary human TBs from first trimester placentas or term placentas and rodent models. Although these models have been extensively used, they all have limitations and may not be appropriate for studying TSC function.

Human primary TSCs derived from first trimester placenta are not only difficult to obtain but also to culture. To address this, many researchers have tried to improve the components of the growth medium for primary TBs. Okae, Toh, Sato et al., optimized the culture conditions and successfully isolated proliferative TSCs [4], which could differentiate into either STBs or EVTs [4]. However, because of ethical problems, it is difficult to acquire human placental tissues and blastocysts.

Over the past decade, many researchers have tried to reprogram human pluripotent stem cells into TBs, including, human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells. These stem cells have been considered as new sources for cell replacement therapy since they can be derived from healthy people as well as from patients [5]. The cells can be differentiated into TB-like cells after treatment with bone morphogenetic protein 4 (BMP4) without the use of fibroblast growth factor-2 (FGF2) [[6], [7], [8], [9]]. Most BMP4-induced cells contain various TB subtypes including STBs, invasive EVTs, and mononuclear cytotrophoblasts [1]. However, BMP4-induced cytotrophoblasts cannot proliferate or directionally differentiate into STBs and EVTs [5]. Thus, the BMP4-induced TB cells are not considered as a good TSC in vitro model. Recently, we have developed a limited-area micromesh culture method to establish hiPSC-derived TSCs (hereafter referred to as TS^hiPSCcells) without BMP4 treatment. These TS^hiPSC cells can proliferate and differentiate into STBs and EVTs. However, the identity of TS^hiPSC cells remains to be characterized.

In this study, to investigate the identity of TS^hiPSC cells, we performed global gene analysis and compared the transcriptional profiles of TS^hiPSC cells with those of BeWo cells, derived from choriocarcinomas considered to represent villous cytotrophoblasts, and those of primary TSCs, established by Okae, Toh, Sato et al. [4], hereafter referred to as CT cells. To better understand the identity of TS^hiPSC cells, we further evaluated whether they can be used as a representative model for studying the placental barrier. Our analysis results indicated that TS^hiPSC and CT cells shared more similarities, compared with BeWo cells. To our knowledge, this is first study to compare TSC lines and provide novel insights into the study of human trophoblast development and functions.