Tanaka Erika, Koyanagi-Aoi Michiyo, Nakagawa So, Shimode Sayumi, Yamada Hideto, Terai Yoshito, Aoi Takashi
Division of Stem Cell Medicine, Graduate School of Medicine, Kobe University, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan.
Division of Advanced Medical Science, Graduate School of Science, Technology and Innovation, Kobe University, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan.
Regen Ther. 2024 Sep 5;26:729-740. doi: 10.1016/j.reth.2024.08.020. eCollection 2024 Jun.
In human placental development, the trophectoderm (TE) appears in blastocysts on day 5 post-fertilization and develops after implantation into three types of trophoblast lineages: cytotrophoblast (CT), syncytiotrophoblast (ST), and extravillous trophoblast (EVT). CDX2/Cdx2 is expressed in the TE, and Cdx2 expression is upregulated by knockdown of Foxo1 in mouse ESCs. However, the significance of FOXO1 in trophoblast lineage differentiation during the early developmental period remains unclear. In this study, we examined the effect of FOXO1 inhibition on the differentiation of naive human induced pluripotent stem cells (iPSCs) into TE and trophoblast lineages.
We induced TE differentiation from naive iPSCs in the presence or absence of a FOXO1 inhibitor, and the resulting cells were subjected to trophoblast differentiation procedures without the FOXO1 inhibitor. The cells obtained in these processes were assessed for morphology, gene expression, and hCG secretion using phase-contrast microscopy, reverse transcription polymerase chain reaction (RT-PCR), quantitative RT-PCR (RT-qPCR), RNA-seq, immunochromatography, and a chemiluminescent enzyme immunoassay.
In the induction of trophoblast differentiation from naive iPSCs, treatment with a FOXO1 inhibitor resulted in the enhanced expression of TE markers, CDX2 and HAND1, but conversely decreased the expression of ST markers, such as ERVW1 (Syncytin-1) and GCM1, and an EVT marker, HLA-G. The proportion of cells positive for an early TE marker TACSTD2 and negative for a late TE marker ENPEP was higher in FOXO1 inhibitor-treated cells than in non-treated cells. The expressions of ERVW1 (Syncytin-1), ERVFRD-1 (Syncytin-2), and other endogenous retrovirus (ERV)-associated genes that have been reported to be expressed in trophoblasts were suppressed in the cells obtained by differentiating the TE cells treated with FOXO1 inhibitor.
Treatment with a FOXO1 inhibitor during TE induction from naive iPSCs promotes early TE differentiation but hinders the progression of differentiation into ST and EVT. The suppression of ERV-associated genes may be involved in this process.
在人类胎盘发育过程中，滋养外胚层（TE）在受精后第5天出现在囊胚中，并在植入后发育为三种滋养层谱系：细胞滋养层（CT）、合体滋养层（ST）和绒毛外滋养层（EVT）。CDX2/Cdx2在TE中表达，并且在小鼠胚胎干细胞中，Foxo1的敲低会上调Cdx2的表达。然而，FOXO1在早期发育阶段滋养层谱系分化中的意义仍不清楚。在本研究中，我们研究了FOXO1抑制对未分化的人类诱导多能干细胞（iPSC）向TE和滋养层谱系分化的影响。
我们在存在或不存在FOXO1抑制剂的情况下诱导未分化的iPSC向TE分化，然后将所得细胞在不存在FOXO1抑制剂的情况下进行滋养层分化程序。使用相差显微镜、逆转录聚合酶链反应（RT-PCR）、定量RT-PCR（RT-qPCR）、RNA测序、免疫层析和化学发光酶免疫测定法对在这些过程中获得的细胞进行形态、基因表达和hCG分泌评估。
在从未分化的iPSC诱导滋养层分化过程中，用FOXO1抑制剂处理导致TE标志物CDX2和HAND1的表达增强，但相反地降低了ST标志物如ERVW1（合胞素-1）和GCM1以及EVT标志物HLA-G的表达。早期TE标志物TACSTD2阳性且晚期TE标志物ENPEP阴性的细胞比例在FOXO1抑制剂处理的细胞中高于未处理的细胞。在用FOXO1抑制剂处理的TE细胞分化获得的细胞中，据报道在滋养层中表达的ERVW1（合胞素-1）、ERVFRD-1（合胞素-2）和其他内源性逆转录病毒（ERV）相关基因的表达受到抑制。
在从未分化的iPSC诱导TE过程中用FOXO1抑制剂处理可促进早期TE分化，但阻碍向ST和EVT的分化进程。ERV相关基因的抑制可能参与了这一过程。
