Primitive endoderm stem cell lines in mice

A blastocyst consists of three distinctive cell types: epiblast (EPI), trophoblast (TB), and primitive endoderm (PrE). Stem cell lines representing EPI and TB (embryonic stem (ES) cells and trophoblast stem (TS) cells) have been derived and they contribute to epiblast derivatives and trophoblast derivatives of stem cell-blastocyst chimeras, respectively. Although derived from PrE, extraembryonic endoderm (XEN) cells contribute to only a limited part of parietal endoderm (PE) but rarely to other PrE derivatives. Here we describe the establishment of primitive endoderm stem (PrES) cell lines in mice. PrES cells were derived and maintained in a serum-free media containing CHIR99021, FGF4, heparin, and PDGF-AA. RNA-seq analysis revealed that the transcriptome of PrES cells is globaly different from XEN cells: PrES cells express not only endoderm markers (Dab2, Gata4, Gata6, Sox17, etc.), but also pluripotent markers (Pou5f1, Cdh1, Nanog, Zfp42, etc.), and resembles in vivo founder PrE. PrES cells were rapidly and efficiently incorporated into PrE after blastocyst injection and efficiently contributed to all PrE derivatives, including PE, VE (visceral endoderm), and MZE (marginal zone endoderm) in chimeras. Importantly, PrES cells rescued embryonic lethality of PrE-depleted blastocysts by complementing all PrE derivatives. PrES cells thus not only contribute to understanding of the mechanisms of PrE specification but also provide a critical resource for artificial embryo reconstitution by stem cells alone. mRNAs were extracted from ES, TS, XEN and PrES stem cell

囊胚（blastocyst）包含三种独特的细胞类型：上胚层（epiblast, EPI）、滋养层（trophoblast, TB）与原始内胚层（primitive endoderm, PrE）。已成功建立代表上胚层与滋养层的干细胞系——胚胎干细胞（embryonic stem, ES细胞）与滋养层干细胞（trophoblast stem, TS细胞），二者可分别参与干细胞-囊胚嵌合体的上胚层衍生物与滋养层衍生物的形成。尽管胚外内胚层（extraembryonic endoderm, XEN细胞）源自原始内胚层，但其仅能有限参与壁内胚层（parietal endoderm, PE）的形成，极少参与其他原始内胚层衍生物的发育。本研究报道了小鼠原始内胚层干细胞（primitive endoderm stem, PrES细胞）系的建立。PrES细胞可在含有CHIR99021、成纤维细胞生长因子4（FGF4）、肝素与血小板衍生生长因子AA（PDGF-AA）的无血清培养基中分离培养并维持存活。RNA测序（RNA-seq）分析显示，PrES细胞的转录组与XEN细胞存在全局差异：PrES细胞不仅表达内胚层标志物（如Dab2、Gata4、Gata6、Sox17等），还表达多能性标志物（如Pou5f1、Cdh1、Nanog、Zfp42等），其转录组特征与体内原始内胚层创始细胞高度相似。囊胚注射后，PrES细胞可快速且高效地整合至宿主原始内胚层中，并可高效参与嵌合体中所有原始内胚层衍生物的形成，包括壁内胚层（PE）、脏内胚层（visceral endoderm, VE）与边缘区内胚层（marginal zone endoderm, MZE）。重要的是，PrES细胞可通过互补所有原始内胚层衍生物，挽救缺失原始内胚层的囊胚的胚胎致死表型。综上，PrES细胞不仅有助于解析原始内胚层特化的分子机制，还可为仅通过干细胞构建人工胚胎重构提供关键实验资源。本研究从ES、TS、XEN及PrES干细胞中提取了mRNA。