Department for Reproductive Medicine, Ghent University Hospital, Ghent, Belgium.
Nat Protoc. 2013 Feb;8(2):254-64. doi: 10.1038/nprot.2012.157. Epub 2013 Jan 10.
Little is known about the true developmental origin of human embryonic stem cells (hESCs) or the events that initiate their generation. Recently, we have shown that hESCs originate from a post-inner cell mass (ICM) intermediate (PICMI), a unique transient epiblast-like structure that is different from both its ICM progenitor and its subsequent hESC fate. As a closer progenitor of hESCs than the ICM, the PICMI could be used to provide further insight into the human pluripotent state. Here we provide a detailed (7-d) protocol for the culture of the human preimplantation embryos in order to derive the PICMI. Subsequent identification and cryopreservation of the PICMI are described, in addition to hESC derivation. The initial hESC outgrowth is visible within 2-7 d after PICMI plating. By using the protocol provided, we observed PICMI formation in 21.3% of plated blastocysts with good-quality ICMs. Of the PICMIs used for hESC derivation, 80.6% showed hESC outgrowth after further culture.
关于人类胚胎干细胞（hESC）的真正发育起源或启动其产生的事件，人们知之甚少。最近，我们已经证明 hESC 起源于一个原内细胞团（ICM）后中间体（PICMI），这是一种独特的短暂胚外样结构，与它的 ICM 前体及其随后的 hESC 命运都不同。作为比 ICM 更接近 hESC 的祖细胞，PICMI 可以用于提供对人类多能性状态的进一步深入了解。在这里，我们提供了一个详细的（7 天）人类胚胎培养方案，以获得 PICMI。此外，还描述了 PICMI 的后续鉴定和冷冻保存，以及 hESC 的衍生。在 PICMI 接种后 2-7 天内，可见初始 hESC 外植体。使用提供的方案，我们观察到在具有良好 ICM 的接种囊胚中，有 21.3%形成了 PICMI。在用于 hESC 衍生的 PICMI 中，有 80.6%在进一步培养后显示出 hESC 外植体。
