Derivation of human embryonic stem cells using a post-inner cell mass intermediate

Little is known about the true developmental origin of human embryonic stem cells (hESCESCESCs) or the events that initiate their generation. Recently, we have shown that hESCESCESCs 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 hESCESCESC fate. As a closer progenitor of hESCESCESCs 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 hESCESCESC derivation. The initial hESCESCESC 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 hESCESCESC derivation, 80.6% showed hESCESCESC outgrowth after further culture