Establishment of human expanded potential stem cell lines via preimplantation embryo cultivation and somatic cell reprogramming

Abstract

<p>We previously reported the derivation of expanded potential stem cells (EPSCs) by modulating signaling pathways involved in preimplantation embryogenesis. These cells exhibit expanded developmental potential into embryonic and extraembryonic lineages, and we have shown that human EPSCs (hEPSCs) possess trophoblast differentiation potency for generating human trophoblast stem cells. Here we report protocols for deriving stable hEPSC lines directly from morula or early blastocyst stages of human preimplantation embryos (hEPSC-em) and by reprogramming human dermal fibroblasts (human induced EPSCs) using six exogenous factors, as an extension to our previous protocols on deriving porcine EPSCs from preimplantation embryos and by reprogramming somatic cells. These hEPSC lines proliferate robustly over long-term passaging and are amenable to both simple indels and precision genome editing. We provide guidance for characterizing these newly established hEPSCs, including cell-cycle analysis, pluripotency validation and karyotyping. The hEPSCs form teratomas with embryonic and extraembryonic cell lineages and readily differentiate into human trophoblast stem cells in vitro. At the molecular level, hEPSCs have unique features such as high expression of core histone genes and low H3K27me3 levels resembling eight-cell/morula stage embryos. These properties make hEPSCs a valuable tool not only for studying early human development but also for potential applications in regenerative medicine. The protocols presented in this manuscript can be readily performed by postgraduate students or postdoctoral fellows and completed within around 2 months.</p>