Laminin111-based defined culture promoting self-renewing human pluripotent stem cells with properties of the early post-implantation epiblast.

In the mammalian embryo, a formative pluripotent phase is proposed to exist at the early post-implantation period, during the transition from the pre-implantation naive-to the post-implantation primed-epiblast. By recapitulating a laminin component of the extracellular matrix niche during embryonic formative transition, and defined culture conditions, we generated cultures highly enriched for self-renewing human pluripotent stem cells (hPSCs), exhibiting properties of early post-implantation epiblast cells. These hPSCs display post-implantation-epiblast gene expression profiles. FGF and TGF-ß signaling maintain their self-renewal for multiple passages. They have inactive canonical Wnt signaling, do not express primitive streak markers, and are competent to initiate differentiation toward germline and somatic fates. hPSCs exhibiting early post-implantation epiblast properties may shed light on human embryonic PSCs development and may serve for initiating somatic and germ cell specification.

Derivation of xeno-free and GMP-grade human embryonic stem cells--platforms for future clinical applications.

Clinically compliant human embryonic stem cells (hESCs) should be developed in adherence to ethical standards, without risk of contamination by adventitious agents. Here we developed for the first time animal-component free and good manufacturing practice (GMP)-compliant hESCs. After vendor and raw material qualification, we derived xeno-free, GMP-grade feeders from umbilical cord tissue, and utilized them within a novel, xeno-free hESC culture system. We derived and characterized three hESC lines in adherence to regulations for embryo procurement, and good tissue, manufacturing and laboratory practices. To minimize freezing and thawing, we continuously expanded the lines from initial outgrowths and samples were cryopreserved as early stocks and banks. Batch release criteria included DNA-fingerprinting and HLA-typing for identity, characterization of pluripotency-associated marker expression, proliferation, karyotyping and differentiation in-vitro and in-vivo. These hESCs may be valuable for regenerative therapy. The ethical, scientific and regulatory methodology presented here may serve for development of additional clinical-grade hESCs.