Derivation of naïve human embryonic stem cells

Abstract

The naïve pluripotent state has been shown in mice to lead to broad and more robust developmental potential relative to primed mouse epiblast cells. The human naïve ES cell state has eluded derivation without the use of transgenes, and forced expression of OCT4, KLF4, and KLF2 allows maintenance of human cells in a naïve state [Hanna J, et al. (2010) Proc Natl Acad Sci USA 107 (20):9222-9227]. We describe two routes to generate nontransgenic naïve human ES cells (hESCs). The first is by reverse toggling of preexisting primed hESC lines by preculture in the histone deacetylase inhibitors butyrate and suberoylanilide hydroxamic acid, followed by culture in MEK/ERK and GSK3 inhibitors (2i) with FGF2. The second route is by direct derivation from a human embryo in 2i with FGF2. We show that human naïve cells meet mouse criteria for the naïve state by growth characteristics, antibody labeling profile, gene expression, X-inactivation profile, mitochondrial morphology, microRNA profile and development in the context of teratomas. hESCs can exist in a naïve state without the need for transgenes. Direct derivation is an elusive, but attainable, process, leading to cells at the earliest stage of in vitro pluripotency described for humans. Reverse toggling of primed cells to naïve is efficient and reproducible.

Naïve Induced Pluripotent Stem Cells Generated From β-Thalassemia Fibroblasts Allow Efficient Gene Correction With CRISPR/Cas9

“…Several studies comparing naïve and primed pluripotent stem cells have indicated that the two types of cells exhibit different molecular and functional properties, including colony morphologies, single-cell passage abilities [7,8], dependent signaling pathways [6,9,10], pluripotent gene expression profiles, and, most important, mouse embryo integration capacity to generate cross-species chimeras [7,11,12]. Previous studies have also suggested that human primed iPSCs and mouse EpiSCs can be converted to the naïve ground state via chemical manipulation or the overexpression of specific transcription factors [12][13][14][15][16][17]. Recently, several groups have further modified culture conditions to maintain human ESCs or iPSCs in the naïve ground state [12,13,15,18].…”

Cdx2Efficiently Induces Trophoblast Stem-Like Cells in Naïve, but Not Primed, Pluripotent Stem Cells

“…For example, since human ESCs bear numerous similarities to EpiSCs [5,6], our model predicts that ectopic Cdx2 would not induce expression of TSC-like cell genes in human ESCs in the conditions we tested here. Recently, totipotent or naïve pluripotent stem cell lines have been derived from both humans and mice [56][57][58][59][60]. In several cases, these cell lines have been shown to contribute to extraembryonic lineages in chimeras.…”

The molecular underpinnings of totipotency

“…These conditions also make ES cell derivation possible from normally recalcitrant mouse strains [33] and rat embryos [34], and most recently a variant of 2i has been used to derive naive human ES cells [15][16][17]. In 2i conditions, ES cells are also heterogeneous and include a significant fraction of cells that can be prospectively isolated based on the expression of the extraembryonic transcription factor Hhex [35] (figure 1b).…”