Derivation of novel human ground state naive pluripotent stem cells

Abstract

Mouse embryonic stem (ES) cells are isolated from the inner cell mass of blastocysts, and can be preserved in vitro in a naive inner-cell-mass-like configuration by providing exogenous stimulation with leukaemia inhibitory factor (LIF) and small molecule inhibition of ERK1/ERK2 and GSK3β signalling (termed 2i/LIF conditions). Hallmarks of naive pluripotency include driving Oct4 (also known as Pou5f1) transcription by its distal enhancer, retaining a pre-inactivation X chromosome state, and global reduction in DNA methylation and in H3K27me3 repressive chromatin mark deposition on developmental regulatory gene promoters. Upon withdrawal of 2i/LIF, naive mouse ES cells can drift towards a primed pluripotent state resembling that of the post-implantation epiblast. Although human ES cells share several molecular features with naive mouse ES cells, they also share a variety of epigenetic properties with primed murine epiblast stem cells (EpiSCs). These include predominant use of the proximal enhancer element to maintain OCT4 expression, pronounced tendency for X chromosome inactivation in most female human ES cells, increase in DNA methylation and prominent deposition of H3K27me3 and bivalent domain acquisition on lineage regulatory genes. The feasibility of establishing human ground state naive pluripotency in vitro with equivalent molecular and functional features to those characterized in mouse ES cells remains to be defined. Here we establish defined conditions that facilitate the derivation of genetically unmodified human naive pluripotent stem cells from already established primed human ES cells, from somatic cells through induced pluripotent stem (iPS) cell reprogramming or directly from blastocysts. The novel naive pluripotent cells validated herein retain molecular characteristics and functional properties that are highly similar to mouse naive ES cells, and distinct from conventional primed human pluripotent cells. This includes competence in the generation of cross-species chimaeric mouse embryos that underwent organogenesis following microinjection of human naive iPS cells into mouse morulas. Collectively, our findings establish new avenues for regenerative medicine, patient-specific iPS cell disease modelling and the study of early human development in vitro and in vivo.

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

“…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]. In particular, the 5i/L/FA system, developed by Jaenisch's group, contains a combination of inhibitors and growth factors sufficient for the induction and maintenance of the naïve ground state [15].…”

Chemically controlled aggregation of pluripotent stem cells

“…In addition to the HES2 line, DS is broadly applicable for aggregation control across different cell lines and culture conditions. We cultured the H9 PSC line in a medium containing DS in both conventional PSC culture conditions and conditions supporting a “naïve” pluripotent state (Gafni et al, 2013) and found that the DS is able to control H9 PSC aggregation in both pluripotent states (Supporting Information Figure S1h).…”

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.…”