Accessing naïve human pluripotency

Abstract

Pluripotency manifests during mammalian development through formation of the epiblast, founder tissue of the embryo proper. Rodent pluripotent stem cells can be considered as two distinct states: naïve and primed. Naïve pluripotent stem cell lines are distinguished from primed cells by self-renewal in response to LIF signaling and MEK/GSK3 inhibition (LIF/2i conditions) and two active X chromosomes in female cells. In rodent cells, the naïve pluripotent state may be accessed through at least three routes: explantation of the inner cell mass, somatic cell reprogramming by ectopic Oct4, Sox2, Klf4, and C-myc, and direct reversion of primed post-implantation-associated epiblast stem cells (EpiSCs). In contrast to their rodent counterparts, human embryonic stem cells and induced pluripotent stem cells more closely resemble rodent primed EpiSCs. A critical question is whether naïve human pluripotent stem cells with bona fide features of both a pluripotent state and naïve-specific features can be obtained. In this review, we outline current understanding of the differences between these pluripotent states in mice, new perspectives on the origins of naïve pluripotency in rodents, and recent attempts to apply the rodent paradigm to capture naïve pluripotency in human cells. Unraveling how to stably induce naïve pluripotency in human cells will influence the full realization of human pluripotent stem cell biology and medicine.

Figure 1

Accessing rodent naïve pluripotency through three different routes. Pluripotent stem cells may be derived from in vivo sources such as the preimplantation blastocyst or the postimplantation epiblast, resulting in naïve mouse ESCs or primed EpiSCs respectively. Naïve iPSCs or primed iEpiSCs may be obtained by altering the cultural conditions during somatic cell reprogramming. Finally, naïve and primed pluripotent stem cells are directly interconvertible by differentiation of naïve pluripotent stem cells to primed pluripotent stem cells, or direct reversion of primed pluripotent stem cells to a naïve state. Therefore, naïve pluripotency may be captured in vitro in the form of (a) embryonic stem cells (ESCs) during ESC derivation, (b) induced pluripotent stem cells (iPSCs) during somatic cell reprogramming, or (c) Epi-iPSCs during reversion of EpiSCs to a naïve mESC-like state.

Figure 2

Attempts to generate naïve human pluripotent stem cells through direct reprogramming or reversion of hESCs/hiPSCs. Several groups have attempted to access the naïve human pluripotent state through (a) direct reprogramming of primary somatic cells or secondary fibroblasts, (b) reversion of conventional ‘primed’ human ESCs, iPSCs, or ‘secondary’ human iPSCs. (a) Direct reprogramming of human cells using constitutive lentiviruses, inducible lentiviruses, retroviruses, or Piggybac Transposon has given rise to transgene-dependent naïve human iPS cells, transgene-independent naïve human iPS cells and primitive neural stem cells. (b) Reversion of conventional human ESCs/iPSCs by stable or transfection of OCT4, KLF2, KLF4 and transfer into LIF/2i or LIF/2i/FK yields transgene-dependent or transgene-independent naïve human iPSCs; transfer and selection of integrationfree human iPS cells in LIF/2i yields primitive neural stem cells. (c) Can primed hESCs be converted directly into a stable naïve pluripotent state? Primed human ESC line H9 cultured in mTESR medium (top) and transgene-dependent naïve human iPS cell line cultured in LIF/2i/DOX conditions. The transgene-dependent nhiPSC line is dependent on continuous induction of a transgene-cassette encoding the four factors OCT4, SOX2, KLF4, and CMYC and was generated from the BJ fibroblast cell line. 2i = MEK inhibitor PD0325901 and GSK3-beta inhibitor CHIR99021. FK = Forskolin. rtTA = reverse tetracycline transactivator. Large star indicates self-renewal is sustained by high levels of transgenes. Small star indicates the presence of residual transgenes.