Capturing Human Naïve Pluripotency in the Embryo and in the Dish

Abstract

Although human embryonic stem cells (hESCs) were first derived almost 20 years ago, it was only recently acknowledged that they share closer molecular and functional identity to postimplantation lineage-primed murine epiblast stem cells than to naïve preimplantation inner cell mass-derived mouse ESCs (mESCs). A myriad of transcriptional, epigenetic, biochemical, and metabolic attributes have now been described that distinguish naïve and primed pluripotent states in both rodents and humans. Conventional hESCs and human induced pluripotent stem cells (hiPSCs) appear to lack many of the defining hallmarks of naïve mESCs. These include important features of the naïve ground state murine epiblast, such as an open epigenetic architecture, reduced lineage-primed gene expression, and chimera and germline competence following injection into a recipient blastocyst-stage embryo. Several transgenic and chemical methods were recently reported that appear to revert conventional human PSCs to mESC-like ground states. However, it remains unclear if subtle deviations in global transcription, cell signaling dependencies, and extent of epigenetic/metabolic shifts in these various human naïve-reverted pluripotent states represent true functional differences or alternatively the existence of distinct human pluripotent states along a spectrum. In this study, we review the current understanding and developmental features of various human pluripotency-associated phenotypes and discuss potential biological mechanisms that may support stable maintenance of an authentic epiblast-like ground state of human pluripotency.

Keywords: blastocyst; epiblast; hESC; human embryonic stem cell; inner cell mass; naive human pluripotency.

FIG. 1.

Embryonic pluripotency in early mouse and human embryonic development. Left: Pluripotent cells arise in the murine embryo during the cleavage stage, following loss of totipotency. Functional capacity to form all three germ layer lineages is retained up to the postimplantation egg cylinder epiblast. Two categories of PSCs have been isolated from murine embryos: mESCs and mEpiSCs. mESC lines can be isolated from postcleavage preimplantation embryos and model the ground state of pluripotency in the ICM of the blastocyst. In contrast, mEpiSC lines can be isolated from postimplantation epiblasts and mimic the continuum of lineage-primed developmental states that proceed to gastrulation. Right: Human embryonic pluripotency follows slower developmental kinetics than the mouse, but can be classified by analogous morphogenetic changes. Similar to mESCs, hESCs have been isolated from postcleavage preimplantation embryonic ICMs, but hESC lines share closer phenotypic and functional similarity with mEpiSCs than mESCs. hESCs may represent an equivalent of the developmentally more advanced human embryonic disc rather than the preimplantation epiblast cells they originate from. Red: pluripotent cells, green: trophectoderm, blue: primitive endoderm. hESCs, human embryonic stem cells; ICM, inner cell mass; mESCs, mouse embryonic stem cells; mEpiSCs, mouse epiblast-derived stem cells; PSCs, pluripotent stem cells.

FIG. 2.

Functional phenotypes of primed and naïve pluripotent states. (a) Functional shifts in the peri-implantation mouse embryo. The mouse pluripotent epiblast progresses from a naïve ground state (red) to a primed lineage-biased state (blue) following implantation. Naïve and primed states exploit distinct signaling pathways and their transition is accompanied by the sequential specification of trophectoderm (green) and primitive endoderm (violet) lineages. Known signaling pathways directing trophectoderm and primitive endoderm are indicated. In the mouse embryo, naïve and primed states can be distinguished by differing telomere lengthening and DNA repair strategies, levels of global repressive epigenetic marks (eg, DNA CpG methylation), and usage of metabolic pathways. Both states also display nonequivalent functional pluripotencies, with only the naïve state showing capacity for germline-competent chimera formation. In contrast, postimplantation epiblast cells have a partially committed lineage bias. In vitro expansion of mouse naïve epiblast cells generates mESC lines, while the postimplantation epiblast can generate lineage-primed mEpiSC lines. Functional capacities that have been demonstrated in vivo (embryo) or using in vitro surrogates (mESC, mEpiSC) are indicated. (b) Functional shifts in the human peri-implantation embryo. Similarly to the mouse, the human pluripotent epiblast is believed to recapitulate a steady progression from a naïve preimplantation state (red) to postimplantation primed lineage-biased states (blue). The signaling pathways that are essential for human naïve and primed states remain a subject of debate and have been extrapolated from hESC or single-cell RNA sequencing of preimplantation human embryos. The progression of human pluripotency is accompanied by the specification of trophectoderm (green) and primitive endoderm (violet) lineages, although the kinetics for emergence of extraembryonic lineages diverge between both species. The human naïve and primed states can also be distinguished by differing telomere lengthening and DNA repair mechanisms, global levels of repressive epigenetic marks, and metabolic pathway usage. The chimeric contribution of the postimplantation epiblast of nonhuman primates remains undetermined. However, nonhuman primate (NH-Primate) studies indicate that chimera formation may be restricted to early cleavage embryos, with possible low engraftment capacity for later preimplantation stages demonstrated by whole ICM transfer experiments. Functional capacities that have been demonstrated in vivo (embryo) or using in vitro surrogates (hESCs) are indicated.

FIG. 3.

Summary of epigenetic and functional phenotypes that are detected in distinct human and mouse pluripotent states. Select human and mouse PSC culture systems are presented with their downstream outcomes on WNT/β-catenin, FGF2/MEK/ERK, LIF/STAT3, and BMP/SMAD circuitries. + and − indicate signaling activities that have been verified to be, respectively, up- and downregulated using the aforementioned cocktails of small molecules, growth factors, and cytokines. The figure lists a series of epigenomic and functional hallmarks that have been associated with and distinguish between primed and naïve pluripotent cell populations. *Non-nuclear β-catenin only, **unpublished data (MEK/ERK) or subject to interline variability (BMP/SMAD), ***directly targeted by culture conditions, but nonverified, ****normal chromosome preparations were only verified between 5 and 17 passages. n/a, not applicable; N.D., not determined. ecto., meso., endo., PGC, and TE indicate reported detections of neuroectoderm, mesoderm (ie, cardiac, hemato-vascular), definitive endoderm, primordial germ cell, and trophectoderm lineages in directed differentiation assays. BMP, bone morphogenetic protein; ERK, extracellular signal-regulated kinase; LIF, leukemia inhibitory factor; MEK, mitogen-activated protein ERK.

FIG. 4.

Schematic summary of four main signaling pathways that regulate the naïve pluripotent state. (a) The BMP/SMAD, LIF/STAT3, FGF2/ERK, and WNT/β-catenin pathways are the four main molecular axes regulating naïve pluripotency. These circuits not only share a few common transcriptional effectors but also act separately to reinforce the core pluripotency network through mechanisms that can involve the KLF circuitry. Fluctuating subcellular distribution of the WNT pathway effector β-catenin may regulate accessibility of the core factors, OCT4 and Nanog, by facilitating their functions in either the nucleus or at the cell membrane (eg, to reinforce E-Cadherin strengthening of STAT3 signaling). (b) Downstream signaling of BMP/SMAD, WNT/β-catenin, LIF/STAT3, or MEK/ERK suppression results in marked reductions of genome-wide chromatin repressive marks (ie, reduced DNMT3a/DNMT3b levels and impaired DNMT1 recruitment following UHRF1 downregulation) as well as downregulation of lineage priming at developmental promoters by mechanisms that involve RNA-pol II pausing and accessibility to the PRC2. Green arrows: activation. Red blunt line: inhibition. Proteins known to reinforce (green) or destabilize (red) naïve pluripotency are shown. KLF, Krüppel-like factor; PRC2, polycomb repressor complex 2.