Unraveling the Spatiotemporal Human Pluripotency in Embryonic Development

Abstract

There have been significant advances in understanding human embryogenesis using human pluripotent stem cells (hPSCs) in conventional monolayer and 3D self-organized cultures. Thus, in vitro models have contributed to elucidate the molecular mechanisms for specification and differentiation during development. However, the molecular and functional spectrum of human pluripotency (i.e., intermediate states, pluripotency subtypes and regionalization) is still not fully understood. This review describes the mechanisms that establish and maintain pluripotency in human embryos and their differences with mouse embryos. Further, it describes a new pluripotent state representing a transition between naïve and primed pluripotency. This review also presents the data that divide pluripotency into substates expressing epiblast regionalization and amnion specification as well as primordial germ cells in primates. Finally, this work analyzes the amnion's relevance as an "signaling center" for regionalization before the onset of gastrulation.

Keywords: amnion; formative; naïve; primed; synthetic biology.

FIGURE 1

Signaling pathways related to the specification and segregation of pluripotent epiblast lineages in mouse and human. (A) In the mouse embryo at 3.5 days post-fertilization (E3.5), the ICM coexpresses NANOG and GATA6. By day 4.5, the ICM segregates into NANOG⁺ EPI and GATA6⁺ PE. EPI specification occurs in cells with low levels or suppression of the FGF-ERK pathway. (B) In human embryos, there is also colocalization of NANOG and GATA before lineage segregation, but the inhibition of FGF-ERK does not induce alterations in the specification of EPI (NANOG⁺, OCT4⁺) or PE (GATA4⁺). In contrast, blocking TGF-β signaling promotes the deficiency of both NANOG and SOX17, so this pathway is essential for the segregation of EPI and EP in the human embryo. ICM, inner cell mass; EPI, epiblast; PE, primitive endoderm; TE, trophoectoderm.

FIGURE 2

Spectrum of pluripotency in the mouse embryo. At 4.5 days post-fertilization (E4.5), the mouse epiblast has acquired a naïve state, the most sublime state of pluripotency. On E5.0 the blastocyst is implanted, where the epiblast forms a rosette structure. It has been proposed that this stage represents an intermediate pluripotency between the pre-implanted naïve and post-implanted primed states. In in vitro studies, this intermediate state has been described as formative pluripotency. On day 5.5 post-implantation, the formative-primed epiblast has a molecular and potential configuration different from that of the naïve state. On day 6.5, pluripotency can be divided into anterior and posterior regionalization of the epiblast. The anterior epiblast maintains a formative pluripotency, while the posterior epiblast acquires a primed pluripotency. The hypothesis proposes that on day 7.5, the posterior epiblast has a potential bias toward mesodermal precursors of the primitive streak, and the anterior epiblast acquires an ectoderm identity; its posteriorization is avoided by signals from the anterior visceral endoderm. Furthermore, mouse primordial germ cells (mPGCs) are specified in the posterior proximal epiblast from a pre-gastrulation subpopulation.

FIGURE 3

Spectrum of pluripotency in the human embryo. On embryonic day 6 (E6), the human blastocyst is pre-implanted, with probable naïve pluripotency. During implantation (E7-8), two lineages emerge in human embryos: the extra-embryonic mesoderm from the visceral endoderm (hypoblast) and the amniotic epithelium from the epiblast. On days 9–10, the amniotic cavity arises, while the post-implanted epiblast still expresses genes related to pluripotency but is no longer in the naïve state. It has been suggested that human primordial germ cell (hPGC) specification occurs at around E11-12, but also both lineages can arise from the same population that possesses germinal pluripotency. It is likely that the human epiblast, similar to the mouse, acquires an anterior-posterior regionalization with different subtypes of pluripotency biased toward specific lineages. On day 16, the onset of human gastrulation is preceded by the appearance of the primitive streak in the posterior region of the epiblast.

FIGURE 4

Hypothesis of the human epiblast regionalization. This hypothesis suggested that human pluripotency is regionalized into anterior and posterior. The posterior subpopulation has a differentiation bias toward mesoderm precursors to initiate gastrulation at E16, probably because of signals (WNT and BMP4) from the extraembryonic mesoderm and amniotic epithelium. On the other hand, the anterior region is still pluripotent and prevents its posteriorization by signals (negative regulators of BMP4 and WNT such as CER1 and DKK1, respectively) from the anterior visceral endoderm (AVE).