The sequential change from totipotency to multipotency occurs during early mammalian embryo development. However, due to the lack of cellular models to recapitulate the distinct potency of stem cells at each stage, their molecular and cellular characteristics remain ambiguous. The establishment of isogenic naïve and primed pluripotent stem cells to represent the pluripotency in the inner cell mass of the pre-implantation blastocyst and in the epiblast from the post-implantation embryo allows the understanding of the distinctive characteristics of two different states of pluripotent stem cells. This review discusses the prominent disparities between naïve and primed pluripotency, including signaling pathways, metabolism, and epigenetic status, ultimately facilitating a comprehensive understanding of their significance during early mammalian embryonic development.

Trophoblast stem cells (TSCs) have recently been derived from human embryos and early-first-trimester placenta; however, aside from ethical challenges, the unknown disease potential of these cells limits their scientific utility. We have previously established a bone morphogetic protein 4 (BMP4)-based two-step protocol for differentiation of primed human pluripotent stem cells (hPSCs) into functional trophoblasts; however, those trophoblasts could not be maintained in a self-renewing TSC-like state. Here, we use the first step from this protocol, followed by a switch to newly developed TSC medium, to derive bona fide TSCs. We show that these cells resemble placenta- and naive hPSC-derived TSCs, based on their transcriptome as well as their in vitro and in vivo differentiation potential. We conclude that primed hPSCs can be used to generate functional TSCs through a simple protocol, which can be applied to a widely available set of existing hPSCs, including induced pluripotent stem cells, derived from patients with known birth outcomes.

Generating patient-specific stem cells representing the onset of development has become possible since the discovery of somatic cell reprogramming into induced pluripotent stem cells. However, human pluripotent stem cells are generally cultured in a primed pluripotent state: they are poised for differentiation and represent a stage of development corresponding to post-implantation epiblast. Here, we describe a protocol to reprogram human fibroblasts into naive pluripotent stem cells by overexpressing the transcription factors OCT4, SOX2, KLF4, and c-MYC using Sendai viruses. The resulting cells represent an earlier stage of development that corresponds to pre-implantation epiblast. We also discuss validation methods for human naive pluripotent stem cells.

Trophoblasts are extraembryonic cells that are essential for maintaining pregnancy. Human trophoblasts arise from the morula as trophectoderm (TE), which, after implantation, differentiates into cytotrophoblasts (CTs), syncytiotrophoblasts (STs), and extravillous trophoblasts (EVTs), composing the placenta. Here we show that naïve, but not primed, human pluripotent stem cells (PSCs) recapitulate trophoblast development. Naive PSC-derived TE and CTs (nCTs) recreated human and monkey TE-to-CT transition. nCTs self-renewed as CT stem cells and had the characteristics of proliferating villous CTs and CTs in the cell column of the first trimester. Notably, although primed PSCs differentiated into trophoblast-like cells (BMP4, A83-01, and PD173074 [BAP]-treated primed PSCs [pBAPs]), pBAPs were distinct from nCTs and human placenta-derived CT stem cells, exhibiting properties consistent with the amnion. Our findings establish an authentic paradigm for human trophoblast development, demonstrating the invaluable properties of naive human PSCs. Our system provides a platform to study the molecular mechanisms underlying trophoblast development and related diseases.

Forty years have passed since the first pluripotent stem cells (PSCs), mouse embryonic stem cells (ESCs), were established. Since then, several PSCs have been reported, including human ESCs in 1998, mouse epiblast stem cells (EpiSCs) in 2007, induced PSCs (iPSCs) in 2006 and 2007, and naïve human PSCs in 2014. Naïve PSCs are thought to correspond to pre-implantation epiblast cells, whereas conventional (or primed) human PSCs correspond to post-implantation epiblast cells. Thus, naïve and primed PSCs are classified by their developmental stages and have stage-specific characteristics, despite sharing the common feature of pluripotency. In this review, we discuss the current status of PSCs and their use to model human peri-implantation development.

Pluripotency refers to the potential of single cells to form all cells and tissues of an organism. The observation that pluripotent stem cells can chimerize the embryos of evolutionarily distant species, albeit at very low efficiencies, could with further modifications, facilitate the production of human-animal interspecies chimeras. The generation of human-animal interspecies chimeras, if achieved, will enable practitioners to recapitulate pathologic human tissue formation in vivo and produce patient-specific organs inside livestock species. However, little is known about the nature of chimera-competent cellular states in primates. Here, I discuss recent advances in our understanding of the pluripotency continuum in humans and non-human primates (NHPs). Although undefined differences between humans and NHPs still justify the utility of studying human cells, the complementary use of NHP PS cells could also allow one to conduct pilot studies testing interspecies chimera generation strategies with reduced ethical concerns associated with human interspecies neurological chimerism. However, the availability of standardized, high-quality and validated NHP PS cell lines covering the spectrum of primate pluripotent states is lacking. Therefore, a clearer understanding of the primate pluripotency continuum will facilitate the complementary use of both human and NHP PS cells for testing interspecies organogenesis strategies, with the hope of one day enabling human organ generation inside livestock species.

Pluripotency refers to the capacity of single cells to form derivatives of the three germ layers-ectoderm, mesoderm, and endoderm. Pluripotency can be captured in vitro as a spectrum of pluripotent stem cell states stabilized in specialized laboratory conditions. The recent discovery that pluripotent stem cells can colonize the embryos of distantly related animal organisms could, with further refinement, enable the generation of chimeric embryos composed of cells of human and animal origin. If achievable, the production of human-animal chimeras will open up new opportunities for regenerative medicine, facilitating human disease modeling and human organ generation inside large animals. However, the generation of human-animal interspecies chimeras is anticipated to require human chimera-competent pluripotent stem cells. Thus, it remains imperative to examine the pluripotency continuum more closely in light of advances that will facilitate the production of human-animal chimeras. This piece will review the current understanding of the pluripotency continuum and interspecies chimeras. © 2019 by John Wiley & Sons, Inc.

The search for a better animal model to simulate human disease has been a \"holy grail\" of biomedical research for decades. Recent identification of different types of pluripotent stem cells (PS cells) and advances in chimera research might soon permit the generation of interspecies chimeras from closely related species, such as those between humans and other primates. Here, we suggest that the creation of human-primate chimeras-specifically, the transfer of human stem cells into (non-ape) primate hosts-could surpass the limitations of current monkey models of neurological and psychiatric disease, but would also raise important ethical considerations concerning the use of monkeys in invasive research. Questions regarding the scientific value and ethical concerns raised by the prospect of human-monkey chimeras are more urgent in light of recent advances in PS cell research and attempts to generate interspecies chimeras between humans and animals. While some jurisdictions prohibit the introduction of human PS cells into monkey preimplantation embryos, other jurisdictions may permit and even encourage such experiments. Therefore, it is useful to consider blastocyst complementation experiments more closely in light of advances that could make these chimeras possible and to consider the ethical and political issues that are raised.

Human pluripotent stem (PS) cells can be isolated from preimplantation embryos or by reprogramming of somatic cells or germline progenitors. Human PS cells are considered the \"holy grail\" of regenerative medicine because they have the potential to form all cell types of the adult body. Because of their similarity to humans, nonhuman primate (NHP) PS cells are also important models for studying human biology and disease, as well as for developing therapeutic strategies and test bed for cell replacement therapy. This chapter describes adjusted methods for cultivation of PS cells from different primate species, including African green monkey, rhesus monkey, chimpanzee, and human. Supplementation of E8 medium and inhibitors of the Tankyrase and GSK3 kinases to various primate PS cell media reduce line-dependent predisposition for spontaneous differentiation in conventional PS cell cultures. We provide methods for basic characterization of primate PS cell lines, which include immunostaining for pluripotency markers such as OCT4 and TRA-1-60, as well as in vivo teratoma formation assay. We provide methods for generating alternative PS cells including region-selective primed PS cells, two different versions of naïve-like cells, and recently reported extended pluripotent stem (EPS) cells. These derivations are achieved by acclimation of conventional PS cells to target media, episomal reprogramming of somatic cells, or resetting conventional PS cells to a naïve-like state by overexpression of KLF2 and NANOG. We also provide methods for isolation of PS cells from human blastocysts. We describe how to generate interspecies primate-mouse chimeras at the blastocyst and postimplantation embryo stages. Systematic evaluation of the chimeric competency of human and primate PS cells will aid in efforts to overcome species barriers and achieve higher grade chimerism in postimplantation conceptuses that could enable organ-specific enrichment of human xenogeneic PS cell derivatives in large animals such as pigs and sheep.

Humans develop from a unique group of pluripotent cells in early embryos that can produce all cells of the human body. While pluripotency is only transiently manifest in the embryo, scientists have identified conditions that sustain pluripotency indefinitely in the laboratory. Pluripotency is not a monolithic entity, however, but rather comprises a spectrum of different cellular states. Questions regarding the scientific value of examining the continuum of pluripotent stem (PS) cell states have gained increased significance in light of attempts to generate interspecies chimeras between humans and animals. In this chapter, I review our ever-evolving understanding of the continuum of pluripotency. Historically, the discovery of two different PS cell states in mice fostered a general conception of pluripotency comprised of two distinct attractor states: naïve and primed. Naïve pluripotency has been defined by competence to form germline chimeras and governance by unique KLF-based transcription factor (TF) circuitry, whereas primed state is distinguished by an inability to generate chimeras and alternative TF regulation. However, the discovery of many alternative PS cell states challenges the concept of pluripotency as a binary property. Moreover, it remains unclear whether the current molecular criteria used to classify human naïve-like pluripotency also identify human chimera-competent PS cells. Therefore, I examine the pluripotency continuum more closely in light of recent advances in PS cell research and human interspecies chimera research.