Understanding stem cell states • Capturing naive human pluripotency • Defining naive human pluripotency • Trophoblast stem cells • Stem-cell-derived trophoblast organoids • Future directions
Understanding different states of pluripotency
Embryonic stem cells (ESCs) have the ability to self-renew indefinitely while maintaining the capacity to differentiate into all cell types found in the body. Due to these unique properties, ESCs have become a versatile tool in wide-ranging biomedical applications, from disease modeling to toxicology testing to clinical trials. In addition, the discovery of induced pluripotent stem cells (iPSCs) provides new possibilities to model complex genetic disorders and a source of autologous cells for transplantation. However, major challenges must be overcome before human ESCs and iPSCs can be used in a realistic way in regenerative medicine. The main challenge is that current human ESCs and iPSCs do not resemble the ground state “naive” pluripotent cells found in the blastocyst, but instead are more similar to “primed” precursors that arise after the embryo has implanted. The naive state is signified by an unrestricted developmental potential, whereas the primed state displays repressive chromatin features and lineage priming (Figure 1).
Figure 1. Overview of pluripotent stem cell states in mouse and human. Two distinct pluripotent stem cell states have been stably isolated from mouse embryos: embryonic stem cells (ESCs) derived from the pre-implantation blastocyst are considered to be in a “naive” pluripotent state, whereas epiblast stem cells (EpiSCs) derived from the post-implantation epiblast are in a “primed” pluripotent state. The naive state has an unbiased developmental potential, while the primed state displays lineage priming and repressive chromatin. Human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) most closely correspond to primate post-implantation embryos. There has been significant interest in isolating human ESCs and iPSCs in a naive state that faithfully resembles the human pre-implantation blastocyst. Figure adapted from Dong, Fischer and Theunissen, Exp. Cell Res., 2019.
While naive stem cells can be derived in rodents, their isolation has long remained elusive in the human system. The discovery of naive human pluripotent stem cells has broad implications for biomedical research. First, naive human cells may offer an enhanced starting point for differentiation into disease-relevant cell types, overcoming the heterogeneity frequently observed in current human ESCs and iPSCs. Second, the isolation of naive human cells may provide a cell culture system to study epigenetic mechanisms of human pre-implantation development that cannot be investigated in primed cells. Such studies are essential to help understand the high percentage of unexplained pregnancy loss. Third, naive induction may correct the erosion of dosage compensation prevalent in female human ESC and iPSC lines, enabling faithful in vitro modeling of X-linked diseases, such as mental retardation and autism spectrum disorders. Fourth, the injection of naive human cells into the blastocyst of an animal host may allow the generation of interspecies chimeras, providing a novel paradigm to study functional cells derived from patient iPSCs in vivo.
Capturing naive human pluripotency
In order to isolate candidate naive hESCs, we first established a naive-specific fluorescent reporter system. Experiments in mice have shown that the expression of OCT4, a master regulator of pluripotency, is controlled by distinct enhancer elements in the naive and primed pluripotent states. By generating hESCs carrying a deletion of the proximal enhancer in an OCT4-GFP reporter allele, activation of the naive-specific distal enhancer can be monitored in real time. Primed mouse EpiSCs can be reprogrammed to naive pluripotency by overexpression of defined transcription factors and switching to naive-specific culture conditions. Using a similar strategy, we found that inducible expression of KLF2 and NANOG could induce a putative naive state in hESCs. By screening a small molecule library, we identified a combination of five kinase inhibitors that could maintain naive reporter activity in the absence of transgene expression. These conditions also enabled the isolation of naive hESCs directly from primed cells, blastocysts or somatic cells. Naive hESCs are characterized by the upregulation of naive-specific transcription factors and display a reduction in repressive chromatin features (Figure 2).
Figure 2. Strategy for isolating naive human pluripotent stem cells. An OCT4-GFP reporter allele containing a deletion of the proximal enhancer was activated by Doxycycline (DOX)-inducible expression of KLF2 and NANOG and switching to 2i/LIF conditions. Screening a small molecule library identified a combination of five kinase inhibitors (5i) that could maintain OCT4-ΔPE-GFP reporter activity upon DOX withdrawal. When combined with recombinant FGF and Activin, these conditions enable the direct transgene-free derivation of naive hESCs and iPSCs from primed cells, explanted blastocysts or somatic cells. Figure adapted from Theunissen et al., Cell Stem Cell, 2014.
The correspondence between stem cells and early human embryos
Studies in recent years have revealed substantial differences in early embryogenesis between mouse and human. These differences include the lack of diapause in human, divergent responses of mouse and human embryos to FGF/Erk inhibition, and differences in the spatiotemporal expression of key developmental regulators and mechanisms of dosage compensation. Based on these observations, an accurate interpretation of human stem cell states must rely on direct comparison with human pluripotent cells in vivo, rather than extrapolation from rodent models. We therefore compared naive and primed hESCs to early human embryos using stringent molecular criteria (Figure 3).
Figure 3. Criteria for defining naive human pluripotency. We have compared candidate naive human PSCs to early embryos using four criteria: (i) The correspondence between transposon expression in naive human ESCs and early stages of human embryogenesis; (ii) The levels of global DNA methylation; (iii) The status of the X chromosome in female cells; (iv) The ability of naive human ESCs to incorporate into mouse morula or blastocyst-stage embryos and contribute to mid-gestation interspecies chimeras. Figure from Theunissen et al., Cell Stem Cell., 2016.
We examined the expression of transposable elements in human pluripotent stem cells and early embryonic stages. Because transposable elements are highly abundant (our genome contains approximately 4 million individual transposable integrants compared to 25,000 genes), the expression of transposons provides a highly sensitive measure of cellular identity. Naive cells in 5i/L/A acquire a transposon transcription signature that closely resembles human morula and blastocyst stage embryos. In addition, base-resolution bisulfite sequencing revealed that naive induction is accompanied by genome-wide depletion in DNA methylation, which is reversible upon differentiation except at imprinted regions. Finally, induction of naive human pluripotency in female cells involves the reactivation of an inactive X chromosome. These results demonstrate that naive human pluripotent stem cells share several defining features with pre-implantation embryos.
Derivation of human trophoblast stem cells (hTSCs) from naive human pluripotent stem cells (hPSCs)
The placenta is a critical organ system that mediates the exchange of nutrients, gases and waste products between the mother and the developing fetus. Placental abnormalities in the first trimester are associated with pregnancy complications such as preeclampsia, miscarriage and fetal growth restriction. However, the placenta is also considered the least understood human organ since access to first-trimester placental tissue is scarce and animal models inadequately recapitulate human placental development. Trophoblast constitutes the predominant epithelial cell type in the placenta and originates from the outer layer of cells in the blastocyst, called the trophectoderm. Given the ethical and legal constraints of studying placental development in vivo, there has been significant interest in establishing reliable in vitro model systems of human trophoblast specification and differentiation.
Figure 4. Derivation of human trophoblast stem cells (hTSCs) from naive human pluripotent stem cells (hPSCs). Isogenic naive and primed hPSCs were treated with hTSC media. While primed hPSCs acquired a neural morphology, naive hPSCs differentiated into cells that closely resembled primary hTSCs based on morphological, transcriptional and epigenetic criteria. Furthermore, naive hPSC-derived hTSCs were capable of undergoing cell-type-specific differentiation into specialized trophoblast cell types. Figure adapted from Dong et al., eLife, 2020.
Since naive human pluripotent stem cells (hPSCs) share gene expression and chromatin accessibility signatures with trophoblast cells, we examined their ability to differentiate into this extra-embryonic lineage. We seeded isogenic naive and primed hPSCs in a culture cocktail that promotes the self-renewal of human trophoblast stem cells (hTSCs) (Figure 4). Naive, but not primed, hPSCs acquired typical hTSC-like morphology within several passages. We proceeded to further characterize the naive hPSC-derived hTSCs (referred to as “naive hTSCs”), and found that they express key trophoblast markers at both the mRNA and protein level. We then subjected these naive hTSCs to conditions that promote terminal differentiation towards the extravillous trophoblast (EVT) and syncytiotrophoblast (STB) lineages. The resulting cells satisfy both morphological and molecular criteria of EVT and STB, respectively. Finally, we found that naive hTSCs closely resemble primary hTSCs based on their transcriptome and chromatin accessibility landscape and correspond to a post-implantation TE identity. We conclude that naive hPSCs harbor enhanced trophoblast potential compared to primed hPSCs. The ability to efficiently acquire trophoblast fate may be an inherent property of naive human pluripotency.
Stem-cell-derived trophoblast organoids (SC-TOs): an accessible 3D model system of human placental developmental and disease
We also explored whether naïve hPSCs may provide an accessible source of 3D trophoblast organoids. While such organoids were previously derived from first-trimester placental tissues, access to these tissues is limited due to ethical and regulatory constraints. Indeed, when transferred to Matrigel in appropriate media, naïve-hPSC-derived hTSCs readily self-organize into 3D organoids that exhibit an outer layer of progenitor cells and an inner syncytial compartment (Figure 5a). Single cell transcriptome profiling on these stem-cell-derived trophoblast organoids (SC-TOs) generated from naïve and primary hTSCs revealed a highly concordant cellular distribution of progenitor and specialized trophoblast states (Figure 5b). Transcriptome profiling also revealed a close alignment to trophoblast subtypes found in the early human post-implantation embryo. We further showed that SC-TOs generated from female naïve hPSCs undergo X chromosome inactivation (XCI) and recapitulate patchy XCI patterns seen in human placental tissues (Figure 5c).
Figure 5. Generation of stem-cell-derived trophoblast organoids (SC-TOs). (a) Human trophoblast stem cells (hTSCs) isolated from naïve hPSCs or primary tissues can self-organize into 3D trophoblast organoids with an inner syncytial compartment and outer shell of cytotrophoblast (CTB) progenitors. (b) Single cell transcriptome studies revealed close alignment between stem-cell-derived trophoblast organoids (SC-TOs) and trophoblast cell types in the early post-implantation embryo (embryonic day 7-14). (c) Female naïve hPSCs undergo X chromosome inactivation upon differentiation into hTSCs and organoid formation results in the clonal expansion of maternal or paternal XCI patterns, as seen in the human placenta. (d) SC-TOs display selective vulnerability to SARS-CoV-2 and Zika viruses, which correlates with relative expression of viral entry factors. This graphical abstract summarizes our recently published findings (Karvas et al., Cell Stem Cell, 2022).
Finally, we sought to investigate whether SC-TOs may be useful to model placental susceptibility to emerging viral infections. Whereas SC-TOs were readily infected by Zika virus, they demonstrated only limited susceptibility to SARS-CoV-2 (Figure 5d). This selective vulnerability correlates with differential expression levels of the entry factors for these two viruses. These studies demonstrate that naïve hPSCs provide an accessible source of 3D trophoblast organoids, which can be used to model the developing placenta and its vulnerability to emerging pathogens. Future efforts in our lab will exploit this 3D organoid model to investigate placental-endometrial crosstalk and the genetic basis of human placental development.
Future Directions
The above studies indicate that naive hPSCs offer a window into mechanisms of human pre-implantation development that are difficult to model in conventional hPSCs, such as the regulation of X chromosome reactivation and inactivation, the activity of early embryonic transposons, and trophoblast specification. However, current naive hPSCs do not perfectly resemble pluripotent cells in the human blastocyst, as exemplified by their global loss of imprinting and genetic instability during long-term culture. Thus, it remains to be determined whether strategies can be devised to capture naive hPSCs that are as robust as their mouse counterparts, which may facilitate wide-ranging applications in regenerative medicine, including more efficient contribution to interspecies chimeras.
Our current research is focused on three broad questions: (i) What are the signaling requirements for naive pluripotency, and how can we manipulate these pathways to create an optimal culture environment for deriving and maintaining human ESCs and iPSCs? (ii) What are the major transcriptional and epigenetic mechanisms underpinning the self-renewal and lineage commitment of pluripotent cells in the embryo and the culture dish? (iii) Can we leverage the unique properties of distinct pluripotent stem cell states to better model early human development and disease?
Theunissen Lab 