Tumor suppressor FRMD3 controls mammary epithelial cell fate determination via notch signaling pathway.

The luminal-to-basal transition in mammary epithelial cells (MECs) is accompanied by changes in epithelial cell lineage plasticity; however, the underlying mechanism remains elusive. Here, we report that deficiency of Frmd3 inhibits mammary gland lineage development and induces stemness of MECs, subsequently leading to the occurrence of triple-negative breast cancer. Loss of Frmd3 in PyMT mice results in a luminal-to-basal transition phenotype. Single-cell RNA sequencing of MECs indicated that knockout of Frmd3 inhibits the Notch signaling pathway. Mechanistically, FERM domain-containing protein 3 (FRMD3) promotes the degradation of Disheveled-2 by disrupting its interaction with deubiquitinase USP9x. FRMD3 also interrupts the interaction of Disheveled-2 with CK1, FOXK1/2, and NICD and decreases Disheveled-2 phosphorylation and nuclear localization, thereby impairing Notch-dependent luminal epithelial lineage plasticity in MECs. A low level of FRMD3 predicts poor outcomes for breast cancer patients. Together, we demonstrated that FRMD3 is a tumor suppressor that functions as an endogenous activator of the Notch signaling pathway, facilitating the basal-to-luminal transformation in MECs.

A single-cell chromatin accessibility dataset of human primed and naïve pluripotent stem cell-derived teratoma.

Teratoma, due to its remarkable ability to differentiate into multiple cell lineages, is a valuable model for studying human embryonic development. The similarity of the gene expression and chromatin accessibility patterns in these cells to those observed in vivo further underscores its potential as a research tool. Notably, teratomas derived from human naïve (pre-implantation epiblast-like) pluripotent stem cells (PSCs) have larger embryonic cell diversity and contain extraembryonic lineages, making them more suitable to study developmental processes. However, the cell type-specific epigenetic profiles of naïve PSC teratomas have not been yet characterized. Using single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq), we analyzed 66,384 cell profiles from five teratomas derived from human naïve PSCs and their post-implantation epiblast-like (primed) counterparts. We observed 17 distinct cell types from both embryonic and extraembryonic lineages, resembling the corresponding cell types in human fetal tissues. Additionally, we identified key transcription factors specific to different cell types. Our dataset provides a resource for investigating gene regulatory programs in a relevant model of human embryonic development.

Stage-specific GATA3 induction promotes ILC2 development after lineage commitment.

Group 2 innate lymphoid cells (ILC2s) are a subset of innate lymphocytes that produce type 2 cytokines, including IL-4, IL-5, and IL-13. GATA3 is a critical transcription factor for ILC2 development at multiple stages. However, when and how GATA3 is induced to the levels required for ILC2 development remains unclear. Herein, we identify ILC2-specific GATA3-related tandem super-enhancers (G3SE) that induce high GATA3 in ILC2-committed precursors. G3SE-deficient mice exhibit ILC2 deficiency in the bone marrow, lung, liver, and small intestine with minimal impact on other ILC lineages or Th2 cells. Single-cell RNA-sequencing and subsequent flow cytometry analysis show that GATA3 induction mechanism, which is required for entering the ILC2 stage, is lost in IL-17RB+PD-1- late ILC2-committed precursor stage in G3SE-deficient mice. Cnot6l, part of the CCR4-NOT deadenylase complex, is a possible GATA3 target during ILC2 development. Our findings implicate a stage-specific regulatory mechanism for GATA3 expression during ILC2 development.

Generation of functional neurons from adult human mucosal olfactory ensheathing glia by direct lineage conversion.

A recent approach to promote central nervous system (CNS) regeneration after injury or disease is direct conversion of somatic cells to neurons. This is achieved by transduction of viral vectors that express neurogenic transcription factors. In this work we propose adult human mucosal olfactory ensheathing glia (hmOEG) as a candidate for direct reprogramming to neurons due to its accessibility and to its well-characterized neuroregenerative capacity. After induction of hmOEG with the single neurogenic transcription factor NEUROD1, the cells under study exhibited morphological and immunolabeling neuronal features, fired action potentials and expressed glutamatergic and GABAergic markers. In addition, after engraftment of transduced hmOEG cells in the mouse hippocampus, these cells showed specific neuronal labeling. Thereby, if we add to the neuroregenerative capacity of hmOEG cultures the conversion to neurons of a fraction of their population through reprogramming techniques, the engraftment of hmOEG and hmOEG-induced neurons could be a procedure to enhance neural repair after central nervous system injury.

Interconnected lineage trajectories link conventional and natural killer (NK)-like exhausted CD8+ T cells beneficial in type 1 diabetes.

Distinct Natural Killer (NK)-like CD57+ and PD-1+ CD8+ exhausted-like T cell populations (Tex) have both been linked to beneficial immunotherapy response in autoimmune type 1 diabetes (T1D) patients. The origins and relationships between these cell types are poorly understood. Here we show that while PD-1+ and CD57+ Tex populations are epigenetically similar, CD57+ Tex cells display unique increased chromatin accessibility of inhibitory Killer Cell Immunoglobulin-like Receptor (iKIR) and other NK cell genes. PD-1+ and CD57+ Tex also show reciprocal expression of Inhibitory Receptors (IRs) and iKIRs accompanied by chromatin accessibility of Tcf1 and Tbet transcription factor target sites, respectively. CD57+ Tex show unappreciated gene expression heterogeneity and share clonal relationships with PD-1+ Tex, with these cells differentiating along four interconnected lineage trajectories: Tex-PD-1+, Tex-CD57+, Tex-Branching, and Tex-Fluid. Our findings demonstrate new relationships between Tex-like populations in human autoimmune disease and suggest that modulating common precursor populations may enhance response to autoimmune disease treatment.

Joint inference of cell lineage and mitochondrial evolution from single-cell sequencing data.

MOTIVATION: Eukaryotic cells contain organelles called mitochondria that have their own genome. Most cells contain thousands of mitochondria which replicate, even in nondividing cells, by means of a relatively error-prone process resulting in somatic mutations in their genome. Because of the higher mutation rate compared to the nuclear genome, mitochondrial mutations have been used to track cellular lineage, particularly using single-cell sequencing that measures mitochondrial mutations in individual cells. However, existing methods to infer the cell lineage tree from mitochondrial mutations do not model "heteroplasmy," which is the presence of multiple mitochondrial clones with distinct sets of mutations in an individual cell. Single-cell sequencing data thus provide a mixture of the mitochondrial clones in individual cells, with the ancestral relationships between these clones described by a mitochondrial clone tree. While deconvolution of somatic mutations from a mixture of evolutionarily related genomes has been extensively studied in the context of bulk sequencing of cancer tumor samples, the problem of mitochondrial deconvolution has the additional constraint that the mitochondrial clone tree must be concordant with the cell lineage tree. RESULTS: We formalize the problem of inferring a concordant pair of a mitochondrial clone tree and a cell lineage tree from single-cell sequencing data as the Nested Perfect Phylogeny Mixture (NPPM) problem. We derive a combinatorial characterization of the solutions to the NPPM problem, and formulate an algorithm, MERLIN, to solve this problem exactly using a mixed integer linear program. We show on simulated data that MERLIN outperforms existing methods that do not model mitochondrial heteroplasmy nor the concordance between the mitochondrial clone tree and the cell lineage tree. We use MERLIN to analyze single-cell whole-genome sequencing data of 5220 cells of a gastric cancer cell line and show that MERLIN infers a more biologically plausible cell lineage tree and mitochondrial clone tree compared to existing methods. AVAILABILITY AND IMPLEMENTATION: https://github.com/raphael-group/MERLIN.

Maximum likelihood phylogeographic inference of cell motility and cell division from spatial lineage tracing data.

MOTIVATION: Recently developed spatial lineage tracing technologies induce somatic mutations at specific genomic loci in a population of growing cells and then measure these mutations in the sampled cells along with the physical locations of the cells. These technologies enable high-throughput studies of developmental processes over space and time. However, these applications rely on accurate reconstruction of a spatial cell lineage tree describing both past cell divisions and cell locations. Spatial lineage trees are related to phylogeographic models that have been well-studied in the phylogenetics literature. We demonstrate that standard phylogeographic models based on Brownian motion are inadequate to describe the spatial symmetric displacement (SD) of cells during cell division. RESULTS: We introduce a new model-the SD model for cell motility that includes symmetric displacements of daughter cells from the parental cell followed by independent diffusion of daughter cells. We show that this model more accurately describes the locations of cells in a real spatial lineage tracing of mouse embryonic stem cells. Combining the spatial SD model with an evolutionary model of DNA mutations, we obtain a phylogeographic model for spatial lineage tracing. Using this model, we devise a maximum likelihood framework-MOLLUSC (Maximum Likelihood Estimation Of Lineage and Location Using Single-Cell Spatial Lineage tracing Data)-to co-estimate time-resolved branch lengths, spatial diffusion rate, and mutation rate. On both simulated and real data, we show that MOLLUSC accurately estimates all parameters. In contrast, the Brownian motion model overestimates spatial diffusion rate in all test cases. In addition, the inclusion of spatial information improves accuracy of branch length estimation compared to sequence data alone. On real data, we show that spatial information has more signal than sequence data for branch length estimation, suggesting augmenting lineage tracing technologies with spatial information is useful to overcome the limitations of genome-editing in developmental systems. AVAILABILITY AND IMPLEMENTATION: The python implementation of MOLLUSC is available at https://github.com/raphael-group/MOLLUSC.

Extraembryonic gut endoderm cells undergo programmed cell death during development.

Despite a distinct developmental origin, extraembryonic cells in mice contribute to gut endoderm and converge to transcriptionally resemble their embryonic counterparts. Notably, all extraembryonic progenitors share a non-canonical epigenome, raising several pertinent questions, including whether this landscape is reset to match the embryonic regulation and if extraembryonic cells persist into later development. Here we developed a two-colour lineage-tracing strategy to track and isolate extraembryonic cells over time. We find that extraembryonic gut cells display substantial memory of their developmental origin including retention of the original DNA methylation landscape and resulting transcriptional signatures. Furthermore, we show that extraembryonic gut cells undergo programmed cell death and neighbouring embryonic cells clear their remnants via non-professional phagocytosis. By midgestation, we no longer detect extraembryonic cells in the wild-type gut, whereas they persist and differentiate further in p53-mutant embryos. Our study provides key insights into the molecular and developmental fate of extraembryonic cells inside the embryo.

Whole-genome landscape of histone H3K4me3 modification during sperm cell lineage development in tomato.

BACKGROUND: During male gametogenesis of flowering plants, sperm cell lineage (microspores, generative cells, and sperm cells) differentiated from somatic cells and acquired different cell fates. Trimethylation of histone H3 on lysine 4 (H3K4me3) epigenetically contributes to this process, however, it remained unclear how H3K4me3 influences the gene expression in each cell type. Here, we conducted chromatin immunoprecipitation sequencing (ChIP-seq) to obtain a genome-wide landscape of H3K4me3 during sperm cell lineage development in tomato (Solanum lycopersicum). RESULTS: We show that H3K4me3 peaks were mainly enriched in the promoter regions, and intergenic H3K4me3 peaks expanded as sperm cell lineage differentiated from somatic cells. H3K4me3 was generally positively associated with transcript abundance and served as a better indicator of gene expression in somatic and vegetative cells, compared to sperm cell lineage. H3K4me3 was mutually exclusive with DNA methylation at 3' proximal of the transcription start sites. The microspore maintained the H3K4me3 features of somatic cells, while generative cells and sperm cells shared an almost identical H3K4me3 pattern which differed from that of the vegetative cell. After microspore division, significant loss of H3K4me3 in genes related to brassinosteroid and cytokinin signaling was observed in generative cells and vegetative cells, respectively. CONCLUSIONS: Our results suggest the asymmetric division of the microspore significantly reshapes the genome-wide distribution of H3K4me3. Selective loss of H3K4me3 in genes related to hormone signaling may contribute to functional differentiation of sperm cell lineage. This work provides new resource data for the epigenetic studies of gametogenesis in plants.

The Endosomal Sorting Complex, ESCRT, has diverse roles in blood progenitor maintenance, lineage choice and immune response.

Most hematological malignancies are associated with reduced expression of one or more components of the Endosomal Sorting Complex Required for Transport (ESCRT). However, the roles of ESCRT in stem cell and progenitor maintenance are not resolved. Parsing signaling pathways in relation to the canonical role of ESCRT poses a challenge. The Drosophila hematopoietic organ, the larval lymph gland, provides a path to dissect the roles of cellular trafficking pathways such as ESCRT in blood development and maintenance. Drosophila has 13 core ESCRT components. Knockdown of individual ESCRTs showed that only Vps28 and Vp36 were required in all lymph gland progenitors. Using the well-conserved ESCRT-II complex as an example of the range of phenotypes seen upon ESCRT depletion, we show that ESCRTs have cell-autonomous as well as non-autonomous roles in progenitor maintenance and differentiation. ESCRT depletion also sensitized posterior lobe progenitors to respond to immunogenic wasp infestation. We also identify key heterotypic roles for ESCRT in position-dependent control of Notch activation to suppress crystal cell differentiation. Our study shows that the cargo sorting machinery determines the identity of progenitors and their adaptability to the dynamic microenvironment. These mechanisms for control of cell fate may tailor developmental diversity in multiple contexts.

Cardiac Development Long Non-Coding RNA (CARDEL) Is Activated during Human Heart Development and Contributes to Cardiac Specification and Homeostasis.

Successful heart development depends on the careful orchestration of a network of transcription factors and signaling pathways. In recent years, in vitro cardiac differentiation using human pluripotent stem cells (hPSCs) has been used to uncover the intricate gene-network regulation involved in the proper formation and function of the human heart. Here, we searched for uncharacterized cardiac-development genes by combining a temporal evaluation of human cardiac specification in vitro with an analysis of gene expression in fetal and adult heart tissue. We discovered that CARDEL (CARdiac DEvelopment Long non-coding RNA; LINC00890; SERTM2) expression coincides with the commitment to the cardiac lineage. CARDEL knockout hPSCs differentiated poorly into cardiac cells, and hPSC-derived cardiomyocytes showed faster beating rates after controlled overexpression of CARDEL during differentiation. Altogether, we provide physiological and molecular evidence that CARDEL expression contributes to sculpting the cardiac program during cell-fate commitment.

ScRNA-Seq Analyses Define the Role of GATA3 in iNKT Cell Effector Lineage Differentiation.

While the transcription factor GATA-3 is well-established for its crucial role in T cell development, its specific influence on invariant natural killer T (iNKT) cells remains relatively unexplored. Using flow cytometry and single-cell transcriptomic analysis, we demonstrated that GATA-3 deficiency in mice leads to the absence of iNKT2 and iNKT17 cell subsets, as well as an altered distribution of iNKT1 cells. Thymic iNKT cells lacking GATA-3 exhibited diminished expression of PLZF and T-bet, key transcription factors involved in iNKT cell differentiation, and reduced production of Th2, Th17, and cytotoxic effector molecules. Single-cell transcriptomics revealed a comprehensive absence of iNKT17 cells, a substantial reduction in iNKT2 cells, and an increase in iNKT1 cells in GATA-3-deficient thymi. Differential expression analysis highlighted the regulatory role of GATA-3 in T cell activation signaling and altered expression of genes critical for iNKT cell differentiation, such as Icos, Cd127, Eomes, and Zbtb16. Notably, restoration of Icos, but not Cd127, expression could rescue iNKT cell development in GATA-3-deficient mice. In conclusion, our study demonstrates the pivotal role of GATA-3 in orchestrating iNKT cell effector lineage differentiation through the regulation of T cell activation pathways and Icos expression, providing insights into the molecular mechanisms governing iNKT cell development and function.

Interplay between Pitx2 and Pax7 temporally governs specification of extraocular muscle stem cells.

Gene regulatory networks that act upstream of skeletal muscle fate determinants are distinct in different anatomical locations. Despite recent efforts, a clear understanding of the cascade of events underlying the emergence and maintenance of the stem cell pool in specific muscle groups remains unresolved and debated. Here, we invalidated Pitx2 with multiple Cre-driver mice prenatally, postnatally, and during lineage progression. We showed that this gene becomes progressively dispensable for specification and maintenance of the muscle stem (MuSC) cell pool in extraocular muscles (EOMs) despite being, together with Myf5, a major upstream regulator during early development. Moreover, constitutive inactivation of Pax7 postnatally led to a greater loss of MuSCs in the EOMs compared to the limb. Thus, we propose a relay between Pitx2, Myf5 and Pax7 for EOM stem cell maintenance. We demonstrate also that MuSCs in the EOMs adopt a quiescent state earlier that those in limb muscles and do not spontaneously proliferate in the adult, yet EOMs have a significantly higher content of Pax7+ MuSCs per area pre- and post-natally. Finally, while limb MuSCs proliferate in the mdx mouse model for Duchenne muscular dystrophy, significantly less MuSCs were present in the EOMs of the mdx mouse model compared to controls, and they were not proliferative. Overall, our study provides a comprehensive in vivo characterisation of MuSC heterogeneity along the body axis and brings further insights into the unusual sparing of EOMs during muscular dystrophy.

Uncovering the role of TET2-mediated ENPEP activation in trophoblast cell fate determination.

Early trophoblast differentiation is crucial for embryo implantation, placentation and fetal development. Dynamic changes in DNA methylation occur during preimplantation development and are critical for cell fate determination. However, the underlying regulatory mechanism remains unclear. Recently, we derived morula-like expanded potential stem cells from human preimplantation embryos (hEPSC-em), providing a valuable tool for studying early trophoblast differentiation. Data analysis on published datasets showed differential expressions of DNA methylation enzymes during early trophoblast differentiation in human embryos and hEPSC-em derived trophoblastic spheroids. We demonstrated downregulation of DNA methyltransferase 3 members (DNMT3s) and upregulation of ten-eleven translocation methylcytosine dioxygenases (TETs) during trophoblast differentiation. While DNMT inhibitor promoted trophoblast differentiation, TET inhibitor hindered the process and reduced implantation potential of trophoblastic spheroids. Further integrative analysis identified that glutamyl aminopeptidase (ENPEP), a trophectoderm progenitor marker, was hypomethylated and highly expressed in trophoblast lineages. Concordantly, progressive loss of DNA methylation in ENPEP promoter and increased ENPEP expression were detected in trophoblast differentiation. Knockout of ENPEP in hEPSC-em compromised trophoblast differentiation potency, reduced adhesion and invasion of trophoblastic spheroids, and impeded trophoblastic stem cell (TSC) derivation. Importantly, TET2 was involved in the loss of DNA methylation and activation of ENPEP expression during trophoblast differentiation. TET2-null hEPSC-em failed to produce TSC properly. Collectively, our results illustrated the crucial roles of ENPEP and TET2 in trophoblast fate commitments and the unprecedented TET2-mediated loss of DNA methylation in ENPEP promoter.

Eomes expression identifies the early bone marrow precursor to classical NK cells.

Natural killer (NK) cells traffic through the blood and mount cytolytic and interferon-γ (IFNγ)-focused responses to intracellular pathogens and tumors. Type 1 innate lymphoid cells (ILC1s) also produce type 1 cytokines but reside in tissues and are not cytotoxic. Whether these differences reflect discrete lineages or distinct states of a common cell type is not understood. Using single-cell RNA sequencing and flow cytometry, we focused on populations of TCF7+ cells that contained precursors for NK cells and ILC1s and identified a subset of bone marrow lineage-negative NK receptor-negative cells that expressed the transcription factor Eomes, termed EomeshiNKneg cells. Transfer of EomeshiNKneg cells into Rag2-/-Il2rg-/- recipients generated functional NK cells capable of preventing metastatic disease. By contrast, transfer of PLZF+ ILC precursors generated a mixture of ILC1s, ILC2s and ILC3s that lacked cytotoxic potential. These findings identified EomeshiNKneg cells as the bone marrow precursor to classical NK cells and demonstrated that the NK and ILC1 lineages diverged early during development.

Distinct developmental pathways generate functionally distinct populations of natural killer cells.

Natural killer (NK) cells function by eliminating virus-infected or tumor cells. Here we identified an NK-lineage-biased progenitor population, referred to as early NK progenitors (ENKPs), which developed into NK cells independently of common precursors for innate lymphoid cells (ILCPs). ENKP-derived NK cells (ENKP_NK cells) and ILCP-derived NK cells (ILCP_NK cells) were transcriptionally different. We devised combinations of surface markers that identified highly enriched ENKP_NK and ILCP_NK cell populations in wild-type mice. Furthermore, Ly49H+ NK cells that responded to mouse cytomegalovirus infection primarily developed from ENKPs, whereas ILCP_NK cells were better IFNγ producers after infection with Salmonella and herpes simplex virus. Human CD56dim and CD56bright NK cells were transcriptionally similar to ENKP_NK cells and ILCP_NK cells, respectively. Our findings establish the existence of two pathways of NK cell development that generate functionally distinct NK cell subsets in mice and further suggest these pathways may be conserved in humans.

Brain Chimeroids reveal individual susceptibility to neurotoxic triggers.

Interindividual genetic variation affects the susceptibility to and progression of many diseases1,2. However, efforts to study how individual human brains differ in normal development and disease phenotypes are limited by the paucity of faithful cellular human models, and the difficulty of scaling current systems to represent multiple people. Here we present human brain Chimeroids, a highly reproducible, multidonor human brain cortical organoid model generated by the co-development of cells from a panel of individual donors in a single organoid. By reaggregating cells from multiple single-donor organoids at the neural stem cell or neural progenitor cell stage, we generate Chimeroids in which each donor produces all cell lineages of the cerebral cortex, even when using pluripotent stem cell lines with notable growth biases. We used Chimeroids to investigate interindividual variation in the susceptibility to neurotoxic triggers that exhibit high clinical phenotypic variability: ethanol and the antiepileptic drug valproic acid. Individual donors varied in both the penetrance of the effect on target cell types, and the molecular phenotype within each affected cell type. Our results suggest that human genetic background may be an important mediator of neurotoxin susceptibility and introduce Chimeroids as a scalable system for high-throughput investigation of interindividual variation in processes of brain development and disease.

Capturing totipotency in human cells through spliceosomal repression.

The cleavage of zygotes generates totipotent blastomeres. In human 8-cell blastomeres, zygotic genome activation (ZGA) occurs to initiate the ontogenesis program. However, capturing and maintaining totipotency in human cells pose significant challenges. Here, we realize culturing human totipotent blastomere-like cells (hTBLCs). We find that splicing inhibition can transiently reprogram human pluripotent stem cells into ZGA-like cells (ZLCs), which subsequently transition into stable hTBLCs after long-term passaging. Distinct from reported 8-cell-like cells (8CLCs), both ZLCs and hTBLCs widely silence pluripotent genes. Interestingly, ZLCs activate a particular group of ZGA-specific genes, and hTBLCs are enriched with pre-ZGA-specific genes. During spontaneous differentiation, hTBLCs re-enter the intermediate ZLC stage and further generate epiblast (EPI)-, primitive endoderm (PrE)-, and trophectoderm (TE)-like lineages, effectively recapitulating human pre-implantation development. Possessing both embryonic and extraembryonic developmental potency, hTBLCs can autonomously generate blastocyst-like structures in vitro without external cell signaling. In summary, our study provides key criteria and insights into human cell totipotency.

Lineage specification in glioblastoma is regulated by METTL7B.

Glioblastomas are the most common malignant brain tumors in adults; they are highly aggressive and heterogeneous and show a high degree of plasticity. Here, we show that methyltransferase-like 7B (METTL7B) is an essential regulator of lineage specification in glioblastoma, with an impact on both tumor size and invasiveness. Single-cell transcriptomic analysis of these tumors and of cerebral organoids derived from expanded potential stem cells overexpressing METTL7B reveal a regulatory role for the gene in the neural stem cell-to-astrocyte differentiation trajectory. Mechanistically, METTL7B downregulates the expression of key neuronal differentiation players, including SALL2, via post-translational modifications of histone marks.

Naive pluripotent stem cell-based models capture FGF-dependent human hypoblast lineage specification.

The hypoblast is an essential extraembryonic tissue set aside within the inner cell mass in the blastocyst. Research with human embryos is challenging. Thus, stem cell models that reproduce hypoblast differentiation provide valuable alternatives. We show here that human naive pluripotent stem cell (PSC) to hypoblast differentiation proceeds via reversion to a transitional ICM-like state from which the hypoblast emerges in concordance with the trajectory in human blastocysts. We identified a window when fibroblast growth factor (FGF) signaling is critical for hypoblast specification. Revisiting FGF signaling in human embryos revealed that inhibition in the early blastocyst suppresses hypoblast formation. In vitro, the induction of hypoblast is synergistically enhanced by limiting trophectoderm and epiblast fates. This finding revises previous reports and establishes a conservation in lineage specification between mice and humans. Overall, this study demonstrates the utility of human naive PSC-based models in elucidating the mechanistic features of early human embryogenesis.