A robust model for cell type-specific interindividual variation in single-cell RNA sequencing data.

Single-cell RNA sequencing (scRNA-seq) has been widely used to characterize cell types based on their average gene expression profiles. However, most studies do not consider cell type-specific variation across donors. Modelling this cell type-specific inter-individual variation could help elucidate cell type-specific biology and inform genes and cell types underlying complex traits. We therefore develop a new model to detect and quantify cell type-specific variation across individuals called CTMM (Cell Type-specific linear Mixed Model). We use extensive simulations to show that CTMM is powerful and unbiased in realistic settings. We also derive calibrated tests for cell type-specific interindividual variation, which is challenging given the modest sample sizes in scRNA-seq. We apply CTMM to scRNA-seq data from human induced pluripotent stem cells to characterize the transcriptomic variation across donors as cells differentiate into endoderm. We find that almost 100% of transcriptome-wide variability between donors is differentiation stage-specific. CTMM also identifies individual genes with statistically significant stage-specific variability across samples, including 85 genes that do not have significant stage-specific mean expression. Finally, we extend CTMM to partition interindividual covariance between stages, which recapitulates the overall differentiation trajectory. Overall, CTMM is a powerful tool to illuminate cell type-specific biology in scRNA-seq.

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.

CellRank 2: unified fate mapping in multiview single-cell data.

Single-cell RNA sequencing allows us to model cellular state dynamics and fate decisions using expression similarity or RNA velocity to reconstruct state-change trajectories; however, trajectory inference does not incorporate valuable time point information or utilize additional modalities, whereas methods that address these different data views cannot be combined or do not scale. Here we present CellRank 2, a versatile and scalable framework to study cellular fate using multiview single-cell data of up to millions of cells in a unified fashion. CellRank 2 consistently recovers terminal states and fate probabilities across data modalities in human hematopoiesis and endodermal development. Our framework also allows combining transitions within and across experimental time points, a feature we use to recover genes promoting medullary thymic epithelial cell formation during pharyngeal endoderm development. Moreover, we enable estimating cell-specific transcription and degradation rates from metabolic-labeling data, which we apply to an intestinal organoid system to delineate differentiation trajectories and pinpoint regulatory strategies.

A single-cell atlas of pig gastrulation as a resource for comparative embryology.

Cell-fate decisions during mammalian gastrulation are poorly understood outside of rodent embryos. The embryonic disc of pig embryos mirrors humans, making them a useful proxy for studying gastrulation. Here we present a single-cell transcriptomic atlas of pig gastrulation, revealing cell-fate emergence dynamics, as well as conserved and divergent gene programs governing early porcine, primate, and murine development. We highlight heterochronicity in extraembryonic cell-types, despite the broad conservation of cell-type-specific transcriptional programs. We apply these findings in combination with functional investigations, to outline conserved spatial, molecular, and temporal events during definitive endoderm specification. We find early FOXA2 + /TBXT- embryonic disc cells directly form definitive endoderm, contrasting later-emerging FOXA2/TBXT+ node/notochord progenitors. Unlike mesoderm, none of these progenitors undergo epithelial-to-mesenchymal transition. Endoderm/Node fate hinges on balanced WNT and hypoblast-derived NODAL, which is extinguished upon endodermal differentiation. These findings emphasise the interplay between temporal and topological signalling in fate determination during gastrulation.

Tissue-intrinsic beta-catenin signals antagonize Nodal-driven anterior visceral endoderm differentiation.

The anterior-posterior axis of the mammalian embryo is laid down by the anterior visceral endoderm (AVE), an extraembryonic signaling center that is specified within the visceral endoderm. Current models posit that AVE differentiation is promoted globally by epiblast-derived Nodal signals, and spatially restricted by a BMP gradient established by the extraembryonic ectoderm. Here, we report spatially restricted AVE differentiation in bilayered embryo-like aggregates made from mouse embryonic stem cells that lack an extraembryonic ectoderm. Notably, clusters of AVE cells also form in pure visceral endoderm cultures upon activation of Nodal signaling, indicating that tissue-intrinsic factors can restrict AVE differentiation. We identify ß-catenin activity as a tissue-intrinsic factor that antagonizes AVE-inducing Nodal signals. Together, our results show how an AVE-like population can arise through interactions between epiblast and visceral endoderm alone. This mechanism may be a flexible solution for axis patterning in a wide range of embryo geometries, and provide robustness to axis patterning when coupled with signal gradients.

TGF-ß modulates cell fate in human ES cell-derived foregut endoderm by inhibiting Wnt and BMP signaling.

Genetic differences between pluripotent stem cell lines cause variable activity of extracellular signaling pathways, limiting reproducibility of directed differentiation protocols. Here we used human embryonic stem cells (hESCs) to interrogate how exogenous factors modulate endogenous signaling events during specification of foregut endoderm lineages. We find that transforming growth factor ß1 (TGF-ß1) activates a putative human OTX2/LHX1 gene regulatory network which promotes anterior fate by antagonizing endogenous Wnt signaling. In contrast to Porcupine inhibition, TGF-ß1 effects cannot be reversed by exogenous Wnt ligands, suggesting that induction of SHISA proteins and intracellular accumulation of Fzd receptors render TGF-ß1-treated cells refractory to Wnt signaling. Subsequently, TGF-ß1-mediated inhibition of BMP and Wnt signaling suppresses liver fate and promotes pancreas fate. Furthermore, combined TGF-ß1 treatment and Wnt inhibition during pancreatic specification reproducibly and robustly enhance INSULIN+ cell yield across hESC lines. This modification of widely used differentiation protocols will enhance pancreatic ß cell yield for cell-based therapeutic applications.

Application of stem cells in the study of developmental and functional toxicity of endodermal-derived organs caused by nanoparticles.

Nanoparticles have unique properties that make them useful in biomedicine. However, their extensive use raises concerns about potential hazards to the body. Therefore, it is crucial to establish effective and robust toxicology models to evaluate the developmental and functional toxicity of nanoparticles on the body. This article discusses the use of stem cells to study the developmental and functional toxicity of organs of endodermal origin due to nanoparticles. The study discovered that various types of nanoparticles have varying effects on stem cells. The application of stem cell models can provide a possibility for studying the effects of nanoparticles on organ development and function, as they can more accurately reflect the toxic mechanisms of different types of nanoparticles. However, stem cell toxicology systems currently cannot fully reflect the effects of nanoparticles on entire organs. Therefore, the establishment of organoid models and other advanced assessment models is expected to address this issue.

Smad4 is essential for epiblast scaling and morphogenesis after implantation, but nonessential before implantation.

Bone morphogenic protein (BMP) signaling plays an essential and highly conserved role in embryo axial patterning in animal species. However, in mammalian embryos, which develop inside the mother, early development includes a preimplantation stage, which does not occur in externally developing embryos. During preimplantation, the epiblast is segregated from extra-embryonic lineages that enable implantation and development in utero. Yet, the requirement for BMP signaling is imprecisely defined in mouse early embryos. Here, we show that, in contrast to previous reports, BMP signaling (SMAD1/5/9 phosphorylation) is not detectable until implantation when it is detected in the primitive endoderm - an extra-embryonic lineage. Moreover, preimplantation development appears to be normal following deletion of maternal and zygotic Smad4, an essential effector of canonical BMP signaling. In fact, mice lacking maternal Smad4 are viable. Finally, we uncover a new requirement for zygotic Smad4 in epiblast scaling and cavitation immediately after implantation, via a mechanism involving FGFR/ERK attenuation. Altogether, our results demonstrate no role for BMP4/SMAD4 in the first lineage decisions during mouse development. Rather, multi-pathway signaling among embryonic and extra-embryonic cell types drives epiblast morphogenesis postimplantation.

Low concentrations of saracatinib promote definitive endoderm differentiation through inhibition of FAK-YAP signaling axis.

Optimizing the efficiency of definitive endoderm (DE) differentiation is necessary for the generation of diverse organ-like structures. In this study, we used the small molecule inhibitor saracatinib (SAR) to enhance DE differentiation of human embryonic stem cells and induced pluripotent stem cells. SAR significantly improved DE differentiation efficiency at low concentrations. The interaction between SAR and Focal Adhesion Kinase (FAK) was explored through RNA-seq and molecular docking simulations, which further supported the inhibition of DE differentiation by p-FAK overexpression in SAR-treated cells. In addition, we found that SAR inhibited the nuclear translocation of Yes-associated protein (YAP), a downstream effector of FAK, which promoted DE differentiation. Moreover, the addition of SAR enabled a significant reduction in activin A (AA) from 50 to 10 ng/mL without compromising DE differentiation efficiency. For induction of the pancreatic lineage, 10 ng/ml AA combined with SAR at the DE differentiation stage yielded a comparative number of PDX1+/NKX6.1+ pancreatic progenitor cells to those obtained by 50 ng/ml AA treatment. Our study highlights SAR as a potential modulator that facilitates the cost-effective generation of DE cells and provides insight into the orchestration of cell fate determination.

A geometrical model of cell fate specification in the mouse blastocyst.

The lineage decision that generates the epiblast and primitive endoderm from the inner cell mass (ICM) is a paradigm for cell fate specification. Recent mathematics has formalized Waddington's landscape metaphor and proven that lineage decisions in detailed gene network models must conform to a small list of low-dimensional stereotypic changes called bifurcations. The most plausible bifurcation for the ICM is the so-called heteroclinic flip that we define and elaborate here. Our re-analysis of recent data suggests that there is sufficient cell movement in the ICM so the FGF signal, which drives the lineage decision, can be treated as spatially uniform. We thus extend the bifurcation model for a single cell to the entire ICM by means of a self-consistently defined time-dependent FGF signal. This model is consistent with available data and we propose additional dynamic experiments to test it further. This demonstrates that simplified, quantitative and intuitively transparent descriptions are possible when attention is shifted from specific genes to lineages. The flip bifurcation is a very plausible model for any situation where the embryo needs control over the relative proportions of two fates by a morphogen feedback.

The migration pattern of cells during the mesoderm and endoderm differentiation from human pluripotent stem cells.

Gastrulation is the first major differentiation process in animal embryos. However, the dynamics of human gastrulation remain mostly unknown owing to the ethical limitations. We studied the dynamics of the mesoderm and endoderm cell differentiation from human pluripotent stem cells for insight into the cellular dynamics of human gastrulation. Human pluripotent stem cells have properties similar to those of the epiblast, which gives rise to the three germ layers. The mesoderm and endoderm were induced with more than 75% purity from human induced pluripotent stem cells. Single-cell dynamics of pluripotent stem cell-derived mesoderm and endoderm cells were traced using time-lapse imaging. Both mesoderm and endoderm cells migrate randomly, accompanied by short-term directional persistence. No substantial differences were detected between mesoderm and endoderm migration. Computer simulations created using the measured parameters revealed that random movement and external force, such as the spread out of cells from the primitive streak area, mimicked the homogeneous discoidal germ layer formation. These results were consistent with the development of amniotes, which suggests the effectiveness of human pluripotent stem cells as a good model for studying human embryogenesis.

PAX1 represses canonical Wnt signaling pathway and plays dual roles during endoderm differentiation.

BACKGROUND: Paired box 1 (PAX1) is a transcription factor and essential for the development of pharyngeal pouches-derived tissues, including thymus. PAX1 mutations are identified in Severe Combined Immunodeficiency (SCID) patients with Otofaciocervical Syndrome Type 2 (OTFCS2). However, despite the critical roles of PAX1 in embryonic development and diseases, detailed insights into its molecular mode of action are critically missing. METHODS: The repressing roles of PAX1 and SCID associated mutants on Wnt signaling pathway were investigated by luciferase reporter assays, qRT-PCR and in situ hybridization in HEK293FT, HCT116 cells and zebrafish embryos, respectively. Co-immunoprecipitation (co-IP) and western blotting assays were carried out to identify the molecular mechanisms underlying PAX1's role on Wnt signaling pathway. hESC based endoderm differentiation, flow cytometry, high-throughput sequencing data analysis, and qRT-PCR assays were utilized to determine the roles of PAX1 during endoderm differentiation. RESULTS: Here, we show that PAX1 represses canonical Wnt signaling pathway in vertebrate cells. Mechanically, PAX1 competes with SUMO E3 ligase PIASy to bind to TCF7L2, thus perturbing TCF7L2 SUMOylation level, further reducing its transcriptional activity and protein stability. Moreover, we reveal that PAX1 plays dual roles in hESC-derived definitive and foregut/pharyngeal endoderm cells, which give rise to the thymus epithelium, by inhibiting Wnt signaling. Importantly, our data show PAX1 mutations found in SCID patients significantly compromise the suppressing ability of PAX1 on Wnt signaling. CONCLUSIONS: Our study presents a novel molecular mode of action of PAX1 in regulation of canonical Wnt signaling and endoderm differentiation, thus providing insights for the molecular basis of PAX1 associated SCID, offering better understanding of the behavior of PAX1 in embryogenesis.

Glycolysis-Stimulated Esrrb Lactylation Promotes the Self-Renewal and Extraembryonic Endoderm Stem Cell Differentiation of Embryonic Stem Cells.

Embryonic stem cells (ESCs) favor glycolysis over oxidative phosphorylation for energy production, and glycolytic metabolism is critical for pluripotency establishment, maintenance, and exit. However, an understanding of how glycolysis regulates the self-renewal and differentiation of ESCs remains elusive. Here, we demonstrated that protein lactylation, regulated by intracellular lactate, contributes to the self-renewal of ESCs. We further showed that Esrrb, an orphan nuclear receptor involved in pluripotency maintenance and extraembryonic endoderm stem cell (XEN) differentiation, is lactylated on K228 and K232. The lactylation of Esrrb enhances its activity in promoting ESC self-renewal in the absence of the LIF and XEN differentiation of ESCs by increasing its binding at target genes. Our studies reveal the importance of protein lactylation in the self-renewal and XEN differentiation of ESCs, and the underlying mechanism of glycolytic metabolism regulating cell fate choice.

Gastrulation: Its Principles and Variations.

As epiblast cells initiate development into various somatic cells, they undergo a large-scale reorganization, called gastrulation. The gastrulation of the epiblast cells produces three groups of cells: the endoderm layer, the collection of miscellaneous mesodermal tissues, and the ectodermal layer, which includes the neural, epidermal, and associated tissues. Most studies of gastrulation have focused on the formation of the tissues that provide the primary route for cell reorganization, that is, the primitive streak, in the chicken and mouse. In contrast, how gastrulation alters epiblast-derived cells has remained underinvestigated. This chapter highlights the regulation of cell and tissue fate via the gastrulation process. The roles and regulatory functions of neuromesodermal progenitors (NMPs) in the gastrulation process, elucidated in the last decade, are discussed in depth to resolve points of confusion. Chicken and mouse embryos, which form a primitive streak as the site of mesoderm precursor ingression, have been investigated extensively. However, primitive streak formation is an exception, even among amniotes. The roles of gastrulation processes in generating various somatic tissues will be discussed broadly.

Ingested soil bacteria breach gut epithelia and prime systemic immunity in an insect.

Insects lack acquired immunity and were thought to have no immune memory, but recent studies reported a phenomenon called immune priming, wherein sublethal dose of pathogens or nonpathogenic microbes stimulates immunity and prevents subsequential pathogen infection. Although the evidence for insect immune priming is accumulating, the underlying mechanisms are still unclear. The bean bug Riptortus pedestris acquires its gut microbiota from ambient soil and spatially structures them into a multispecies and variable community in the anterior midgut and a specific, monospecies Caballeronia symbiont population in the posterior region. We demonstrate that a particular Burkholderia strain colonizing the anterior midgut stimulates systemic immunity by penetrating gut epithelia and migrating into the hemolymph. The activated immunity, consisting of a humoral and a cellular response, had no negative effect on the host fitness, but on the contrary protected the insect from subsequent infection by pathogenic bacteria. Interruption of contact between the Burkholderia strain and epithelia of the gut weakened the host immunity back to preinfection levels and made the insects more vulnerable to microbial infection, demonstrating that persistent acquisition of environmental bacteria is important to maintain an efficient immunity.

SOX17 enables immune evasion of early colorectal adenomas and cancers.

A hallmark of cancer is the avoidance of immune destruction. This process has been primarily investigated in locally advanced or metastatic cancer1-3; however, much less is known about how pre-malignant or early invasive tumours evade immune detection. Here, to understand this process in early colorectal cancers (CRCs), we investigated how naive colon cancer organoids that were engineered in vitro to harbour Apc-null, KrasG12D and Trp53-null (AKP) mutations adapted to the in vivo native colonic environment. Comprehensive transcriptomic and chromatin analyses revealed that the endoderm-specifying transcription factor SOX17 became strongly upregulated in vivo. Notably, whereas SOX17 loss did not affect AKP organoid propagation in vitro, its loss markedly reduced the ability of AKP tumours to persist in vivo. The small fraction of SOX17-null tumours that grew displayed notable interferon-γ (IFNγ)-producing effector-like CD8+ T cell infiltrates in contrast to the immune-suppressive microenvironment in wild-type counterparts. Mechanistically, in both endogenous Apc-null pre-malignant adenomas and transplanted organoid-derived AKP CRCs, SOX17 suppresses the ability of tumour cells to sense and respond to IFNγ, preventing anti-tumour T cell responses. Finally, SOX17 engages a fetal intestinal programme that drives differentiation away from LGR5+ tumour cells to produce immune-evasive LGR5- tumour cells with lower expression of major histocompatibility complex class I (MHC-I). We propose that SOX17 is a transcription factor that is engaged during the early steps of colon cancer to orchestrate an immune-evasive programme that permits CRC initiation and progression.

Human primordial germ cell-like cells specified from resetting precursors develop in human hindgut organoids.

Human primordial germ cells (hPGCs), the precursors of eggs and sperm, start their complex development shortly after specification and during their migration to the primitive gonads. Here, we describe protocols for specifying hPGC-like cells (hPGCLCs) from resetting precursors and progressing them with the support of human hindgut organoids. Resetting hPGCLCs (rhPGCLCs) are specified from human embryonic stem cells (hESCs) transitioning from the primed into the naive state of pluripotency. Hindgut organoids are also derived from hESCs after a sequential differentiation into a posterior endoderm/hindgut fate. Both rhPGCLCs and hindgut organoids are combined and co-cultured for 25 d. The entire procedure takes ~1.5 months and can be successfully implemented by a doctoral or graduate student with basic skills and experience in hESC cultures. The co-culture system supports the progression of rhPGCLCs at a developmental timing analogous to that observed in vivo. Compared with previously developed hPGCLC progression protocols, which depend on co-cultures with mouse embryonic gonadal tissue, our co-culture system represents a developmentally relevant model closer to the environment that hPGCs first encounter after specification. Together with the potential for investigations of events during hPGC specification and early development, these protocols provide a practical approach to designing efficient models for in vitro gametogenesis. Notably, the rhPGCLC-hindgut co-culture system can also be adapted to study failings in hPGC migration, which are associated with the etiology of some forms of infertility and germ cell tumors.

SETD3 regulates endoderm differentiation of mouse embryonic stem cells through canonical Wnt signaling pathway.

With self-renewal and pluripotency features, embryonic stem cells (ESCs) provide an invaluable tool to investigate early cell fate decisions. Pluripotency exit and lineage commitment depend on precise regulation of gene expression that requires coordination between transcription (TF) and chromatin factors in response to various signaling pathways. SET domain-containing 3 (SETD3) is a methyltransferase that can modify histones in the nucleus and actin in the cytoplasm. Through an shRNA screen, we previously identified SETD3 as an important factor in the meso/endodermal lineage commitment of mouse ESCs (mESC). In this study, we identified SETD3-dependent transcriptomic changes during endoderm differentiation of mESCs using time-course RNA-seq analysis. We found that SETD3 is involved in the timely activation of the endoderm-related gene network. The canonical Wnt signaling pathway was one of the markedly altered signaling pathways in the absence of SETD3. The assessment of Wnt transcriptional activity revealed a significant reduction in Setd3-deleted (setd3∆) mESCs coincident with a decrease in the nuclear pool of the key TF ß-catenin level, though no change was observed in its mRNA or total protein level. Furthermore, a proximity ligation assay (PLA) found an interaction between SETD3 and ß-catenin. We were able to rescue the differentiation defect by stably re-expressing SETD3 or activating the canonical Wnt signaling pathway by changing mESC culture conditions. Our results suggest that alterations in the canonical Wnt pathway activity and subcellular localization of ß-catenin might contribute to the endoderm differentiation defect of setd3∆ mESCs.

Assessment of human embryonic stem cells differentiation into definitive endoderm lineage on the soft substrates.

Pluripotent stem cells (PSCs) hold enormous potential for treating multiple diseases owing to their ability to self-renew and differentiate into any cell type. Albeit possessing such promising potential, controlling their differentiation into a desired cell type continues to be a challenge. Recent studies suggest that PSCs respond to different substrate stiffness and, therefore, can differentiate towards some lineages via Hippo pathway. Human PSCs can also differentiate and self-organize into functional cells, such as organoids. Traditionally, human PSCs are differentiated on stiff plastic or glass plates towards definitive endoderm and then into functional pancreatic progenitor cells in the presence of soluble growth factors. Thus, whether stiffness plays any role in differentiation towards definitive endoderm from human pluripotent stem cells (hPSCs) remains unclear. Our study found that the directed differentiation of human embryonic stem cells towards endodermal lineage on the varying stiffness did not differ from the differentiation on stiff plastic dishes. We also observed no statistical difference between the expression of yes-associated protein (YAP) and phosphorylated YAP. Furthermore, we demonstrate that lysophosphatidic acid, a YAP activator, enhanced definitive endoderm formation, whereas verteporfin, a YAP inhibitor, did not have the significant effect on the differentiation. In summary, our results suggest that human embryonic stem cells may not differentiate in response to changes in stiffness, and that such cues may not have as significant impact on the level of YAP. Our findings indicate that more research is needed to understand the direct relationship between biophysical forces and hPSCs differentiation.

HBO1 determines SMAD action in pluripotency and mesendoderm specification.

TGF-ß signaling family plays an essential role to regulate fate decisions in pluripotency and lineage specification. How the action of TGF-ß family signaling is intrinsically executed remains not fully elucidated. Here, we show that HBO1, a MYST histone acetyltransferase (HAT) is an essential cell intrinsic determinant for TGF-ß signaling in human embryonic stem cells (hESCs). HBO1-/- hESCs fail to response to TGF-ß signaling to maintain pluripotency and spontaneously differentiate into neuroectoderm. Moreover, HBO1 deficient hESCs show complete defect in mesendoderm specification in BMP4-triggered gastruloids or teratomas. Molecularly, HBO1 interacts with SMAD4 and co-binds the open chromatin labeled by H3K14ac and H3K4me3 in undifferentiated hESCs. Upon differentiation, HBO1/SMAD4 co-bind and maintain the mesoderm genes in BMP4-triggered mesoderm cells while lose chromatin occupancy in neural cells induced by dual-SMAD inhibition. Our data reveal an essential role of HBO1, a chromatin factor to determine the action of SMAD in both human pluripotency and mesendoderm specification.