The SMAD2/3 interactome reveals that TGFß controls m6A mRNA methylation in pluripotency.

The TGFß pathway has essential roles in embryonic development, organ homeostasis, tissue repair and disease. These diverse effects are mediated through the intracellular effectors SMAD2 and SMAD3 (hereafter SMAD2/3), whose canonical function is to control the activity of target genes by interacting with transcriptional regulators. Therefore, a complete description of the factors that interact with SMAD2/3 in a given cell type would have broad implications for many areas of cell biology. Here we describe the interactome of SMAD2/3 in human pluripotent stem cells. This analysis reveals that SMAD2/3 is involved in multiple molecular processes in addition to its role in transcription. In particular, we identify a functional interaction with the METTL3-METTL14-WTAP complex, which mediates the conversion of adenosine to N6-methyladenosine (m6A) on RNA. We show that SMAD2/3 promotes binding of the m6A methyltransferase complex to a subset of transcripts involved in early cell fate decisions. This mechanism destabilizes specific SMAD2/3 transcriptional targets, including the pluripotency factor gene NANOG, priming them for rapid downregulation upon differentiation to enable timely exit from pluripotency. Collectively, these findings reveal the mechanism by which extracellular signalling can induce rapid cellular responses through regulation of the epitranscriptome. These aspects of TGFß signalling could have far-reaching implications in many other cell types and in diseases such as cancer.

Cell cycle regulators control mesoderm specification in human pluripotent stem cells.

The mesoderm is one of the three germ layers produced during gastrulation from which muscle, bones, kidneys, and the cardiovascular system originate. Understanding the mechanisms that control mesoderm specification could inform many applications, including the development of regenerative medicine therapies to manage diseases affecting these tissues. Here, we used human pluripotent stem cells to investigate the role of cell cycle in mesoderm formation. To this end, using small molecules or conditional gene knockdown, we inhibited proteins controlling G1 and G2/M cell cycle phases during the differentiation of human pluripotent stem cells into lateral plate, cardiac, and presomitic mesoderm. These loss-of-function experiments revealed that regulators of the G1 phase, such as cyclin-dependent kinases and pRb (retinoblastoma protein), are necessary for efficient mesoderm formation in a context-dependent manner. Further investigations disclosed that inhibition of the G2/M regulator cyclin-dependent kinase 1 decreases BMP (bone morphogenetic protein) signaling activity specifically during lateral plate mesoderm formation while reducing fibroblast growth factor/extracellular signaling-regulated kinase 1/2 activity in all mesoderm subtypes. Taken together, our findings reveal that cell cycle regulators direct mesoderm formation by controlling the activity of key developmental pathways.

Optimized inducible shRNA and CRISPR/Cas9 platforms for in vitro studies of human development using hPSCs.

Inducible loss of gene function experiments are necessary to uncover mechanisms underlying development, physiology and disease. However, current methods are complex, lack robustness and do not work in multiple cell types. Here we address these limitations by developing single-step optimized inducible gene knockdown or knockout (sOPTiKD or sOPTiKO) platforms. These are based on genetic engineering of human genomic safe harbors combined with an improved tetracycline-inducible system and CRISPR/Cas9 technology. We exemplify the efficacy of these methods in human pluripotent stem cells (hPSCs), and show that generation of sOPTiKD/KO hPSCs is simple, rapid and allows tightly controlled individual or multiplexed gene knockdown or knockout in hPSCs and in a wide variety of differentiated cells. Finally, we illustrate the general applicability of this approach by investigating the function of transcription factors (OCT4 and T), cell cycle regulators (cyclin D family members) and epigenetic modifiers (DPY30). Overall, sOPTiKD and sOPTiKO provide a unique opportunity for functional analyses in multiple cell types relevant for the study of human development.

Brachyury and SMAD signalling collaboratively orchestrate distinct mesoderm and endoderm gene regulatory networks in differentiating human embryonic stem cells.

The transcription factor brachyury (T, BRA) is one of the first markers of gastrulation and lineage specification in vertebrates. Despite its wide use and importance in stem cell and developmental biology, its functional genomic targets in human cells are largely unknown. Here, we use differentiating human embryonic stem cells to study the role of BRA in activin A-induced endoderm and BMP4-induced mesoderm progenitors. We show that BRA has distinct genome-wide binding landscapes in these two cell populations, and that BRA interacts and collaborates with SMAD1 or SMAD2/3 signalling to regulate the expression of its target genes in a cell-specific manner. Importantly, by manipulating the levels of BRA in cells exposed to different signalling environments, we demonstrate that BRA is essential for mesoderm but not for endoderm formation. Together, our data illuminate the function of BRA in the context of human embryonic development and show that the regulatory role of BRA is context dependent. Our study reinforces the importance of analysing the functions of a transcription factor in different cellular and signalling environments.

A novel piperidine identified by stem cell-based screening attenuates pulmonary arterial hypertension by regulating BMP2 and PTGS2 levels.

Genetic defects in bone morphogenetic protein type II receptor (BMPRII) signalling and inflammation contribute to the pathogenesis of pulmonary arterial hypertension (PAH). The receptor is activated by bone morphogenetic protein (BMP) ligands, which also enhance BMPR2 transcription. A small-molecule BMP upregulator with selectivity on vascular endothelium would be a desirable therapeutic intervention for PAH.We assayed compounds identified in the screening of BMP2 upregulators for their ability to increase the expression of inhibitor of DNA binding 1 (Id1), using a dual reporter driven specifically in human embryonic stem cell-derived endothelial cells. These assays identified a novel piperidine, BMP upregulator 1 (BUR1), that increased endothelial Id1 expression with a half-maximal effective concentration of 0.098 µmol·L-1 Microarray analyses and immunoblotting showed that BUR1 induced BMP2 and prostaglandin-endoperoxide synthase 2 (PTGS2) expression. BUR1 effectively rescued deficient angiogenesis in autologous BMPR2+/R899X endothelial cells generated by CRISPR/Cas9 and patient cells.BUR1 prevented and reversed PAH in monocrotaline rats, and restored BMPRII downstream signalling and modulated the arachidonic acid pathway in the pulmonary arterial endothelium in the Sugen 5416/hypoxia PAH mouse model.In conclusion, using stem cell technology we have provided a novel small-molecule compound which regulates BMP2 and PTGS2 levels that might be useful for the treatment of PAH.

Epigenetic and transcriptional regulations prime cell fate before division during human pluripotent stem cell differentiation.

Stem cells undergo cellular division during their differentiation to produce daughter cells with a new cellular identity. However, the epigenetic events and molecular mechanisms occurring between consecutive cell divisions have been insufficiently studied due to technical limitations. Here, using the FUCCI reporter we developed a cell-cycle synchronised human pluripotent stem cell (hPSC) differentiation system for uncovering epigenome and transcriptome dynamics during the first two divisions leading to definitive endoderm. We observed that transcription of key differentiation markers occurs before cell division, while chromatin accessibility analyses revealed the early inhibition of alternative cell fates. We found that Activator protein-1 members controlled by p38/MAPK signalling are necessary for inducing endoderm while blocking cell fate shifting toward mesoderm, and that enhancers are rapidly established and decommissioned between different cell divisions. Our study has practical biomedical utility for producing hPSC-derived patient-specific cell types since p38/MAPK induction increased the differentiation efficiency of insulin-producing pancreatic beta-cells.

Retinoic acid signaling modulation guides in vitro specification of human heart field-specific progenitor pools.

Cardiogenesis relies on the precise spatiotemporal coordination of multiple progenitor populations. Understanding the specification and differentiation of these distinct progenitor pools during human embryonic development is crucial for advancing our knowledge of congenital cardiac malformations and designing new regenerative therapies. By combining genetic labelling, single-cell transcriptomics, and ex vivo human-mouse embryonic chimeras we uncovered that modulation of retinoic acid signaling instructs human pluripotent stem cells to form heart field-specific progenitors with distinct fate potentials. In addition to the classical first and second heart fields, we observed the appearance of juxta-cardiac field progenitors giving rise to both myocardial and epicardial cells. Applying these findings to stem-cell based disease modelling we identified specific transcriptional dysregulation in first and second heart field progenitors derived from stem cells of patients with hypoplastic left heart syndrome. This highlights the suitability of our in vitro differentiation platform for studying human cardiac development and disease.

Microwell-mediated control of embryoid body size regulates embryonic stem cell fate via differential expression of WNT5a and WNT11.

Recently, various approaches for controlling the embryonic stem (ES) cell microenvironment have been developed for regulating cellular fate decisions. It has been reported that the lineage specific differentiation could be affected by the size of ES cell colonies and embryoid bodies (EBs). However, much of the underlying biology has not been well elucidated. In this study, we used microengineered hydrogel microwells to direct ES cell differentiation and determined the role of WNT signaling pathway in directing the differentiation. This was accomplished by forming ES cell aggregates within microwells to form different size EBs. We determined that cardiogenesis was enhanced in larger EBs (450 microm in diameter), and in contrast, endothelial cell differentiation was increased in smaller EBs (150 microm in diameter). Furthermore, we demonstrated that the EB-size mediated differentiation was driven by differential expression of WNTs, particularly noncanonical WNT pathway, according to EB size. The higher expression of WNT5a in smaller EBs enhanced endothelial cell differentiation. In contrast, the increased expression of WNT11 enhanced cardiogenesis. This was further validated by WNT5a-siRNA transfection assay and the addition of recombinant WNT5a. Our data suggest that EB size could be an important parameter in ES cell fate specification via differential gene expression of members of the noncanonical WNT pathway. Given the size-dependent response of EBs to differentiate to endothelial and cardiac lineages, hydrogel microwell arrays could be useful for directing stem cell fates and studying ES cell differentiation in a controlled manner.

Generation of functional hepatocytes by forward programming with nuclear receptors.

Production of large quantities of hepatocytes remains a major challenge for a number of clinical applications in the biomedical field. Directed differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells (HLCs) provides an advantageous solution and a number of protocols have been developed for this purpose. However, these methods usually follow different steps of liver development in vitro, which is time consuming and requires complex culture conditions. In addition, HLCs lack the full repertoire of functionalities characterising primary hepatocytes. Here, we explore the interest of forward programming to generate hepatocytes from hPSCs and to bypass these limitations. This approach relies on the overexpression of three hepatocyte nuclear factors (HNF1A, HNF6, and FOXA3) in combination with different nuclear receptors expressed in the adult liver using the OPTi-OX platform. Forward programming allows for the rapid production of hepatocytes (FoP-Heps) with functional characteristics using a simplified process. We also uncovered that the overexpression of nuclear receptors such as RORc can enhance specific functionalities of FoP-Heps thereby validating its role in lipid/glucose metabolism. Together, our results show that forward programming could offer a versatile alternative to direct differentiation for generating hepatocytes in vitro.

Single-cell atlas of human liver development reveals pathways directing hepatic cell fates.

The liver has been studied extensively due to the broad number of diseases affecting its vital functions. However, therapeutic advances have been hampered by the lack of knowledge concerning human hepatic development. Here, we addressed this limitation by describing the developmental trajectories of different cell types that make up the human liver at single-cell resolution. These transcriptomic analyses revealed that sequential cell-to-cell interactions direct functional maturation of hepatocytes, with non-parenchymal cells playing essential roles during organogenesis. We utilized this information to derive bipotential hepatoblast organoids and then exploited this model system to validate the importance of signalling pathways in hepatocyte and cholangiocyte specification. Further insights into hepatic maturation also enabled the identification of stage-specific transcription factors to improve the functionality of hepatocyte-like cells generated from human pluripotent stem cells. Thus, our study establishes a platform to investigate the basic mechanisms directing human liver development and to produce cell types for clinical applications.

TGFß signalling is required to maintain pluripotency of human naïve pluripotent stem cells.

The signalling pathways that maintain primed human pluripotent stem cells (hPSCs) have been well characterised, revealing a critical role for TGFß/Activin/Nodal signalling. In contrast, the signalling requirements of naive human pluripotency have not been fully established. Here, we demonstrate that TGFß signalling is required to maintain naive hPSCs. The downstream effector proteins - SMAD2/3 - bind common sites in naive and primed hPSCs, including shared pluripotency genes. In naive hPSCs, SMAD2/3 additionally bind to active regulatory regions near to naive pluripotency genes. Inhibiting TGFß signalling in naive hPSCs causes the downregulation of SMAD2/3-target genes and pluripotency exit. Single-cell analyses reveal that naive and primed hPSCs follow different transcriptional trajectories after inhibition of TGFß signalling. Primed hPSCs differentiate into neuroectoderm cells, whereas naive hPSCs transition into trophectoderm. These results establish that there is a continuum for TGFß pathway function in human pluripotency spanning a developmental window from naive to primed states.

Cholangiocyte organoids can repair bile ducts after transplantation in the human liver.

Organoid technology holds great promise for regenerative medicine but has not yet been applied to humans. We address this challenge using cholangiocyte organoids in the context of cholangiopathies, which represent a key reason for liver transplantation. Using single-cell RNA sequencing, we show that primary human cholangiocytes display transcriptional diversity that is lost in organoid culture. However, cholangiocyte organoids remain plastic and resume their in vivo signatures when transplanted back in the biliary tree. We then utilize a model of cell engraftment in human livers undergoing ex vivo normothermic perfusion to demonstrate that this property allows extrahepatic organoids to repair human intrahepatic ducts after transplantation. Our results provide proof of principle that cholangiocyte organoids can be used to repair human biliary epithelium.

Analysis of endothelial-to-haematopoietic transition at the single cell level identifies cell cycle regulation as a driver of differentiation.

BACKGROUND: Haematopoietic stem cells (HSCs) first arise during development in the aorta-gonad-mesonephros (AGM) region of the embryo from a population of haemogenic endothelial cells which undergo endothelial-to-haematopoietic transition (EHT). Despite the progress achieved in recent years, the molecular mechanisms driving EHT are still poorly understood, especially in human where the AGM region is not easily accessible. RESULTS: In this study, we take advantage of a human pluripotent stem cell (hPSC) differentiation system and single-cell transcriptomics to recapitulate EHT in vitro and uncover mechanisms by which the haemogenic endothelium generates early haematopoietic cells. We show that most of the endothelial cells reside in a quiescent state and progress to the haematopoietic fate within a defined time window, within which they need to re-enter into the cell cycle. If cell cycle is blocked, haemogenic endothelial cells lose their EHT potential and adopt a non-haemogenic identity. Furthermore, we demonstrate that CDK4/6 and CDK1 play a key role not only in the transition but also in allowing haematopoietic progenitors to establish their full differentiation potential. CONCLUSION: We propose a direct link between the molecular machineries that control cell cycle progression and EHT.

Single-Cell Sequencing of Developing Human Gut Reveals Transcriptional Links to Childhood Crohn's Disease.

Human gut development requires the orchestrated interaction of differentiating cell types. Here, we generate an in-depth single-cell map of the developing human intestine at 6-10 weeks post-conception. Our analysis reveals the transcriptional profile of cycling epithelial precursor cells; distinct from LGR5-expressing cells. We propose that these cells may contribute to differentiated cell subsets via the generation of LGR5-expressing stem cells and receive signals from surrounding mesenchymal cells. Furthermore, we draw parallels between the transcriptomes of ex vivo tissues and in vitro fetal organoids, revealing the maturation of organoid cultures in a dish. Lastly, we compare scRNA-seq profiles from pediatric Crohn's disease epithelium alongside matched healthy controls to reveal disease-associated changes in the epithelial composition. Contrasting these with the fetal profiles reveals the re-activation of fetal transcription factors in Crohn's disease. Our study provides a resource available at www.gutcellatlas.org, and underscores the importance of unraveling fetal development in understanding disease.

Naive Pluripotent Stem Cells Exhibit Phenotypic Variability that Is Driven by Genetic Variation.

Variability among pluripotent stem cell (PSC) lines is a prevailing issue that hampers not only experimental reproducibility but also large-scale applications and personalized cell-based therapy. This variability could result from epigenetic and genetic factors that influence stem cell behavior. Naive culture conditions minimize epigenetic fluctuation, potentially overcoming differences in PSC line differentiation potential. Here we derived PSCs from distinct mouse strains under naive conditions and show that lines from distinct genetic backgrounds have divergent differentiation capacity, confirming a major role for genetics in PSC phenotypic variability. This is explained in part through inconsistent activity of extra-cellular signaling, including the Wnt pathway, which is modulated by specific genetic variants. Overall, this study shows that genetic background plays a dominant role in driving phenotypic variability of PSCs.

Method to Synchronize Cell Cycle of Human Pluripotent Stem Cells without Affecting Their Fundamental Characteristics.

Cell cycle progression and cell fate decisions are closely linked in human pluripotent stem cells (hPSCs). However, the study of these interplays at the molecular level remains challenging due to the lack of efficient methods allowing cell cycle synchronization of large quantities of cells. Here, we screened inhibitors of cell cycle progression and identified nocodazole as the most efficient small molecule to synchronize hPSCs in the G2/M phase. Following nocodazole treatment, hPSCs remain pluripotent, retain a normal karyotype and can successfully differentiate into the three germ layers and functional cell types. Moreover, genome-wide transcriptomic analyses on single cells synchronized for their cell cycle and differentiated toward the endoderm lineage validated our findings and showed that nocodazole treatment has no effect on gene expression during the differentiation process. Thus, our synchronization method provides a robust approach to study cell cycle mechanisms in hPSCs.

Conditional Manipulation of Gene Function in Human Cells with Optimized Inducible shRNA.

The difficulties involved in conditionally perturbing complex gene expression networks represent major challenges toward defining the mechanisms controlling human development, physiology, and disease. We developed an OPTimized inducible KnockDown (OPTiKD) platform that addresses the limitations of previous approaches by allowing streamlined, tightly-controlled, and potent loss-of-function experiments for both single and multiple genes. The method relies on single-step genetic engineering of the AAVS1 genomic safe harbor with an optimized tetracycline-responsive cassette driving one or more inducible short hairpin RNAs (shRNAs). OPTiKD provides homogeneous, dose-responsive, and reversible gene knockdown. When implemented in human pluripotent stem cells (hPSCs), the approach can be then applied to a broad range of hPSC-derived mature cell lineages that include neurons, cardiomyocytes, and hepatocytes. Generation of OPTiKD hPSCs in commonly used culture conditions is simple (plasmid based), rapid (two weeks), and highly efficient (>95%). Overall, this method facilitates the functional annotation of the human genome in health and disease. © 2018 by John Wiley & Sons, Inc.

Human axial progenitors generate trunk neural crest cells in vitro.

The neural crest (NC) is a multipotent embryonic cell population that generates distinct cell types in an axial position-dependent manner. The production of NC cells from human pluripotent stem cells (hPSCs) is a valuable approach to study human NC biology. However, the origin of human trunk NC remains undefined and current in vitro differentiation strategies induce only a modest yield of trunk NC cells. Here we show that hPSC-derived axial progenitors, the posteriorly-located drivers of embryonic axis elongation, give rise to trunk NC cells and their derivatives. Moreover, we define the molecular signatures associated with the emergence of human NC cells of distinct axial identities in vitro. Collectively, our findings indicate that there are two routes toward a human post-cranial NC state: the birth of cardiac and vagal NC is facilitated by retinoic acid-induced posteriorisation of an anterior precursor whereas trunk NC arises within a pool of posterior axial progenitors.

Variability of human pluripotent stem cell lines.

Human pluripotent stem cells derived from embryos (human Embryonic Stem Cells or hESCs) or generated by direct reprogramming of somatic cells (human Induced Pluripotent Stem Cells or hiPSCs) can proliferate almost indefinitely in vitro while maintaining the capacity to differentiate into a broad diversity of cell types. These two properties (self-renewal and pluripotency) confers human pluripotent stem cells a unique interest for clinical applications since they could allow the production of infinite quantities of cells for disease modelling, drug screening and cell based therapy. However, recent studies have clearly established that human pluripotent stem cell lines can display variable capacity to differentiate into specific lineages. Consequently, the development of universal protocols of differentiation which could work efficiently with any human pluripotent cell line is complicated substantially. As a consequence, each protocol needs to be adapted to every cell line thereby limiting large scale applications and precluding personalised therapies. Here, we summarise our knowledge concerning the origin of this variability and describe potential solutions currently available to bypass this major challenge.

Inducible and Deterministic Forward Programming of Human Pluripotent Stem Cells into Neurons, Skeletal Myocytes, and Oligodendrocytes.

The isolation or in vitro derivation of many human cell types remains challenging and inefficient. Direct conversion of human pluripotent stem cells (hPSCs) by forced expression of transcription factors provides a potential alternative. However, deficient inducible gene expression in hPSCs has compromised efficiencies of forward programming approaches. We have systematically optimized inducible gene expression in hPSCs using a dual genomic safe harbor gene-targeting strategy. This approach provides a powerful platform for the generation of human cell types by forward programming. We report robust and deterministic reprogramming of hPSCs into neurons and functional skeletal myocytes. Finally, we present a forward programming strategy for rapid and highly efficient generation of human oligodendrocytes.