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Background Association of chromatin with lamin proteins at the nuclear periphery has emerged as a potential mechanism to coordinate cell type-specific gene expression and maintain cellular identity via gene silencing. Unlike many histone modifications and chromatin-associated proteins, lamina-associated domains (LADs) are mapped genome-wide in relatively few genetically normal human cell types, which limits our understanding of the role peripheral chromatin plays in development and disease. Results To address this gap, we map LAMIN B1 occupancy across twelve human cell types encompassing pluripotent stem cells, intermediate progenitors, and differentiated cells from all three germ layers. Integrative analyses of this atlas with gene expression and repressive histone modification maps reveal that lamina-associated chromatin in all twelve cell types is organized into at least two subtypes defined by differences in LAMIN B1 occupancy, gene expression, chromatin accessibility, transposable elements, replication timing, and radial positioning. Imaging of fluorescently labeled DNA in single cells validates these subtypes and shows radial positioning of LADs with higher LAMIN B1 occupancy and heterochromatic histone modifications primarily embedded within the lamina. In contrast, the second subtype of lamina-associated chromatin is relatively gene dense, accessible, dynamic across development, and positioned adjacent to the lamina. Most genes gain or lose LAMIN B1 occupancy consistent with cell types along developmental trajectories; however, we also identify examples where the enhancer, but not the gene body and promoter, changes LAD state. Conclusions Altogether, this atlas represents the largest resource to date for peripheral chromatin organization studies and reveals an intermediate chromatin subtype.

Pluripotent cells generate all types of cells in the body and have largely been classified dichotomously into two types: naive and primed. Arguing against a binary classification system, a study now discovers a unique transition state between naive and primed pluripotency and describes the signals that control this transition.

Multipotent self-renewing haematopoietic stem cells (HSCs) regenerate the adult blood system after transplantation1, which is a curative therapy for numerous diseases including immunodeficiencies and leukaemias2. Although substantial effort has been applied to identifying HSC maintenance factors through the characterization of the in vivo bone-marrow HSC microenvironment or niche3–5, stable ex vivo HSC expansion has previously been unattainable6,7. Here we describe the development of a defined, albumin-free culture system that supports the long-term ex vivo expansion of functional mouse HSCs. We used a systematic optimization approach, and found that high levels of thrombopoietin synergize with low levels of stem-cell factor and fibronectin to sustain HSC self-renewal. Serum albumin has long been recognized as a major source of biological contaminants in HSC cultures8; we identify polyvinyl alcohol as a functionally superior replacement for serum albumin that is compatible with good manufacturing practice. These conditions afford between 236- and 899-fold expansions of functional HSCs over 1 month, although analysis of clonally derived cultures suggests that there is considerable heterogeneity in the self-renewal capacity of HSCs ex vivo. Using this system, HSC cultures that are derived from only 50 cells robustly engraft in recipient mice without the normal requirement for toxic pre-conditioning (for example, radiation), which may be relevant for HSC transplantation in humans. These findings therefore have important implications for both basic HSC research and clinical haematology. An albumin-free culture system for the long-term ex vivo expansion of mouse haematopoietic stem cells produces 236- to 899-fold expansion, and generates cultures that robustly engraft in recipient mice without toxic pre-conditioning.

Rationale: Coronary artery disease is the leading cause of death worldwide, but there are currently no methods to stimulate artery growth or regeneration in diseased hearts. Studying how arteries are built during development could illuminate strategies for re-building these vessels during ischemic heart disease. We previously found that Dach1 deletion in mouse embryos resulted in small coronary arteries. However, it was not known whether Dach1 gain-of-function would be sufficient to increase arterial vessels and whether this could benefit injury responses. Objective: We investigated how Dach1 overexpression in endothelial cells affected transcription and artery differentiation, and how it influenced recovery from myocardial infarction. Methods and Results: Dach1 was genetically overexpressed in coronary endothelial cells in either developing or adult hearts using ApjCreER. This increased the length and number of arterial end branches expanded arteries during development, in both the heart and retina, by inducing capillary endothelial cells to differentiate and contribute to growing arteries. Single-cell RNA sequencing of endothelial cells undergoing Dach1 -induced arterial specification indicated that it potentiated normal artery differentiation, rather than functioning as a master regulator of artery cell fate. Single-cell RNA sequencing also showed that normal arterial differentiation is accompanied by repression of lipid metabolism genes, which were also repressed by Dach1. In adults, Dach1 overexpression did not cause a statistically significant change artery structure before injury, but increased the number of perfused arteries in the injury zone post-myocardial infarction. Conclusions: Our data demonstrate that increasing Dach1 is a novel method for driving artery specification and extending arterial branches, which could be explored as a means of mitigating the effects of coronary artery disease.

SummaryMammalian SWI/SNF (BAF) chromatin remodelers play dosage-sensitive roles in many human malignancies and neurologic disorders. The gene encoding the BAF subunit, ACTL6A, is amplified at an early stage in the development of squamous cell carcinomas (SCCs), but its oncogenic role remains unclear. Here we demonstrate that ACTL6A overexpression leads to its stoichiometric assembly into BAF complexes and drives its interaction and engagement with specific regulatory regions in the genome. In normal epithelial cells, ACTL6A was sub-stoichiometric to other BAF subunits. However, increased ACTL6A levels by ectopic expression or in SCC cells led to near-saturation of ACTL6A within BAF complexes. Increased ACTL6A occupancy enhanced polycomb opposition over the genome activating SCC genes and also enhanced the recruitment of transcription factor TEAD with its co-activator YAP, promoting their chromatin binding and enhancer accessibility. Both of these mechanisms appeared to be critical and function as a molecular AND gate for SCC initiation and maintenance, thereby explaining the specificity of the role of ACTL6A amplification in SCCs.HighlightsACTL6A occupancy within BAF complexes is sub-stoichiometric in normal epithelial cells.SCC cells upregulate ACTL6A thus increasing ACTL6A assembly with BAF complexes.Genome-wide chromatin profiling identifies ACTL6A-dependent regulatory regions.Increasing ACTL6A incorporation enhances TEAD-YAP binding to BAF complexes.ACTL6A overexpression counteracts polycomb-mediated repression at SCC signature genes.

Mammalian SWI/SNF (BAF) chromatin remodelers play dosage-sensitive roles in many human malignancies and neurologic disorders. The gene encoding the BAF subunit actin-like 6a (ACTL6A) is amplified early in the development of many squamous cell carcinomas (SCCs), but its oncogenic role remains unclear. Here we demonstrate that ACTL6A overexpression leads to its stoichiometric assembly into BAF complexes and drives their interaction and engagement with specific regulatory regions in the genome. In normal epithelial cells, ACTL6A was substoichiometric to other BAF subunits. However, increased ACTL6A levels by ectopic expression or in SCC cells led to near saturation of ACTL6A within BAF complexes. Increased ACTL6A occupancy enhanced polycomb opposition genome-wide to activate SCC genes and facilitated the co-dependent loading of BAF and TEAD-YAP complexes on chromatin. Both mechanisms appeared to be critical and function as a molecular AND gate for SCC initiation and maintenance, thereby explaining the specificity of the role of ACTL6A amplification in SCCs.

As our closest living relatives, non-human primates uniquely enable explorations of human health, disease, development, and evolution. Considerable effort has thus been devoted to generating induced pluripotent stem cells (iPSCs) from multiple non-human primate species. Here we establish improved culture methods for chimpanzee (Pan troglodytes) and pig-tailed macaque (Macaca nemestrina) iPSCs. Such iPSCs spontaneously differentiate in conventional culture conditions, but can be readily propagated by inhibiting endogenous WNT signaling. Chimpanzee and pig-tailed macaque iPSCs cultured in these enhanced conditions can differentiate into cardiomyocytes and neural crest in vitro and form teratomas in vivo. As a unique functional test of these iPSCs, we injected them into the pre-implantation embryos of another non-human species, rhesus macaques (Macaca mulatta). Ectopic expression of anti-apoptotic gene BCL2 enhances the survival and proliferation of chimpanzee and pig-tailed macaque iPSCs within the pre-implantation embryo, although the identity and long-term contribution of the transplanted cells warrants further investigation. In summary, we disclose transcriptomic, cell lines, and cell culture resources that may be broadly enabling for non-human primate iPSC research.

Stem cell research endeavors to generate specific subtypes of classically defined "cell types." Here, we generate90% pure human artery or vein endothelial cells from pluripotent stem cells within 3-4 days. We specified artery cells by inhibiting vein-specifying signals and vice versa. These cells modeled viral infection of human vasculature by Nipah and Hendra viruses, which are extraordinarily deadly (∼57%-59% fatality rate) and require biosafety-level-4 containment. Generating pure populations of artery and vein cells highlighted that Nipah and Hendra viruses preferentially infected arteries; arteries expressed higher levels of their viral-entry receptor. Virally infected artery cells fused into syncytia containing up to 23 nuclei, which rapidly died. Despite infecting arteries and occupying ∼6%-17% of their transcriptome, Nipah and Hendra largely eluded innate immune detection, minimally eliciting interferon signaling. We thus efficiently generate artery and vein cells, introduce stem-cell-based toolkits for biosafety-level-4 virology, and explore the arterial tropism and cellular effects of Nipah and Hendra viruses.

Generating primordial germ cells (PGCs) from human pluripotent stem cells (hPSCs) advances studies of human reproduction and development of infertility treatments, but currently entails complex 3D aggregates. Here we develop a simplified, monolayer method to differentiate hPSCs into PGCs within 3.5 days. We used our simplified differentiation platform and single-cell RNA-sequencing to uncover new insights into PGC specification. Transient WNT activation for 12 hours followed by WNT inhibition specified PGCs; by contrast, sustained WNT instead induced primitive streak. Thus, somatic (primitive streak) and PGCs are related—yet distinct—lineages segregated by temporally-dynamic signaling. Pluripotency factors including NANOG are continuously expressed during the transition from pluripotency to posterior epiblast to PGCs, thus bridging pluripotent and germline states. Finally, hPSC-derived PGCs can be easily purified by virtue of their CXCR4+PDGFRA−GARP− surface-marker profile and single-cell RNA-sequencing reveals that they harbor strong transcriptional similarities with fetal PGCs.

Association of chromatin with lamin proteins at the nuclear periphery has emerged as a potential mechanism to coordinate cell type-specific gene expression and maintain cellular identity via gene silencing. Unlike many histone modifications and chromatin-associated proteins, lamin-associated domains (LADs) have yet to be mapped genome-wide in a diverse panel of human cell types, which has limited our understanding of the role peripheral chromatin plays in development and disease. To address this gap, we mapped LAMIN B1 (LB1) across twelve human cell types encompassing pluripotent stem cells, intermediate progenitors, and differentiated cells from all three germ layers. Integrative analyses of this atlas of peripheral chromatin with publicly available genomic data, as well as gene expression and repressive histone maps generated for this study, revealed that in all twelve cellular contexts lamin-associated chromatin is organized into at least two subtypes defined by differences in LB1 occupancy, gene expression, chromatin accessibility, transposable elements, replication timing, and radial positioning. Most genes gain or lose LB1 occupancy consistent with their cell type along developmental trajectories; however, we also identified examples where the enhancer, but not the gene body and promoter, change LAD state. Imaging of fluorescently labeled DNA in single cells validated these transitions and showed intermediate radial positioning of LADs that are gene dense, relatively accessible, and dynamic across development. This atlas represents the largest resource to date for peripheral chromatin organization studies.

Multiple adult tissues are maintained by stem cells of restricted developmental potential which can only form a subset of lineages within the tissue. For instance, the two adult lung epithelial compartments (airways and alveoli) are separately maintained by distinct lineage-restricted stem cells. A challenge has been to obtain multipotent stem cells and/or progenitors that can generate all epithelial cell types of a given tissue. Here we show that mouse Sox9+ multipotent embryonic lung progenitors can be isolated and expanded long term in 3D culture. Cultured Sox9+ progenitors transcriptionally resemble their in vivo counterparts and generate both airway and alveolar cell types in vitro. Sox9+ progenitors that were transplanted into injured adult mouse lungs differentiated into all major airway and alveolar lineages in vivo in a region-appropriate fashion. We propose that a single expandable embryonic lung progenitor population with broader developmental competence may eventually be used as an alternative for region-restricted adult tissue stem cells in regenerative medicine.

Liver diseases afflict millions of patients worldwide. Currently, the only long-term treatment for liver failure is the transplantation of a new liver. However, intravenously transplanting a suspension of human hepatocytes might be a less-invasive approach to partially reconstitute lost liver functions in human patients as evinced by promising outcomes in clinical trials. The purpose of this essay is to emphasize outstanding questions that continue to surround hepatocyte transplantation. While adult primary human hepatocytes are the gold standard for transplantation, hepatocytes are heterogeneous. Whether all hepatocytes engraft equally and what specifically defines an "engraftable" hepatocyte capable of long-term liver reconstitution remains unclear. To this end, mouse models of liver injury enable the evaluation of human hepatocytes and their behavior upon transplantation into a complex injured liver environment. While mouse models may not be fully representative of the injured human liver and human hepatocytes tend to engraft mice less efficiently than mouse hepatocytes, valuable lessons have nonetheless been learned from transplanting human hepatocytes into mouse models. With an eye to the future, it will be crucial to eventually detail the optimal biological source (whether in vivo- or in vitro-derived) and presumptive heterogeneity of human hepatocytes and to understand the mechanisms through which they engraft and regenerate liver tissue in vivo.

Water deprivation produces a drive to seek and consume water. How neural activity creates this motivation remains poorly understood. We used activity-dependent genetic labeling to characterize neurons activated by water deprivation in the hypothalamic median preoptic nucleus (MnPO). Single-cell transcriptional profiling revealed that dehydration-activated MnPO neurons consist of a single excitatory cell type. After optogenetic activation of these neurons, mice drank water and performed an operant lever-pressing task for water reward with rates that scaled with stimulation frequency. This stimulation was aversive, and instrumentally pausing stimulation could reinforce lever-pressing. Activity of these neurons gradually decreased over the course of an operant session. Thus, the activity of dehydration-activated MnPO neurons establishes a scalable, persistent, and aversive internal state that dynamically controls thirst-motivated behavior.

Mbd3, a member of Nucleosome Remodeling and Deacetylase (NuRD) co-repressor complex, was previously identified as an inhibitor for deterministic iPSC reprogramming where up to 100% of donor cells successfully complete the process. NuRD can assume multiple mutually-exclusive conformations, and it remains unclear whether this deterministic phenotype can be attributed to a specific Mbd3/NuRD sub-complex. Moreover, since complete ablation of Mbd3 blocks somatic cell proliferation, we aimed to explore functionally relevant alternative ways to neutralize Mbd3-dependent NuRD activity. We identify Gatad2a, a NuRD-specific subunit, whose complete deletion specifically disrupts Mbd3/NuRD repressive activity on the pluripotency circuitry during iPSC differentiation and reprogramming without disrupting somatic cell proliferation. Ablation of Gatad2a facilitates deterministic murine iPSC reprogramming within 8-days. We validate a distinct molecular axis, Gatad2a-Chd4-Mbd3, within Mbd3/NuRD as being critical for blocking re-establishment of naïve pluripotency, and further highlight signaling-dependent and post-translational modifications of Mbd3/NuRD that influence its interactions and assembly.

Too many choices can be problematic. This is certainly the case for human pluripotent stem cells (hPSCs): they harbor the potential to differentiate into hundreds of cell types; yet it is highly challenging to exclusively differentiate hPSCs into a single desired cell type. This review focuses on unresolved and fundamental questions regarding hPSC differentiation and critiquing the identity and purity of the resultant cell populations. These are timely issues in view of the fact that hPSC-derived cell populations have or are being transplanted into patients in over 30 ongoing clinical trials. While many in vitro differentiation protocols purport to "mimic development," the exact number and identity of intermediate steps that a pluripotent cell takes to differentiate into a given cell type in vivo remains largely unknown. Consequently, most differentiation efforts inevitably generate a heterogeneous cellular population, as revealed by single-cell RNA-sequencing and other analyses. The presence of unwanted cell types in differentiated hPSC populations does not portend well for transplantation therapies. This provides an impetus to precisely control differentiation to desired ends-for instance, by logically blocking the formation of unwanted cell types or by overexpressing lineage-specifying transcription factors-or by harnessing technologies to selectively purify desired cell types. Conversely, approaches to differentiate three-dimensional "organoids" from hPSCs intentionally generate heterogeneous cell populations. While this is intended to mimic the rich cellular diversity of developing tissues, whether all such organoids are spatially organized in a manner akin to native organs (and thus, whether they fully qualify as organoids) remains to be fully resolved. This article is categorized under: Adult Stem Cells > Tissue Renewal > Regeneration: Stem Cell Differentiation and Reversion Gene Expression > Transcriptional Hierarchies: Cellular Differentiation Early Embryonic Development: Gastrulation and Neurulation.

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure-which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults-is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

SummaryThrough the production of chimeric animals, induced pluripotent stem cells (iPSCs) can generate personalized organs with diverse applications for both basic research and translational medicine. This concept was first validated in rodents by forming a rat pancreas in mice and vice versa. However, the potential use of human iPSCs to generate xenogenic organs in other species is technically and ethically difficult. Recognizing these concerns, we explored the generation of chimeric nonhuman primates (NHP) embryos, by injecting either chimpanzee or pig-tailed macaque iPSCs into rhesus macaque embryos. We first derived iPSCs from chimpanzees and pig-tailed macaques. We found that the chimpanzee iPSCs mixed well with human iPSCs duringin vitroco-culture and differentiation. The differentiation of mixed human and chimpanzee iPSCs formed functioning cardiomyocyte layers in vitro, whereas human or chimpanzee iPSC mixed with pig-tailed macaque or mouse cells do not; these results indicate that chimpanzee and human cells are closely related in function. Considering the ethical aspects of injecting human iPSCs into nonhuman primate blastocysts, we tested whether chimpanzee iPSCs injected into 99 macaque 5-day-old embryos formed cross-species chimeras two days after injection. Strikingly, the chimpanzee iPSCs survived, proliferated and integrated near the inner cell mass (ICM) of rhesus macaque embryos. These findings highlight the broad potential of primate iPSCs in forming cross-species chimeras beyond rodents and provides a foundational basis for organ generation using human iPSCs.

Cell types more advanced in development than embryonic stem cells, such as EpiSCs, fail to contribute to chimeras when injected into pre-implantation-stage blastocysts, apparently because the injected cells undergo apoptosis. Here we show that transient promotion of cell survival through expression of the anti-apoptotic gene BCL2 enables EpiSCs and Sox17+ endoderm progenitors to integrate into blastocysts and contribute to chimeric embryos. Upon injection into blastocyst, BCL2-expressing EpiSCs contributed to all bodily tissues in chimeric animals while Sox17+ endoderm progenitors specifically contributed in a region-specific fashion to endodermal tissues. In addition, BCL2 expression enabled rat EpiSCs to contribute to mouse embryonic chimeras, thereby forming interspecies chimeras that could survive to adulthood. Our system therefore provides a method to overcome cellular compatibility issues that typically restrict chimera formation. Application of this type of approach could broaden the use of embryonic chimeras, including region-specific chimeras, for basic developmental biology research and regenerative medicine.

The ability to harness the processes by which complex tissues arise during embryonic development would improve the ability to engineer complex tissuelike constructs in vitro—a longstanding goal of tissue engineering and regenerative medicine. In embryos, uniform populations of stem cells are exposed to spatial gradients of diffusible extracellular signaling proteins, known as morphogens. Varying levels of these signaling proteins induce stem cells to differentiate into distinct cell types at different positions along the gradient, thus creating spatially patterned tissues. Here, the authors describe two straightforward and easy-to-adopt microfluidic strategies to expose human pluripotent stem cells in vitro to spatial gradients of desired differentiation-inducing extracellular signals. Both approaches afford a high degree of control over the distribution of extracellular signals, while preserving the viability of the cultured stem cells. The first microfluidic platform is commercially available and entails static culture, whereas the second microfluidic platform requires fabrication and dynamic fluid exchange. In each platform, the authors first computationally modeled the spatial distribution of differentiation-inducing extracellular signals. Then, the authors used each platform to expose human pluripotent stem cells to a gradient of these signals (in this case, inducing a cell type known as the primitive streak), resulting in a regionalized culture with differentiated primitive streak cells predominately localized on one side and undifferentiated stem cells at the other side of the device. By combining this approach with a fluorescent reporter for differentiated cells and live-cell fluorescence imaging, the authors characterized the spatial and temporal dynamics of primitive streak differentiation within the induced signaling gradients. Microfluidic approaches to create precisely controlled morphogen gradients will add to the stem cell and developmental biology toolkit, and may eventually pave the way to create increasingly spatially patterned tissuelike constructs in vitro.

SummaryHematopoietic stem cell (HSC) transplantation can replace diseased blood systems with a healthy one, thereby treating or curing genetic blood and immune disorders including autoimmune diseases and immunodeficiencies. However, toxic chemotherapy or radiation is necessary to ablate an animal’s existing blood system prior to HSC transplantation, leading to significant morbidity. To accomplish safer blood-system replacement we developed a combination of six monoclonal antibodies to safely and specifically deplete the HSCs, T cells and NK cells of immune-competent mice. Remarkably, immunologically-foreign (allogeneic) HSCs mismatched at half or all the MHC genes could engraft these antibody-treated mice, generating donor blood systems that stably co-existed with host blood cells. These chimeric immune systems were immunologically tolerant to heart tissue from the HSC donor, providing a safe platform for HSC transplantation as a means to solid organ transplantation. The ability to transplant MHC-mismatched HSCs without chemotherapy or radiation has significant ramifications for regenerative medicine.

There were multiple prerequisites to the evolution of multicellular animal life, including the generation of multiple cell fates ("cellular diversity") and their patterned spatial arrangement ("spatial form"). Wnt proteins operate as primordial symmetry-breaking signals. By virtue of their short-range nature and their capacity to activate both lineage-specifying and cell-polarizing intracellular signaling cascades, Wnts can polarize cells at their site of contact, orienting the axis of cell division while simultaneously programming daughter cells to adopt diverging fates in a spatially stereotyped way. By coupling cell fate to position, symmetry-breaking Wnt signals were pivotal in constructing the metazoan body by generating cellular diversity and spatial form.

Summary How are closely related lineages, including liver, pancreas, and intestines, diversified from a common endodermal origin? Here, we apply principles learned from developmental biology to rapidly reconstitute liver progenitors from human pluripotent stem cells (hPSCs). Mapping the formation of multiple endodermal lineages revealed how alternate endodermal fates (e.g., pancreas and intestines) are restricted during liver commitment. Human liver fate was encoded by combinations of inductive and repressive extracellular signals at different doses. However, these signaling combinations were temporally re-interpreted: cellular competence to respond to retinoid, WNT, TGF-β, and other signals sharply changed within 24 hr. Consequently, temporally dynamic manipulation of extracellular signals was imperative to suppress the production of unwanted cell fates across six consecutive developmental junctures. This efficiently generated 94.1% ± 7.35% TBX3+ HNF4A+ human liver bud progenitors and 81.5% ± 3.2% FAH+ hepatocyte-like cells by days 6 and 18 of hPSC differentiation, respectively; the latter improved short-term survival in the Fah–/– Rag2–/– Il2rg–/– mouse model of liver failure., Graphical abstract Ang et al. chart how human liver progenitors develop from pluripotent stem cells through six developmental steps, including extracellular signals and surface markers associated with liver formation. This knowledge enables efficient generation of liver bud progenitors and, subsequently, hepatocyte-like cells that could function in vivo and in vitro.

Despite decisive progress in differentiating pluripotent stem cells (PSCs) into diverse cell-types, the often-lengthy differentiation and functional immaturity of such cell-types remain pertinent issues. Here we address the first challenge of prolonged differentiation in the generation of hepatocyte-like cells from PSCs. We delineate a roadmap describing the extracellular signals controlling six sequential branching lineage choices leading from pluripotency to endoderm, foregut, and finally, liver progenitors. By blocking formation of unwanted cell-types at each lineage juncture and manipulating temporally-dynamic signals, we accelerated generation of 89.0±3.1% AFP+human liver bud progenitors and 87.3±9.4% ALBUMIN+hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. 81.5±3.2% of hepatocyte-like cells expressed metabolic enzyme FAH (as assayed by a new knock-in reporter line) and improved short-term survival in theFah-/-Rag2-/-Il2rg-/-mouse model of liver failure. Collectively the timed signaling interventions indicated by this developmental roadmap enable accelerated production of human liver progenitors from PSCs.

It is imperative to unveil the full range of differentiated cell types into which human pluripotent stem cells (hPSCs) can develop. The need is twofold: it will delimit the therapeutic utility of these stem cells and is necessary to place their position accurately in the developmental hierarchy of lineage potential. Accumulated evidence suggested that hPSC could developin vitrointo an extraembryonic lineage (trophoblast (TB)) that is typically inaccessible to pluripotent embryonic cells during embryogenesis. However, whether these differentiated cells are truly authentic TB has been challenged. In this debate, we present a case for and a case against TB differentiation from hPSCs. By analogy to other differentiation systems, our debate is broadly applicable, as it articulates higher and more challenging standards for judging whether a given cell type has been genuinely produced from hPSC differentiation.

The self-renewal of multipotent hematopoietic stem cells (HSCs) is key for life-long maintenance of the blood system and the curative capacity of clinical HSC transplantation. However, while in vivo HSC self-renewal capacity has been well-described, existing culture conditions poorly support ex vivo HSC self-renewal and afford only a very limited window to study or modify HSCs in-a-dish. By taking a reductionist optimization approach, we have developed a simple culture platform that supports functional mouse HSCs ex vivo over 1-2 months. Importantly, we identified the synthetic polymer polyvinyl alcohol as a superior, inexpensive, and chemically-defined alternative to serum albumin supplements, which have long represented a major source of biological contaminants and batch-to-batch variability in HSC cultures. Limiting dilution transplantation analysis of day-28 HSC cultures estimated a ∼900-fold expansion of functional HSCs (based on >1% multilineage engraftment at 16-weeks post-transplantation) with secondary transplantation analysis estimating >200-fold expansion of serially-engraftable long-term HSCs. HSCs could also be expanded clonally using this system, demonstrating bona fide ex vivo HSC self-renewal. The large numbers of functional HSCs generated by this long-term ex vivo expansion protocol even enabled HSC transplantation in nonconditioned immunocompetent and immunodeficient recipients. Finally, this albumin-free and GMP-compatible culture system also supported human HSCs ex vivo, as determined by long-term engraftment in NSG mice. Thus, our novel HSC culture platform provides an important new tool to interrogate HSC self-renewal and lineage commitment, and also suggests a novel approach in clinical HSC transplantation.

Summary Hematopoietic cell transplantation can correct hematological and immunological disorders by replacing a diseased blood system with a healthy one, but this currently requires depleting a patient’s existing hematopoietic system with toxic and non-specific chemotherapy, radiation, or both. Here we report an antibody-based conditioning protocol with reduced toxicity and enhanced specificity for robust hematopoietic stem cell (HSC) transplantation and engraftment in recipient mice. Host pre-treatment with six monoclonal antibodies targeting CD47, T cells, NK cells, and HSCs followed by donor HSC transplantation enabled stable hematopoietic system reconstitution in recipients with mismatches at half (haploidentical) or all major histocompatibility complex (MHC) genes. This approach allowed tolerance to heart tissue from HSC donor strains in haploidentical recipients, showing potential applications for solid organ transplantation without immune suppression. Fully mismatched chimeric mice developed antibody responses to nominal antigens, showing preserved functional immunity. These findings suggest approaches for transplanting immunologically mismatched HSCs and solid organs with limited toxicity.

SummaryHuman pluripotent stem cell (hPSC) differentiation typically yields heterogeneous populations. Knowledge of signals controlling embryonic lineage bifurcations could efficiently yield desired cell types through exclusion of alternate fates. Therefore, we revisited signals driving induction and anterior-posterior patterning of definitive endoderm to generate a coherent roadmap for endoderm differentiation. With striking temporal dynamics, BMP and Wnt initially specified anterior primitive streak (progenitor to endoderm), yet, 24 hr later, suppressed endoderm and induced mesoderm. At lineage bifurcations, cross-repressive signals separated mutually exclusive fates; TGF-β and BMP/MAPK respectively induced pancreas versus liver from endoderm by suppressing the alternate lineage. We systematically blockaded alternate fates throughout multiple consecutive bifurcations, thereby efficiently differentiating multiple hPSC lines exclusively into endoderm and its derivatives. Comprehensive transcriptional and chromatin mapping of highly pure endodermal populations revealed that endodermal enhancers existed in a surprising diversity of “pre-enhancer” states before activation, reflecting the establishment of a permissive chromatin landscape as a prelude to differentiation.

Multiple adult tissues are maintained by stem cells of restricted developmental potential which can only form a subset of lineages within the tissue. For instance, the two adult lung epithelial compartments (airways and alveoli) are separately maintained by distinct lineage-restricted stem cells. A challenge has been to obtain multipotent stem cells and/or progenitors that can generate all epithelial cell types of a given tissue. Here we show that mouse Sox9

Stem-cell differentiation to desired lineages requires navigating alternating developmental paths that often lead to unwanted cell types. Hence, comprehensive developmental roadmaps are crucial to channel stem-cell differentiation toward desired fates. To this end, here, we map bifurcating lineage choices leading from pluripotency to 12 human mesodermal lineages, including bone, muscle, and heart. We defined the extrinsic signals controlling each binary lineage decision, enabling us to logically block differentiation toward unwanted fates and rapidly steer pluripotent stem cells toward 80%-99% pure human mesodermal lineages at most branchpoints. This strategy enabled the generation of human bone and heart progenitors that could engraft in respective in vivo models. Mapping stepwise chromatin and single-cell gene expression changes in mesoderm development uncovered somite segmentation, a previously unobservable human embryonic event transiently marked by HOPX expression. Collectively, this roadmap enables navigation of mesodermal development to produce transplantable human tissue progenitors and uncover developmental processes. VIDEO ABSTRACT.

Understanding the basis of the unrestricted multilineage differentiation potential of pluripotent cells will be of developmental and translational consequence. We propose that pluripotency transcription factors are lineage specifiers that direct commitment to specific fetal lineages. Individual factors bestow the ability to differentiate into particular cell types, and concomitant expression of multiple lineage specifiers within pluripotent cells enables differentiation into every fetal lineage. Moreover, we speculate that, rather than being an intrinsically stable "ground state," pluripotency is an inherently precarious condition in which rival lineage specifiers continually compete to specify differentiation along mutually exclusive lineages.

SummaryThe combined activity of three transcription factors can reprogram adult cells into induced pluripotent stem cells (iPSCs). However, the transgenic methods used for delivering reprogramming factors have raised concerns regarding the future utility of the resulting stem cells. These uncertainties could be overcome if each transgenic factor were replaced with a small molecule that either directly activated its expression from the somatic genome or in some way compensated for its activity. To this end, we have used high-content chemical screening to identify small molecules that can replace Sox2 in reprogramming. We show that one of these molecules functions in reprogramming by inhibiting Tgf-β signaling in a stable and trapped intermediate cell type that forms during the process. We find that this inhibition promotes the completion of reprogramming through induction of the transcription factor Nanog.

Recent efforts have attempted to convert non-blood cells into hematopoietic stem cells (HSCs) with the goal of generating blood lineages de novo. Here we show that hematopoietic transcription factors Scl, Lmo2, Runx1 and Bmi1 can convert a developmentally distant lineage (fibroblasts) into ‘induced hematopoietic progenitors' (iHPs). Functionally, iHPs generate acetylcholinesterase+ megakaryocytes and phagocytic myeloid cells in vitro and can also engraft immunodeficient mice, generating myeloerythoid and B-lymphoid cells for up to 4 months in vivo. Molecularly, iHPs transcriptionally resemble native Kit+ hematopoietic progenitors. Mechanistically, reprogramming factor Lmo2 implements a hematopoietic programme in fibroblasts by rapidly binding to and upregulating the Hhex and Gfi1 genes within days. Moreover the reprogramming transcription factors also require extracellular BMP and MEK signalling to cooperatively effectuate reprogramming. Thus, the transcription factors that orchestrate embryonic hematopoiesis can artificially reconstitute this programme in developmentally distant fibroblasts, converting them into engraftable blood progenitors., Direct reprogramming of closely-related lineages can generate hematopoietic stem cells. Here, the authors show hematopoietic transcription factors Scl, Lmo2, Runx1 and Bmi1 can reprogram fibroblasts into induced hematopoietic progenitors (iHPs), which are engraftable blood progenitors.

Pluripotency, the ability to generate any cell type of the body, is an evanescent attribute of embryonic cells. Transitory pluripotent cells can be captured at different time points during embryogenesis and maintained as embryonic stem cells or epiblast stem cells in culture. Since ontogenesis is a dynamic process in both space and time, it seems counterintuitive that these two temporal states represent the full spectrum of organismal pluripotency. Here we show that by modulating culture parameters, a stem-cell type with unique spatial characteristics and distinct molecular and functional features, designated as region-selective pluripotent stem cells (rsPSCs), can be efficiently obtained from mouse embryos and primate pluripotent stem cells, including humans. The ease of culturing and editing the genome of human rsPSCs offers advantages for regenerative medicine applications. The unique ability of human rsPSCs to generate post-implantation interspecies chimaeric embryos may facilitate our understanding of early human development and evolution.

Human pluripotent stem cells (hPSCs) have been perennially paraded as a source of cells for cell replacement therapies because they can (theoretically) give rise to any single cell type within the human body 1. Hence, they can create in vitro a vast number of any human cell type to replace the diseased cell population that a patient might require — this is a salient goal that regenerative medicine aspires to deliver on 2. However, despite the ever-expanding menagerie of therapeutically relevant differentiated lineages being created from hPSCs, usage of these stem cell-derived progeny for regenerative medicine still remains an uncertainty. The question here is whether hPSC lines are faithful to their parental pluripotent antecedents in the embryo from which they were derived. During the enterprise of embryonic development, native pluripotent cells housed within the blastocyst give rise to the entire repertoire of differentiated cell types within the human body that successfully subserve important physiological responsibilities. When put to the test, will hPSCs properly differentiate in vitro to produce physiologically functional differentiated cell types that are equatable to the bona fide differentiated lineages that we actually find in the human body? Or will they produce differentiated progeny that partially resemble the cells that we see in vivo? Concerns have surfaced — for example, hPSC-derived pancreatic β-cells sometimes fail to secrete insulin in response to glucose, unlike authentic pancreatic β-cells found in vivo3 and hPSC-generated hematopoietic stem cells rarely contribute to the hematopoietic systems of recipient animals 4 — which potentially disqualify usage of these cells to treat diabetes or hematological deficiencies. In a recent paper published in Cell Research, Patterson et al. investigate the fidelity of differentiated cell types created from hPSCs — were they made in the likeness of actual bodily cells found in the human embryo? To this end, the authors differentiated hPSCs into cell types representative of the three fetal germ layers from which all of the embryo proper arises — neural progenitors (definitive ectoderm), fibroblasts (mesoderm), and hepatocytes (definitive endoderm) — and then they used microarrays to interrogate whether hPSC-derived cell types were transcriptionally similar to cells from the same lineage taken from actual human fetuses (Figure 1) 5. While pervasive transcriptional congruities were found between fetus-derived and hESC/hiPSC-derived cell types, some striking differences were reported — around ∼10% of assayed genes were differentially expressed between embryo-derived and hPSC-derived cells (Figure 1). Moreover, no matter if hESCs or hiPSCs were specified into neural progenitors, fibroblasts, or hepatocytes, their resultant differentiated offspring continued to express pluripotency-associated genes LIN28A, LIN28B, and DPPA4, amongst others (Figure 1). Rarely if ever did genuine fetus-derived differentiated cells express any of these three pluripotency markers — hPSC-derived cells generally expressed these genes thousands of times higher than their embryonic counterparts. It is possible that some malingering undifferentiated hPSCs persisted throughout the differentiation protocols, consequently contributing to the elevated expression of these pluripotency genes in hPSC-derived lineages. Whatever the reason, enduring expression of pluripotency genes in stem cell-derived progeny is translationally troublesome; for example, one notes that expression of LIN28 in differentiated cells is tumorigenic 6. Might hPSC-derived differentiated cells be prone to oncogenic subversion? If so, this could indeed pose a serious impediment to the exploitation of differentiated derivatives of hPSCs for cell-based therapies in human patients. Figure 1 To assess the clinical fitness of differentiated cell types generated from human pluripotent stem cells (hPSCs; both embryonic stem cells and induced pluripotent stem cells), Patterson et al. systematically differentiated hPSCs into neural progenitors, ... In light of their findings that hPSC-derived cells were similar but not identical to bona fide embryo-derived cells, the authors posited that these discrepancies might be an issue of “developmental maturation” — the immediate differentiated progeny of hESCs and hiPSCs might resemble very early differentiated cell types found in the nascent fetus, not more matured differentiated cell types found at later developmental stages. Indeed, this appeared to be partially the case — while hPSC-derived neural progenitors differentially expressed ∼10% of assayed genes as compared to neural progenitors taken from week 16 human fetal spinal cord, the number of differentially expressed genes was halved when they were compared against spinal cord progenitors sourced from weeks 6.5-8 of fetal development (although a subset of genes remained to be differentially expressed regardless of what week of fetal development was chosen). Based on these findings, Patterson et al. advance that hPSC-derived lineages might resemble the respective cell types taken from human fetuses prior to developmental week 6. The main import to be abstracted from the findings of Patterson et al. is that hPSC-derived differentiated cell types transcriptionally resemble but are not identical to their embryonic counterparts; and also those hPSC-derived lineages continue to aberrantly express select pluripotency genes. The reasons for these transcriptional incongruities are unclear — for example, we have now become cognizant of the fact that hPSCs in vitro differentially express a large number of genes ( > 1 700) as compared to native pluripotent cells found in human blastocysts 7. These precocious differences between hPSCs and authentic embryonic pluripotent cells might have contributed to the differences in gene expression seen in their differentiated endpoints — if there are already transcriptional issues with the hPSCs that we are starting out with altogether in the beginning of these in vitro differentiation regimens. Or perhaps hPSCs are not in themselves defective, but our current differentiation procedures are yet to be fully optimized and do not completely recapitulate developmental cell type specification programs found in the embryo, yielding “incompletely programmed” differentiated cell types that are similar yet different from fetal cells 9. What do these findings portend for the usage of hPSCs for regenerative medicine? Are transcriptional incongruences between hPSC-derived and fetal cells causes for concern? One utilitarian argument to be waged is that it is inconsequential that hPSC-derived progeny are transcriptionally divergent from their in vivo counterparts — as long as they can physiologically perform. This also invokes another challenge. The apparent “developmental naivety" of hPSC-derived cells also remains an important clinical concern — to frame it in the above example of β-cells; adult β-cells secrete insulin in response to glucose, but fetal β-cells do not 3. Clearly, should hPSCs be capable of differentiating into cell types similar to those found in the fetus, procedures must be elaborated to “mature” these cells into adult-like cells with the appropriate physiological functionalities that are appropriate to transplant into diseased adult patients 8. Nevertheless, in any event, this present report by Patterson et al. is timely, beckoning concern in regards to the filial piety of hPSCs and their differentiated offspring. This is the first experimental demonstration that there are clear transcriptional differences between multiple hPSC-derived cell types and their in vivo counterparts. Perhaps further studies delving into their epigenetic differences might yield interesting insights as well. More importantly, whether these cells are still physiologically functional enough to be therapeutically exploited or the differentiation procedures need be improved are the questions now.

Pluripotent cells in embryos are situated near the apex of the hierarchy of developmental potential. They are capable of generating all cell types of the mammalian body proper. Therefore, they are the exemplar of stem cells. In vivo, pluripotent cells exist transiently and become expended within a few days of their establishment. Yet, when explanted into artificial culture conditions, they can be indefinitely propagated in vitro as pluripotent stem cell lines. A host of transcription factors and regulatory genes are now known to underpin the pluripotent state. Nonetheless, how pluripotent cells are equipped with their vast multilineage differentiation potential remains elusive. Consensus holds that pluripotency transcription factors prevent differentiation by inhibiting the expression of differentiation genes. However, this does not explain the developmental potential of pluripotent cells. We have presented another emergent perspective, namely, that pluripotency factors function as lineage specifiers that enable pluripotent cells to differentiate into specific lineages, therefore endowing pluripotent cells with their multilineage potential. Here we provide a comprehensive overview of the developmental biology, transcription factors, and extrinsic signaling associated with pluripotent cells, and their accompanying subtypes, in vitro heterogeneity and chromatin states. Although much has been learned since the appreciation of mammalian pluripotency in the 1950s and the derivation of embryonic stem cell lines in 1981, we will specifically emphasize what currently remains unclear. However, the view that pluripotency factors capacitate differentiation, recently corroborated by experimental evidence, might perhaps address the long-standing question of how pluripotent cells are endowed with their multilineage differentiation potential.

Stem cells fuel tissue development, renewal, and regeneration, and these activities are controlled by the local stem cell microenvironment, the "niche." Wnt signals emanating from the niche can act as self-renewal factors for stem cells in multiple mammalian tissues. Wnt proteins are lipid-modified, which constrains them to act as short-range cellular signals. The locality of Wnt signaling dictates that stem cells exiting the Wnt signaling domain differentiate, spatially delimiting the niche in certain tissues. In some instances, stem cells may act as or generate their own niche, enabling the self-organization of patterned tissues. In this Review, we discuss the various ways by which Wnt operates in stem cell control and, in doing so, identify an integral program for tissue renewal and regeneration.

BACKGROUND In adult mammalian organisms, multiple tissues—including the skin, blood, stomach, and intestines—are entrapped in a state of permanent regeneration; older cells are constantly shed, and the tissue is continuously being regenerated from resident stem cells. This phenomenon of “tissue renewal” was appreciated by Leblond in 1956, but the underlying mechanism has been unclear. It is now evident that a class of extracellular developmental signaling proteins, known as Wnt signals, animate the continued renewal of several mammalian tissues by fuelling stem cell activity. If the Wnt pathway is inhibited, tissue renewal is crippled. This signaling pathway is an ancient evolutionary program dating from when Wnt signals arose in the simplest multicellular organisms, in which Wnts acted as primordial symmetry-breaking signals crucial for the generation of patterned tissues during embryogenesis. In vertebrates, these signals also function in pattern maintenance: They sustain tissue renewal, enabling tissues to be continuously replenished and maintained over a lifetime. Multiple adult organs are in a state of continual regeneration. In tissues such as the skin, intestines, brain, and mammary glands, Wnt signaling proteins sustain this constant regeneration by inducing stem cells (green cells in the illustration) to grow. This leads to the robust supply of new cells (green) in order to replenish and maintain the tissue. [Image credits are available in the full article online.] ADVANCES In contrast to traditional “long-range” developmental signals, Wnts seem to act as short-range intercellular signals—acting mostly between adjacent cells. Lending credence to this notion, a membrane-tethered Wnt protein variant can fulfill most functions of a normal Wnt protein in Drosophila . Likely explaining the short-range nature of these signals, Wnt proteins are attached to a lipid and therefore are hydrophobic; they cannot freely traverse the extracellular space by themselves. This provides insight into how tissue renewal is regulated. It implies that Wnt signals emanating from the stem cell microenvironment (the “niche”) may influence adjacent stem cells without affecting a broad field of cells located farther away. The concept of an external niche, however, may have to be refined because it is clear that stem cells can sometimes act as their own niche and have unexpected developmental self-organizing capacities. Last, the widespread importance of Wnt signaling in driving tissue renewal has been revealed by the identification of Axin2 and Lgr5 , genes expressed in cells that are responding to Wnt signals. Genetically labeling Axin2 + or Lgr5 + cells in a variety of tissues has revealed that such cells fuel tissue renewal in the intestines, mammary gland, skin, and brain, among other organs. OUTLOOK The amazing continuous self-regeneration of various mammalian tissues over years and decades continues to be an enigmatic terra incognita in biology. For instance, visualization of stem cells in real-time in vivo (through intravital microscopy) has shown that when some stem cells are ablated, they are replaced by more differentiated cells that are recalled to the stem cell niche, whereupon they regain stem cell identity to effect tissue repair. Therefore, lineage barriers between stem cell and differentiated fates are not always stringent and can be traversed during times of tissue damage. Reactivated Wnt signals may be instrumental in this process, and perhaps such signals could be exploited in order to enkindle tissue regeneration after injury or disease. From a pragmatic perspective, Wnt signals have already found practical use in manipulating stem cells, enabling propagation of stem cells in vitro as self-renewing cell populations and as organoids.