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Summary A library of well-characterized human induced pluripotent stem cell (hiPSC) lines from clinically healthy human subjects could serve as a useful resource of normal controls for in vitro human development, disease modeling, genotype-phenotype association studies, and drug response evaluation. We report generation and extensive characterization of a gender-balanced, racially/ethnically diverse library of hiPSC lines from 40 clinically healthy human individuals who range in age from 22 to 61 years. The hiPSCs match the karyotype and short tandem repeat identities of their parental fibroblasts, and have a transcription profile characteristic of pluripotent stem cells. We provide whole-genome sequencing data for one hiPSC clone from each individual, genomic ancestry determination, and analysis of mendelian disease genes and risks. We document similar transcriptomic profiles, single-cell RNA-sequencing-derived cell clusters, and physiology of cardiomyocytes differentiated from multiple independent hiPSC lines. This extensive characterization makes this hiPSC library a valuable resource for many studies on human biology., Highlights • A library of induced pluripotent stem cells from 40 healthy human subjects • Racially/ethnically diverse subjects of clinically well-characterized health • Whole-genome sequencing identifies variants of mild common phenotypes or incomplete penetrance • Similar physiology of cardiomyocytes from independent hiPSC clones and individuals, In this resource, Schaniel et al. report the generation and characterization of a gender-balanced, racially/ethnically diverse library of hiPSC lines from 40 clinically well-characterized healthy human individuals. The authors provide whole-genome sequencing data, ancestry determination, and analysis of mendelian disease genes and risks. Furthermore, similar physiology of cardiomyocytes differentiated from independent hiPSC clones derived from two individuals is documented.

Insulin resistance (IR) precedes the development of type 2 diabetes (T2D) and increases cardiovascular disease risk. Although genome wide association studies (GWAS) have uncovered new loci associated with T2D, their contribution to explain the mechanisms leading to decreased insulin sensitivity has been very limited. Thus, new approaches are necessary to explore the genetic architecture of insulin resistance. To that end, we generated an iPSC library across the spectrum of insulin sensitivity in humans. RNA-seq based analysis of 310 induced pluripotent stem cell (iPSC) clones derived from 100 individuals allowed us to identify differentially expressed genes between insulin resistant and sensitive iPSC lines. Analysis of the co-expression architecture uncovered several insulin sensitivity-relevant gene sub-networks, and predictive network modeling identified a set of key driver genes that regulate these co-expression modules. Functional validation in human adipocytes and skeletal muscle cells (SKMCs) confirmed the relevance of the key driver candidate genes for insulin responsiveness., Author summary Insulin resistance is characterized by a defective response (“resistance”) to normal insulin concentrations to uptake the glucose present in the blood, and is the underlying condition that leads to type 2 diabetes (T2D) and increases the risk of cardiovascular disease. It is estimated that 25–33% of the US population are insulin resistant enough to be at risk of serious clinical consequences. For more than a decade, large population studies have tried to discover the genes that participate in the development of insulin resistance, but without much success. It is now increasingly clear that the complex genetic nature of insulin resistance requires novel approaches centered in patient specific cellular models. To fill this gap, we have generated an induced pluripotent stem cell (iPSC) library from individuals with accurate measurements of insulin sensitivity, and performed gene expression and key driver analyses. Our work demonstrates that iPSCs can be used as a revolutionary technology to model insulin resistance and to discover key genetic drivers. Moreover, they can develop our basic knowledge of the disease, and are ultimately expected to increase the therapeutic targets to treat insulin resistance and type 2 diabetes.

Mosaicism due to somatic mutations can cause multiple diseases including cancer, developmental and overgrowth syndromes, neurodevelopmental disorders, autoinflammatory diseases, and atrial fibrillation. With the increased use of next generation sequencing technology, multiple tools have been developed to identify low-frequency variants, specifically from matched tumor-normal tissues in cancer studies. To investigate whether mosaic variants are implicated in congenital heart disease (CHD), we developed a pipeline using the cancer somatic variant caller MuTect to identify mosaic variants in whole-exome sequencing (WES) data from a cohort of parent/affected child trios (n = 715) and a cohort of healthy individuals (n = 416). This is a novel application of the somatic variant caller designed for cancer to WES trio data. We identified two cases with mosaic KMT2D mutations that are likely pathogenic for CHD, but conclude that, overall, mosaicism detectable in peripheral blood or saliva does not account for a significant portion of CHD etiology.

For effective treatment, induced pluripotent stem cell (iPSC)-retinal pigment epithelium (RPE) must recapitulate the physiology of native human RPE cells. A set of physiologically relevant functional assays that assess the polarized functional activity and maturation state of the intact RPE monolayer is provided. The study data show that donor-to-donor variability exceeds the tissue-to-tissue variability for a given donor and provides, for the first time, criteria necessary to identify iPSC-RPE cells most suitable for clinical application., Induced pluripotent stem cells (iPSCs) can be efficiently differentiated into retinal pigment epithelium (RPE), offering the possibility of autologous cell replacement therapy for retinal degeneration stemming from RPE loss. The generation and maintenance of epithelial apical-basolateral polarity is fundamental for iPSC-derived RPE (iPSC-RPE) to recapitulate native RPE structure and function. Presently, no criteria have been established to determine clonal or donor based heterogeneity in the polarization and maturation state of iPSC-RPE. We provide an unbiased structural, molecular, and physiological evaluation of 15 iPSC-RPE that have been derived from distinct tissues from several different donors. We assessed the intact RPE monolayer in terms of an ATP-dependent signaling pathway that drives critical aspects of RPE function, including calcium and electrophysiological responses, as well as steady-state fluid transport. These responses have key in vivo counterparts that together help determine the homeostasis of the distal retina. We characterized the donor and clonal variation and found that iPSC-RPE function was more significantly affected by the genetic differences between different donors than the epigenetic differences associated with different starting tissues. This study provides a reference dataset to authenticate genetically diverse iPSC-RPE derived for clinical applications. Significance The retinal pigment epithelium (RPE) is essential for maintaining visual function. RPE derived from human induced pluripotent stem cells (iPSC-RPE) offer a promising cell-based transplantation therapy for slowing or rescuing RPE-induced visual function loss. For effective treatment, iPSC-RPE must recapitulate the physiology of native human RPE. A set of physiologically relevant functional assays are provided that assess the polarized functional activity and maturation state of the intact RPE monolayer. The present data show that donor-to-donor variability exceeds the tissue-to-tissue variability for a given donor and provides, for the first time, criteria necessary to identify iPSC-RPE most suitable for clinical application.

Hemoglobin production during mammalian development is characterized by temporal switches of the genes coding for the α- and ß-globin chains. Defects in this controlled process can lead to hemoglobinapathies such as sickle cell disease and ß-thalassemia. The ability of human embryonic stem cells (hESC) to proceed through hematopoiesis could provide a clinically useful source of red blood cells. However, hESC-derived red cells exhibit an embryonic/fetal, but not adult, mode of hemoglobin expression. The resource described here is a hESC line engineered to express a reporter from its adult globin promoter, providing a screening platform for small molecules that lead to efficient induction of adult globin.

Background Human-induced pluripotent stem cells (hiPSCs) show a great promise as a renewable source of cells with broad biomedical applications. Since insulin has been used in the maintenance of hiPSCs, in this study we explored the role of insulin in culture of these cells. Methods We report conditions for insulin starvation and stimulation of hiPSCs. Crystal violet staining was used to study the adhesion and proliferation of hiPSCs. Apoptosis and cell cycle assays were performed through flow cytometry. Protein arrays were used to confirm phosphorylation targets, and mRNA sequencing was used to evaluate the effect of transcriptome. Results Insulin improved the seeding and proliferation of hiPSCs. We also observed an altered cell cycle profile and increase in apoptosis in hiPSCs in the absence of insulin. Furthermore, we confirmed phosphorylation of key components of insulin signaling pathway in the presence of insulin and demonstrated the significant effect of insulin on regulation of the mRNA transcriptome of hiPSCs. Conclusion Insulin is a major regulator of seeding, proliferation, phosphorylation and mRNA transcriptome in hiPSCs. Collectively, our work furthers our understanding of human pluripotency and paves the way for future studies that use hiPSCs for modeling genetic ailments affecting insulin signaling pathways. Electronic supplementary material The online version of this article (10.1186/s13287-019-1319-5) contains supplementary material, which is available to authorized users.

Research in photobiology is currently limited by a lack of devices capable of delivering precise and tunable irradiation to cells in a high-throughput format. This limits researchers to using expensive commercially available or custom-built light sources which make it difficult to replicate, standardize, optimize, and scale experiments. Here we present an open-source Microplate Photoirradiation System (MPS) developed to enable high-throughput light experiments in standard 96 and 24-well microplates for a variety of applications in photobiology research. This open-source system features 96 independently controlled LEDs (4 LEDs per well in 24-well), Wi-Fi connected control and programmable graphical user interface (GUI) for control and programming, automated calibration GUI, and modular control and LED boards for maximum flexibility. A web-based GUI generates light program files containing irradiation parameters for groups of LEDs. These parameters are then uploaded wirelessly, stored and used on the MPS to run photoirradiation experiments inside any incubator. A rapid and semi-quantitative porphyrin metabolism assay was also developed to validate the system in wild-type fibroblasts. Protoporphyrin IX (PpIX) fluorescence accumulation was induced by incubation with 5-aminolevulinic acid (ALA), a photosensitization method leveraged clinically to destroy malignant cell types in a process termed photodynamic therapy (PDT), and cells were irradiated with 405nm light with varying irradiance, duration and pulsation parameters. Immediately after light treatment with the MPS, subsequent photobleaching was measured in live, adherent cells in both 96-well and a 24-well microplates using a microplate reader. Results demonstrate the utility and reliability of the Microplate Photoirradiation System to irradiate cells with precise irradiance and timing parameters in order to measure PpIx photobleaching kinetics in live adherent cells and perform comparable experiments with both 24 and 96 well microplate formats. The high-throughput capability of the MPS enabled measurement of enough irradiance conditions in a single microplate to fit PpIX fluorescence to a bioexponential decay model of photobleaching, as well as reveal a dependency of photobleaching on duty-cycle-but not frequency-in a pulsed irradiance regimen. We thank the Graduate School of Biological Sciences and the Black Family Stem Cell Institute at Icahn School of Medicine for providing financial support for the project. Chris Merck is affiliated with his own LLC (Merck Engineering LLC). Merck Engineering LLC did not contribute funding to the development of the MPS or its biological validation and has no pecuniary interest in this study. Chris Merck worked as a volunteer collaborator not representing any company or institution. Merck Engineering LLC did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific role of this author is articulated in the 'author contributions' section.

Variability in induced pluripotent stem cell (iPSC) lines remains a concern for disease modeling and regenerative medicine. We have used RNA-sequencing analysis and linear mixed models to examine the sources of gene expression variability in 317 human iPSC lines from 101 individuals. We found that ∼50% of genome-wide expression variability is explained by variation across individuals and identified a set of expression quantitative trait loci that contribute to this variation. These analyses coupled with allele-specific expression show that iPSCs retain a donor-specific gene expression pattern. Network, pathway, and key driver analyses showed that Polycomb targets contribute significantly to the non-genetic variability seen within and across individuals, highlighting this chromatin regulator as a likely source of reprogramming-based variability. Our findings therefore shed light on variation between iPSC lines and illustrate the potential for our dataset and other similar large-scale analyses to identify underlying drivers relevant to iPSC applications.

Induced pluripotent stem cell (iPSC) therapeutics are a promising treatment for genetic and infectious diseases. To assess engraftment, risk of neoplastic formation, and therapeutic benefit in an autologous setting, testing iPSC therapeutics in an appropriate model, such as the pigtail macaque (Macaca nemestrina; Mn), is crucial. Here, we developed a chemically defined, scalable, and reproducible specification protocol with bone morphogenetic protein 4, prostaglandin-E2 (PGE2), and StemRegenin 1 (SR1) for hematopoietic differentiation of Mn iPSCs. Sequential coculture with bone morphogenetic protein 4, PGE2, and SR1 led to robust Mn iPSC hematopoietic progenitor cell formation. The combination of PGE2 and SR1 increased CD34+CD38−Thy1+CD45RA−CD49f+ cell yield by 6-fold. CD34+CD38−Thy1+CD45RA−CD49f+ cells isolated on the basis of CD34 expression and cultured in SR1 expanded 3-fold and maintained this long-term repopulating HSC phenotype. Purified CD34high cells exhibited 4-fold greater hematopoietic colony-forming potential compared with unsorted hematopoietic progenitors and had bilineage differentiation potential. On the basis of these studies, we calculated the cell yields that must be achieved at each stage to meet a threshold CD34+ cell dose that is required for engraftment in the pigtail macaque. Our protocol will support scale-up and testing of iPSC-derived CD34high cell therapies in a clinically relevant nonhuman primate model.

Human high-grade gliomas (hHGG) remain a therapeutic challenge in neuro-oncology despite current multimodality treatments. We recently demonstrated that murine embryonic stem cell (mESC)-derived astrocytes conditionally expressing proapoptotic genes can successfully be used to induce apoptosis and tumor shrinkage of hHGG tumor in vitro and in an in vivo mouse model. The first step in the translation of these results to the clinical settings, however, requires availability of human embryonic stem cells (hESC)- and/or induced pluripotent cell (hiPSC)-derived astrocytes engineered to express proapoptotic genes. The potential for directed differentiation of hESCs and hiPSCs to functional postmitotic astrocytes is not fully characterized. In this study, we show that once specified to neuro-epithelial lineage, hiPSC could be differentiated to astrocytes with a similar efficiency as hESC. However, our analyses of 2 hESC and 2 hiPSC cell lines showed some variability in differentiation potential into astrocytic lineages. Both the hESC- and hiPSC-derived astrocytes appeared to follow the functional properties of mESC-derived astrocytes, namely, migration and tropism for hHGG. This work provides evidence that hESC- and hiPSC-derived cells are able to generate functionally active astrocytes. These results demonstrate the feasibility of using iPSC-derived astrocytes, a new potential source for therapeutic use for brain tumors and other neurological diseases.

Directed differentiation of human embryonic stem (hES) cells and human induced pluripotent stem (hiPS) cells captures in vivo developmental pathways for specifying lineages in vitro, thus avoiding perturbation of the genome with exogenous genetic material. Thus far, derivation of endodermal lineages has focused predominantly on hepatocytes, pancreatic endocrine cells and intestinal cells1–5. The ability to differentiate pluripotent cells into anterior foregut endoderm (AFE) derivatives would expand their utility for cell therapy and basic research to tissues important for immune function, such as the thymus; for metabolism, such as thyroid and parathyroid; and for respiratory function, such as trachea and lung. We find that dual inhibition of transforming growth factor (TGF)-β and bone morphogenic protein (BMP) signaling after specification of definitive endoderm from pluripotent cells results in a highly enriched AFE population that is competent to be patterned along dorsoventral and anteroposterior axes. These findings provide an approach for the generation of AFE derivatives.

The generation of reprogrammed induced pluripotent stem cells (iPSCs) from patients with defined genetic disorders holds the promise of increased understanding of the aetiologies of complex diseases and may also facilitate the development of novel therapeutic interventions. We have generated iPSCs from patients with LEOPARD syndrome (an acronym formed from its main features; that is, lentigines, electrocardiographic abnormalities, ocular hypertelorism, pulmonary valve stenosis, abnormal genitalia, retardation of growth and deafness), an autosomal-dominant developmental disorder belonging to a relatively prevalent class of inherited RAS-mitogen-activated protein kinase signalling diseases, which also includes Noonan syndrome, with pleomorphic effects on several tissues and organ systems. The patient-derived cells have a mutation in the PTPN11 gene, which encodes the SHP2 phosphatase. The iPSCs have been extensively characterized and produce multiple differentiated cell lineages. A major disease phenotype in patients with LEOPARD syndrome is hypertrophic cardiomyopathy. We show that in vitro-derived cardiomyocytes from LEOPARD syndrome iPSCs are larger, have a higher degree of sarcomeric organization and preferential localization of NFATC4 in the nucleus when compared with cardiomyocytes derived from human embryonic stem cells or wild-type iPSCs derived from a healthy brother of one of the LEOPARD syndrome patients. These features correlate with a potential hypertrophic state. We also provide molecular insights into signalling pathways that may promote the disease phenotype.

Lung and airway epithelial cells generated in vitro from human pluripotent stem cells (hPSCs) have applications in regenerative medicine, modeling of lung disease, drug screening and studies of human lung development. Here we describe a strategy for directed differentiation of hPSCs into developmental lung progenitors, and their subsequent differentiation into predominantly distal lung epithelial cells. The protocol entails four stages that recapitulate lung development, and it takes ∼50 d. First, definitive endoderm (DE) is induced in the presence of high concentrations of activin A. Subsequently, lung-biased anterior foregut endoderm (AFE) is specified by sequential inhibition of bone morphogenetic protein (BMP), transforming growth factor-β (TGF-β) and Wnt signaling. AFE is then ventralized by applying Wnt, BMP, fibroblast growth factor (FGF) and retinoic acid (RA) signaling to obtain lung and airway progenitors. Finally, these are further differentiated into more mature epithelial cells types using Wnt, FGF, cAMP and glucocorticoid agonism. This protocol is conducted in defined conditions, it does not involve genetic manipulation of the cells and it results in cultures in which the majority of the cells express markers of various lung and airway epithelial cells, with a predominance of cells identifiable as functional type II alveolar epithelial cells.

In this report, we have defined the stage at which Scl functions in the establishment of the hematopoietic system and provide evidence that its primary role is in the generation of the hematopoietic lineages from a progenitor called the blast colony-forming cell (BL-CFC), a cell considered to be the in vitro equivalent of the hemangioblast. Using an embryonic stem (ES) cell line in which lacZ cDNA has been targeted to the Scl locus, we show that most of the BL-CFCs are detected in the SCL/lacZ- population, indicating that this progenitor does not express Scl. In the blast colony assay, Scl-/- cells initiate colony growth but are unable to generate endothelial and hematopoietic progeny and thus form colonies consisting of vascular smooth muscle cells only. The capacity to give rise to blast colonies can be rescued by retroviral transduction of a wild-type Scl gene into Scl-/- FLK-1+ cells, suggesting that the BL-CFC is generated in this population. Finally, we show that Scl-/- endothelial cells display a growth deficiency in monolayer cultures that can be partially overcome by maintaining this population as 3-dimensional aggregates indicating that specific cellular interactions are required for maintenance of the Scl-/- endothelial lineage in vitro.

The study of complex cardiovascular disease (CVD) has been hampered by the lack of appropriate human cellular model systems. In response, the NHBLI sponsored the NextGen Consortium, which encompasses 9 independent efforts spanning the portfolio of NHLBI related phenotypes. The goals of the consortium include: 1. To develop and improve methods for large-scale production and characterization of induced pluripotent stem cell (iPSC) models for CVD; 2. To create a resource of iPSC lines from a large number of phenotypically and genotypically characterized individuals. Our GENESiPS project is focused on insulin resistance (IR), a condition that affects 25-33% of the US population with serious health consequences including risk of type II diabetes and CVD. Although much is known about the physiological changes occurring during IR, little is known about the molecular pathways that drive the appearance of IR. Certain mature cell types as adipocytes, endothelial cells and skeletal muscle cells have been associated with the origin, maintenance and progression of IR. IPSCs offer an unprecedented opportunity of modeling human disease in vitro. We have created iPSC lines on insulin resistant and insulin sensitive patient groups with prior GWAS genotyping. Differentiation of these iPSCs to relevant cell types is providing the opportunity to correlate insulin sensitivity and high-density genetic variation data with specific cell-based profiling. We will validate our in vitro model and study the molecular pathways that define IR and its relationship to endothelial dysfunction. Relevant to the larger scientific community the establishment of iPSC lines on over 150 individuals (3 to 6 clones per patient) that reflect the range of insulin resistance in the general population. The iPSC lines were created from erythroblasts using the non-integrative Sendai virus system, passaged to allow clearance of Sendai virus and growth in feeder free conditions. The lines have been extensively characterized for markers of pluripotency (Tra1-60), sample identity and genomic integrity. Through the NextGen consortium, these lines, as well as phenotypic and genome-wide genotyping data will be available to qualified investigators.

Cord blood (CB) cells that express CD34 have extensive hematopoietic capacity and rapidly divide ex vivo in the presence of cytokine combinations; however, many of these CB CD34+ cells lose their marrow-repopulating potential. To overcome this decline in function, we treated dividing CB CD34+ cells ex vivo with several histone deacetylase inhibitors (HDACIs). Treatment of CB CD34+ cells with the most active HDACI, valproic acid (VPA), following an initial 16-hour cytokine priming, increased the number of multipotent cells (CD34+CD90+) generated; however, the degree of expansion was substantially greater in the presence of both VPA and cytokines for a full 7 days. Treated CD34+ cells were characterized based on the upregulation of pluripotency genes, increased aldehyde dehydrogenase activity, and enhanced expression of CD90, c-Kit (CD117), integrin α6 (CD49f), and CXCR4 (CD184). Furthermore, siRNA-mediated inhibition of pluripotency gene expression reduced the generation of CD34+CD90+ cells by 89%. Compared with CB CD34+ cells, VPA-treated CD34+ cells produced a greater number of SCID-repopulating cells and established multilineage hematopoiesis in primary and secondary immune-deficient recipient mice. These data indicate that dividing CB CD34+ cells can be epigenetically reprogrammed by treatment with VPA so as to generate greater numbers of functional CB stem cells for use as transplantation grafts.

The use of human stem cell-derived cardiomyocytes to study atrial biology and disease has been restricted by the lack of a reliable method for stem cell-derived atrial cell labeling and purification. The goal of this study was to generate an atrial-specific reporter construct to identify and purify human stem cell-derived atrial-like cardiomyocytes. We have created a bacterial artificial chromosome (BAC) reporter construct in which fluorescence is driven by expression of the atrial-specific gene sarcolipin (SLN). When purified using flow cytometry, cells with high fluorescence specifically express atrial genes and display functional calcium handling and electrophysiological properties consistent with atrial cardiomyocytes. Our data indicate that SLN can be used as a marker to successfully monitor and isolate hiPSC-derived atrial-like cardiomyocytes. These purified cells may find many applications, including in the study of atrial-specific pathologies and chamber-specific lineage development.

The aim of the study was to ascertain whether Ce, a lanthanide that has been implicated in the pathogenesis of tropical endomyocardial fibrosis, interferes with the biosynthetic repertoire of the cardiac muscle in vivo. Female Sprague-Dawley rats received Ce chloride iv at 1.3 mg/kg body wt.; controls received an equal volume of physiological saline. Rates of protein synthesis and transcription in cardiac muscle, measured in terms of incorporation of (3H)-phenylalanine and (3H)-uridine, respectively, into trichloroacetic acid-insoluble material were found to be significantly higher in Ce-treated animals. As low levels of Ce were earlier shown to stimulate collagen as well as noncollagen protein synthesis in cardiac fibroblasts in vitro, the stimulatory effect of the element in vivo reported here supports the speculation that it may influence the expression of proteins like collagen in the heart and contribute to their accumulation as in endomyocardial fibrosis.

The breakthrough development of induced pluripotent stem cell (iPSC) technology is not only revolutionizing basic stem cell science but is also spurring efforts to reprogram one somatic cell type directly into another. Induced pluripotent stem cells provide scientists with a self-renewing and, thus, unlimited, source of pluripotent cells for targeted differentiation, in principle, into the entire range of cell types found in the body. Therefore, iPSC technology and the increasingly refined abilities to differentiate iPSCs into disease-relevant mature cells has far reaching implications for understanding disease etiology and promoting drug discovery and other advances in regenerative medicine. In this review, we summarize the latest progress in the application of patient-specific iPSCs for disease modeling, drug screening and cell replacement therapy, and discuss their impact on precision personalized medicine.

The onset of hematopoiesis in the mouse embryo and in the embryonic stem (ES) cell differentiation model is defined by the emergence of the hemangioblast, a progenitor with both hematopoietic and vascular potential. While there is evidence for the existence of a hemangioblast in the mouse, it is unclear if this progenitor develops during the establishment of the human hematopoietic system. In this report, we have mapped hematopoietic development in human ES cell (hESC) differentiation cultures and demonstrated that a comparable hemangioblast population exists. The human hemangioblasts were identified by their capacity to generate blast colonies that display both hematopoietic and vascular potential. These colony-forming cells express the receptor tyrosine kinase KDR (VEGF receptor 2) and represent a transient population that develops in BMP-4–stimulated embryoid bodies (EBs) between 72 and 96 hours of differentiation, prior to the onset of the primitive erythroid program. Two distinct types of hemangioblasts were identified, those that give rise to primitive erythroid cells, macrophages, and endothelial cells and those that generate only the primitive erythroid population and endothelial cells. These findings demonstrate for the first time the existence of the human hemangioblast and in doing so identify the earliest stage of hematopoietic commitment.