Abstract

Significant advances in cellular reprogramming technologies and hematopoietic differentiation from human pluripotent stem cells (hPSCs) have already enabled the routine production of multiple lineages of blood cells in vitro and opened novel opportunities to study hematopoietic development, model genetic blood diseases, and manufacture immunologically matched cells for transfusion and cancer immunotherapy. However, the generation of hematopoietic cells with robust and sustained multilineage engraftment has not been achieved. Here, we highlight the recent advances in understanding the molecular and cellular pathways leading to blood development from hPSCs and discuss potential approaches that can be taken to facilitate the development of technologies for de novo production of hematopoietic stem cells.

Introduction

Pluripotent stem cells (PSCs) are defined as cells capable of self-renewal and differentiation into derivatives of all 3 germ layers. The first successful derivation of human PSCs (hPSCs), embryonic stem cells (hESCs), by James Thomson in 19981  dramatically elevated the interest in PSC biology because many viewed hESCs as a novel unlimited source of human cells for cell replacement therapies, drug screening, and developmental studies. In 2006, advances in understanding of the core transcriptional regulatory circuitry in mouse and human ESCs led to another crucial discovery by Shinya Yamanaka,2  who identified the set of reprogramming factors capable of inducing ESC-like cells (induced PSCs [iPSCs]) from mouse somatic fibroblasts. One year later, iPSCs were obtained from human somatic cells.3-5  Human iPSCs (hiPSCs) offer a novel tool to study and treat diseases because they capture the entire genome of a particular patient and provide an inexhaustible supply of immunologically compatible cells for experimentation and transplantation. Although initially iPSCs were generated from fibroblasts using retroviral vectors, multiple strategies for generating transgene-free iPSCs from fibroblasts and other cell types, including blood, have been developed within a short period (reviewed by Hussein and Nagy6  and Gonzalez et al7 ). With the iPSC field progressing very rapidly, the next challenge will be to demonstrate the functional usefulness of iPSC-derived cells in preclinical models of various human diseases and eventually move this technology into the clinic.

Hematopoietic stem cell (HSC) transplantation has become the standard of care for otherwise incurable blood cancers and deadly genetic diseases. The expansion of HSC donor registries, along with the development of alternative sources for HSC transplantation, including cord blood and haploidentical donors, and the use of novel conditioning regimens have significantly improved access to transplantation for patients with hematologic diseases.8,9  However, transplant engraftment failure, graft-versus-host disease, and delayed reconstitution still remain significant causes of morbidity and mortality after bone marrow transplantation8,9  leaving ∼50% of patients with a permanent disability or without a cure.10  Because iPSCs can be expanded indefinitely ex vivo and potentially differentiated into hematopoietic cells with blood-reconstituting capability,11,12  they open a unique opportunity to improve the outcomes of bone marrow transplantation by providing a supply of unlimited number of immunologically matched HSCs.13,14  Patients with monogenic hematologic and immune diseases would benefit the most from a iPSC-based bone marrow transplantation procedure. Currently, a lack of methodology for efficient expansion and genetic modifications of somatic HSCs and the risk for insertional mutagenesis with viral vectors remain the major limitations for HSC-based gene therapy.15  As shown in Figure 1, autologous iPSC lines can be generated from patients with genetic defects, precisely corrected with the wild-type gene by homologous recombination and then used to produce healthy hematopoietic cells for transplantation without the risk for graft-versus-host disease. The successful treatment of sickle cell anemia in a mouse model using gene-corrected iPSCs provided proof-of-principle that the clinical application of iPSCs to treat geneticblood diseases is feasible.16  In the setting of leukemia, iPSCs can be used to produce immunologically matched HSCs as well as T cells targeted to leukemia antigens and antigen-loaded dendritic cells to induce an anti-leukemia immune response.17,18  In addition, autologous panmyeloid progenitors can be generated form iPSCs19  for the management of cytopenias in patients with delayed engraftment.

Figure 1

Therapeutic potential of hPSCs for blood diseases. iPSCs can be potentially used to treat patients with monogenic genetic blood diseases such as sickle cell anemia, β-thalassemia, Fanconi anemia, or SCID (upper panel). Autologous skin or blood cells from these patients can be reprogrammed into iPSCs. The defective gene in iPSCs can be repaired using homologous recombination. De novo generation of HSCs from gene-corrected iPSCs would provide immunologically matched cells for bone marrow transplantation. For cancer therapy, autologous iPSCs could be generated from skin fibroblasts or other somatic cells lacking leukemia mutation and used to generate HSCs for bone marrow transplantation as well as immune cells to induce an anti-leukemia immune response (lower panel). Professional illustration by Paulette Dennis.

Figure 1

Therapeutic potential of hPSCs for blood diseases. iPSCs can be potentially used to treat patients with monogenic genetic blood diseases such as sickle cell anemia, β-thalassemia, Fanconi anemia, or SCID (upper panel). Autologous skin or blood cells from these patients can be reprogrammed into iPSCs. The defective gene in iPSCs can be repaired using homologous recombination. De novo generation of HSCs from gene-corrected iPSCs would provide immunologically matched cells for bone marrow transplantation. For cancer therapy, autologous iPSCs could be generated from skin fibroblasts or other somatic cells lacking leukemia mutation and used to generate HSCs for bone marrow transplantation as well as immune cells to induce an anti-leukemia immune response (lower panel). Professional illustration by Paulette Dennis.

In recent years, major progress has been made in developing systems for hematopoietic differentiation and producing major types of blood cells from hPSCs (reviewed by Kaufman14 ). However, the generation of hematopoietic cells with robust long-term reconstitution potential from hPSCs remains a significant challenge. The identification of sequential progenitors and molecular mechanisms leading to formation of various blood lineages from hPSCs is critical in overcoming this limitation. In this review, I focus on recent progress made in understanding cellular and molecular pathways leading to hematopoietic specification from hPSCs and discuss key approaches that could be undertaken to induce the formation of engraftable blood cells from hPSCs.

Translating embryonic hematopoiesis to PSC differentiation

Mesodermal development and HSC specification in the embryo

The knowledge gained from studies of embryogenesis and mouse ESC differentiation provided major insights into key pathways that regulate the sequential commitment of PSCs to blood cells and laid the foundation for the development of hematopoietic differentiation protocols for hPSCs. During embryogenesis, gastrulation is the first critical step in specification of pluripotent embryonic cells into blood. The beginning of gastrulation is marked by formation of primitive streak (PS). Epiblast cells ingress into the PS to give rise to the mesoderm and definitive endoderm.20  Although the entire PS expresses the T gene (also known as Brachyury),21  the subset of cells with hematoendothelial potential within the PS can be identified by the expression of inert domain-containing receptor (KDR) (Flk1 or vascular endothelial growth factor [VEGF] receptor 2).22,23  KDR+ cells migrate into the yolk sac, where they form vascular plexus and blood islands. Nodal, bone morphogenetic protein 4 (BMP4), WNT3, and fibroblast growth factor 2 (FGF2) are the most critical factors required for PS and mesoderm induction.24-27  Importantly, FGF2 upregulates expression of KDR on mesodermal precursors and makes them sensitive to VEGF.28  At this stage, the interaction of VEGF-A produced by the visceral endoderm with KDR becomes essential for the normal development of endothelial and blood lineages.23,29  Manipulation of these pathways in mouse ESC cultures helped to optimize mesodermal differentiation and provided additional knowledge regarding molecular regulation of hematopoietic mesoderm (reviewed by Keller30  and Murry and Keller31 ).

In the mouse embryo, the first blood progenitors are formed in the yolk sac where they can be identified using hematopoietic colony-forming assays as early as embryonic day 7.25 (E7.25). The yolk sac initially generates primitive hematopoietic cells, including nucleated red blood cells expressing embryonic hemoglobin, macrophages, and megakaryocytes.32,33  The second wave of yolk sac hematopoiesis, termed definitive erythromyeloid hematopoiesis, emerges when distinct blood islands can be recognized morphologically at E8.25.32,34  This wave is associated with an expansion of erythroid precursors expressing adult β-globins and unilineage and multilineage myeloid precursors.32  The first HSCs capable of reconstituting the entire hematopoietic system of wild-type adult animals are observed in the aorta-gonado-mesonephros (AGM) region, vitelline and umbilical arteries and placenta by E10.5-11.35-40  At E11.5, it is estimated that the AGM region contains ∼1 HSC.41  Emerging HSCs undergo expansion in the fetal liver and subsequently migrate to bone marrow, which becomes the predominant site of hematopoiesis in postnatal life.42 

Hemogenic specification: concept of hemangioblast and hemogenic endothelium

During embryogenesis, endothelial and hematopoietic cells develop in parallel. The close spatial and temporal relationships between these 2 lineages were first noted by early embryologists in the late 19th century.43  In 1917, Florence Sabin postulated the existence of a common precursor for blood and endothelial cells based on her observation of blood development within the yolk sac of chicken embryos.44  This common precursor was later named hemangioblast by Murray, who defined it as aggregates of yolk sac mesenchyme (mesoderm) from which endothelial and hematopoietic cells develop.45  Although the term hemangioblast initially designated the mesodermal precursor, the modern literature applies it very broadly to describe any type of cell that can produce both endothelial and blood cells after culture in specific conditions in vitro. It became clear, however, that cells with hemangioblastic activity represent a very heterogeneous group of progenitors, which include cells at the mesodermal stage of development in the yolk sac and cells with typical endothelial characteristics in the AGM region.46-48 

Within the AGM, hematopoietic cells were found to bud off from the endothelium lining the wall of the aorta.49  Recent studies in mice have provided direct evidence that this process represents the formation of definitive blood cells and HSCs from a unique population of endothelial cells on the ventral wall of the dorsal aorta, defined as hemogenic endothelium (HE), through an endothelial-hematopoietic transition.50-52  Dynamic tracing and imaging studies conducted in vivo have demonstrated that endothelial-hematopoietic transition represents a continuous process in which endothelial cells gradually acquire hematopoietic morphology and phenotype.50,53,54  Although the concept of HE was initially developed based on AGM studies, it became clear that endothelium in other embryonic and extraembryonic sites possess hemogenic potential. Among these sites are the vitelline and umbilical arteries,55  placenta,38  head vasculature,56  endocardium,57  and nascent yolk sac capillaries.58 

Hematopoietic development from hPSCs

In general, the approaches for hESC differentiation into blood cells are similar to those employed for mouse ESCs. The first successful differentiation of hESCs was achieved by Kaufman et al using coculture with the S17 mouse stromal cell line.59  Since then, several embryoid body formation and 2D culture protocols, including serum- and feeder-free, have been developed for hematopoietic differentiation of hESCs.60-66  After successful reprogramming of human somatic cells to pluripotency, hESC protocols have also been applied to differentiate hiPSCs. It has been shown that the patterns of hematopoietic differentiation from hESCs and iPSCs are very similar.66-69  Many factors that are important for hematopoietic specification of mouse PSCs appear to play critical roles in induction of mesoderm and hematopoietic commitment in the hPSCs as well. As expected from murine studies, BMP4, WNT, FGF2, and VEGF have been shown to promote hematopoiesis from hESCs (Figure 2A).60,62,65,70-74  Hematopoietic cytokines are important components of hPSC differentiation systems and are required for amplification of emerging hematopoietic colony-forming cells (CFCs) and specification toward lymphoid lineages. The protocols for hematopoietic differentiation of hPSCs are extensively reviewed by Kardel and Eaves.75  Overall, hESC studies have revealed many similarities in hematopoietic differentiation with mouse ESCs, including requirements for intrinsic and extrinsic signaling and hierarchical organization of hematopoietic precursors. However, several differences have also been noted and will be discussed below.

Figure 2

A model of hematopoietic development from hPSCs. (A) The most critical factors involved in specification of hematovascular precursors from PSCs and regulation of blood formation from HE. (B) Stages of hematopoietic development from hPSCs. Mesodermal stage of development is defined as expression of the mesodermal markers, APLNR and KDR.62,77,80  The lack of expression of typical endothelial (CD31, VE-cadherin), endothelial/mesenchymal (CD73, CD105), and hematopoietic (CD43, CD45) markers, ie, EMHlin phenotype, separates mesoderm from lineage-committed cells.86,90  The most primitive mesodermal precursors with hematopoietic potential arise in coculture with OP9 or an embryoid body system on day 3 of differentiation. These cells have features of a posterior PS, coexpress KDR, APLNR, and PDGFRα and capable of forming BL (hemangioblast) colonies in the presence of FGF2 and VEGF.62,77,80,86  The formation of BL colonies in clonogenic medium proceeds through VE-cadherin+ endothelial intermediates, which generate primitive hematopoietic cells with erythroid, megakaryocytic, and macrophage potentials.77,86  Progressive mesodermal commitment to endothelial and hematopoietic cells is associated with downregulation of PDGFRα71,86  and PS genes, and upregulation of KDR, TAL1, and GATA2 genes associated with angiohematopoietic development leading to formation of EMHlinKDRbrightAPLNR+PDGFRαlow/− hematovascular mesodermal precursors (HVMPs).86  HVMPs lack BL-CFC potential, but are highly enriched in cells that form hematoendothelial clusters on OP9.86  The endothelial stage of development was defined as expression of the typical endothelial markers VE-cadherin, CD31, and CD34 and the absence of the panhematopoietic marker CD43 (supplemental Table 1; see the Blood Web site).66,86,90,101,102  Within the VE-cadherin+CD43 population, HE cells (ie, cells lacking hematopoietic CFC potential but capable of forming blood cells after culture with stromal cells) were discriminated from non-HE cells based on lack of CD73 expression.86,102  The first hematopoietic progenitors emerging from the VE-cadherin+ population express CD235a, low levels of CD43, and lack CD41a expression. These cells have a unique potential to form hematopoietic colonies in the presence of FGF2 and hematopoietic cytokines, but also retain endothelial potential and therefore were designated as angiogenic hematopoietic progenitors (AHPs).86  Advanced hematopoietic development is associated with upregulation of CD43 expression; segregation of all hematopoietic CFCs to the CD43+ fraction66,100,128 ; and establishment of distinct subsets of CD43+ hematopoietic cells, including CD41a+CD235a+ erythro-megakaryocytic progenitors100,115,117  and linCD34+CD43+CD45+/− multipotent myelolymphoid progenitors.66,100,128  Progressive acquisition of the angiogenic and hematopoietic program by differentiated cells is emphasized by green and red colors, respectively.

Figure 2

A model of hematopoietic development from hPSCs. (A) The most critical factors involved in specification of hematovascular precursors from PSCs and regulation of blood formation from HE. (B) Stages of hematopoietic development from hPSCs. Mesodermal stage of development is defined as expression of the mesodermal markers, APLNR and KDR.62,77,80  The lack of expression of typical endothelial (CD31, VE-cadherin), endothelial/mesenchymal (CD73, CD105), and hematopoietic (CD43, CD45) markers, ie, EMHlin phenotype, separates mesoderm from lineage-committed cells.86,90  The most primitive mesodermal precursors with hematopoietic potential arise in coculture with OP9 or an embryoid body system on day 3 of differentiation. These cells have features of a posterior PS, coexpress KDR, APLNR, and PDGFRα and capable of forming BL (hemangioblast) colonies in the presence of FGF2 and VEGF.62,77,80,86  The formation of BL colonies in clonogenic medium proceeds through VE-cadherin+ endothelial intermediates, which generate primitive hematopoietic cells with erythroid, megakaryocytic, and macrophage potentials.77,86  Progressive mesodermal commitment to endothelial and hematopoietic cells is associated with downregulation of PDGFRα71,86  and PS genes, and upregulation of KDR, TAL1, and GATA2 genes associated with angiohematopoietic development leading to formation of EMHlinKDRbrightAPLNR+PDGFRαlow/− hematovascular mesodermal precursors (HVMPs).86  HVMPs lack BL-CFC potential, but are highly enriched in cells that form hematoendothelial clusters on OP9.86  The endothelial stage of development was defined as expression of the typical endothelial markers VE-cadherin, CD31, and CD34 and the absence of the panhematopoietic marker CD43 (supplemental Table 1; see the Blood Web site).66,86,90,101,102  Within the VE-cadherin+CD43 population, HE cells (ie, cells lacking hematopoietic CFC potential but capable of forming blood cells after culture with stromal cells) were discriminated from non-HE cells based on lack of CD73 expression.86,102  The first hematopoietic progenitors emerging from the VE-cadherin+ population express CD235a, low levels of CD43, and lack CD41a expression. These cells have a unique potential to form hematopoietic colonies in the presence of FGF2 and hematopoietic cytokines, but also retain endothelial potential and therefore were designated as angiogenic hematopoietic progenitors (AHPs).86  Advanced hematopoietic development is associated with upregulation of CD43 expression; segregation of all hematopoietic CFCs to the CD43+ fraction66,100,128 ; and establishment of distinct subsets of CD43+ hematopoietic cells, including CD41a+CD235a+ erythro-megakaryocytic progenitors100,115,117  and linCD34+CD43+CD45+/− multipotent myelolymphoid progenitors.66,100,128  Progressive acquisition of the angiogenic and hematopoietic program by differentiated cells is emphasized by green and red colors, respectively.

Mesoderm induction from hPSCs

Understanding the mechanisms regulating induction and specification of mesoderm to hematovascular progenitors is essential in tracing the development of pre-HSCs and in defining factors required for their specification. Similar to mouse ESCs, the early stages of mesodermal development from hESCs can be monitored by expression of KDR (FLK1) and platelet-derived growth factor receptor alpha (PDGFRα).62,70,71,76,77  However, in contrast to mouse ESCs, low levels of KDR can be detected in undifferentiated hESCs.77-79  The successful targeting of green fluorescent protein (GFP) reporter to the locus of MIXL1, a gene transiently expressed in the PS, enabled the more accurate identification and isolation of the mesodermal populations at the PS stage in hESC cultures.76  Molecular profiling of MIXL1-GFP cells or wild-type hESCs at early stages of differentiation revealed apelin receptor (APLNR) as a novel marker of the PS population in hPSC cultures.77,80  APLNR was found to be expressed in cells with features of posterior mesoderm and anterior mesendoderm.77,80  In contrast to KDR, undifferentiated hESCs do not express APLNR, and because APLNR expression is homogeneous, positive and negative populations can be clearly separated by flow cytometry.

Using the MIXL1-GFP cell line, Davis et al76  demonstrated the essential role of BMP4 in induction of PS mesoderm from hESCs. The formation of mesoderm is also dependent on Activin A and FGF signaling.81-83  Inhibition of these pathways using small molecules completely blocks mesoderm development from hESCs.70  Several studies have documented the important role of canonical WNT signaling in blood development from hESC,71,72  which can be at least partially attributed to the enhancement of mesoderm formation after WNT signaling activation. Canonical WNT/β-catenin signaling is required for establishing nascent PS/mesendoderm from hESCs, whereas specification of nascent PS to anterior and posterior PS is regulated by the balance between BMP and Activin/Nodal signaling.84  Furthermore, activation of canonical WNT signaling promotes the development of more mature hematovascular mesoderm expressing a high level of KDR (KDRbright mesoderm) and hematoendothelial progenitors from hESCs.71,72  Vijayaragavan et al reported that mesoderm formation from hESCs is also affected by noncanonical WNT signaling.85 

Hemangioblastic potential of hPSC-derived mesodermal precursors

Similar to mouse ESCs, the onset of hematopoiesis in hESC and hiPSC cultures is marked by the emergence of blast CFCs (BL-CFCs).62,77,80,86  Because BL-CFCs consist of vascular and hematopoietic progenitors, they are commonly referred as hemangioblasts. BL-CFCs are detected at the mesodermal stage of development, before the appearance of hematopoietic progenitors that form colonies in response to hematopoietic cytokines. Development of BL colonies from mesodermal cells requires FGF2 and VEGF but not hematopoietic cytokines.62,77  In defined serum-free clonogenic medium, FGF2 alone is sufficient to induce BL colonies from APLNR+ mesodermal cells.77,86  The formation of BL-CFCs is also promoted by the addition of apelin peptides to differentiation cultures or clonogenic medium.80,86  Similar to findings in mouse systems,87  human hemangioblasts (BL-CFCs) generate hematopoietic colonies through endothelial intermediates.77,86  Using time-lapse studies, we demonstrated that development of BL colonies in clonogenic cultures proceed through a core stage at which highly motile mesodermal cells undergo several divisions, upregulate expression of KDR and other endothelial genes (including CDH5, PECAM, and ESAM), and form immotile tight aggregates composed of ∼30 VE-cadherin+ epithelioid cells (cores). The core stage of differentiation is readily identifiable after 3 days of culture of mesodermal cells in semisolid clonogenic medium. Subsequently, VE-cadherin+ cells undergo endothelial-hematopoietic transition leading to the formation of CD235a+/−/CD41+/− cells with erythroblast morphology. The hematopoietic potential of BL-CFCs is mostly restricted to primitive erythroid and megakaryocytic cells, and macrophages.62,86  Interestingly, the development of BL colonies in culture closely recapitulates events leading to blood formation in vivo. In chicken embryo, FGF produced by endodermal cells induces the aggregation of migrating PS cells adjacent to the endoderm, upregulation of KDR, and formation of angioblasts and hemangioblasts.28,88  In differentiating hPSC cultures (Figure 2A), the BL-CFCs with hemangioblastic activity are highly enriched within the KDR+ and APLNR+PDGFRα+ nascent mesodermal population expressing MIXL1 and other PS genes.62,77,80,86  However, the proportion of BL-CFCs within isolated KDR+ or APLNR+ cells remains low at 1.5% to 4%.62,77,80 

Stepwise specification toward hematopoietic and endothelial lineages in mouse ESC cultures proceeds through conversion of KDR+PDGFRα+ primitive mesodermal cells into KDR+PDGFRα cells with properties of lateral plate mesoderm.89  To define the mesodermal subsets of differentiating hPSCs, we analyzed the kinetics of expression of APLNR, PDGFRα, and KDR mesodermal markers in hPSCs differentiated on the OP9 bone marrow stromal cell line.77  Because these markers could also be found on differentiated cells at postmesodermal stages, we demarcated mesodermal stage of development as EMHlin, ie, the stage at which cells lack the expression of endothelial (CD31, VE-cadherin), endothelial/mesenchymal (CD73, CD105), and hematopoietic (CD43, CD45) lineage markers.86,90  On the basis of these analyses, we identified 2 distinct phases of mesodermal development. EMHlinAPLNR+PDGFRα+ cells, the most primitive mesodermal cells, emerge at days 2 to 3 of differentiation in OP9 coculture (Figure 2). These cells are reminiscent of the primitive posterior mesoderm in the embryo and express genes associated with PS (T, MIXL1, EOMES) and lateral plate/extraembryonic mesoderm (FOXF1, WNT5a, BMP4). As discussed above, the day 3 EMHlinAPLNR+PDGFRα+ cells have the potential to form BL colonies in response to FGF2.77,86  The next step of more advanced mesodermal commitment is associated with the emergence of HVMPs, which can be detected based on high expression of KDR and low to no expression of PDGFRα in EMHlinAPLNR+ cells, ie, EMHlinKDRbrightAPLNR+PDGFRalow/− phenotype. Similar to day 3 posterior mesoderm cells, HVMPs express lateral plate/extraembryonic mesoderm genes. However, they upregulate expression of TAL1, HHEX, LMO2, GATA2, and ETV2 genes associated with angiohematopoietic development, and downregulate expression of PS genes (Figure 2). HVMPs lack BL-CFC potential but are highly enriched in bipotential cells that can form hematoendothelial clusters when cocultured on OP9 and produce the entire spectrum of myeloid progenitors.86  It has been shown that KDR+PDGFRαlow mesodermal population with angiohematopoietic potential is also present in hESC cultures differentiated using the embryoid body method, and that generation of such cells is significantly elevated by the addition of BMP4 and WNT3a.71  Collectively, these results indicate that at least 2 distinct mesodermal subsets are formed during hESC specification to endothelial and hematopoietic lineages. Both of these populations (posterior mesoderm cells and HVMPs) have endothelial and hematopoietic potentials when cultured in vitro (ie, possess hemangioblastic activity). Primitive hematopoietic potential can be detected within immature posterior mesoderm cells, whereas more mature HVMPs generate blood cells with definitive features.

Formation of HE in hPSC cultures

The evidence that HSCs originate from HE with definitive hematopoietic potential underscores the need to identify HE progenitors in hPSC cultures.91  In embryos, HE can be reliably identified based on morphology and anatomical location.49  However, identification of HE in hESC differentiation cultures faces significant challenges because of the considerable overlap in expression of surface markers by endothelial and hematopoietic cells. One of the most specific markers for endothelial cells, VE-cadherin,92-94  is commonly used to isolate endothelial cells from mouse embryo and ESC cultures.95-97  In mouse systems, VE-cadherin staining is typically combined with blood cell–specific antibodies CD41a (early marker of embryonic hematopoietic progenitors) and CD45 (panhematopoietic marker) to separate endothelial and hematopoietic phases of development and identify a transient population of HE emerging around the onset of blood cell generation.87,97,98  Initially, Wang et al99  demonstrated that hESC-derived VE-cadherin+ cells expressing CD31 (PECAM) and KDR (FLK1) but lacking CD45 have hematopoietic and endothelial potentials. Recently, major progress has been made in further dissection of this population to enrich toward HE activity and separate HE more precisely from already established blood cells and non-HE (supplemental Table 1). Identification of CD43 as a marker that covers the entire population of hematopoietic cells, including CD45 CFCs, in hPSC cultures,67,100  made it possible to accurately segregate the endothelial stage from the hematopoietic stage. CD31+CD43 or CD34+CD43 cells isolated from differentiated hESC cells entirely lacked hematopoietic CFC potential but had the capacity to generate blood and endothelial cells after culture with OP9 stromal cells (ie, displayed HE properties).100,101  Recently, Rafii et al102  engineered hESCs to express fluorescent reporters under the VE-cadherin and CD41a promoter (Pr). This system makes it possible to directly observe the formation of round CD41aPr+ blood cells from VE-cadherinPr+CD41aPr epithelioid precursors. Round cells developed from VE-cadherinPr+ cells expressed CD43 and possessed broad hematopoietic CFC potential. Although CD43 enables the precise separation of VE-cadherin+ blood cells from endothelium, further narrowing down the HE phenotype requires the identification of markers that distinguish HE from non-HE. By analyzing the kinetics of VE-cadherin and other endothelial marker expression in hPSC coculture with OP9, we identified 2 distinct populations of cells within VE-cadherin+CD43 endothelial cells on the basis of expression of CD73 (5′-nucleotidase; Figure 2B).86  Both VE-cadherin+CD43CD73+ and CD73 populations expressed KDR, CD31, ESAM, CD34, and other typical endothelial molecules and were capable of generating endothelial cells but lacked hematopoietic colony-forming activity (formation of blood colonies in semisolid medium supplemented with hematopoietic cytokines). However, only VE-cadherin+CD43CD73 cells displayed HE properties (ie, the ability to generate blood cells after secondary coculture with OP9). In contrast to non-HE, HE expressed high levels of RUNX1, which is known to mark HE in the embryo,103  and was capable of generating the entire spectrum of myeloid progenitors, including erythroid cells expressing adult β-hemoglobin. The segregation of HE from non-HE on the basis of expression of CD73 was also shown using VE-cadherinPr/CD41Pr dual reporter transgenic hESCs.102  Kennedy et al66  found that cells with HE phenotype possessed T lymphoid potential and demonstrated the critical role of Activin/Nodal signaling in establishing a lymphoid program in HE. It has also been found, that, in parallel with the acquisition of endothelial markers, differentiated hESCs begin to express angiotensin-converting enzyme (CD143). When CD143+CD43CD45CD41a cells were isolated and analyzed, they showed hemangioblastic properties.104  Angiotensin-converting enzyme activity was found to be important for regulation of hematopoietic vs endothelial differentiation of isolated CD143+ cells. Inhibition of angiotensin II type 1 receptor enhanced hematopoietic potential of CD143+ cells, whereas inhibition of angiotensin II type 2 receptor skewed their differentiation toward endothelial cells.104 

The mechanisms regulating HE development and endothelial-hematopoietic transition remain largely unknown. Mouse studies have identified Runx152  as a positive regulator and HoxA3105  as a negative regulator of blood formation from endothelium, and showed the pivotal role of Sox17 in HE expansion106  and Hes-mediated Notch signaling in HSC formation from HE in the AGM region.107  The critical role of SOX17 in regulation of endothelial-hematopoietic transition has been also demonstrated in hPSCs.101  Conditional expression of SOX17 in hESC-derived CD34+CD43 cells promoted the formation of a unique population of VE-cadherin+CD43+CD45 cells with HE properties while knockdown of its expression inhibited blood formation, indicating that SOX17 is essential for induction and maintenance of the hemogenic program in endothelial cells. This conclusion was also supported by findings that overexpressed SOX17 binds to promoters and regulatory regions of a large number of hematopoietic and endothelial genes.101  Lee et al108  found that formation of hematopoietic progenitors from hPSC-derived CD31+CD45 cells can be enhanced by HES1-mediated activation of NOTCH signaling using Jag1. The developmental progression from HE to hematopoietic progenitors depends on transforming growth factor beta (TGFβ) signaling. In an embryoid body differentiation system, treatment of hESCs at the CD31+CD43 stage of development with TGFβ1 suppressed formation of CD43+ cells, whereas TGFβ inhibitors exerted the opposite effect.74 

Specification and diversification of hematopoietic lineages from hPSCs

In hPSC differentiation cultures, the cells that are already committed to a hematopoietic fate and capable of forming various colonies of blood cells in response to hematopoietic cytokines arise within the CD34+ population before expression of a CD45 typical panhematopoietic marker.59,61,78,109  Although CD34 specifically identifies hematopoietic progenitors and stem cells within the somatic hematopoietic compartment, in hPSC cultures, CD34 is expressed by a variety of cell types, including endothelial and mesenchymal stem cells.100,110  Thus, the use of CD34 alone may not be sufficient to select for a pure population of hematopoietic progenitors from hPSCs. By analyzing the kinetics of expression of specific hematopoietic markers in hESCs differentiated on OP9, Vodyanik et al100  identified leukosialin (CD43) as a marker that reliably separates CD43+ colony-forming hematopoietic progenitors from CD43CD31+ endothelial cells and CD43CD31 cells with mesenchymal characteristics within the CD34+ population generated from hESCs in coculture with OP9. Similarly, selection of CD43+ cells from hiPSCs differentiated on OP9 or in embryoid bodies made it possible to separate hematopoietic CFCs from nonhematopoietic cells.66,67  Recent time-lapse video recordings of endothelial-hematopoietic transition demonstrated that the gain of CD43 expression tightly correlated with the transformation of epithelioid endothelial cells into round blood cells.86  Because CD43 provides a repulsive barrier around the cell,111  acquisition of CD43 expression may have functional significance for budding and separation of blood cells from the endothelium.

In hPSC cultures differentiated on OP9, the first CD43+ cells were detected within emerging VE-cadherin+ cells as early as day 4 of differentiation.86,100  These cells were CD41a and expressed the erythroid marker, CD235a (glycophorin A), and low levels of CD43, which was best detected using highly sensitive fluorochromes. The emerging VE-cadherin+CD43lowCD235a+CD41a cells had primarily hematopoietic characteristics and colony-forming potential and lacked the expression of CLDN5 and CAV1, typical endothelial genes; however, they retained the capacity to grow endothelial cells (Figure 2B). Because of these properties, VE-cadherin+CD43lowCD235a+CD41a cells were defined as AHPs.86,90  Although AHPs expressed the erythroid cell marker CD235a, they had a unique potential to form a broad spectrum of hematopoietic colonies in serum-free medium supplemented with hematopoietic cytokines and FGF2.86  Gross morphologic examinations of some of these colonies resemble hemangioblast/BL colonies formed by BL-CFCs. However, in contrast to BL colonies, colonies generated by AHPs do not develop through an endothelial core stage. Whether AHPs represent a unique transition stage between HE and blood or a distinct wave of hematopoietic progenitors remains to be determined.

In mouse embryo and ESC cultures, CD41a (GPIIb) expression marks the initiation of hematopoiesis and precedes the expression of the erythroid marker, TER119.112-114  However, in hPSC cultures, the first CD41a+ cells were detected within the already established CD43+CD235a+ population.86  Thus, the vast majority of CD41a+ cells coexpress CD235a. Although early CD41a+CD235a+ cells express high levels of VE-cadherin, they are completely devoid of endothelial potential.86  Analysis of the colony-forming capacity of CD41a+CD235a+ cells revealed that they are highly enriched in erythro-megakaryocytic progenitors,100,115  but also show some myeloid potential in serum-free clonogenic medium.86  hESC- and hiPSCs-derived CD235a+CD41a+ progenitors with erythro-megakaryocytic potential can be expanded and differentiated into erythroid or megakaryocytic cells.116,117  The expansion and fate switch of CD235a+CD41a+ cells is regulated by the aryl hydrocarbon receptor.117  Multiple studies have demonstrated the feasibility of high-scale production of red blood cells and platelets in bulk hPSC cultures (reviewed by Chang et al118  and Takayama and Eto119 ). However, erythroid cells generated in these cultures have primitive and fetal-like characteristics.120,121  They express mostly embryonic and fetal hemoglobins with low levels of adult hemoglobin and do not enucleate efficiently. Although several studies have demonstrated that hPSC-derived platelets are capable of initiating thrombus formation, the hemostatic properties of these cells remain to be evaluated in animal models of thrombocytopenia.

The progenitors with broad myelolymphoid potential and linCD34+CD43+CD45 phenotype can be detected in hPSC cultures shortly after emergence of CD235a+CD41a+ cells. Acquisition of CD45 expression by lin cells is associated with progressive myeloid commitment.100  The linCD34+CD43+CD45+/− hematopoietic progenitors are capable of forming the entire spectrum of hematopoietic colonies in serum-containing CFC medium. The CFC potentials of these cells was comparable or even higher than that of phenotypically similar cord blood cells.122  The hESC- and hiPSC-derived linCD34+CD43+CD45+/− cells display many phenotypical features of HSCs, including CD90 and c-kit expression and the lack of CD38 and CD45RA expression.67,100  These cells have high aldehyde dehydrogenase activity, the ability to efflux rhodamine-123, and LTC-IC potential.78,100,123  The linCD34+CD43+CD45+ cells could be expanded with granulocyte-macrophage colony-stimulating factor and differentiated into myelomonocytic cells including neutrophils, eosinophils, macrophages, dendritic and Langerhans cells, and osteoclasts.19  CD34+ and CD43+ cells generated from hESCs or hiPSCs also possess natural killer and B lymphoid potentials.78,100,124-126  Natural killer cells generated from hPSCs have cytolytic function and can be generated in large numbers using defined conditions.125,126  However, B lymphoid cultures produced very low numbers of CD19+CD10+ cells. Although these cells exhibited multiple genomic D-JH rearrangements, they did not express IgM or CD5, indicating that their development failed to progress beyond the pre–B-cell stage.124  The T-cell potential of hESCs was initially demonstrated using in vivo studies, which found that hESC-derived CD34+ cells transplanted into human thymus/fetal liver grafts in SCID-hu mice generated T cells, including CD4+CD8+ T-cell precursors.127  Later, T cells were generated from hESCs and iPSCs in vitro using OP9 cells expressing the NOTCH ligands δ-like 1 (DLL1) or 4 (DLL4).18,66,128,129  By analyzing the T-cell potential of CD43+ populations expressing different levels of CD34 and CD43, Timmermans et al found that T cells were derived exclusively from the CD34highCD43low population of hematopoietic progenitors.128  T-cell potential could also be detected when NOTCH signaling is activated in CD34+CD43CD45 cells expressing endothelial markers.66  Collectively, these studies indicate that cells with panmyeloid and lymphoid potentials (ie, definitive hematopoietic cells) can be generated from hPSCs.

Hematopoietic engraftment potential of hPSC-derived cells

Following the initial studies demonstrating the presence of CD34+ cells with hematopoietic colony-forming potential in hESC cultures, there was significant interest in evaluating the hematopoietic repopulation potential of these cells. Several reports have detected human hematopoietic cells in the bone marrow of immunocompromised mice and sheep many months after intravenous or intrafemoral injection of an entire population of differentiated hESCs or hESC-derived CD34+ cells.130-134  However, bone marrow engraftment observed in these studies was low (within 0.1%-2%) and was mostly restricted to the myeloid lineage. Similarly low levels of engraftment were reported after intrafemoral transplantation of differentiated hiPSCs.135  It was suggested that hPSC-derived cells have limited migratory potential and seem unable to complete maturation within the adult bone marrow environment.131,135  To find out whether the neonatal environment provides a better support for hematopoietic engraftment of differentiating hESCs, Tian et al transplanted CD34+ cells obtained from luciferase-expressing hESCs into newborn NSG mice.136  Although the authors detected luciferase+ cells several months after transplantation, they found that these cells were mostly endothelial and not hematopoietic. Recently, 2 studies have demonstrated that hematopoietic differentiation could be achieved in vivo during teratoma formation from hiPSCs.137,138  The generation of blood cells within teratomas was enhanced by injecting hiPSCs together with OP9 stromal cells ectopically expressing DLL1 and WNT3a. When CD34+CD45+ cells were isolated from these teratomas and transplanted into NSG mice, the pattern of hematopoietic engraftment was very similar to that observed after transplantation of in vitro differentiated hPSCs (ie, very limited 0.1%-2% hematopoietic chimerism in the bone marrow with predominant myeloid engraftment).137  In another study, hematopoietic differentiation within teratomas was amplified by administration of human SCF and TPO via an micro-osmotic pump. However, only ∼4% NOD/SCID and ∼30% of NOD/SCID/JAK3null mice showed a low level of hematopoietic chimerism after transplantation of 600 CD34+CD45+ teratoma-derived cells.138  Although these studies demonstrated the feasibility of generating hematopoietic cells with limited engraftment potential from hPSCs, they also indicate that current differentiation conditions do not reproduce the complexity of embryonic hematopoietic development that leads to HSC specification and expansion.

Defining conditions that allow engineering HSCs

Why do in vitro hPSC cultures fail to yield cells with robust hematopoietic engraftment potential? Because HSC specification in the AGM region of an embryo is a very rare event (1 HSC per AGM at E11.5)41  followed by HSC expansion in the fetal liver, one can argue that hPSC differentiation cultures produce rare HSCs but fail to expand them. However, this situation is probably unlikely. During embryogenesis, the hematopoietic hierarchy appears in reverse sequence as compared with an adult one,139  ie, the formation of progenitors with more restricted hematopoietic potential precedes emergence of HSCs (Figure 3). In mice, panmyeloid progenitors and T and B lymphoid precursors were first identified in the yolk sac before HSC potential could be detected in the embryo proper.139-141  In addition, clusters of multipotential blood cells are formed at many sites within the aorta; however, HSCs are restricted to the ventral wall of the dorsal aorta.55,142  This indicates that finely tuned local signaling is required for multipotential blood cells to acquire repopulation potential. Therefore, it is more likely that multipotential blood cells generated in hPSC cultures represent a pre-HSC stage of development and that blood cells arising in hPSC cultures simply fail to complete the HSC specification after transition from the endothelial to the hematopoietic stage.

Figure 3

Schematic diagram demonstrating the opposite sequence of blood cell development between embryos and adults. In embryos, cells with restricted hematopoietic potential appear before HSC specification. In adults, hematopoiesis proceeds through gradual maturation of HSCs, leading to formation of progenitors with more restricted potential.

Figure 3

Schematic diagram demonstrating the opposite sequence of blood cell development between embryos and adults. In embryos, cells with restricted hematopoietic potential appear before HSC specification. In adults, hematopoiesis proceeds through gradual maturation of HSCs, leading to formation of progenitors with more restricted potential.

Currently, 2 major models exist to explain the relationship between adult and embryonic hematopoietic hierarchies.139  According to the first model, the embryonic and adult hierarchies arise from a common hematopoietic ancestor that gives rise to both primitive yolk sac and definitive embryonic hematopoiesis. The other model suggests that primitive and definitive hierarchies arise independently. Studies in Xenopus found that separation of adult and embryonic hematopoiesis occurs as early as the 32-cell blastomere stage.143  Independent origins of yolk and definitive embryonic hematopoiesis are also supported by most of the mouse studies.35-37  Nevertheless, the contribution of the yolk sac precursors to definitive adult hematopoiesis cannot be entirely excluded.144,145  Regardless of which model more accurately predicts the in vivo process, the most critical question in relationship to hPSC differentiation studies is whether the more primitive yolk sac precursors can acquire adult HSC properties after exposure to particular signaling events. Studies by Yoder et al strongly support the hypothesis that local environment can induce HSC potential in yolk sac cells.146,147  In contrast to AGM, yolk sac organ cultures are unable to initiate and support HSC development ex vivo or repopulate adult recipients.41  However, CD34+c-kit+ or CD34+c-kit+CD41+ cells isolated from the yolk sac of E9-9.5 embryos are capable of reconstituting multilineage long-term hematopoiesis when transplanted into busulfan-conditioned neonates.112,146,147  Similarly, hematopoietic progenitors with multilineage potential that engraft in neonates can be isolated from the para-aortic splanchnopleura (the AGM primordium) of an E9 mouse embryo, before definitive HSC potential can be detected in an adult recipient repopulation assay.146  Thus, embryonic cells with detectable myeloid and lymphoid potentials in vitro, but lacking adult repopulation potential, can mature into HSCs in the neonatal environment. On the basis of these studies, induction of the equivalent of yolk sac/para-aortic splanchnopleura multilineage newborn-repopulating cells in hPSC cultures is considered a critical step toward establishing protocols for generating HSCs ex vivo.30  By using T-cell potential as a major criterion for the identification of such cells in hPSC cultures, Kennedy et al found that progenitors with T lymphoid potential reside within the CD34+CD43CD45 population of cells expressing CD34 and other endothelial markers.66  Importantly, the production of these T lymphoid progenitors could be enhanced by manipulation of Activin/Nodal signaling pathways during the first 4 days of hPSC differentiation,66  indicating that the definitive hematopoietic program can be enhanced during the mesodermal stage. Further engraftment studies will be required to determine whether hPSC-derived cells with lymphoid potential can repopulate newborn mice, similar to cells produced in vivo during embryogenesis.

To produce HSCs from hPSCs, it is also necessary to better understand the mechanisms regulating the formation of and specification of blood cells from HE cells. The acquisition of arterial identity through activation of NOTCH signaling and activation of hematopoietic programing by RUNX1 are the most critical factors for the establishment of HE cells in vivo (reviewed by Zape and Zovein148 ). However, it has become clear that HE cells are heterogeneous and not all HE cells can produce HSCs. Recent evidence indicates that at least 2 distinct populations of HE cells with erythromyeloid and HSC potentials exist in the mouse embryo, and that Ly6a (Sca1) specifically marks HSC-producing HE cells.149  It is already known that at least 2 distinct types of VE-cadherin+ cells with primitive and broad erythromyeloid hematopoietic potentials can be produced from hPSCs.66,86,91,102  However, the specification of cells with broad lymphomyeloid potential directly from HE cells remains to be shown using clonal studies. The recent identification of HE progenitors in hPSC cultures66,86  and the generation of genetically engineered cell lines to trace endothelial-hematopoietic transition102  have already provided a platform for assaying upstream factors required for HE formation with HSC potential and downstream factors that promote HSC expansion in the human system.

The reprogramming studies provided strong evidence that cellular identities are defined by gene regulatory networks controlled by few master regulatory factors. Identification of the master factors required for the specification of definitive/adult type HSCs from embryonic precursors would be one of the essential prerequisites to establishing a protocol for generating HSCs ex vivo. Mouse studies discovered HoxB4 or its upstream regulator Cdx4 as chief factors capable of inducing a self-renewal program in ESC-derived hematopoietic progenitors.12,150  The rationale for selecting HoxB4 to engineer HSCs from ESCs came from embryonic studies that demonstrated the lack of expression of several critical homeobox regulators of definitive HSCs, including HoxB4, in the yolk sac.151  Nevertheless, HoxB4-induced cells do not entirely mimic the function and phenotype of somatic HSCs and produce mostly myeloid engraftment. Recently, the Daley group152  performed comprehensive molecular profiling studies to compare in vitro–generated hematopoietic cells from mouse ESCs with cells from embryonic hematopoietic sites. These studies revealed that HoxB4-induced HSCs lack HoxA9 expression and a Notch signaling signature,152  which may explain their limited lymphoid potential. Although attempts have been made to induce HSCs from hPSCs using HOXB4, it was found that hESC-derived hematopoietic cells already express HOXB4, and its forced expression does not enhance engraftment of human cells.131,153  In a search for the intrinsic determinants required for HSC specification from hPSCs, several groups performed molecular profiling studies of ESC-derived and in vivo–produced human primitive hematopoietic cells. These studies revealed an apparent similarity between the transcriptomes of phenotypically identical fetal liver and hESC-derived primitive hematopoietic populations,122,154  although distinctive differences in the expression of genes regulating HSC self-renewal, homing, and chromatin remodeling were also noted.122  Interestingly, compared with the fetal liver primitive hematopoietic cells, hESC-derived hematopoietic progenitors showed much higher levels of expression of genes from the HOXB cluster, but significantly lower levels of genes from the HOXA cluster.122,154  Differences in genes involved in NOTCH signaling pathways,155  polycomb and trithorax complexes were also noted.122,156  Capacity to home to bone marrow is one of the critical features of HSCs. However, hESC-derived linCD34+CD43+CD45+/− hematopoietic progenitors express very low levels of the HSC-homing molecule CXCR4, which may indicate that these cells have yet to acquire competent homing capability.122  Overall, molecular profiling studies have identified several unique features of the transcriptome of human and mouse PSC-derived hematopoietic precursors. Insights gained from these studies can be further explored to define novel molecular targets capable of activating an adult-type HSC program during hPSC differentiation.

Concluding remarks

The de novo generation of HSCs remains a significant challenge. Achieving this goal requires collaborative efforts of developmental, stem cell, and molecular biologists. Embryonic studies have identified the essential role of Notch, Wnt, Hedgehog, and TGFβ/Smad signaling pathways in HSC development and the maintenance of an HSC program. Further understanding the molecular mechanisms and niche factors critical for specifying HE vs non-HE cells and HSCs vs non-HSC precursors from HE cells in the embryo will be an essential prerequisite to the development of optimal conditions for HSC production in vitro. The reconstruction of gene regulatory networks and identification of master regulatory factors that control HSC identity and drive HSC precursors to adopt HSC fate will ultimately enable conversion of hPSC-derived cells into adult-type HSCs (Figure 4). Recently, a hemogenic program was successfully induced in mouse and human fibroblasts by forced expression of Gata2, Gfi1b, cFos, and Etv6157  and OCT4, respectively.158  Although these reports demonstrated the feasibility of generating hematopoietic hierarchy for clinical application by direct transformation of somatic cells and bypassing the pluripotency stage, the next challenging step is to determine factors capable of inducing the hematopoietic self-renewal program in directly transformed somatic cells and achieve the scalability of the direct reprogramming process. Nevertheless, both approaches, PSC-based and direct reprogramming of somatic cells to HSCs, have to be pursued to better understand the genetic and epigenetic factors governing hematopoietic specification and the self-renewal program. No doubt, these studies will continue to provide fascinating insights into the fundamental questions of HSC biology, and eventually will lead to the development of novel stem cell therapies.

Figure 4

Potential approaches for de novo induction of HSCs.

Figure 4

Potential approaches for de novo induction of HSCs.

The online version of this article contains a data supplement.

Acknowledgments

I thank Gene Uenishi and Derek Theisen for proofreading the manuscript.

This work is supported by funds from the National Institutes of Health, National Heart, Lung, and Blood Institute (U01HL099773 and R01 HL116221) and the Charlotte Geyer Foundation. The author declares no competing financial interests.

Authorship

Contribution: I.S. wrote the paper.

Conflict-of-interest disclosure: The author declares no competing financial interest relating to the topic of this article.

Correspondence: Igor I. Slukvin, Department of Pathology and Laboratory Medicine, University of Wisconsin, 1220 Capitol Court, Madison, WI 53715; e-mail: islukvin@wisc.edu.

References

References
1
Thomson
 
JA
Itskovitz-Eldor
 
J
Shapiro
 
SS
, et al. 
Embryonic stem cell lines derived from human blastocysts.
Science
1998
, vol. 
282
 
5391
(pg. 
1145
-
1147
)
2
Takahashi
 
K
Yamanaka
 
S
Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.
Cell
2006
, vol. 
126
 
4
(pg. 
663
-
676
)
3
Takahashi
 
K
Tanabe
 
K
Ohnuki
 
M
, et al. 
Induction of pluripotent stem cells from adult human fibroblasts by defined factors.
Cell
2007
, vol. 
131
 
5
(pg. 
861
-
872
)
4
Yu
 
J
Vodyanik
 
MA
Smuga-Otto
 
K
, et al. 
Induced pluripotent stem cell lines derived from human somatic cells.
Science
2007
, vol. 
318
 
5858
(pg. 
1917
-
1920
)
5
Park
 
IH
Zhao
 
R
West
 
JA
, et al. 
Reprogramming of human somatic cells to pluripotency with defined factors.
Nature
2008
, vol. 
451
 
7175
(pg. 
141
-
146
)
6
Hussein
 
SM
Nagy
 
AA
Progress made in the reprogramming field: new factors, new strategies and a new outlook.
Curr Opin Genet Dev
2012
, vol. 
22
 
5
(pg. 
435
-
443
)
7
González
 
F
Boué
 
S
Izpisúa Belmonte
 
JC
Methods for making induced pluripotent stem cells: reprogramming à la carte.
Nat Rev Genet
2011
, vol. 
12
 
4
(pg. 
231
-
242
)
8
Spitzer
 
TR
Dey
 
BR
Chen
 
YB
Attar
 
E
Ballen
 
KK
The expanding frontier of hematopoietic cell transplantation.
Cytometry B Clin Cytom
2012
, vol. 
82
 
5
(pg. 
271
-
279
)
9
Copelan
 
EA
Hematopoietic stem-cell transplantation.
N Engl J Med
2006
, vol. 
354
 
17
(pg. 
1813
-
1826
)
10
Daley
 
GQ
The promise and perils of stem cell therapeutics.
Cell Stem Cell
2012
, vol. 
10
 
6
(pg. 
740
-
749
)
11
Sancho-Martinez
 
I
Li
 
M
Izpisua Belmonte
 
JC
Disease correction the iPSC way: advances in iPSC-based therapy.
Clin Pharmacol Ther
2011
, vol. 
89
 
5
(pg. 
746
-
749
)
12
Kyba
 
M
Perlingeiro
 
RC
Daley
 
GQ
HoxB4 confers definitive lymphoid-myeloid engraftment potential on embryonic stem cell and yolk sac hematopoietic progenitors.
Cell
2002
, vol. 
109
 
1
(pg. 
29
-
37
)
13
Daley
 
GQ
Towards the generation of patient-specific pluripotent stem cells for combined gene and cell therapy of hematologic disorders.
Hematology Am Soc Hematol Educ Program
2007
(pg. 
17
-
22
)
14
Kaufman
 
DS
Toward clinical therapies using hematopoietic cells derived from human pluripotent stem cells.
Blood
2009
, vol. 
114
 
17
(pg. 
3513
-
3523
)
15
Rivière
 
I
Dunbar
 
CE
Sadelain
 
M
Hematopoietic stem cell engineering at a crossroads.
Blood
2012
, vol. 
119
 
5
(pg. 
1107
-
1116
)
16
Hanna
 
J
Wernig
 
M
Markoulaki
 
S
, et al. 
Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin.
Science
2007
, vol. 
318
 
5858
(pg. 
1920
-
1923
)
17
Iwamoto
 
H
Ojima
 
T
Hayata
 
K
, et al. 
 
Antitumor immune response of dendritic cells (DCs) expressing tumor-associated antigens derived from induced pluripotent stem cells: In comparison to bone marrow-derived DCs. Int J Cancer. 2013. Jul 3. doi: 10.1002/ijc.28367. [Epub ahead of print]
18
Themeli
 
M
Kloss
 
CC
Ciriello
 
G
, et al. 
 
Generation of tumor-targeted human T lymphocytes from induced pluripotent stem cells for cancer therapy. Nature Biotechnol. 2013;31(10):928-933
19
Choi
 
KD
Vodyanik
 
MA
Slukvin
 
II
Generation of mature human myelomonocytic cells through expansion and differentiation of pluripotent stem cell-derived lin-CD34+CD43+CD45+ progenitors.
J Clin Invest
2009
, vol. 
119
 
9
(pg. 
2818
-
2829
)
20
Kinder
 
SJ
Tsang
 
TE
Quinlan
 
GA
Hadjantonakis
 
AK
Nagy
 
A
Tam
 
PP
The orderly allocation of mesodermal cells to the extraembryonic structures and the anteroposterior axis during gastrulation of the mouse embryo.
Development
1999
, vol. 
126
 
21
(pg. 
4691
-
4701
)
21
Wilkinson
 
DG
Bhatt
 
S
Herrmann
 
BG
Expression pattern of the mouse T gene and its role in mesoderm formation.
Nature
1990
, vol. 
343
 
6259
(pg. 
657
-
659
)
22
Yamaguchi
 
TP
Dumont
 
DJ
Conlon
 
RA
Breitman
 
ML
Rossant
 
J
flk-1, an flt-related receptor tyrosine kinase is an early marker for endothelial cell precursors.
Development
1993
, vol. 
118
 
2
(pg. 
489
-
498
)
23
Shalaby
 
F
Ho
 
J
Stanford
 
WL
, et al. 
A requirement for Flk1 in primitive and definitive hematopoiesis and vasculogenesis.
Cell
1997
, vol. 
89
 
6
(pg. 
981
-
990
)
24
Winnier
 
G
Blessing
 
M
Labosky
 
PA
Hogan
 
BL
Bone morphogenetic protein-4 is required for mesoderm formation and patterning in the mouse.
Genes Dev
1995
, vol. 
9
 
17
(pg. 
2105
-
2116
)
25
Saxton
 
TM
Pawson
 
T
Morphogenetic movements at gastrulation require the SH2 tyrosine phosphatase Shp2.
Proc Natl Acad Sci USA
1999
, vol. 
96
 
7
(pg. 
3790
-
3795
)
26
Liu
 
P
Wakamiya
 
M
Shea
 
MJ
Albrecht
 
U
Behringer
 
RR
Bradley
 
A
Requirement for Wnt3 in vertebrate axis formation.
Nat Genet
1999
, vol. 
22
 
4
(pg. 
361
-
365
)
27
Conlon
 
FL
Lyons
 
KM
Takaesu
 
N
, et al. 
A primary requirement for nodal in the formation and maintenance of the primitive streak in the mouse.
Development
1994
, vol. 
120
 
7
(pg. 
1919
-
1928
)
28
Flamme
 
I
Breier
 
G
Risau
 
W
Vascular endothelial growth factor (VEGF) and VEGF receptor 2 (flk-1) are expressed during vasculogenesis and vascular differentiation in the quail embryo.
Dev Biol
1995
, vol. 
169
 
2
(pg. 
699
-
712
)
29
Carmeliet
 
P
Ferreira
 
V
Breier
 
G
, et al. 
Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele.
Nature
1996
, vol. 
380
 
6573
(pg. 
435
-
439
)
30
Keller
 
G
Embryonic stem cell differentiation: emergence of a new era in biology and medicine.
Genes Dev
2005
, vol. 
19
 
10
(pg. 
1129
-
1155
)
31
Murry
 
CE
Keller
 
G
Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development.
Cell
2008
, vol. 
132
 
4
(pg. 
661
-
680
)
32
Palis
 
J
Robertson
 
S
Kennedy
 
M
Wall
 
C
Keller
 
G
Development of erythroid and myeloid progenitors in the yolk sac and embryo proper of the mouse.
Development
1999
, vol. 
126
 
22
(pg. 
5073
-
5084
)
33
Xu MJ
 
Matsuoka
 
S
Yang
 
FC
, et al. 
Evidence for the presence of murine primitive megakaryocytopoiesis in the early yolk sac.
Blood
2001
, vol. 
97
 
7
(pg. 
2016
-
2022
)
34
Silver
 
L
Palis
 
J
 
Initiation of murine embryonic erythropoiesis: a spatial analysis. Blood. 1997;89(4):1154-1164
35
de Bruijn
 
MF
Ma
 
X
Robin
 
C
Ottersbach
 
K
Sanchez
 
MJ
Dzierzak
 
E
Hematopoietic stem cells localize to the endothelial cell layer in the midgestation mouse aorta.
Immunity
2002
, vol. 
16
 
5
(pg. 
673
-
683
)
36
Medvinsky
 
A
Dzierzak
 
E
 
Definitive hematopoiesis is autonomously initiated by the AGM region. Cell. 1996;86(6):897-906
37
Müller
 
AM
Medvinsky
 
A
Strouboulis
 
J
Grosveld
 
F
Dzierzak
 
E
Development of hematopoietic stem cell activity in the mouse embryo.
Immunity
1994
, vol. 
1
 
4
(pg. 
291
-
301
)
38
Gekas
 
C
Dieterlen-Lièvre
 
F
Orkin
 
SH
Mikkola
 
HK
The placenta is a niche for hematopoietic stem cells.
Dev Cell
2005
, vol. 
8
 
3
(pg. 
365
-
375
)
39
Ottersbach
 
K
Dzierzak
 
E
The murine placenta contains hematopoietic stem cells within the vascular labyrinth region.
Dev Cell
2005
, vol. 
8
 
3
(pg. 
377
-
387
)
40
de Bruijn
 
MF
Speck
 
NA
Peeters
 
MC
Dzierzak
 
E
Definitive hematopoietic stem cells first develop within the major arterial regions of the mouse embryo.
EMBO J
2000
, vol. 
19
 
11
(pg. 
2465
-
2474
)
41
Kumaravelu
 
P
Hook
 
L
Morrison
 
AM
, et al. 
Quantitative developmental anatomy of definitive haematopoietic stem cells/long-term repopulating units (HSC/RUs): role of the aorta-gonad-mesonephros (AGM) region and the yolk sac in colonisation of the mouse embryonic liver.
Development
2002
, vol. 
129
 
21
(pg. 
4891
-
4899
)
42
Cumano
 
A
Godin
 
I
Ontogeny of the hematopoietic system.
Annu Rev Immunol
2007
, vol. 
25
 
25
(pg. 
745
-
785
)
43
Maximow
 
AA
Relation of blood cells to connective tissues and endothelium.
Physiol Rev
1924
, vol. 
4
 
4
(pg. 
533
-
563
)
44
Sabin
 
F
Origin and development of the primitive vessels of the chick and of the pig.
Carnegie Inst Wash Publ Contribs Embryol
1917
, vol. 
6
 (pg. 
61
-
124
)
45
Murray
 
PDF
The development in vitro of the blood of the early chick embryo.
Proc R Soc Lond, B
1932
, vol. 
111
 
773
(pg. 
497
-
521
)
46
Lacaud
 
G
Gore
 
L
Kennedy
 
M
, et al. 
Runx1 is essential for hematopoietic commitment at the hemangioblast stage of development in vitro.
Blood
2002
, vol. 
100
 
2
(pg. 
458
-
466
)
47
Choi
 
K
Kennedy
 
M
Kazarov
 
A
Papadimitriou
 
JC
Keller
 
G
A common precursor for hematopoietic and endothelial cells.
Development
1998
, vol. 
125
 
4
(pg. 
725
-
732
)
48
Huber
 
TL
Kouskoff
 
V
Fehling
 
HJ
Palis
 
J
Keller
 
G
Haemangioblast commitment is initiated in the primitive streak of the mouse embryo.
Nature
2004
, vol. 
432
 
7017
(pg. 
625
-
630
)
49
Jaffredo
 
T
Gautier
 
R
Eichmann
 
A
Dieterlen-Lièvre
 
F
Intraaortic hemopoietic cells are derived from endothelial cells during ontogeny.
Development
1998
, vol. 
125
 
22
(pg. 
4575
-
4583
)
50
Boisset
 
JC
van Cappellen
 
W
Andrieu-Soler
 
C
Galjart
 
N
Dzierzak
 
E
Robin
 
C
In vivo imaging of haematopoietic cells emerging from the mouse aortic endothelium.
Nature
2010
, vol. 
464
 
7285
(pg. 
116
-
120
)
51
Zovein
 
AC
Hofmann
 
JJ
Lynch
 
M
, et al. 
Fate tracing reveals the endothelial origin of hematopoietic stem cells.
Cell Stem Cell
2008
, vol. 
3
 
6
(pg. 
625
-
636
)
52
Chen
 
MJ
Yokomizo
 
T
Zeigler
 
BM
Dzierzak
 
E
Speck
 
NA
Runx1 is required for the endothelial to haematopoietic cell transition but not thereafter.
Nature
2009
, vol. 
457
 
7231
(pg. 
887
-
891
)
53
Kissa
 
K
Herbomel
 
P
Blood stem cells emerge from aortic endothelium by a novel type of cell transition.
Nature
2010
, vol. 
464
 
7285
(pg. 
112
-
115
)
54
Bertrand
 
JY
Chi
 
NC
Santoso
 
B
Teng
 
S
Stainier
 
DY
Traver
 
D
Haematopoietic stem cells derive directly from aortic endothelium during development.
Nature
2010
, vol. 
464
 
7285
(pg. 
108
-
111
)
55
Yokomizo
 
T
Dzierzak
 
E
Three-dimensional cartography of hematopoietic clusters in the vasculature of whole mouse embryos.
Development
2010
, vol. 
137
 
21
(pg. 
3651
-
3661
)
56
Li
 
Z
Lan
 
Y
He
 
W
, et al. 
Mouse embryonic head as a site for hematopoietic stem cell development.
Cell Stem Cell
2012
, vol. 
11
 
5
(pg. 
663
-
675
)
57
Nakano
 
H
Liu
 
X
Arshi
 
A
, et al. 
 
Haemogenic endocardium contributes to transient definitive haematopoiesis. Nature Commun. 2013;4:1564
58
Li
 
W
Ferkowicz
 
MJ
Johnson
 
SA
Shelley
 
WC
Yoder
 
MC
Endothelial cells in the early murine yolk sac give rise to CD41-expressing hematopoietic cells.
Stem Cells Dev
2005
, vol. 
14
 
1
(pg. 
44
-
54
)
59
Kaufman
 
DS
Hanson
 
ET
Lewis
 
RL
Auerbach
 
R
Thomson
 
JA
Hematopoietic colony-forming cells derived from human embryonic stem cells.
Proc Natl Acad Sci USA
2001
, vol. 
98
 
19
(pg. 
10716
-
10721
)
60
Chadwick
 
K
Wang
 
L
Li
 
L
, et al. 
Cytokines and BMP-4 promote hematopoietic differentiation of human embryonic stem cells.
Blood
2003
, vol. 
102
 
3
(pg. 
906
-
915
)
61
Zambidis
 
ET
Peault
 
B
Park
 
TS
Bunz
 
F
Civin
 
CI
Hematopoietic differentiation of human embryonic stem cells progresses through sequential hematoendothelial, primitive, and definitive stages resembling human yolk sac development.
Blood
2005
, vol. 
106
 
3
(pg. 
860
-
870
)
62
Kennedy
 
M
D’Souza
 
SL
Lynch-Kattman
 
M
Schwantz
 
S
Keller
 
G
Development of the hemangioblast defines the onset of hematopoiesis in human ES cell differentiation cultures.
Blood
2007
, vol. 
109
 
7
(pg. 
2679
-
2687
)
63
Salvagiotto
 
G
Burton
 
S
Daigh
 
CA
Rajesh
 
D
Slukvin
 
II
Seay
 
NJ
A defined, feeder-free, serum-free system to generate in vitro hematopoietic progenitors and differentiated blood cells from hESCs and hiPSCs.
PLoS ONE
2011
, vol. 
6
 
3
pg. 
e17829
 
64
Niwa
 
A
Heike
 
T
Umeda
 
K
, et al. 
A novel serum-free monolayer culture for orderly hematopoietic differentiation of human pluripotent cells via mesodermal progenitors.
PLoS ONE
2011
, vol. 
6
 
7
pg. 
e22261
 
65
Pick
 
M
Azzola
 
L
Mossman
 
A
Stanley
 
EG
Elefanty
 
AG
Differentiation of human embryonic stem cells in serum-free medium reveals distinct roles for bone morphogenetic protein 4, vascular endothelial growth factor, stem cell factor, and fibroblast growth factor 2 in hematopoiesis.
Stem Cells
2007
, vol. 
25
 
9
(pg. 
2206
-
2214
)
66
Kennedy
 
M
Awong
 
G
Sturgeon
 
CM
, et al. 
 
T lymphocyte potential marks the emergence of definitive hematopoietic progenitors in human pluripotent stem cell differentiation cultures. Cell Rep. 2012;2(6):1722-1735
67
Choi
 
KD
Yu
 
J
Smuga-Otto
 
K
, et al. 
Hematopoietic and endothelial differentiation of human induced pluripotent stem cells.
Stem Cells
2009
, vol. 
27
 
3
(pg. 
559
-
567
)
68
Woods
 
NB
Parker
 
AS
Moraghebi
 
R
, et al. 
Brief report: efficient generation of hematopoietic precursors and progenitors from human pluripotent stem cell lines.
Stem Cells
2011
, vol. 
29
 
7
(pg. 
1158
-
1164
)
69
Park
 
TS
Zimmerlin
 
L
Zambidis
 
ET
 
Efficient and simultaneous generation of hematopoietic and vascular progenitors from human induced pluripotent stem cells. Cytometry A. 2013;83(1):114-126
70
Zhang
 
P
Li
 
J
Tan
 
Z
, et al. 
Short-term BMP-4 treatment initiates mesoderm induction in human embryonic stem cells.
Blood
2008
, vol. 
111
 
4
(pg. 
1933
-
1941
)
71
Wang
 
Y
Nakayama
 
N
 
WNT and BMP signaling are both required for hematopoietic cell development from human ES cells. Stem Cell Res. 2009;3(2-3):113-125
72
Woll
 
PS
Morris
 
JK
Painschab
 
MS
, et al. 
Wnt signaling promotes hematoendothelial cell development from human embryonic stem cells.
Blood
2008
, vol. 
111
 
1
(pg. 
122
-
131
)
73
Cerdan
 
C
Rouleau
 
A
Bhatia
 
M
VEGF-A165 augments erythropoietic development from human embryonic stem cells.
Blood
2004
, vol. 
103
 
7
(pg. 
2504
-
2512
)
74
Wang
 
C
Tang
 
X
Sun
 
X
, et al. 
TGFβ inhibition enhances the generation of hematopoietic progenitors from human ES cell-derived hemogenic endothelial cells using a stepwise strategy.
Cell Res
2012
, vol. 
22
 
1
(pg. 
194
-
207
)
75
Kardel
 
MD
Eaves
 
CJ
Modeling human hematopoietic cell development from pluripotent stem cells.
Exp Hematol
2012
, vol. 
40
 
8
(pg. 
601
-
611
)
76
Davis
 
RP
Ng
 
ES
Costa
 
M
, et al. 
Targeting a GFP reporter gene to the MIXL1 locus of human embryonic stem cells identifies human primitive streak-like cells and enables isolation of primitive hematopoietic precursors.
Blood
2008
, vol. 
111
 
4
(pg. 
1876
-
1884
)
77
Vodyanik
 
MA
Yu
 
J
Zhang
 
X
, et al. 
A mesoderm-derived precursor for mesenchymal stem and endothelial cells.
Cell Stem Cell
2010
, vol. 
7
 
6
(pg. 
718
-
729
)
78
Vodyanik
 
MA
Bork
 
JA
Thomson
 
JA
Slukvin
 
II
Human embryonic stem cell-derived CD34+ cells: efficient production in the coculture with OP9 stromal cells and analysis of lymphohematopoietic potential.
Blood
2005
, vol. 
105
 
2
(pg. 
617
-
626
)
79
Yang
 
L
Soonpaa
 
MH
Adler
 
ED
, et al. 
Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population.
Nature
2008
, vol. 
453
 
7194
(pg. 
524
-
528
)
80
Yu
 
QC
Hirst
 
CE
Costa
 
M
, et al. 
APELIN promotes hematopoiesis from human embryonic stem cells.
Blood
2012
, vol. 
119
 
26
(pg. 
6243
-
6254
)
81
D’Amour
 
KA
Agulnick
 
AD
Eliazer
 
S
Kelly
 
OG
Kroon
 
E
Baetge
 
EE
Efficient differentiation of human embryonic stem cells to definitive endoderm.
Nat Biotechnol
2005
, vol. 
23
 
12
(pg. 
1534
-
1541
)
82
Chng
 
Z
Teo
 
A
Pedersen
 
RA
Vallier
 
L
SIP1 mediates cell-fate decisions between neuroectoderm and mesendoderm in human pluripotent stem cells.
Cell Stem Cell
2010
, vol. 
6
 
1
(pg. 
59
-
70
)
83
Vallier
 
L
Touboul
 
T
Chng
 
Z
, et al. 
Early cell fate decisions of human embryonic stem cells and mouse epiblast stem cells are controlled by the same signalling pathways.
PLoS ONE
2009
, vol. 
4
 
6
pg. 
e6082
 
84
Sumi
 
T
Tsuneyoshi
 
N
Nakatsuji
 
N
Suemori
 
H
Defining early lineage specification of human embryonic stem cells by the orchestrated balance of canonical Wnt/beta-catenin, Activin/Nodal and BMP signaling.
Development
2008
, vol. 
135
 
17
(pg. 
2969
-
2979
)
85
Vijayaragavan
 
K
Szabo
 
E
Bossé
 
M
Ramos-Mejia
 
V
Moon
 
RT
Bhatia
 
M
Noncanonical Wnt signaling orchestrates early developmental events toward hematopoietic cell fate from human embryonic stem cells.
Cell Stem Cell
2009
, vol. 
4
 
3
(pg. 
248
-
262
)
86
Choi
 
KD
Vodyanik
 
MA
Togarrati
 
PP
, et al. 
Identification of the hemogenic endothelial progenitor and its direct precursor in human pluripotent stem cell differentiation cultures.
Cell Rep
2012
, vol. 
2
 
3
(pg. 
553
-
567
)
87
Lancrin
 
C
Sroczynska
 
P
Stephenson
 
C
Allen
 
T
Kouskoff
 
V
Lacaud
 
G
The haemangioblast generates haematopoietic cells through a haemogenic endothelium stage.
Nature
2009
, vol. 
457
 
7231
(pg. 
892
-
895
)
88
Risau
 
W
Flamme
 
I
Vasculogenesis.
Annu Rev Cell Dev Biol
1995
, vol. 
11
 (pg. 
73
-
91
)
89
Sakurai
 
H
Era
 
T
Jakt
 
LM
, et al. 
In vitro modeling of paraxial and lateral mesoderm differentiation reveals early reversibility.
Stem Cells
2006
, vol. 
24
 
3
(pg. 
575
-
586
)
90
Slukvin
 
II
Deciphering the hierarchy of angiohematopoietic progenitors from human pluripotent stem cells.
Cell Cycle
2013
, vol. 
12
 
5
(pg. 
720
-
727
)
91
Sturgeon
 
CM
Ditadi
 
A
Clarke
 
RL
Keller
 
G
Defining the path to hematopoietic stem cells.
Nat Biotechnol
2013
, vol. 
31
 
5
(pg. 
416
-
418
)
92
Breier
 
G
Breviario
 
F
Caveda
 
L
, et al. 
Molecular cloning and expression of murine vascular endothelial-cadherin in early stage development of cardiovascular system.
Blood
1996
, vol. 
87
 
2
(pg. 
630
-
641
)
93
Lampugnani
 
MG
Resnati
 
M
Raiteri
 
M
, et al. 
A novel endothelial-specific membrane protein is a marker of cell-cell contacts.
J Cell Biol
1992
, vol. 
118
 
6
(pg. 
1511
-
1522
)
94
Vittet
 
D
Buchou
 
T
Schweitzer
 
A
Dejana
 
E
Huber
 
P
Targeted null-mutation in the vascular endothelial-cadherin gene impairs the organization of vascular-like structures in embryoid bodies.
Proc Natl Acad Sci USA
1997
, vol. 
94
 
12
(pg. 
6273
-
6278
)
95
Nishikawa
 
SI
Nishikawa
 
S
Hirashima
 
M
Matsuyoshi
 
N
Kodama
 
H
Progressive lineage analysis by cell sorting and culture identifies FLK1+VE-cadherin+ cells at a diverging point of endothelial and hemopoietic lineages.
Development
1998
, vol. 
125
 
9
(pg. 
1747
-
1757
)
96
Nishikawa
 
SI
Nishikawa
 
S
Kawamoto
 
H
, et al. 
In vitro generation of lymphohematopoietic cells from endothelial cells purified from murine embryos.
Immunity
1998
, vol. 
8
 
6
(pg. 
761
-
769
)
97
Eilken
 
HM
Nishikawa
 
S
Schroeder
 
T
Continuous single-cell imaging of blood generation from haemogenic endothelium.
Nature
2009
, vol. 
457
 
7231
(pg. 
896
-
900
)
98
Hashimoto
 
K
Fujimoto
 
T
Shimoda
 
Y
Huang
 
X
Sakamoto
 
H
Ogawa
 
M
Distinct hemogenic potential of endothelial cells and CD41+ cells in mouse embryos.
Dev Growth Differ
2007
, vol. 
49
 
4
(pg. 
287
-
300
)
99
Wang
 
L
Li
 
L
Shojaei
 
F
, et al. 
Endothelial and hematopoietic cell fate of human embryonic stem cells originates from primitive endothelium with hemangioblastic properties.
Immunity
2004
, vol. 
21
 
1
(pg. 
31
-
41
)
100
Vodyanik
 
MA
Thomson
 
JA
Slukvin
 
II
Leukosialin (CD43) defines hematopoietic progenitors in human embryonic stem cell differentiation cultures.
Blood
2006
, vol. 
108
 
6
(pg. 
2095
-
2105
)
101
Nakajima-Takagi
 
Y
Osawa
 
M
Oshima
 
M
, et al. 
Role of SOX17 in hematopoietic development from human embryonic stem cells.
Blood
2013
, vol. 
121
 
3
(pg. 
447
-
458
)
102
Rafii
 
S
Kloss
 
CC
Butler
 
JM
, et al. 
Human ESC-derived hemogenic endothelial cells undergo distinct waves of endothelial to hematopoietic transition.
Blood
2013
, vol. 
121
 
5
(pg. 
770
-
780
)
103
North
 
T
Gu
 
TL
Stacy
 
T
, et al. 
Cbfa2 is required for the formation of intra-aortic hematopoietic clusters.
Development
1999
, vol. 
126
 
11
(pg. 
2563
-
2575
)
104
Zambidis
 
ET
Park
 
TS
Yu
 
W
, et al. 
Expression of angiotensin-converting enzyme (CD143) identifies and regulates primitive hemangioblasts derived from human pluripotent stem cells.
Blood
2008
, vol. 
112
 
9
(pg. 
3601
-
3614
)
105
Iacovino
 
M
Chong
 
D
Szatmari
 
I
, et al. 
HoxA3 is an apical regulator of haemogenic endothelium.
Nat Cell Biol
2011
, vol. 
13
 
1
(pg. 
72
-
78
)
106
Clarke
 
RL
Yzaguirre
 
AD
Yashiro-Ohtani
 
Y
, et al. 
The expression of Sox17 identifies and regulates haemogenic endothelium.
Nat Cell Biol
2013
, vol. 
15
 
5
(pg. 
502
-
510
)
107
Guiu
 
J
Shimizu
 
R
D’Altri
 
T
, et al. 
Hes repressors are essential regulators of hematopoietic stem cell development downstream of Notch signaling.
J Exp Med
2013
, vol. 
210
 
1
(pg. 
71
-
84
)
108
Lee
 
JB
Werbowetski-Ogilvie
 
TE
Lee
 
JH
, et al. 
Notch-HES1 signaling axis controls hemato-endothelial fate decisions of human embryonic and induced pluripotent stem cells.
Blood
 
2013. 122(7):1162-1173
109
Ng
 
ES
Davis
 
RP
Azzola
 
L
Stanley
 
EG
Elefanty
 
AG
Forced aggregation of defined numbers of human embryonic stem cells into embryoid bodies fosters robust, reproducible hematopoietic differentiation.
Blood
2005
, vol. 
106
 
5
(pg. 
1601
-
1603
)
110
Kopher
 
RA
Penchev
 
VR
Islam
 
MS
Hill
 
KL
Khosla
 
S
Kaufman
 
DS
Human embryonic stem cell-derived CD34+ cells function as MSC progenitor cells.
Bone
2010
, vol. 
47
 
4
(pg. 
718
-
728
)
111
Manjunath
 
N
Correa
 
M
Ardman
 
M
Ardman
 
B
Negative regulation of T-cell adhesion and activation by CD43.
Nature
1995
, vol. 
377
 
6549
(pg. 
535
-
538
)
112
Ferkowicz
 
MJ
Starr
 
M
Xie
 
X
, et al. 
CD41 expression defines the onset of primitive and definitive hematopoiesis in the murine embryo.
Development
2003
, vol. 
130
 
18
(pg. 
4393
-
4403
)
113
Mikkola
 
HK
Fujiwara
 
Y
Schlaeger
 
TM
Traver
 
D
Orkin
 
SH
Expression of CD41 marks the initiation of definitive hematopoiesis in the mouse embryo.
Blood
2003
, vol. 
101
 
2
(pg. 
508
-
516
)
114
Otani
 
T
Inoue
 
T
Tsuji-Takayama
 
K
, et al. 
Progenitor analysis of primitive erythropoiesis generated from in vitro culture of embryonic stem cells.
Exp Hematol
2005
, vol. 
33
 
6
(pg. 
632
-
640
)
115
Klimchenko
 
O
Mori
 
M
Distefano
 
A
, et al. 
A common bipotent progenitor generates the erythroid and megakaryocyte lineages in embryonic stem cell-derived primitive hematopoiesis.
Blood
2009
, vol. 
114
 
8
(pg. 
1506
-
1517
)
116
Dias
 
J
Gumenyuk
 
M
Kang
 
H
, et al. 
Generation of red blood cells from human induced pluripotent stem cells.
Stem Cells Dev
2011
, vol. 
20
 
9
(pg. 
1639
-
1647
)
117
Smith
 
BW
Rozelle
 
SS
Leung
 
A
, et al. 
The aryl hydrocarbon receptor directs hematopoietic progenitor cell expansion and differentiation.
Blood
2013
, vol. 
122
 
3
(pg. 
376
-
385
)
118
Chang
 
KH
Bonig
 
H
Papayannopoulou
 
T
 
Generation and characterization of erythroid cells from human embryonic stem cells and induced pluripotent stem cells: an overview. Stem Cells Int. 2011;2011:791604
119
Takayama
 
N
Eto
 
K
Pluripotent stem cells reveal the developmental biology of human megakaryocytes and provide a source of platelets for clinical application.
Cell Mol Life Sci
2012
, vol. 
69
 
20
(pg. 
3419
-
3428
)
120
Chang
 
CJ
Mitra
 
K
Koya
 
M
, et al. 
Production of embryonic and fetal-like red blood cells from human induced pluripotent stem cells.
PLoS ONE
2011
, vol. 
6
 
10
pg. 
e25761
 
121
Kobari
 
L
Yates
 
F
Oudrhiri
 
N
, et al. 
Human induced pluripotent stem cells can reach complete terminal maturation: in vivo and in vitro evidence in the erythropoietic differentiation model.
Haematologica
2012
, vol. 
97
 
12
(pg. 
1795
-
1803
)
122
Salvagiotto
 
G
Zhao
 
Y
Vodyanik
 
M
, et al. 
Molecular profiling reveals similarities and differences between primitive subsets of hematopoietic cells generated in vitro from human embryonic stem cells and in vivo during embryogenesis.
Exp Hematol
2008
, vol. 
36
 
10
(pg. 
1377
-
1389
)
123
Ran
 
D
Shia
 
WJ
Lo
 
MC
, et al. 
RUNX1a enhances hematopoietic lineage commitment from human embryonic stem cells and inducible pluripotent stem cells.
Blood
2013
, vol. 
121
 
15
(pg. 
2882
-
2890
)
124
Carpenter
 
L
Malladi
 
R
Yang
 
CT
, et al. 
Human induced pluripotent stem cells are capable of B-cell lymphopoiesis.
Blood
2011
, vol. 
117
 
15
(pg. 
4008
-
4011
)
125
Woll
 
PS
Martin
 
CH
Miller
 
JS
Kaufman
 
DS
Human embryonic stem cell-derived NK cells acquire functional receptors and cytolytic activity.
J Immunol
2005
, vol. 
175
 
8
(pg. 
5095
-
5103
)
126
Knorr
 
DA
Ni
 
Z
Hermanson
 
D
, et al. 
 
Clinical-scale derivation of natural killer cells from human pluripotent stem cells for cancer therapy. Stem Cells Transl Med. 2013;2(4):274-283
127
Galic
 
Z
Kitchen
 
SG
Kacena
 
A
, et al. 
 
T lineage differentiation from human embryonic stem cells. Proc Natl Acad Sci U S A. 2006;103(31):11742-11747. Epub 12006 Jul 11714
128
Timmermans
 
F
Velghe
 
I
Vanwalleghem
 
L
, et al. 
Generation of T cells from human embryonic stem cell-derived hematopoietic zones.
J Immunol
2009
, vol. 
182
 
11
(pg. 
6879
-
6888
)
129
Vizcardo
 
R
Masuda
 
K
Yamada
 
D
, et al. 
Regeneration of human tumor antigen-specific T cells from iPSCs derived from mature CD8(+) T cells.
Cell Stem Cell
2013
, vol. 
12
 
1
(pg. 
31
-
36
)
130
Lu
 
M
Kardel
 
MD
O’Connor
 
MD
Eaves
 
CJ
Enhanced generation of hematopoietic cells from human hepatocarcinoma cell-stimulated human embryonic and induced pluripotent stem cells.
Exp Hematol
2009
, vol. 
37
 
8
(pg. 
924
-
936
)
131
Wang
 
L
Menendez
 
P
Shojaei
 
F
, et al. 
Generation of hematopoietic repopulating cells from human embryonic stem cells independent of ectopic HOXB4 expression.
J Exp Med
2005
, vol. 
201
 
10
(pg. 
1603
-
1614
)
132
Ledran
 
MH
Krassowska
 
A
Armstrong
 
L
, et al. 
Efficient hematopoietic differentiation of human embryonic stem cells on stromal cells derived from hematopoietic niches.
Cell Stem Cell
2008
, vol. 
3
 
1
(pg. 
85
-
98
)
133
Narayan
 
AD
Chase
 
JL
Lewis
 
RL
, et al. 
Human embryonic stem cell-derived hematopoietic cells are capable of engrafting primary as well as secondary fetal sheep recipients.
Blood
2006
, vol. 
107
 
5
(pg. 
2180
-
2183
)
134
Tian
 
X
Woll
 
PS
Morris
 
JK
Linehan
 
JL
Kaufman
 
DS
Hematopoietic engraftment of human embryonic stem cell-derived cells is regulated by recipient innate immunity.
Stem Cells
2006
, vol. 
24
 
5
(pg. 
1370
-
1380
)
135
Risueño
 
RM
Sachlos
 
E
Lee
 
JH
, et al. 
Inability of human induced pluripotent stem cell-hematopoietic derivatives to downregulate microRNAs in vivo reveals a block in xenograft hematopoietic regeneration.
Stem Cells
2012
, vol. 
30
 
2
(pg. 
131
-
139
)
136
Tian
 
X
Hexum
 
MK
Penchev
 
VR
Taylor
 
RJ
Shultz
 
LD
Kaufman
 
DS
Bioluminescent imaging demonstrates that transplanted human embryonic stem cell-derived CD34(+) cells preferentially develop into endothelial cells.
Stem Cells
2009
, vol. 
27
 
11
(pg. 
2675
-
2685
)
137
Amabile
 
G
Welner
 
RS
Nombela-Arrieta
 
C
, et al. 
In vivo generation of transplantable human hematopoietic cells from induced pluripotent stem cells.
Blood
2013
, vol. 
121
 
8
(pg. 
1255
-
1264
)
138
Suzuki
 
N
Yamazaki
 
S
Yamaguchi
 
T
, et al. 
 
Generation of engraftable hematopoietic stem cells from induced pluripotent stem cells by way of teratoma formation. Mol Ther. 2013;21(7):1424-1431
139
Medvinsky
 
A
Rybtsov
 
S
Taoudi
 
S
Embryonic origin of the adult hematopoietic system: advances and questions.
Development
2011
, vol. 
138
 
6
(pg. 
1017
-
1031
)
140
Yoshimoto
 
M
Porayette
 
P
Glosson
 
NL
, et al. 
Autonomous murine T-cell progenitor production in the extra-embryonic yolk sac before HSC emergence.
Blood
2012
, vol. 
119
 
24
(pg. 
5706
-
5714
)
141
Yoshimoto
 
M
Montecino-Rodriguez
 
E
Ferkowicz
 
MJ
, et al. 
Embryonic day 9 yolk sac and intra-embryonic hemogenic endothelium independently generate a B-1 and marginal zone progenitor lacking B-2 potential.
Proc Natl Acad Sci USA
2011
, vol. 
108
 
4
(pg. 
1468
-
1473
)
142
Taoudi
 
S
Medvinsky
 
A
Functional identification of the hematopoietic stem cell niche in the ventral domain of the embryonic dorsal aorta.
Proc Natl Acad Sci USA
2007
, vol. 
104
 
22
(pg. 
9399
-
9403
)
143
Ciau-Uitz
 
A
Walmsley
 
M
Patient
 
R
Distinct origins of adult and embryonic blood in Xenopus.
Cell
2000
, vol. 
102
 
6
(pg. 
787
-
796
)
144
Samokhvalov
 
IM
Samokhvalova
 
NI
Nishikawa
 
S
Cell tracing shows the contribution of the yolk sac to adult haematopoiesis.
Nature
2007
, vol. 
446
 
7139
(pg. 
1056
-
1061
)
145
Weissman
 
IL
Papaioannou
 
V
Grdner
 
R
Clarkson
 
B
Marks
 
PA
Till
 
JE
Fetal hematopoietic origins of the adult hematolymphoid cells.
Differentiation of Normal and Neoplastic Cells
1978
New York
Cold Spring Harbor Laboratory Press
(pg. 
33
-
47
)
146
Yoder
 
MC
Hiatt
 
K
Dutt
 
P
Mukherjee
 
P
Bodine
 
DM
Orlic
 
D
Characterization of definitive lymphohematopoietic stem cells in the day 9 murine yolk sac.
Immunity
1997
, vol. 
7
 
3
(pg. 
335
-
344
)
147
Yoder
 
MC
Hiatt
 
K
Mukherjee
 
P
In vivo repopulating hematopoietic stem cells are present in the murine yolk sac at day 9.0 postcoitus.
Proc Natl Acad Sci USA
1997
, vol. 
94
 
13
(pg. 
6776
-
6780
)
148
Zape
 
JP
Zovein
 
AC
Hemogenic endothelium: origins, regulation, and implications for vascular biology.
Semin Cell Dev Biol
2011
, vol. 
22
 
9
(pg. 
1036
-
1047
)
149
Chen
 
MJ
Li
 
Y
De Obaldia
 
ME
, et al. 
Erythroid/myeloid progenitors and hematopoietic stem cells originate from distinct populations of endothelial cells.
Cell Stem Cell
2011
, vol. 
9
 
6
(pg. 
541
-
552
)
150
Wang
 
Y
Yates
 
F
Naveiras
 
O
Ernst
 
P
Daley
 
GQ
Embryonic stem cell-derived hematopoietic stem cells.
Proc Natl Acad Sci USA
2005
, vol. 
102
 
52
(pg. 
19081
-
19086
)
151
McGrath
 
KE
Palis
 
J
Expression of homeobox genes, including an insulin promoting factor, in the murine yolk sac at the time of hematopoietic initiation.
Mol Reprod Dev
1997
, vol. 
48
 
2
(pg. 
145
-
153
)
152
McKinney-Freeman
 
S
Cahan
 
P
Li
 
H
, et al. 
The transcriptional landscape of hematopoietic stem cell ontogeny.
Cell Stem Cell
2012
, vol. 
11
 
5
(pg. 
701
-
714
)
153
Lee
 
GS
Kim
 
BS
Sheih
 
JH
Moore
 
M
Forced expression of HoxB4 enhances hematopoietic differentiation by human embryonic stem cells.
Mol Cells
2008
, vol. 
25
 
4
(pg. 
487
-
493
)
154
Dou
 
DR
Minasian
 
A
Sierra
 
M
, et al. 
Inability to express HOXA cluster and BCL11A genes compromises self-renewal and multipotency of hESC-derived hematopoietic cells.
 
In: Proceedings from American Society of Hematology 54th Annual Meeting; December 8-11, 2012; Atlanta, GA. Abstract 1190
155
Shojaei
 
F
Menendez
 
P
Molecular profiling of candidate human hematopoietic stem cells derived from human embryonic stem cells.
Exp Hematol
2008
, vol. 
36
 
11
(pg. 
1436
-
1448
)
156
Schnerch
 
A
Lee
 
JB
Graham
 
M
Guezguez
 
B
Bhatia
 
M
Human embryonic stem cell-derived hematopoietic cells maintain core epigenetic machinery of the polycomb group/Trithorax Group complexes distinctly from functional adult hematopoietic stem cells.
Stem Cells Dev
2013
, vol. 
22
 
1
(pg. 
73
-
89
)
157
Pereira
 
CF
Chang
 
B
Qiu
 
J
, et al. 
Induction of a hemogenic program in mouse fibroblasts.
Cell Stem Cell
2013
, vol. 
13
 
2
(pg. 
205
-
218
)
158
Szabo
 
E
Rampalli
 
S
Risueño
 
RM
, et al. 
Direct conversion of human fibroblasts to multilineage blood progenitors.
Nature
2010
, vol. 
468
 
7323
(pg. 
521
-
526
)