Efforts to derive hematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) are complicated by the fact that embryonic hematopoiesis consists of two programs, primitive and definitive, that differ in developmental potential. As only definitive hematopoiesis generates HSCs, understanding how this program develops is essential for being able to produce this cell population in vitro. Here we show that both hematopoietic programs transition through hemogenic endothelial intermediates and develop from KDR+CD34−CD144− progenitors that are distinguished by CD235a expression. Generation of primitive progenitors (KDR+CD235a+) depends on stage-specific Activin-nodal signaling and inhibition of the Wnt-β-catenin pathway, whereas specification of definitive progenitors (KDR+CD235a−) requires Wnt-β-catenin signaling during this same time frame. Together, these findings establish simple selective differentiation strategies for the generation of primitive or definitive hematopoietic progenitors via Wnt-β-catenin manipulation, and in doing so provide access to enriched populations for future studies on hPSC-derived hematopoietic development.

The efficient generation of hematopoietic stem cells from human pluripotent stem cells is dependent on the appropriate specification of the definitive hematopoietic program during differentiation. In this study, we used T lymphocyte potential to track the onset of definitive hematopoiesis from human embryonic and induced pluripotent stem cells differentiated with specific morphogens in serum- and stromal-free cultures. We show that this program develops from a progenitor population with characteristics of hemogenic endothelium, including the expression of CD34, VE-cadherin, GATA2, LMO2, and RUNX1. Along with T cells, these progenitors display the capacity to generate myeloid and erythroid cells. Manipulation of Activin/Nodal signaling during early stages of differentiation revealed that development of the definitive hematopoietic progenitor population is not dependent on this pathway, distinguishing it from primitive hematopoiesis. Collectively, these findings demonstrate that it is possible to generate T lymphoid progenitors from pluripotent stem cells and that this lineage develops from a population whose emergence marks the onset of human definitive hematopoiesis.

A variety of tissue lineages can be differentiated from pluripotent stem cells by mimicking embryonic development through stepwise exposure to morphogens, or by conversion of one differentiated cell type into another by enforced expression of master transcription factors. Here, to yield functional human haematopoietic stem cells, we perform morphogen-directed differentiation of human pluripotent stem cells into haemogenic endothelium followed by screening of 26 candidate haematopoietic stem-cell-specifying transcription factors for their capacity to promote multi-lineage haematopoietic engraftment in mouse hosts. We recover seven transcription factors (ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1 and SPI1) that are sufficient to convert haemogenic endothelium into haematopoietic stem and progenitor cells that engraft myeloid, B and T cells in primary and secondary mouse recipients. Our combined approach of morphogen-driven differentiation and transcription-factor-mediated cell fate conversion produces haematopoietic stem and progenitor cells from pluripotent stem cells and holds promise for modelling haematopoietic disease in humanized mice and for therapeutic strategies in genetic blood disorders.

The generation of haematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) will depend on the accurate recapitulation of embryonic haematopoiesis. In the early embryo, HSCs develop from the haemogenic endothelium (HE) and are specified in a Notch-dependent manner through a process named endothelial-to-haematopoietic transition (EHT). As HE is associated with arteries, it is assumed that it represents a subpopulation of arterial vascular endothelium (VE). Here we demonstrate at a clonal level that hPSC-derived HE and VE represent separate lineages. HE is restricted to the CD34+CD73−CD184− fraction of day 8 embryoid bodies (EBs) and it undergoes a NOTCH-dependent EHT to generate RUNX1C+ cells with multilineage potential. Arterial and venous VE progenitors, by contrast, segregate to the CD34+CD73medCD184+ and CD34+CD73hiCD184− fractions, respectively. Together, these findings identify HE as distinct from VE and provide a platform for defining the signalling pathways that regulate their specification to functional HSCs.

We identified TGFbeta3 as the disease gene involved in ARVD1. The identification of a novel ARVC gene will increase the power of the genetic screening for early diagnosis of asymptomatic carriers among relatives of ARVC patients.

Corresponding author: m.cavazzana@nck.aphp.fr, Professor Marina Cavazzana-Calvo, Biotherapy Department, Hopital Necker EnfantsMalades, 149 rue de Sevres, F-75015 Paris, France; tel. +33 1 44 49 50 68; fax +33 1 44 49 25 05. 18 These authors contributed equally to this work 19 These authors contributed equally to this work Author contributions: C.L.-P. and E.M.S contributed equally to this study by performing most of the experimental work and analysis, with the assistance of C.D.-C. and E.M. C.Picard and F.R.-L. performed apoptotic tests on the fibroblasts, gave critical advices and comments in designing the experiments. Experiments shown in Fig. 4 were performed by V.M. A.D. performed the RNA interference experiments. F.V. provided expertise in histological examination. K.L.S.-S. mapped P6 deletion and found P7 mutation. J.C.M. performed the sequencing project. C.B. performed the genome-wide linkage scan. C.Picard, L.M.N., N.M.W., A.F., M.M.A. and M.C.-C. recruited and diagnosed the RD patients and provide materials for them. F.Calvo gave critical comments in designing the experiments and helped to sequence the healthy and pathological samples. C.Petit contributed to the design of the inner ear experiments, F.Candotti designed and coordinated the sequencing project. C. Picard and L.A. performed the Lodscore analysis. L.A. and A.F. contributed equally to this study. M.C.-C. supervised the overall project. M.C.-C., C.L.-P., E.M.S L.A. and A.F. wrote the paper and added the comments from all authors. Author information: All authors declare that there is no competing financial interests. Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. Correspondence and requests for materials should be addressed to Marina Cavazzana-Calvo (m.cavazzana@nck.aphp.fr). NIH Public Access Author ManuscriptNat Genet. Author manuscript; available in PMC 2010 January 1. Published in final edited form as:Nat Genet. We have identified biallelic mutations in the adenylate kinase 2 (AK2) gene in seven patients affected with RD. These mutations resulted in the absence or a strong decrease in protein expression. We then demonstrated that restoration of AK2 expression in the bone marrow cells of RD patients overcomes the neutrophil differentiation arrest underlining its specific requirement in the development of a restricted set of haematopoietic lineages. Lastly, we established that AK2 is specifically expressed in the stria vascularis region of the inner ear, which provides an explanation to the sensorineural deafness. These results suggest a novel mechanism regulating haematopoetic cell differentiation, and involved in one of the most severe human immunodeficiency syndromes.The term "reticular dysgenesis" (RD), was coined in 1959 by de Vall and Seyneheve 1 and relates to the histological findings in primary and secondary lymphohaematopoietic organs, where the scarcity of cells highlights the prominent reticular tissue framework. The lack of polymorphonuclear neutrophils (PMNs) in affected patien...

Hematopoietic stem cells (HSCs) develop from a specialized subpopulation of endothelial cells known as hemogenic endothelium (HE). Although the HE origin of HSCs is now well established in different species, the signaling pathways that control this transition remain poorly understood. Here, we show that activation of retinoic acid (RA) signaling in aorta-gonad-mesonephros-derived HE ex vivo dramatically enhanced its HSC potential, whereas conditional inactivation of the RA metabolizing enzyme retinal dehydrogenase 2 in VE-cadherin expressing endothelial cells in vivo abrogated HSC development. Wnt signaling completely blocked the HSC inductive effects of RA modulators, whereas inhibition of the pathway promoted the development of HSCs in the absence of RA signaling. Collectively, these findings position RA and Wnt signaling as key regulators of HSC development and in doing so provide molecular insights that will aid in developing strategies for their generation from pluripotent stem cells.

Human pluripotent stem cells (hPSCs) provide an unparalleled opportunity to establish in vitro differentiation models that will transform our approach to the study of human development. In the case of the blood system, these models will enable investigation of the earliest stages of human embryonic haematopoiesis that was previously not possible. In addition, they will provide platforms for studying the origins of human blood cell diseases and for generating de novo haematopoietic stem and progenitor cell populations for cell-based regenerative therapies.