NOTCH signaling specifies arterial-type definitive hemogenic endothelium from human pluripotent stem cells.

NOTCH signaling is required for the arterial specification and formation of hematopoietic stem cells (HSCs) and lympho-myeloid progenitors in the embryonic aorta-gonad-mesonephros region and extraembryonic vasculature from a distinct lineage of vascular endothelial cells with hemogenic potential. However, the role of NOTCH signaling in hemogenic endothelium (HE) specification from human pluripotent stem cell (hPSC) has not been studied. Here, using a chemically defined hPSC differentiation system combined with the use of DLL1-Fc and DAPT to manipulate NOTCH, we discover that NOTCH activation in hPSC-derived immature HE progenitors leads to formation of CD144+CD43-CD73-DLL4+Runx1 + 23-GFP+ arterial-type HE, which requires NOTCH signaling to undergo endothelial-to-hematopoietic transition and produce definitive lympho-myeloid and erythroid cells. These findings demonstrate that NOTCH-mediated arterialization of HE is an essential prerequisite for establishing definitive lympho-myeloid program and suggest that exploring molecular pathways that lead to arterial specification may aid in vitro approaches to enhance definitive hematopoiesis from hPSCs.

BCR/ABL oncogene-induced PI3K signaling pathway leads to chronic myeloid leukemia pathogenesis by impairing immuno-modulatory function of hemangioblasts.

An increasing number of studies indicate that during development, endothelial and hematopoietic cells derive from common progenitors named hemangioblasts that have important roles in the pathogenesis. This is particularly true in chronic myeloid leukemia (CML). Here, we isolated fetal liver kinase-1-positive (Flk1(+)) cells from CML patients and found they expressed BCR/ABL-specific CML oncogene. We examined their biological characteristics as well as immunological functions and further detected the possible molecular mechanism involved in the leukemia genesis. We showed that CML patient-derived Flk1(+)CD31(-)CD34(-) mesenchymal stem cells (MSCs) had normal morphology, phenotype and karyotype but appeared impaired immuno-modulatory function. The capacity of Flk1(+)CD31(-)CD34(-) MSCs from CML patients to inhibit T lymphocyte activation and proliferation was impaired in vitro. CML patient-derived MSCs have dampening immuno-modulatory functions, suggesting that the dysregulation of hematopoiesis and immune response might originate from MSCs rather than hematopoietic stem cells (HSCs). These Ph(+) putative CML hemangioblast upregulated TGF-ß1 and resultantly activated matrix metalloproteinase-9 (MMP-9) to enhance s-KitL and s-ICAM-1 secretion, which activated c-kit(+) HSCs from the quiescent state to the proliferative state. Further studies showed that phosphatidylinositol-3 kinase (PI3K)/Akt/nuclear factor (NF)-κB signaling pathway was involved in CML pathogenesis. Flk1(+)CD31(-)CD34(-) MSCs that express BCR/ABL leukemia oncogene are hemangioblasts and they have a critical role in the progression of CML through PI3K/Akt/NF-κB signaling pathway.

Adult human circulating CD34⁻Lin⁻CD45⁻CD133⁻ cells can differentiate into hematopoietic and endothelial cells.

A precise identification of adult human hemangioblast is still lacking. To identify circulating precursors having the developmental potential of the hemangioblast, we established a new ex vivo long-term culture model supporting the differentiation of both hematopoietic and endothelial cell lineages. We identified from peripheral blood a population lacking the expression of CD34, lineage markers, CD45 and CD133 (CD34⁻Lin⁻CD45⁻CD133⁻ cells), endowed with the ability to differentiate after a 6-week culture into both hematopoietic and endothelial lineages. The bilineage potential of CD34⁻Lin⁻CD45⁻CD133⁻ cells was determined at the single-cell level in vitro and was confirmed by transplantation into NOD/SCID mice. In vivo, CD34⁻Lin⁻CD45⁻CD133⁻ cells showed the ability to reconstitute hematopoietic tissue and to generate functional endothelial cells that contribute to new vessel formation during tumor angiogenesis. Molecular characterization of CD34⁻Lin⁻D45⁻CD133⁻ cells unveiled a stem cell profile compatible with both hematopoietic and endothelial potentials, characterized by the expression of c-Kit and CXCR4 as well as EphB4, EphB2, and ephrinB2. Further molecular and functional characterization of CD34⁻Lin⁻CD45⁻CD133⁻ cells will help dissect their physiologic role in blood and blood vessel maintenance and repair in adult life.

Original immunophenotype of blood endothelial progenitor cells and microparticles in patients with isolated arterial erectile dysfunction and late onset hypogonadism: effects of androgen replacement therapy.

OBJECTIVE: Blood endothelial progenitor cells (EPCs) and endothelial microparticles (EMPs) have been proposed as markers of endothelial dysfunction. Aim of this study was to evaluate an original immunophenotype of EPCs and EMPs in patients with isolated arterial erectile dysfunction (ED) and late onset hypogonadism (LOH) before and after androgen replacement therapy. MATERIALS AND METHODS: Fifty patients (50-64 years) with ED and LOH were selected. EPC (CD45(neg)/CD34(pos)/CD144(pos)) and EMP (CD45(neg)/CD34(neg)/CD144(pos)) blood concentrations were evaluated by flow cytometry. Thirty patients received androgen replacement therapy (Tostrex® ProStrakan) for 6 months (group A), other 20 patients not received androgen therapy for the contraindications in their clinical history (group B). RESULTS: After 6 months, group B showed IIEF-5 score, peak systolic velocity and acceleration time significantly worse than group A; in addition EPCs and EMPs were significantly higher in group B compared to group A. CONCLUSIONS: Patients with isolated arterial ED and LOH not treated with androgen therapy showed worst vascular parameters measured by penile Doppler and higher EPCs and EMPs compared to treated hypogonadal patients, hence, LOH appears to be an additional vascular risk factor, and these markers may be considered as predictors of cavernous artery disease. Finally, androgen therapy improves endothelial dysfunction.

Major histocompatibility complex-I expression on embryonic stem cell-derived vascular progenitor cells is critical for syngeneic transplant survival.

Donor-recipient cell interactions are essential for functional engraftment after nonautologous cell transplantation. During this process, transplant engraftment is characterized and defined by interactions between transplanted cells with local and recruited inflammatory cells. The outcome of these interactions determines donor cell fate. Here, we provide evidence that lineage-committed embryonic stem cell (ESC)-derived vascular progenitor cells are the target of major histocompatibility complex (MHC) class I-dependent, natural killer (NK) cell-mediated elimination in vitro and in vivo. Treatment with interferon γ was found to significantly upregulate MHC class I expression on ESC-derived vascular progenitor cells, rendering them less susceptible to syngeneic NK cell-mediated killing in vitro and enhancing their survival and differentiation potential in vivo. Furthermore, in vivo ablation of NK cells led to enhanced progenitor cell survival after transplantation into a syngeneic murine ischemic hindlimb model, providing additional evidence that NK cells mediate ESC-derived progenitor cell transplant rejection. These data highlight the importance of recipient immune-donor cell interactions, and indicate a functional role for MHC-I antigen expression during successful ESC-derived syngeneic transplant engraftment.

Human embryonic stem cells hemangioblast express HLA-antigens.

BACKGROUND: It has been suggested that the initial differentiation of endothelial and hematopoietic cells during embryogenesis occurs from a common progenitor, called hemangioblast (hB). We hypothesized that these cells with dual hematopoietic/endothelial potential could be used in future regenerative medicine. METHODS: We used the two-step differentiation technology to generate bipotential blast cells from human embryonic stem cells (hES). This involved short differentiation in our in vitro EB system followed by differentiation in semisolid culture medium supplemented with mixture of cytokines. RESULTS: The occurrence of blast-colony-forming cells (BL-CFC) during EB differentiation (day 0-6) was transient and peaked on day 3. The emergence of this event was associated with expression of mesoderm gene T, and inversely correlated with expression of endoderm gene FoxA2. Similarly, the highest BL-CFC number was associated with increase in expression of early hematopoietic/endothelial genes: CD34, CD31 and KDR. The derived colonies were composed of 30-50 blast cells on day 6 in culture. These cells had homogenous appearance in Wright-Giemsa stain, but to a different extent expressed markers of immature hematopoietic and endothelial cells (CD31, CD34, VE-cadherin, Flt-1) and mature differentiated cells (CD45, CD33, CD146). We found that some of them expressed fetal and embryonic globin genes. Interestingly, these cells expressed also HLA class I molecules, however at very low levels compared to endothelial and hematopoietic cells. The blast cells could be successfully differentiated to hematopoietic cells in a CFU assay. In these conditions, blast cells formed CFU-M colonies (63.4 +/- 0.8%) containing macrophages, BFU-E colonies (19.5 +/- 3.5%) containing nucleated red blood cells, and CFU-EM colonies (17.1 +/- 2.7%) composed of macrophages and nucleated erythrocytes. Cells of CFU-EM and BFU-E colonies expressed both epsilon - and gamma- globin genes, but not adult-type gamma-globin. When in endothelial cell culture conditions, blast cells differentiated to endothelial cells which had the ability to take up Dil-Ac-LDL and to form complex vascular networks in Matrigel. CONCLUSION: 1) Hematoendothelial precursors exist transiently in early embryonic development and form single cell-derived colonies; 2) their differentiation can be tracked by the use of chosen molecular markers; 3) blast colonies consist of cells having properties of endothelial and hematopoietic precursors, however the issue of their ability to maintain dual properties over time needs to be further explored; 4) blast cells can potentially be used in regenerative medicine due to their low expression of HLA molecules.