Rescue of hematopoietic stem/progenitor cells formation in plcg1 zebrafish mutant.

Hematopoietic stem/progenitor cells (HSPC) in zebrafish emerge from the aortic hemogenic endothelium (HE) and migrate towards the caudal hematopoietic tissue (CHT), where they expand and differentiate during definitive hematopoiesis. Phospholipase C gamma 1 (Plcγ1) has been implicated for hematopoiesis in vivo and in vitro and is also required to drive arterial and HSPC formation. Genetic mutation in plcg1-/- (y10 allele) completely disrupts the aortic blood flow, specification of arterial fate, and HSPC formation in zebrafish embryos. We previously demonstrated that ginger treatment promoted definitive hematopoiesis via Bmp signaling. In this paper, we focus on HSPC development in plcg1-/- mutants and show that ginger/10-gingerol (10-G) can rescue the expression of arterial and HSPC markers in the HE and CHT in plcg1-/- mutant embryos. We demonstrate that ginger can induce scl/runx1 expression, and that rescued HE fate is dependent on Bmp and Notch. Bmp and Notch are known to regulate nitric oxide (NO) production and NO can induce hematopoietic stem cell fate. We show that ginger produces a robust up-regulation of NO. Taken together, we suggest in this paper that Bmp, Notch and NO are potential players that mediate the effect of ginger/10-G for rescuing the genetic defects in blood vessel specification and HSPC formation in plcg1-/- mutants. Understanding the molecular mechanisms of HSPC development in vivo is critical for understanding HSPC expansion, which will have a positive impact in regenerative medicine.

Ethanol exposure leads to disorder of blood island formation in early chick embryo.

Ethanol's effect on embryonic vasculogenesis and its underlying mechanism is obscure. Using VE-cadherin in situ hybridization, we found blood islands formation was inhibited in area opaca, but abnormal VE-cadherin+ cells were seen in area pellucida. We hypothesise ethanol may affect blood island progenitor cell migration and differentiation. DiI and in vitro experiments revealed ethanol inhibited cell migration, Quantitative PCR analysis revealed that ethanol exposure enhanced cell differentiation in area pellucida of HH5 chick embryos and repressed cell differentiation in area pellucida of HH8 chick embryos. By exposing to 2,2'-azobis-amidinopropane dihydrochloride, a ROS inducer, which gave a similar anti-vasculogenesis effect as ethanol and this anti-vasculogenesis effect could be reversed by vitamin C. Overall, exposing early chick embryos to ethanol represses blood island progenitor cell migration but disturbed differentiation at a different stage, so that the disorder of blood island formation occurs through excess ROS production and altered vascular-associated gene expression.

Nitric Oxide Donor Molsidomine Positively Modulates Myogenic Differentiation of Embryonic Endothelial Progenitors.

Embryonic VE-Cadherin-expressing progenitors (eVE-Cad+), including hemogenic endothelium, have been shown to generate hematopoietic stem cells and a variety of other progenitors, including mesoangioblasts, or MABs. MABs are vessel-associated progenitors with multilineage mesodermal differentiation potential that can physiologically contribute to skeletal muscle development and regeneration, and have been used in an ex vivo cell therapy setting for the treatment of muscular dystrophy. There is currently a therapeutic need for molecules that could improve the efficacy of cell therapy protocols; one such good candidate is nitric oxide. Several studies in animal models of muscle dystrophy have demonstrated that nitric oxide donors provide several beneficial effects, including modulation of the activity of endogenous cell populations involved in muscle repair and the delay of muscle degeneration. Here we used a genetic lineage tracing approach to investigate whether the therapeutic effect of nitric oxide in muscle repair could derive from an improvement in the myogenic differentiation of eVE-Cad+ progenitors during embryogenesis. We show that early in vivo treatment with the nitric oxide donor molsidomine enhances eVE-Cad+ contribution to embryonic and fetal myogenesis, and that this effect could originate from a modulation of the properties of yolk sac hemogenic endothelium.

An expandable, inducible hemangioblast state regulated by fibroblast growth factor.

During development, the hematopoietic and vascular lineages are thought to descend from common mesodermal progenitors called hemangioblasts. Here we identify six transcription factors, Gata2, Lmo2, Mycn, Pitx2, Sox17, and Tal1, that "trap" murine cells in a proliferative state and endow them with a hemangioblast potential. These "expandable" hemangioblasts (eHBs) are capable, once released from the control of the ectopic factors, to give rise to functional endothelial cells, multilineage hematopoietic cells, and smooth muscle cells. The eHBs can be derived from embryonic stem cells, from fetal liver cells, or poorly from fibroblasts. The eHBs reveal a central role for fibroblast growth factor, which not only promotes their expansion, but also facilitates their ability to give rise to endothelial cells and leukocytes, but not erythrocytes. This study serves as a demonstration that ephemeral progenitor states can be harnessed in vitro, enabling the creation of tractable progenitor cell lines.

Human haemato-endothelial precursors: cord blood CD34+ cells produce haemogenic endothelium.

Embryologic and genetic evidence suggest a common origin of haematopoietic and endothelial lineages. In the murine embryo, recent studies indicate the presence of haemogenic endothelium and of a common haemato-endothelial precursor, the haemangioblast. Conversely, so far, little evidence supports the presence of haemogenic endothelium and haemangioblasts in later stages of development. Our studies indicate that human cord blood haematopoietic progenitors (CD34+45+144-), triggered by murine hepatocyte conditioned medium, differentiate into adherent proliferating endothelial precursors (CD144+CD105+CD146+CD31+CD45-) capable of functioning as haemogenic endothelium. These cells, proven to give rise to functional vasculature in vivo, if further instructed by haematopoietic growth factors, first switch to transitional CD144+45+ cells and then to haematopoietic cells. These results highlight the plasticity of haemato-endhothelial precursors in human post-natal life. Furthermore, these studies may provide highly enriched populations of human post-fetal haemogenic endothelium, paving the way for innovative projects at a basic and possibly clinical level.

APELIN promotes hematopoiesis from human embryonic stem cells.

Transcriptional profiling of differentiating human embryonic stem cells (hESCs) revealed that MIXL1-positive mesodermal precursors were enriched for transcripts encoding the G-protein-coupled APELIN receptor (APLNR). APLNR-positive cells, identified by binding of the fluoresceinated peptide ligand, APELIN (APLN), or an anti-APLNR mAb, were found in both posterior mesoderm and anterior mesendoderm populations and were enriched in hemangioblast colony-forming cells (Bl-CFC). The addition of APLN peptide to the media enhanced the growth of embryoid bodies (EBs), increased the expression of hematoendothelial genes in differentiating hESCs, and increased the frequency of Bl-CFCs by up to 10-fold. Furthermore, APLN peptide also synergized with VEGF to promote the growth of hESC-derived endothelial cells. These studies identified APLN as a novel growth factor for hESC-derived hematopoietic and endothelial cells.

TGFß inhibition enhances the generation of hematopoietic progenitors from human ES cell-derived hemogenic endothelial cells using a stepwise strategy.

Embryonic hematopoiesis is a complex process. Elucidating the mechanism regulating hematopoietic differentiation from pluripotent stem cells would allow us to establish a strategy to efficiently generate hematopoietic cells. However, the mechanism governing the generation of hematopoietic progenitors from human embryonic stem cells (hESCs) remains unknown. Here, on the basis of the emergence of CD43(+) hematopoietic cells from hemogenic endothelial (HE) cells, we demonstrated that VEGF was essential and sufficient, and that bFGF was synergistic with VEGF to specify the HE cells and the subsequent transition into CD43(+) hematopoietic cells. Significantly, we identified TGFß as a novel signal to regulate hematopoietic development, as the TGFß inhibitor SB 431542 significantly promoted the transition from HE cells into CD43(+) hematopoietic progenitor cells (HPCs) during hESC differentiation. By defining these critical signaling factors during hematopoietic differentiation, we can efficiently generate HPCs from hESCs. Our strategy could offer an in vitro model to study early human hematopoietic development.

Incremental increase in VEGFR1⁺ hematopoietic progenitor cells and VEGFR2⁺ endothelial progenitor cells predicts relapse and lack of tumor response in breast cancer patients.

Animal models have demonstrated the critical role of bone marrow-derived VEGFR1(+) hematopoietic progenitor cells (HPCs) and VEGFR2(+) endothelial progenitor cells (EPCs) in metastatic progression. We explored whether these cells could predict relapse and response in breast cancer (BC) patients. One hundred and thirty-two patients with stages 1-4 BC were enrolled on 2 studies. Circulating CD45(+)/CD34(+)/VEGFR1(+) HPCs and CD45(dim)/CD133(+)/VEGFR2(+) EPCs were assessed from peripheral blood mononuclear cells using flow cytometry. Changes in HPCs and EPCs were analyzed in (1) patients without overt disease that relapsed and (2) metastatic patients according to response by RECIST. At study entry, 102 patients were without evidence of disease and 30 patients had metastatic BC. Seven patients without evidence of BC by exam, labs, and imaging developed recurrence while on study. Median HPC/ml (range) increased from 645.8 (23.5-1,914) to 2,899 (1,176-37,336), P = 0.016, followed by an increase in median EPC/ml from 21.3 (4.7-42.5) to 94.7 (28.2-201.3), P = 0.016, prior to clinical relapse. In metastatic patients with progressive disease, median HPC/ml increased from 1,696 (10-16,470) to 5,124 (374-77,605), P = 0.0009, and median EPC/ml increased from 26 (0-560) to 71 (0-615) prior to progression, P = 0.10. In patients with responding disease, median HPC/ml decreased from 6,147 (912-85,070) to 633 (47-18,065), P = 0.05, and EPC/ml decreased from 46 (0-197) to 23 (0-105), P = 0.41, at response. There were no significant changes in these cells over time in patients with stable disease. Circulating bone marrow-derived HPCs and EPCs predict relapse and disease progression in BC patients.

Influence of the oxygen microenvironment on the proangiogenic potential of human endothelial colony forming cells.

Therapeutic angiogenesis is a promising strategy to promote the formation of new or collateral vessels for tissue regeneration and repair. Since changes in tissue oxygen concentrations are known to stimulate numerous cell functions, these studies have focused on the oxygen microenvironment and its role on the angiogenic potential of endothelial cells. We analyzed the proangiogenic potential of human endothelial colony-forming cells (hECFCs), a highly proliferative population of circulating endothelial progenitor cells, and compared outcomes to human dermal microvascular cells (HMVECs) under oxygen tensions ranging from 1% to 21% O2, representative of ischemic or healthy tissues and standard culture conditions. Compared to HMVECs, hECFCs (1) exhibited significantly greater proliferation in both ischemic conditions and ambient air; (2) demonstrated increased migration compared to HMVECs when exposed to chemotactic gradients in reduced oxygen; and (3) exhibited comparable or superior proangiogenic potential in reduced oxygen conditions when assessed using a vessel-forming assay. These data demonstrate that the angiogenic potential of both endothelial populations is influenced by the local oxygen microenvironment. However, hECFCs exhibit a robust angiogenic potential in oxygen conditions representative of physiologic, ischemic, or ambient air conditions, and these findings suggest that hECFCs may be a superior cell source for use in cell-based approaches for the neovascularization of ischemic or engineered tissues.

Proteomics identifies thymidine phosphorylase as a key regulator of the angiogenic potential of colony-forming units and endothelial progenitor cell cultures.

Endothelial progenitor cell (EPC) cultures and colony-forming units (CFUs) have been extensively studied for their therapeutic and diagnostic potential. Recent data suggest a role for EPCs in the release of proangiogenic factors. To identify factors secreted by EPCs, conditioned medium from EPC cultures and CFUs was analyzed using a matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometer combined with offline peptide separation by nanoflow liquid chromatography. Results were verified by RT-PCR and multiplex cytokine assays and complemented by a cellular proteomic analysis of cultured EPCs and CFUs using difference in-gel electrophoresis. This extensive proteomic analysis revealed the presence of the proangiogenic factor thymidine phosphorylase (TP). Functional experiments demonstrated that inhibition of TP by 5-bromo-6-amino-uracil or gene silencing resulted in a significant increase in basal and oxidative stress-induced apoptosis, whereas supplementation with 2-deoxy-D-ribose-1-phosphate (dRP), the enzymatic product of TP, abrogated this effect. Moreover, dRP produced in EPC cultures stimulated endothelial cell migration in a paracrine manner, as demonstrated by gene-silencing experiments in transmigration and wound repair assays. RGD peptides and inhibitory antibodies to integrin alphavbeta3 attenuated the effect of conditioned medium from EPC cultures on endothelial migration. Finally, the effect of TP on angiogenesis was investigated by implantation of Matrigel plugs in mice. In these in vivo experiments, dRP strongly promoted neovascularization. Our data support the concept that EPCs exert their proangiogenic activity in a paracrine manner and demonstrate a key role of TP activity in their survival and proangiogenic potential.