Bone marrow endothelial cells (ECs) are essential for reconstitution of hematopoiesis, but their role in self-renewal of long term-hematopoietic stem cells (LT-HSCs) is unknown. We have developed angiogenic models to demonstrate that EC-derived angiocrine growth factors support in vitro self-renewal and in vivo repopulation of authentic LT-HSCs. In serum/cytokine-free co-cultures, ECs through direct cellular contact, stimulated incremental expansion of repopulating CD34−Flt3−cKit+Lineage−Sca1+ LT-HSCs, which retained their self-renewal ability, as determined by single cell and serial transplantation assays. Angiocrine expression of Notch-ligands by ECs promoted proliferation and prevented exhaustion of LT-HSCs derived from wild-type, but not Notch1/Notch2 deficient mice. In transgenic notch-reporter (TNR.Gfp) mice, regenerating TNR.Gfp+ LT-HSCs were detected in cellular contact with sinusoidal ECs and interfering with angiocrine, but not perfusion function, of SECs impaired repopulation of TNR.Gfp+ LT-HSCs. ECs establish an instructive vascular niche for clinical scale expansion of LT-HSCs and a cellular platform to identify stem cell-active trophogens.

The stable introduction of a functional beta-globin gene in haematopoietic stem cells could be a powerful approach to treat beta-thalassaemia and sickle-cell disease. Genetic approaches aiming to increase normal beta-globin expression in the progeny of autologous haematopoietic stem cells might circumvent the limitations and risks of allogeneic cell transplants. However, low-level expression, position effects and transcriptional silencing hampered the effectiveness of viral transduction of the human beta-globin gene when it was linked to minimal regulatory sequences. Here we show that the use of recombinant lentiviruses enables efficient transfer and faithful integration of the human beta-globin gene together with large segments of its locus control region. In long-term recipients of unselected transduced bone marrow cells, tetramers of two murine alpha-globin and two human betaA-globin molecules account for up to 13% of total haemoglobin in mature red cells of normal mice. In beta-thalassaemic heterozygous mice higher percentages are obtained (17% to 24%), which are sufficient to ameliorate anaemia and red cell morphology. Such levels should be of therapeutic benefit in patients with severe defects in haemoglobin production.

Patients affected by ␤-thalassemia major require lifelong transfusions because of insufficient or absent production of the ␤ chain of hemoglobin (Hb). A minority of patients are cured by allogeneic bone marrow transplantation. In the most severe of the hitherto available mouse models of ␤-thalassemia, a model for human ␤-thalassemia intermedia, we previously demonstrated that globin gene transfer in bone marrow cells is curative, stably raising Hb levels from 8.0-8.5 to 11.0-12.0 g/dL in long-term chimeras. To fully assess the therapeutic potential of gene therapy in the context of a lethal anemia, we now have created an adult model of ␤ 0 -thalassemia major. In this novel model, mice engrafted with ␤-globin-null (Hbb th3/th3 ) fetal liver cells succumb to ineffective erythropoiesis within 60 days. These mice rapidly develop severe anemia (2-4 g/dL), massive splenomegaly, extramedullary hematopoiesis (EMH), and hepatic iron overload. Remarkably, most mice (11 of 13) treated by lentivirus-mediated globin gene transfer were rescued. Long-term chimeras with an average 1.0-2. IntroductionThe ␤-thalassemias are caused by more than 200 mutations that lead to decreased or absent production of the ␤ chain of hemoglobin (Hb). 1-3 Most of the mutations are point mutations in the ␤-globin gene that either decrease the level of transcription or mRNA stability or lead to nonfunctional mRNA, as is the case in nonsense and frameshift mutations. Other mutations include deletions in the ␤-globin gene, as well as rare deletions located distally to the ␤-globin gene itself. 1,3,4 There is a large spectrum in the severity of the disease found in homozygotes and compound heterozygotes. Patients affected by ␤-thalassemia intermedia do not require chronic transfusion therapy in the first years of life, but they often worsen over time, eventually developing hypersplenism, osteopenia, extramedullary hematopoiesis (EMH), and iron overload. Some patients eventually require splenectomy and blood transfusions. In patients with ␤-thalassemia major, or Cooley anemia, the absent or extremely reduced production of the ␤ chain of Hb causes severe ineffective erythropoiesis, massive erythroid hyperplasia in the bone marrow and extramedullary sites, and hemolysis. The ensuing iron overload can lead to endocrine deficiencies, cirrhosis, and cardiac failure. In the absence of lifelong transfusions, the disease is lethal. 5 Current disease management consists of prenatal diagnosis, transfusion therapy, or allogeneic bone marrow transplantation. 1,3,4 Only the latter is curative, but this option is limited to a minority of patients for whom a histocompatible donor can be identified.New approaches aimed at ameliorating the condition of patients with severe ␤-thalassemia are greatly needed. Genetic approaches based on the transfer of a regulated human ␤-globin gene in autologous hematopoietic stem cells (HSCs) represent an attractive potential treatment. 6 Their implementation has been hampered by the difficulty of appropriately regulating expression o...