Enforced expression of IntroductionHematopoiesis relies on the unique abilities of relatively few hematopoietic stem cells to self-renew and generate progenitors that will differentiate into the mature cells forming the blood system. This dynamic process is tightly regulated by a complex of internal and external signals, such as transcription factors, growth factors, and cell cycle regulators (for reviews, see Orkin 1 and Verfaillie 2 ). Many transcription factors, including homeobox (Hox) transcription factors, have been shown to be key players in the proliferation and differentiation of early progenitor cells. 3,[4][5][6] Specific expression patterns of multiple Hox genes have been detected in normal and leukemic hematopoiesis. 7,8 Enforced expression of Hox genes has been shown to affect the ability of progenitors and stem cells to proliferate and differentiate. [9][10][11][12][13][14][15][16][17] One of these genes, Hoxb4, has been implicated in the regulation of hematopoietic stem cell regeneration, 8 and retrovirally engineered overexpression in murine bone marrow cells dramatically increases the stem cell pool ex vivo and in vivo, resulting in faster, more complete recovery of the stem cells in transplantation studies with no adverse effect on differentiation or lineage distribution. 14,[18][19][20][21] This is in contrast to the overexpression of other Hox genes, which can perturb the proliferation and lineage commitment of primitive progenitors and can give rise to hematopoietic malignancies. 10,11,13,15,16,[22][23][24] However, recent studies have suggested that the effect of Hoxb4 is concentration dependent and is not necessarily restricted to proliferation. [25][26][27] Thus, the level of Hoxb4 expression has to be within a specific range for Hoxb4 to increase stem cell proliferation without adverse effects on differentiation. Although enforced expression of Hoxb4 in hematopoietic cells has been studied in detail, its physiologic role in hematopoiesis is poorly understood. Recently, we described a mouse model deficient in Hoxb3 and Hoxb4, showing reduced proliferative capacity of the stem cell pool without otherwise perturbing hematopoiesis. 28 Here we report a novel mouse model in which the Hoxb4 gene alone has been completely removed through the Cre/loxP technique. Hoxb4-deficient mice have a phenotype similar to that of double Hoxb3/Hoxb4 knockout (KO) mice, although the effects are milder in the Hoxb4 Ϫ/Ϫ mice. The phenotype observed seems mainly confined to the stem cell pool, resulting in reduced proliferative capacity of bone marrow and fetal liver hematopoietic stem cells (HSCs) without affecting differentiation or lineage choice. Deficiency of Hoxb4 or Hoxb3 and Hoxb4 affects the expression of other Hox genes and the expression of cell cycle regulators, indicating a complex regulatory role of these Hox genes. Collectively, these findings indicate that Hoxb4 improves proliferative recruitment of HSCs in settings demanding high proliferation, such as transplantation, but that it has a less pro...

The Homeobox (Hox) transcription factors are important regulators of normal and malignant hematopoiesis because they control proliferation, differentiation, and self-renewal of hematopoietic cells at different levels of the hematopoietic hierarchy. In transgenic mice we show that the expression of HOXA10 is tightly regulated by doxycycline. Intermediate concentrations of HOXA10 induced a 15-fold increase in the repopulating capacity of hematopoietic stem cells (HSCs) after 13 days of in vitro culture. Notably, the proliferation induction of HSC by HOXA10 was dependent on the HOXA10 concentration, because high levels of HOXA10 had no effect on HSC proliferation. Furthermore, high levels of HOXA10 blocked erythroid and megakaryocyte development, demonstrating that tight regulation of HOXA10 is critical for normal development of the erythroid and megakaryocytic lineages. The HOXA10-mediated effects on hemato-poietic cells were associated with altered expression of genes that govern stem-cell self-renewal and lineage commitment (eg, hepatic leukemia factor [HlF], Dick-kopf-1 [Dkk-1], growth factor independent -1 [Gfi-1], and Gata-1). Interestingly, binding sites for HOXA10 were found in HLF, Dkk-1, and Gata-1, and Dkk-1 and Gfi-1 were transcriptionally activated by HOXA10. These findings reveal novel molecular pathways that act downstream of HOXA10 and identify HOXA10 as a master regulator of postnatal hematopoietic development. (Blood. 2007;109:3687-3696)

Several homeobox transcription factors, such as HOXB3 and HOXB4, have been implicated in regulation of hematopoiesis. In support of this, studies show that overexpression of HOXB4 strongly enhances hematopoietic stem cell regeneration. Here we find that mice deficient in both Hoxb3 and Hoxb4 have defects in endogenous hematopoiesis with reduced cellularity in hematopoietic organs and diminished number of hematopoietic progenitors without perturbing lineage commitment. Analysis of embryonic day 14.5 fetal livers revealed a significant reduction in the hematopoietic stem cell pool, suggesting that the reduction in cellularity observed postnatally is due to insufficient expansion during fetal development. Primitive Lin ؊ ScaI ؉ c-kit ؉ hematopoietic progenitors lacking Hoxb3 and Hoxb4 displayed impaired proliferative capacity in vitro. Similarly, in vivo repopulating studies of Hoxb3/Hoxb4-deficient hematopoietic cells resulted in lower repopulating capability compared to normal littermates. Since no defects in homing were observed, these results suggest a slower regeneration of mutant HSC. Furthermore, treatment with cytostatic drugs demonstrated slower cell cycle kinetics of hematopoietic stem cells deficient in Hoxb3 and Hoxb4, resulting in increased tolerance to antimitotic drugs. Collectively, these data suggest a direct physiological role of Hoxb4 and Hoxb3 in regulating stem cell regeneration and that these genes are required for maximal proliferative response.Class I Homeobox (Hox) genes encode a family of 39 transcription factors sharing a highly conserved DNA-binding domain. In mammals they play a major role in specifying position and tissue fate in the embryo, as has been demonstrated by several lack-of-function Hox gene mutants that exhibit various developmental abnormalities (see, for example, references 6, 29, 30, 38, 41, 49, and 58). Hox genes are also expressed postnatally, and several of them are expressed in primitive hematopoietic cells and committed progenitors but downregulated upon differentiation to mature cells (44).Murine models have been generated where enforced expression of Hox genes is used to determine the effect of overexpression on self-renewal, differentiation, and other cell fate decisions during hematopoiesis (for reviews, see references 10 and 55). Such models include overexpression of HOXA10, as well as HOXA9, which both affected myelo-and lymphopoiesis and ultimately lead to myeloid leukemia (5, 9, 23, 52, 53). Expression of HOXB3 and HOXB4 is found in the primitive CD34 ϩ population that is highly enriched for human hematopoietic stem cells (HSCs) but is rapidly downregulated as the cells differentiate into committed progenitors (44). Despite very similar expression pattern of HOXB3 and HOXB4, suggesting a common role or collaboration between these factors, the consequences from overexpressing these genes are very different. Although enforced expression of HOXB3 blocks both T-and B-cell development and causes a myeloproliferative disorder (46), overexpression of HOXB4 greatly...

Diamond-Blackfan anemia (DBA) is a congenital bone marrow failure syndrome characterized by a specific deficiency in erythroid progenitors. Forty percent of the patients are blood transfusion-dependent. Recent reports show that the ribosomal protein S19 (RPS19) gene is mutated in 25% of all patients with DBA. We constructed oncoretroviral vectors containing the RPS19 gene to develop gene therapy for RPS19-deficient DBA. These vectors were used to introduce the RPS19 gene into CD34 ؉ bone marrow (BM) cells from 4 patients with DBA with RPS19 gene mutations. Overexpression of the RPS19 transgene increased the number of erythroid colonies by almost 3-fold. High expression levels of the RPS19 transgene improved erythroid colony-forming ability substantially whereas low expression levels had no effect. Overexpression of RPS19 had no detrimental effect on granulocyte-macrophage colony formation. Therefore, these findings suggest that gene therapy for RPS19-deficient patients with DBA using viral vectors that express the RPS19 gene is feasible. IntroductionDiamond-Blackfan anemia (DBA) is a congenital or early-onset pure red cell aplasia/hypoplasia. The disease is characterized by a moderate-to-severe aregenerative anemia and erythroblastopenia in an otherwise normocellular bone marrow. 1,2 Typically, the disorder may present with severe chronic normochromic, macrocytic anemia, and reticulocytopenia. 3,4 In approximately 30% to 40% of patients, there are associated physical malformations, including prenatal or postnatal growth retardation, hand and thumb malformations, and congenital heart defects. 5,6 Most of the reported cases of DBA are sporadic but 10% to 25% have a positive family history. 7,8 Seventy percent of patients respond initially to corticosteroid treatment, 2,9 but 40% become transfusion-dependent. 9 Allogeneic bone marrow transplantation has been shown to be an effective cure for the disease, which demonstrates that the cause of the disease is intrinsic to the bone marrow. [10][11][12] However, the mainstay of therapy for transfusion-dependent patients is frequent blood transfusions, which lead to iron overload. As a consequence, hemosiderosis is a major cause of death among transfusiondependent patients with DBA. In vitro hematopoietic progenitor culture studies indicate that DBA results from an intrinsic defect in erythroid progenitors, erythroid burst-forming units (BFU-Es) and erythroid colony-forming units (CFU-Es), 5,13 and not from a defect in the bone marrow microenvironment, 14 which is consistent with successful marrow transplantation as a treatment for DBA.Several studies have ruled out a number of candidate genes for DBA, including those encoding interleukin 9, the erythropoietin receptor, stem cell factor (SCF) and its receptor, c-kit. 3,[15][16][17][18] Recently, a balanced translocation (X;19) was identified in a patient with DBA and the translocation breakpoint was shown to disrupt the ribosomal protein S19 (RPS19) gene. 8,19,20 Subsequent analysis of the RPS19 gene revealed mutations i...

Diamond-Blackfan anemia (DBA) is a congenital bone marrow failure syndrome characterized by a specific deficiency in erythroid progenitors. Since some patients with DBA develop a reduction in thrombocytes and granulocytes with age, we asked whether multipotent hematopoietic progenitors from DBA patients had normal proliferative capacity in liquid expansion cultures. CD34(+) cells derived from DBA patients showed deficient proliferation in liquid culture containing IL-3, IL-6, and SCF. Single CD34(+) CD38(-) cells from DBA patients exhibited deficient proliferation recruitment in a limiting dilution assay containing IL-3, IL-6, SCF, Tpo, FL, and G-CSF or containing IL-3, IL-6, and SCF. Our findings suggest that the underlying hematopoietic defect in DBA may not be limited to the erythroid lineage. Since a fraction of DBA patients have a deficiency in ribosomal protein S19 (RPS19), we constructed lentiviral vectors containing the RPS19 gene for overexpression in hematopoietic progenitors from RPS19-deficient DBA patients. Enforced expression of the RPS19 transgene improved the proliferation of CD34(+) cells from DBA patients with RPS19 mutation. Similarly, enforced expression of RPS19 improved erythroid development of RPS19-deficient hematopoietic progenitors as determined by colony assays and erythroid differentiation cultures. These findings suggest that gene therapy for RPS19-deficient DBA is feasible.

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Infantile malignant osteopetrosis (IMO) is a fatal disease caused by lack of functional osteoclasts, and the only available treatment is hematopoietic stem cell (HSC) transplantation. In the majority of patients, the TCIRG1 gene, coding for a subunit of a proton pump essential for bone resorption, is mutated. Oc/oc mice have a deletion in the homologue gene

Enforced expression of the HOXB4 transcription factor and downregulation of p21Cip1/Waf (p21) can each independently increase proliferation of murine hematopoietic stem cells (HSCs). We asked whether the increase in HSC self-renewal generated by overexpression of HOXB4 is enhanced in p21-deficient HSCs.