The phenotype induced by the GATA-1 low (neo␦HS) mutation is here further characterized by analyzing the hemopoietic system during the aging (up to 20 months) of a GATA-1 low colony (135 mutants and 40 normal littermates). Mutants expressed normal hematocrit values (Hct ‫؍‬ 45.9 ؎ 4.0) until 12 months but became anemic from 15 months on (Hct ‫؍‬ 30.9 ؎ 3.9; P < .05). Anemia was associated with several markers of myelofibrosis such as the presence of tear-drop poikilocytes and progenitor cells in the blood, collagen fibers in the marrow and in the spleen, and hemopoietic foci in the liver. Semiquantitative reverse transcription-polymerase chain reaction showed that growth factor genes implicated in the development of myelofibrosis (such as osteocalcin, transforming growth factor-␤1, platelet-derived growth factor, and vascular endothelial growth factor) were all expressed in the marrow from the mutants at higher levels than in corresponding normal tissues. The GATA-1 low mutants experienced a slow progression of the disease because the final exitus was not observed until at least 15 months with a probability of survival more favorable than that of W/W v mice concurrently kept in the animal facility (P < .001, by Kaplan-Meier analysis). In conclusion, impaired GATA-1 expression may contribute to the development of myelofibrosis, and the GATA-1 low mutants may represent a suitable animal model for the human disease that may shed light on its pathogenesis. (Blood.

Here it is shown that the phenotype of adult mice lacking the first enhancer (DNA hypersensitive site I) and the distal promoter of the GATA-1 gene (neoΔHS or GATA-1low mutants) reveals defects in mast cell development. These include the presence of morphologically abnormal alcian blue+ mast cells and apoptotic metachromatic− mast cell precursors in connective tissues and peritoneal lavage and numerous (60–70% of all the progenitors) “unique” trilineage cells committed to erythroid, megakaryocytic, and mast pathways in the bone marrow and spleen. These abnormalities, which were mirrored by impaired mast differentiation in vitro, were reversed by retroviral-mediated expression of GATA-1 cDNA. These data indicate an essential role for GATA-1 in mast cell differentiation.

We describe a new two-step culture method for mass production in vitro of erythroid cells from either CD34+ (10(5) cells/mL) or light-density (10(6) cells/mL) cells purified from the blood of normal donors and thalassemic patients. The method includes (i) culture of the cells in the presence of dexamethasone and estradiol (10(-6) M each) and (ii) the growth factors SCF (50 ng/mL), IL-3 (1 ng/mL), and EPO (1 U/mL). In their proliferative phase, these cultures generated approximately 1.2 x 10(7) erythroblasts for each milliliter of blood collected from normal donors or thalassemic patients. They were composed mostly (90%) of CD45(low)/glycophorin (GPA)(neg)/CD71(1ow) cells at day 7, 50-60\% of which became CD45(neg)/GPA+/CD71high by days 15-20. However, when cells from days 7 to 12 of the proliferative phase were transferred in differentiation medium containing EPO and insulin, they progressed to mature erythroblasts (g90% benzidine(pos) and CD45(neg)/GPA+/CD71medium) in 4 days. Because of the high number of erythroid cells that are generated from modest volumes of blood, this method will prove useful in donor-specific studies of erythroid differentiation.