Hemoglobinopathies, such as β-thalassemias and sickle cell anemia (SCA), are among the most common inherited gene defects. Novel models of human erythropoiesis that result in terminally differentiated red blood cells (RBCs) would be able to address the pathophysiological abnormalities in erythrocytes in congenital RBC disorders and to test the potential of reversing these problems by gene therapy. We have developed an in vitro model of production of human RBCs from normal CD34+ hematopoietic progenitor cells, using recombinant growth factors to promote terminal RBC differentiation. Enucleated RBCs were then isolated to a pure population by flow cytometry in sufficient numbers for physiological studies. Morphologically, the RBCs derived in vitro ranged from early polylobulated forms, resembling normal reticulocytes to smooth biconcave discocytes. The hemoglobin pattern in the in vitro-derived RBCs mimicked the in vivo adult or postnatal pattern of β-globin production, with negligible γ-globin synthesis. To test the gene therapy potential using this model, CD34+ cells were genetically marked with a retroviral vector carrying a cell-surface reporter. Gene transfer into CD34+ cells followed by erythroid differentiation resulted in expression of the marker gene on the surface of the enucleated RBC progeny. This model of human erythropoiesis will allow studies on pathophysiology of congenital RBC disorders and test effective therapeutic strategies.

The role of the homeobox gene HOXA5 in normal human hematopoiesis was studied by constitutively expressing theHOXA5 cDNA in CD34+ and CD34+CD38− cells from bone marrow and cord blood. By using retroviral vectors that contained both HOXA5and a cell surface marker gene, pure populations of progenitors that expressed the transgene were obtained for analysis of differentiation patterns. Based on both immunophenotypic and morphological analysis of cultures from transduced CD34+ cells, HOXA5expression caused a significant shift toward myeloid differentiation and away from erythroid differentiation in comparison to CD34+ cells transduced with Control vectors (P= .001, n = 15 for immunophenotypic analysis; and P < .0001, n = 19 for morphological analysis). Transduction of more primitive progenitors (CD34+CD38− cells) resulted in a significantly greater effect on differentiation than did transduction of the largely committed CD34+ population (P = .006 for difference between HOXA5 effect on CD34+v CD34+CD38−cells). Erythroid progenitors (burst-forming unit-erythroid [BFU-E]) were significantly decreased in frequency among progenitors transduced with the HOXA5 vector (P = .016, n = 7), with no reduction in total CFU numbers. Clonal analysis of single cells transduced with HOXA5 or control vectors (cultured in erythroid culture conditions) showed that HOXA5expression prevented erythroid differentiation and produced clones with a preponderance of undifferentiated blasts. These studies show that constitutive expression of HOXA5 inhibits human erythropoiesis and promotes myelopoiesis. The reciprocal inhibition of erythropoiesis and promotion of myelopoiesis in the absence of any demonstrable effect on proliferation suggests that HOXA5 diverts differentiation at a mulitpotent progenitor stage away from the erythroid toward the myeloid pathway.

Platelet function was investigated in three patients with the Lesch-Nyhan syndrome. Platelet count, morphology and size distribution was normal in all patients. Platelet turnover was normal. Electron microscopy did not reveal any ultrastructural abnormality. Template bleeding times were normal and prolonged after aspirin ingestion in two out of the three patients: the patient that failed to respond to the aspirin challenge also had decreased retention of platelets on a glass bead column. Biochemical studies revealed that total platelet ATP was reduced by 34% in the presence of a normal level of ADP in the storage pool. These platelets failed to incorporate radioactive hypoxanthine but did incorporate radioactive adenine to produce adenine nucleotides and a trace amount of guanine nucleotides. The results indicate that normal platelets have a functionally intact pathway for utilizing hypoxanthine as a source of preformed purine, and that the failure to salvage this purine, as in the Lesch-Nyhan syndrome, results in a decreased level of total platelet ATP. These findings suggest that platelets can function normally despite a one third reduction in total ATP content.