HOXB GENE-EXPRESSION AND FUNCTION IN DIFFERENTIATING PURIFIED HEMATOPOIETIC PROGENITORS

Intensive efforts have led to the development of methods for stringent purification of adult hematopoietic progenitor cells (HPCs), particul arly from peripheral blood (PB). The purification procedure previously reported by our group (Science, 1990) provided a high HPC frequency, but yielded a low HPC recovery (less than or equal to 5-10%). We there fore developed an improved purification methodology based on ''potenti ated'' negative immunobead selection (Step IIIP) by addition of anti-C D45, -11a and -71 monoclonal antibodies (mAbs) to the previously utili zed panel of mAbs. This simplified procedure consistently allows not o nly high level purification but also abundant recovery of early HPCs: the final Step IIIP cell population (0.95 x 10(6) cells/4 PB donors, m ean value) features an 81% HPC frequency and a recovery of 45% of the initial HPCs. The purified HPCs bear the primitive HPC phenotype, i.e. , they are consistently CD34(+), largely CD33(-)/45RA(-), and in part HLA-DR(-/low)/CD38(-/low)/Thy-1(+). In optimized semisolid culture, th e purified erythroid/multipotent HPCs give rise to macroscopic colonie s (10,000-150,000 cells/clone, >0.5 mm size colonies). This purificati on methodology compares favorably with previously reported procedures in terms of combined HPC frequency and recovery: availability of a lar ge number of highly purified, early HPCs will provide an experimental tool for analysis of the molecular/cellular basis of early hematopoies is. We have investigated by reverse transcription-polymerase chain rea ction (RT-PCR) the mRNA expression of homeobox B (HOXB) cluster genes in purified HPCs induced in liquid suspension culture to gradual eryth roid or granulopoietic (largely eosinophilic) differentiation and matu ration by differential growth factor (GF) stimulus. Only B3 is express ed in quiescent HPCs. After GF treatment B3 expression is enhanced in the initial 24 h and then through erythroid and granulopoietic differe ntiation and maturation. HOXB4 and B5 are induced at slightly later ti mes and expressed through maturation in both lineages, while B6 is sel ectively induced in granulocytic differentiation. B2 is transiently ex pressed at low level in the granulopoietic pathway, while it is detect ed only in advanced stages of erythropoiesis; B7, B8 and B9 are essent ially not detected. Functional studies were performed with antisense p hosphorothioate oligomers to HOX mRNAs including: 1) anti-B3 oligomer (alpha-B3) treatment of purified HPCs induces a striking blockade of b oth erythroid and granulomonocgtic colony formation, 2) alpha-B6 selec tively and markedly inhibits granulomonocytic colony formation, 3) alp ha-B4 and alpha-B5 cause a significant, less pronounced decrease of bo th colony types and finally, 4) alpha-B2 and alpha-B7, alpha-B9 exert little and no effect respectively. These studies provide novel evidenc e on the coordinate expression of selected HOXB cluster genes in eryth ro- and granulopoiesis, particularly in the early stages of differenti ation; B3 apparently functions as a master gene in early hematopoiesis , while B6 exerts a key selective function in the granulopoietic pathw ay.