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), particularly 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 (< or = 5-10%). We therefore developed an improved purification methodology based on "potentiated" negative immunobead selection (Step IIIP) by addition of anti-CD45, -11a and -71 monoclonal antibodies (mAbs) to the previously utilized panel of mAbs. This simplified procedure consistently allows not only 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, mean 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 semi-solid culture, the purified erythroid/multipotent HPCs give rise to macroscopic colonies (10,000-150,000 cells/clone, > 0.5 mm size colonies). This purification methodology compares favorably with previously reported procedures in terms of combined HPC frequency and recovery: availability of a large number of highly purified, early HPCs will provide an experimental tool for analysis of the molecular/cellular basis of early hematopoiesis. We have investigated by reverse transcription-polymerase chain reaction (RT-PCR) the mRNA expression of homeobox B (HOXB) cluster genes in purified HPCs induced in liquid suspension culture to gradual erythroid or granulopoietic (largely eosinophilic) differentiation and maturation by differential growth factor (GF) stimulus. Only B3 is expressed in quiescent HPCs. After GF treatment B3 expression is enhanced in the initial 24 h and then through erythroid and granulopoietic differentiation and maturation. HOXB4 and B5 are induced at slightly later times and expressed through maturation in both lineages, while B6 is selectively induced in granulocytic differentiation. B2 is transiently expressed at low level in the granulopoietic pathway, while it is detected only in advanced stages of erythropoiesis; B7, B8 and B9 are essentially not detected. Functional studies were performed with antisense phosphorothioate oligomers to HOX mRNAs including: 1) anti-B3 oligomer (alpha-B3) treatment of purified HPCs induces a striking blockade of both erythroid and granulomonocytic colony formation, 2) alpha-B6 selectively and markedly inhibits granulomonocytic colony formation, 3) alpha-B4 and alpha-B5 cause a significant, less pronounced decrease of both colony types and finally, 4) alpha-B2 and alpha-B7, alpha-B9 exert little and no effect respectively.