Biological characterization of CD34(+) cells mobilized into peripheral blood

We review here the functional and kinetic characteristics of highly purifie d hematopoietic CD34(+) mobilized into peripheral blood (PB) by granulocyte colony-stimulating factor (G-CSF) with or without chemotherapy for autolog ous or allogeneic transplantation. Circulating CD34(+) cells were evaluated for their colony-forming capacity and trilineage proliferative response to selected recombinant human (rh) CSF in vitro, and the content of very prim itive long-term culture initiating cells (LTC-IC). In addition, the cycling status of PB CD34(+) cells, including committed clonogenic progenitor cell s and the more immature LTC-IC, was determined by the cytosine arabinoside (Ara-C) suicide test and the acridine orange (AO) flow cytometric technique . By comparison, bone marrow (BM) CD34(+) cells from the same individuals w ere studied under steady-state conditions and during G-CSF administration, Clonogenic assays in methylcellulose showed the same frequency of colony-fo rming unit cells (CFU-C) when PB primed-CD34(+) cells and BM cells were sti mulated with phytohemagglutinin-lymphocyte-conditioned medium (PHA-LCM). Ho wever, mobilized CD34(+) cells were significantly more responsive than thei r steady-state BM counterparts to interleukin-3 (IL-3) and stem cell factor (SCF) combined with G-CSF or IL-3 in the presence of erythropoietin (Epo). Conversely, circulating and BM megakaryocyte precursors (CFU-MK) showed th e same clonogenic efficiency in response to IL-3, GM-CSF and IL-3, IL-6 and Epo. Interestingly, very few CD34(+) cells expressed the Mpl receptor and this finding resulted in the lower proliferative response of mobilized CFU- MK to the Mpl-ligand (megakaryocyte growth and development factor; MGDF), a s compared to BM cells, After 5 weeks of liquid culture supported by the en gineered murine stromal cell line M2-10B4 to produce G-CSF and IL-3, we rep orted a similar frequency of LTC-IC in PB and steady-state BM, Kinetic stud ies on PB and BM CD34(+) cells, including LTC-IC, showed the low number of circulating progenitor cells in S and G(2)M phase whereas simultaneous DNA/ RNA analysis and the Ara-C suicide assay demonstrated that the majority of PB CD34(+) cells and LTC-IC are not quiescent (ie in G(o) phase) being in G (1) phase. Moreover, G-CSF administration prevented apoptosis in a small bu t significant proportion of mobilized CD34(+) cells. Thus, our results indi cate that mobilized and BM CD34(+) cells can be considered equivalent for t he frequency of both committed and more immature hematopoietic progenitor c ells, although they show different kinetic and functional profiles. A furth er set of experiments indicated that G-CSF treatment did not alter the allo antigen presenting function of CD34+ cells which was mainly mediated by the upregulation of costimulatory molecules upon coincubation with allogeneic T cells. Taken together, these findings should allow a better understanding of PBSC transplantation.