New insights into the characterization and ex vivo manipulation of CD34+ cells

Background and Objective. The CD34 antigen represents to date the only molecule whose expression is restricted to a small population of primitive hematopoietic progenitor cells. In the last few years,a great progress in the characterization, enumeration, and ex-vivo expansion of CD34+ cells was made. To address these issues, the Italian Society of' Experimental Hematoogy organized a Meeting in Florence on February 26, 1998. Inf'ormation Sources. The material examined in the present reyiew includes full papers and abstracts published in journals covered by the Science Citation lndex and Medline. All the participants to the Meeting in Florence have been actively working in the field of biology and clinical application of CD34- cells. Summaries of their oral presentation at the norence Meeting are reported in the Appendix to this paper. State of Art and Perspectives. Bone marrow, mobilized peripheral blood and cord blood are the commonly used sources ot hematopoietic cells tor transplant. Recently some guidelines for the flow cytometry enumeration of CD34+ cells were proposed by the European Yorking Group on Clinical Cell Analysis, and we herewith summarize them as follows : 1) single platform assay (absolute count and CD34* cell estimation on a single instrument, i.e. flow cytometer); 2) whole blood staining, NH4CI based erythrocyte lysis, no fixation, no washing; 3) class II or III conjugated CD34 McAbs (class II CD34 McAbs only with phycoerythrin); 4) counterstaining with CD45 McAb; 5) use of a nucleic acid dye to select viable nucleated cells; 6) acquisition of list mode data containing at least 50-100 CD34• cells during data acquisition; 7) sequential gating strategy during list mode data analysis. As far as the ex vivo expansion of cord blood stem cells is concerned, it can be stated that this technique could mafte the use of cord blood transplant feasible also for adult patients. Recently we developed a stroma-free culture system in which a progressive, increasingly greater production of hemopoietic progenitors belonging to all the hematopoietic lineages was sustained for over six months. A similar sustained and prolonged expansion of the most primitive stem cells that can be detected in vitro (LTC-IC), was also documented. The extremely prolonged maintenance and the massive expansions suggest that extensive self-renewal and little differentiation can be triggered in vitro by FLT3/FLK2 ligand (FL) plur c-mpl ligand (throm- bopoietin) and this could represent a first step towards the implementation of clinical expansion- transplantation strategies.