A novel protocol that allows short-term stem cell expansion of both committed and pluripotent hematopoietic progenitor cells suitable for clinical use

To obtain long-term engraftment and hematopoiesis in myeloablated patients, the cell population used for hematopoietic reconstitution should include a sufficient number of early pluripotent hematopoietic stem cells (HSCs), al ong with committed cells from the various lineages. For this purpose, the s mall subset of CD34+ cells purified from different sources must be expanded ex vivo. Since cytokines may induce both proliferation and differentiation , expansion would provide a cell population comprising committed as well as uncommitted cells. Optimization of HSC expansion methods could be obtained by a combination of cytokines able to sustain renewal of pluripotent cells yet endowed with poor differentiation potential. We used variations of the combinations of cytokines described by Brugger et al. [W. Brugger, S. Heim fels, R. J. Berenson, R. Mertelsmann, and L. Kanz (1995) N. Engl. J. Med. 3 33, 283-287] and Piacibello et al. [W. Piacibello, F. Sanavio, L. Garetto, A. Severino, D. Bergandi, J. Ferrario, F. Fagioli, M. Berger, and M. Agliet ta (1997) Blood 89, 2644-2653] to expand UCB CD34+ cells and monitored prol iferation rate and phenotype after 14 days of culture. Several hematopoieti c lineage-associated surface antigens were evaluated. Our data show that fl t3L and thrombopoietin in combination with IL-3, while sustaining a high CD 34+ proliferation rate, provide a relatively low enrichment in very early u ncommitted CD34+/CD38- cells. Conversely, in the absence of IL-3, they are less effective in inducing proliferation yet significantly increase the num ber of CD34+/CD38- cells. A combination of the above protocols, applied sim ultaneously to aliquots of the same sample, would allow expansion of both c ommitted and pluripotent HSC. This strategy may represent a significant imp rovement for clinical applications. (C) 2001 Academic Press.