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

• Published in: Blood cells, molecules, & diseases molecules, & diseases, 2001-07, Vol.27 (4), p.715-724
• Main Authors: Astori, Giuseppe, Malangone, Walter, Adami, Valentina, Risso, Angela, Dorotea, Laura, Falasca, Elisabetta, Marini, Luisa, Spizzo, Riccardo, Bigi, Leonardo, Sala, Pierguido, Tonutti, Elio, Biffoni, Franco, Rinaldi, Cristina, Del Frate, Giovanni, Pittino, Marco, Degrassi, Alberto
• Format: Article
• Language: English
• Subjects: ADP-ribosyl Cyclase; ADP-ribosyl Cyclase 1; Antigens, CD; Antigens, CD34 - analysis; Antigens, Differentiation - analysis; CD34+ cells; Cell Culture Techniques - methods; Cell Differentiation; Cell Division - drug effects; Drug Synergism; Erythropoietin - pharmacology; ex vivo expansion; Fetal Blood - cytology; Flow Cytometry; Hematopoietic Stem Cell Transplantation - methods; hematopoietic stem cells; Hematopoietic Stem Cells - classification; Hematopoietic Stem Cells - cytology; Hematopoietic Stem Cells - drug effects; Humans; Immunomagnetic Separation; Immunophenotyping; Infant, Newborn; Interleukin-3 - pharmacology; Interleukin-6 - pharmacology; Membrane Glycoproteins; Membrane Proteins - pharmacology; NAD+ Nucleosidase - analysis; Stem Cell Factor - pharmacology; Thrombopoietin - pharmacology; umbilical cord blood
• ISSN: 1079-9796; 1096-0961
• DOI: 10.1006/bcmd.2001.0439

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), along with committed cells from the various lineages. For this purpose, the small 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. Heimfels, R. J. Berenson, R. Mertelsmann, and L. Kanz (1995) N. Engl. J. Med. 333, 283–287] and Piacibello et al. [W. Piacibello, F. Sanavio, L. Garetto, A. Severino, D. Bergandi, J. Ferrario, F. Fagioli, M. Berger, and M. Aglietta (1997) Blood 89, 2644–2653] to expand UCB CD34+ cells and monitored proliferation rate and phenotype after 14 days of culture. Several hematopoietic lineage-associated surface antigens were evaluated. Our data show that flt3L and thrombopoietin in combination with IL-3, while sustaining a high CD34+ proliferation rate, provide a relatively low enrichment in very early uncommitted CD34+/CD38- cells. Conversely, in the absence of IL-3, they are less effective in inducing proliferation yet significantly increase the number of CD34+/CD38- cells. A combination of the above protocols, applied simultaneously to aliquots of the same sample, would allow expansion of both committed and pluripotent HSC. This strategy may represent a significant improvement for clinical applications.