title: A novel protocol that allows short-term stem cell expansion of both committed and pluripotent hematopoietic progenitor cells suitable for clinical use.
abstract: 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.
authors.affiliation: Dideco SpA, Mirandola, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Astori
authors.forename: W
authors.initials: W
authors.lastname: Malangone
authors.forename: V
authors.initials: V
authors.lastname: Adami
authors.forename: A
authors.initials: A
authors.lastname: Risso
authors.forename: L
authors.initials: L
authors.lastname: Dorotea
authors.forename: E
authors.initials: E
authors.lastname: Falasca
authors.forename: L
authors.initials: L
authors.lastname: Marini
authors.forename: R
authors.initials: R
authors.lastname: Spizzo
authors.forename: L
authors.initials: L
authors.lastname: Bigi
authors.forename: P
authors.initials: P
authors.lastname: Sala
authors.forename: E
authors.initials: E
authors.lastname: Tonutti
authors.forename: F
authors.initials: F
authors.lastname: Biffoni
authors.forename: C
authors.initials: C
authors.lastname: Rinaldi
authors.forename: G
authors.initials: G
authors.lastname: Del Frate
authors.forename: M
authors.initials: M
authors.lastname: Pittino
authors.forename: A
authors.initials: A
authors.lastname: Degrassi
mesh_terms: D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D015703:Antigens, CD; D018952:Antigens, CD34 / Q000032:analysis; D000943:Antigens, Differentiation / Q000032:analysis; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002455:Cell Division / Q000187:drug effects; D004357:Drug Synergism; D004921:Erythropoietin / Q000494:pharmacology; D005312:Fetal Blood / Q000166:cytology; D005434:Flow Cytometry; D018380:Hematopoietic Stem Cell Transplantation / Q000379:methods*; D006412:Hematopoietic Stem Cells / Q000145:classification / Q000166:cytology* / Q000187:drug effects; D006801:Humans; D018189:Immunomagnetic Separation; D016130:Immunophenotyping; D007231:Infant, Newborn; D007377:Interleukin-3 / Q000494:pharmacology; D015850:Interleukin-6 / Q000494:pharmacology; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000494:pharmacology; D009244:NAD+ Nucleosidase / Q000032:analysis; D019089:Stem Cell Factor / Q000494:pharmacology; D013926:Thrombopoietin / Q000494:pharmacology
chemical_list: D015703:Antigens, CD; D018952:Antigens, CD34; D000943:Antigens, Differentiation; D007377:Interleukin-3; D015850:Interleukin-6; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D019089:Stem Cell Factor; C084881:flt3 ligand protein; D004921:Erythropoietin; D013926:Thrombopoietin; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; D009244:NAD+ Nucleosidase; D051997:ADP-ribosyl Cyclase 1