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), 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.
Astori
G
G
Dideco SpA, Mirandola, Italy.
Malangone
W
W
Adami
V
V
Risso
A
A
Dorotea
L
L
Falasca
E
E
Marini
L
L
Spizzo
R
R
Bigi
L
L
Sala
P
P
Tonutti
E
E
Biffoni
F
F
Rinaldi
C
C
Del Frate
G
G
Pittino
M
M
Degrassi
A
A
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
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