Preclinical ex vivo expansion of peripheral blood CD34+ selected cells from cancer patients mobilized with combination chemotherapy and granulocyte colony-stimulating factor.
OBJECTIVE
Ex vivo peripheral blood progenitor cell (PBPC) expansion has been proposed as a strategy to increase the number of haematopoietic progenitors available for cell transplantation. We have expanded CD34+ cells from PBPCs obtained from four patients with haematological malignancies and one patient with an Ewing's sarcoma.


METHODS
Cells were expanded in the Dideco 'Pluricell system'. After 12 days in culture, we evaluated cell phenotype, total nucleated cells, CD34+ fold increase, cell apoptosis and colony assay of expanded cells. Cell engraftment has been evaluated by transplanting two groups of irradiated non-obese diabetic/severe combined immunodeficient (NOD-SCID) mice with expanded and non-expanded cell populations.


RESULTS
Total nucleated cells and CD34+ cells increased 59.5 and 4.0 times, respectively. The expanded cells were mainly constituted of myeloid and megakaryocytic cells. A significant increase in the number of colony-forming unit-granulocyte macrophage (CFU-GM) was observed in the CFU assay. Ten mice transplanted with expanded cells showed a best overall survival (80%) compared to 10 mice transplanted with non-expanded cells (20%). Human CD45+ cells were detected by flow cytometry and polymerase chain reaction in bone marrow and spleen of transplanted animals. The relative low engraftment level obtained with the expanded cells suggests a loss of SCID repopulating cells maybe due to cell differentiation during expansion.


CONCLUSIONS
We have demonstrated the feasibility of the ex vivo expansion of mobilized PBPCs from cancer patients, evidencing a clonal expansion of CFUs and the ability of the expanded cells to engraft the bone marrow and spleen of immunosuppressed mice. The differentiation of the CD34+ stem cell compartment could be further minimized by ameliorating the expansion conditions.
Lorenzon
D
D
Clinical and Experimental Haematology Research Unit, Centro di Riferimento Oncologico, IRCCS, Aviano, PN, Italy.
Mazzucato
M
M
Abbruzzese
L
L
Cilli
M
M
De Angeli
S
S
Degan
M
M
Mambrini
G
G
Piccardi
F
F
Rupolo
M
M
Michieli
M
M
De Marco
L
L
Gattei
V
V
Astori
G
G
D000328:Adult; D000818:Animals; D018952:Antigens, CD34; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods; D002478:Cells, Cultured; D005240:Feasibility Studies; D005260:Female; D006085:Graft Survival; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology; D019650:Hematopoietic Stem Cell Mobilization / Q000379:methods; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology; D007937:Leukapheresis; D008297:Male; D051379:Mice; D036102:Peripheral Blood Stem Cell Transplantation / Q000379:methods*
D018952:Antigens, CD34; D036341:Intercellular Signaling Peptides and Proteins; D016179:Granulocyte Colony-Stimulating Factor