Ex Vivo Expansion and Differentiation of Unselected Peripheral Blood Progenitor Cells in Serum-Free Media

1998 ◽  
Vol 7 (6) ◽  
pp. 481-491 ◽  
Author(s):  
RONALD L. PAQUETTE ◽  
ELISA GONZALES ◽  
RYAN YOSHIMURA ◽  
LAWRENCE TRAN ◽  
RUTH CHOI ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Serum Free ◽  
Peripheral Blood Progenitor Cells ◽  
Free Media

scholarly journals Maintenance of transplantation potential in ex vivo expanded CD34(+)- selected human peripheral blood progenitor cells

Blood ◽  
1994 ◽  
Vol 84 (9) ◽  
pp. 2898-2903 ◽  
Author(s):  
R Henschler ◽  
W Brugger ◽  
T Luft ◽  
T Frey ◽  
R Mertelsmann ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Peripheral Blood ◽  
Large Scale ◽  
Ex Vivo ◽  
Human Peripheral Blood ◽  
Ex Vivo Expansion ◽  
High Dose ◽  
Peripheral Blood Progenitor Cells ◽  
Limiting Dilution

Abstract CD34(+)-selected hematopoietic progenitor cells are being increasingly used for autotransplantation, and recent evidence indicates that these cells can be expanded ex vivo. Of 15 patients with solid tumors undergoing a phase I/II clinical trial using CD34(+)-selected peripheral blood progenitor cells (PBPCs) after high-dose chemotherapy, we analyzed the frequency of long-term culture-initiating cells (LTCIC) as a measure of transplantation potential before and after ex vivo expansion of CD34+ cells. PBPCs were mobilized by combination chemotherapy and granulocyte colony-stimulating factor (G-CSF). The original unseparated leukapheresis preparations, the CD34(+)-enriched transplants, as well as nonabsorbed fractions eluting from the CD34 immunoaffinity columns (Ceprate; CellPro, Bothell, WA) were monitored for their capacity to repopulate irradiated allogeneic stroma in human long-term bone marrow cultures. We found preservation of more than three quarters of fully functional LTCIC in the CD34(+)-selected fractions. Quantitation of LTCIC by limiting dilution analysis showed a 53-fold enrichment of LTCIC from 1/9,075 in the unseparated cells to an incidence of 1/169 in the CD34+ fractions. Thus, in a single apheresis, it was possible to harvest a median of 1.65 x 10(4) LTCIC per kg body weight (range, 0.71 to 3.72). In addition, in six patients, large-scale ex vivo expansions were performed using a five-factor cytokine combination consisting of stem cell factor (SCF), interleukin-1 (IL-1), IL-3, IL-6, and erythropoietin (EPO), previously shown to expand committed progenitor cells. LTCIC were preserved, but not expanded during the culture period. Optimization of ex vivo expansion growth factor requirements using limiting dilution assays for LTCIC estimation indicated that the five-factor combination using SCF, IL-1, IL-3, IL-6, and EPO together with autologous plasma was the most reliable combination securing both high progenitor yield and, at the same time, optimal preservation of LTCIC. Our data suggest that ex vivo-expanded CD34+ PBPCs might be able to allow long-term reconstitution of hematopoiesis.

scholarly journals Maintenance of transplantation potential in ex vivo expanded CD34(+)- selected human peripheral blood progenitor cells

Blood ◽  
1994 ◽  
Vol 84 (9) ◽  
pp. 2898-2903 ◽  
Author(s):  
R Henschler ◽  
W Brugger ◽  
T Luft ◽  
T Frey ◽  
R Mertelsmann ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Peripheral Blood ◽  
Large Scale ◽  
Ex Vivo ◽  
Human Peripheral Blood ◽  
Ex Vivo Expansion ◽  
High Dose ◽  
Peripheral Blood Progenitor Cells ◽  
Limiting Dilution

CD34(+)-selected hematopoietic progenitor cells are being increasingly used for autotransplantation, and recent evidence indicates that these cells can be expanded ex vivo. Of 15 patients with solid tumors undergoing a phase I/II clinical trial using CD34(+)-selected peripheral blood progenitor cells (PBPCs) after high-dose chemotherapy, we analyzed the frequency of long-term culture-initiating cells (LTCIC) as a measure of transplantation potential before and after ex vivo expansion of CD34+ cells. PBPCs were mobilized by combination chemotherapy and granulocyte colony-stimulating factor (G-CSF). The original unseparated leukapheresis preparations, the CD34(+)-enriched transplants, as well as nonabsorbed fractions eluting from the CD34 immunoaffinity columns (Ceprate; CellPro, Bothell, WA) were monitored for their capacity to repopulate irradiated allogeneic stroma in human long-term bone marrow cultures. We found preservation of more than three quarters of fully functional LTCIC in the CD34(+)-selected fractions. Quantitation of LTCIC by limiting dilution analysis showed a 53-fold enrichment of LTCIC from 1/9,075 in the unseparated cells to an incidence of 1/169 in the CD34+ fractions. Thus, in a single apheresis, it was possible to harvest a median of 1.65 x 10(4) LTCIC per kg body weight (range, 0.71 to 3.72). In addition, in six patients, large-scale ex vivo expansions were performed using a five-factor cytokine combination consisting of stem cell factor (SCF), interleukin-1 (IL-1), IL-3, IL-6, and erythropoietin (EPO), previously shown to expand committed progenitor cells. LTCIC were preserved, but not expanded during the culture period. Optimization of ex vivo expansion growth factor requirements using limiting dilution assays for LTCIC estimation indicated that the five-factor combination using SCF, IL-1, IL-3, IL-6, and EPO together with autologous plasma was the most reliable combination securing both high progenitor yield and, at the same time, optimal preservation of LTCIC. Our data suggest that ex vivo-expanded CD34+ PBPCs might be able to allow long-term reconstitution of hematopoiesis.

CD34-positive cells isolated from cryopreserved peripheral-blood progenitor cells can be expanded ex vivo and used for transplantation with little or no toxicity.

1996 ◽  
Vol 14 (6) ◽  
pp. 1839-1847 ◽  
Author(s):  
M J Alcorn ◽  
T L Holyoake ◽  
L Richmond ◽  
C Pearson ◽  
E Farrell ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Peripheral Blood ◽  
Large Scale ◽  
Ex Vivo ◽  
Interleukin 1 ◽  
Cell Number ◽  
Autologous Serum ◽  
Platelet Recovery ◽  
Peripheral Blood Progenitor Cells ◽  
Historical Controls

PURPOSE The objectives of this phase I study were to assess the feasibility of using cryopreserved peripheral-blood progenitor cells (PBPC) for large-scale CD34 selection and subsequent expansion, and the safety of their use for reinfusion following chemoradiotherapy. PATIENTS AND METHODS For 10 patients with nonmyeloid malignancy, an aliquot from a PBPC harvest was recovered from liquid nitrogen, and CD34 selected using the Isolex system (Baxter Healthcare, Newbury, United Kingdom) and expanded for 8 days ex vivo in a medium free of animal proteins but supplemented with autologous serum, stemcell factor (SCF), interleukin-1 beta (IL-1 beta), IL-3, IL-6, and erythropoietin. RESULTS The mean increase for cell number was 21-fold, for colony-forming units-granulocyte/macrophage (CFU-GM) 139-fold, and for burst-forming units-erythroid (BFU-E) 114-fold. The expanded cells were reinfused in tandem with unmanipulated material (> or = 25 x 10(4) CFU-GM/kg). The patients did not experience any adverse effects immediately on cell infusion or within 48 hours. The 10 index patients were compared with 10 historical controls for parameters of myelosuppressive morbidity. In this small study, there were no differences in either neutrophil or platelet recovery between the patients who received expanded cells and historical controls. CONCLUSION These data demonstrate that CD34 cells can successfully be selected from cryopreserved material, expanded ex vivo on a large scale, and safely reinfused following myeloablative conditioning regimens.

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