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.

Ex vivo expanded peripheral blood progenitor cells provide rapid neutrophil recovery after high-dose chemotherapy in patients with breast cancer

Blood ◽  
2000 ◽  
Vol 96 (9) ◽  
pp. 3001-3007 ◽  
Author(s):  
Ian McNiece ◽  
Roy Jones ◽  
Scott I. Bearman ◽  
Pablo Cagnoni ◽  
Yago Nieto ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
High Dose Chemotherapy ◽  
Control Group ◽  
High Dose ◽  
Platelet Recovery ◽  
Peripheral Blood Progenitor Cells ◽  
Neutrophil Engraftment

Abstract Ex vivo expanded peripheral blood progenitor cells (PBPCs) have been proposed as a source of hematopoietic support to decrease or eliminate the period of neutropenia after high-dose chemotherapy. CD34 cells were selected from rhG-CSF mobilized PBPCs from patients with breast cancer and were cultured for 10 days in defined media containing 100 ng/mL each of rhSCF, rhG-CSF, and PEG-rhMGDF in 1 L Teflon bags at 20 000 cells/mL. After culture the cells were washed and reinfused on day 0 of transplantation. On day +1, cohort 1 patients (n = 10) also received an unexpanded CD34-selected PBPC product. These patients engrafted neutrophils (absolute neutrophil count, >500/μL) in a median of 6 (range, 5-14) days. Cohort 2 patients (n = 11), who received expanded PBPCs only, engrafted neutrophils in a median of 8 (range, 4-16) days. In comparison, the median time to neutrophil engraftment in a historical control group of patients (n = 100) was 9 days (range, 7-30 days). All surviving patients are now past the 15-month posttransplantation stage with no evidence of late graft failure. The total number of nucleated cells harvested after expansion culture was shown to be the best predictor of time to neutrophil engraftment, with all patients receiving more than 4 × 107 cells/kg, engrafting neutrophils by day 8. No significant effect on platelet recovery was observed in any patient. These data demonstrate that PBPCs expanded under the conditions defined can shorten the time to engraftment of neutrophils compared with historical controls and that the rate of engraftment is related to the dose of expanded cells transplanted.

Ex vivo expanded peripheral blood progenitor cells provide rapid neutrophil recovery after high-dose chemotherapy in patients with breast cancer

Blood ◽  
2000 ◽  
Vol 96 (9) ◽  
pp. 3001-3007 ◽  
Author(s):  
Ian McNiece ◽  
Roy Jones ◽  
Scott I. Bearman ◽  
Pablo Cagnoni ◽  
Yago Nieto ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
High Dose Chemotherapy ◽  
Control Group ◽  
High Dose ◽  
Platelet Recovery ◽  
Peripheral Blood Progenitor Cells ◽  
Neutrophil Engraftment

Ex vivo expanded peripheral blood progenitor cells (PBPCs) have been proposed as a source of hematopoietic support to decrease or eliminate the period of neutropenia after high-dose chemotherapy. CD34 cells were selected from rhG-CSF mobilized PBPCs from patients with breast cancer and were cultured for 10 days in defined media containing 100 ng/mL each of rhSCF, rhG-CSF, and PEG-rhMGDF in 1 L Teflon bags at 20 000 cells/mL. After culture the cells were washed and reinfused on day 0 of transplantation. On day +1, cohort 1 patients (n = 10) also received an unexpanded CD34-selected PBPC product. These patients engrafted neutrophils (absolute neutrophil count, >500/μL) in a median of 6 (range, 5-14) days. Cohort 2 patients (n = 11), who received expanded PBPCs only, engrafted neutrophils in a median of 8 (range, 4-16) days. In comparison, the median time to neutrophil engraftment in a historical control group of patients (n = 100) was 9 days (range, 7-30 days). All surviving patients are now past the 15-month posttransplantation stage with no evidence of late graft failure. The total number of nucleated cells harvested after expansion culture was shown to be the best predictor of time to neutrophil engraftment, with all patients receiving more than 4 × 107 cells/kg, engrafting neutrophils by day 8. No significant effect on platelet recovery was observed in any patient. These data demonstrate that PBPCs expanded under the conditions defined can shorten the time to engraftment of neutrophils compared with historical controls and that the rate of engraftment is related to the dose of expanded cells transplanted.

Ex vivo expanded unselected peripheral blood: progenitor cells reduce posttransplantation neutropenia, thrombocytopenia, and anemia in patients with breast cancer

Blood ◽  
2000 ◽  
Vol 96 (7) ◽  
pp. 2385-2390 ◽  
Author(s):  
Ronald L. Paquette ◽  
Sanaa T. Dergham ◽  
Ellen Karpf ◽  
He-Jing Wang ◽  
Dennis J. Slamon ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
Conditioning Regimen ◽  
High Dose Chemotherapy ◽  
High Dose ◽  
Cell Dose ◽  
Development Factor ◽  
Peripheral Blood Progenitor Cells

The safety and efficacy of administering ex vivo expanded peripheral blood progenitor cells (PBPC) to patients with breast cancer who undergo high-dose chemotherapy and PBPC transplantation was investigated. Unselected PBPC were cultured in gas-permeable bags containing 1-L serum-free media, granulocyte colony-stimulating factor, stem cell factor, and pegylated megakaryocyte growth and development factor for 9 days. Cell dose cohorts were assigned to have between 2 and 24 × 109 PBPC cultured at 1, 2, or 3 × 106 cells/mL. Twenty-four patients received high-dose chemotherapy followed by infusion of the cultured PBPC and at least 5 × 106 CD34+ uncultured cryopreserved PBPC per kilogram. No toxicities resulted from infusions of the ex vivo expanded PBPC. The study patients had shorter times to neutrophil (P = .0001) and platelet (P = .01) recovery and fewer red cell transfusions (P = .02) than 48 historical controls who received the same conditioning regimen and posttransplantation care and at least 5 × 106CD34+ PBPC per kilogram. Improvements in all these endpoints were significantly correlated with the expanded cell dose. Nine of 24 (38%) patients recovered neutrophil counts above 500/μL by day 5 or 6 after transplantation, whereas none of the controls had neutrophil recovery before the eighth day. Seven (29%) patients had neutropenia for 3 or fewer days, and 9 (38%) patients did not experience neutropenic fevers or require broad-spectrum antibiotics. Therefore, ex vivo expanded PBPC are capable of ameliorating posttransplantation neutropenia, thrombocytopenia, and anemia in patients receiving high-dose chemotherapy.

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

EPCs Derived from Ex Vivo Expansion of Unmobilized Human Peripheral Blood Co-Express Hematopoietic and Endothelial-Specific Markers and Can Be Derived from Culture of Highly Purified Populations of CD14-Positive Monocytes.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2682-2682
Author(s):  
Emerson E. Sharpe ◽  
Amylynn A. Teleron ◽  
Bin Li ◽  
Pampee P. Young
Keyword(s):  
Progenitor Cells ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
Human Peripheral Blood ◽  
Ex Vivo Expansion ◽  
Adherent Cells ◽  
Hematopoietic Stem ◽  
Endothelial Markers ◽  
Cell Based Therapy

Abstract An increasing amount of data has suggested a more dynamic role of vasculogenesis, whereby bone marrow (BM)-derived circulating endothelial progenitor cells (EPCs) home and contribute to new blood vessel formation during tumor growth, ischemic injury, and wound healing. EPCs can be obtained by isolating hematopoietic progenitor cells from BM or cord blood. Alternatively, ex vivo expansion of unmobilized human peripheral blood (PB) can generate adherent cells, PB-EPCs, that express endothelial markers and also, upon administration, incorporate into developing neovasculature. The relative ease of obtaining unmobilized human PB has made PB-EPCs an attractive candidate with which to develop cell based therapy to treat ischemia. In parallel with clinical trials designed to understand their therapeutic potential, there is a continued effort to better characterize the PB-EPC and understand its biology. It is currently thought that EPCs are directly derived from a CD34+/lin- faction of hematopoietic stem cells (HSCs). However, in our current study, we have confirmed prior reports that ex vivo expansion of human PB generates similar numbers of EPCs as compared to plating unfractionated human BM, which contains >50-fold higher CD34+/lin- content, suggesting that PB-EPCs may not be derived from the CD34+/lin- population. We used immunofluorescence and FACs analysis to further show that PB-EPCs not only express endothelial markers such as vWF, Vascular Endothelial Growth Factor Receptor 1 and 2 (also known as flt-1 and flk-1, respectively), VE-cadherin, UEA-1 lectin, Tie-1 and Tie-2 but also hematopoietic markers such as CD45 and CD14, a marker enriched on monocytes. To test if PB-derived CD14-positive cells can give rise to PB-EPCs, we isolated them from human PB to >98% purity and plated them on fibronectin-coated coverslips. In vitro culture of CD14-positive cells generated adherent clusters of spindle shaped cells morphologically similar to EPCs. Culture of the CD14-negative fraction failed to yield any adherent cells. After ten days, the coverslips were removed and the cells were stained with various endothelial (flk-1, vWF, uptake of DiI-AcLDL, and UEA-1 lectin) and monocyte/hematopoietic (CD14 and CD45) cell markers. In analysis of these slides, the EPCs derived from the purified CD14 fraction stained positive for all six markers. These observations suggest that PB-EPCs can differentiate from cells of the monocytic lineage in vitro without the necessity of interaction from cells contained in the CD14-negative population. Further experiments will test the possibility that monocytes may be an intermediate in the differentiation of EPCs in vivo.

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