scholarly journals Human multidrug resistance-1 gene transfer to long-term repopulating human mobilized peripheral blood progenitor cells

2000 ◽  
Vol 25 (S2) ◽  
pp. S118-S124 ◽  
Author(s):  
B Schiedlmeier ◽  
K Wermann ◽  
K Kühlcke ◽  
HG Eckert ◽  
C Baum ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Gene Transfer ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Peripheral Blood Progenitor Cells ◽  
Multidrug Resistance 1 Gene ◽  
Mobilized Peripheral Blood

Quantitative assessment of retroviral transfer of the human multidrug resistance 1 gene to human mobilized peripheral blood progenitor cells engrafted in nonobese diabetic/severe combined immunodeficient mice

Blood ◽  
2000 ◽  
Vol 95 (4) ◽  
pp. 1237-1248 ◽  
Author(s):  
B. Schiedlmeier ◽  
K. Kühlcke ◽  
H. G. Eckert ◽  
C. Baum ◽  
W. J. Zeller ◽  
...  
Keyword(s):  
Stem Cell ◽  
Multidrug Resistance ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Autologous Transplantation ◽  
Embryonic Stem ◽  
Scid Mice ◽  
Nonobese Diabetic ◽  
Peripheral Blood Progenitor Cells ◽  
Mobilized Peripheral Blood

Mobilized peripheral blood progenitor cells (PBPC) are a potential target for the retrovirus-mediated transfer of cytostatic drug-resistance genes. We analyzed nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse-repopulating CD34+ PBPC from patients with cancer after retroviral transduction in various cytokine combinations with the hybrid vector SF-MDR, which is based on the Friend mink cell focus-forming/murine embryonic stem-cell virus and carries the human multidrug resistance 1 (MDR1) gene. Five to 13 weeks after transplantation of CD34+ PBPC into NOD/SCID mice (n = 84), a cell dose-dependent multilineage engraftment of human leukocytes up to an average of 33% was observed. The SF-MDR provirus was detected in the bone marrow (BM) and in its granulocyte fractions in 96% and 72%, respectively, of chimeric NOD/SCID mice. SF-MDR provirus integration assessed by quantitative real-time polymerase chain reaction (PCR) was optimal in the presence of Flt-3 ligand/thrombopoietin/stem-cell factor, resulting in a 6-fold (24% ± 5% [mean ± SE]) higher average proportion of gene-marked human cells in NOD/SCID mice than that achieved with IL-3 alone (P < .01). A population of clearly rhodamine-123dull human myeloid progeny cells could be isolated from BM samples from chimeric NOD/SCID mice. On the basis of PCR and rhodamine-123 efflux data, up to 18% ± 4% of transduced cells were calculated to express the transgene. Our data suggest that the NOD/SCID model provides a valid assay for estimating the gene-transfer efficiency to repopulating human PBPC that may be achievable in clinical autologous transplantation. P-glycoprotein expression sufficient to prevent marrow aplasia in vivo may be obtained with this SF-MDR vector and an optimized transduction protocol.

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 Polyclonal Long-Term MFGS-gp91phox Marking in Rhesus Macaques after Nonmyeloablative Transplantation with Transduced Autologous Peripheral Blood Progenitor Cells

2006 ◽  
Vol 14 (2) ◽  
pp. 202-211 ◽  
Author(s):  
Sebastian Brenner ◽  
Martin F. Ryser ◽  
Uimook Choi ◽  
Narda Whiting-Theobald ◽  
Eberhard Kuhlisch ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Peripheral Blood ◽  
Rhesus Macaques ◽  
Peripheral Blood Progenitor Cells

In Vitro Manipulation of Peripheral Blood Progenitor Cell Collections

1998 ◽  
Vol 21 (6_suppl) ◽  
pp. 1-10
Author(s):  
C. Carlo-Stella ◽  
V. Rizzoli
Keyword(s):  
Progenitor Cells ◽  
Peripheral Blood ◽  
Progenitor Cell ◽  
High Dose ◽  
Hematopoietic Stem ◽  
Peripheral Blood Progenitor Cells ◽  
Stem And Progenitor Cells ◽  
Therapy Strategies ◽  
Mobilized Peripheral Blood

Mobilized peripheral blood progenitor cells (PBPC) are increasingly used to reconstitute hematopoiesis in patients undergoing high-dose chemoradiotherapy. PBPC collections comprise a heterogeneous population containing both committed progenitors and pluripotent stem cells and can be harvested (i) in steady state, (ii) after chemotherapeutic conditioning, (iii) growth factor priming, or (iv) both. The use of PBPC has opened new therapeutic perspectives mainly related to the availability of large amounts of mobilized hematopoietic stem and progenitor cells. Extensive manipulation of the grafts, including the possibility of exploiting these cells as vehicles for gene therapy strategies, are now possible and will be reviewed.

Effects of long-term cryopreservation on peripheral blood progenitor cells

Cytotherapy ◽  
2012 ◽  
Vol 14 (10) ◽  
pp. 1228-1234 ◽  
Author(s):  
Gregory S. Vosganian ◽  
Jill Waalen ◽  
Kevin Kim ◽  
Sejal Jhatakia ◽  
Ethan Schram ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Peripheral Blood ◽  
Peripheral Blood Progenitor Cells

Efficient Gene Transfer into Human CD34+ Peripheral Blood Progenitor Cells Using Pseudotyped Recombinant Adeno-Associated Viral Vectors

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4627-4627
Author(s):  
Leopold Sellner ◽  
Marlon Veldwijk ◽  
Marius Stiefelhagen ◽  
Jurgen A. Kleinschmidt ◽  
Stephanie Laufs ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Progenitor Cells ◽  
Safety Profile ◽  
Peripheral Blood ◽  
Vector System ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Peripheral Blood Progenitor Cells ◽  
The Difference ◽  
Relevant Gene

Abstract Due to their pluripotency, human CD34+ hematopoietic stem cells are targets of interest for the treatment of many acquired and inherited disorders of the hematopoietic system using gene therapeutic approaches. Human CD34+ peripheral blood progenitor cells (PBPC) can be readily mobilized into the blood and harvested by leukapheresis, providing an easily accessible source of hematopoietic progenitor cells. Unfortunately, for gene transfer into CD34+ PBPC, most current vector systems either lack sufficient transduction efficiency or an acceptable safety profile. Standard adeno-associated virus-based vectors have an advantageous safety profile, yet lack the required efficiency. Therefore a panel of pseudotyped recombinant adeno-associated viral (rAAV2/1 - rAAV2/6) vectors expressing the eGFP gene was screened on human G-CSF-mobilized CD34+ PBPC to determine their efficacy. In addition, the difference in transgene expression between conventional single-stranded rAAV and self complementary rAAV (scAAV) vectors was determined. For each vector n≥6 was performed and data are shown as mean ± SD. Of all screened conventional rAAV vectors, rAAV2/6 proved to be the most efficient (13.5% ± 9.8% GFP+ and CD34+ PBPC; p<0.001 vs other vectors) on human CD34+ PBPC, followed by rAAV2/2 (2.6% ± 2.0% GFP+ cells) and rAAV2/1 (1.4% ± 1.2% GFP+ cells). For rAAV2/3, rAAV2/4 and rAAV2/5 no relevant gene transfer efficiency (<1% GFP+ cells) was observed. Furthermore, the relevance of the single-to-double-strand conversion block in transduction of human PBPC could be shown using scAAV vectors. scAAV2/6 and scAAV2/2 (both p<0.001) showed significantly higher gene expression (38.4% ± 12.2% and 11.8% ± 5.7% GFP+ cells, respectively) compared to their conventional counterparts in this cell entity. Similar results were observed for scAAV2/1 vectors (2.8% ± 1.9% GFP+ cells), though the difference was not significant. Of note, as previously observed using AAV peptide library-derived rAAV vectors (Sellner et al., 2008, Exp Hematol. 36), also here inter-patient variances in CD34+ PBPC susceptibility were found. For the first time we were able to obtain clinically relevant gene transfer and expression levels (>10%) with expression rates up to 60% in human CD34+ PBPC using an AAV-based vector system, thereby providing an efficient alternative vector system for gene transfer into this clinically important target cell.

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