scholarly journals Retroviral transduction of human progenitor cells: use of granulocyte colony-stimulating factor plus stem cell factor to mobilize progenitor cells in vivo and stimulation by Flt3/Flk-2 ligand in vitro

Blood ◽  
1996 ◽  
Vol 88 (12) ◽  
pp. 4452-4462 ◽  
Author(s):  
NJ Elwood ◽  
H Zogos ◽  
T Willson ◽  
CG Begley
Keyword(s):  
Gene Transfer ◽  
Progenitor Cells ◽  
Stem Cell Factor ◽  
Fold Increase ◽  
Transduction Efficiency ◽  
Granulocyte Colony Stimulating Factor ◽  
Retroviral Transduction ◽  
Stimulating Factor

The clinical application of gene transfer is hindered by the availability of the multipotential stem cells and the difficulty in obtaining efficient retroviral transduction. To assess potential means by which gene transfer into human hemopoietic stem cells might be enhanced, the retroviral transduction efficiency of human bone marrow cells (BM) or peripheral blood progenitor cells (PBPC) was compared at multiple time points after in vivo administration of granulocyte colony- stimulating factor (G-CSF). This was further compared with the transduction efficiency of cells mobilized with G-CSF plus stem cell factor (SCF) in a cohort of patients randomized to receive either one or two growth factors and with normal BM function. Using the LNL6 retrovirus, retroviral transduction efficiencies of up to 19% were observed for both PBPC and BM (n = 26 patients). There was at least a 100-fold increase in PBPC with G-CSF alone and a further 30-fold increase in the total number of progenitor cells available for retroviral transduction using the combination of SCF plus G-CSF. However, pretreatment of patients with G-CSF with or without SCF did not enhance the retroviral infectability of growth factor-mobilized progenitor cells. The effect of the growth factor, Flk-2/Flt3 ligand (FL), was also examined with respect to retroviral transduction efficiency of human progenitor cells. FL plus IL-3 in vitro increased the retroviral transduction efficiency up to eightfold compared with results observed using other combinations of cytokines tested (P < .001). These findings have clinical implications both for increasing the number of target cells for in vivo gene-marking/gene-therapy studies and improving the efficiency of gene transfer.

scholarly journals Retroviral transduction and expression of the human alkyltransferase cDNA provides nitrosourea resistance to hematopoietic cells

Blood ◽  
1995 ◽  
Vol 85 (11) ◽  
pp. 3342-3351 ◽  
Author(s):  
JA Allay ◽  
LL Dumenco ◽  
ON Koc ◽  
L Liu ◽  
SL Gerson
Keyword(s):  
Gene Transfer ◽  
High Efficiency ◽  
Hematopoietic Cells ◽  
K562 Cells ◽  
Fold Increase ◽  
Transduction Efficiency ◽  
Retroviral Transduction ◽  
Alkyl Moiety

Myelosuppression is the dose-limiting toxicity for nitrosourea chemotherapy. This toxicity predominantly involves modification of the O6 position of guanine with an alkyl moiety. The enzyme responsible for repair of O6-alkylguanine adducts, O6-alkylguanine-DNA alkyltransferase (alkyltransferase), is expressed at low levels in bone marrow (BM) cells. High alkyltransferase expression prevents the cytotoxicity and carcinogenicity of nitrosoureas in several transgenic and in vitro gene transfer models. We used gene transfer using a novel myeloproliferative sarcoma virus (MPSV) based retrovirus (vM5MGMT) to express the human alkyltransferase cDNA (MGMT) in human and murine hematopoietic cells. Transduced K562 cells had very high levels of alkyltransferase expression and significantly increased resistance to 1,3-bis (2-chloroethyl) nitrosourea (BCNU) as compared with untransduced K562 cells. Primary murine BM progenitors showed a high transduction efficiency with vM5MGMT and have increased BCNU resistance in vitro. After BM transplantation with vM5MGMT-transduced BM cells and BCNU treatment of these mice, BM, spleen and thymus had a 10- to 40-fold increase in alkyltransferase expression that persisted for at least 23 weeks posttransplantation. Progenitor cells procured from mice expressing high levels of alkyltransferase also had increased resistance to BCNU. Thus, an MPSV-based retroviral vector transduces mouse and human hematopoietic cells at high efficiency and results in high levels of gene expression both in vitro and in vivo. Overexpression of the alkyltransferase protein may protect hematopoietic progenitors from nitrosourea-induced myelosuppression.

scholarly journals Recombinant rat stem cell factor synergizes with recombinant human granulocyte colony-stimulating factor in vivo in mice to mobilize peripheral blood progenitor cells that have enhanced repopulating potential

Blood ◽  
1993 ◽  
Vol 82 (6) ◽  
pp. 1720-1723 ◽  
Author(s):  
RA Briddell ◽  
CA Hartley ◽  
KA Smith ◽  
IK McNiece
Keyword(s):  
Stem Cell ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Stem Cell Factor ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Peripheral Blood Progenitor Cells ◽  
Stimulating Factor ◽  
Synergistic Increase

Abstract Splenectomized mice treated for 7 days with pegylated recombinant rat stem cell factor (rrSCF-PEG) showed a dose-dependent increase in peripheral blood progenitor cells (PBPC) that have enhanced in vivo repopulating potential. A dose of rrSCF-PEG at 25 micrograms/kg/d for 7 days produced no significant increase in PBPC. However, when this dose of rrSCF-PEG was combined with an optimal dose of recombinant human granulocyte colony-stimulating factor (rhG-CSF; 200 micrograms/kg/d), a synergistic increase in PBPC was observed. Compared with treatment with rhG-CSF alone, the combination of rrSCF-PEG plus rhG-CSF resulted in a synergistic increase in peripheral white blood cells, in the incidence and absolute numbers of PBPC, and in the incidence and absolute numbers of circulating cells with in vivo repopulating potential. These data suggest that low doses of SCF, which would have minimal, if any, effects in vivo, can synergize with optimal doses of rhG-CSF to enhance the mobilization of PBPC stimulated by rhG-CSF alone.

scholarly journals Recombinant rat stem cell factor synergizes with recombinant human granulocyte colony-stimulating factor in vivo in mice to mobilize peripheral blood progenitor cells that have enhanced repopulating potential

Blood ◽  
1993 ◽  
Vol 82 (6) ◽  
pp. 1720-1723 ◽  
Author(s):  
RA Briddell ◽  
CA Hartley ◽  
KA Smith ◽  
IK McNiece
Keyword(s):  
Stem Cell ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Stem Cell Factor ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Peripheral Blood Progenitor Cells ◽  
Stimulating Factor ◽  
Synergistic Increase

Splenectomized mice treated for 7 days with pegylated recombinant rat stem cell factor (rrSCF-PEG) showed a dose-dependent increase in peripheral blood progenitor cells (PBPC) that have enhanced in vivo repopulating potential. A dose of rrSCF-PEG at 25 micrograms/kg/d for 7 days produced no significant increase in PBPC. However, when this dose of rrSCF-PEG was combined with an optimal dose of recombinant human granulocyte colony-stimulating factor (rhG-CSF; 200 micrograms/kg/d), a synergistic increase in PBPC was observed. Compared with treatment with rhG-CSF alone, the combination of rrSCF-PEG plus rhG-CSF resulted in a synergistic increase in peripheral white blood cells, in the incidence and absolute numbers of PBPC, and in the incidence and absolute numbers of circulating cells with in vivo repopulating potential. These data suggest that low doses of SCF, which would have minimal, if any, effects in vivo, can synergize with optimal doses of rhG-CSF to enhance the mobilization of PBPC stimulated by rhG-CSF alone.

scholarly journals In vivo synergy between recombinant human stem cell factor and recombinant human granulocyte colony-stimulating factor in baboons enhanced circulation of progenitor cells

Blood ◽  
1994 ◽  
Vol 84 (3) ◽  
pp. 800-810 ◽  
Author(s):  
RG Andrews ◽  
RA Briddell ◽  
GH Knitter ◽  
T Opie ◽  
M Bronsden ◽  
...  
Keyword(s):  
Stem Cell ◽  
Progenitor Cells ◽  
Stem Cell Factor ◽  
Low Dose ◽  
Granulocyte Colony Stimulating Factor ◽  
Human Stem Cell ◽  
Colony Stimulating Factor ◽  
Stimulating Factor ◽  
Human Stem Cell Factor

Abstract Recombinant human stem cell factor (rhSCF) and recombinant human granulocyte colony-stimulating factor (rhG-CSF) are synergistic in vitro in stimulating the proliferation of hematopoietic progenitor cells and their precursors. We examined the in vivo synergy of rhSCF with rhG-CSF for stimulating hematopoiesis in vivo in baboons. Administration of low-dose (LD) rhSCF (25 micrograms/kg) alone did not stimulate changes in circulating WBCs. In comparison, administration of LD rhSCF in combination with rhG-CSF at 10 micrograms/kg or 100 micrograms/kg stimulated increases in circulating WBCs of multiple types up to twofold higher than was stimulated by administration of the same dose of rhG-CSF alone. When the dose of rhG-CSF is increased to 250 micrograms/kg, the administration of LD rhSCF does not further increase the circulating WBC counts. Administration of LD rhSCF in combination with rhG-CSF also stimulated increased circulation of hematopoietic progenitors. LD rhSCF alone stimulated less of an increase in circulating progenitors, per milliliter of blood, than did administration of rhG-CSF alone at 100 micrograms/kg. Baboons administered LD rhSCF together with rhG-CSF at 10, 100, or 250 micrograms/kg had 3.5- to 16-fold higher numbers per milliliter of blood of progenitors cells of multiple types, including colony-forming units granulocyte/macrophage (CFU-GM), burst-forming unit-erythroid (BFU-E), and colony-forming and burst-forming units-megakaryocyte (CFU- MK and BFU-MK) compared with animals given the same dose of rhG-CSF without rhSCF, regardless of the rhG-CSF dose. The increased circulation of progenitor cells stimulated by the combination of rhSCF plus rhG-CSF was not necessarily directly related to the increase in WBCs, as this effect on peripheral blood progenitors was observed even at an rhG-CSF dose of 250 micrograms/kg, where coadministration of LD rhSCF did not further increase WBC counts. Administration of very-low- dose rhSCF (2.5 micrograms/kg) with rhG-CSF, 10 micrograms/kg, did not stimulate increases in circulating WBCs, but did increase the number of megakaryocyte progenitor cells in blood compared with rhG-CSF alone. LD rhSCF administered alone for 7 days before rhG-CSF did not result in increased levels of circulating WBCs or progenitors compared with rhG- CSF alone. Thus, the synergistic effects of rhSCF with rhG-CSF were both dose- and time-dependent. The doses of rhSCF used in these studies have been tolerated in vivo in humans.(ABSTRACT TRUNCATED AT 400 WORDS).

scholarly journals In vivo synergy between recombinant human stem cell factor and recombinant human granulocyte colony-stimulating factor in baboons enhanced circulation of progenitor cells

Blood ◽  
1994 ◽  
Vol 84 (3) ◽  
pp. 800-810 ◽  
Author(s):  
RG Andrews ◽  
RA Briddell ◽  
GH Knitter ◽  
T Opie ◽  
M Bronsden ◽  
...  
Keyword(s):  
Stem Cell ◽  
Progenitor Cells ◽  
Stem Cell Factor ◽  
Low Dose ◽  
Granulocyte Colony Stimulating Factor ◽  
Human Stem Cell ◽  
Colony Stimulating Factor ◽  
Stimulating Factor ◽  
Human Stem Cell Factor

Recombinant human stem cell factor (rhSCF) and recombinant human granulocyte colony-stimulating factor (rhG-CSF) are synergistic in vitro in stimulating the proliferation of hematopoietic progenitor cells and their precursors. We examined the in vivo synergy of rhSCF with rhG-CSF for stimulating hematopoiesis in vivo in baboons. Administration of low-dose (LD) rhSCF (25 micrograms/kg) alone did not stimulate changes in circulating WBCs. In comparison, administration of LD rhSCF in combination with rhG-CSF at 10 micrograms/kg or 100 micrograms/kg stimulated increases in circulating WBCs of multiple types up to twofold higher than was stimulated by administration of the same dose of rhG-CSF alone. When the dose of rhG-CSF is increased to 250 micrograms/kg, the administration of LD rhSCF does not further increase the circulating WBC counts. Administration of LD rhSCF in combination with rhG-CSF also stimulated increased circulation of hematopoietic progenitors. LD rhSCF alone stimulated less of an increase in circulating progenitors, per milliliter of blood, than did administration of rhG-CSF alone at 100 micrograms/kg. Baboons administered LD rhSCF together with rhG-CSF at 10, 100, or 250 micrograms/kg had 3.5- to 16-fold higher numbers per milliliter of blood of progenitors cells of multiple types, including colony-forming units granulocyte/macrophage (CFU-GM), burst-forming unit-erythroid (BFU-E), and colony-forming and burst-forming units-megakaryocyte (CFU- MK and BFU-MK) compared with animals given the same dose of rhG-CSF without rhSCF, regardless of the rhG-CSF dose. The increased circulation of progenitor cells stimulated by the combination of rhSCF plus rhG-CSF was not necessarily directly related to the increase in WBCs, as this effect on peripheral blood progenitors was observed even at an rhG-CSF dose of 250 micrograms/kg, where coadministration of LD rhSCF did not further increase WBC counts. Administration of very-low- dose rhSCF (2.5 micrograms/kg) with rhG-CSF, 10 micrograms/kg, did not stimulate increases in circulating WBCs, but did increase the number of megakaryocyte progenitor cells in blood compared with rhG-CSF alone. LD rhSCF administered alone for 7 days before rhG-CSF did not result in increased levels of circulating WBCs or progenitors compared with rhG- CSF alone. Thus, the synergistic effects of rhSCF with rhG-CSF were both dose- and time-dependent. The doses of rhSCF used in these studies have been tolerated in vivo in humans.(ABSTRACT TRUNCATED AT 400 WORDS).

Functional analysis of human hematopoietic repopulating cells mobilized with granulocyte colony-stimulating factor alone versus granulocyte colony-stimulating factor in combination with stem cell factor

Blood ◽  
2002 ◽  
Vol 100 (3) ◽  
pp. 869-878 ◽  
Author(s):  
David A. Hess ◽  
Krysta D. Levac ◽  
Francis N. Karanu ◽  
Michael Rosu-Myles ◽  
Martin J. White ◽  
...  
Keyword(s):  
Stem Cell ◽  
Stem Cell Factor ◽  
Fold Increase ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Cell Content ◽  
Serum Free ◽  
Nonobese Diabetic ◽  
Stimulating Factor

Abstract Using in vitro progenitor assays, serum-free in vitro cultures, and the nonobese diabetic/severe combined immune-deficient (NOD/SCID) ecotropic murine virus knockout xenotransplantation model to detect human SCID repopulating cells (SRCs) with multilineage reconstituting function, we have characterized and compared purified subpopulations harvested from the peripheral blood (PB) of patients receiving granulocyte colony-stimulating factor (G-CSF) alone or in combination with stem cell factor (SCF). Mobilized G-CSF plus SCF PB showed a 2-fold increase in total mononuclear cell content and a 5-fold increase in CD34-expressing cells depleted for lineage-marker expression (CD34+Lin−) as compared with patients treated with G-CSF alone. Functionally, G-CSF plus SCF–mobilized CD34+CD38−Lin−cells contained a 2-fold enhancement in progenitor frequency as compared with G-CSF–mobilized subsets. Despite enhanced cellularity and progenitor capacity, G-CSF plus SCF mobilization did not increase the frequency of SRCs as determined by limiting dilution analysis by means of unfractionated PB cells. Purification of SRCs from these sources demonstrated that as few as 1000 CD34+CD38−Lin− cells from G-CSF–mobilized PB contained SRC capacity while G-CSF plus SCF–mobilized CD34+CD38−Lin−cells failed to repopulate at doses up to 500 000 cells. In addition, primitive CD34−CD38−AC133+Lin−cells derived from G-CSF plus SCF–mobilized PB were capable of differentiation into CD34-expressing cells, while the identical subfractions from G-CSF PB were unable to produce CD34+cells in serum-free cultures. Our study defines qualitative and quantitative distinctions among subsets of primitive cells mobilized by means of G-CSF plus SCF versus G-CSF alone, and therefore has implications for the utility of purified repopulating cells from these sources.

Abstract 41: Explant-Derived Cells from Chronic Heart Failure Activated Endothelial-Mesenchymal Transition and Attenuated Pluripotency Genes Expression

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Liudmila Zakharova ◽  
Hikmet Nural ◽  
Mohamed A Gaballa
Keyword(s):  
Gene Expression ◽  
Progenitor Cells ◽  
Smooth Muscle Actin ◽  
Fold Increase ◽  
Gene Expressions ◽  
Mesenchymal Transition ◽  
Cell Outgrowth ◽  
Pluripotency Genes

Cardiac progenitor cells are generated from atria explants; however the cellular origin and the mechanisms of cell outgrowth are unclear. Using transgenic tamoxifen-induced Willms tumor 1 (Wt1)-Cre/ERT and Cre-activated GFP reporter mice, we found approximately 40% of explant-derived cells and 74% of explant-derived c-Kit+ cells originated from the epicardium. In atria from sham hearts, Wt1+ cells were located in a thin epicardial layer, while c-Kit+ cells were primarily found within both the sub-epicardium and the myocardium, albeit at low frequency. No overlap between c-Kit+ and Wt1+ cells was observed, suggesting that epicardial Wt1+ cells do not express c-Kit marker in vivo, but more likely the c-Kit marker was acquired in culture. Compared with 4 days in culture, at day 21 we observed 7 folds increase in Snail gene expression; 32% increase in α-smooth muscle actin (SMA) marker, and 30% decrease in E-cadherin marker, suggesting that the explant-derived cells underwent epithelial to mesenchymal transition (EMT) in vitro. Cell outgrowths released TGF-β (1036.4 ± 1.18 pm/ml) and exhibited active TGF-β signaling, which might triggered the EMT. Compared to shams, CHF cell outgrowths exhibited elevated levels of EMT markers, SMA (49% vs. 34%) and Snail (2 folds), and reduced level of Wt1 (11% vs. 22%). In addition, CHF cell outgrowths had two folds increase in Pai1 gene expression, a direct target of TGF-β signaling. In c-Kit+ cells derived from CHF explants, Nanog gene expression was 4 folds lower and Sox 2 was 2 folds lower compared with cells from shams. Suppression of EMT in cell outgrowth increased the percentage of c-Kit+ and Wt1+ cells by 17%, and 15%, respectively. Also suppression of EMT in c-Kit+ cells resulted in 4 folds increase in Nanog and 3 fold increase in Sox2 gene expressions. Our results showed that CHF may further exuberates EMT while diminishes the re-activation of pluripotency genes. Thus, EMT modulation in CHF is a possible strategy to regulate both the yield and the pluripotency of cardiac-explant-derived progenitor cells.

scholarly journals In vitro and in vivo hematopoietic effect of mutant human granulocyte colony-stimulating factor [see comments]

Blood ◽  
1990 ◽  
Vol 75 (9) ◽  
pp. 1788-1793 ◽  
Author(s):  
M Okabe ◽  
M Asano ◽  
T Kuga ◽  
Y Komatsu ◽  
M Yamasaki ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Intravenous Injection ◽  
Specific Activity ◽  
High Specific Activity ◽  
Daily Injection ◽  
Total Leukocyte Count ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Stimulating Factor

About 100 derivatives of human recombinant granulocyte colony- stimulating factor (rhG-CSF) were created by various gene-mutagenic techniques, and KW-2228, in which amino acids were replaced at five positions of N-terminal region of intact rhG-CSF, was picked up and evaluated for its biologic and physicochemical properties in comparison with intact rhG-CSF. KW-2228 showed two to four times higher specific activity than that of intact rhG-CSF in mouse and/or human bone marrow progenitor cells by colony-forming unit assay in soft agar, and by cell- proliferation assay in liquid culture. KW-2228 showed a potency to increase peripheral neutrophil counts when it was administered to normal C3H/He mice by single intravenous injection. Increase of total leukocyte count and neutrophils was observed, with peak level at 8 to 12 hours at low doses (0.5 to 1.0 micrograms/mouse), and the highest level was maintained for 24 to 30 hours at high doses (5 to 10 micrograms/mouse). The granulopoietic effect of KW-2228 was examined by several doses of single course (once daily for 10 days) or multiple courses (twice daily injection for 5 days followed by cessation for 9 days on one cycle, 3 cycles in total) of treatment. KW-2228 showed higher activity than that of rhG-CSF, especially at sub-optimal doses of multiple courses of treatment. Furthermore, KW-2228 was found to be more stable physicochemically and biologically than intact rhG-CSF, especially under thermal conditions at 56 degrees C and in the human plasma at 37 degrees C, suggesting a protease resistancy. Pharmacokinetic study showed that plasma concentration of KW-2228 assayed for its bioactivity maintained a higher level than that of intact rhG-CSF for 60 minutes after intravenous injection of this protein to normal mice. Those results suggest that KW-2228 might show a superior in vivo hematopoietic effect to intact rhG-CSF due to its high specific activity to progenitor cells, and also due to its improved physicochemical, biologic, and pharmacokinetic stability in host animals.

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