Stable oncoretroviral gene transfer into hematopoietic stem cells (HSCs) provides permanent genetic disease correction. It is crucial to transplant enough transduced HSCs to compete with and replace ...the defective host hemopoiesis. To increase the number of transduced cells, the role of ex vivo expansion was investigated. For a possible clinical application, all experiments were carried out in serum-free media. A low-affinity nerve growth factor receptor (LNGFR) pseudotyped murine retroviral vector was used to transduce cord blood CD34(+) cells, which were then expanded ex vivo. These cells engrafted up to three generations of serially transplanted nonobese diabetic/severe combined immunodeficiency mice: 54.26% +/- 5.59%, 19.05% +/- 2.01%, and 6.15% +/- 5.16% CD45(+) cells from primary, secondary, and tertiary recipient bone marrow, respectively, were LNGFR(+). Repopulation in secondary and tertiary recipients indicates stability of transgene expression and long-term self-renewal potential of transduced HSCs, suggesting that retroviral gene transfer into HSCs, followed by ex vivo expansion, could facilitate long-term engraftment of genetically modified HSCs.
Because mobilized peripheral blood (MPB) represents an attractive source of cells for gene therapy, we investigated the ability of advanced-generation lentiviral vectors (LV) to transfer the Enhanced ...Green Fluorescent Protein (EGFP) gene into CD34+ cells isolated from MPB in culture conditions that allowed expansion of transplantable human HSC. So far, a few studies reported transduction of MPB cells with LV pseudotyped with VSV-G. However, the critical issue remains whether primitive, hematopoietic repopulating cells have indeed been transduced.
In vitro and in vivo experiments provided here show that MPB CD34+ cells can be effectively transduced by advanced generation LV: they do not lose their proliferation potential, self renewal and multilineage differentiation ability, moreover transgene expression is maintained. Following 48 hour-exposure to FL+TPO+SCF+IL6 and overnight incubation with vector particles, MPB CD34+ cells were further cultured for up to six weeks. During this period of culture the CD34+ cells population, the CD34+/GFP+ fraction and the CFC output were maintained.
Transduction efficiency of NOD/SCID repopulating cells (SRC) was assessed by serial transplants into NOD/SCID mice. Primary mice transplanted with transduced cells showed high level of engraftment (11,5 % CD45+ cells): within the CD45+ cells 11,4% expressed GFP (n=15). Moreover, mice transplanted with transduced, week-1 expanded cells showed higher levels of human engraftment than those transplanted with transduced, non-expanded cells (range from 9.25 % to 10.5 % for transduced basal cells; range from 14, 3 % to 38,19 % for transduced 1-week expanded cells). Engraftment was multilineage, with GFP+/lineage+ cells. Serial transplants were performed with transduced, week 1-expanded cells. Secondary engraftment levels were 1.6 %; 19.3 % of human cells were GFP+ (n=3). Moreover, levels of human engraftment were higher in mice injected with transduced, 1- week expanded cells (3.5 %) rather than in those injected with transduced, non expanded cells (1.2 %). Engraftment was multilineage with GFP+/lINEAGE+ cells.
These results show that LV efficiently transduced HSC, that maintain proliferation and self-renewal ability.
Stable oncoretroviral gene transfer into hematopoietic stem cells (HSC) provides permanent genetic disease correction. It is crucial to transplant enough transduced HSC to compete with and replace ...the defective host hemopoiesis. To increase the number of transduced cells the role of ex-vivo expansion was investigated. For a possible clinical application all experiments were done according to good anufacturing practice (GMP) guidelines. The combination of Flt3 ligand (FL), Stem Cell Factor (SCF), Thrombopoietin (TPO), and Interleukin-6 (IL6) has been shown to stimulate proliferation and self-renewal of very primitive (SCID-repopulating cells, SRC) hematopoietic cells. We asked whether it is possible to efficiently transduce HSC with oncoretroviral vector, to expand them and whether transduced cells retain their self-renewal potential, as demonstrated by their capacity to efficiently and serially engraft NOD/SCID mice A Gibbon ape leukemia virus (GALV)-pseudotyped vectors already approved for clinical application has been used to efficiently and durably deliver a defective, non functional form of the cell surface marker truncated low affinity nerve growth factor receptor (LNGFR) into primitive cord blood (CB) HSC. The transduction was performed, following an up-to 24 hour exposure to FL, TPO, IL-6 and SCF, in the presence of the growth factors in serum-free (SF) medium on retronectin (RT) coated plates. At day 3 post-transduction, total cells and CD34+ cells were expanded 24-fold and 8.5-fold respectively. More than 40% of the cells were CD34+. Transduction efficiency was >55%. Serial transplantation is the most reliable method to assess the stable expression of a gene in cells with high proliferative potential. Mice transplanted with transduced or mock-transduced, expanded cells showed higher levels of human engraftment than those transplanted with unmanipulated cells (56.7%, 55% and 39.9% respectively). LNGFR expression of CD45+ cells was 14.35± 4.27%. All secondary mice transplanted with cells from primary recipient BM resulted engrafted (21.7%, 13.5 and 2.8% respectively). LNGFR expression was 47.54± 3.1% respectively of human CD45+ cells. BM cells from secondary recipients were used for tertiary transplants. Only mice transplanted with expanded cells were positively engrafted. Two mice out of five transplanted with secondary recipient BM cells derived from mice transplanted with transduced and expanded cells, showed good levels of human engraftment (6.15% CD45+). LNGFR expression was 49.1± 4.4% of human CD45+ cells. FACS analysis of the different subpopulations showed LNGFR expression within the progenitor (CD34+), B (CD19+), myeloid (CD14+), erythroid (GpA+) and megakaryocyte cells (CD41+) in equivalent proportion. BM of the engrafted mice was placed in a human colony assay. Human colonies also were generated from the murine BM. In conclusion, we have validated a SF-protocol for efficient gene transfer into human CB HSC using a retroviral vector. Under these conditions, transduced and expanded cells repopulated NOD/SCID mice for 3 generations with a human multilineage graft stably expressing the transgene. In a view of future clinical applications, this protocol represents a major step towards the achievement of this goal.
The ability of advanced-generation lentiviral vectors to transfer the green fluorescent protein (GFP) gene into human hematopoietic stem cells (HSCs) was studied in culture conditions that allowed ...expansion of transplantable human HSCs. Following 96 hours' exposure to flt3/flk2 ligand (FL), thrombopoietin (TPO), stem cell factor (SCF), and interleukin-6 (IL-6) and overnight incubation with vector particles, cord blood (CB) CD34(+) cells were further cultured for up to 4 weeks. CD34(+) cell expansion was similar for both transduced and control cells. Transduction efficiency of nonobese diabetic/severe combined immunodeficient (NOD/SCID) repopulating cells (SRCs) was assessed by transplants into NOD/SCID mice. Mice that received transplants of transduced week 1 and week 4 expanded cells showed higher levels of human engraftment than mice receiving transplants of transduced nonexpanded cells (with transplants of 1 x 10(5) CD34(+) cells, the percentages of CD45(+) cells were 20.5 +/- 4.5 week 1, expanded and 27.2 +/- 8.2 week 4, expanded vs 11.7 +/- 2.5 nonexpanded; n = 5). The GFP(+)/CD45(+) cell fraction was similar in all cases (12.5% +/- 2.9% and 12.2% +/- 2.7% vs 12.7% +/- 2.1%). Engraftment was multilineage, with GFP(+)/lineage(+) cells. Clonality analysis performed on the bone marrow of mice receiving transduced and week 4 expanded cells suggested that more than one integrant likely contributed to the engraftment of GFP-expressing cells. Serial transplantations were performed with transduced week 4 expanded CB cells. Secondary engraftment levels were 10.7% +/- 4.3% (n = 12); 19.7% +/- 6.2% of human cells were GFP(+). In tertiary transplants the percentage of CD45(+) cells was lower (4.3% +/- 1.7%; n = 10); 14.8% +/- 5.9% of human cells were GFP(+), and human engraftment was multilineage. These results show that lentiviral vectors efficiently transduce HSCs, which can undergo expansion and maintain proliferation and self-renewal ability.
The expression, activity and functions of mitogen-activated protein (MAP) kinases in primary human hematopoietic progenitors (HP) have not yet been fully clarified.
To perform our experiments we used ...a stroma-free cell culture system in which the combination of FLT3 ligand (FL), stem cell factor (SCF) and thrombopoietin (TPO) induces massive expansion and proliferation of cord blood HP. The addition of IL-3 results in a rapid decrease of HP due to the prevalence of maturation and cell death. To detect extracellular regulated kinase (ERK) immunoenzymatic activity we recovered HP from FL, SCF and TPO stimulated long term cultures (LTC) after four weeks of culture. Some samples were recovered 16 h after addition of IL-3 to the LTC. We selectively immunoprecipitated p44/42 ERK kinase from 245 microg of cell lysates. We then analysed dual-phosphorylation of ERK-activating kinase-kinase (p45 MEK1/2) and of p44 ERK1 and p42 ERK2, and investigated MEK and ERK expression.
ERK activity, MEK1, and p42 and p44 ERK dual-phosphorylation were undetectable in the expanding, greatly proliferating and self-renewing HP. However, after addition of IL-3 sustained (still detectable 16 h after the stimulus) and high levels of ERK activity and dual-phosphorylation of the kinases were seen. The levels of MEK and ERK expression were stable in the different phases.
These findings add new information on the intracellular mechanisms of HP and help explain the very low levels of hematopoietic toxicity recently seen when treating cancer with down-modulators of ERK activity.
Identification of culture conditions that support expansion or even long-term maintenance of in vivo repopulating human hematopoietic stem cells is still a major challenge. Using a combination of ...FLT3 ligand (FL), Stem Cell Factor (SCF), Thrombopoietin (TPO) and Interleukin 6 (IL6), we cultured cord blood (CB) CD34+ cells for up to 12 weeks and transplanted their progeny into sublethally irradiated NOD/SCID mice. Bone marrow engraftment was considered successful when recipients contained measurable numbers of human CD45+, CD71+ and Glycophorin A+(GpA) cells 8 weeks after transplantation. Twelve-week expanded cells with FL+SCF+TPO+IL6 successfully engrafted all of the recipients and human CD45(+)+CD71(+)+GpA(+) cells represented 4.3 to 22.4% of bone marrow. Substitution of IL6 with IL3 led to an even better expansion of cells and a similar clonogenic progenitor output in the first 8 weeks of culture; however, LTC-IC output increased up to week 6 and then decreased and disappeared. By contrast, with FL+SCF+TPO+IL6, LTC-IC kept increasing up to week 12. Four-week cultured cells with FL+SCF+TPO+IL3 less efficiently engrafted NOD/SCID mice, both as measured by frequency of positive recipients (4 out of 10) and percentage of engrafted human cells (< or =2%). Six-week expanded cells failed to engraft. This study provides evidence that many, but not all, of the so-called "early acting" cytokines, can sustain long-term maintenance and even expansion of human primitive in vivo repopulating stem cells. In particular, in the culture conditions used in this study, the presence of IL3 greatly reduces the repopulating potential of expanded CD34+ CB cells.
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