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TISSUE-SPECIFIC STEM CELLS

Sustained Long-Term Engraftment and Transgene Expression of Peripheral Blood CD34⁺ Cells Transduced with Third-Generation Lentiviral Vectors

^aLaboratory of Clinical Oncology, Department of Oncological Sciences, University of Torino Medical School, Institute for Cancer Research and Treatment, Candiolo (Torino) Italy;
^cDepartment of Public Health and Microbiology, University of Torino Medical School, Torino, Italy;
^bPediatric Onco-Haematology Unit, Stem Cell Transplantation and Cellular Therapy Center, Regina Margherita Children's Hospital, Torino, Italy;
^dHematology and Clinical Immunology Section and
^eInternal Medicine and Oncological Science Section, University of Perugia, Perugia, Italy

Key Words. Ex vivo gene transfer • Hematopoietic stem cells • Enhanced green fluorescent protein • Expansion • Mobilized peripheral blood

Correspondence: Correspondence: Wanda Piacibello, M.D., Laboratory of Clinical Oncology, IRCC Institute for Cancer Research and Treatment, Provinciale 142, 10060 Candiolo, Torino, Italy. Telephone: 390119933349; Fax: 390119933522; e-mail: wanda.piacibello{at}ircc.it

Received on February 19, 2008; accepted for publication on March 14, 2008.

First published online in STEM CELLS EXPRESS  March 27, 2008.

	ABSTRACT

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
As mobilized peripheral blood (MPB) represents an attractive cell source for gene therapy, we investigated the ability of third-generation lentiviral vectors (LVs) to transfer the enhanced green fluorescent protein gene into MPB CD34⁺ cells in culture conditions allowing expansion of transplantable human hematopoietic stem cells. To date, few studies have reported transduction of MPB cells with vesicular stomatitis virus G pseudotyped LVs. The critical issue remains whether primitive, hematopoietic repopulating cells have, indeed, been transduced. In vitro (5 weeks' culture in FLT3 ligand + thrombopoietin + stem cell factor + interleukin 6) and in vivo (serial transplantation in NOD/SCID mice) experiments show that MPB CD34⁺ cells can be effectively long-term transduced by LV and maintain their proliferation, self-renewal, and multilineage differentiation potentials. We show that expansion following transduction improves the engraftment of transduced MPB CD34⁺ (4.6-fold expansion of SCID repopulating cells by limiting dilution studies). We propose ex vivo expansion after transduction as an effective tool to improve gene therapy protocols with MPB.

Disclosure of potential conflicts of interest is found at the end of this article.

	INTRODUCTION

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
Gene transfer into hematopoietic stem cells (HSCs) using retroviral vectors might provide a permanent correction of genetic diseases as the retroviruses can introduce and permanently express genes in the host cells. HSCs are important targets for gene therapy because of their self-renewal and multilineage differentiation abilities. These combined properties are reflected in the ability of an HSC to completely and durably reconstitute hematopoiesis of a myeloablated recipient and maintain it throughout the entire life span.

Lentiviral vectors (LVs) are instrumental tools for the genetic modification of HSCs as they can stably integrate into the host-cell genome. Since LVs do not require cell division for stable integration into the host genome, they represent a good tool to deliver genes in primitive HSCs, which usually reside in a quiescent status. Although many species-specific lentiviruses exist, most preclinical gene therapy experiments have been performed using vectors based on the human immunodeficiency virus type-1 (HIV-1) [1–5].

HSCs reside in cord blood (CB), bone marrow (BM), or mobilized peripheral blood (MPB). Most of the work with lentiviral transduction was performed using CB HSCs. Efficient transduction of primitive repopulating stem cells was demonstrated by secondary and tertiary transplantation of NOD/SCID mice with lentivirus-transfected CB CD34⁺ cells [4, 5]. Despite the successful lentiviral gene transfer into HSCs from CB, less is known about the gene transfer efficiency into adult HSCs. Several findings highlight that CB, adult BM, and MPB HSCs show important biological differences. Compared with their CB counterparts, adult HSCs are hard to manipulate ex vivo, possess less proliferation potential, have different cytokine requirements, have a much higher proportion of G₀ cells, and are more refractory to transduction [6–8]. Therefore, as a result of these factors, gene transfer efficiency in these cells might be suboptimal. Nevertheless, autologous BM cells or MPB cells are more therapeutically relevant stem cell sources for human gene therapy trials. In particular, granulocyte colony-stimulating factor (G-CSF)-MPB has become the major source of HSC for autologous and allogeneic transplantation, as it leads to a faster hematopoietic engraftment after transplantation compared with BM cells not primed by G-CSF [9]. Thus, an important issue to be addressed is whether it is possible to obtain efficient lentiviral gene transfer into MPB CD34⁺ cells, which are now those most commonly used in clinical settings. In this study we addressed this point by evaluating the ability of third-generation LVs to efficiently and durably deliver the enhanced green fluorescent protein (EGFP) reporter gene into primitive MPB HSCs that were subsequently subjected to ex vivo expansion.

	MATERIALS AND METHODS

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
Human Cells
After written informed consent was obtained, human MPB cells were collected from leftovers of leukaphereses from normal donors who had received G-CSF for 5 days prior to the procedure.

CD34⁺ Cell Isolation
CD34⁺ cells were isolated by magnetic immunoseparation (miniMACS) (Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com). Purification efficiency was 87%–92%.

Recombinant Human Cytokines
Recombinant human stem cell factor, recombinant human FLT3-ligand, and recombinant human granulocyte colony-stimulating factor (a gift from Amgen, Thousand Oaks, CA, http://www.amgen.com); recombinant human thrombopoietin (a gift from Kirin Brewery Co., Tokyo, http://www.kirin.co.jp/english); recombinant human granulocyte monocyte colony-stimulating factor and recombinant human interleukin 3 (rhIL-3) (Novartis International, Holzkirchen, Germany, http://www.novartis.com); recombinant human erythropoietin (EPREX) (from Cilag AG, Milan, Italy, http://www.cilag.ch/indexE.htm); and rhIL-6 (from Peprotech, Rocky Hill, NJ, http://www.peprotech.com) were used.

Production and Characterization of the Vector
Replication-defective self-inactivating HIV-1 vectors were constructed as described [1, 5, 10]. Vectors were produced by transient transfection of four plasmids in 293T cells. The four plasmids used were the transfer vector pRRLsin.PPT.hPGK.eGFP.Wpre [10], the VSV-G envelope-encoding plasmid pMD.G [5], and the packaging plasmids CMVR8.74 [2] and pRSV-Rev. Viral supernatants were concentrated by ultracentrifugation.

Titers of vector preparations were determined by transduction of HeLa cells with serial dilutions of vector supernatants, followed by cytometric analysis 3 days later. Final vector titers were in the range of 10⁹ transducing units (TU) per milliliter.

Transduction of CD34⁺ Progenitor Cells with Lentiviral Vectors
To induce cell cycling prior to transduction, 1 x 10⁵ CD34⁺ cells per milliliter were prestimulated for 48 hours with FL, stem cell factor (50 ng/ml), thrombopoietin, and interleukin 6 (10 ng/ml) (FST6). The transduction was carried out as previously described [5]. Prestimulated CD34⁺ cells (1 x 10⁵ cells) were resuspended in 100 µl of Iscove's modified Dulbecco's medium (IMDM) (Gibco, Grand Island, NY, http://www.invitrogen.com) with 10% fetal calf serum (FCS) (HyClone, Logan, UT, http://www.hyclone.com). Prestimulated cells were transduced with LVs at multiplicities of infection (MOIs) of 50 and 100 (corresponding to concentrations of the transducing units of 50 x 10⁶ and 100 x 10⁶ cells per milliliter, respectively) for 12 hours in the presence of the same cytokine combination and incubated at 37°C and 5% CO₂. Then the cells were harvested, washed twice, and used to initiate both stroma-free expansion cultures and semisolid cultures (described in Cell Culture Assays).

Cell Culture Assays

Clonogenic Assays.   Assays for granulopoietic, erythroid, megakaryocyte, and multilineage granulocyte-erythroid-macrophage-megakaryocyte colony-forming units were performed as previously described [11–13]. Colonies were scored after 14 days' culture, and EGFP⁺ (fluorescent) colonies were identified by fluorescence microscopy.

Stroma-Free Expansion Cultures.   Stroma-free long-term expansion cultures were performed in 24-well plates for 5 weeks as reported [11–14]. Briefly, after transduction, 2 x 10⁴ MPB CD34⁺ cells were cultured in quadruplicate flat-bottomed 24-well plates in 1 ml of IMDM + 10% FCS with FST6. Every week, all wells were demidepopulated by removing half of the cell suspension, which was replaced with fresh medium and growth factors. Harvested cells were used to assess colony content and immunophenotype (CD34⁺, CD34⁺/EGFP⁺ cells). Large-scale expansion cultures for mouse transplantations were performed as reported [5]: 1 x 10⁵ CD34⁺ cells per milliliter (mock or transduced) were seeded in tissue culture T25 flasks in the above-mentioned medium plus growth factors and allowed to grow for an additional 7 days.

Immunophenotyping by Flow Cytometry.   After purification, aliquots of CD34⁺ MPB cells were stained with anti-CD34-phycoerythrin (PE) (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com) or the corresponding control monoclonal antibody (MoAb) as described [5]. After transduction and then once a week, aliquots of cultured cells were washed and then evaluated for CD34 and EGFP expression. Flow cytometry was performed with a FACSVantage SE (Becton Dickinson). At least 10,000 events were acquired for each analysis, performed with CellQuest software (Becton Dickinson).

Animals
NOD/LtSz scid/scid (NOD/SCID) mice from Jackson Laboratory (Bar Harbor, ME, http://www.jax.org) were maintained in the animal facilities of Immunology and Experimental Oncology Centre and Institute for Cancer Research and Treatment, Candiolo, Torino, Italy. Eight-week-old mice were irradiated with 350 cGy of total body irradiation from a ¹³⁷Cs source and, 24 hours later, given a single i.v. injection of CD34⁺ MPB cells harvested after transduction followed or not by an additional 7 days' expansion. The mice were sacrificed 8 weeks after transplantation to assess the number and types of human cells detectable in the marrow.

Flow Cytometric Detection of Human Cells in Murine Tissues
Mouse BM was flushed from the long bones; the human engraftment and the EGFP expression were assessed by flow cytometry after the cells were stained with human-specific MoAb as previously described [13]. The presence of 0.5% of human CD45⁺ cells in the mouse BM defined a positive engraftment. Additional cell aliquots were stained with anti-human CD14-PE, CD19-PE, CD41-PE, and CD34-PE in combination with anti-human CD45-peridinin-chlorophyll-protein complex to allow discrimination of subpopulations within the CD45 gate.

DNA Extraction and Analysis of Human Cell Engraftment
DNA was extracted from the mouse BM by the NucleoSpin Blood Kit (Macherey-Nagel GmbH and Co. KG, Düren, Germany, http://www.macherey-nagel.com). The presence of human-specific DNA within the BM of transplanted mice was confirmed by polymerase chain reaction (PCR) amplification of an 850-base pair (bp) fragment of the -satellite region of the human chromosome 17 [15]. The presence of the marker gene was confirmed by the amplification of a 604-bp EGFP fragment (forward primer, 5'-GCTGGACGGCGACGTAAAC; reverse primer, 5'CCATGTGATCGCGCTTCTC). PCR amplification of the housekeeping gene GAPDH was used as a DNA control. To enhance the signal, PCR products were transferred by Southern blot to a nylon membrane (Hybond-N) (Amersham Biosciences, Milan, Italy, http://www.amersham.com) and hybridized in the presence of Rapid-hyp buffer (Amersham Biosciences) with forward and reverse primers previously [³²P]-labeled using 20 U of T4 polynucleotide kinase (New England Biolabs, Celbio, Italy, http://www.neb.com) and [³²P]-adenosine-5'-triphosphate for 1 hour at 37°C.

Real-Time Quantitative PCR
Quantitation of green fluorescent protein DNA was obtained by real-time quantitative polymerase chain reaction (qPCR) as previously described [5]. Briefly, vector copies per genome were quantified by real-time qPCR from 50 ng of template DNA extracted from the MPB CD34⁺ culture cell by a commercial kit (Qiagen, Hilden, Germany, http://www1.qiagen.com), using two sets of primers and probes to detect the LV backbone: LV forward primer, 5'-TGAAAGCGAAAGGGAAACCA-3'; LV reverse primer, 5'-CCGTGCGCGCTTCAG-3'; LV probe, 5'-(VIC)-CTCTCTCGACGCAGGACT-(TAMRA)-3'; and human genomic DNA RNaseP gene (Applied Biosystems, Weiterstadt, Germany, http://europe.appliedbiosystems.com). Serial dilutions of DNA from a human cell line with known number of lentiviral vector integrations (determined by Southern blot) were used for standard curves. Reactions were carried out in triplicate according to the manufacturer's instructions and analyzed by using the ABI Prism 7900 HT sequence detection system (Applied Biosystems).

Limiting Dilution Assays
The SCID repopulating cell (SRC) frequency in a population of cells (transduced, or transduced and subsequently expanded for a week) was determined by injecting cohorts of mice with several dilutions of cells. The SRC frequency was calculated from the proportions of negative mice in each cohort, using the L-Calc T program (StemCell Technologies, Vancouver, BC, Canada, http://www.stemcell.com), which uses Poisson statistics and the method of maximum likelihood [14]. After a log transformation, the ratio of frequencies was computed, and the difference from a ratio of 1 was analyzed with a test for the proportions (L-Calc T software [14]).

Statistical Analysis
Because of the non-normal distribution of the CD45 and EGFP values, the differences among groups were analyzed by nonparametric tests; the Mann-Whitney test was used to compare the engraftment levels between the MOI doses of 50 and 100 and between expanded and unexpanded groups.

The expansion of transduced MPB populations recorded in a 5-week period was analyzed considering the cumulative number of expanded cells or colonies for each week. A Kaplan-Meier curve was estimated for MOI 50 and MOI 100 for total cells and for CD34⁺ and colony forming cells (CFCs) separately, and the differences between doses were analyzed using the log-rank test. Differences were considered statistically significant with a p value <.05. The statistical analyses were performed using SPSS 14.0 for Windows (SPSS, Chicago, http://www.spss.com).

	RESULTS

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
Ex Vivo Expansion of Transduced MPB CD34⁺ Cells
After prestimulation with FST6 for 48 hours, MPB CD34⁺ cells were transduced with MOIs of 50 and 100 TU per cell with the lentiviral vector carrying the EGFP reporter gene in the presence of the same cytokines. Three days after transduction, cells were 68.5% ± 9.3% CD34⁺. Transduction efficiencies with both MOIs were 53.3 ± 7.5 and 21.15 ± 8.3, respectively (Fig. 1A; Table 1). To determine the effect of the transduction on the proliferation potential of CD34⁺ cells, transduced cells were used to initiate stroma-free long-term cultures in FST6. The total cells, the CD34⁺ cells, and the CFC populations were monitored each week. Table 1 shows robust cell proliferation for at least 5 weeks. CD34⁺ and EGFP⁺/CD34⁺ cells were detected over the entire culture period. Hemopoietic colonies, a good proportion of which were fluorescent, were generated for 5 weeks' culture, quite a long period of time for primary adult hemopoietic progenitor/stem cells [14]. Table 1 shows the values of each cell population during the observation period. In general, cells transduced with a MOI of 100 seemed to proliferate more slowly than those transduced with an MOI of 50. This was evident when total cell numbers and CFC numbers were evaluated. However, the proportion of CD34⁺/EGFP⁺ cells at weeks 1 and 2 was greater in cultures treated with a MOI of 100 than in cultures treated with a MOI of 50. To better illustrate the growth of the transduced cell populations with the passing of time, cell expansion was analyzed considering the cumulative number of expanded cells or colonies for each week. A Kaplan-Meier curve was estimated for MOIs of 50 and 100 for total cells, CD34⁺ cells, and CFCs separately, and the differences between MOIs were analyzed using the log-rank test (supplemental online Fig. 1). Although the cumulative production of EGFP⁺ cells and of CD34⁺/EGFP⁺ cells showed no clear-cut differences, the fluorescent colony output was clearly less abundant when the cells were transduced with an MOI of 100 (p < .001), probably because hemopoietic colonies are a more homogeneous cell subpopulation than total cells or CD34⁺ cells. This phenomenon, which was observed in liquid cultures soon after transduction and over a period of 5 weeks, might be due to an excessive viral concentration that could be toxic for less-differentiated primary human cells.

View larger version (42K): [in this window] [in a new window]   Figure 1. Transduction of MPB CD34+. (A): Transduction efficiency of mobilized peripheral blood (MPB) CD34+ cells exposed to lentiviral vectors. Representative flow cytometric analysis showing, on the left, the transduction efficiency of MPB CD34+ cells exposed to lentiviral vectors in the doses of 50 tu per cell and, on the right, the transduction efficiency of CD34+ cells, originated from the same MPB, exposed to lentiviral vectors in the doses of 100 tu per cell. Isotype control-nonspecific IgG1 staining is shown at the top. (B): Quantitative real-time polymerase chain reaction (PCR) of vector DNA from MPB CD34+ cells 2 weeks post-transduction. Results are presented as the mean of three experiments. PCR was performed in a final volume of 10 µl of TaqMan Universal PCR Master Mix (Applied Biosystems), and forward and reverse primers for the amplification of the backbone of the lentiviral vector were used at a concentration of 5 pmol/µl. The probe for the backbone of lentiviral vector was 5'-labeled with the reporter fluorescent dye 5-fluorescein and a fluorescence dye quencher, 6-car- boxy-tetramethyl-rhodamine, at the 3' end, hybridized to the target. A 5 pmol/µl concentration was used. The thermal cycling conditions included 2 minutes at 50°C and 10 minutes at 95°C. Thermal cycling proceeded with 40 cycles at 95°C for 0.5 minutes and 60°C for 2 minutes. For each sample, the ABI Prism 7900 HT software provided an amplification curve constructed by relating the fluorescence signal intensity to the cycle number. Collection and analysis software was developed at Applied Biosystems. Abbreviations: C/G, vector copies per genome; EGFP, enhanced green fluorescent protein; FITC, fluorescein isothiocyanate; GFP, green fluorescent protein; MOI, multiplicity of infection; tu, transducing units.

In Vivo Engraftment and Survival of Transduced Cells
These data prompted us to attempt to verify whether the transduced cells retained the long-term and multilineage repopulating ability, together with sustained transgene expression. To do this, we transplanted sublethally irradiated NOD/SCID mice, which are a highly reliable assay to assess the presence of in vivo repopulating stem cells [8, 12, 13]. In four separate experiments, 22 sublethally irradiated NOD/SCID mice were transplanted with different sources of MPB CD34⁺ previously transduced with MOI 100 TU per cell or 50 TU per cell. Eight weeks after transplantation, we evaluated the level of human engraftment and transgene expression in the murine BM. The results are summarized in Table 2. All transplanted animals were engrafted at different levels according to the MPB samples and the MOI. The levels of human CD45⁺ cells in the 11 mice transplanted with 1 x 10⁶ cells transduced with a MOI of 100 and in the 11 mice transplanted with the same number of cells transduced with a MOI of 50 were 7.4 ± 1.7 and 14.3 ± 2.9, respectively. Such a difference was statistically significant (p < .001). Conversely, EGFP⁺ cells (within the gate of CD45⁺ cells) in the 11 mice transplanted with 1 x 10⁶ cells transduced with 100 TU per cell were 17.4 ± 2; those of the 11 mice transplanted with the same number of cells transduced with 50 TU per cell were 17.2% ± 2.2%. These values were not significantly different (Table 2). In all mice, the engraftment was multilineage. Furthermore, the transduction procedure using an MOI 50 TU per cell did not impair the engraftment levels, since mice transplanted with similar numbers of mock and transduced cells presented similar levels of engraftment (Table 3). We concluded that 50 TU per cell is the most reliable MOI that can guarantee higher engraftment levels with a similar proportion of EGFP⁺ positivity. Therefore, we decided to use this MOI in the next sets of experiments.

View larger version (64K): [in this window] [in a new window]   Figure 2. Repopulation capacity of transduced, unexpanded and expanded, MPB CD34+ cells in primary and secondary mice. (A): Top, a representative fluorescence-activated cell sorting (FACS) analysis of bone marrow (BM) cells from an individual primary mouse transplanted with 1 x 106 transduced but unexpanded MPB CD34+ cells; bottom, a representative FACS analysis of BM cells from an individual primary mouse transplanted with the progeny of initial 1 x 106 MPB CD34+ cells expanded for 1 week after the transduction in FL, thrombopoietin, stem cell factor, and interleukin 6. The isotype controls are shown in the left panels. The right panels represent the percentage of EGFP+/CD45+ cells in total BM. The percentages of EGFP+ cells within the CD45+ cell gate are 11.3% and 17.7% for transduced unexpanded cells and transduced 1-week-expanded cells, respectively. (B): Representative molecular analysis of human and transgene engraftment. Polymerase chain reaction (PCR) amplification of an 850-pb fragment on the human -satellite; the negative control is represented by a nontransplanted mouse BM, and the positive control is represented by HeLa EGFP-transfected cells. The presence of the transgene was confirmed by a PCR amplification of a 604-pb fragment of the EGFP. The PCR products were subsequently subjected to Southern blot with a radiolabeled probe. Amplification of a sequence from the human GAPDH gene was used to control for the presence of DNA. (C–E): Limiting dilution assay to determine SRC frequency in mice transplanted with decreasing doses of transduced and unexpanded (C) or 1-week-expanded (D) cells. The number of mice, the cell doses, and the statistical analysis are summarized in (E). (F): Representative FACS profile of human CD45+ and EGFP+ cells in the total BM of a secondary mouse that was injected with 20 x 106 unfractionated BM cells harvested from a primary mouse sacrificed at week 8 after transplantation of unexpanded transduced cells (top panels) and a secondary mouse transplanted with 20 x 106 unfractionated BM cells harvested from a primary mouse transplanted with 1-week-expanded transduced cells (bottom panels). Primary and secondary mice were sacrificed 8 weeks after transplantation. The isotype controls are shown in the left panels. The right panels represent the percentage of EGFP+/CD45+ cells in total BM. The percentages of EGFP+ cells within the CD45+ cell gate are 19.1% and 25.5% for transduced unexpanded cells and transduced 1-week-expanded cells, respectively. (G): Representative molecular analysis of human and transgene engraftment. PCR amplification of an 850-pb fragment on the human -satellite confirmed the human engraftment. PCR amplification of a 604-pb fragment of the EGFP confirmed the presence of the transgene. The PCR products were subsequently subjected to Southern blot with a radiolabeled probe. Amplification of a sequence from the human GAPDH gene was used to control the presence of DNA. Shown are unexpanded and expanded cells derived from the same MPB sample. Abbreviations: C+, positive control of expression; C–, negative control of expression; C.I., confidence interval; EGFP, enhanced green fluorescent protein; FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; MPB, mobilized peripheral blood; SRC, Scid repopulating cell.

We conclude that (a) the transduction procedure does not impair the ability of the cells to undergo expansion, and (b) the expansion of the transduced cells represents a good tool to increase the number of MPB HSC expressing the transgene and improve their engraftment.

In Vivo Engraftment of Secondary Mice
We performed secondary transplants to prove that primitive stem cells had indeed been transduced and had not lost their self-renewal ability or multilineage differentiation capacity. To this end, we took 15–20 x 10⁶ of unfractionated BM cells from primary recipients and inoculated them into sublethally irradiated secondary recipients. All mice were engrafted. The BM of secondary mice (n= 7) transplanted with transduced and unexpanded cells contained 1.5% ± 0.6% huCD45⁺ cells (0.28 ± 0.06 CD45⁺/EGFP⁺; n= 7); 18.5% ± 1.5% huCD45⁺ cells were EGFP⁺. Human engraftment was significantly higher in secondary mice transplanted with transduced and 1-week-expanded cells (n= 9) (4.3% ± 0.3% huCD45⁺; 0.89 ± 0.3 CD45⁺/EGFP⁺; p < .005 and p < .001, respectively); 20.3% ± 8.6% EGPF⁺ cells within the CD45⁺ population than in secondary mice transplanted with transduced and now-expanded cells; (p= not significant) (Table 3; Fig. 2F). There was multilineage engraftment in all the animals, and the presence of human cells and EGFP⁺ cells was confirmed at a molecular level (Fig. 2G).

	DISCUSSION

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
The results presented in this study demonstrate that MPB CD34⁺ cells can be transduced by third-generation LV and do not lose their proliferation potential, self-renewal, or multilineage differentiation ability. We show a sustained in vitro production of cells and progenitors expressing the transgene and to our knowledge, for the first time, in vivo hemopoietic reconstitution by EGFP-expressing progeny for two sequential generations of mice. After the 7 days' expansion, we observed a fourfold amplification of total CD34⁺ cell number and of CD34⁺/EGFP⁺ cells and a sevenfold expansion of EGFP⁺ committed progenitors (supplemental online Table 1). Above all, limiting dilution assays show that SRCs are increased 4.6-fold, meaning that this growth factor combination is particularly favorable to the proliferation of more primitive, long-term repopulating stem cells. This result is supported by previous studies that showed, in BM and MPB CD34⁺ cells, a sixfold expansion after 1–3 weeks' expansion containing the same growth factors used in the present study [14]. The frequency of SRC in transduced MPB CD34⁺ cells is only slightly lower than that reported for freshly isolated CD34⁺ cells, indicating that the cell manipulation used in this study to transduce the cells is not too harmful for long-term repopulating human stem cells. Moreover, we previously showed that during the first week of expansion, the telomere length of MPB CD34⁺ cells does not shorten, indicating an unchanged residual proliferation potential of the expanded cells [14].

Serial transplantation is the most reliable method to assess the stable expression of a transgene in cells with high proliferation and self-renewal potential. The BM cells of mice transplanted with transduced and expanded MPB cells indeed sustained secondary transplants in which the engraftment levels of the total cell population and the transduced population were significantly higher than secondary transplants obtained with cells transplanted soon after transduction, indicating that transduced and expanded SRC retained a stable transgene expression, as it generated a progeny of HSCs that was still carrying the transgene in a stable way.

Although other reports demonstrate transduction of human CD34⁺ cells without cytokine prestimulation [16], our group and others [14, 17] have consistently observed higher levels of gene transfer if the CD34⁺ cells are cultured with well-defined cytokines. Here, we show that such a cytokine combination, previously known to induce SRC expansion [14], can improve not only the engraftment levels but also the engraftment of transduced cells, which can durably express the transgene. In support of this observation, a recent study has shown that proteasome activity, a critical step limiting the efficiency of HSC transduction by LVs, is downregulated in hematopoietic progenitors by cytokine prestimulation [18]. An MOI of 50 has been used, as in the first part of the studies, which appeared less toxic for transplantable stem cells than an MOI of 100. After 2 weeks' culture, cells transduced with MOI 50 contained 0.4 viral copies per cell, and those transduced with MOI 100 contained 0.9 copies. The use of a low MOI is warranted for clinical trials, as the risk of insertional mutagenesis, also for lentiviral vectors, is present. A number of studies show that a higher MOI is needed for MPB than for cord blood to reach a sufficient transduction efficiency and that with an MOI of 100, gene transfer rates were obtained with a reasonable number of integration sites (a figure in the neighborhood of three to four copies per cell), thus limiting the risk of insertional oncogenesis [19]. In the present protocol, with an MOI of 50 we obtained a reasonable proportion of transduced cells, in which only 0.4 vector copies are integrated in the genome. Moreover, the transduced population durably expresses the transgene. Such a proportion of cells remains stable with time, particularly in in vivo experiments, indicating the stable transduction of a proportion of stem cells that repopulate myeloablated recipient BM together with nontransduced stem cells, in keeping with the observation that myelopoiesis is sustained by a number of clones.

	CONCLUSION

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
We demonstrated here that the ex vivo expansion can even increase the number of transduced MPB cells. This is a particularly relevant aspect if we consider that transplanting enough transduced HSCs to compete with and replace the defective host hemopoiesis is crucial for clinical gene therapy protocols [20].

	DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
We thank Kirin and Amgen for a continuous supply of growth factors, Luigi Naldini and Elisa Vigna for help with real-time quantitative PCR, Tsvee Lapidot for continuous scientific support, and Catherine Tighe and Andrew Martin Garvey for editing the manuscript. This work was supported by grants from Associazione Italiana per la Ricerca sul Cancro (Milan, Italy) (to M.A. and W.P.), Istituto Superiore della Sanità (National Program on Stem Cells), the Ministero dell'Istruzione, dell'Università e della Ricerca (Rome, Italy), Ricerca Finalizzata Regione Piemonte (to W.P. and M.A.), EUROCORD III-European Community (contract no. QLK3-CT-2002-01918) (to W.P.), and Associazione Donatrici Italiane Sangue del Cordone Ombelicale. M.T. and L.G. contributed equally to this work.

	FOOTNOTES

 
Author contributions: M.T. and L.G.: conception and design, manuscript writing, animal models; M.G. and L.C.: collection and/or assembly of data; V.L.: vector production; Y.P. and N.J.: molecular analysis; S.C.: in vitro experiments; C.C.: vector production; G.M.: data analysis and interpretation; F.F.: financial support, provision of study material; A.T.: provision of study material; M.A.: financial support, final approval of manuscript; W.P.: conception and design, financial support, manuscript writing, final approval of manuscript.

	REFERENCES

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 

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