Serial Transplantations in Nonobese Diabetic ... - Wiley Online Library

Download PDF
2 downloads 0 Views 477KB Size Report
Comment
nome requires stimulation with hematopoietic growth factors/cyto- kines for the quiescent HSCs to enter the cell cycle and undergo division ex vivo [3]. In many ...
TRANSLATIONAL AND CLINICAL RESEARCH Serial Transplantations in Nonobese Diabetic/Severe Combined Immunodeficiency Mice of Transduced Human CD34ⴙ Cord Blood Cells: Efficient Oncoretroviral Gene Transfer and Ex Vivo Expansion Under Serum-Free Conditions LORETTA GAMMAITONI,a SIMONA LUCCHI,b,c STEFANIA BRUNO,a MELANIA TESIO,a MONICA GUNETTI,a YMERA PIGNOCHINO,a GIORGIA MIGLIARDI,a LORENZA LAZZARI,b MASSIMO AGLIETTA,a PAOLO REBULLA,a WANDA PIACIBELLOa a

Laboratory of Medical Oncology, Institute for Cancer Research and Treatment, Candiolo, Italy; bCell Factory, Centro Trasfusionale e di Immunologia dei Trapianti, Fondazione Istituto di Ricovero e Cura a Carattere Scientifico Ospedale Maggiore, Policlinico, Mangiagalli e Regina Elena, Milan, Italy; cIstituto Auxologico Italiano, Cusano Milanino, Milan, Italy Key Words. Cord blood • Xenotransplantations • Gene marking • Oncoretroviral vectors Nonobese diabetic/severe combined immunodeficiency mice • Hematopoietic stem cells • Expansion Ex vivo gene transfer

ABSTRACT 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 CD34ⴙ 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% ⴞ 5.59%, 19.05% ⴞ 2.01%, and 6.15% ⴞ 5.16% CD45ⴙ cells from primary, secondary, and tertiary recipient bone marrow, respectively, were LNGFRⴙ. 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. STEM CELLS 2006;24:1201–1212

INTRODUCTION

ability to engraft and repopulate the host as a consequence of negative effects on the survival and, perhaps, the long-term in vivo repopulation ability of stem cells [3, 4], which may explain the disappointing results obtained in the large number of clinical trials of gene transfer to HSCs [5]. Only a few studies have reported suitable culture conditions for the expansion of transplantable human cord blood (CB) stem cells [6 – 8]. Among these, the combination of Flt3 ligand (FL), stem cell factor (SCF), thrombopoietin (TPO), interleukin-6 (IL-6), and FL, TPO, IL-6, and IL-11 have been shown to stimulate proliferation and self-renewal of very primitive (severe combined immunodeficiency-repopulating cells

For the correction of many inherited or acquired defects of the hematopoietic system, the relevant gene must be delivered, integrated, and stably expressed in hematopoietic stem cells (HSCs) [1]. Gene transfer into HSCs using oncoretroviral vectors has the potential to provide permanent correction of genetic diseases, as the retroviruses can introduce and permanently express genes in the host cells [2]. However, integration of these vectors into the genome requires stimulation with hematopoietic growth factors/cytokines for the quiescent HSCs to enter the cell cycle and undergo division ex vivo [3]. In many cases, this leads to a decrease in their

Correspondence: Wanda Piacibello, M.D., University of Torino Medical School Department of Oncological Sciences, IRCC, Institute for Cancer Research and Treatment, Laboratory of Clinical Oncology, Prov. 142, 10060 Candiolo, Torino, Italy. Telephone: ⫹39-011-9933349; Fax: ⫹39-011-9933522; e-mail: [email protected] Received August 24, 2005; accepted for publication January 1, 2006; first published online in STEM CELLS EXPRESS January 12, 2006. ©AlphaMed Press 1066-5099/2006/$20.00/0 doi: 10.1634/stemcells.2005-0408

STEM CELLS 2006;24:1201–1212 www.StemCells.com

Oncoretrovirally Transduced HSC Transplantation

1202

[SRCs]) hematopoietic cells both in vitro and in vivo [9 –14]. The transplantation assay available in sublethally irradiated nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice has been instrumental in defining and characterizing the most primitive cells of the hemopoietic system [15–18]. Gibbon ape leukemia virus (GALV)-pseudotyped vectors obtained from a stable packaging cell line (NIH 3T3-derived packaging cell line PG13) and already approved for clinical application have been used to deliver efficiently and durably a defective, nonfunctional form [19, 20] of the cell surface marker low-affinity nerve growth factor receptor, truncated of its intracytoplasmatic domain (⌬LNGFR) [21–24] into primitive CB HSCs. The aim of our study was to assess the feasibility of a transduction plus expansion preclinical protocol. We investigated whether it is possible to efficiently transduce human HSCs with a ⌬LNGFR-GALV-pseudotyped vector and to expand them and whether transduced and expanded cells retain their self-renewal potential, as well as the ability to express stably the transgene, as demonstrated by their capacity to efficiently and serially engraft NOD/SCID mice with ⌬LNGFR⫹ hemopoietic progeny. CB is an established source of HSCs for allogeneic or autologous transplantation [25], as in the case of children with adenosine deaminase deficiency [26]. CB is regarded as a valid alternative to adult stem cell sources. The main limitation in the widespread use of CB stem cells is its small physiological volume [27]. Therefore, an insufficient number of genetically modified transplanted HSCs might result in an inability to compete with and eventually replace the defective host hemopoiesis. Thus, expansion of transplantable stem cells in vitro might prove extremely useful. We prove the feasibility of a preclinical protocol of retroviral gene transfer associated with extensive ex vivo manipulation of human primitive HSCs, which retain their stem cell properties under serum-free conditions in the presence of two different cytokine combinations (FL, TPO, IL-6, IL-11; and FL, TPO, IL-6, SCF). Therefore, retroviral gene transfer into HSCs, followed by their ex vivo expansion, might provide the means to facilitate long-term engraftment of genetically modified HSCs.

MATERIALS

AND

METHODS

Sample Collection and Isolation of CD34ⴙ Cells Umbilical CB was obtained, after written informed consent, at the end of full-term pregnancies, after clamping and cutting of the cord, by draining the blood into sterile collection tubes containing the anticoagulant citratephosphate dextrose.

CD34ⴙ Cell Purification Mononuclear cells (MNCs) were isolated from CB using Lymphoprep (Sentinel, Milan, Italy, http://sentinel.it/index.asp) density gradient centrifugation. CD34⫹ cells were isolated using a magnetic immunoseparation device (miniMACS; Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec. com). Purification efficiency was verified by flow cytometry counter staining with a CD34-phycoerythrin (PE; HPCA-2; Becton, Dickinson and Company, Franklin Lakes, NJ, http:// www.bd.com) antibody (87%–92% CD34⫹).

Recombinant Human Cytokines The following recombinant purified human cytokines were used: recombinant human (rh) stem cell factor (rhSCF), Flt3ligand (rhFL), and granulocyte-colony stimulating factor (rhGCSF) were a gift from Amgen (Thousand Oaks, CA, http:// www.amgen.com); thrombopoietin (rhTPO) was a generous gift from Kirin Brewery (Tokyo, http://www.kirin.co.jp/english); granulocyte monocyte colony-stimulating factor (rhGM-CSF) and interleukin 3 (rhIL-3) were from Sandoz (Novartis, Holzkirchen, Germany, http://www.sandoz.com/site/en/index.shtml); erythropoietin (rhEPO; EPREX) was from Cilag (Milan, Italy, http:// www.cilag.ch); rhIL-6 and rhIL-11 were purchased from PeproTech (Rocky Hill, NJ, http://www.peprotech.com).

Production and Characterization of the Vector

The modified ⌬LNGFR coding sequence was obtained from MgSLdelS by polymerase chain reaction (PCR) (10 minutes of denaturation at 94°C, followed by 35 cycles of 1 minute at 94°C, 1 minute at 65°C, 2.5 minutes at 72°C, and the final 10 minutes at 72°C), using the following primers: 5⬘GAGGCGGGCCATGGGGGCAGGTGCCACCGGCCGCGCAATGGACGG-3⬘ and 5⬘-GACTCTAGAGGATCCCCCTGTT-3⬘. The forward primer contains two silent mutations (bases in bold type), to create a Ncol site encompassing the first ATG of the ⌬LNGFR coding sequence, and to suppress that localized at the second ATG. The PCR product was subsequently cut with Ncol/BamHI restriction enzymes and the resulting fragment was cloned into Ncol/BamHI sites of the MFG backbone by ligation. The correct insertion was confirmed by restriction endonuclease mapping.

Generation of ⌬LNGFR Amphotropic Packaging Clones and Viral Titer The MFG-⌬LNGFR vector was transfected into the Phoenix Eco packaging cell line containing gag and pol of the Moloney murine leukemia virus (MoMLV) and ecotropic env, using polyethylenimine (Sigma-Aldrich, St. Louis, http://www. sigmaaldrich.com). After 48 hours, the virus containing supernatant was harvested and used to transduce (three cycles of 12 hours each) the amphotropic PG13 packaging cell line in the presence of polybrene (Sigma-Aldrich). This cell line contains gag and pol of MoMLV and env of the GALV [28 –30]. After transduction, the ⌬LNGFR-PG13 expressing cells were selected by sterile fluorescence-activated cell sorting (FACS) after incubation with an anti-human LNGFR [31] (Chemicon, Temecula, CA, http://www.chemicon.com), and clones were obtained from each cell. The viral titer of the clones was estimated by transducing HeLa cells with the retrovirus containing supernatant in the presence of polybrene. The clone with the highest viral titer was chosen to produce the retroviral supernatant for transduction.

Transduction of CD34ⴙ Progenitor Cells

For the transduction of human CD34⫹ cells, retroviral supernatant was harvested from confluent PG-13 monolayers after 6 –16 hours of cultivation in serum-free CellGro medium (BioWhittaker Molecular Applications, Rockland, ME, http://www. bmaproducts.com) supplemented with 1% penicillin/streptomycin and passed through a 0.45-␮m filter (Millipore, Molsheim, France, http://www.millipore.com) to remove cellular debris before transduction. CB CD34⫹ cells (1 ⫻ 105 cells per ml)

Gammaitoni, Lucchi, Bruno et al. isolated from several CB samples pooled together in order to obtain enough CD34⫹ cells, were prestimulated for 24 hours in serum-free CellGro supplemented with 1% penicillin/streptomycin in the presence of (a) SCF (50 ng/ml) ⫹ FL (50 ng/ml) ⫹ TPO (10 ng/ml) ⫹ IL-6 (10 ng/ml) or (b) FL (50 ng/ml) ⫹TPO (10 ng/ml) ⫹ IL-6 (10 ng/ml) ⫹ IL-11 (10 ng/ml). Multiwell non-tissue culture-treated plates (Becton Dickinson) were coated with retronectin (Takara, Otsu, Japan, http://www. takara.co.jp) and the recombinant fibronectin fragment CH-296 (15 ␮g/cm2), and preloaded by centrifuging the plates with filtered retroviral supernatant at 2,500 rpm for 30 minutes [22]. After prestimulation, every 12 hours, half of the medium was replaced with fresh viral supernatant (titer: 1 ⫻ 105/0.5 ml) containing the cytokine cocktails mentioned above and incubated at 37°C, 5% CO2; three infection cycles were performed. At day 5, the cells were harvested, washed, counted, and analyzed for expression of the LNGFR and CD34 by flow cytometry.

Cell Culture Assays Clonogenic Assays. Assays for granulopoietic colony-forming units (CFU-GM), erythroid CFUs (BFU-E), megakaryocytic CFUs (CFU-Mk), and multilineage granulocyte-erythroidmacrophage-megakaryocyte CFUs (CFU-GEMM) were performed as previously described [8, 32, 33]. Hematopoietic colonies were scored after 14 days of culture by optical microscopy. The long-term culture-initiating cell (LTC-IC) content of the initial CD34⫹ population and of the CD34⫹ cells isolated from long-term cultures at different time points (1.5 ⫻ 103 CD34⫹ cells) was determined by assaying for secondary colony-forming cells (CFCs) in methylcellulose culture after 6 weeks of stromal coculture, as described [8, 10, 32]. Stroma-Free Expansion Cultures. Stroma-free expansion cultures for extended periods were performed in 24-well plates, as previously reported [8]. Briefly, CB CD34⫹ cells (0.2 ⫻ 105), unmanipulated or after transduction, were cultured in quadruplicate flat-bottomed 24-well plates in 1 ml of CellGro with FL, TPO, SCF, and IL-6. Each week, all wells, after vigorous pipetting, were demidepopulated by removing half of the cell suspension, which was replaced with fresh medium and growth factors. Harvested cells were used to assay the CFC content and the CD34⫹, CD34⫹/LNGFR⫹ expression. Expansion cultures for mouse transplantations were performed, and 0.5 ⫻ 105 to 1 ⫻ 105 CB CD34⫹ cells per ml, resuspended in the same medium plus growth factors described above, were seeded in tissue culture T25 flasks.

Immunophenotyping by Flow Cytometry

After purification, aliquots of CD34⫹ CB cells were stained with anti-CD34-PE (Becton Dickinson) or the corresponding control antibody as previously described [8]. After transduction of CD34⫹ cells, and then once a week, aliquots of cultured cells were washed and then subjected to the same procedure to evaluate CD34 and LNGFR expression using an unconjugated mouse antihuman LNGFR antibody (Chemicon) which was detected with a goat antimouse F(ab)-fluorescein isothiocyanate (FITC) (EuroClone Celbio, Milan, Italy, http://www.celbio.it). Flow cytometric analysis was performed with a FACSVantage www.StemCells.com

1203

SE (Becton Dickinson). At least 10,000 events were acquired for each analysis. Analysis was performed with CellQuest software (Becton Dickinson).

Animals NOD/LtSz scid/scid (NOD/SCID) mice were obtained from Jackson Laboratory (Bar Harbor, ME, http://www.jax.org) and maintained in the animal facilities of Centro di Immunogenetica ed Oncologia Sperimentale (Torino, Italy). Mice were irradiated at 6 – 8 weeks of age with 350 cGy total body irradiation from a 137Cs source and, 24 hours later, given a single intravenous injection of human CD34⫹ CB cells harvested from expansion cultures or after transduction, as described. Mice were sacrificed 6 – 8 weeks after transplantation to assess the number and types of human cells detectable in femurs and tibias.

Flow Cytometric Detection of Human Cells in Murine Tissues Bone marrow (BM) cells were flushed from the femurs and tibias with a syringe and 26-gauge needle, and flow cytometric analysis was performed using a FACSVantage cytometer after staining the cells with human-specific monoclonal antibodies. FACS analysis of human CD45 and LNGFR expression in the BM of primary, secondary, and tertiary mice was performed on total BM cells after staining the cells with PerCP-labeled MoAb specific for human CD45 (Becton Dickinson) or PE-glycophorin-A (GpA; DAKO, Glostrup, Denmark, http://www.dako. com) in combination with an unconjugated mouse antihuman LNGFR antibody (Chemicon) which was detected with a goat antimouse F(ab)-FITC (EuroClone) or, alternatively, a PE-conjugated antihuman LNGFR antibody (BD Pharmingen, Milan, Italy, http://www.bdbiosciences.com/pharmingen). Additional aliquots of cells were stained with antihuman CD14-PE, CD19PE, CD41-PE (Dako), and CD34-PE (Becton Dickinson) in combination with antihuman CD45-PerCP and LNGFR antibodies to allow discrimination of subpopulations within the CD45 gate. The presence of ⱖ0.5% of human CD45⫹ and GpA⫹, cells in the BM of NOD/SCID mice defined a positive engraftment.

DNA Extraction and Analysis of Human Cell Engraftment High molecular weight DNA was extracted from the BM of mice by the NucleoSpin Blood Kit (Machery-Nagel, GmbH and Co. KG, Du¨ren, Germany, http://www.macherey-nagel.com). The presence of human-specific DNA in the murine BM of transplanted mice was confirmed by PCR amplifying an 850base pair (bp) fragment of the ␣-satellite region of the human chromosome 17 [34].

Polymerase Chain Reaction for Human LNGFR The presence of LNGFR provirus in NOD/SCID BM was determined by polymerase chain reaction (PCR) amplifying the specific 425-bp fragment of the LNGFR gene [35].

RESULTS In Vitro CB CD34ⴙ Cell Expansion A GALV-pseudotyped Moloney murine leukemia virus retroviral vector containing the low-affinity nerve growth factor receptor (LNGFR) was used to transduce CB CD34⫹ cells. The

1204

Oncoretrovirally Transduced HSC Transplantation

Figure 1. Ex vivo long-term expansion of CD34⫹ cord blood (CB) cells after gene transfer with a Gibbon ape leukemia virus (GALV)-pseudotyped retroviral vector. CB CD34⫹ cells were prestimulated for 24 hours with stem cell factor (SCF) (50 ng/ml) ⫹ Flt3 ligand (FL) (50 ng/ml) ⫹ thrombopoietin (TPO) (10 ng/ml) ⫹ interleukin (IL)-6 (10 ng/ml); then 24-hour prestimulated cells (1 ⫻ 105 cells per ml) were transduced in serum-free CellGro supplemented with 1% penicillin/streptomycin in the presence of FL, TPO, SCF, and IL-6 for 3 consecutive days by replacing half of the cell culture medium with a GALV-pseudotyped retroviral supernatant (titer: 1 ⫻ 106 to 5 ⫻ 106) supplemented with the cytokine combination mentioned above. Plates were precoated with the recombinant fibronectin fragment CH296. Cells were then washed and seeded in new 24-well plates and cultured in the presence of the same growth factors in serum-free conditions. (A): Flow cytometric analysis of one representative transduction experiment into CD34⫹ cells derived from umbilical CB. The left panels show the isotype controls for nonspecific IgG1 staining. The CD34 and LNGFR expression on LNGFR-transduced cells is shown in the right panels. Aliquots of cells from weekly demidepopulated wells were counted (B) and plated in semisolid cultures to assess the clonogenic progenitor output (C, D). Values represent the number of cells and colonies present in a single well. Abbreviations: CFC, colony-forming cell; CFU-GM, colony-forming units-granulopoietic; FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; LTC-IC, long-term culture-initiating cell; NGFR, nerve growth factor receptor; SSC, side scatter.

transduction was performed, following an exposure of up to 24 hours 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 the end of the transduction procedure (day 3 post-transduction), a 2.59-fold expansion in total cell numbers and a 1.2-fold expansion in CD34⫹ cell numbers were observed, with ⬎50% of the cells retaining expression of the CD34 cell surface antigen (Fig. 1A; Table 1). Transduction efficiency was determined by flow cytometric analysis of LNGFR expression on day 3 post-transduction. Representative profiles of transduced CBderived CD34⫹ cells are shown (Fig. 1A). Transduction efficiency was always ⬎50% (Fig. 1A; Table 1). In addition, half of the CD34⫹ cells coexpressed LNGFR (Fig. 1A; Table 1). LNGFR expression on mock-transduced cells was absent. After transduction, LNGFR-transduced and mock-transduced cells were plated, in the presence of FL, TPO, IL-6, and SCF, in 24-well plates (0.1 ⫻ 105 cells per ml) in SF conditions to compare the in vitro expansion duration and extent. Each week, all the wells were demidepopulated, after vigorous pipetting and resuspension of the cells, by removing half of the culture volume, which was replaced with fresh medium and

growth factors. The harvested media cells were counted, and suitable aliquots of the cell suspension were assayed for immunophenotype analysis and colony assays. The results of the in vitro experiments are summarized in Figure 1B–1D and in Table 1. After 1 week of culture, a 50.75-fold expansion in total cell numbers was observed, with a good (22.66%) percentage of the cells retaining expression of the CD34 cell surface antigen (Fig. 1B; Table 1). The percentage of the CD34⫹ cells coexpressing LNGFR was also high (54.04%) (Table 1). The CD34⫹ cell number and the number of the CD34⫹ cells coexpressing LNGFR remained very high until week 3 (Table 1). CD34⫹ cells coexpressing LNGFR were still present at a good level for up to 12 weeks (data not shown). Clonogenic assays showed an increase in CFC production up to week 7 without a significant difference between LNGFR- and mock-transduced cells (Fig. 1C). LTC-IC output remained quite high up to week 4 (Fig. 1D).

In Vivo Repopulation Ability of Transduced CB CD34ⴙ Cells

The CB CD34⫹ cell transduction was performed, following an exposure of up to 24 hours, to FL, TPO, IL-6, and SCF or FL, TPO, IL-6, and IL-11, in the presence of the growth factors in

Gammaitoni, Lucchi, Bruno et al.

1205

Table 1. Transduced cord blood CD34⫹ cell in vitro expansion, in serum-free conditions, in the presence of Flt3 ligand, thrombopoietin, stem cell factor, and interleukin-6 6

Total cell no. (⫻10 ) of the bulk culture Percentage of CD34⫹b CD34⫹ cell no. (⫻106)c Percentage of LNGFR⫹/CD34⫹d LNGFR⫹/CD34⫹ cell no. (⫻106)c Percentage of LNGFR⫹b

Day 3a

Week 1a

Week 2a

Week 3a

Week 4a

Week 5a

Week 6a

2.59 46.39 1.2 56.39 0.68 63.46

55.75 22.66 11.5 54.04 6.215 63.79

114.5 12.66 14.5 54.47 7.9 63.29

140.6 12.8 18 52.69 9.48 45.1

450.2 5.62 25.3 36.06 9.1 38.65

306.6 4.99 15.3 42.54 6.51 38.195

897.6 1.66 14.9 31.86 4.9 34.31

Mean of two different experiments performed in triplicate. Time after transduction. b Percentage calculated on the total. c Number of positive cells generated from 1 ⫻ 106 CD34⫹ cells at time ⫽ 0. d Percentage calculated on the CD34⫹ gate. Abbreviation: LNGFR, low-affinity nerve growth factor receptor. a

SF medium on RT-coated plates. At the end of transduction, in both culture conditions, ⬎50% of CD34⫹ cells were also LNGFR⫹ (Fig. 1A). We transplanted into sublethally irradiated NOD/SCID mice 200,000 CB CD34⫹ cells just after isolation with Mini-MACS (unmanipulated) or after transduction process (transduced). Six weeks after transplantation, the murine BM was harvested and flow-cytometric analysis was performed to evaluate human engraftment and transgene expression. The results of these experiments are summarized in Table 2 and Figures 2 and 3. Mice transplanted with transduced or mock-transduced cells in the presence of FL, TPO, IL-6, and SCF showed high levels of human engraftment (28.42% ⫾ 13.76% for unmanipulated vs. 21.54% ⫾ 7.71% and 43.35% ⫾ 13.29% human CD45⫹ in murine BM for transduced and mock-transduced cells, respectively) (Table 2; Figs. 2A, 3A). Mice transplanted with transduced or mock-transduced cells in the presence of FL, TPO, IL-6, and IL-11 also showed high levels of human engraftment (28.42% ⫾ 13.76% for unmanipulated vs. 28.4% ⫾ 4.39% and 38.5% ⫾ 6.36% human CD45⫹ in murine BM for transduced and mock-transduced cells, respectively) (Table 2; Figs. 2A, 3A). The difference in human engraftment level between unmanipulated and transduced cells was not statistically significant. The difference in the human engraftment level between the two different culture systems was not statistically significant. The difference between LNGFR- and mock-transduced cells was not statistically significant in spite of a possible toxic effect of the virus during the transduction procedure. Human CD45⫹ cells that coexpressed LNGFR in the BM of NOD/SCID transplanted with cells transduced in the presence of FL, TPO, IL-6, and SCF or FL, TPO, IL-6, and IL-11 were 11% ⫾ 3.42% and 11.51% ⫾ 3.61%, respectively (Table 2; Figs. 2A, 3A). The difference between the two different culture systems was not statistically significant. FACS analysis of the different subpopulations showed LNGFR expression within the progenitor (CD34⫹), B cells (CD19⫹), myeloid cells (CD14⫹), erythroid cells (GpA⫹), and megakaryocyte cells (CD41⫹) in equivalent proportions for both culture systems without a statistically significant difference (data not shown). The BM of the engrafted mice was placed in a human colony assay. High numbers of both granulocyte-macrophage and erythoid colonies were detected showing that progenitor cells were maintained in the BM of NOD/SCID mice (Table 2). www.StemCells.com

Human CFU-GM and BFU-E output starting from 1 ⫻ 106 BM cells of NOD/SCID mice transplanted with unmanipulated CB CD34⫹ cells was not significantly higher than those generated from NOD/SCID mice transplanted with CB CD34⫹ cells LNGFR- and mock-transduced in the presence of FL, TPO, IL-6, and SCF (CFU-GM number, 280 ⫾ 33.62 vs. 219.33 ⫾ 48.69 and 227 ⫾ 28.58, respectively; BFU-E number, 90.58 ⫾ 12.73 vs. 70 ⫾ 13 and 100.67 ⫾ 22.14, respectively). Human CFU-GM output starting from 1 ⫻ 106 BM cells of NOD/SCID mice transplanted with unmanipulated CB CD34⫹ cells was not significantly higher than those generated from NOD/SCID mice transplanted with CB CD34⫹ cells LNGFR- and mock-transduced in the presence of FL, TPO, IL-6, and IL-11 (280 ⫾ 33.62 colonies vs. 224 ⫾ 62.07 and 250 ⫾ 72.12 colonies, respectively). Conversely, BFU-E generated by 1 ⫻ 106 BM cells of NOD/SCID mice transplanted with CB CD34⫹ cells LNGFRand mock-transduced in the presence of FL, TPO, IL-6, and IL-11 were significantly lower than in BM of mice transplanted with unmanipulated CB CD34⫹ cells (90.58 ⫾ 12.73 colonies vs. 10 ⫾ 0.8 and 8 ⫾ 0.71 colonies, respectively). BM cells from primary recipients were used for secondary transplants. Four out of six mice transplanted with BM cells derived from primary recipients injected with unmanipulated CB CD34⫹ cells, showed rather good but variable levels of human engraftment (6.15% ⫾ 5.87%) (Table 2; Figs. 2A, 2B, 3A, 3B). All mice transplanted with BM cells from primary recipients injected with CB CD34⫹ cells mock-transduced, both in the presence of FL, TPO, IL-6, and IL-11 and in the presence of FL, TPO, IL-6, and SCF were engrafted (2.35% ⫾ 0.07% and 2.3% ⫾ 1.37%, respectively) (Table 2; Figs. 2A, 2B, 3A, 3B). Three out of five secondary recipients and three out of three secondary recipients transplanted with BM cells derived from primary recipients injected with CB CD34⫹ cells LNGFRtransduced in the presence of FL, TPO, IL-6, and IL-11 and FL, TPO, IL-6, and SCF, respectively, resulted engrafted (1.44% ⫾ 0.54% and 1.42% ⫾ 0.23%, respectively) (Table 2; Figs. 2A, 2B, 3A, 3B). Human CD45⫹ cells in BM of NOD/SCID transplanted with cells transduced in the presence of FL, TPO, IL-6, and SCF or FL, TPO, IL-6, and IL-11, which coexpressed LNGFR, were 25.05% ⫾ 1.09% and 14.1% ⫾ 3.4%, respectively (Table 2; Figs. 2A, 3A). In all mice, multilineage engraftment (CD19, CD14, CD34, CD41, GpA) was found with Lin⫹/ LNGFR⫹ cells (data not shown). Human colonies (CFU-GM,

Oncoretrovirally Transduced HSC Transplantation

1206

Table 2. Comparison of engraftment in NOD/SCID mice of unmanipulated, transduced, or mock-transduced (FL ⫹ TPO ⫹ IL-6 ⫹ SCF or FL ⫹ TPO ⫹ IL-6 ⫹ IL-11) cells under serum-free conditions Percentage of huCD45ⴙ ⴙ huGpAⴙ (no. of engrafted mice/no. of transplanted mice)a

Treatment Unmanipulatede Primary recipientsf Secondary recipientsg FL ⴙ TPO ⴙ IL-6 ⴙ IL-11e Primary recipientsf Secondary recipientsg FL ⴙ TPO ⴙ IL-6 ⴙ SCFi Primary recipientsf Secondary recipientsg

Multilineage engraftmentb

Percentage of huCD45ⴙ coexpressing LNGFRⴙc

⫹ ⫹

A A

280 ⫾ 33.62 N.D.

28.42 ⫾ 13.76 (6) 6.15 ⫾ 5.87 (4/6)

CFU-GM (ⴛ106)d

MOCK LNGFR MOCK LNGFR

38.5 ⫾ 6.36h (2/2) 28.4 ⫾ 4.39h (5/5) 2.35 ⫾ 0.07h (2/2) 1.44 ⫾ 0.54h (3/5)

⫹ ⫹ ⫹ ⫹

A 11.51 ⫾ 3.61 A 14.1 ⫾ 3.4

250 ⫾ 72.12h 224 ⫾ 62.07h N.D. N.D.

MOCK LNGFR MOCK LNGFR

43.35 ⫾ 13.3h,j (3/3) 21.54 ⫾ 7.71h,j (3/3) 2.3 ⫾ 1.37h,j (3/3) 1.42 ⫾ 0.23h,j (3/3)

⫹ ⫹ ⫹ ⫹

A 11 ⫾ 3.42 A 25.05 ⫾ 1.09k

227 ⫾ 28.58h,j 219 ⫾ 48.69h,j N.D. N.D.

BFU-E (ⴛ106)d 90.58 ⫾ 12.73 N.D. 8 ⫾ 0.71 10 ⫾ 0.8 N.D. N.D. 100.67 ⫾ 22.14h 70 ⫾ 13h 2.5 ⫾ 0.21 2.5 ⫾ 0.21

Mean ⫾ SD of the percentage of human CD45⫹ and GpA⫹ cells (the presence of ⱖ0.5% of human CD45⫹ and GpA⫹ cells in the BM of NOD/SCID mice defined a positive engraftment) detected by flow cytometric analysis and confirmed by human chromosome 17 ␣-satellite polymerase chain reaction. b Mutilineage engraftment was defined by the presence of CD19⫹, CD34⫹, CD33⫹, CD14⫹, and CD41⫹ cells within the CD45 gate. c Mean ⫾ SD of the percentage of LNGFR⫹ cells calculated on the CD45⫹ cell gate detected by flow cytometric analysis. d Mean ⫾ SD of the colony-forming cell number generated by 1 ⫻ 106 total BM cells of the secondary transplanted mice seeded in triplicate dishes for semisolid assays. Recombinant human IL-3, granulocyte monocyte colony-stimulating factor, SCF, and erythropoietin were added at the start of cultures. Dishes were scored after 14 days of incubation, after staining with human-specific monoclonal antibody. Only fluorescent colonies were considered to be of human origin. e Mean of two different experiments. f Cord blood CD34⫹ cells (2 ⫻ 105 per mouse) were transplanted as they were (“unmanipulated”) or after transduction under procedure in serum-free conditions in the presence of FL, TPO, IL-6, and SCF or FL, TPO, IL-6, and IL-11 and then transplanted. g Unseparated BM cells of a primary mouse (15 ⫻ 106 to 30 ⫻ 106) were injected into sublethally irradiated NOD/SCID recipients, which were sacrificed 6 weeks later. Engraftment was evaluated on BM cells of the sacrificed secondary animals with the same parameters used for primary engraftment. h p ⬎ .05 vs. unmanipulated. i Mean of three different experiments. j p ⬎ .05 vs. FL, TPO, IL-6, and IL-11. k p ⬍ .01 vs. primary recipient. Abbreviations: A, absent; BFU-E, colony-forming units-erythroid; BM, bone marrow; CFU-GM, colony-forming units-granulopoietic; FL, Flt3 ligand; hu, human; IL, interleukin; LNGFR, low-affinity nerve growth factor receptor; N.D., not detectable; NOD/SCID, nonobese diabetic/severe combined immunodeficiency; SCF, stem cell factor; TPO, thrombopoietin. a

BFU-E) were not detectable in the BM of NOD/SCID mice transplanted with CB CD34⫹ cells LNGFR- and mock-transduced in the presence of FL, TPO, IL-6, and IL-11. Although human CFU-GM were not detectable, some human BFU-E were detected in the BM of NOD/SCID mice transplanted with CB CD34⫹ cells NGFR- and mock-transduced in the presence of FL, TPO, IL-6, and SCF. BM cells from secondary recipients were used for tertiary transplants. Nevertheless, no tertiary recipients resulted engrafted.

In Vivo Repopulation Ability of Transduced and Expanded CB CD34ⴙ Cells

The CD34⫹ cell number after a single CB collection is limited: an insufficient number of genetically modified transplanted HSCs might be unable to compete with and eventually replace the defective host hemopoiesis. Our aim in this second part of

the study was to establish stable gene transfer into long-term repopulating CB cells under SF conditions and to expand transduced transplantable stem cells in vitro. As both cytokine combinations had similar effects in terms of HSC transduction efficiency and preservation, only FL, TPO, IL-6, and SCF combination was used for expansion cultures. At the end of transduction, some of the transduced cells were directly transplanted into NOD/SCID mice, whereas the remaining transduced cells were plated, in the presence of FL, TPO, IL-6, and SCF, into T75 cell culture flasks in SF conditions (0.5 or 1 ⫻ 105 cells per ml) to perform expansions for the NOD/SCID mouse transplants. To assess whether transduced and ex vivo expanded cells retained the long-term and multilineage repopulating ability, we intravenously injected the progeny of 200,000 CD34⫹ cells transduced and 1 week expanded into sublethally irradiated NOD/SCID mice (Fig. 4A).

Gammaitoni, Lucchi, Bruno et al.

Figure 2. LNGFR expression on CB-derived CD34⫹ cells transduced with a Gibbon ape leukemia virus-pseudotyped retroviral vector in the presence of FL, TPO, IL-6, and IL-11 and serial transplantations in NOD/SCID mice. FACS profile (A) and PCR analysis (B) of marrow cells from a representative NOD/SCID mouse that, 6 weeks earlier, had received a transplant of 2 ⫻ 105 transduced CB CD34⫹ in the presence of FL, TPO, IL-6, and IL-11. The BM of the primary mouse was injected into a secondary sublethally irradiated NOD/SCID mouse. (A): FACS analysis of human CD45 and LNGFR expression in the BM of primary (human engraftment, 28% CD45⫹/total BM cells) and secondary (human engraftment, 2.3% CD45⫹/total BM cells) mice was performed on total BM cells. The numbers in the top right quadrants show the percentages of LNGFR⫹ cells within the CD45⫹ population. (B): The positive control is represented by human CB mononuclear cells for ␣-satellite PCR and by LNGFR-expressing cell line PG13 for LNGFR PCR. The negative control is represented by a nontransplanted mouse BM. Amplification of a sequence from the human GAPDH gene was used to control for the presence of DNA. Mice 1 through 5 were transplanted with LNGFR-transduced CD34⫹ cells; mice 6 and 7 were transplanted with mock-transduced CD34⫹ cells. Abbreviations: C⫺, negative control; C⫹, positive control; CB, cord blood; FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; FL, Flt3 ligand; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL, interleukin; LNGFR, low-affinity nerve growth factor receptor; NGF-R, nerve growth factor receptor; NOD/SCID, nonobese diabetic/severe combined immunodeficiency; PCR, polymerase chain reaction; TPO, thrombopoietin.

Six weeks after transplantation, the murine BM was harvested; flow cytometric analysis was performed to evaluate human engraftment and transgene expression. The results of these experiments are summarized in Table 3 and Figure 4B and 4C. Mice transplanted with LNGFR or mock-transduced and 1 week expanded cells showed higher levels of human www.StemCells.com

1207

Figure 3. LNGFR expression on CB-derived CD34⫹ cells transduced with a Gibbon ape leukemia virus-pseudotyped retroviral vector in the presence of FL, TPO, IL-6, and SCF and serial transplantations in NOD/SCID mice. FACS profile (A) and PCR analysis (B) of marrow cells from a representative NOD/SCID mouse that 6 weeks earlier had received a transplant of 2 ⫻ 105 transduced CB CD34⫹ cells in the presence of FL, TPO, IL-6, and SCF. The bone marrow (BM) of the primary mouse was injected into a secondary sublethally irradiated NOD/SCID mouse. (A): FACS analysis of human CD45 and LNGFR expression in the BM of primary (human engraftment, 22.7% CD45⫹/ total BM cells) and secondary (human engraftment, 3.3% CD45⫹/total BM cells) mice was performed on total BM cells. The numbers in the top right quadrants show the percentages of LNGFR⫹ cells within the CD45⫹ population. (B): The positive control is represented by human CB MNC for ␣-satellite PCR and by LNGFR-expressing cell line PG13 for LNGFR PCR. The negative control is represented by a nontransplanted mouse BM. Amplification of a sequence from the human GAPDH gene was used as a control for the presence of DNA. Mice 1 through 3 were transplanted with mock-transduced CD34⫹ cells; mice 4 through 6 were transplanted with LNGFR-transduced CD34⫹ cells. Abbreviations: C⫺, negative control; C⫹, positive control; CB, cord blood; FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; FL, Flt3 ligand; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL, interleukin; LNGFR, low-affinity nerve growth factor receptor; NGFR, nerve growth factor receptor; NOD/SCID, nonobese diabetic/severe combined immunodeficiency; PCR, polymerase chain reaction; SCF, stem cell factor; TPO, thrombopoietin.

1208

Oncoretrovirally Transduced HSC Transplantation engraftment (54.26% ⫾ 5.59% and 61.12% ⫾ 6.99%, respectively) than those transplanted with unmanipulated or transduced and nonexpanded cells (Tables 2, 3; Fig. 4B, 4C). Previous exposure to the viral supernatant and the transduction protocol do not affect the repopulating ability of 1 week expanded cells. In the BM of NOD/SCID transplanted with LNGFR-transduced cells, human CD45⫹ cells that coexpressed LNGFR were 15.39% ⫾ 7.41%. LNGFR expression on mocktransduced human engrafted cells was negligible (Table 3; Fig. 4B, 4C). FACS analysis of the different subpopulations showed LNGFR expression within the progenitor (CD34⫹), B cells (CD19⫹), myeloid cells (CD14⫹), erythroid cells (GpA⫹) and megakaryocyte cells (CD41⫹) in equivalent proportions (data not shown). The BM of the engrafted mice was placed in a human colony assay. High numbers of both granulocyte-macrophage and erythroid colonies were detected (Table 3), showing that progenitor cells were maintained in the BM of NOD/SCID mice transplanted with LNGFR-transduced (CFU-GM number, 711.45 ⫾ 281.8 per 1 ⫻ 106 cells; BFU-E number, 262.3 ⫾ 159.9 per 1 ⫻ 106 cells; LTC-IC, 44.5 ⫾ 5.79 per 1 ⫻ 106 cells) and mock-transduced CB CD34⫹ cells (CFU-GM number, 704.4 ⫾ 257.7 per 1 ⫻ 106 cells; BFU-E number, 409.8 ⫾ 226.5 per 1 ⫻ 106 cells; LTC-IC number, 41.75 ⫾ 1.69 per 1 ⫻ 106 cells). BM cells from primary recipients were used for serial transplants. All mice transplanted with cells from primary recipient BM were engrafted (19.05% ⫾ 2.01% and 19.25% ⫾ 4.64% of human engraftment level, respectively, for LNGFR- or mocktransduced) (Table 3; Fig. 4B, 4C). Human CD45⫹ cells coexpressing LNGFR in secondary mouse BM transplanted with cells transduced and expanded for 1 week were 29.55 ⫾ 1.55% (Table 3; Fig. 4B, 4C). In all mice, multilineage engraftment (CD45, CD19, CD14, CD34, CD41, Glycophorin A) was found with Lin⫹/LNGFR⫹ cells. Human colonies were detected in the BM of NOD/SCID mice transplanted with LNGFR-transduced (CFU-GM number, 50.1 ⫾ 5.9 per 1 ⫻ 106 cells; BFU-E number, 9.4 ⫾ 1.4 per 1 ⫻ 106 cells) and mock-transduced CB CD34⫹ cells (CFU-GM number, 77.5 ⫾ 9.5 per 1 ⫻ 106 cells; BFU-E number, 23.2 ⫾ 2.7 per 1 ⫻ 106 cells) (Table 3).

Figure 4. LNGFR expression on 1-week ex vivo expanded CB-derived CD34⫹ cells transduced with a Gibbon ape leukemia virus-pseudotyped retroviral vector and serial transplantations in NOD/SCID mice. (A): Flow cytometric analysis of transduced CD34⫹ cells derived from umbilical CB after 1 week of ex vivo expansion in the presence of FL, TPO, IL-6, and SCF. Quadrants on the left show the isotype controls for nonspecific IgG1 staining. The CD34 and LNGFR expression on LNGFR-transduced cells is shown in the right-hand quadrants. FACS profile (B) and PCR analysis (C) of marrow cells from a representative NOD/SCID mouse that, 6 weeks earlier, had received a transplant of 2 ⫻ 105 transduced CB CD34⫹ cells that had been expanded for an additional week after transduction. The BM of the primary mouse was injected into a secondary sublethally irradiated NOD/SCID mouse; the

BM of this mouse was injected into a tertiary mouse. (B): FACS analysis of human CD45 and LNGFR expression in the BM of primary (human engraftment, 88.8% CD45⫹/total BM cells), secondary (human engraftment, 32.4% CD45⫹/total BM cells), and tertiary (human engraftment, 9.8% CD45⫹/total BM cells) mice was performed on total BM cells. The numbers in the top right quadrants show the percentages of LNGFR⫹ cells within the CD45⫹ population. (C): The positive control is represented by human CB MNCs for ␣-satellite PCR and by LNGFR-expressing cell line PG13 for LNGFR PCR. The negative control is represented by a nontransplanted mouse BM. Amplification of a sequence from the human GAPDH gene was used as a control for the presence of DNA. Mock-transduced mouse engraftment not shown. Only 9 of 11 representative primary and five of six representative secondary LNGFR-transduced mice are shown. Abbreviations: C⫺, negative control; C⫹, positive control; CB, cord blood; FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; FL, Flt3 ligand; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL, interleukin; LNGFR, low-affinity nerve growth factor receptor; NGFR, nerve growth factor receptor; NOD/SCID, nonobese diabetic/severe combined immunodeficiency; PCR, polymerase chain reaction; PE, phycoerythrin; SCF, stem cell factor; SSC, side scatter; TPO, thrombopoietin.

Gammaitoni, Lucchi, Bruno et al.

1209

Table 3. Comparison of engraftment in NOD/SCID mice of transduced or mock-transduced cells after 1-week expansion in serum-free conditions Primary Recipientsa MOCK Percentage of huCD45⫹⫹huGpA⫹c No. of engrafted mice/no. of transplanted mice Multilineage engraftmentg Percentage of huCD45⫹h coexpressing LNGFR CFU-GMl BFU-El LTC-ICl

LNGFR

61.12 ⫾ 6.99

d,e

5/5 ⫹ N.D.

Secondary Recipientsb MOCK

54.26 ⫾ 5.59

d,e

11/11 ⫹ 15.39 ⫾ 7.41

704.4 ⫾ 257.7d,e 711.45 ⫾ 281.8d,e,m 409.8 ⫾ 226.5d,e 262.3 ⫾ 159.9d,e,m 41.75 ⫾ 1.69 44.5 ⫾ 5.79

Tertiary Recipientsb

LNGFR

19.25 ⫾ 4.64

d,f

19.05 ⫾ 2.01

d,f

MOCK

LNGFR

7.48 ⫾ 3.66

6.15 ⫾ 5.16

5/5

6/6

2/4

2/5

⫹ N.D.

⫹ 29.55 ⫾ 1.55i,j

⫹ N.D.

⫹ 25 ⫾ 9.9k

50.1 ⫾ 5.9m 9.4 ⫾ 1.4m N.D.

43.6 ⫾ 16.8 18.2 ⫾ 6.3 N.D.

66.8 ⫾ 27.5m 10.4 ⫾ 7.9m N.D.

77.5 ⫾ 9.5 23.2 ⫾ 2.7 N.D.

Mean of three different experiments. a The progeny of 2 ⫻ 105 CB CD34⫹ cells/mouse were transplanted after MOCK or LNGFR transduction and 1 week of expansion under serum-free conditions in the presence of FL, TPO, SCF, and IL-6, corresponding to 2.75 ⫻ 106 CD34⫹ cells transplanted for each mouse on average. b Unseparated BM cells (15 ⫻ 106 to 30 ⫻ 106) of a primary or secondary mouse were injected into sublethally irradiated NOD/SCID recipients, which were sacrificed 6 weeks later. Engraftment was evaluated on BM cells of the sacrificed animals (secondary and tertiary recipients) with the same parameters used for primary engraftment. c Mean ⫾ SD of the percentage of human CD45⫹ and GpA⫹ cells (the presence of ⱖ0.5% of human CD45⫹ and GpA⫹ cells in the BM of NOD/SCID mice defined a positive engraftment) detected by flow cytometric analysis and confirmed by human chromosome 17 ␣-satellite polymerase chain reaction. d p ⬍ .01 vs. unmanipulated (data shown in Table 2). e p ⬍ .01 vs. primary recipients transplanted with LNGFR-transduced in the presence of FL, TPO, SCF, and IL-6 and nonexpanded CB CD34⫹ cells (data shown in Table 2). f p ⬍ .01 vs. secondary recipient transplanted with LNGFR-transduced in the presence of FL, TPO, SCF, and IL-6 and nonexpanded CB CD34⫹ cells (data shown in Table 2). g Mutilineage engraftment was defined by the presence of CD19⫹, CD34⫹, CD33⫹, CD14⫹, and CD41⫹ cells in the CD45 gate. h Mean ⫾ SD of the percentage of LNGFR⫹ cells calculated on the CD45⫹ cell gate detected by flow cytometric analysis. i p ⬍ .01 vs. secondary recipient transplanted with LNGFR-transduced in the presence of FL, TPO, SCF, and IL-6 and nonexpanded CB CD34⫹ cells (data shown in Table 2). j p ⬍ .01 vs. primary recipient transplanted with LNGFR-transduced and CB CD34⫹ cells expanded for 1 week. k p ⬎ .05 vs. secondary recipient transplanted with LNGFR-transduced and CB CD34⫹ cells expanded for 1 week. l Mean ⫾ SD of the colony-forming cell number generated from 1 ⫻ 106 total BM cells of the secondary transplanted mice seeded in triplicate dishes for semisolid assays. Recombinant human IL-3, granulocyte monocyte colony-stimulating factor, SCF, and erythropoietin were added at the start of cultures. Dishes were scored after 14 days of incubation, after staining with human-specific monoclonal antibody. Only fluorescent colonies were considered of human origin. m p ⬎ .05 vs. corresponding recipient transplanted with CB CD34⫹ cells mock-transduced and expanded for 1 week. Abbreviations: BFU-E, colony-forming units-erythroid; BM, bone marrow; CB, cord blood; CFU-GM, colony-forming units-granulopoietic; FL, Flt3 ligand; hu, human; IL, interleukin; LNGFR, low-affinity nerve growth factor receptor; LTC-IC, long-term culture-initiating cell; N.D., not detectable; NOD/SCID, nonobese diabetic/severe combined immunodeficiency; SCF, stem cell factor; TPO, thrombopoietin.

BM cells from secondary recipients were used for tertiary transplants. Two mice out of five transplanted with secondary recipient BM cells derived from mice transplanted with transduced and 1-week expanded cells showed rather good levels of human engraftment (6.15% CD45⫹) (Table 3; Fig. 4B, 4C). LNGFR expression was 25% ⫾ 9.9% of human CD45⫹ cells (Table 3, Fig. 4B, 4C). In these mice, multilineage engraftment (CD19, CD14, CD34, CD41, GpA) was found by flow cytometric analysis. All subpopulations had similar percentages of LNGFR⫹ cells. Human colonies were also generated from the murine BM mice transplanted with LNGFR-transduced (CFU-GM number, 66.8 per 1 ⫻ 106 cells; BFU-E number, 10.4 per 1 ⫻ 106 cells) and mock-transduced CB CD34⫹ cells (CFU-GM number, 43.6 ⫾ 16.8 per 1 ⫻ 106 cells; BFU-E number, 18.2 ⫾ 6.3 per 1 ⫻ 106 cells) (Table 3). www.StemCells.com

DISCUSSION

The CB CD34⫹ cell number after a single CB collection is limited, due to its small physiological volume. An insufficient number of genetically modified transplanted HSCs might lead to an inability to compete with and eventually replace the defective host hemopoiesis. Expansion of transduced transplantable stem cells in vitro might prove extremely useful. Our aim was to establish stable gene transfer into long-term repopulating CB cells under SF conditions and to expand transduced transplantable stem cells in vitro. Multiple factors probably contribute to the inefficiency of gene transfer to human repopulating cells. Most HSCs are quiescent [36] and therefore refractory to transduction by the current generation of murine retroviral vectors; integration of these vectors into the genome requires stimulation with

1210

hematopoietic growth factors/cytokines for the quiescent HSCs to enter the cell cycle and undergo division ex vivo [3]. GALVpseudotyped retroviruses have been shown to mediate higher levels of gene transfer to CD34⫹ and CD34⫹CD38⫺ cells, but the transduced cells have not been evaluated for their long-term repopulating ability after additional expansion procedure in order to increase the number of immature progenitors carrying the transgene [3, 19 –21, 23, 24, 37]. The NOD/SCID model system, with serial transplantations, has been used to study the long-term engraftment of primitive human hematopoietic cells, which retain extensive proliferation and multilineage differentiation potential [7, 17, 18, 38, 39]. Unlike the majority of LTC-ICs, which are incapable of repopulation, SRCs are found predominantly in the CD34⫹CD38⫺ cell fraction [31]. Furthermore, kinetic experiments indicate that engraftment of SRCs is followed by a large expansion of LTC-ICs in vivo, suggesting that these are derived from a more primitive cell [17]. Although both CFCs and LTC-ICs are readily transduced, the efficiency of gene transfer to SRCs has generally been very low, and the repopulating potential has been markedly compromised by ex vivo culture [28]. Furthermore, current gene transfer protocols require the removal of HSCs from their natural microenvironmental niches and their manipulation in ex vivo conditions, which may alter the integrity and functionality of these cells. These functions, at least partially, can be maintained by appropriate combinations of cytokines and growth factors. FL, SCF, TPO, IL-6, and IL-11, in particular, have been shown to act synergistically to stimulate the proliferation and amplification of SRC cells both in vitro and in vivo [8, 10, 13, 14]. Ideally, ex vivo manipulation of HSCs should preserve the intrinsic properties of these cells. In vitro experiments performed under SF conditions showed that a three-step transduction protocol, preceded by 24-hour cytokine prestimulation, in the presence of FL, TPO, IL-6, and SCF allowed a transduction efficiency ranging from 50%–70%, with ⬎50% of the cells retaining expression of the CD34 cell surface antigen. At the end of the transduction procedure, total and CD34⫹ cells were not only maintained but also slightly amplified (2.59-fold and 1.2-fold expansion, respectively). A high number of CD34⫹ cells also coexpressing LNGFR were maintained for up to 6 weeks. The transgene expression was high until the exhaustion of culture. In previously published papers, we demonstrated that high levels of human engraftment in three generations of NOD/SCID mice were obtained upon transplantation of CB CD34⫹ cells expanded in the presence of fetal calf serum and of a cytokine combination optimal for SRC [10, 40]. The same culture system was successfully used in gene transfer protocols with lentiviral vectors. In these protocols, transduced and expanded HSCs engrafted three serial generations of NOD/SCID mice. The grafted cells demonstrated a stable and prolonged transgene expression [40]. In another study, the effect of a cytokine cocktail containing FL, TPO, IL-6, and IL-11, combined with serum-replete or serum-free conditions, on the ex vivo expansion of CB CD34⫹ cells was investigated [23, 24]. Expansion might be slightly improved using serum, but the use of serum-free medium could be essential to apply good manufacturing practice conditions suitable for clinical use; this demonstrates that a long-term expansion of CB HSCs in a stroma- and serum-free system was

Oncoretrovirally Transduced HSC Transplantation possible. In addition, long-term expanded cells retained their ability of sustained and multilineage engraftment in NOD/SCID mice [13, 14]. On the basis of these data, the transduction was performed in the presence of FL, TPO, IL-6, and SCF or FL, TPO, IL-6, and IL-11, in SF medium. The transgene integration is mediated by a preintegration complex (PIC) comprising viral DNA, reverse transcriptase and integrase as well as poorly characterized host proteins [41]. Lentiviral vector PIC can penetrate into the nucleus so that integration can occur in nondividing cells that are in the G1 phase of the cell cycle. A major limitation of retroviral vectors is that their PIC requires that the nucleus membrane is dissolved so that PIC can come in direct contact with host cell DNA; hence, there is efficient integration only in dividing cells [41]. Our main goal was to transduce HSCs with long-term repopulation ability and to obtain a high transduction efficiency using a retroviral vector similarly to what was obtained using a lentiviral vector. This task was achieved by culturing HSCs with early acting cytokines for 24 hours before transduction in order to trigger cell division and maintain the stemness potential. The in vitro results demonstrated that 24-hour prestimulation with FL, TPO, IL-6, IL-11 or FL, TPO, IL-6, SCF can sustain a very high transduction efficiency of CB CD34⫹ cells, even with a retroviral vector. To assess whether transduced cells retained the long-term and multilineage repopulating ability together with a sustained transgene expression, we transplanted NOD/SCID mice with transduced cells, compared with unmanipulated CD34⫹ cells. The difference in the repopulating potential between the two different culture protocols was negligible. However, the human engraftment level was significantly greater in mice transplanted with mock-transduced cells than in mice engrafted with unmanipulated CD34⫹ cells, probably because of the amplification occurring in culture. In contrast, LNGFR-transduced cell repopulation potential was similar to that of unmanipulated cells. The difference between LNGFR- and mock-transduced was probably due to some virus toxicity in the transduction procedure. After transduction, aliquots of transduced cells were then ex vivo expanded for 1 additional week in the presence of FL, TPO, IL-6, and SCF and transplanted into sublethally irradiated NOD/SCID mice. Mice were transplanted with LNGFR- or mock-transduced, and subsequently, 1 week expanded cells showed higher levels of human engraftment (54.26% ⫾ 2.39% and 61.12% ⫾ 4.99%, respectively) than those transplanted with unmanipulated or transduced and nonexpanded cells; a high number of human CD45⫹ cells coexpressed LNGFR. These data indicate not only that the toxicity due to the viral supernatant exposure and the transduction procedure did not affect the repopulating ability of cells but also that subsequent ex vivo expansion significantly increased the human engraftment ability, probably because of the expansion of transplantable cells. Serial transplantation is the most reliable method to assess the stable expression of a transgene in cells with high proliferation and self-renewal potential. BM cells of mice transplanted with LNGFR-transduced or mock-transduced cells indeed sustain a secondary transplant. Most importantly, the BM of mice transplanted with transduced and expanded cells could even sustaining even tertiary transplants. This result may be explained with the about 10-fold amplification of total CD34⫹ cell

Gammaitoni, Lucchi, Bruno et al. number and of CD34⫹ coexpressing LNGFR cell number occurring after the additional 1 week of expansion that followed the transduction procedure. In such a way, a higher number of genetically modified SRCs, capable of sustaining a complete and long-term hematopoietic reconstitution in NOD/SCID recipients, were transplanted. Moreover, during the first week of expansion, the telomere length of CB CD34⫹ cells not only does not shorten but actually increases, indicating a great residual proliferation potential of the expanded cells [42]. In conclusion, we have validated a serum-free protocol for efficient gene transfer into human CB HSCs using a retroviral vector and clearly demonstrated the feasibility of a protocol for transducing HSCs with the same efficiency obtained with lentiviral vectors. All was accomplished exclusively by the addition of a prestimulation step in order to trigger cell division, followed by 1 week of expansion with FL, SCF, TPO, and IL-6. The stem cell feature of the cells obtained at the end of the transduction and expansion protocol was demonstrated by: 1) their ability to longterm repopulate three generations of NOD/SCID mice with cells belonging to all hemopoietic lineages; 2) the stable expression of the transgene in a similar proportion of the multilineage engrafted cells along three generations of mice. This result was obtained by transducing and then expanding CB CD34⫹ cells for 7 days. Demonstrating the feasibility of a sound gene transfer protocol under serum-free conditions is an essential prerequisite for the implementation of any clinical application. Once the longterm multilineage engraftment of cells expressing the transgene is proved, a very important issue is to test its safety. To this end, the issue of insertional leukemogenesis has to be addressed. Indeed, it has been shown that retroviral transduction can lead to the inappropriate activation of oncogenes and subsequent leukemogenesis [43–50]. These experiments are warranted, and suitable gene transfer transplantation experiments are being implemented in a large number of animal models. Cumulative data obtained from ⬎300 mice transplanted with HSCs transduced and not expanded with ⌬LNGFR-expressing retroviral vectors showed normal engraftment and the persistence of ⌬LNGFR-expressing HSCs in primary, secondary, and tertiary recipients, with no adverse events [51]. To date, 22 primary, secondary, and tertiary mice have been transplanted with transduced and expanded cells, and 16 have been trans-

REFERENCES 1

Williams DA, Nienhuis AW, Hawley RG et al. Gene therapy 2000. In: Schechter D, ed. American Society of Hematology Education Program. Philadelphia: W.B. Saunders, 2000:376 –393.

2

Karlsson S. Treatment of genetic defects in hematopoietic cell function by gene transfer. Blood 1991;78:2481–2492.

3

Miller DG, Adam MA, Miller AD. Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection. Mol Cell Biol 1990;10:4239 – 4242.

1211

planted with transduced, nonexpanded cells. In none of these animals do we have evidence of leukemia. Moreover, the reconstituted human hemopoiesis in murine BM was multilineage in all mice; furthermore, differentiated human hemopoietic cells were always present with the same proportion detected in recipients transplanted with nontransduced cells (W.P., personal communication). A second important point would be using such a retroviral transduction protocol to follow the fate of different clones during the serial transplants. These experiments have been addressed, using both retroviral and lentiviral vectors, by a number of groups, including ours, for lentiviral vectors [40], with the evidence of an oligoclonal hemopoiesis [52]. A future task will be also able to set up technological resources that can lead to the identification and isolation of stem cell clones with gene integration not adjacent to an oncogene, with subsequent amplification of such potentially “safe” clones, followed by their engraftment for clinical use.

ACKNOWLEDGMENTS We thank Kirin and Amgen for continuous supply of growth factors. We thank L. Ramini for invaluable secretarial assistance. We thank Andrew M. Garvey, B.A., L.T.C.L., for editorial assistance. This work was supported by grants from Associazione Italiana per la Ricerca sul Cancro (Milan, Italy), Istituto Superiore della Sanita` (National Program On Stem Cells), Consiglio Nazionale delle Ricerche (Progetto Strategico Oncologia), and the Ministero dell’Istruzione, dell’Universita` e della Ricerca (Rome, Italy) (to W.P. and M.A.); European Community grant no. PL99-00859 (to W.P.); EUROCORD III-European Community contract no. QLK3-CT-2002-01918 (to W.P.); Associazioni Donatrici Italiane Cordone Ombelicale; Fondazione Cariplo; Ministero della Salute (Ricerca Finalizzata 2002, 2003); Cariplo, Ministero della Salute (Progetto Ricerca Finalizzata 2002 e 2003, Malattie Neurodegenerative, ex art. 56 Anno 2003); Ministero dell’Istruzione, dell’Universita` e della Ricerca (Fondo per gli Investimenti della Ricerca di Base 2001) (to L.L. and R.P.); and Programma Nazionale Cellule Staminali 2003-Istituto Superiore di Sanita` and Fondazione I. Monzino (to L.L. and R.P.). G.L., L.S., and B.S. contributed equally to this work.

DISCLOSURES The authors indicate no potential conflicts of interest.

7

Conneally E, Cashman J, Petzer A et al. Expansion in vitro of transplantable human cord blood stem cells demonstrated using a quantitative assay of their lympho-myeloid repopulating activity in non obese diabetic-scid/scid mice. Proc Natl Acad Sci U S A 1997;94:9836 –9841.

8

Piacibello W, Sanavio F, Garetto L et al. Extensive amplification and self-renewal of human primitive hematopoietic stem cells from cord blood. Blood 1997;89:2644 –2653.

9

Kollet O, Aviram R, Chevath J et al. The soluble interleukin-6 (IL-6) receptor/IL-6 fusion protein enhances in vitro maintenance and proliferation of human CD34(⫹)CD38(⫺/Low) cells capable of repopulating severe combined immunodeficiency mice. Blood 1999;94:923–931.

4

Richter J. Gene transfer to hematopoietic cells: The clinical experience. Eur J Haematol 1997;59:67–75.

5

Gothot A, Van Der Loo JC, Clapp DW et al. Cell cycle-related changes in repopulating capacity of human mobilized peripheral blood CD341 cells in non-obese diabetic/severe combined immune-deficient mice. Blood 1998;92:2641–2649.

10 Piacibello W, Sanavio F, Severino A et al. Engraftment in non obese diabetic severe combined immunodeficient mice of human CD34(⫹) cord blood cells after ex vivo expansion: Evidence for the amplification and self-renewal of repopulating stem cells. Blood 1999;93:3736 –3749.

6

Guenechea G, Segovia JC, Albella B et al. Delayed engraftment of nonobese diabetic/severe combined immunodeficient mice transplanted with ex vivoexpanded human CD341 cord blood cells. Blood 1999;93:1097–1105.

11 Ueda T, Tsuji K, Yoshino H et al. Expansion of human NOD/SCIDrepopulating cells by stem cell factor, Flk2/Flt3 ligand, thrombopoietin, IL-6, and soluble IL-6 receptor. J Clin Invest 2000;105:1013–1021.

www.StemCells.com

1212

12 Gilmore GL, Depasquale DK, Lister J et al. Ex vivo expansion of human umbilical cord blood CD34(⫹) hematopoietic stem cells. Exp Hematol 2000;28:297–305. 13 Lazzari L, Lucchi S, Rebulla P et al. Long-term expansion and maintenance of cord blood haematopoietic stem cells using thrombopoietin, Flt3-ligand, interleukin (IL)-6 and IL-11 in a serum-free and stroma-free culture system. Br J Haematol 2001;112:397– 404. 14 Blundell MP, Demaison C, Brouns G et al. Quality of repopulation in nonobese diabetic severe combined immunodeficient mice engrafted with expanded cord blood CD34⫹ cells. Blood 1999;94:3269 –3270. 15 Larochelle A, Vormoor J, Lapidot T et al. Engraftment of immunedeficient mice with primitive hematopoietic cells from beta-thalassemia and sickle cell anemia patients: Implications for evaluating human gene therapy protocols. Hum Mol Genet 1995;4:163–172. 16 Lapidot T, Pflumio F, Doedens M et al. Cytokine stimulation of multilineage hematopoiesis from immature human cells engrafted in SCID mice. Science 1992;255:2406 –2411. 17 Cashman JD, Lapidot T, Wang JCY et al. Kinetic evidence of the regeneration of multilineage hematopoiesis from primitive cells in normal human bone marrow transplanted into immunodeficient mice. Blood 1997;89:4307– 4316. 18 Pflumio F, Izac B, Katz A et al. Phenotype and function of human hematopoietic cells engrafting immune-deficient CB 7-severe combined immunodeficiency mice and non obese diabetic-severe combined immunodeficiency mice after transplantation of human cord blood mononuclear cells. Blood 1996;88:3731–3740. 19 Chevalier S, Praloran V, Smith C et al. Expression and functionality of the trkA proto-oncogene product/NGF receptor in undifferentiated hematopoietic cells. Blood 1994;6:1479 –1485. 20 Torcia M, Bracci-Laudiero L, Lucibello M et al. Nerve growth factor is an autocrine survival factor for memory B lymphocytes. Cell 1996;85: 354 –356. 21 Bonini C, Ferrari G, Verzeletti S et al. HSV-TK gene transfer into donor lymphocytes for control of allogeneic graft-versus-leukemia. Science 1997;276:1719 –1724. 22 Ruggieri L, Aiuti A, Salomoni M et al. Cell-surface marking of CD34⫹ restricted phenotypes of human hematopoietc progenitor cells by retrovirus-mediated gene transfer. Hum Gene Ther 1997;8:1611–1623. 23 Giaretta I, Madeo D, Bonaguro R et al. A comparative evaluation of gene transfer into blood cells using the same retroviral backbone for independent expression of the EGFP and deltaLNFGR marker genes. Haematologica 2000;85:680 – 689. 24 Movassagh M, Desmyter C, Baillou C et al. High level gene transfer to cord blood progenitors using gibbon ape leukemia virus pseudotype retroviral vectors and an improved clinically applicable protocol. Hum Gene Ther 1998;9:225–234. 25 Gluckman E. Hematopoietic stem cell transplant using umbilical cordblood. N Engl J Med 2001;24:1860 –1861. 26 Kohn D, Weinberg K, Nolta J et al. Engraftment of gene-modified umbilical cord blood cells in neonateds with adenoise deaminase deficiency. Nat Med 1995;1:1017–1023. 27 Cavazzana-Calvo M, Hacein-Bey S, De Saint Basile G et al. Gene therapy of human severe combined immunodeficiency (Scid)-X1 disease. Science 2000;28:627– 629. 28 Kiem HP, Heyward S, Winkler A et al. Gene transfer into marrow repopulating cells: Comparison between amphotropic and gibbon ape leukemia virus pseudotyped retroviral vectors in a competititve repopulation assay in baboons. Blood 1997;90:4638 – 4645. 29 Miller AD, Garcia JV, von Suhr N et al. Construction and properties of retrovirus packaging cells based on gibbon ape leukemia virus. J Virol 1991;65:2220 –2224. 30 Kavanaugh MP, Miller DG, Zhang W et al. Cell-surface receptors for gibbon ape leukemia virus and amphotropic murine retrovirus are inducible sodium-dependent phosphate symporters. Proc Natl Acad Sci U S A 1994;91:7071–7075.

Oncoretrovirally Transduced HSC Transplantation

31 McCowage GB, Phillips KL, Gentry TL et al. Multiparameter-fluorescence activated cell sorting analysis of retroviral vector gene transfer into primitive umbilical cord blood cells. Exp Hematol 1998;26:288 –298. 32 Piacibello W, Sanavio F, Garetto L et al. Differential growth factor requirement of primitive cord blood hematopoietic stem cell for selfrenewal and amplification vs proliferation and differentiation. Leukemia 1998;12:718 –727. 33 Piacibello W, Garetto L, Sanavio F et al. The effects of FLT3 ligand on in vitro human megakaryocytopoiesis. Exp Hematol 1996;24:340 –346. 34 Becker M, Nitsche A, Neumann C et al. Sensitive PCR method for the detection and real-time quantification of human cells in xenotransplantation systems. Br J Cancer 2002;87:1328 –1335. 35 Schilz AJ, Brouns G, Knoss H et al. High efficiency gene transfer to human hematopoietic SCID-repopulating cells under serum-free conditions. Blood 1998;92:3163–3171. 36 Hao QL, Thiemann FT, Petersen D et al. Extended long-term culture reveals a highly quiescent and primitive human hematopoietic progenitor population Blood 1996;88:3306 –3313. 37 Glimm H, Kiem HP, Darovsky B et al. Efficient gene transfer in primitive CD34⫹/CD38lo human bone marrow cells reselected after long-term exposure to GALV-pseudotyped retroviral vector. Hum Gene Ther 1997;8:2079 –2086. 38 Vormoor J, Lapidot T, Pflumio F et al. Immature human cord blood progenitors engraft and proliferate to high levels in severe combined immunodeficient mice. Blood 1994;83:2489 –2497. 39 Bhatia M, Wang JCY, Kapp U et al. Purification of primitive human hematopoietic cells capable of repopulating immune-deficient mice. Proc Natl Acad Sci U S A 1997;94:5320 –5325. 40 Piacibello W, Bruno S, Sanavio F et al. Lentiviral gene transfer and ex vivo expansion of human primitive stem cells capable of primary, secondary, and tertiary multilineage repopulation in NOD/SCID mice. Nonobese diabetic/ severe combined immunodeficient. Blood 2002;100:4391–4400. 41 Fisher A, Cavazzana-Calvo M. Integration of retroviruses: A fine balance between efficiency and danger. PLoS Med 2005;2:e10. 42 Gammaitoni L, Weisel KC, Gunetti M et al. Elevated telomerase activity and minimal telomere loss in cord blood long-term cultures with extensive stem cell replication. Blood 2004;103:4440 – 4448. 43 Hacein-Bey-Abina S, Le Deist F, Carlier F et al. Sustained correction of X-linked severe combined immunodeficiency by ex vivo gene therapy. N Engl J Med 2002;18:346:1185–1193. 44 Hacein-Bey-Abina S, von Kalle C, Schmidt M et al. A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. N Engl J Med 2003;348:255–256. 45 Hacein-Bey-Abina S, Von Kalle C, Schmidt M et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCIDX1. Science 2003;302:415– 419. 46 Schmidt M, Hacein-Bey-Abina S, Wissler M et al. Clonal evidence for the transduction of CD34⫹ cells with lymphomyeloid differentiation potential and self-renewal capacity in the SCID-X1 gene therapy trial. Blood 2005;105:2699 –2706. 47 Kiem HP, Sellers S, Thomasson B et al. Long-term clinical and molecular follow-up of large animals receiving retrovirally transduced stem and progenitor cells: No progression to clonal hematopoiesis or leukemia. Mol Ther 2004;9:389 –395. 48 Kustikova O, Fehse B, Modlich U et al. Clonal dominance of hematopoietic stem cells triggered by retroviral gene marking. Science 2005;308:1171–1174. 49 De Palma M, Montini E, de Sio FR et al. Promoter trapping reveals significant differences in integration site selection between MLV and HIV vectors in primary hematopoietic cells. Blood 2005;105:2307–2315. 50 Laufs S, Nagy KZ, Giordano FA et al. Insertion of retroviral vectors in NOD/SCID repopulating human peripheral blood progenitor cells occurs preferentially in the vicinity of transcription start regions and in introns. Mol Ther 2004;10:874 – 881. 51 Bonini C, Grez M, Traversari C et al. Safety of retroviral gene marking with a truncated NGF receptor. Nat Med 2003;9:367–369. 52 Mazurier F, Gan OI, McKenzie JL et al. Lentivector-mediated clonal tracking reveals intrinsic heterogeneity in the human hematopoietic stem cell compartment and culture-induced stem cell impairment. Blood 2004;103:545–552.