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1.  In vivo selection of hematopoietic stem cells transduced at a low multiplicity-of-infection with a foamy viral MGMTP140K vector 
Experimental hematology  2008;36(3):283-292.
Using a clinically relevant transduction strategy, we investigated to what extent hematopoietic stem cells in lineage-negative bone marrow (Linneg BM) could be genetically modified with a FV vector that expresses the DNA repair protein, O6-methylguanine DNA methyltransferase (MGMTP140K) and selected in vivo with submyeloablative versus myeloablative alkylator therapy.
Linneg BM was transduced at a low multiplicity-of-infection (MOI), with the FV vector, MD9-P140K, that co-expresses MGMTP140K and the enhanced green fluorescent protein, transplanted into C57BL/6 mice, and mice treated with submyeloablative or myeloablative alkylator therapy. The BM was analyzed for the presence of in vivo selected, MD9-P140K-transduced cells at 6 months post-transplantation and subsequently transplanted into secondary recipient animals.
Following submyeloablative therapy, 55% of the mice expressed MGMTP140K in the BM. Proviral integration was observed in ∼50% of committed BM-derived progenitors and analysis of proviral insertion sites indicated up to 2 integrations per transduced progenitor colony. Transduced BM cells selected with submyeloablative therapy reconstituted secondary recipient mice for up to 6 months post-transplantation. In contrast, following delivery of myeloablative therapy to primary recipient mice, only 25% survived. Hematopoietic stem cells were transduced since BM cells from the surviving animals reconstituted secondary recipients with MGMTP140K positive cells for 5-6 months.
In vivo selection of MD9-P140K-transduced BM cells was more efficient following submyeloablative versus myeloablative therapy. These data indicate that a critical number of transduced-stem cells must be present to produce sufficient numbers of genetically modified progeny to protect against the acute toxicity associated with myeloablative therapy.
PMCID: PMC2699892  PMID: 18279716
gene therapy; hematopoietic stem cells; foamy virus vector; O6-methylguanine DNA methyltransferase (MGMT)
2.  Stable Transgene Expression in Primitive Human CD34+ Hematopoietic Stem/Progenitor Cells, Using the Sleeping Beauty Transposon System 
Human Gene Therapy  2009;20(12):1607-1626.
Sleeping Beauty (SB) transposon-mediated integration has been shown to achieve long-term transgene expression in a wide range of host cells. In this study, we improved the SB transposon-mediated gene transfer system for transduction of human CD34+ stem/progenitor cells by two approaches: (1) to increase the transposition efficacy, a hyperactive mutant of SB, HSB, was used; (2) to improve the expression of the SB transposase and the transgene cassette carried by the transposon, different viral and cellular promoters were evaluated. SB components were delivered in trans into the target cells by Nucleoporation. The SB transposon-mediated integration efficacy was assessed by integrated transgene (enhanced green fluorescent protein [eGFP]) expression both in vitro and in vivo. In purified human cord blood CD34+ cells, HSB achieved long-term transgene expression in nearly 7-fold more cells than the original SB transposase. Significantly brighter levels of eGFP expression (5-fold) were achieved with the human elongation factor 1α (EF1-α) promoter in Jurkat human T cells, compared with that achieved with the modified myeloproliferative sarcoma virus long terminal repeat enhancer–promoter (MNDU3); in contrast, the MNDU3 promoter expressed eGFP at the highest level in K-562 myeloid cells. In human CD34+ cord blood cells studied under conditions directing myeloid differentiation, the highest transgene integration and expression were achieved using the EF1-α promoter to express the SB transposase combined with the MNDU3 promoter to express the eGFP reporter. Stable transgene expression was achieved at levels up to 27% for more than 4 weeks of culture after improved gene transfer to CD34+ cells (average, 17%; n = 4). In vivo studies evaluating engraftment and differentiation of the SB-modified human CD34+ cells demonstrated that SB-modified human CD34+ cells engrafted in NOD/SCID/γ chainnull (NSG) mice and differentiated into multilineage cell types with eGFP expression. More importantly, secondary transplantation studies demonstrated that the integrated transgene was stably expressed in more primitive CD34+ hematopoietic stem cells (HSCs) with long-term repopulating capability. This study demonstrates that an improved HSB gene transfer system can stably integrate genes into primitive human HSCs while maintaining the pluripotency of the stem cells, which shows promise for further advancement of non-virus-based gene therapy using hematopoietic stem cells.
PMCID: PMC4094131  PMID: 19689196
3.  Previously undetected human hematopoietic cell populations with short-term repopulating activity selectively engraft NOD/SCID-β2 microglobulin–null mice 
Journal of Clinical Investigation  2001;107(2):199-206.
Increasing use of purified or cultured human hematopoietic cells as transplants has revealed an urgent need for better methods to predict the speed and durability of their engraftment potential. We now show that NOD/SCID-β2 microglobulin–null (NOD/SCID-β2m–/–) mice are sequentially engrafted by two distinct and previously unrecognized populations of transplantable human short-term repopulating hematopoietic cells (STRCs), neither of which efficiently engraft NOD/SCID mice. One is predominantly CD34+CD38+ and is myeloid-restricted; the other is predominantly CD34+CD38– and has broader lymphomyeloid differentiation potential. In contrast, the long-term repopulating human cells that generate lymphoid and myeloid progeny in NOD/SCID mice engraft and self-renew in NOD/SCID-β2m–/– mice equally efficiently. In short-term expansion cultures of adult bone marrow cells, myeloid-restricted STRCs were preferentially amplified (greater than tenfold) and, interestingly, both types of STRC were found to be selectively elevated in mobilized peripheral blood harvests. These results suggest an enhanced sensitivity of STRCs to natural killer cell–mediated rejection. They also provide new in vivo assays for different types of human STRC that may help to predict the engraftment potential of clinical transplants and facilitate future investigation of early stages of human hematopoietic stem cell differentiation.
PMCID: PMC199177  PMID: 11160136
4.  Noninvasive Bioluminescent Imaging Demonstrates Long-Term Multilineage Engraftment of Ex Vivo-Expanded CD34-Selected Umbilical Cord Blood Cells 
Stem Cells (Dayton, Ohio)  2009;27(8):1932-1940.
The use of umbilical cord blood (UCB) grafts for hematopoietic stem cell transplantation (HSCT) is a promising technique that permits a degree of human leukocyte antigen mismatch between the graft and the host without the concomitant higher rate of graft-versus-host disease that would be observed between an adult marrow graft and a mismatched host. A disadvantage to the use of UCB for HSCT is that immune reconstitution may be significantly delayed because of the low stem cell dose available in the graft. Ex vivo expansion of UCB CD34 cells would provide a greater number of stem cells; however, there are persistent concerns that ex vivo-expanded CD34 cells may lose pluripotency and the ability to contribute meaningfully to long-term engraftment. To address this issue, we transduced CD34-selected UCB cells with a lentiviral construct expressing luciferase, and determined homing and engraftment patterns in vivo by noninvasive bioluminescent imaging in sublethally irradiated NOD/SCID/IL-2Rγ−/− (NSG) mice. Graft contribution to multilineage commitment was also confirmed by analysis of primary and secondary transplants by flow cytometry and immunohistochemistry. Our results demonstrate that, other than a mild delay at the onset of engraftment, there were no significant differences in lineage repopulation or in long-term or secondary engraftment between culture-expanded and unexpanded UCB CD34-selected cells. The results suggest that multipotent stem cells can be expanded ex vivo and can contribute meaningfully to long-term hematopoietic engraftment.
PMCID: PMC3348864  PMID: 19544439
Bioluminescent imaging; Umbilical cord blood; Hematopoiesis; Transplantation; Engraftment; Luciferase; Calvarium
5.  Stem Cell Selection In Vivo Using Foamy Vectors Cures Canine Pyruvate Kinase Deficiency 
PLoS ONE  2012;7(9):e45173.
Hematopoietic stem cell (HSC) gene therapy has cured immunodeficiencies including X-linked severe combined immunodeficiency (SCID-X1) and adenine deaminase deficiency (ADA). For these immunodeficiencies corrected cells have a selective advantage in vivo, and low numbers of gene-modified cells are sufficient to provide therapeutic benefit. Strategies to efficiently transduce and/or expand long-term repopulating cells in vivo are needed for treatment of diseases that require higher levels of corrected cells, such as hemoglobinopathies. Here we expanded corrected stem cells in vivo in a canine model of a severe erythroid disease, pyruvate kinase deficiency.
Methodology/Principal Findings
We used a foamy virus (FV) vector expressing the P140K mutant of methylguanine methyltransferase (MGMTP140K) for in vivo expansion of corrected hematopoietic repopulating cells. FV vectors are attractive gene transfer vectors for hematopoietic stem cell gene therapy since they efficiently transduce repopulating cells and may be safer than more commonly used gammaretroviral vectors. Following transplantation with HSCs transduced ex vivo using a tri-cistronic FV vector that expressed EGFP, R-type pyruvate kinase, and MGMTP140K, we were able to increase marking from approximately 3.5% to 33% in myeloid long-term repopulating cells resulting in a functional cure.
Here we describe in one affected dog a functional cure for a severe erythroid disease using stem cell selection in vivo. In addition to providing a potential cure for patients with pyruvate kinase deficiency, in vivo selection using foamy vectors with MGMTP140K has broad potential for several hematopoietic diseases including hemoglobinopathies.
PMCID: PMC3441638  PMID: 23028826
6.  In vivo selection of MGMT(P140K) lentivirus–transduced human NOD/SCID repopulating cells without pretransplant irradiation conditioning 
Journal of Clinical Investigation  2003;112(10):1561-1570.
Infusion of transduced hematopoietic stem cells into nonmyeloablated hosts results in ineffective in vivo levels of transduced cells. To increase the proportion of transduced cells in vivo, selection based on P140K O6-methylguanine-DNA-methyltransferase (MGMT[P140K]) gene transduction and O6-benzylguanine/1,3-bis(2-chloroethyl)-1-nitrosourea (BG/BCNU) treatment has been devised. In this study, we transduced human NOD/SCID repopulating cells (SRCs) with MGMT(P140K) using a lentiviral vector and infused them into BG/BCNU–conditioned NOD/SCID mice before rounds of BG/BCNU treatment as a model for in vivo selection. Engraftment was not observed until the second round of BG/BCNU treatment, at which time human cells emerged to compose up to 20% of the bone marrow. Furthermore, 99% of human CFCs derived from NOD/SCID mice were positive for provirus as measured by PCR, compared with 35% before transplant and 11% in untreated irradiation-preconditioned mice, demonstrating selection. Bone marrow showed BG-resistant O6-alkylguanine-DNA-alkyltransferase (AGT) activity, and CFUs were stained intensely for AGT protein, indicating high transgene expression. Real-time PCR estimates of the number of proviral insertions in individual CFUs ranged from 3 to 22. Selection resulted in expansion of one or more SRC clones containing similar numbers of proviral copies per mouse. To our knowledge, these results provide the first evidence of potent in vivo selection of MGMT(P140K) lentivirus–transduced human SRCs following BG/BCNU treatment.
PMCID: PMC259124  PMID: 14617757
7.  Intratibial Injection of Human Multiple Myeloma Cells in NOD/SCID IL-2Rγ(Null) Mice Mimics Human Myeloma and Serves as a Valuable Tool for the Development of Anticancer Strategies 
PLoS ONE  2013;8(11):e79939.
We systematically analyzed multiple myeloma (MM) cell lines and patient bone marrow cells for their engraftment capacity in immunodeficient mice and validated the response of the resulting xenografts to antimyeloma agents.
Design and Methods
Using flow cytometry and near infrared fluorescence in-vivo-imaging, growth kinetics of MM cell lines L363 and RPMI8226 and patient bone marrow cells were investigated with use of a murine subcutaneous bone implant, intratibial and intravenous approach in NOD/SCID, NOD/SCID treated with CD122 antibody and NOD/SCID IL-2Rγ(null) mice (NSG).
Myeloma growth was significantly increased in the absence of natural killer cell activity (NSG or αCD122-treated NOD/SCID). Comparison of NSG and αCD122-treated NOD/SCID revealed enhanced growth kinetics in the former, especially with respect to metastatic tumor sites which were exclusively observed therein. In NSG, MM cells were more tumorigenic when injected intratibially than intravenously. In NOD/SCID in contrast, the use of juvenile long bone implants was superior to intratibial or intravenous cancer cell injection. Using the intratibial NSG model, mice developed typical disease symptoms exclusively when implanted with human MM cell lines or patient-derived bone marrow cells, but not with healthy bone marrow cells nor in mock-injected animals. Bortezomib and dexamethasone delayed myeloma progression in L363- as well as patient-derived MM cell bearing NSG. Antitumor activity could be quantified via flow cytometry and in vivo imaging analyses.
Our results suggest that the intratibial NSG MM model mimics the clinical situation of the disseminated disease and serves as a valuable tool in the development of novel anticancer strategies.
PMCID: PMC3819303  PMID: 24223204
8.  Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model 
Small animal models such as mice have been extensively used to study human disease and to develop new therapeutic interventions. Despite the wealth of information gained from these studies, the unique characteristics of mouse immunity as well as the species specificity of viral diseases such as human immunodeficiency virus (HIV) infection led to the development of humanized mouse models. The earlier models involved the use of C. B 17 scid/scid mice and the transplantation of human fetal thymus and fetal liver termed thy/liv (SCID-hu) 1, 2 or the adoptive transfer of human peripheral blood leukocytes (SCID-huPBL) 3. Both models were mainly utilized for the study of HIV infection.
One of the main limitations of both of these models was the lack of stable reconstitution of human immune cells in the periphery to make them a more physiologically relevant model to study HIV disease. To this end, the BLT humanized mouse model was developed. BLT stands for bone marrow/liver/thymus. In this model, 6 to 8 week old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) immunocompromised mice receive the thy/liv implant as in the SCID-hu mouse model only to be followed by a second human hematopoietic stem cell transplant 4. The advantage of this system is the full reconstitution of the human immune system in the periphery. This model has been used to study HIV infection and latency 5–8.
We have generated a modified version of this model in which we use genetically modified human hematopoietic stem cells (hHSC) to construct the thy/liv implant followed by injection of transduced autologous hHSC 7, 9. This approach results in the generation of genetically modified lineages. More importantly, we adapted this system to examine the potential of generating functional cytotoxic T cells (CTL) expressing a melanoma specific T cell receptor. Using this model we were able to assess the functionality of our transgenic CTL utilizing live positron emission tomography (PET) imaging to determine tumor regression (9).
The goal of this protocol is to describe the process of generating these transgenic mice and assessing in vivo efficacy using live PET imaging. As a note, since we use human tissues and lentiviral vectors, our facilities conform to CDC NIH guidelines for Biosafety Level 2 (BSL2) with special precautions (BSL2+). In addition, the NSG mice are severely immunocompromised thus, their housing and maintenance must conform to the highest health standards (
PMCID: PMC3576416  PMID: 23271478
Cancer Biology; Issue 70; Stem Cell Biology; Immunology; Biomedical Engineering; Medicine; Bioengineering; Genetics; Oncology; Humanized mice; stem cell transplantation; stem cells; in vivo animal imaging; T cells; cancer; animal model
9.  An Improved Protocol for Efficient Engraftment in NOD/LTSZ-SCIDIL-2RγNULL Mice Allows HIV Replication and Development of Anti-HIV Immune Responses 
PLoS ONE  2012;7(6):e38491.
Cord blood hematopoietic progenitor cells (CB-HPCs) transplanted immunodeficient NOD/LtsZ-scidIL2Rγnull (NSG) and NOD/SCID/IL2Rγnull (NOG) mice need efficient human cell engraftment for long-term HIV-1 replication studies. Total body irradiation (TBI) is a classical myeloablation regimen used to improve engraftment levels of human cells in these humanized mice. Some recent reports suggest the use of busulfan as a myeloablation regimen to transplant HPCs in neonatal and adult NSG mice. In the present study, we further ameliorated the busulfan myeloablation regimen with fresh CB-CD34+cell transplantation in 3–4 week old NSG mice. In this CB-CD34+transplanted NSG mice engraftment efficiency of human CD45+cell is over 90% in peripheral blood. Optimal engraftment promoted early and increased CD3+T cell levels, with better lymphoid tissue development and prolonged human cell chimerism over 300 days. These humanized NSG mice have shown long-lasting viremia after HIV-1JRCSF and HIV-1Bal inoculation through intravenous and rectal routes. We also saw a gradual decline of the CD4+T cell count, widespread immune activation, up-regulation of inflammation marker and microbial translocation after HIV-1 infection. Humanized NSG mice reconstituted according to our new protocol produced, moderate cellular and humoral immune responses to HIV-1 postinfection. We believe that NSG mice reconstituted according to our easy to use protocol will provide a better in vivo model for HIV-1 replication and anti-HIV-1 therapy trials.
PMCID: PMC3366932  PMID: 22675567
Immune-deficient mice serve as critical hosts for transplantation of xenogeneic cells for in vivo analysis of various biological processes. Since investigators typically select one or two immune-deficient mouse strains as recipients, no comprehensive study has been published documenting differences in human tumor engraftment. Taking advantage of the increased metastatic potential of RhoC-expressing human (A375) melanoma cells, we evaluate 4 immune-deficient mouse strains: scid, NOD-scid, NOD-scid β2mnull, and NOD-scid IL2Rγnull as xenograft tumor recipients.
Experimental design
Bioluminescence, magnetic resonance imaging and histopathology was employed to monitor serial tumor growth. NK cell function was examined in each mouse strain using standard 51 Chromium release assays.
Melanoma metastases growth is delayed and variable in scid, and NOD-scid mice. In contrast, NOD-scid β2mnull and NOD-scid IL2Rγnull mice show rapid tumor engraftment, although tumor growth is variable in NOD-scid β2mnull mice. NK cells were detected in all strains except NOD-scid IL2Rγnull, and in vitro activated scid, NOD-scid and NOD-scid β2mnull NK cells kill human melanoma lines and primary melanoma cells. Expression of human NKG2D ligands MHC class I chain-related A and B molecules renders melanoma susceptible to murine NK cell-mediated cytotoxicity and killing is inhibited by antibody blockade of murine NKG2D.
Murine NKG2D recognition of MICA/B is an important receptor-ligand interaction employed by NK cells in immune-deficient strains to limit engraftment of human tumors. The absolute NK deficiency in NOD-scid IL2Rγnull animals makes this strain an excellent recipient of melanoma and potentially other human malignancies.
PMCID: PMC2697956  PMID: 19447870
11.  Suppression of HLA Expression by Lentivirus-mediated Gene Transfer of siRNA Cassettes and In Vivo Chemoselection to Enhance Hematopoietic Stem Cell Transplantation 
Immunologic research  2009;44(1-3):112-126.
Current approaches for hematopoietic stem cell (HSC) and organ transplantation are limited by donor and host-mediated immune responses to allo-antigens. Application of these therapies is limited by the toxicity of preparative and post-transplant immunosuppressive regimens and a shortage of appropriate HLA-matched donors. We have been exploring two complementary approaches for genetically modifying donor cells that achieve long-term suppression of cellular proteins that elicit host immune responses to mismatched donor antigens, and provide a selective advantage to genetically engineered donor cells after transplantation.
The first approach is based on recent advances that make feasible targeted down-regulation of HLA expression. Suppression of HLA expression could help to overcome limitations imposed by extensive HLA polymorphisms that restrict the availability of suitable donors. Accordingly, we have recently investigated whether knockdown of HLA by RNA interference (RNAi) enables allogeneic cells to evade immune recognition. For efficient and stable delivery of short hairpin-type RNAi constructs (shRNA), we employed lentivirus-based gene transfer vectors that integrate into genomic DNA, thereby permanently modifying transduced donor cells. Lentivirus-mediated delivery of shRNA targeting pan-Class I and allele-specific HLA achieved efficient and dose-dependent reduction in surface expression of HLA in human cells, and enhanced resistance to allo-reactive T lymphocyte-mediated cytotoxicity, while avoiding non-MHC restricted killing.
Complementary strategies for genetic engineering of HSC that would provide a selective advantage for transplanted donor cells and enable successful engraftment with less toxic preparative and immunosuppressive regimens would increase the numbers of individuals to whom HLA suppression therapy could be offered. Our second strategy is to provide a mechanism for in vivo selection of genetically modified HSC and other donor cells. We have uniquely combined transplantation during the neonatal period, when tolerance may be more readily achieved, with a positive selection strategy for in vivo amplification of drug-resistant donor HSC. This model system enables the evaluation of mechanisms of tolerance induction to neo-antigens, and allogeneic stem cells during immune ontogeny. HSC are transduced ex vivo by lentivirus-mediated gene transfer of P140K-O6-methylguanine-methyltransferase (MGMTP140K). The MGMTP140K DNA repair enzyme confers resistance to benzylguanine, an inhibitor of endogenous MGMT, and to chloroethylating agents such as BCNU. In vivo chemoselection enables enrichment of donor cells at the stem cell level. Using complementary approaches of in vivo chemoselection and RNAi-induced silencing of HLA expression may enable the generation of histocompatibility-enhanced, and eventually, perhaps “universally” compatible cellular grafts.
PMCID: PMC2938774  PMID: 19048410
HLA – Human Leukocyte Antigen; MHC – Major Histocompability Complex; shRNA - short hairpin-type RNAi; RNAi – RNA interference; MGMT - O6-methylguanine-DNA-methyltransferase; MAG - MGMTP140K-2A-GFP lentivirus vector; BU - Busulfan; BG - Benzylguanine; BCNU - 1,3-bis(2-chloroethyl1-nitrosourea); Lentivirus
12.  Cocultivation of umbilical cord blood CD34+ cells with retro-transduced hMSCs leads to effective amplification of long-term culture-initiating cells 
AIM: To establish a novel coculture system for ex vivo expansion of umbilical cord blood(UCB) hematopoietic progenitors using thrombopoietin (TPO)/Flt-3 ligand (FL)-transduced human marrow-derived mesenchymal stem cells (tfhMSCs) as feeder.
METHODS: UCB CD34+ cells were isolated and cultured using four culture systems in serum-containing or serum-free medium. Suitable aliquots of cultured cells were used to monitor cell production, clonogenic activity, and long-term culture-initiating culture (LTC-IC) output. Finally, the severe-combined immunodeficient (SCID) mouse-repopulating cell (SRC) assay was performed to confirm ability of the cultured cells to reconstitute long-term hematopoiesis.
RESULTS: There were no significant differences in the number of total nucleated cells among different culture systems in serum-containing medium during 21-d culture. However, on d 14, the outputs of CD34+ cells, CFU-C and CFU-GEMM in tfhMSCs coculture system were significantly enhanced. LTC-IC assay demonstrated that the tfhMSCs coculture system had the most powerful activity. The severe-combined immunodeficient (SCID) mouse repopulating cell (SRC) assay confirmed extensive ability of the expanded cells to reconstitute long-term hematopoiesis. Furthermore, PCR analysis demonstrated the presence of human hematopoietic cells in the bone marrow and peripheral blood cells of NOD/SCID mice.
CONCLUSION: The TPO/FL-transduced hMSCs, in combination with additive cytokines, can effectively expand hematopoietic progenitors from UCB in vitro and the tfhMSCs coculture system may be a suitable system for ex vivo manipulation of primitive progenitor cells under contact culture conditions.
PMCID: PMC4066057  PMID: 16489638
Mesenchymal stem cells; Thrombopoietin; Flt-3 ligand; Hematopoiesis
13.  Human Interleukin-15 Improves Engraftment of Human T Cells in NOD-SCID Mice 
Clinical and Vaccine Immunology  2006;13(2):227-234.
Human nonobese diabetic-severe combined immune deficiency (NOD-SCID) mouse chimeras have been widely used as an in vivo model to assess human immune function. However, only a small fraction of transferred human T lymphocytes can be detected in human peripheral blood lymphocyte (huPBL)-NOD-SCID chimeras. To improve the reconstitution of human T lymphocytes in NOD-SCID mice, the use of recombinant human interleukin-15 (rhIL-15) as a stimulator of human lymphocytes was explored. Administration of rhIL-15 after transplantation of huPBLs into NOD-SCID mice increased reconstitution of human T lymphocytes in a dose-dependent manner, with an optimal dosage of 1 μg/mouse. The number of human T lymphocytes (HLA-ABC+ CD3+) in the lymphoid organs or tissue of rhIL-15-treated huPBL-NOD-SCID mice increased 11- to 80-fold, and phytohemagglutinin-induced T-lymphocyte proliferation and cytokine production were significantly enhanced. Additionally, although mature human cells have not been thought to enter the murine thymus, human T lymphocytes were detected in the huPBL-NOD-SCID thymus after rhIL-15 treatment. Thus, rhIL-15 can be used to optimize long-term peripheral T-cell engraftment in these human-mouse chimeras and may also be useful in clinical treatment of T-cell deficiencies.
PMCID: PMC1391933  PMID: 16467330
14.  Knockdown of HPRT for Selection of Genetically Modified Human Hematopoietic Progenitor Cells 
PLoS ONE  2013;8(3):e59594.
The inability to obtain sufficient numbers of transduced cells remains a limitation in gene therapy. One strategy to address this limitation is in vivo pharmacologic selection of transduced cells. We have previously shown that knockdown of HPRT using lentiviral delivered shRNA facilitates efficient selection of transduced murine hematopoietic progenitor cells (HPC) using 6-thioguanine (6TG). Herein, we now extend these studies to human HPC. We tested multiple shRNA constructs in human derived cell lines and identified the optimal shRNA sequence for knockdown of HPRT and 6TG resistance. We then tested this vector in human umbilical cord blood derived HPC in vitro and in NOD/SCID recipients. Knockdown of HPRT effectively provided resistance to 6TG in vitro. 6TG treatment of mice resulted in increased percentages of transduced human CD45+ cells in the peripheral blood and in the spleen in particular, in both myeloid and lymphoid compartments. 6TG treatment of secondary recipients resulted in higher percentages of transduced human cells in the bone marrow, confirming selection from the progeny of long-term repopulating HPCs. However, the extent of selection of cells in the bone marrow at the doses of 6TG tested and the toxicity of higher doses, suggest that this strategy may be limited to selection of more committed progenitor cells. Together, these data suggest that human HPC can be programmed to be resistant to purine analogs, but that HPRT knockdown/6TG-based selection may not be robust enough for in vivo selection.
PMCID: PMC3598703  PMID: 23555045
15.  Isoform-Specific Potentiation of Stem and Progenitor Cell Engraftment by AML1/RUNX1  
PLoS Medicine  2007;4(5):e172.
AML1/RUNX1 is the most frequently mutated gene in leukaemia and is central to the normal biology of hematopoietic stem and progenitor cells. However, the role of different AML1 isoforms within these primitive compartments is unclear. Here we investigate whether altering relative expression of AML1 isoforms impacts the balance between cell self-renewal and differentiation in vitro and in vivo.
Methods and Findings
The human AML1a isoform encodes a truncated molecule with DNA-binding but no transactivation capacity. We used a retrovirus-based approach to transduce AML1a into primitive haematopoietic cells isolated from the mouse. We observed that enforced AML1a expression increased the competitive engraftment potential of murine long-term reconstituting stem cells with the proportion of AML1a-expressing cells increasing over time in both primary and secondary recipients. Furthermore, AML1a expression dramatically increased primitive and committed progenitor activity in engrafted animals as assessed by long-term culture, cobblestone formation, and colony assays. In contrast, expression of the full-length isoform AML1b abrogated engraftment potential. In vitro, AML1b promoted differentiation while AML1a promoted proliferation of progenitors capable of short-term lymphomyeloid engraftment. Consistent with these findings, the relative abundance of AML1a was highest in the primitive stem/progenitor compartment of human cord blood, and forced expression of AML1a in these cells enhanced maintenance of primitive potential both in vitro and in vivo.
These data demonstrate that the “a” isoform of AML1 has the capacity to potentiate stem and progenitor cell engraftment, both of which are required for successful clinical transplantation. This activity is consistent with its expression pattern in both normal and leukaemic cells. Manipulating the balance of AML1 isoform expression may offer novel therapeutic strategies, exploitable in the contexts of leukaemia and also in cord blood transplantation in adults, in whom stem and progenitor cell numbers are often limiting.
The truncated "a" isoform of AML1 is shown to have the capacity to potentiate stem and progenitor cell engraftment, both of which are required for successful clinical transplantation.
Editors' Summary
Blood contains red blood cells (which carry oxygen round the body), platelets (which help the blood to clot), and white blood cells (which fight off infections). All these cells, which are regularly replaced, are derived from hematopoietic stem cells, blood-forming cells present in the bone marrow. Like all stem cells, hematopoietic stem cells self-renew (reproduce themselves) and produce committed progenitor cells, which develop into mature blood cells in a process called hematopoiesis. Many proteins control hematopoiesis, some of which are called transcription factors; these factors bind to DNA through their DNA-binding domain and then control the expression of genes (that is, how DNA is turned into proteins) through particular parts of the protein (their transcription regulatory domains). An important hematopoietic transcription factor is AML1—a protein first identified because of its involvement in acute myelogenous leukemia (AML, a form of blood cancer). Mutations (changes) in the AML1 gene are now known to be present in other types of leukemia, which are often characterized by overproliferation of immature blood cells.
Why Was This Study Done?
Because of AML1′s crucial role in hematopoiesis, knowing more about which genes it regulates and how its activity is regulated could provide clues to treating leukemia and to improving hematopoietic cell transplantation. Many cancer treatments destroy hematopoietic stem cells, leaving patients vulnerable to infection. Transplants of bone marrow or cord blood (the cord that links mother and baby during pregnancy contains peripheral blood stem cells) can replace the missing cells, but cord blood in particular often contains insufficient stem cells for successful transplantation. It would be useful, therefore, to expand the stem cell content of these tissues before transplantation. In this study, the researchers investigated the effect of AML1 on self-renewal and differentiation of hematopoietic stem and progenitor cells in the laboratory (in vitro) and in animals (in vivo). In particular, they have asked how two isoforms (closely related versions) of AML1 affect the ability of these cells to grow and differentiate (engraft) in mice after transplantation.
What Did the Researchers Do and Find?
The researchers artificially expressed AML1a and AML1b (both isoforms contain a DNA binding domain, but only AML1b has transcription regulatory domains) in mouse hematopoietic stem and progenitor cells and then tested the cells' ability to engraft in mice. AML1a-expressing cells engrafted better than unaltered cells and outgrew unaltered cells when transplanted as a mixture. AML1b-expressing cells, however, did not engraft. In vitro, AML1a-expressing cells grew more than AML1b-expressing cells, whereas differentiation was promoted in AML1b-expressing cells. To investigate whether the isoforms have the same effects in human cells, the researchers measured the amount of AML1a and AML1b mRNA (the template for protein production) made by progenitor cells in human cord blood. Although AML1b (together with AML1c, an isoform with similar characteristics) mRNA predominated in all the progenitor cell types, the relative abundance of AML1a was greatest in the stem and progenitor cells. Furthermore, forced expression of AML1a in these cells improved their ability to divide in vitro and to engraft in mice.
What Do These Findings Mean?
These findings indicate that AML1a expression increases the self-renewal capacity of hematopoietic stem and progenitor cells and consequently improves their ability to engraft in mice, whereas AML1b expression encourages the differentiation of these cell types. These activities are consistent with the expression patterns of the two isoforms in normal hematopoietic cells and in leukemic cells—the mutated AML made by many leukemic cells resembles AML1a. Because the AML1 isoforms were expressed at higher than normal levels in these experiments, the physiological relevance of these findings needs to be confirmed by showing that normal levels of AML1a and AML1b produce similar results. Nevertheless, these results suggest that manipulating the balance of AML1 isoforms made by hematopoietic cells might be useful clinically. In leukemia, a shift toward AML1b expression might slow the proliferation of leukemic cells and encourage their differentiation. Conversely, in cord blood transplantation, a shift toward AML1a expression might improve patient outcomes by expanding the stem and progenitor cell populations.
Additional Information.
Please access these Web sites via the online version of this summary at
Wikipedia has pages on hematopoiesis and hematopoietic stem cells (note: Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
The US National Cancer Institute has a fact sheet on bone marrow and peripheral blood stem cell transplantation (in English and Spanish) and information for patients and professionals on leukemia (in English)
The American Society of Hematology provides patient information about blood diseases, including information on bone marrow and stem cell transplantation
PMCID: PMC1868041  PMID: 17503961
16.  Mesenchymal Stem Cells Secreting Angiopoietin-Like-5 Support Efficient Expansion of Human Hematopoietic Stem Cells Without Compromising Their Repopulating Potential 
Stem Cells and Development  2010;20(8):1371-1381.
Clinical and preclinical applications of human hematopoietic stem cells (HSCs) are often limited by scarcity of cells. Expanding human HSCs to increase their numbers while maintaining their stem cell properties has therefore become an important area of research. Here, we report a robust HSC coculture system wherein cord blood CD34+ CD133+ cells were cocultured with mesenchymal stem cells engineered to express angiopoietin-like-5 in a defined medium. After 11 days of culture, SCID repopulating cells were expanded ∼60-fold by limiting dilution assay in NOD-scid Il2rg−/− (NSG) mice. The cultured CD34+ CD133+ cells had similar engraftment potential to uncultured CD34+ CD133+ cells in competitive repopulation assays and were capable of efficient secondary reconstitution. Further, the expanded cells supported a robust multilineage reconstitution of human blood cells in NSG recipient mice, including a more efficient T-cell reconstitution. These results demonstrate that the expanded CD34+ CD133+ cells maintain both short-term and long-term HSC activities. To our knowledge, this ∼60-fold expansion of SCID repopulating cells is the best expansion of human HSCs reported to date. Further development of this coculture method for expanding human HSCs for clinical and preclinical applications is therefore warranted.
PMCID: PMC3148832  PMID: 21142526
17.  Expansion of human NOD/SCID-repopulating cells by stem cell factor, Flk2/Flt3 ligand, thrombopoietin, IL-6, and soluble IL-6 receptor 
Journal of Clinical Investigation  2000;105(7):1013-1021.
Here, we demonstrate a significant ex vivo expansion of human hematopoietic stem cells capable of repopulating in NOD/SCID mice. Using a combination of stem cell factor (SCF), Flk2/Flt3 ligand (FL), thrombopoietin (TPO), and a complex of IL-6 and soluble IL-6 receptor (IL-6/sIL-6R), we cultured cord blood CD34+ cells for 7 days and transplanted these cells into NOD/SCID mice. Bone marrow engraftment was judged successful when recipient animals contained measurable numbers of human CD45+ cells 10–12 weeks after transplantation. When cells were cultured with SCF+FL+TPO+IL-6/sIL-6R, 13 of 16 recipients were successfully engrafted, and CD45+ cells represented 11.5% of bone marrow cells in engrafted recipients. Cells cultured with a subset of these factors were less efficiently engrafted, both as measured by frequency of successful transplantations and prevalence of CD45+ cells. In animals receiving cells cultured with all 4 factors, human CD45+ cells represented various lineages, including a large number of CD34+ cells. The proportion of CD45+ cells in recipient marrow was 10 times higher in animals receiving these cultured cells than in those receiving comparable numbers of fresh CD34+ cells, and the expansion rate was estimated at 4.2-fold by a limiting dilution method. Addition of IL-3 to the cytokine combination abrogated the repopulating ability of the expanded cells. The present study may provide a novel culture method for the expansion of human transplantable hematopoietic stem cells suitable for clinical applications.
PMCID: PMC377479  PMID: 10749580
18.  Human CD34+ CD133+ Hematopoietic Stem Cells Cultured with Growth Factors Including Angptl5 Efficiently Engraft Adult NOD-SCID Il2rγ−/− (NSG) Mice 
PLoS ONE  2011;6(4):e18382.
Increasing demand for human hematopoietic stem cells (HSCs) in clinical and research applications necessitates expansion of HSCs in vitro. Before these cells can be used they must be carefully evaluated to assess their stem cell activity. Here, we expanded cord blood CD34+ CD133+ cells in a defined medium containing angiopoietin like 5 and insulin-like growth factor binding protein 2 and evaluated the cells for stem cell activity in NOD-SCID Il2rg−/− (NSG) mice by multi-lineage engraftment, long term reconstitution, limiting dilution and serial reconstitution. The phenotype of expanded cells was characterized by flow cytometry during the course of expansion and following engraftment in mice. We show that the SCID repopulating activity resides in the CD34+ CD133+ fraction of expanded cells and that CD34+ CD133+ cell number correlates with SCID repopulating activity before and after culture. The expanded cells mediate long-term hematopoiesis and serial reconstitution in NSG mice. Furthermore, they efficiently reconstitute not only neonate but also adult NSG recipients, generating human blood cell populations similar to those reported in mice reconstituted with uncultured human HSCs. These findings suggest an expansion of long term HSCs in our culture and show that expression of CD34 and CD133 serves as a marker for HSC activity in human cord blood cell cultures. The ability to expand human HSCs in vitro should facilitate clinical use of HSCs and large-scale construction of humanized mice from the same donor for research applications.
PMCID: PMC3084708  PMID: 21559522
19.  Changes in the Proliferative Activity of Human Hematopoietic Stem Cells in NOD/SCID Mice and Enhancement of Their Transplantability after In Vivo Treatment with Cell Cycle Inhibitors 
The Journal of Experimental Medicine  2002;196(9):1141-1150.
Human hematopoietic tissue contains rare stem cells with multilineage reconstituting ability demonstrable in receptive xenogeneic hosts. We now show that within 3 wk nonobese diabetic severe combined immunodeficiency (NOD/SCID) mice transplanted with human fetal liver cells regenerate near maximum levels of daughter human hematopoietic stem cells (HSCs) able to repopulate secondary NOD/SCID mice. At this time, most of the human HSCs (and other primitive progenitors) are actively proliferating as shown by their sensitivity to treatments that kill cycling cells selectively (e.g., exposure to high specific-activity [3H]thymidine in vitro or 5-fluorouracil in vivo). Interestingly, the proliferating human HSCs were rapidly forced into quiescence by in vivo administration of stromal-derived factor-1 (SDF-1) and this was accompanied by a marked increase in the numbers of human HSCs detectable. A similar result was obtained when transforming growth factor-β was injected, consistent with a reversible change in HSCs engrafting potential linked to changes in their cell cycle status. By 12 wk after transplant, most of the human HSCs had already entered Go and treatment with SDF-1 had no effect on their engrafting activity. These findings point to the existence of novel mechanisms by which inhibitors of HSC cycling can regulate the engrafting ability of human HSCs executing self-renewal divisions in vivo.
PMCID: PMC2194104  PMID: 12417625
TGF-β; SDF-1; cell cycle; stem cells; engraftment
20.  Factors Affecting Human T Cell Engraftment, Trafficking and Associated Xenogeneic Graft-Versus-Host Disease in NOD/SCID beta2mnull Mice 
Experimental hematology  2007;35(12):1823-1838.
Graft-versus-host disease (GVHD) is the major cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation. Models of immunodeficient mice that consistently and efficiently reconstitute with xenoreactive human T cells would be a valuable tool for the in vivo study of GVHD, as well as other human immune responses.
Materials and Methods
We developed a consistent and sensitive model of human GVHD by retro-orbitally injecting purified human T cells into sublethally irradiated NOD/SCID-β2mnull recipients. In addition, we characterized for the first time the trafficking patterns and expansion profiles of xenoreactive human T cells in NOD/SCID-β2mnull recipients using in vivo bioluminescence imaging.
All NOD/SCID-β2mnull mice conditioned with 300 cGy of total body irradiation and injected with 1 × 107 human T cells exhibited human T cell engraftment, activation, and expansion, with infiltration of multiple target tissues and a subsequent greater than 20% loss of pretransplant body weight. Importantly, histological examination of the GVHD target tissues revealed changes consistent with human GVHD. Furthermore, we also showed by in vivo bioluminescence imaging that the development of lethal GVHD in the NOD/SCID-β2mnull recipients was dependent upon the initial retention and early expansion of human T cells in the retroorbital sinus cavity.
Our NOD/SCID-β2mnull mouse model provides a system to study the pathophysiology of acute GVHD induced by human T cells and aids in the development of more effective therapies for human GVHD.
PMCID: PMC2238776  PMID: 17764813
21.  Co-expression of MGMTP140K and α-l-iduronidase in primary hepatocytes from mucopolysaccharidosis type I mice enables efficient selection with metabolic correction 
The journal of gene medicine  2008;10(3):249-259.
Systemic in vivo gene therapy has resulted in widespread correction in animal models when treated at birth. However, limited improvement was observed in postnatally treated animals with mainly targeting to the liver and bone marrow. It has been shown that an O6-methylguanine-DNA-methyltransferase variant (MGMTP140K) mediated in vivo selection of transduced hematopoietic stem cells (HSC) in animals.
We investigated the feasibility of MGMTP140K-mediated selection in primary hepatocytes from a mouse model of mucopolysaccharidosis type I (MPS I) in vitro using lentiviral vectors.
We found that multiple cycles of O6-benzylguanine (BG)/1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) treatment at a dosage effective for ex vivo HSC selection led to a two-fold increase of MGMT-expressing primary hepatocytes under culture conditions with minimum cell expansion. This enrichment level was comparable to that obtained after selection at a hepatic maximal tolerated dose of BCNU. Similar levels of increase were observed regardless of initial transduction frequency, or the position of MGMT (upstream or downstream of internal ribosome entry site) in the vector constructs. In addition, we found that elongation factor 1α promoter was superior to the long-terminal repeat promoter from spleen focus-forming virus with regard to transgene expression in primary hepatocytes. Moreover, the levels of therapeutic transgene expression in transduced, enzyme-deficient hepatocytes directly correlated with the doses of BCNU, leading to metabolic correction in transduced hepatocytes and metabolic cross-correction in neighbouring non-transduced MPS I cells.
These results demonstrate that MGMTP140K expression confers successful protection/selection in primary hepatocytes, and provide ‘proof of concept’ to the prospect of MGMTP140K-mediated co-selection for hepatocytes and HSC using BG/BCNU treatment.
PMCID: PMC3987669  PMID: 18076130
ex-vivo selection; lentivirus vector; lysosomal storage diseases; metabolic cross correction; methylguanine-DNA-methyltransferase; primary hepatocytes
22.  Fucosyltransferase VII improves the function of selectin ligands on cord blood hematopoietic stem cells 
Glycobiology  2013;23(10):1184-1191.
Selectins and their carbohydrate ligands mediate the homing of hematopoietic stem/progenitor cells (HSPCs) to the bone marrow. We have previously shown that ex vivo fucosylation of selectin ligands on HSPCs by α1,3 fucosyltransferase VI (FUT6) leads to improved human cord blood (CB)-HSPC engraftment in non-obese diabetic (NOD)/severe combined immune deficient (SCID) mice. In the present study, we determined whether surface fucosylation with α1,3 fucosyltransferase VII (FUT7), which is primarily expressed by hematopoietic cells, improves the function of selectin ligands on CB-HSPCs in comparison with FUT6. A saturating amount of either FUT6 or FUT7, which generates comparable levels of expression of fucosylated epitopes on CB CD34+ cells, was used for these experiments. In vitro, FUT7-treated CB CD34+ cells exhibited greater binding to P- or E-selectin than that of FUT6-treated CB CD34+ cells under static or physiological flow conditions. In vivo, FUT7 treatment, like FUT6, improved the early engraftment of CB CD34+ cells in the bone marrow of sublethally irradiated NOD/SCID interleukin (IL)-2Rγnull (NSG) mice. FUT7 also exhibited marginally—yet statistically significant—increased engraftment at 4 and 6 weeks after transplantation. In addition, FUT7-treated CB CD34+ cells exhibited increased homing to the bone marrow of irradiated NSG mice relative to sham-treated cells. These data indicate that FUT7 is effective at improving the function of selectin ligands on CB-HSPCs in vitro and enhancing early engraftment of treated CB-HSPCs in the bone marrow of recipients.
PMCID: PMC3766281  PMID: 23899669
CD34+; cord blood hematopoietic stem cell transplantation; fucosyltransferase; NSG mice; selectin; selectin ligands
23.  Xenogeneic Graft-versus-Host-Disease in NOD-scid IL-2Rγnull Mice Display a T-Effector Memory Phenotype 
PLoS ONE  2012;7(8):e44219.
The occurrence of Graft-versus-Host Disease (GvHD) is a prevalent and potentially lethal complication that develops following hematopoietic stem cell transplantation. Humanized mouse models of xenogeneic-GvHD based upon immunodeficient strains injected with human peripheral blood mononuclear cells (PBMC; “Hu-PBMC mice”) are important tools to study human immune function in vivo. The recent introduction of targeted deletions at the interleukin-2 common gamma chain (IL-2Rγnull), notably the NOD-scid IL-2Rγnull (NSG) and BALB/c-Rag2null IL-2Rγnull (BRG) mice, has led to improved human cell engraftment. Despite their widespread use, a comprehensive characterisation of engraftment and GvHD development in the Hu-PBMC NSG and BRG models has never been performed in parallel. We compared engrafted human lymphocyte populations in the peripheral blood, spleens, lymph nodes and bone marrow of these mice. Kinetics of engraftment differed between the two strains, in particular a significantly faster expansion of the human CD45+ compartment and higher engraftment levels of CD3+ T-cells were observed in NSG mice, which may explain the faster rate of GvHD development in this model. The pathogenesis of human GvHD involves anti-host effector cell reactivity and cutaneous tissue infiltration. Despite this, the presence of T-cell subsets and tissue homing markers has only recently been characterised in the peripheral blood of patients and has never been properly defined in Hu-PBMC models of GvHD. Engrafted human cells in NSG mice shows a prevalence of tissue homing cells with a T-effector memory (TEM) phenotype and high levels of cutaneous lymphocyte antigen (CLA) expression. Characterization of Hu-PBMC mice provides a strong preclinical platform for the application of novel immunotherapies targeting TEM-cell driven GvHD.
PMCID: PMC3429415  PMID: 22937164
24.  Superior Human Leukocyte Reconstitution and Susceptibility to Vaginal HIV Transmission in Humanized NOD-scid IL-2R −/− (NSG) BLT Mice 
Virology  2011;417(1):154-160.
Humanized Bone marrow/Liver/Thymus (BLT) mice recapitulate the mucosal transmission of HIV, permitting study of early events in HIV pathogenesis and evaluation of preexposure prophylaxis methods to inhibit HIV transmission. Human hematopoiesis is reconstituted in NOD-scid mice by implantation of human fetal liver and thymus tissue to generate human T cells plus intravenous injection of autologous liver-derived CD34+ hematopoietic stem cells to engraft the mouse bone marrow. In side-by-side comparisons, we show that NOD-scid mice homozygous for a deletion of the IL-2Rγ-chain (NOD-scid IL-2Rγ−/−) are far superior to NOD-scid mice in both their peripheral blood reconstitution with multiple classes of human leukocytes (e.g., a mean of 182 versus 14 CD4+ T cells per μl 12 weeks after CD34+ injection) and their susceptibility to intravaginal HIV exposure (84% versus 11% viremic mice at 4 weeks). These results should speed efforts to obtain preclinical animal efficacy data for new HIV drugs and microbicides.
PMCID: PMC3152643  PMID: 21684569
HIV/AIDS pathogenesis; HIV vaginal transmission; Humanized mice; HIV animal models; Hematopoietic stem cells; BLT mice; NOD-scid mice; NOD-scid IL-2Rγ−/− mice
25.  Transduction of Human CD34+ Repopulating Cells with a Self-Inactivating Lentiviral Vector for SCID-X1 Produced at Clinical Scale by a Stable Cell Line 
Human Gene Therapy Methods  2012;23(5):297-308.
Self-inactivating (SIN)-lentiviral vectors have safety and efficacy features that are well suited for transduction of hematopoietic stem cells (HSCs), but generation of vector at clinical scale has been challenging. Approximately 280 liters of an X-Linked Severe Combined Immunodeficiency Disorder (SCID-X1) SIN-lentiviral vector in two productions from a stable cell line were concentrated to final titers of 4.5 and 7.2×108 tu/ml. These two clinical preparations and three additional development-scale preparations were evaluated in human CD34+ hematopoietic cells in vitro using colony forming cell (CFU-C) assay and in vivo using the NOD/Lt-scid/IL2Rγnull (NSG) mouse xenotransplant model. A 40-hour transduction protocol using a single vector exposure conferred a mean NSG repopulating cell transduction of 0.23 vector genomes/human genome with a mean myeloid vector copy number of 3.2 vector genomes/human genome. No adverse effects on engraftment were observed from vector treatment. Direct comparison between our SIN-lentiviral vector using a 40-hour protocol and an MFGγc γ-retroviral vector using a five-day protocol demonstrated equivalent NSG repopulating cell transduction efficiency. Clonality survey by linear amplification-mediated polymerase chain reaction (LAM-PCR) with Illumina sequencing revealed common clones in sorted myeloid and lymphoid populations from engrafted mice demonstrating multipotent cell transduction. These vector preparations will be used in two clinical trials for SCID-X1.
Greene and colleagues report that they have successfully produced a self-inactivating (SIN) lentiviral vector at a large enough scale for two clinical trials for SCID-X1. They demonstrate that this vector has reproducible transduction efficiency both in vitro and in vivo and that no adverse effects on engraftment were observed in NOD/Lt-scid/IL2Rγnull (NSG) mice.
PMCID: PMC3732136  PMID: 23075105

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