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.
gene therapy; hematopoietic stem cells; foamy virus vector; O6-methylguanine DNA methyltransferase (MGMT)
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.
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.
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.
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.
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.
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.
HIV/AIDS pathogenesis; HIV vaginal transmission; Humanized mice; HIV animal models; Hematopoietic stem cells; BLT mice; NOD-scid mice; NOD-scid IL-2Rγ−/− mice
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.
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.
Foamy virus (FV) vectors have shown great promise for hematopoietic stem cell (HSC) gene therapy. Their ability to efficiently deliver transgenes to multi-lineage long-term repopulating cells in large animal models suggests they will be effective for several human hematopoietic diseases. Here, we review FV vector studies in large animal models, including the use of FV vectors with the mutant O6-methylguanine-DNA methyltransferase, MGMTP140K to increase the number of genetically modified cells after transplantation. In these studies, FV vectors have mediated efficient gene transfer to polyclonal repopulating cells using short ex vivo transduction protocols designed to minimize the negative effects of ex vivo culture on stem cell engraftment. In this regard, FV vectors appear superior to gammaretroviral vectors, which require longer ex vivo culture to effect efficient transduction. FV vectors have also compared favorably with lentiviral vectors when directly compared in the dog model. FV vectors have corrected leukocyte adhesion deficiency and pyruvate kinase deficiency in the dog large animal model. FV vectors also appear safer than gammaretroviral vectors based on a reduced frequency of integrants near promoters and also near proto-oncogenes in canine repopulating cells. Together, these studies suggest that FV vectors should be highly effective for several human hematopoietic diseases, including those that will require relatively high percentages of gene-modified cells to achieve clinical benefit.
foamy virus vectors; gene therapy; hematopoietic stem cells; animal models
Transplantation of marrow-derived mesenchymal stem cells (MSCs), expanded by culture in addition to whole bone marrow, has been shown to enhance engraftment of human hematopoietic stem cells (HSCs). Our hypothesis was that there might be an optimum ratio range that could enhance engraftment. We examined the percent donor chimerism according to the ratio of HSCs to MSCs in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. We tested a series of ratios of co-transplanted CD34+-selected bone marrow cells, and marrow-derived MSCs into sublethally irradiated NOD/SCID mice. In all experiments, 1×105 bone marrow derived human CD34+ cells were administered to each mouse and human MSCs from different donors were infused concomitantly. We repeated the procedure three times and evaluated engraftment with flow cytometry four weeks after each transplantation. Serial ratios of HSCs to MSCs were 1:0, 1:1, 1:2 and 1:4, in the first experiment, 1:0, 1:1, 1:2, 1:4 and 1:8 in the second and 1:0, 1:1, 1:4, 1:8 and 1:16 in the third. Cotransplantation of HSCs and MSCs enhanced engraftment as the dose of MSCs increased. Our results suggest that the optimal ratio of HSCs and MSCs for cotransplantation might be in the range of 1:8-1:16; whereas, an excessive dose of MSCs might decrease engraftment efficiency.
Hematopoietic Stem Cells; Mesenchymal Stem Cells; Transplantation; Mice, SCID; Engraftment
Xenotransplantation of human acute myeloid leukemia (AML) in immunocompromised animals has been critical for defining leukemic stem cells. However, existing immunodeficient strains of mice have short life spans and low levels of AML cell engraftment, hindering long-term evaluation of primary human AML biology. A recent study suggested that NOD/LtSz-scid IL2Rγc null (NSG) mice have enhanced AML cell engraftment, but this relied on technically challenging neonatal injections. Here, we performed extensive analysis of AML engraftment in adult NSG mice using tail vein injection. Of the 35 AML samples analyzed, 66% showed bone marrow engraftment over 0.1%. Further, 37% showed high levels of engraftment (>10%), with some as high as 95%. A 2–44-fold expansion of AML cells was often seen. Secondary and tertiary recipients showed consistent engraftment, with most showing further AML cell expansion. Engraftment did not correlate with French–American–British subtype or cytogenetic abnormalities. However, samples with FLT3 mutations showed a higher probability of engraftment than FLT3 wild type. Importantly, animals developed organomegaly and a wasting illness consistent with advanced leukemia. We conclude that the NSG xenotransplantation model is a robust model for human AML cell engraftment, which will allow better characterization of AML biology and testing of new therapies.
AML; NOD/SCID/gamma chain deficient; xenotransplantation
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.
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.
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.
Human cord blood (CB) is a potential source of hematopoietic stem cells (HSC) for gene therapy to treat patients with hematopoietic disorders. However, limited numbers of CB CD34+ cells, low transduction efficiency with lentiviral vectors (LVs), and low engraftment efficiency of NOD/SCID repopulating cells (SRC), a measure of HSC, are blocks to this procedure. To optimize culture and transduction conditions, we compared various lengths of time for pre-stimulation before transduction, transduction duration, and post-transduction cell culture.
Materials and methods
We used a lentiviral vector to transduce human cord blood CD34+ cells followed by engraftment into NOD/SCID mice. We evaluated the effects of pre-stimulation and transduction time, and optimized the ex vivo cell culture duration before transplantation.
We were able to achieve up to 40% transduction efficiency and up to 50% engraftment efficiency of SRC in CB CD34+ cells when CD34+ CB cells were either not pre-stimulated or pre-stimulated in 1% FBS medium for 1 hr, followed by 5 hr transduction and 3 days culture in a cocktail of growth factors after transduction. No apparent functional changes of CB CD34+ cells were noted under these conditions.
This gene transduction/cell expansion protocol is the first systematic study to optimize pre-stimulation time, transduction time and very importantly, ex vivo culture time after transduction, and may be of use for LV gene transduction in a gene therapy setting.
In the present study, a novel adeno-associated virus (AAV) vector-mediated gene delivery approach was taken to improve the reconstitution of functional CD8+ T cells in humanized mice, thereby mimicking the human immune system (HIS). Human genes encoding HLA-A2 and selected human cytokines (A2/hucytokines) were introduced to an immune-deficient mouse model [NOD/SCID/IL2rγnull (NSG) mice] using AAV serotype 9 (AAV9) vectors, followed by transplantation of human hematopoietic stem cells. NSG mice transduced with AAV9 encoding A2/hucytokines resulted in higher levels of reconstitution of human CD45+ cells compared to NSG mice transduced with AAV9 encoding HLA-A2 alone or HLA-A2-transgenic NSG mice. Furthermore, this group of HIS mice also mounted the highest level of antigen-specific A2-restricted human CD8+ T-cell response upon vaccination with recombinant adenoviruses expressing human malaria and HIV antigens. Finally, the human CD8+ T-cell response induced in human malaria vaccine-immunized HIS mice was shown to be functional by displaying cytotoxic activity against hepatocytes that express the human malaria antigen in the context of A2 molecules. Taken together, our data show that AAV vector-mediated gene delivery is a simple and efficient method to transfer multiple human genes to immune-deficient mice, thus facilitating successful reconstitution of HIS in mice. The HIS mice generated in this study should ultimately allow us to swiftly evaluate the T-cell immunogenicity of various human vaccine candidates in a pre-clinical setting.
Transplantation of human skin on immunodeficient mice that support engraftment with functional human immune systems would be an invaluable tool for investigating mechanisms involved in wound healing and transplantation. NOD-scid IL2rγnull (NSG) readily engraft with human immune systems but human skin graft integrity is poor. In contrast, human skin graft integrity is excellent on CB17-scid bg (SCID.bg) mice, but they engraft poorly with human immune systems.
Human skin grafts transplanted onto immunodeficient NSG, SCID.bg, and other immunodeficient strains were evaluated for graft integrity, preservation of graft endothelium and their ability to be rejected following engraftment of allogeneic peripheral blood mononuclear cells (PBMC).
Human skin transplanted onto NSG mice develops an inflammatory infiltrate, consisting predominately of host Gr1+ cells, that is detrimental to the survival of human endothelium in the graft. Treatment of graft recipients with anti-Gr1 antibody reduces this cellular infiltrate, preserves graft endothelium, and promotes wound healing, tissue development and graft remodeling. Excellent graft integrity of the transplanted skin includes multilayered stratified human epidermis, well developed human vasculature, human fibroblasts and passenger leukocytes. Injection of unfractionated, CD4 or CD8 allogeneic human PBMC induces a rapid destruction of the transplanted skin graft.
NSG mice treated with anti-Gr1 antibody provide a model optimized for both human skin graft integrity and engraftment of a functional human immune system. This model provides the opportunity to investigate mechanisms orchestrating inflammation, wound healing, revascularization, tissue remodeling, and allograft rejection and can provide guidance for improving outcomes following clinical transplantation.
SCID; IL2rg; skin transplantation; humanized mice; NOD/SCID; NSG
Leukemia initiating cells (LICs) have been the subject of considerable investigation because of their ability to self-renew and maintain leukemia. Thus, selective targeting and killing of LIC would provide highly efficient and novel therapeutic strategies. Here we explored whether we could use a canine leukemia cell line (G374) derived from a dog that received HOXB4 transduced repopulating cells to study leukemia in the murine xenograft model and the dog.
Materials and Methods
G374 cells were infused in dogs intravenously (IV) and in NOD/SCID and NOD/SCID/IL2Rγnull mice either IV or directly into the bone cavity (IF). Animals were bled to track engraftment and proliferation of G374 cells, and were sacrificed when they appeared ill.
We found that canine LICs are capable of sustained in vitro self-renewal while maintaining their ability to induce AML that resembles human disease in both dogs and mice. Furthermore, we developed a novel strategy for the quantification of LIC frequency in large animals and showed that this frequency was highly comparable to that determined by limited dilution in mouse xenotransplants. We also demonstrated, using single cell analysis, that LICs are heterogenous in their self-renewal capacity and regenerate a leukemic cell population consistent with a hierarchical leukemia model.
The availability of this novel framework should accelerate the characterization of LICs and the translation of animal studies into clinical trials.
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.
TGF-β; SDF-1; cell cycle; stem cells; engraftment
Regulatory T cells are essential to maintain immune homeostasis and prevent autoimmunity. Therapy with in vitro expanded human nTRegs is being tested to prevent graft versus host disease, which is a major cause for morbidity and mortality associated with hematopoietic stem cell transplantation. Their usefulness in therapy will depend on their capacity to survive, migrate appropriately and retain suppressive activity when introduced into a transplant recipient. The lack of a suitable animal model for studying the in vivo reconstitutive capability of human nTRegs is a major impediment for investigating the behavior of adoptively transferred nTRegs
in vivo. We show that injection of a plasmid encoding human IL-2 is necessary and sufficient for long term engraftment of in vitro expanded nTRegs in NOD-SCID IL2rγcnull mice. We also demonstrate that these in vivo reconstituted TRegs traffic to different organs of the body and retain suppressive function. Finally, in an IL-2 accelerated GVHD model, we show that these in vivo reconstituted TRegs are capable of preventing severe xenogenic response of human PBMCs. Thus, this novel ‘hu-TReg mouse’ model offers a pre-clinical platform to study the in vivo function and stability of human nTRegs and their ability to modulate autoimmune diseases and GVHD.
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.
The major targets of HIV infection in humans are CD4+ T cells. CD4+ T cell depletion is a hallmark of AIDS. Previously, the SCID-hu thy/liv model was used to study the effect of HIV on thymopoeisis in vivo. However, these mice did not develop high levels of peripheral T cell reconstitution and required invasive surgery for infection and analysis. Here, we describe a novel variant of this model in which thy/liv implantation results in systemic reconstitution with human T cells in the absence of any other human hematopoietic lineages.
NOD/SCID-hu thy/liv and NSG-hu thy/liv mice were created by implanting human fetal thymus and liver tissues under the kidney capsule of either NOD/SCID or NSG mice. In contrast to NOD/SCID-hu thy/liv mice that show little or no human cells in peripheral blood or tissues, substantial systemic human reconstitution occurs in NSG-hu thy/liv. These mice are exclusively reconstituted with human T cells (i.e. T-cell only mice or TOM). Despite substantial levels of human T cells no signs of graft-versus-host disease (GVHD) were noted in these mice over a period of 14 months. TOM are readily infected after parenteral exposure to HIV-1. HIV replication is sustained in peripheral blood at high levels and results in modest reduction of CD4+ T cells. HIV-1 replication in TOM responds to daily administration of combination antiretroviral therapy (ART) resulting in strong suppression of virus replication as determined by undetectable viral load in plasma. Latently HIV infected resting CD4+ T cells can be isolated from suppressed mice that can be induced to express HIV ex-vivo upon activation demonstrating the establishment of latency in vivo.
NSG-hu thy/liv mice are systemically reconstituted with human T cells. No other human lymphoid lineages are present in these mice (i.e. monocytes/macrophages, B cells and DC are all absent). These T cell only mice do not develop GVHD, are susceptible to HIV-1 infection and can efficiently maintain virus replication. HIV infected TOM undergoing ART harbor latently infected, resting CD4+ T cells.
HSC transplantation using genetically modified autologous cells is a promising therapeutic strategy for various genetic diseases, cancer, and HIV. However, for many of these conditions, the current efficiency of gene transfer to HSCs is not sufficient for clinical use. The ability to increase the percentage of gene-modified cells following transplantation is critical to overcoming this obstacle. In vivo selection with mutant methylguanine methyltransferase (MGMTP140K) has been proposed to overcome low gene transfer efficiency to HSCs. Previous studies have shown efficient in vivo selection in mice and dogs but only transient selection in primates. Here, we report efficient and stable MGMTP140K-mediated multilineage selection in both macaque and baboon nonhuman primate models. Treatment consisting of both O6-benzylguanine (O6BG) and N,N′-bis(2-chloroethyl)-N-nitroso-urea (BCNU) stably increased the percentage of transgene-expressing cells from a range of initial levels of engrafted genetically modified cells, with the longest follow-up after drug treatment occurring over 2.2 years. Drug treatment was well tolerated, and selection occurred in myeloid, lymphoid, and erythroid cells as well as platelets. Retrovirus integration site analysis before and after drug treatments confirmed the presence of multiple clones. These nonhuman primate studies closely model a clinical setting and should have broad applications for HSC gene therapy targeting human diseases of malignant, genetic, and infectious nature, including HIV.
Currently used mouse models fail to fully reflect human immunity to tuberculosis (TB), which hampers progress in research and vaccine development. Bone marrow-liver-thymus (BLT) mice, generated by engrafting human fetal liver, thymus, and hematopoietic stem cells in severely immunodeficient NOD/SCID/IL-2Rγ-/- (NSG) mice, have shown potential to model human immunity to infection. We engrafted HLA-A2-positive fetal tissues into NSG mice transgenically expressing human leukocyte antigen (HLA)-A2.1 (NSG-A2) to generate NSG-A2-BLT mice and characterized their human immune response to Mycobacterium bovis bacillus Calmette-Guerin (BCG) infection to assess the utility of this model for investigating human TB.
NSG-A2-BLT mice were infected intravenously with BCG and the immune response of engrafted human immune cells was characterized. After ex vivo antigenic stimulation of splenocytes, interferon (IFN)-γ-producing cells were detected by ELISPOT from infected, but not uninfected NSG-A2-BLT mice. However, the levels of secreted IFN-γ, determined by ELISA, were not significantly elevated by antigenic stimulation. NSG-A2-BLT mice were susceptible to BCG infection as determined by higher lung bacillary load than the non-engrafted control NSG-A2 mice. BCG-infected NSG-A2-BLT mice developed lung lesions composed mostly of human macrophages and few human CD4+ or CD8+ T cells. The lesions did not resemble granulomas typical of human TB.
Engrafted human immune cells in NSG-A2-BLT mice showed partial function of innate and adaptive immune systems culminating in antigen-specific T cell responses to mycobacterial infection. The lack of protection was associated with low IFN-γ levels and limited numbers of T cells recruited to the lesions. The NSG-A2-BLT mouse is capable of mounting a human immune response to M. tuberculosis in vivo but a quantitatively and possibly qualitatively enhanced effector response will be needed to improve the utility of this model for TB research.
Animal model; BCG; Tuberculosis; BLT mice; NSG mice
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.
We recently reported that chronic myelogenous leukemia (CML) cells converted into myofibroblasts to create a microenvironment for proliferation of CML cells in vitro. To analyze a biological contribution of CML-derived myofibroblasts in vivo, we observed the characters of leukemic nonobese diabetes/severe combined immunodeficiency (NOD/SCID) mouse. Bone marrow nonadherent mononuclear cells as well as human CD45-positive cells obtained from CML patients were injected to the irradiated NOD/SCID mice. When the chimeric BCR-ABL transcript was demonstrated in blood, human CML cells were detected in NOD/SCID murine bone marrow. And CML-derived myofibroblasts composed with the bone marrow-stroma, which produced significant amounts of human vascular endothelial growth factor A. When the parental CML cells were cultured with myofibroblasts separated from CML cell-engrafted NOD/SCID murine bone marrow, CML cells proliferated significantly. These observations indicate that CML cells make an adequate microenvironment for their own proliferation in vivo.