Biological background data up to 11 weeks of age and tumorigenic susceptibility to xenotransplantation with HeLa cells were compared between severely immuno-deficient NOG and NSG mice. The body weight was lower in NOG mice than in NSG mice. Severe depletion of peripheral blood lymphocytes and lymphoid hypoplasia that are well-known characteristics of these mice were equally observed. No lymphoproliferative lesions developed in any mouse of either strain. The occurrence of ectopic exocrine gland and cyst was a common finding in the thymus of both strains. In addition, minimal spongiotic change was observed in the medulla oblongata and spinal cord in both strains, and its incidence in female NOG mice was a little higher than that in NSG mice. In the adrenal, subcapsular cell hyperplasia that is known as an age-related change in non-genetically modified mice developed earlier and its incidence was higher in NSG mice than in NOG mice. The development of female genital organs of NOG mice was slightly retarded in comparison with that of NSG mice. To evaluate tumorigenic susceptibility to xenotransplantation, female mice were implanted in the dorsal subcutis with 1×103 to 1×106 cells/head of HeLa cells, and were checked up to 16 weeks after implantation. As a result, there was no significant strain difference on tumor formation rate and tumor volume. In conclusion, the present study clearly demonstrated that NOG and NSG mice showed no distinct strain differences in either biological features or biological disadvantages.

Natural killer (NK) cell-deficient mice are useful models in biomedical research. NOD/SCID mice have been used as a model of this type in research. However, the actual status of NK cells in NOD/SCID mice and CB17/SCID mice in comparison with that in BALB/c mice has not been sufficiently evaluated.

Following bone fracture, the repair process starts with an inflammatory reaction at the fracture site. Fracture healing is disturbed when the initial inflammation is increased or prolonged, whereby, a balanced inflammatory response is anticipated to be crucial for fracture healing, because it may induce down-stream responses leading to tissue repair. However, the impact of the immune response on fracture healing remains poorly understood. Here, we investigated bone healing in NOD/scid-IL2Rγcnull mice, which exhibit severe defects in innate and adaptive immunity, by biomechanical testing, histomorphometry and micro-computed tomography. We demonstrated that NOD/scid-IL2Rγcnull mice exhibited normal skeletal anatomy and a mild bone phenotype with a slightly reduced bone mass in the trabecular compartment in comparison to immunocompetent Balb/c mice. Fracture healing was impaired in immunodeficient NOD/scid-IL2Rγcnull mice. Callus bone content was unaffected during the early healing stage, whereas it was significantly reduced during the later healing period. Concomitantly, the amount of cartilage was significantly increased, indicating delayed endochondral ossification, most likely due to the decreased osteoclast activity observed in cells isolated from NOD/scid-IL2Rγcnull mice. Our results suggest that--under aseptic, uncomplicated conditions--the immediate immune response after fracture is non-essential for the initiation of bone formation. However, an intact immune system in general is important for successful bone healing, because endochondral ossification is delayed in immunodeficient NOD/scid-IL2Rγcnull mice.

We have used homozygous albumin enhancer/promoter-driven urokinase-type plasminogen activator/severe combined immunodeficient (uPA/SCID) mice as hosts for chimeric mice with humanized livers. However, uPA/SCID mice show four disadvantages: the human hepatocytes (h-heps) replacement index in mouse liver is decreased due to deletion of uPA transgene by homologous recombination, kidney disorders are likely to develop, body size is small, and hemizygotes cannot be used as hosts as more frequent homologous recombination than homozygotes. To solve these disadvantages, we have established a novel host strain that has a transgene containing albumin promoter/enhancer and urokinase-type plasminogen activator cDNA and has a SCID background (cDNA-uPA/SCID). We applied the embryonic stem cell technique to simultaneously generate a number of transgenic lines, and found the line with the most appropriate levels of uPA expression-not detrimental but with a sufficiently damaged liver. We transplanted h-heps into homozygous and hemizygous cDNA-uPA/SCID mice via the spleen, and monitored their human albumin (h-alb) levels and body weight. Blood h-alb levels and body weight gradually increased in the hemizygous cDNA-uPA/SCID mice and were maintained until they were approximately 30 weeks old. By contrast, blood h-alb levels and body weight in uPA/SCID chimeric mice decreased from 16 weeks of age onwards. A similar decrease in body weight was observed in the homozygous cDNA-uPA/SCID genotype, but h-alb levels were maintained until they were approximately 30 weeks old. Microarray analyses revealed identical h-heps gene expression profiles in homozygous and hemizygous cDNA-uPA/SCID mice were identical to that observed in the uPA/SCID mice. Furthermore, like uPA/SCID chimeric mice, homozygous and hemizygous cDNA-uPA/SCID chimeric mice were successfully infected with hepatitis B virus and C virus. These results indicate that hemizygous cDNA-uPA/SCID mice may be novel and useful hosts for producing chimeric mice for use in future long-term studies, including hepatitis virus infection analysis or drug toxicity studies.

Generation of orthotopic xenograft mouse models of leukemia is important to understand the mechanisms of leukemogenesis, cancer progression, its cross talk with the bone marrow microenvironment, and for preclinical evaluation of drugs. In these models, following intravenous injection, leukemic cells home to the bone marrow and proliferate there before infiltrating other organs, such as spleen, liver, and the central nervous system. Moreover, such models have been shown to accurately recapitulate the human disease and correlate with patient response to therapy and prognosis. Thus, various immune-deficient mice strains have been used with or without recipient preconditioning to increase engraftment efficiency. Mice homozygous for the severe combined immune deficiency (SCID) mutation and with non-obese diabetic background (NOD/SCID) have been used in the majority of leukemia xenograft studies. Later, NOD/SCID mice deficient for interleukin 2 receptor gamma chain (IL2Rγ) gene called NSG mice became the model of choice for leukemia xenografts. However, engraftment of leukemia cells without irradiation preconditioning still remained a challenge. In this study, we used NSG mice with null alleles for major histocompatibility complex class I beta2-microglobulin (β2m) called NSG-B2m. This is a first report describing the 100% engraftment efficiency of pediatric leukemia cell lines and primary samples in NSG-B2m mice in the absence of host preconditioning by sublethal irradiation. We also show direct comparison of the engraftment efficiency and growth rate of pediatric acute leukemia cells in NSG-B2m and NOD/SCID mice, which showed 80-90% engraftment efficiency. Secondary and tertiary xenografts in NSG-B2m mice generated by injection of cells isolated from the spleens of leukemia-bearing mice also behaved similar to the primary patient sample. We have successfully engrafted 25 acute lymphoblastic leukemia (ALL) and 5 acute myeloid leukemia (AML) patient samples with distinct cytogenetic characteristics in NSG-B2m mice, with the purpose of generating pediatric ALL and AML xenografts for preclinical evaluation of drugs. Thus, our data support the use of NSG-B2m mouse model for leukemia engraftment and in vivo preclinical drug efficacy studies.

CD1 molecules are glycoproteins that present lipids and glycolipids for recognition by T cells. CD1-dependent immune activation has been implicated in a wide range of immune responses, however, our understanding of the role of this pathway in human disease remains limited because of species differences between humans and other mammals: whereas humans express five different CD1 gene products (CD1a, CD1b, CD1c, CD1d, and CD1e), muroid rodents express only one CD1 isoform (CD1d). Here we report that immune deficient mice engrafted with human fetal thymus, liver, and CD34(+) hematopoietic stem cells develop a functional human CD1 compartment. CD1a, b, c, and d isoforms were highly expressed by human thymocytes, and CD1a(+) cells with a dendritic morphology were present in the thymic medulla. CD1(+) cells were also detected in spleen, liver, and lungs. APCs from spleen and liver were capable of presenting bacterial glycolipids to human CD1-restricted T cells. ELISpot analyses of splenocytes demonstrated the presence of CD1-reactive IFN-γ producing cells. CD1d tetramer staining directly identified human iNKT cells in spleen and liver samples from engrafted mice, and injection of the glycolipid antigen α-GalCer resulted in rapid elevation of human IFN-γ and IL-4 levels in the blood indicating that the human iNKT cells are biologically active in vivo. Together, these results demonstrate that the human CD1 system is present and functionally competent in this humanized mouse model. Thus, this system provides a new opportunity to study the role of CD1-related immune activation in infections to human-specific pathogens.

In our previous study, lycorine, a natural alkaloid extracted from Amaryllidaceae, exhibited anti-leukemia effects in vitro. To determine whether lycorine has an anti-tumor effect in vivo, a series of experiments were carried out in this study. HL-60 cells (5 x 10(6)) were inoculated i.v. into severe combined immuno-deficiency (SCID) mice after these mice had been irradiated (total body receiving 200cGy chi irradiation). Treatment was given once a day from day 2 to 6, and from day 14 to 18. Lycorine (5 or 10 mg/kg/day i.p.) was found to decrease the percentages of immature granular leukocytes and of monocytes among the peripheral blood cells, and the mean survival time of both lycorine-treated groups was longer than that of the control group. Compared with the asynchronous and cytosine arabinoside- (Ara-C) treated (20 mg/kg/day i.p.) group, treatment with lycorine was more effective. Lycorine was also found to alleviate the infiltration of tumor cells into the liver, bone, and marrow. When SCID mice inoculated with HL-60 cells were then treated with lycorine, no severe adverse effects were observed. This study revealed that lycorine, when tested in the human leukemia xenograft models, appears to exhibit anti-tumor activity in vivo and is a useful therapy against acute promyelocytic leukemia.

Immune cells and stromal cells regulate wound healing and regeneration through complex activation patterns with spatiotemporal variation. The scarless regeneration of Spiny mice (Acomys species) is no exception; differential activation of immune and stromal cell populations seems to play a role in its remarkable regenerative capacity. To elucidate the role and interplay of Acomys immune cells in mammalian regeneration, we sought to create Acomys-Mus chimeras by transplanting bone marrow (BM) from Acomys into NOD Scid Gamma (NSG), a severely immunodeficient mouse line often used in creating humanized mice. Here, we report that Acomys BM cells fail to reconstitute and differentiate when transferred to irradiated NSG adults and neonates. In addition, we did not detect the presence of donor cells nor observe the onset of Graft versus Host Disease (GvHD)-like pathology, even after transplanting Acomys splenocytes in Acomys-Mus chimeras suggesting early graft failure. Overall, these results demonstrate the adoptive transfer of Acomys BM alone is not sufficient to establish Acomys hematopoietic system in NSG mouse.

We have investigated to develop novel vaccines against SARS CoV using cDNA constructs encoding the structural antigen; spike protein (S), membrane protein (M), envelope protein (E), or nucleocapsid (N) protein, derived from SARS CoV. Mice vaccinated with SARS-N or -M DNA using pcDNA 3.1(+) plasmid vector showed T cell immune responses (CTL induction and proliferation) against N or M protein, respectively. CTL responses were also detected to SARS DNA-transfected type II alveolar epithelial cells (T7 cell clone), which are thought to be initial target cells for SARS virus infection in human. To determine whether these DNA vaccines could induce T cell immune responses in humans as well as in mice, SCID-PBL/hu mice was immunized with these DNA vaccines. As expected, virus-specific CTL responses and T cell proliferation were induced from human T cells. SARS-N and SARS-M DNA vaccines and SCID-PBL/hu mouse model will be important in the development of protective vaccines.

Many studies have demonstrated that anti-dsDNA IgG is closely associated with lupus nephritis. Recently, it was found that activation of the fibroblast growth factor-inducible 14 (Fn14) signaling pathway damages glomerular filtration barrier in MRL/lpr lupus-prone mice. However, MRL/lpr mice have high titers of serum autoantibodies other than anti-dsDNA IgG. The aim of this study was to further explore the effect of Fn14 deficiency on anti-dsDNA IgG-induced glomerular damage in severe combined immunodeficiency (SCID) mice that have no endogenous IgG. Fn14 deficiency was generated in SCID mice. The murine hybridoma cells producing control IgG or anti-dsDNA IgG were intraperitoneally injected into mice. In two weeks, the urine, serum, and kidney tissue samples were harvested from mice at sacrifice. It showed that the injection of anti-dsDNA IgG, but not control IgG hybridoma cells, induced proteinuria and glomerular damage in SCID mice. Between the wild-type (WT) and knockout (KO) mice injected with anti-dsDNA IgG hybridoma cells, the latter showed a decrease in both proteinuria and glomerular IgG deposition. The histopathological changes, inflammatory cell infiltration, and proinflammatory cytokine production were also attenuated in the kidneys of the Fn14-KO mice upon anti-dsDNA IgG injection. Therefore, Fn14 deficiency effectively protects SCID mice from anti-dsDNA IgG-induced glomerular damage.

We compare for the first time, the metastatic aggressiveness of the parental MDA-MB-231 breast cancer cell line and two luciferase-tagged in vivo-derived and selected pro-metastatic variants (LM2-4/luc⁺ and 164/8-1B/luc⁺ in SCID, NOD-SCID and NOD-SCID-IL-2Rγ(null) (NSG) mice following orthotopic implantation and primary tumour resection. The variants are known to be more aggressively metastatic in SCID mice, compared to the parental line which has limited spontaneous metastatic competence in these mice. When 2×10⁶ cells were injected into the mammary fat pad, the growth of the resultant primary tumours was identical for the various cell lines in the three strains of mice. However, metastatic spread of all three cell lines, including the MDA-MB-231 parental cell line, was strikingly more aggressive in the highly immunocompromised NSG mice compared to both NOD-SCID and SCID mice, resulting in extensive multi-organ metastases and a significant reduction in overall survival. While these studies were facilitated by monitoring post-surgical spontaneous metastases using whole body bioluminescence imaging, we observed that the luciferase-tagged parental line showed altered growth and diminished metastatic properties compared to its untagged counterpart. Our results are the first to show that host immunity can have a profound impact on the spread of spontaneous visceral metastases and survival following resection of a primary tumour in circumstances where the growth of primary tumours is not similarly affected; as such they highlight the importance of immunity in the metastatic process, and by extension, suggest certain therapeutic strategies that may have a significant impact on reducing metastasis.

Recently we have established a new culture condition enabling the derivation of extended pluripotent stem (EPS) cells, which, compared to conventional pluripotent stem cells, possess superior developmental potential and germline competence. However, it remains unclear whether this condition permits derivation of EPS cells from mouse strains that are refractory or non-permissive to pluripotent cell establishment. Here, we show that EPS cells can be robustly generated from non-permissive NOD-scid Il2rg-/- mice through de novo derivation from blastocysts. Furthermore, these cells can also be efficiently generated by chemical reprogramming from embryonic NOD-scid Il2rg-/- fibroblasts. NOD-scid Il2rg-/- EPS cells can be expanded for more than 20 passages with genomic stability and can be genetically modified through gene targeting. Notably, these cells contribute to both embryonic and extraembryonic lineages in vivo. More importantly, they can produce chimeras and integrate into the E13.5 genital ridge. Our study demonstrates the feasibility of generating EPS cells from refractory mouse strains, which could potentially be a general strategy for deriving mouse pluripotent cells. The generation of NOD-scid Il2rg-/- EPS cell lines permits sophisticated genetic modification in NOD-scid Il2rg-/- mice, which may greatly advance the optimization of humanized mouse models for biomedical applications.

Previously, we found that rapid leukemia engraftment (short time to leukemia, TTL(short)) in the NOD/SCID/huALL (non-obese diabetic/severe combined immuno-deficiency/human acute lymphoblastic leukemia) xenograft model is indicative of early patient relapse. As earlier intact apoptosis sensitivity was predictive for good prognosis in patients, we investigated the importance of apoptosis signaling on NOD/SCID/huALL engraftment. Intact apoptosome function as reflected by cytochrome c-related activation of caspase-3 (CRAC-positivity) was strongly associated with prolonged NOD/SCID engraftment (long time to leukemia, TTL(long)) of primary leukemia cells, good treatment response and superior patient survival. Conversely, deficient apoptosome function (CRAC-negativity) was associated with rapid engraftment (TTL(short)) and early relapse. Moreover, an intact apoptosis signaling was associated with high transcript and protein levels of the pro-apoptotic death-associated protein kinase1 (DAPK1). Our data strongly emphasize the impact of intrinsic apoptosis sensitivity of ALL cells on the engraftment phenotype in the NOD/SCID/huALL model, and most importantly also on patient outcome.

Deficiency of adenosine deaminase (ADA, EC3.5.4.4), a housekeeping enzyme intrinsic to the purine salvage pathway, leads to severe combined immunodeficiency (SCID) both in humans and mice. Lack of ADA results in the intracellular accumulation of toxic metabolites which have effects on T cell development and function. While untreated ADA-SCID is a fatal disorder, there are different therapeutic options available to restore ADA activity and reconstitute a functioning immune system, including enzyme replacement therapy (ERT). Administration of ERT in the form of pegylated bovine ADA (PEG-ADA) has proved a life-saving though non-curative treatment for ADA-SCID patients. However, in many patients treated with PEG-ADA, there is suboptimal immune recovery with low T and B cell numbers. Here, we show reduced thymus cellularity in ADA-SCID mice despite weekly PEG-ADA treatment. This was associated with lack of effective adenosine (Ado) detoxification in the thymus. We also show that thymocyte development in ADA-deficient thymi is arrested at the DN3-to-DN4 stage transition with thymocytes undergoing dATP-induced apoptosis rather than defective TCRβ rearrangement or β-selection. Our studies demonstrate at a detailed level that exogenous once-a-week enzyme replacement does not fully correct intra-thymic metabolic or immunological abnormalities associated with ADA deficiency.

X-linked severe combined immunodeficiency (X-SCID) has been successfully treated by hematopoietic stem cell (HSC) transduction with retroviral vectors expressing the interleukin-2 receptor subunit gamma gene (IL2RG), but several patients developed malignancies due to vector integration near cellular oncogenes. This adverse side effect could in principle be avoided by accurate IL2RG gene editing with a vector that does not contain a functional promoter or IL2RG gene. Here, we show that adeno-associated virus (AAV) gene editing vectors can insert a partial Il2rg cDNA at the endogenous Il2rg locus in X-SCID murine bone marrow cells and that these ex vivo-edited cells repopulate transplant recipients and produce CD4+ and CD8+ T cells. Circulating, edited lymphocytes increased over time and appeared in secondary transplant recipients, demonstrating successful editing in long-term repopulating cells. Random vector integration events were nearly undetectable, and malignant transformation of the transplanted cells was not observed. Similar editing frequencies were observed in human hematopoietic cells. Our results demonstrate that therapeutically relevant HSC gene editing can be achieved by AAV vectors in the absence of site-specific nucleases and suggest that this may be a safe and effective therapy for hematopoietic diseases where in vivo selection can increase edited cell numbers.

Tissue engineering for cartilage regeneration provides an alternative to surgery for degenerative osteoarthritis. Recently, a highly organized three-dimensional (3D) alginate scaffold was prepared using a microfluidic device; this scaffold is effective for chondrocyte culture in vitro. The performance of this scaffold was further demonstrated; an alginate scaffold seeded with porcine chondrocytes was implanted in the dorsal subcutaneous site of SCID mice. The recipients were sacrificed at 2, 4, and 6 weeks after transplantation. The grafted implants retrieved from the subcutaneous site were analyzed with histologic examinations. Real-time PCR was used to identify the gene expression patterns of the chondrocytes. The hematoxylin and eosin staining showed that the chondrocytes survived normally in SCID mice; cartilage-like structures were formed after 4 weeks implantation. Immunohistochemical staining revealed cells secreted type II collagen, produced glycosaminoglycans (proved by alcian blue stain), and maintained the expression of S-100. On the other hand, the cells were negative for type I and type X collagen staining. PCR showed that the mRNA expressions of aggrecan and type II collagen were up-regulated at weeks two and four, while type I and type X collagen were down-regulated during the study period. In summary, this highly organized 3D alginate scaffold provided a suitable environment and maintained functional phenotypes for chondrocytes in this animal study.

Scid hematopoietic stem cells (HSCs) have an intrinsic defect in their maintenance within the bone marrow (BM) niche which facilitates HSC transplantation without the absolute requirement of prior conditioning. Nevertheless, NOD scid mice have a significantly altered life span due to early development of thymic lymphomas, which compromises the ability to study the long-term fate of exogenous HSCs and their progeny. Here, we present data on the transplantation of HSCs into NOD scid gamma (NSG) mice to achieve long-term engraftment without prior conditioning. We transplanted allogeneic HSCs constitutively expressing the mCherry fluorescent marker into age-matched NSG mice and assessed donor chimerism 6 months post-transplantation. All transplanted NSG mice showed long-term myeloid and lymphoid cell chimerism. Also, in vivo irradiated HSCs showed long-term engraftment, although overall white blood cell (WBC) donor chimerism was lower compared with non-irradiated HSCs. Using this novel NSG transplantation model, we will be able to study the effects of low dose in vivo X-ray exposure on the long-term fate of HSCs, without the requirement of prior radio-ablation of the recipient, and thus leaving the recipient's BM microenvironment uncompromised. In conclusion, we demonstrated for the first time that allogeneic HSCs from a different inbred strain can compete for niches in the BM compartment of NSG mice.

Yes-associated protein (YAP) is constitutively activated in numerous types of cancer, including gastric carcinoma. The aim of the present study was to investigate the effects of YAP silencing on proliferation, apoptosis, metastasis and angiogenesis in a gastric orthotopic implantation cancer model of severe combined immunodeficiency mice. Small-hairpin RNA (shRNA) targeting the YAP gene was employed to inhibit YAP expression. SGC7901 cells transfected with YAP shRNA demonstrated significantly decreased gastric cancer growth and metastasis in the orthotopic implantation mouse model. Silencing of YAP additionally promoted tumor cell apoptosis, and inhibited tumor cell proliferation and angiogenesis. Notably, YAP shRNA also downregulated the expression of TEA domain family member 1, cyclinD1, vascular endothelial growth factor and fibroblast growth factor-2. The results of the present study suggested that YAP may have a significant role in the proliferation, metastasis and angiogenesis of gastric cancer. RNA interference-mediated silencing of YAP may provide an opportunity to develop a novel treatment strategy for gastric cancer.

No lung cancer xenograft model using non-obese diabetic (NOD)-scid Il2rg (-/-) mice has been reported. The purpose of this study is to select a suitable mouse strain as a xenogenic host for testing tumorigenicity of lung cancer.

Ex vivo expanded erythroblasts (EBs) may serve as advanced transfusion products provided that lodgment occurs in the macrophage-niche of the marrow permitting maturation. EBs expanded from adult and cord blood expressed the receptors (CXCR4, VLA-4, and P-selectin ligand 1) necessary for interaction with macrophages. However, 4-days following transfusion to intact NOD/SCID/IL2Rγ(null) mice, CD235a(pos) EBs were observed inside CD235a(neg) splenic cells suggesting that they underwent phagocytosis. When splenectomized and intact NOD/SCID/IL2Rγ(null) mice were transfused using retrovirally labeled human EBs, human cells were visualized by bioluminescence imaging only in splenectomized animals. Four days after injection, human CD235a(pos) cells were detected in marrow and liver of splenectomized mice but only in spleen of controls. Human CD235a(pos) erythrocytes in blood remained low in all cases. These studies establish splenectomized NOD/SCID/IL2Rγ(null) mice as a suitable model for tracking and quantification of human EBs in vivo.