Transplantation Of Human CD34 Research Articles

Preclinical evaluation of the efficacy of an antibody to human SIRPα for cancer immunotherapy in humanized mouse models.

Tumor-associated macrophages (TAMs) are abundant in the tumor microenvironment and are considered potential targets for cancer immunotherapy. To examine the antitumor effects of agents targeting human TAMs in vivo, we here established preclinical tumor xenograft models based on immunodeficient mice that express multiple human cytokines and have been reconstituted with a human immune system by transplantation of human CD34+ hematopoietic stem and progenitor cells (HIS-MITRG mice). HIS-MITRG mice supported the growth of both human cell line (Raji)- and patient-derived B cell lymphoma as well as the infiltration of human macrophages into their tumors. We examined the potential antitumor action of an antibody to human SIRPα (SE12C3) that inhibits the interaction of CD47 on tumor cells with SIRPα on human macrophages and thereby promotes Fcγ receptor-mediated phagocytosis of the former cells by the latter. Treatment with the combination of rituximab (antibody to human CD20) and SE12C3 inhibited Raji tumor growth in HIS-MITRG mice to a markedly greater extent than did rituximab monotherapy. This enhanced antitumor effect was dependent on human macrophages and attributable to enhanced rituximab-dependent phagocytosis of lymphoma cells by human macrophages. Treatment with rituximab and SE12C3 also induced reprogramming of human TAMs toward a proinflammatory phenotype. Furthermore, the combination treatment essentially prevented the growth of patient-derived diffuse large B cell lymphoma in HIS-MITRG mice. Our findings thus support the study of HIS-MITRG mice as a model for the preclinical evaluation in vivo of potential therapeutics, such as antibodies to human SIRPα, that target human TAMs.

Preclinical Evaluation of the Efficacy of Human Sirpα Antibodies for B-Cell Lymphoma Immunotherapy in Humanized Mouse Models

Tumor-associated macrophages (TAMs) are abundant in the tumor microenvironment and are considered potential targets to enhance cancer immunotherapy against B-cell lymphomas. However, faithful and predictive preclinical tumor models of human B-cell lymphomas for the evaluation of therapeutics targeting human TAMs have not been established yet. To examine the antitumor effects of agents targeting human TAMs in vivo, we here established preclinical tumor xenograft models based on Rag2-/-Il2rg-/- immunodeficient mice that express multiple human cytokines, such as M-CSF, IL-3, GM-CSF, and THPO (MITRG mice), and have been reconstituted with a human immune system (HIS) by transplantation of human CD34+ hematopoietic stem and progenitor cells (HIS-MITRG mice). HIS-MITRG mice supported the growth of both the human cell line (Raji)-derived and diffuse large B cell lymphoma (DLBCL) patient-derived xenograft tumors as well as the infiltration of human macrophages into these tumors. We examined the potential antitumor effect of an antibody to human SIRPα (SE12C3) that inhibits the interaction of CD47 on tumor cells with SIRPα on human macrophages and thereby promotes Fcγ receptor-mediated phagocytosis of tumor cells by human macrophages. Combined treatment with rituximab and SE12C3 inhibited Raji tumor growth in HIS-MITRG mice to a markedly greater extent than did rituximab monotherapy in a therapeutic model started after the engraftment of the tumor. This enhanced antitumor effect was dependent on human macrophages and attributable to enhanced rituximab-dependent phagocytosis of lymphoma cells by human macrophages. Treatment with rituximab and SE12C3 also induced reprogramming of human TAMs towards a proinflammatory phenotype. Furthermore, the combination treatment essentially prevented the growth of DLBCL patient-derived xenograft tumors in HIS-MITRG mice. Our findings thus support the utility of HIS-MITRG mice as a model for the preclinical in vivo evaluation of potential therapeutics, such as antibodies to human SIRPα, that target human TAMs.

Posttransplant Hemophagocytic Lymphohistiocytosis Driven by Myeloid Cytokines and Vicious Cycles of T-Cell and Macrophage Activation in Humanized Mice.

Hemophagocytic lymphohistiocytosis (HLH) has recently been increasingly reported as an important complication after stem cell transplantation, in line with the increase in the number of HLA-mismatched transplantation. Although previous clinical studies have shown an elevation of inflammatory cytokines in patients with HLH after hematopoietic stem cell transplantation, as well as those after viral infection or autoimmune disease, the disease pathogenesis remains poorly understood. Here we explored this issue in humanized mice with functional human lymphohematopoietic systems, which were constructed by transplantation of human CD34+ cells alone, or along with human fetal thymus into NOD/SCID/γc−/− (NSG) or NSG mice carrying human SCF/GM-CSF/IL-3 transgenes (SGM3). In comparison with humanized NSG (huNSG) mice, huSGM3 mice had higher human myeloid reconstitution and aggressive expansion of human CD4+ memory T cells, particularly in the absence of human thymus. Although all huNSG mice appeared healthy throughout the observation period of over 20 weeks, huSGM3 mice developed fatal disease characterized by severe human T cell and macrophage infiltrations to systemic organs. HuSGM3 mice also showed severe anemia and thrombocytopenia with hypoplastic bone marrow, but increased reticulocyte counts in blood. In addition, huSGM3 mice showed a significant elevation in human inflammatory cytokines including IL-6, IL-18, IFN-α, and TNF-γ, faithfully reproducing HLH in clinical situations. Our study suggests that posttransplant HLH is triggered by alloresponses (or xenoresponses in our model), driven by myeloid cytokines, and exacerbated by vicious cycles of T-cell and macrophage activation.

Abstract 3569: Humanized mouse model of MAGE-A1-targeted anti-melanoma T cell therapy

Abstract Adoptive cellular therapy (ACT) has resulted in durable anti-tumor responses for both solid and hematological malignancies, mediated by the persistence and activation of transferred T cells. Unfortunately most patients, especially those with solid tumors, continue to progress despite persistent T cells. Thus revealing challenges specific to the hostile microenvironment of solid tumors and a critical need to identify factors that limit ACT efficacy. Furthermore, serial patient biopsies are challenging to obtain and accessible peripheral blood does not reflect relevant cell interactions at the tumor site. Xenograft models that lack infiltrating human macrophages, NK cells and lymphocytes cannot faithfully recapitulate critical interactions within the tumor microenvironment. To overcome this limitation, we initiated ACT in the MISTRG mouse that supports hematopoiesis after transplantation of human CD34+ stem cells resulting in functional human innate and adaptive immune cells. Co-transplantation of the melanoma cell line (Me275) results in tumor infiltration by human macrophages (TAMs) supporting tumor progression. The cancer/testis antigen Melanoma associated Antigen Gene (MAGE)-A1 is an attractive ACT target due to broad expression in tumors and limited expression in normal tissues rendering off-tumor tissue toxicities unlikely. We have identified a high-affinity HLA-A*02:01-restricted TCR that recognizes MAGE-A1278-286 peptide (TCRMA1) and confers effector functions in transduced CD8 and CD4 T cells when co-transduced with CD8αβ. Ex vivo analysis of MISTRG T cell cytolysis and proliferation suggest that CD4+ T cell help is required in this model. After two weeks of in vitro expansion, CD4+ and CD8+ T cells from humanized MISTRG spleens were transduced and transferred into MISTRG littermates harboring MAGE-A1+ Me275. Although TCRMA1 treated mice subtly controlled the growth of Me275, the changes were not significant. However, compared to an irrelevant TCR, only TCRMA1 CD8+ T cells localized to tumor and persisted in blood and spleen 8 and 28 days post transfer. TCRMA1 CD8 T cells displayed an effector phenotype, with high levels of PD-1 expression characteristic of recently activated and/or exhausted T cells. These results suggest that TCRMA1 cells recognized the tumor and were consequently activated, but could not overcome suppression in the microenvironment, mirroring human studies. Additional studies are being initiated to overcome the specific factors associated with the TAMs that limit in vivo T cell function. This model provides an ideal environment to test next generation T cell therapies, such as addition of intrinsic co-stimulation, to enhance responses against solid tumors. Humanized MISTRG mice are a powerful tool to study suppression of ACT in vivo, offering streamlined approaches to overcome hostile tumor microenvironments before clinical translation. Citation Format: Megan S. McAfee, Trisha Sippel, Daniel Hunter, Jean Campbell, Thomas Schmitt, Robert Pierce, Anthony Rongvaux, Aude Chapuis. Humanized mouse model of MAGE-A1-targeted anti-melanoma T cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3569.

Generation of Humanized Mice for Analysis of Human Dendritic Cells.

Transplantation of human CD34(+) hematopoietic stem and progenitor cells into severe immunocompromised newborn mice allows the development of a human hemato-lymphoid system (HHLS) including dendritic cells (DCs) in vivo. Therefore, it can be a powerful tool to study human DC subsets, residing in different lymphoid and nonlymphoid organs. We have recently generated novel mouse strains called human cytokine knock-in mice in which human versions of several cytokines are knocked into Rag2(-/-)γC(-/-) strains. In addition, human SIRPα, which is a critical factor to prevent donor cell to be eliminated by host macrophages, is expressed as transgene. These mice efficiently support human myeloid cell development and, indeed, allow the analysis of three major subsets of human DC lineages, plasmacytoid DCs and CD1c(+) and CD141(+) classical DCs. Moreover, these strains also support cytokine-mobilized peripheral blood CD34(+) cell engraftment and subsequent DC development. Here we describe our standard methods to characterize DCs developed in human cytokine knock-in mice.

Effects of anti-NKG2A antibody administration on leukemia and normal hematopoietic cells.

Natural killer cells are key cells of the innate immune system. Natural killer cell receptor repertoires are diversified by a stochastic expression of killer-cell-immunoglobulin-like receptors and lectin-like receptors such as NKG2 receptors. All individuals harbor a subset of natural killer cells expressing NKG2A, the inhibitory checkpoint receptor for HLA-E. Most neoplastic and normal hematopoietic cells express HLA-E, the inhibitory ligand of NKG2A. A novel anti-human NKG2A antibody induced tumor cell death, suggesting that the antibody could be useful in the treatment of cancers expressing HLA-E. We found that immunodeficient mice, co-infused with human primary leukemia or Epstein-Barr virus cell lines and NKG2A(+) natural killer cells, pre-treated with anti-human NKG2A, were rescued from disease progression. Human NKG2A(+) natural killer cells reconstituted in immunodeficient mice after transplantation of human CD34(+) cells. These natural killer cells are able to kill engrafted human primary leukemia or Epstein-Barr virus cell lines by lysis after intraperitoneal administration of anti-human NKG2A. Thus, this anti-NKG2A may exploit the anti-leukemic action of the wave of NKG2A(+) natural killer cells recovering after hematopoietic stem cell transplants or adoptive therapy with natural killer cell infusions from matched or mismatched family donors after chemotherapy for acute leukemia, without the need to search for a natural killer cell alloreactive donor.

Adult Donor-Derived Human CD34+ Cell Engraftment and Hemato-Lymphoid System Development in 3rd Generation Humanized Mice

Transplantation of human CD34+ hematopoietic stem and progenitor cells into severe immunocompromised newborn mice allows the development of a human hemato-lymphoid system (HHLS) in vivo (Rongvaux et al. Ann. Rev. Immunol. 2013). While fetal liver- or cord blood- derived CD34+ cells lead to high levels of engraftment, adult donor-derived CD34+ cell transplantation usually led to low levels of engraftment in existing humanized mice models. We recently generated novel mouse strains called 3rd generation humanized mice (3rd gen. huMice) in which human versions of cytokines (M-CSF and TPO with or without IL-3/GM-CSF) are knocked into Balb/c Rag2-/-γC-/- strains (MISTRG or MSTRG, respectively). In addition, human Sirpα, which is a critical factor to prevent donor cell to be eliminated by host macrophages, is expressed as transgene in both strains (Rongvaux et al., Nat. Biotechnol. 2014).To evaluate human adult CD34+ cell engraftment in 3rd gen. huMice, CD34+ cells obtained from peripheral blood after G-CSF administration (3.0 – 5.5 x105 cells) were i.h. injected into sub-lethally irradiated newborn MISTRG or MSTRG and NOD/scid/γC-/- (NSG) mice or Rag2-/-γC-/-hSirpαTg (RGS) mice as controls. Seventeen of 18 (94%) MISTRG/MSTRG mice showed human CD45+ cell engraftment (>1% of total CD45+ cells in BM) 10-16 weeks after injection, whereas 4 of 11 (36%) NSG/RGS mice supported human cell engraftment. Percentages of human cells in the BM of the engrafted MISTRG/MSTRG were 7- to 8 fold higher than in the BM of engrafted NSG/RGS mice (30.2% ± 6.9 vs 4.1% ± 0.9, respectively). MISTRG/MSTRG mice supported significantly increased numbers of non-classical monocytes and NKp46+ cells in BM compared with NSG/RGS mice. Moreover, we observed significantly increased numbers of CD34+ and CD34+CD38- cells, a population enriched for human early progenitor cells and HSCs, in the BM of MISTRG/MSTRG mice. In addition, MISTRG/MSTRG mice supported higher level of human thymocyte development compared to NSG/RGS mice. Besides lymphoid organs, we further observed increased human CD45+ cells, mostly myeloid lineage cells, in the liver and lung of MISTRG/MSTRG mice compared to NSG/RGS mice.Taken together, this study demonstrates that our 3rd gen. huMice models support adult donor-derived HSC engraftment and development of myeloid as well as lymphoid lineage cells at high levels in primary lymphoid and non-lymphoid organs. These models thus have the potential for personalized studies of healthy hematopoiesis as well as hemato-immune system diseases from adult individuals. DisclosuresNo relevant conflicts of interest to declare.

Establishment of a Human Allergy Model Using Human IL-3/GM-CSF–Transgenic NOG Mice

The development of animal models that mimic human allergic responses is crucial to study the pathophysiology of disease and to generate new therapeutic methodologies. Humanized mice reconstituted with human immune systems are essential to study human immune reactions in vivo and are expected to be useful for studying human allergies. However, application of this technology to the study of human allergies has been limited, largely because of the poor development of human myeloid cells, especially granulocytes and mast cells, which are responsible for mediating allergic diseases, in conventional humanized mice. In this study, we developed a novel transgenic (Tg) strain, NOD/Shi-scid-IL2rγ(null) (NOG), bearing human IL-3 and GM-CSF genes (NOG IL-3/GM-Tg). In this strain, a large number of human myeloid cells of various lineages developed after transplantation of human CD34⁺ hematopoietic stem cells. Notably, mature basophils and mast cells expressing FcεRI were markedly increased. These humanized NOG IL-3/GM-Tg mice developed passive cutaneous anaphylaxis reactions when administered anti-4-hydroxy-3-nitrophenylacetyl IgE Abs and 4-hydroxy-3-nitrophenylacetyl. More importantly, a combination of serum from Japanese cedar pollinosis patients and cedar pollen extract also elicited strong passive cutaneous anaphylaxis responses in mice. Thus, to our knowledge, our NOG IL-3/GM-Tg mice are the first humanized mouse model to enable the study of human allergic responses in vivo and are excellent tools for preclinical studies of allergic diseases.

Osteosclerosis and inhibition of human hematopoiesis in NOG mice expressing human Delta-like 1 in osteoblasts

NOD/Shi-scid IL2rγnull (NOG) mice with severe immunodeficiency are excellent recipients to generate "humanized" mice by the transplantation of human CD34(+) hematopoietic stem cells (HSCs). In this study, we developed NOG mice carrying a human Delta-like1 (DLL1) gene, which is a ligand of the Notch receptor and is known to be important in HSC maintenance and self-renewal. We also analyzed the effect of DLL1 signaling on human hematopoiesis and HSC maintenance using humanized DLL1 transgenic NOG mice. To develop DLL1 transgenic NOG (NOG-D1-Tg) mice, a transgenic vector consisting of a human DLL1 complementary DNA fragment placed downstream of the α1(I) collagen (Col1a1) promoter for expression specifically in osteoblasts was constructed. Human CD34(+) HSCs were transplanted into NOG-D1-Tg mice, and differentiation of lymphoid or myeloid lineage cells from human HSCs and maintenance of HSCs in bone marrow were analyzed. Severe osteosclerosis accompanied by increased bone mass and a decreased number of bone marrow cells were observed in NOG-D1-Tg mice. After human HSC transplantation, development of human B lymphocytes, but not T lymphocytes, was significantly suppressed in both bone marrow and the periphery of NOG-D1-Tg mice. Contrary to the initial expectation, retention of human CD34(+) HSCs was inhibited in the bone marrow of NOG-D1-Tg mice. In conclusion, our data suggest that the development of human B lymphocytes and HSC maintenance in osteosclerotic bone may be suppressed by introducing DLL1. These unique humanized mice with sclerotic bone reconstituted by human HSCs are useful models of hematopoiesis in patients with osteosclerosis, such as osteopetrosis, and for investigation of osteogenesis via Notch signaling.

Partial clearance of a hepatotropic virus infection in HLA class I and II double transgenic human immune system mice (105.19)

Abstract Humanized mice have emerged as a promising model to study human immunity in vivo. While they are susceptible to many human lymphotropic pathogens, immune responses to infection remain functionally impaired in these mice. It has been shown that the expression of HLA class I or II improves human immunity in this model. However, little is known about the extent of functional human immune responses in non-lymphoid tissues, such as in the liver, and the role of HLA expression in this context. In this study, we analyzed human antiviral immune responses in humanized mice during a hepatotropic adenovirus infection. Specifically, we compared human immune responses of conventional humanized NSG (NOD-SCID IL2RγNULL) mice to those of a novel NSG strain transgenic for HLA-A*0201 and a chimeric HLA-DR*0101. Humanized mice were generated by transplantation of human CD34+ hematopoietic stem cells (HSC) into newborn, sublethally irradiated mice. The development of a human immune system was comparable in both mouse strains. However, the expression of HLA transgenes resulted in more efficient clearance of an adenovirus infection from the liver, which correlated with liver-infiltration and activation of T cells and the detection of antigen-specific adaptive immune responses. While additional modifications to the xeno-recipient may further improve human immunity this study provides first evidence for functional antiviral immunity against a hepatotropic pathogen

Studying human immunology and immunopathology in humanized mice transplanted with human lymphoid tissues and immune cells

The major bottleneck of understanding human immunology and immunopathology is the lack of relevant animal models. A robust small animal model for hypothesis testing and mechanistic research on human immunology is needed and should meet the following criteria. First, it has well-studied hematolymphoid organs and target cells which are human. Second, human immune pathology including infection diseases can be modeled in vivo. Third, it is genetically inbred and can be manipulated via immunological, genetic and pharmacological means. The immunodeficient mouse transplanted with human immune organs and cells is the obvious candidate. A number of human–mouse chimeric models were developed in the late 1980s, but with only limited success, due to a lack of significant human immune responses in the SCID-hu Thy/Liv mouse1, 2 or selective engraftment of xeno-reactive human T cells in the hu-PBL-SCID mouse.3, 4

Characterization of human B cells in umbilical cord blood-transplanted NOD/SCID mice

Humanized mice are crucially important for preclinical studies. However, the development and potential function of human B cells in chimeras remain unclear. Here, we describe the study of human B cells in NOD/LtSzPrkdcscid/J (NOD/SCID) mice. In this study, we transplanted 1.0×105 human CD34+ cells from umbilical cord blood (UCB) into NOD/SCID mice after pretreatment with anti-asialo GM1 antiserum and sublethal irradiation. Human CD45+ cells were detected in the peripheral blood of the recipient mice from 6weeks after transplantation. CD19+ B cells accounted for the greater part of the CD45+ cells in the human UCB-chimeric mice, but their maturational stages differed in different organs. Most of the bone marrow (BM) CD19+ cells were immature IgM−IgD−CD24hiCD38hi B cells, whereas the mature CD5+IgM+IgD+CD24intCD38intCD19+ B cells were predominantly present in the spleen and peripheral blood. Human immunoglobulin (Ig) M was detected in mouse plasma. The human B cells also secreted human interleukin-10 after stimulation with LPS in vitro. These results show that human CD34+ cells can differentiate into human B cells in NOD/SCID mice, with development and functions that are similar to those of B cell subsets in humans. The transplantation of human CD34+ cells into NOD/SCID mice may provide a useful tool to study the development and function of human B cells.

Abstract P019: Exosomes from Human CD34+ Cells: Critical Mediators of Proangiogenic Paracrine Effect of EPCs

Local transplantation of human CD34+ hematopoietic stem cells has been shown to promote neovascularization in pre-clinical studies in models of myocardial and limb ischemia. In early phase clinical trials, transplantation of CD34+ cells has been associated with reduced angina, improved exercise time and reduced amputation rates. Several studies have suggested that paracrine effects by these pro-angiogenic cells mediate the effects induced by cell transplantation. We hypothesized that CD34+ cells secrete exosomes (Exo), which mediate at least a part of the therapeutic function of the cells. Methods and Results: We isolated Exo from the conditioned media of adult human peripheral blood (PB) CD34+ cells. The angiogenic and therapeutic potency of CD34+ Exo was compared with the intact CD34+ cells and also with PB mononuclear cell (MNC) Exo. Exo from both CD34+ cells and MNC are 50–90nm in size, have cup shaped morphology, and carry known Exo-marker proteins such as CD63, TSG101 and Annexin V as shown by electron microscopy, Western blot and flow cytometry. Compared to CD34+ cells or MNC Exo, CD34+ Exo significantly induces in vitro angiogenic activities such as viability, proliferation and tube formation of HUVECs on matrigel- in a dose dependent manner. In vivo, CD34+ Exo stimulated significant neovascularization in mouse corneal angiogenesis assay (14±4 mm v MNC Exo, 4±1 mm, p<0.01) and incorporation of endothelial (CD31+) cells in mouse matrigel-plug assay (6±1.7% v CD34+ cells, 2±0.8%, p<0.01). Finally, in a mouse model of hind limb ischemia (HLI), CD34+ Exo significantly improved perfusion (ratio: 1.01±0.04 v 0.57±0.1, P<0.05), increased capillary density (1.8±0.3/HPF v 0.9±0.1/HPF, p<0.001) and prevented ischemic leg amputation (16% v 100%), as compared with MNC Exo. Conclusions: These data demonstrate that CD34+ Exo induce angiogenic activity and ischemic tissue repair in the absence of CD34+ cells, and suggest that Exo represent important mediators of the therapeutic effects associated with CD34+ cell therapy. We speculate that Exo derived from CD34+ cells may represent a significant component of the paracrine effect of progenitor-cell transplantation for therapeutic angiogenesis.

Exosomes From Human CD34 + Stem Cells Mediate Their Proangiogenic Paracrine Activity

Transplantation of human CD34(+) stem cells to ischemic tissues has been associated with reduced angina, improved exercise time, and reduced amputation rates in phase 2 clinical trials and has been shown to induce neovascularization in preclinical models. Previous studies have suggested that paracrine factors secreted by these proangiogenic cells are responsible, at least in part, for the angiogenic effects induced by CD34(+) cell transplantation. Our objective was to investigate the mechanism of CD34(+) stem cell-induced proangiogenic paracrine effects and to examine if exosomes, a component of paracrine secretion, are involved. Exosomes collected from the conditioned media of mobilized human CD34(+) cells had the characteristic size (40 to 90 nm; determined by dynamic light scattering), cup-shaped morphology (electron microscopy), expressed exosome-marker proteins CD63, phosphatidylserine (flow cytometry) and TSG101 (immunoblotting), besides expressing CD34(+) cell lineage marker protein, CD34. In vitro, CD34(+) exosomes replicated the angiogenic activity of CD34(+) cells by increasing endothelial cell viability, proliferation, and tube formation on Matrigel. In vivo, the CD34(+) exosomes stimulated angiogenesis in Matrigel plug and corneal assays. Interestingly, exosomes from CD34(+) cells but not from CD34(+) cell-depleted mononuclear cells had angiogenic activity. Our data demonstrate that human CD34(+) cells secrete exosomes that have independent angiogenic activity both in vitro and in vivo. CD34(+) exosomes may represent a significant component of the paracrine effect of progenitor cell transplantation for therapeutic angiogenesis.

Busulfan Conditioning In Sheep In Utero Transplantation Confers Engraftment Potential on Human Hematopoietic Stem Cells as Efficiently as HoxB4 Transduction.

AbstractAbstract 3716 Background:In utero transplantation (IUT) of hematopoietic stem cells (HSCs) has been pursued as a treatment for congenital hematologic and genetic disorders. Although the engraftment of HSCs following IUT has been achieved, the levels of engraftment have been generally low. In order to achieve clinically relevant levels of HSC engraftment, there are many potential ways including the HSC expansion via HoxB4 transduction, and the niche expansion either by co-transplantation with mesenchymal stem cells (MSCs) or by myeloablation using busulfan (BU). BU is an alkylating agent and has application as an alternative to total body irradiation to create open niches for HSC transplantation. However, BU conditioning in IUT has not been reported. Here, we examined the safety and efficacy of BU conditioning in sheep IUT. Moreover, we compared the efficacy of BU conditioning with that of HoxB4 transduction or co-transplantation with MSCs in the context of sheep IUT. Methods:To determine the optimal dose of BU in sheep IUT, fetal sheep at 40–47 gestation days (full term, 147 days) were treated with BU at 0, 3, or 7.5 mg/kg (calculated by maternal body weight) via maternal vein. (BU can pass through placentas.) In the fetuses receiving 3 mg/kg BU, the numbers of colony-forming units (CFUs) in the fetal liver at 2 weeks after BU administration significantly decreased compared with those of the control group (p < 0.01), and there was no trouble in maintenance of pregnancy, or no developmental abnormalities in the fetuses. In the fetuses receiving 7.5 mg/kg BU, four out of the five fetuses died, with severe suppression in the maternal hematopoiesis. We therefore determined that the optimal dose of BU for sheep IUT was 3 mg/kg. Next, to determine the appropriate timing of BU treatment in IUT, BU treatment at 3 mg/kg via maternal vein was conducted at 2 or 6 days prior to IUT, followed by transplantation of human CD34+ cells from umbilical cord blood, into the fetal liver (6-day-interval group [BU-6], n = 4; 2-day-interval group [BU-2], n = 4; control group, n = 6). Furthermore, we performed IUT of CD34+ cells together with MSCs in addition to 6-day-interval BU treatment (BU-6 + MSC co-transplantation group, n = 4). IUT of CD34+ cells transduced with HoxB4 by Sendai virus vector was also conducted (HoxB4 group, n = 4), as reported in the previous annual meeting by our group. After birth, the engraftment of human hematopoietic cells in the lambs was quantitatively evaluated by colony PCR of the bone marrow. Results:Human CFUs were detected in the lamb bone marrow at 1 week after birth as shown in Table. In summary, the BU-6 group showed increased levels of engraftment compared to the control group (p < 0.01), although the BU-2 group did not show increased levels of the engraftment. Thus, the engraftment was improved by transplanting cells at 6 days, not at 2 days, after BU treatment. The failure to improve engraftment in the BU-2 group might be attributable to the effects of remaining BU on transplanted cells, resulting from not fully developed enzymes of drug metabolism in fetuses as is especially known in sheep. Notably, the BU conditioning resulted in improved engraftment to the similar levels as HoxB4 transduction (cf. HoxB4 group). There was no additional effect of MSCs on engraftment (cf. BU-6 + MSC co-transplantation group), presumably because hematopoietic niches had already restored at 6 days after BU administration.Table 1Engraftment of human HSCs in sheep IUTGroupEngraftment (% of human CFUs)P value (compared with control group)BU-6 group (n = 4)1.9 ± 1.1%< 0.01BU-2 group (n = 4)0.6 ± 1.1%not significantHoxB4 group (n = 4)1.7 ± 1.4%< 0.05BU-6 + MSC co-transplantation group (n = 4)1.4 ± 0.6%< 0.05Control group (n = 6)0.0 ± 0.0%– Discussion:BU treatment at 3 mg/kg via maternal vein 6 days prior to transplant can enhance the engraftment of human HSCs after sheep IUT, the efficacy of which would be comparable to that of HoxB4 transduction of human HSCs. In the context of IUT, we suggest that non cell-autonomous enhancement of human HSC engraftment by treatment with BU would be as effective as cell-autonomous enhancement such as HoxB4 transduction. The synergistic effects by both methods remain to be elucidated. Disclosures:No relevant conflicts of interest to declare.

Establishment of a rhabdomyosarcoma xenograft model in human-adapted mice

The outcome of patients with advanced stage rhabdomyosarcoma (RMS) is still sobering. This outcome has not improved through conservative treatments. Therefore, novel treatment approaches such as immunotherapy need to be evaluated in human-adapted animal models. The aim of this study was to develop a humanized mouse model of childhood RMS as a basis for the study of immunotherapeutic approaches. Therefore, NOD/LtSz-scid IL2rgammanull-mice were used for all the experiments (n=19). The animals underwent sublethal irradiation on days 1 and 2 (1 x 300 cGy). After irradiation, the transplantation of human CD34+-cells (1,000,000 cells per animal i.v.) was carried out. Five animals served as the control and did not undergo stem cell transplantation. The engraftment of human cells was assessed in peripheral blood on days 21 and 55 by FACS analysis. Eight weeks after transplantation, the subcutaneous xenotransplantation of human alveolar and embryonal RMS cell lines was carried out. Tumor growth was monitored and tumors were resected 93 days after CD34+-transplantation. The tumor specimens were evaluated histologically. The successful engraftment of human cells with the establishment of a human immune system was observed in 12 out of 14 animals. B and T cells were mostly detected in the peripheral blood. There were only a few monocytes and almost no natural killer cells. The xenotransplantation of alveolar RMS resulting in subcutaneous tumor growth was feasible in 7 animals. The xenotransplantation of embryonal RMS was performed in 5 animals and led to tumor growth in 1 animal. A histological work up showed either alveolar or embryonal RMS cells with central necrosis. This is the first time a xenotransplantation model of human RMS has been developed in a humanized mouse model. The establishment of subcutaneous tumor xenografts was more effective in the alveolar subtype. This model offers a basic tool for further analyzing novel immunotherapeutic approaches in RMS, and could possibly be used in other solid pediatric tumors.

Establishment of a rhabdomyosarcoma xenograft model in human-adapted mice

The outcome of patients with advanced stage rhabdomyosarcoma (RMS) is still sobering. This outcome has not improved through conservative treatments. Therefore, novel treatment approaches such as immunotherapy need to be evaluated in human-adapted animal models. The aim of this study was to develop a humanized mouse model of childhood RMS as a basis for the study of immunotherapeutic approaches. Therefore, NOD/LtSz-scid IL2rγnull-mice were used for all the experiments (n=19). The animals underwent sublethal irradiation on days 1 and 2 (1 x 300 cGy). After irradiation, the transplantation of human CD34+-cells (1,000,000 cells per animal i.v.) was carried out. Five animals served as the control and did not undergo stem cell transplantation. The engraftment of human cells was assessed in peripheral blood on days 21 and 55 by FACS analysis. Eight weeks after transplantation, the subcutaneous xenotransplantation of human alveolar and embryonal RMS cell lines was carried out. Tumor growth was monitored and tumors were resected 93 days after CD34+-transplantation. The tumor specimens were evaluated histologically. The successful engraftment of human cells with the establishment of a human immune system was observed in 12 out of 14 animals. B and T cells were mostly detected in the peripheral blood. There were only a few monocytes and almost no natural killer cells. The xenotransplantation of alveolar RMS resulting in subcutaneous tumor growth was feasible in 7 animals. The xenotransplantation of embryonal RMS was performed in 5 animals and led to tumor growth in 1 animal. A histological work up showed either alveolar or embryonal RMS cells with central necrosis. This is the first time a xenotransplantation model of human RMS has been developed in a humanized mouse model. The establishment of subcutaneous tumor xenografts was more effective in the alveolar subtype. This model offers a basic tool for further analyzing novel immunotherapeutic approaches in RMS, and could possibly be used in other solid pediatric tumors.

Abstract 4196: Successful establishment of human rhabdomyosarcoma xenografts in a human-adapted mouse model

Abstract Purpose: The outcome of patients with advanced stage rhabdomyosarcoma is still sobering. Treatment results cannot be improved by conservative treatment options. Therefore, novel treatment approaches such as immunotherapy need to be evaluated in human-adapted animal models. The aim of this study was to develop a humanized mouse model of childhood rhabdomyosarcoma as a basis for studies of immunotherapeutic approaches. Methods: NOD/LtSz-scid IL2R gamma null mice were used for all experiments (n =14). Animals underwent subletal irradiation (1 × 300cGy). Afterwards, transplantation of human CD34+-cells (1.000.000 cells per animal i.v.) was carried out. Eight weeks after transplantation, subcutaneous xenotransplantation of human alveolar (Rh30) and embryonal rhabdomyosarcoma (RD) cell lines was carried out. Engraftment of human cells was assessed in peripheral blood at day 21 and at day 55 of the experiment. Animals were sacrified and tumors were resected 93 days after CD34+-transplantation. Results: Successful engraftment of human cells with establishment of a human immune system was observed in 12 of 14 animals. B-cells and T-cells were mostly detected in the peripheral blood. There were only a few monocytes and nearly no NK-cells. Xenotransplantation of alveolar RMS resulting in subcutaneous tumor growth was feasible in 7 animals. Xenotransplantation of embryonal RMS was performed in 5 animals and led to tumor growth in 1 animal. Histological work up showed either alveolar or embryonal RMS cells with central necrosis. Conclusion: For the first time, we developed a xenotransplantation model of human rhabdomyosarcoma in a humanized mouse model. Establishment of subcutaneous tumor xenografts was more effective in alveolar subtype. This model will serve as basic research tool for further analysis of novel immunotherapeutic approaches in solid pediatric tumors. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4196.

Transplantation of human CD34+ stem cells from umbilical cord blood to rats with thioacetamide‐induced liver cirrhosis

Liver fibrosis results from accumulation of extracellular matrix components and is associated with many chronic hepatic diseases. There is to date no specific therapy for this disease, and patients receive treatment for its associated complications. Specific progenitor cells, known as oval cells, are present in the liver. As oval cells express markers such as CD34, they are thought to arise from a hematopoietic precursor. The aim of this work was to investigate whether transplantation of hematopoietic CD34(+) stem cells could improve hepatic fibrosis by their differentiation into hepatocytes. CD34(+) stem cells from human umbilical cord blood were purified, transduced with a lentiviral vector containing the green fluorescent protein (GFP) gene and injected via portal vein into rats with liver cirrhosis induced by the 4-month administration of thioacetamide. Rats were killed 15 and 60 days post-transplantation. Up to 37% and 22% fluorescent cells were observed in the blood of control and cirrhotic rats, respectively, at 15 days post-transplantation. At 60 days post-transplantation, however, fluorescent cells were completely absent from the blood. Fluorescence was not detected in liver sections at either 15 or 60 days post-transplantation. Polymerase chain-reaction study to detect the GFP gene ruled out silencing of the transgene. These results suggest that the transplanted cells did not engraft in the liver and were eliminated from the rats.

Heart infarct in NOD-SCID mice: therapeutic vasculogenesis by transplantation of human CD34+ cells and low dose CD34+KDR+ cells.

Hematopoietic (Hem) and endothelial (End) lineages derive from a common progenitor cell, the hemangioblast: specifically, the human cord blood (CB) CD34+KDR+ cell fraction comprises primitive Hem and End cells, as well as hemangioblasts. In humans, the potential therapeutic role of Hem and End progenitors in ischemic heart disease is subject to intense investigation. Particularly, the contribution of these cells to angiogenesis and cardiomyogenesis in myocardial ischemia is not well established. In our studies, we induced myocardial infarct (MI) in the immunocompromised NOD-SCID mouse model, and monitored the effects of myocardial transplantation of human CB CD34+ cells on cardiac function. Specifically, we compared the therapeutic effect of unseparated CD34+ cells vs. PBS and mononuclear cells (MNCs); moreover, we compared the action of the CD34+KDR+ cell subfraction vs. the CD34+KDR- subset. CD34+ cells significantly improve cardiac function after MI, as compared with PBS/MNCs. Similar beneficial actions were obtained using a 2-log lower number of CD34+KDR+ cells, while the same number of CD34+KDR- cells did not have any effects. The beneficial effect of CD34+KDR+ cells may mostly be ascribed to their notable resistance to apoptosis and to their angiogenic action, since cardiomyogenesis was limited. Altogether, our results indicate that, within the CD34+ cell population, the CD34+KDR+ fraction is responsible for the improvement in cardiac hemodynamics and hence represents the candidate active CD34+ cell subset.