Correlation between the immature characteristics of umbilical cord blood-derived mesenchymal stem cells and engraftment of hematopoietic stem cells in NOD/SCID mice.

Umbilical cord blood (UCB)-derived mesenchymal stem cells (MSC) facilitate the engraftment of human (h) hematopoietic stem cells when transplanted simultaneously in animal and human studies. However, the type of MSCs that preferentially enhance the engraftment of HSCs is unknown. Recent studies have shown that MSCs derived from a single source are heterogeneous in terms of cell size, morphology, proliferation rate, and differentiation potential. This study was designed to investigate the properties of UCB-MSCs, which influence the engraftment of hHSCs in a NOD/SCID mouse model. We categorized MSCs as being the most effective (UCB-352 MSCs) or the least effective (UCB-156 MSCs) at promoting the homing and engraftment of HSCs, and compared the characteristics of these 2 MSC populations. We observed that the 2 populations showed differences in characteristics typical of immature MSCs, and related to proliferation potential. We showed that UCB-352 MSCs, which proliferate quickly, preferentially enhanced the engraftment of HSCs in NOD/SCID mice. In addition, we observed differences in the pattern of both PODXL and Oct4 expression, and in the levels of cytokines such as SDF-1 and SCF using flow cytometry and membrane arrays. The more effective UCB-352 MSCs expressed higher levels of PODXL and Oct4, which were associated with immaturity, than did the UCB-156 MSCs. Furthermore, UCB-352 cells secreted greater levels of SDF-1 and SCF, both of which are required for hematopoiesis. We propose that the proliferation potential of UCB-MSCs, coupled with their immature characteristics, may serve as a novel standard to promote the homing and engraftment of HSCs.

BMI1 sustains human glioblastoma multiforme stem cell renewal.

Glioblastoma multiforme (GBM) is one of the most common and aggressive types of brain tumors. In GBM, a subpopulation of CD133-positive cancer initiating cells displays stem cell characteristics. The Polycomb group (PcG) and oncogene BMI1 is part of the Polycomb repressive complex 1 (PRC1) that regulates gene expression by modifying chromatin organization. Here we show that BMI1 is expressed in human GBM tumors and highly enriched in CD133-positive cells. Stable BMI1 knockdown using short hairpin RNA-expressing lentiviruses resulted in inhibition of clonogenic potential in vitro and of brain tumor formation in vivo. Cell biology studies support the notion that BMI1 prevents CD133-positive cell apoptosis and/or differentiation into neurons and astrocytes, depending on the cellular context. Gene expression analyses suggest that BMI1 represses alternate tumor suppressor pathways that attempt to compensate for INK4A/ARF/P53 deletion and PI(3)K/AKT hyperactivity. Inhibition of EZH2, the main component of the PRC2, also impaired GBM tumor growth. Our results reveal that PcG proteins are involved in GBM tumor growth and required to sustain cancer initiating stem cell renewal.

Coxiella burnetii infection in C.B-17 scid-bg mice xenotransplanted with fetal bovine tissue.

Two from a group of approximately 50 C.B-17 scid-bg mice were examined because of lethargy, dehydration, and rough coat. Three months prior to development of clinical signs of disease, mice of this study had been surgically implanted with fetal bovine liver, thymus, and lymph node. At necropsy, marked splenomegaly and mild hepatomegaly were observed in both animals. Large areas of necrosis and inflammation, with associated intracytoplasmic granular basophilic inclusions, were observed in histologic sections of multiple organs. Aerobic and anaerobic culturing of the liver yielded negative results. Six months after the initial case, four more reconstituted scid-bg mice from a different fetal donor had identical clinical, gross, and histologic signs of disease. To determine whether the basophilic inclusions represented an infective agent, 4-month-old immune-naive C.B-17 scid-bg mice were inoculated intraperitoneally with a liver and spleen homogenate from an affected mouse. Two weeks after inoculation, mice developed clinical signs of disease and lesions identical to those seen in the signal mice. On ultrastructural examination of the liver, pleomorphic bacteria were found in large cytoplasmic vacuoles of hepatocytes. Bacterial DNA was amplified from the liver, using primers that amplify a segment of the 16S rRNA gene from many bacterial species. Sequencing of the polymerase chain reaction (PCR) product revealed gene sequence identical to that of Coxiella burnetii, the agent of Q-fever. These results highlight the need to consider infective agents of the donor species when working with xenografted animals.

Donor stromal cells from human blood engraft in NOD/SCID mice.

Little is known about the presence, frequency, and in vivo proliferative potential of stromal cells within blood-derived hematopoietic transplants. In this study, nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice were injected with human CD34(+) peripheral blood cells (PBCs) or cord blood cells (CBCs, either enriched for CD34 or density-gradient separated mononuclear cells). Flow cytometric analysis 5 to 11 weeks after transplantation revealed the presence of a human lymphomyeloid hematopoiesis within the murine bone marrow. Immunohistochemical staining of bone marrow cell suspensions using human-specific antibodies showed human cells staining positive for human fibroblast markers, human von Willebrand factor (vWF) and human KDR (vascular endothelial growth factor receptor-2) in mice transplanted with CD34(+) PBCs or CBCs, with mean frequencies between 0.6% and 2.4%. In stromal layers of bone marrow cultures established from the mice, immunohistochemical staining using human-specific antibodies revealed flattened reticular cells or spindle-shaped cells staining positive with human-specific antifibroblast antibodies (mean frequency, 2.2%). Cell populations of more rounded cells stained positive with human-specific antibodies recognizing CD34 (1.5%), vWF (2.2%), and KDR (1.6%). Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and subsequent complementary DNA sequencing detected transcripts of human KDR (endothelial specific) and human proline hydroxylase-alpha (fibroblast specific) within the bone marrow and spleen of transplanted mice. Analysis of nontransplanted control mice yielded negative results in immunocytochemistry and RT-PCR. Cells expressing endothelial and fibroblast markers were also detected in the grafts before transplantation, and their numbers increased up to 3 log in vivo after transplantation. These results indicate that stromal progenitor cells are present in human cytokine-mobilized peripheral blood or cord blood that engraft in NOD/SCID mice. (Blood. 2000;96:3971-3978)

Transplantation and growth characteristics of human fetal lymph node in immunodeficient mice.

OBJECTIVE: The lymph node is an integral component of the immune system and the major site of antigen-dependent lymphocyte proliferation and differentiation. Development of animal models possessing functional primary human lymph nodes will have a significant impact on research in lymphopoiesis and immune response. To date, successful transplantation of primary human lymph nodes in rodents has not yet been reported. This work was undertaken to develop a reliable methodology to engraft primary human fetal lymph nodes in immunodeficient mice. MATERIALS AND METHODS: Three different sets of parameters, including three different transplantation sites in the mice, two different strains of immunodeficient mice, and two different preconditioning regimens, were evaluated. The growth characteristics of the implanted primary human fetal lymph nodes were examined 3 months after transplantation by histologic, immunocytochemical, and flow cytometric methods. RESULTS: Transplantation of primary human fetal lymph nodes into subcutaneous pouches in the ears in severe combined immunodeficiency (SCID) mice preconditioned with etoposide reproducibly give rise to >80% engraftment. The engrafted primary human fetal lymph nodes undergo massive growth (>200-fold) and retain the same histology and cellular composition as fresh human fetal lymph nodes from the same donors. CONCLUSIONS: We report, for the first time, the development of a reliable methodology to successfully engraft human fetal lymph node in SCID mice. The engrafted human lymph nodes are visible and accessible to experimental manipulations. This SCID-hu mouse model with human lymph node should provide a physiologically relevant system to investigate lymphopoiesis, immunologic response, and virus-mediated immunosuppression.

Human renal cell carcinoma xenografts in SCID mice: tumorigenicity correlates with a poor clinical prognosis.

To establish human renal cell carcinoma (RCC) xenografts for preclinical studies, 55 renal tumors (33 primary and 22 metastatic lesions) were transplanted subcutaneously into severe combined immunodeficient mice. Twenty of 49 evaluable tumors (40.8%) grew with a median latency period of 89 days (36 to 209 days) from the day of engraftment. Tumor growth was stabilized after the fifth passage with a median time between passages of 38 days (19 to 80 days). Tumorigenicity was correlated with the metastatic phenotype of the tumor (54% success rate, p = 0.007) and with reduced survival of patients. Despite a possible evolution of histological features and tumor grading, established RCC xenografts were comparable to parental tumors, as assessed by karyotype and DNA-ploidy analyses. Molecular cytogenetic analysis also revealed specific genetic alterations characterizing distinct RCC types that were constant in parental and corresponding xenografts. In addition, this xenograft model has permitted the selection of minor tumor subclones with a proliferative advantage and minimal overexpressed chromosomal regions. We conclude that severe combined immunodeficient mice are useful recipients for the establishment of long-term RCC xenografts that can be used as valuable tools to evaluate the activity of new therapeutic approaches and to study biological parameters determining in vivo aggressiveness of human RCC.

Induction of human gamma delta T cells in myasthenia gravis thymus transplanted SCID mice.

BACKGROUND: TCR-gamma delta cells develop by extrathymic differentiation and might play an important role in thymectomy-resistant myasthenia gravis (MG) patients. In this study, we show development of human TCR-gamma delta cells in periphery of SCID mice by transplantation of human MG thymus or thymoma. METHODS: Three pieces of thymoma tissue and four of non-thymoma thymic tissues were obtained from MG patients by thymectomy. Each tissue was transplanted into 5-6 weeks old female SCID mice by intraperitoneal injection or surgical implantation. Rate of human TCR-positive cell development and its subtypes (TCR-alpha beta, TCR-gamma delta) were measured in the mice blood at one, three, and six weeks after the transplantation. RESULTS: Human TCR-positive cells were detected three weeks after transplantation. Rate of TCR-gamma delta T cell development got higher in thymoma transplanted group than in non-thymoma transplanted group. CONCLUSIONS: We could successfully develop human mature T cells in SCID mice by transplantation of human MG thymus or thymoma.

Engraftment of severe combined immune deficient/beige mice with bovine foetal lymphoid tissues.

To develop a model of bovine thymus and lymph node growth in vivo, we have implanted bovine foetal tissues (16-23 weeks gestation) under the renal capsule of severe combined immune deficient (SCID)/beige (BG) mice and assayed for graft growth and characteristics 2-18 weeks after engraftment. Bovine foetal thymus and lymph node grew considerably following engraftment of SCID/BG mice. Growth was optimal if bovine foetal tissues were used before gestation Week 17. Bovine-mouse chimerism was confirmed using glucose phosphate isomerase analysis. Bovine thymus grew during the entire 18 weeks of study. Growth of bovine lymph node was initially rapid, reaching a maximum at 2 weeks after transplantation followed by a progressive decrease in size. Transplanted bovine lymph node and thymus were morphologically similar to age-matched bovine foetal tissue for a limited time period. Fibrosis, degeneration and depletion of lymphocytes were evident 6 weeks after engraftment; changes were more severe in lymph node than in thymus whereas increases in lymphocytes, lymphopoiesis and follicle formation were evident in age-matched bovine foetal tissue. Despite growth and morphological similarities of the transplanted tissue, blood counts suggested there was no peripheralization of bovine leucocytes. Bovine immunoglobulins (IgG1 and IgG2) were detected in serum of some SCID/BG chimeric mice for a limited time. The appearance of bovine immunoglobulins at 2 weeks in SCID/BG chimeric mice depended on the age of the foetal donor (> 18 weeks) and coincided with the appearance of morphologically mature lymphocytes in the donor foetus lymph nodes. The ability to produce bovine immunoglobulins decreased 8 weeks after engraftment, coinciding with the depletion of lymphocytes in the engrafted lymph node. Lymphocyte depletion and loss of function of engrafted tissues appear the result of a lack of lymphoid progenitors normally derived from hematopoietic stem cells in the bone marrow.