Survival of Free and Encapsulated Human and Rat Islet Xenografts Transplanted into the Mouse Bone Marrow

Bone marrow was recently proposed as an alternative and potentially immune-privileged site for pancreatic islet transplantation. The aim of the present study was to assess the survival and rejection mechanisms of free and encapsulated xenogeneic islets transplanted into the medullary cavity of the femur, or under the kidney capsule of streptozotocin-induced diabetic C57BL/6 mice. The median survival of free rat islets transplanted into the bone marrow or under the kidney capsule was 9 and 14 days, respectively, whereas that of free human islets was shorter, 7 days (bone marrow) and 10 days (kidney capsule). Infiltrating CD8(+) T cells and redistributed CD4(+) T cells, and macrophages were detected around the transplanted islets in bone sections. Recipient mouse splenocytes proliferated in response to donor rat stimulator cells. One month after transplantation under both kidney capsule or into bone marrow, encapsulated rat islets had induced a similar degree of fibrotic reaction and still contained insulin positive cells. In conclusion, we successfully established a small animal model for xenogeneic islet transplantation into the bone marrow. The rejection of xenogeneic islets was associated with local and systemic T cell responses and macrophage recruitment. Although there was no evidence for immune-privilege, the bone marrow may represent a feasible site for encapsulated xenogeneic islet transplantation.

The role of MYC in bone and in the hematopoietic stem cell niche

Maike Jaworski

The nuclear protein c-Myc is one of the most potent proto-oncogenes described. Its expression is found to be deregulated in more than 50 percent of human cancers, where it has been shown to control a number of biological processes including driving proliferation, cell growth and angiogenesis while inhibiting differentiation. The effects of c-Myc are highly cell type specific and include cell autonomous and non-cell autonomous processes. In order to examine the effect of excess amounts of c-Myc in bone, we have generated a mouse line expressing the tTA transactivator under the control of the mouse 2.3 kb col1a1 promoter (named Col1a1::tTA), which directs transgene expression to mature osteoblasts. By combining this transgene with the Tet-O-Myc allele, we generated a Col1a1::tTA; Tet-O-Myc double transgenic mouse model in which it is possible to inducibly express human c-MYC in osteoblasts during development and in the adult. We document that the used 'tet-off' system directs transgene expression to bones and that c-MYC can be completely repressed by treatment with doxycycline. Using this mouse model, we demonstrate that overexpression of c-MYC leads to remarkable changes in mutant bones, which depend greatly in their severity on the developmental time point of c-MYC induction. Unrestricted c-MYC expression, starting with the first emergence of mature osteoblasts in the fetal skeleton (∼E14.0), leads to lethality of the mutants at birth; their bones are shortened and thicker than in controls. Later induction of c-MYC allows survival, but leads to significant changes in bone morphology. Mutant bones develop a very irregular bone structure with signs of excessive bone formation. Surprisingly, this also correlates with definite signs of excessive osteoclast activity, which explains the formation of cavities seen in the bone matrix. Osteoblasts not only produce bone matrix, they are also very important niche cells for hematopoietic cells, especially for hematopoietic stem cells and B cells. We find that c-MYC overexpression severely impairs B lymphopoiesis and also observe signs of extramedullary hematopoiesis in the spleen, which is indicative of a compromised HSC environment in the bone marrow. Most strikingly, these animals develop frank osteosarcomas leading to a much reduced life span. Tumors arise in various bones, with the ribs and hips being the most prominent sites. These c-MYC driven tumors are very aggressive as is evident by their capacity to spontaneously metastasize to lung and liver, a rather rare phenomenon in mouse models, indicating that these tumors share important aspects of human osteosarcomas. Molecular analysis of c-MYC overexpressing osteoblasts has revealed a defect in their terminal differentiation program, rendering them incapable to progress from a relatively immature proliferative to a quiescent, calcified matrix producing state. To our knowledge this is the first report of experimental osteosarcoma formation, originating from a mature and lineage committed osteoblast population indicating that c-MYC may be able to de-differentiate more mature cells. In summary, our novel conditional mouse model showed that c-MYC expression in mature osteoblasts can lead to the formation of highly aggressive and metastatic osteosarcomas, a lethal disease in humans. This model will be the basis for further studies elucidating the molecular effects of c-MYC on de-differentiation and metastasis formation, processes, which remain poorly understood but are critical events in highly malignant cancers.

EPFL2009

Hes1 as a Mediator of Notch Signalling in T-cell Development and Notch1- Induced T-ALL

Agnieszka Anna Wendorff

The evolutionarily conserved Notch signalling pathway controls a broad spectrum of cell fate decisions in organisms as diverse as fruit flies and mice. Whithin the murine haematopoietic system, Notch1 is indispensable for T cell development as conditional inactivation of Notch1 in bone marrow (BM) precursors results in a complete block in T-cell development and ectopic B-cell development in the thymus. Conversely, constitutive activation of Notch1 signalling plays a causative role in the development of acute T-cell lymphoblastic leukaemia (T-ALL). Numerous mutations within the Notch1 gene, which result in aberrent activation of Notch1 signalling, have been identified in > 50% of human T-ALL patients. Therefore, gain-of-function studies in murine models of T-ALL have been employed by many laboratories to elucidate the molecular mechanisms acting either cooperatively or downstream of oncogenic Notch1 signalling in development and progression of T-ALL. The best-characterized Notch1 target gene to date is a member of the bHLH family of transcriptional repressors, Hes1. This study focuses on the role of Hes1 as a mediator of Notch1 signalling in adult murine haematopoietic development, as well as the requirement for Hes1 in T-ALL development and maintenance. Conditional inactivation of Hes1 in adult murine BM cells results in severe thymic hypoplasia, while myeloid and B cell development remain unperturbed. Upon transplantation in congenic recipients, Hes1-/- progenitors give rise to only a small number of mature T cells. Hes1-inactivation does not however cause ectopic B-cell development in the thymus, and therefore does not phenocopy the loss of Notch1. Thus, while Notch1 signalling is indispensable for T versus B-cell fate specification, this function is not mediated exclusively via Hes1. Strikingly, in a competitive BM chimera setting, Hes1-/- progenitors completely fail to generate T cells. The mechanism by which Hes1 inactivation results in depleted T cell numbers remains to be elucidated, although we have shown that loss of Hes1 does not result in enhanced cell death. In addition, preliminary data are indicative of cell-cycle perturbation in Hes1-deficient immature T cells. Finally, we have demonstrated that upon IT transplantation, Hes1-deficient progenitors successfully give rise to T-cells at all stages of development. This data strongly indicates that Hes1 deficiency does not result in an intrinsic developmental defect but rather in the inability of T cell progenitors to efficiently home to the thymus from the BM. Upregulation of Hes1 expression has been observed in both human T-ALL cell lines as well as murine primary Notch1-induced T-ALL samples. However, the function of Hes1 as an effector of oncogenic Notch1 signalling in modulating disease onset or progression remained to be investigated. The requirement for Hes1 as a mediator of Notch1-induced T-ALL was addressed by inducing simultaneous deletion of Hes1 and expression of a dominant active form of Notch1 (NICD) in a murine model of T-ALL. Our data demonstrate that Hes1 is essential for the development of NICD-induced T-ALL. Loss of Hes1 prevents ectopic development of DP (CD4+CD8+) leukemic T cells in the BM and the subsequent accumulation of DP T cells in peripheral tissues, a hallmark of T-ALL, and consequently results in 10-fold increased life expectancy of experimental animals. We have further determined that Hes1 is required for the maintenance of T-ALL by inactivating Hes1 in a retrovirally pre-induced T-ALL model. Deletion of Hes1 in leukemic DP cells results in rapid cell death and enhanced survival. These data demonstrate that Hes1 is required as a mediator of Notch1 signalling in normal T cell development, and is indispensable to mediate the oncogenic effect of constitutive Notch1 activation in the induction of T-ALL.

EPFL2010

Microencapsulated human mesenchymal stem cells decrease liver fibrosis in mice

Redouan Mahou, Yannick Müller, Christine Wandrey

Background & Aims: Mesenchymal stem cell (MSC) transplantation was shown to be effective for the treatment of liver fibrosis, but the mechanisms of action are not yet fully understood. We transplanted encapsulated human MSCs in two mouse models of liver fibrosis to determine the mechanisms behind the protective effect. Methods: Human bone marrow-derived MSCs were microencapsulated in novel alginate-polyethylene glycol microspheres. In vitro, we analyzed the effect of MSC-conditioned medium on the activation of hepatic stellate cells and the viability, proliferation, cytokine secretion, and differentiation capacity of encapsulated MSCs. The level of fibrosis induced by bile duct ligation (BDL) or carbon tetrachloride (CCl4) was assessed after intraperitoneal transplantation of encapsulated MSCs, encapsulated human fibroblasts, and empty microspheres. Results: MSC-conditioned medium inhibited hepatic stellate cell activation and release of MSC secreted anti-apoptotic (IL-6, IGFBP-2) and anti-inflammatory (IL-1Ra) cytokines. Viability, proliferation, and cytokine secretion of microencapsulated MSCs were similar to those of non-encapsulated MSCs. Within the microspheres, MSCs maintained their capacity to differentiate into adipocytes, chondrocytes, and osteocytes. 23% (5/22) of the MSC clones were able to produce anti-inflammatory IL-1Ra in vitro. Microencapsulated MSCs significantly delayed the development of BDL-and CCl4-induced liver fibrosis. Fibroblasts had an intermediate effect against CCl4-induced fibrosis. Mice transplanted with encapsulated MSCs showed lower mRNA levels of collagen type I, whereas levels of matrix metalloproteinase 9 were significantly higher. Human IL-1Ra was detected in the serum of 36% (4/11) of the mice transplanted with microencapsulated MSCs. Conclusions: MSC-derived soluble molecules are responsible for an anti-fibrotic effect in experimental liver fibrosis. (C) 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Elsevier Science Bv2015