No description available.

No description available.

Despite the clonal origins of tumors, the majority of neoplasms are composed of a heterogeneous population of cells. The origins of this phenotype these cells have the potential to get can be associated with cancer stem cells or tumor initiating cells have the potential to self-renew and to differentiate giving rise to all cell types compromising a heterogeneous malignancy. These cells are clinically important as they preferentially give rise to tumors and are therefore hypothesized to account for the longevity and recurrence of neoplastic lesions. Cancer stem cells have been identified from a broad range of hematopoietic, neural and epithelia tumors; however, their function in mesenchymal neoplasms is less well defined. Using the side population assay, we identified a subpopulation of cells within mesenchymal neoplasms, referred to as side population cells, which are enhanced for tumor initiating potential. Importantly, we show a correlation between the percentage of side population cells and tumor grade suggesting clinical prognostic value as the proportion of side population cells may be a predictor of patient outcome. Interestingly side population cells show distinct molecular features when compared to non-side population cells and manipulation of these molecular mechanisms reduces the ability of side population cells to initiate tumor formation in osteosarcoma cell lines. In conjunction with these experiments, we also sought to determine the cellular origins of the mesenchymal neoplasm, aggressive fibromatosis. Using mouse models we show the influence of a mesenchymal precursor cells in the development of this malignancy. These results identify important biological features of mesenchymal neoplasms from which the development of targeted treatment strategies can begin.

Despite the clonal origins of tumors, the majority of neoplasms are composed of a heterogeneous population of cells. The origins of this phenotype these cells have the potential to get can be associated with cancer stem cells or tumor initiating cells have the potential to self-renew and to differentiate giving rise to all cell types compromising a heterogeneous malignancy. These cells are clinically important as they preferentially give rise to tumors and are therefore hypothesized to account for the longevity and recurrence of neoplastic lesions. Cancer stem cells have been identified from a broad range of hematopoietic, neural and epithelia tumors; however, their function in mesenchymal neoplasms is less well defined. Using the side population assay, we identified a subpopulation of cells within mesenchymal neoplasms, referred to as side population cells, which are enhanced for tumor initiating potential. Importantly, we show a correlation between the percentage of side population cells and tumor grade suggesting clinical prognostic value as the proportion of side population cells may be a predictor of patient outcome. Interestingly side population cells show distinct molecular features when compared to non-side population cells and manipulation of these molecular mechanisms reduces the ability of side population cells to initiate tumor formation in osteosarcoma cell lines. In conjunction with these experiments, we also sought to determine the cellular origins of the mesenchymal neoplasm, aggressive fibromatosis. Using mouse models we show the influence of a mesenchymal precursor cells in the development of this malignancy. These results identify important biological features of mesenchymal neoplasms from which the development of targeted treatment strategies can begin.

Thrombocytopenia is one of the most common hematologic presentations of active human cytomegalovirus (HCMV) infection, especially in recipients of allogeneic hematopoietic stem cell transplantations and newborns of congenital HCMV infection. However, mechanisms of HCMV-induced thrombocytopenia have not been well understood. The precursor of circulating platelets – megakaryocyte, is derived from hematopoietic stem/progenitor cell in bone marrow. We postulate that inhibition to megakaryocytic development is the major pathogenesis of HCMV-induced thrombocytopenia. Megakaryocytic cells as well as supportive microenvironment in bone marrow are major targets of HCMV infection. Presented study mainly focused on the impacts of HCMV to megakaryocytic cells and multipotent mesenchymal stromal cells (MSCs) - the precursor of bone marrow stromal cells. Based on a megakaryocytic cell model challenged by HCMV in vitro, inhibited megakaryocytic endomitosis, proliferation, and cellular expression were respectively demonstrated as decreased polyploidy population, decreased colony formation, and reduced c-Mpl (thrombopoietin receptor) expressing cells. Evoked apoptosis of megakaryocytic cells was also evidenced with increased phosphatidylserine exposure on cell surface and intracellular caspase-3 activation after HCMV infection. Involvement of mitochondrial-mediated intrinsic apoptosis was further shown as losing JC-1 fluorescent signal in infected megakaryocytic cells. These results suggest that inhibition induced by HCMV is exerted through multiple processes directly affecting the megakaryopoietic development. Functional failure of bone marrow microenvironment was demonstrated in bone marrow derived MSCs infected by HCMV in vitro. Suppressed cytokine production, impaired cellular migration, and hindered differentiation of HCMV-infected MSCs were respectively demonstrated by lowered level of stromal cell-derived factor 1 in culture medium, decreased number of cells passed through a porous membrane in a transwell culture, and reduced differentiated cells in either adipogenic or osteogenic induction cultures. Alongside with these changes, HCMV-induced programmed cell death further contributed to the supportive failure. Autophagic cell death in infected MSCs was demonstrated as massive accumulation of vacuoles with double membrane structure and LC-3b II molecules followed by viability loss. De novo apoptosis was also observed as another process of programmed cell death, shown as increased phosphatidylserine exposure on cell surface and intracellular caspase-3 activation of infected MSCs. Increased programmed cell death appeared to be associated with extensive HCMV replication in MSCs, which was featured with typical cytopathic morphology, expression of viral tegument protein pp65, and massive accumulation of various viral particles including mature virions. Sustained activation of extracellular signal-regulated kinases likely represented a signal transduction network connecting viral expression or replication with programmed cell death. In a “MSCs-dependent” megakaryopoiesis model, HCMV-infected MSCs failed to support survival and maintenance of megakaryocytic cells. Taken together, these results suggest that active HCMV expression or replication inhibits multiple cellular functions and induces multiple processes of programmed cell death of MSCs. Such inhibition compromises supportive functions of bone marrow microenvironment, and subsequently reduces platelet production in an indirect manner. In summary, HCMV suppresses cellular function and induced apoptosis on both megakaryocytic cells and their supportive cells, MSCs. Therefore, the inhibitory effects of HCMV on megakaryopoiesis are operated via both direct and indirect mechanisms. / published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

The potential applications of mesenchymal stem cells (MSCs) have been widely advocated, however, many barriers hinder their clinical utilization. Enhancement of the homing of human MSCs (hMSCs) to the target tissues remains a clinical challenge. To overcome this hurdle, the mechanisms responsible for migration and engraftment of hMSCs have to be defined. My study aimed to explore both the underlying mechanisms and means of enhancing the migration of hMSCs. A graft versus host disease (GvHD) injury model and a novel orthotopic neuroblastoma model were established to delineate the distinct property of hMSCs homing towards either injured or cancerous tissues. This highly specific homing process was further revealed to be in a CXCR4-dependent manner. Notably, a novel gene, exchange protein directly activated by cAMP (Epac), was demonstrated to be actively involved in the hMSCs homing process. hMSCs expressed functional Epac and its activation significantly enhanced the migration and adhesion of hMSCs. Furthermore, Epac activation directly contributed to the chemotactic response of hMSCs to SDF-1, suggesting that Epac is linked to the stromal cell derived factor-1 (SDF-1) signaling cascades. Importantly, the homing of hMSCs towards injured tissues in vivo could be dramatically increased by Epac activation. hMSCs are adherent cells and their migration to distant tissues thus requires detachment into a suspension state. This disruption of cell-extracellular matrix interaction, known as anoikis stress, triggers programmed cell death, leading to a marked decrease in the efficiency of cell trafficking and engraftment. Anoikis stress induced massive cell death has emerged as the major challenge in the application of hMSCs. How some of the hMSCs can overcome this adversity and migrate towards distant destinations remains largely unexplored. It was observed that the surviving hMSCs circumvented anoikis stress by forming self-supporting cellular aggregates. Compared to adherent hMSCs, aggregated-hMSCs had better migratory response to both SDF-1α and SDF-1α analogue (CTCE-0214). Such enhanced migratory effect was proven to be CXCR4-dependent both in vitro and in vivo by using a CXCR4 specific antagonist (AMD3100). Although the viability of hMSCs under anoikis stress dramatically decreased, CTCE-0214 could promote cell survival and facilitate the migration of hMSCs towards injured targets. This phenomenon could be partially explained by the increase in anti-apoptosis effect via up-regulated Bcl-2 expression and autophagy activation under CTCE-0214 treatment. The exact effects of hMSCs on tumor growth and progression have long been controversial. Significant fasten growth and promoted metastasis of neuroblastoma in vivo was observed in hMSCs co-transplanted mice in this study. Reciprocally, hMSCs could not only be recruited by primary tumor, but also be selectively attracted by metastatic loci. This recruitment was significantly reduced when hMSCs were pre-treated with AMD3100, suggesting that the SDF-1/CXCR4 axis was a prime mover in this process. In summary, my study demonstrated that the migratory property of hMSCs could be enhanced by novel intrinsic and extrinsic factors using both in vitro and in vivo models. This study provides a new prospective on MSCs biology during the ex vivo manipulation process and I proposed means to overcome some of these hindrance so we can maximize the efficacy of clinical MSCs application in the future. / published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

Myelin is essential for neuronal survival and maintenance of normal functions of the nervous system. Demyelinating disorders are debilitating and are often associated with failure of resident oligodendrocyte precursor cells (OPCs) to differentiate into mature, myelinating oligodendrocytes. Derivation of OPCs, from a safe source that evades ethical issues offers a solution to remyelination therapy. We therefore hypothesized that bone marrow stromal cells (BMSCs) harbour neural progenitor cells at a pre-commitment stage and that in vitro conditions can be exploited to direct differentiation of these cells along the oligodendroglial lineage. For the current study, adult rat BMSCs used were >90% immunopositive for CD90, CD73, STRO-1 (stromal cell markers), 10% for nestin (neural progenitor marker) but negligible for CD45 (haematopoietic cell marker) as measured by flow cytometry. Transfer of the culture from a highly adhesive substratum to a moderately adhesive substratum resulted in increase in proportion of p75-positive cells but CD49b-positive cells remained at 97% and Sox 10-positive cells remained negligible. Transfer of the culture to a non-adherent substratum fostered the generation of neurospheres comprising cells that were positive for the neural stem/progenitor cell (NP) marker, nestin, and for the neural crest markers CD49b, p75 and Sox10. Prior to this stage, the BMSCs were not yet committed to the neural lineage even though transient upregulation of occasional marker may suggest a bias towards the neural crest cell lineage. The BM-NPs were then maintained in adherent culture supplemented with beta-Heregulin (β-Her), basic fibroblast growth factor (bFGF) and platelet-derived growth factor-AA (PDGF-AA) to direct differentiation along the oligodendroglial lineage. Within two weeks of glial induction, cells expressing the OPC markers - NG2, Olig2, PDGFRa and Sox10, were detectable and these could be expanded in culture for up to 3 months with no observable decline in marker expression. These BM-OPCs matured into myelinating oligodendrocytes after 2 weeks in co-culture with either dorsal root ganglion neurons or cortical neurons. In vivo myelination by BM-OPCs was demonstrated by exploitation of the non-myelinated axons of retinal ganglion cells of adult rats. By 8 weeks post-injection of BM-OPCs into the retina, myelin basic protein-positive processes were also observable along the retinal axons. The results not only suppport our hypothesis, but also provide pointers to the adult bone marrow as a safe and accessible source for the derivation of OPCs towards transplantation therapy in acute demyelinating disorders. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Mesenchymal stem cell (MSC) transplantation has achieved only modest success in the treatment of ischemic heart disease due to poor cell viability in the diseased microenvironment. Genetic manipulation on the MSCs holds promising prospects in enhancing cell tolerance against adverse environmental conditions. Recent studies demonstrate that the activation of the NRG1 (neuregulin 1) - ERBB4 (v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 4) pathway can enhance pro-survival signaling, stimulate mature cardiomyocyte cell cycle re-entry and cell division. In this study, I aimed to determine whether activating NRG1-ERBB4 in MSCs can enhance their cardioprotective effects following myocardial infarction. In chapter 3, I determined that MSC endogenously expresses NRG1, but not ERBB4. Considering the absence of ERBB4 in the MSCs might lead to mute response to its ligand NRG1, I exogenously manipulated ERBB4 into MSCs. In chapter 4, MSCs, with or without ERBB4 overexpression were transplanted into mice following myocardial infarction. The transplantation of MSCs with ERBB4 expression considerably improved left ventricular ejection fraction and reduced infarctsize, compared to unmodified MSCs and direct NRG1 injection. ERBB4 overexpression induced greater MSC survival following infarction. The transduction of ERBB4 in MSCs increased cell mobility and apoptotic resistance via a PI3K/Akt pathway under hypoxic conditions in the presence of NRG1. The transplantation of MSCs with ERBB4 expression induced cardiomyocyte division and protected them against apoptosis during early phase of infarction. In chapter 5, a novel autocrine loop regarding to NRG1-ERBB4-NRG1 signaling was identified. MSCs with ERBB4 overexpression in turn increased NRG1 synthesis and secretion. Conditioned medium of ERBB4-expressing MSCs containing elevated NRG1, promoted cardiomyocyte growth, division and anti-senescence, whereas neutralization of NRG1 blunted these effects. Injecting ERBB4-expressing MSCs restored NRG1 in the infarcted myocardium to a level comparable with that of the normal myocardium. These findings collectively suggest overexpressing ERBB4 in MSCs enhances the effectiveness of MSCtherapy following myocardial in farction through potentiating MSC survival and revitalizing endogenous repair and regeneration. The combination of ERBB4 and MSC is more efficient than naïve MSC or solely recombinant NRG1 injection, emerging as potential target for developing novel strategy in treating myocardial diseases. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

No description available.

Liver failure, which is the result of liver injury and pathological inflammation, is currently only successfully treated by organ transplantation. However donor organ shortages preclude transplantation for many patients in need. Thus, bioartificial liver systems (BALS) are being developed as a bridge to transplantation, or to create an environment conducive to liver regeneration. Hepatocytes, the main functional cells of the liver, are the cells of choice for BALSs, but in standard conditions ex vivo, they rapidly suffer from a reduction of their functionality and viability. Coculture with stromal cells, for example bone marrow mesenchymal stromal cells (BM-MSC), has been shown to improve, and extend, hepatocyte function ex vivo up to 21 days. But, only small numbers of BM-MSCs can be harvested from adult volunteers. We have previously described an alternative, more plentiful, source of MSCs — human umbilical cord perivascular cells (HUCPVC) — that are easily expanded and non-alloreactive. Our hypothesis was that HUCPVCs are putative stromal cells for hepatocytes. Our results show that HUCPVCs improved hepatocyte albumin secretion, urea synthesis and maintained hepatocyte cytochrome activity and the expression of hepato-specific genes. Furthermore, there was a net proliferation of hepatocytes, which were polarized in coculture with HUCPVCs, as judged by functional bile canaliculi that were present for up to 40 days. We found that both soluble and non-soluble factors contributed to these effects, while neither was able to allow net proliferation individually. Moreover, HUCPVCs expressed both hepato-trophic and anti-inflammatory factors, at different levels to BM-MSCs, indicating the potential for differential hepato-therapeutics. We conclude that HUCPVCs are putative stromal cells for hepatocytes; they improve hepatocyte functionality, polarity, morphology and net proliferation, and thus present an opportunity for the improvement of both BALS function and liver therapy.