In pathological states such as osteoarthritis, the complex metabolic balance of cartilage is disrupted, leading to a loss in the integrity and biomechanical function of cartilage. Osteoarthritis affects more than 20 million Americans, costing the United States economy over $60 billion yearly. Risk factors for osteoarthritis include age, excessive joint loading, and joint injury. Tissue engineering offers a potential solution for the replacement of diseased and/or damaged cartilage. Unfortunately, plentiful donor cell populations are difficult to assemble, as chondrocytes have a well characterized lack of expansion potential. Mesenchymal progenitor cells offer an alternative with a high expansion potential capable of supplying large quantities of cells. Using an immature bovine model, the chondrogenic differentiation of articular chondrocytes and bone marrow stromal cells was found to be scaffold, media and mechanical stimulation dependent. TGF-beta signaling participated in the response of articular chondrocytes to dynamic compressive loading, as well as enhanced the chondrogenesis of bovine BMSCs, through interactions between loading and TGF-beta/Smad signaling. Also, dynamic loading altered gene expression, matrix synthesis rates and intracellular phosphorylation for bovine BMSCs. However the response of the cells to dynamic loading depends on both media supplementation and the duration of unloaded culture. These studies establish signaling through the TGF-beta pathway as a mechanotransduction pathway for chondrocytes and chondroprogenitors in 3D culture.

Breast cancer is a very heterogeneous disease. This heterogeneity can be observed at many levels, including gene expression, chromosomal aberrations, and disease pathology. A clear understanding of these differences is important since they impact upon treatment efficacy and clinical outcome. Recent studies have demonstrated that the tumour microenvironment also plays a critical role in cancer initiation and progression. Genomic technologies have been used to gain a better understanding of the impact of gene expression heterogeneity on breast cancer, and have identified gene expression signatures associated with clinical outcome, histopathological breast cancer subtypes, and a variety of cancer-related pathways and processes. However, little work has been done in this context to examine the role of the tumour microenvironment in determining breast cancer outcome, or in defining breast cancer heterogeneity. Additionally, little is known about gene expression in histologically normal tissue adjacent to breast tumour, if this is influenced by the tumour, and how this compares with non-tumour-bearing breast tissue. By applying laser--capture microdissection and gene expression profiling to clinical breast cancer specimens the research presented in this thesis addresses these questions. / We have generated gene expression profiles of morphologically normal epithelial and stromal tissue, isolated using laser capture microdissection, from patients with breast cancer or undergoing breast reduction mammoplasty. We determined that morphologically normal epithelium and stroma exhibited distinct expression profiles, but molecular signatures that distinguished breast reduction tissue from tumour-adjacent normal tissue were absent. Stroma isolated from morphologically normal ducts adjacent to tumour tissue contained two distinct expression profiles that correlated with stromal cellularity, and shared similarities with soft tissue tumors with favourable outcome. Adjacent normal epithelium and stroma from breast cancer patients showed no significant association between expression profiles and standard clinical characteristics, but did cluster ER/PR/HER2-negative breast cancers with basal-like subtype expression profiles with poor prognosis. Our data reveal that morphologically normal tissue adjacent to breast carcinomas has not undergone significant gene expression changes when compared to breast reduction tissue, and provide an important gene expression data set for comparative studies of tumour expression profiles. / We compared gene expression profiles of tumour stroma from primary breast tumors and derived signatures strongly associated with clinical outcome. We present a new stroma-derived prognostic predictor (SDPP) that stratifies disease outcome independently of standard clinical prognostic factors and published expression-based predictors. The SDPP predicts outcome in several published whole tumour--derived expression data sets, identifies poor-outcome individuals from multiple clinical subtypes, including lymph node--negative tumors, and shows increased accuracy with respect to previously published predictors, especially for HER2-positive tumors. Prognostic power increases substantially when the predictor is combined with existing outcome predictors. Genes represented in the SDPP reveal the strong prognostic capacity of differential immune responses as well as angiogenic and hypoxic responses, highlighting the importance of stromal biology in tumour progression. / We show that gene expression in the breast tumour microenvironment is highly heterogeneous, identifying at least six different classes of tumour stroma with distinct expression patterns and distinct biological processes. Two of these classes recapitulate the processes identified in the stroma-derived prognostic predictor, while the others are new classes of stroma associated with distinct clinical outcomes. One of these is associated with matrix remodelling and is strongly associated with the basal molecular subtype of breast cancer. The remainder are independent of the previously published molecular subtypes of breast cancer. Additionally, based on independent data from over 800 tumors, the combinations of stroma classes and breast cancer subtypes identify new subgroups of breast tumors that show better discrimination between good and poor outcome individuals than the molecular breast cancer subtypes or the stroma classes alone, suggesting a novel classification scheme for breast cancer. This research demonstrates an important role for the tumour microenvironment in defining breast cancer heterogeneity, with a consequent impact upon clinical outcome. Novel therapies could be targeted at the processes that define the stroma classes suggesting new avenues for individualized treatment.

名古屋大学博士学位論文 学位の種類 : 博士(医学)(課程) 学位授与年月日:平成25年1月31日 匠原龍太郎氏の博士論文として提出された

Understanding the molecular networks that maintain articular cartilage and regulate chondrogenic differentiation of mesenchymal stromal cells (MSCs) are important prerequisites for the improvement of cartilage repair strategies. The first study within this thesis demonstrates that equine cord blood-derived MSCs induced towards a chondrogenic phenotype showed significantly increased miR-140 expression from day 0 to day 14, which was accompanied by decreased expression of previously identified miR-140 targets; ADAMTS-5 and CXCL12. The second study shows that in vitro chondrogenesis on fibronectin coated-PTFE inserts results in more homogeneous hyaline-like cartilage with an increased number of differentiated cells compared with pellet cultures. Finally, the expression of miR-140, miR-9, miR-155 and miR-146a was investigated in an in vitro model of osteoarthritis and suggests a possible role for miR-146a. These results suggest that microRNAs may be useful for directing or enhancing eCB-MSC chondrogenic differentiation and for developing novel biomarker panels of in vivo joint health. / Danish Agency for Science, Technology and Innovation; Equine Guelph; Grayson-Jockey Club Research Foundation; BioE.

No description available.

Tissue engineering and ex vivo gene therapy can be used synergically as tool to regenerate bone, which overcome the problems of currently available bone replacements. Recently, a new source of mesenchymal stromal cells (MSCs) has been found in the umbilical cord; human umbilical cord perivascular cells (HUCPVCs) provide an alternative to bone marrow derived MSCs and due to their easy harvest, fast expansion, and non-immunogeneic and immunomodulatory phenotype we hypothesized that HUCPVCs are a putative candidate cell source for osteogenic ex vivo gene therapy. This work proposes the generation of cocktails of genetically modified HUCPVCs and their cryopreservation as an “off the shelf” therapeutic. This approach involves the engineering of osteogenic cell populations, by genetically modifying HUCPVCs using recombinant adenoviruses to deliver four fundamental genes for bone formation: bone morphogenetic protein 2 (BMP-2), runt-related transcription factor 2 (Runx2), Osterix (OSX/SP7) transcription factor and vascular endothelial growth factor (VEGF). Our results show that HUCPVCs can be efficiently modified by adenoviruses and can be cryopreserved without affecting the production efficiency and bioactivity of proteins of interest produced by the cells. Moreover, overexpression of BMP2, Runx2 and SP7 enhances ALP activity levels in HUCPVCs and upregulates ALP, OPN, COL1A1 and OCN gene expression; data that provides the first evidence of the effects of combinational expression of BMP2, Runx2 and SP7. Furthermore, we report for the first time the genetic modification of human BMSCs to express SP7 and Runx2, which enhances their ALP activity and matrix mineralization capacity.

Background. Recently rodent and porcine bone marrow stromal cells (MSCs) have been reported to be uniquely immune tolerant. In order to confirm these findings in human cells, we tested the hypothesis that human MSCs are also immune tolerant, such that they can be useful as "universal donor cells" for myocardial regenerative therapy. / Methods. Immunocompetent female rats underwent left coronary ligations (n=90). They were randomized into 3 groups. In Group I, lac-Z labeled male human MSCs were implanted into the peri-infarcted area. In Group II and III isogenic rat MSCs or culture medium were injected respectively. Echocardiography was carried out to assess cardiac function, and the specimens were examined serially for up to 8 weeks with immunohistochemistry, FISH and PCR to examine MSCs survival and differentiation. / Results. Human MSCs were found to survive within the rat myocardium without immunosuppression. This was confirmed by PCR and FISH test. No cellular infiltration characteristic of immune rejection was noted. Some of these cells appeared to express cardiomyocyte-specific markers such as troponin-Ic and connexin-43. Furthermore, the implanted MSCs significantly contributed to the improvement in ventricular function and attenuated LV remodeling. / Conclusions. Human MSC survived within this xenogeneic environment, and contributed to the improvement in cardiac function. Our findings support the feasibility of using these cells as "universal donor cells" for xeno- or allo-geneic cell therapy, as they can be tested, prepared and stored well in advance for urgent use. Allogeneic MSCs from healthy donors may be particularly useful for severely ill or elderly patients whose own MSCs could be dysfunctional. / Plusieurs études ont récemment démontré la tolérance immunologiquedes cellules souches stromales (CSS) issues de rongeurs et de porcinés. Pour confirmer cesrésultats chez les cellules humaines, l'étude actuelle évalue l'effet des CSS humaines sur larégénération du myocarde chez des rats immunocompétents et étudie la possibilité d'utiliserces CSS comme « donatrices universelles» à la suite d'un infarctus.

Tissue engineering and ex vivo gene therapy can be used synergically as tool to regenerate bone, which overcome the problems of currently available bone replacements. Recently, a new source of mesenchymal stromal cells (MSCs) has been found in the umbilical cord; human umbilical cord perivascular cells (HUCPVCs) provide an alternative to bone marrow derived MSCs and due to their easy harvest, fast expansion, and non-immunogeneic and immunomodulatory phenotype we hypothesized that HUCPVCs are a putative candidate cell source for osteogenic ex vivo gene therapy. This work proposes the generation of cocktails of genetically modified HUCPVCs and their cryopreservation as an “off the shelf” therapeutic. This approach involves the engineering of osteogenic cell populations, by genetically modifying HUCPVCs using recombinant adenoviruses to deliver four fundamental genes for bone formation: bone morphogenetic protein 2 (BMP-2), runt-related transcription factor 2 (Runx2), Osterix (OSX/SP7) transcription factor and vascular endothelial growth factor (VEGF). Our results show that HUCPVCs can be efficiently modified by adenoviruses and can be cryopreserved without affecting the production efficiency and bioactivity of proteins of interest produced by the cells. Moreover, overexpression of BMP2, Runx2 and SP7 enhances ALP activity levels in HUCPVCs and upregulates ALP, OPN, COL1A1 and OCN gene expression; data that provides the first evidence of the effects of combinational expression of BMP2, Runx2 and SP7. Furthermore, we report for the first time the genetic modification of human BMSCs to express SP7 and Runx2, which enhances their ALP activity and matrix mineralization capacity.

Bone marrow-derived mesenchymal stem cells (MSC) have been shown to provide neuroprotection after transplantation into the injured nervous system. The present thesis investigates whether adult human and rat MSC differentiated along a Schwann cell lineage could increase their expression of neurotrophic factors and promote regeneration after transplantation into the injured peripheral nerve and spinal cord. Human and rat mesenchymal stem cells (hMSC and rMSC) expressed characteristic stem cell surface markers, mRNA transcripts for different neurotrophic factors and demonstrated multi-lineage differentiation potential. Following treatment with a cocktail of growth factors, the hMSC and rMSC expressed typical Schwann cells markers at both the transcriptional and translational level and significantly increased production of brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF). Age and time in culture are of relevance for clinical settings and growth-promoting effects of hMSC from young donors (16-18 years) and old donors (67-75 years) were compared. Undifferentiated hMSC from both young and old donors increased total neurite length of cultured dorsal root ganglion (DRG) neurons. Differentiation of hMSC from the young donors, but not the eldery donors, further enhanced the neurite outgrowth. Undifferentiated hMSC were cultured for eleven weeks in order to examine the effect of in vitro expansion time on neurite outgrowth. hMSC from the young donors maintained their proliferation rate and their ability to enhance neurite outgrowth from DRG neurons. Using a sciatic nerve injury model, a 10mm gap was bridged with either an empty tubular fibrin glue conduit, or conduits containing hMSC, with and without cyclosporine treatment. Cells were labeled with PKH26 prior to transplantation. At 3 weeks after injury the conduits with cells and immunosuppression increased regeneration compared with an empty conduit. PKH26 labeled human cells survived in the rat model and the inflammatory reaction could be suppressed by cyclosporine. After cervical C4 hemisection, BrdU/GFP-labeled rMSC were injected into the lateral funiculus rostral and caudal to the spinal cord lesion site. Spinal cords were analyzed 2-8 weeks after transplantation. Transplanted MSC remained at the injection sites and in the trauma zone for several weeks and were often associated with numerous neurofilament-positive axons. Transplanted rMSC induced up-regulation of vascular endothelial growth factor in spinal cord tissue rostral to the injury site, but did not affect expression of brain-derived neurotrophic factor. Although rMSC provided neuroprotection for rubrospinal neurons and significantly attenuated astroglial and microglial reaction, cell transplantation caused aberrant sprouting of calcitonin gene-related peptide immunostained sensory axons in the dorsal horn. In summary these results demonstrate that both rat and human MSC can be differentiated towards the glial cell lineage, and show functional characteristics similar to Schwann cells. hMSC from the young donors represent a more favorable source for neurotransplantation since they maintain proliferation rate and preserve their growth-promoting effects in long-term cultures. The data also suggest that differentiated MSC increase expression of neurotrophic factors and support regeneration after peripheral nerve and spinal cord injury.

Malignant glioma is a highly lethal and destructive disease with no proper cure. We have investigated some of the hallmarks of cancer in connection to glioma and found ways to disrupt these and prevent tumor growth. The work is done within the context of a glioma subtype distinguished by activation of PDGF signaling termed the proneural subtype. In two of the studies we have investigated mechanisms regulating the glioma cells themselves, and in the other two we have focused on the tumor stroma. In the first study, glioma-initiating cells were isolated in defined serum free culture medium from PDGF-B driven murine glioma and shown to be independent of EGF and FGF2 for self-renewal and proliferation. When cultured in serum the GICs displayed an aberrant differentiation pattern that was reversible. Specific depletion of the transduced PDGF-B caused a loss of self-renewal and tumorigenicity and induced oligodendrocyte differentiation. The transcription factor S-SOX5 has previously been shown to have a tumor suppressive effect on PDGF-B induced murine glioma, and to induce cellular senescence in PDGF-B stimulated cells in vitro. We found that S-SOX5 had a negative effect on proliferation of newly established human glioma cells cultured under stem cell conditions. We also revealed a connection between alterations causing up-regulation of SOX5 with the proneural subgroup and a tendency towards co-occurrence with PDGFRA alterations. Angiogenesis, the formation of new blood vessels from existing ones, is an important hallmark for glioma malignancy. We found that the anti-angiogenic protein HRG had a negative effect on glioma progression in PDGF-B induced experimental tumors and that HRG was able to completely prevent formation of glioblastomas. Subsequently it was shown that HRG could skew pro-tumorigenic tumor associated macrophages into an anti-tumorigenic phenotype. Stromal cells had not previously been fully investigated in gliomas. We observed a correlation between tumor malignancy and increased numbers of tumor-associated macrophages as well as pericytes in PDGF-B induced gliomas. There was also a correlation between tumor grade and vessel functionality that had not previously been shown. Our results offer further understanding of gliomagenesis and present possible future therapies.