Mesenchymal stromal cells (MSCs) are being explored for treatment of inflammatory, ischemic, autoimmune, and degenerative diseases. More and more of these diseases require MSCs to be delivered locally to the diseased site rather than systemically injected into patients. However, little is understood about whether cell cryopreservation or prelicensing will affect the efficacy of the locally injected product or how the local injection environment affects MSC expression of trophic factors and interactions with patient immune cells. Several groups have disagreed on whether cryopreservation hinders MSC potency and therefore it is important to understand the effects of cryopreservation on MSC function and in what contexts cryopreservation can be used. Therefore, a better understanding of MSC phenotype after local injection is needed so that cryopreservation and prelicensing can be optimized to modulate cell potency for more efficacious MSC products. Currently, it has been shown that in vivo there are rapid drastic shifts in gene expression by MSCs which have been locally injected. One of the most prominent gene changes is in the enzyme COX-2 which leads to the production of bioactive lipids called prostaglandins, namely PGE2. PGE2 has several functions depending on the context in which other cells encounter it. In order to model the gene changes that occur in vivo, in vitro cell aggregates termed spheroids have been utilized to study the effects of local injection of MSCs. MSC spheroids have shown more potency than their 2D counterparts in shifting macrophage polarization and rescue of cells from ischemic damage. This thesis examines how process variables like cryopreservation and prelicensing affect the efficacy of the MSC product in the context of local injection. Additionally, it shows how spheroid formation alters therapeutic factor expression and activity and how drug treatment and biomaterials can be utilized to modify potency of these cells. In Chapter 2, we demonstrate that cryopreservation in the context of an ischemia/reperfusion injury in the eye does not significantly decrease MSCs effectiveness in salvaging neuronal cells. However, IFN-γ, a commonly used prelicensing cytokine to increase MSC potency, led to a decrease in the effectiveness of MSCs in this model. Chapters 3 and 4 define the changes that occur to several of MSCs’ trophic factors including immunomodulatory and growth factors and how these alterations affect MSC interactions with macrophages and T cells. Because validation and tracking of locally injected products can be cost-prohibitive for many research groups, Chapter 5 lays out a low-cost method to track fluorescently labeled cells in local injections to skin to aid in minimization of variability in results obtained from animal wound healing models. These findings demonstrate that initial preparation of MSC therapeutics is critical to their efficacy in local injection. Therefore, careful testing of potency for large-scale MSC production pipelines should be evaluated to ensure the efficacy of the resulting product. Additionally, spheroids exhibit differences in the mechanisms of action due to alterations in their secretome which can be partly overcome with co-administration of steroids such as budesonide. Therefore, steroid co-administration with MSCs being considered for local application should be further explored for use in local delivery of MSCs for the treatment of inflammatory conditions. Finally, this research demonstrates the need to further understand the mechanisms by which spheroids alter their gene and trophic factor production to better tailor MSC therapies for disease specific localized injection.

Les syndromes myélodysplasiques (MDS) sont un groupe de pathologies myéloïdes caractérisées par une hématopoïèse inefficace. Le rôle du microenvironnement médullaire (MM) dans l’histoire naturelle de ces pathologies reste incertain. Des anomalies du MM ont été décrites au cours des myélodysplasies et des modèles murins récemment publiés font penser qu’une altération du MM pourrait jouer un rôle dans le déclenchement et/ou l’évolution de ces maladies.Nous avons tenté de développer un modèle in vivo récapitulant l’histoire naturelle des myélodysplasies par des xénogreffes chez des souris NSG et NSG-S. Le faible taux de prise de greffe nous a amenés à développer un modèle in vitro de co-culture en 2D. Ce modèle est une bonne alternative pour les études de nouvelles stratégies thérapeutiques pour les patients atteints de myélodysplasies.Au cours de ce travail, nous avons également réalisé une étude systématique du stroma médullaire de patients atteints de syndromes myélodysplasiques dans le but d’identifier les anomalies fonctionnelles et moléculaires des cellules souches mésenchymateuses (CSMs), cellules centrales du MM pour leur interaction avec les cellules souches hématopoïétiques (CSHs).Les CSMs de MDS ont une clonogénécité diminuée. Nous n’avons pas observé de modification significative de leurs capacités de différenciation en ostéoblastes, adipocytes et chondrocytes ni dans leur capacité à supporter une hématopoïèse normale. Les CSMs de MDS présentent des modifications au niveau épigénétique et transcriptionnel pouvant expliquer l’altération des relations observées grâce à de l’imagerie enregistrée entre les CSMs de MDS et les CSHs dans un modèle de co-culture en 3D.Ces résultats montrent que les CSMs de MDS ont des modifications fonctionnelles et moléculaires et que ces anomalies perturbent leur relation avec les CSHs. / Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal myeloid pathologies characterized by an impaired hematopoiesis. The role of the bone marrow microenvironment (BMM) remains unclear in the natural history of these diseases. Abnormalities of the BMM have been observed in myelodysplasia and a recent published murine model implies that alterations of the BMM could play a role in the trigger/progression of these diseases.Firstly, we tried to develop an in vivo model of MDS in NSG and NSG-S mice. The low rate of engraftment pushed us to develop a 2D co-culture model in vitro. This model is a good alternative to test new therapeutic strategies for MDS patients.In this study, we analysed mesenchymal stromal cells (MSCs) from the bone marrow of pretreated MDS patients in order to identify the functional and molecular abnormalities in those cells of the BMM, central for their interactions with the hematopoietic stem cells (HSCs).MDS MSCs have an impaired clonogenic capacity. We didn’t observed modifications of their differentiation toward osteogenic, adipogenic and chondrogenic pathways and capacity to support of a normal hematopoiesis. MDS MSCs display epigenetic and transcriptomic modifications that could explain the alteration of the relationships between these cells and HSCs observed in imagery in a 3D co-culture model.These results showed that MDS MSCs have functional and molecular abnormalities and that these alterations could impair their relationship with HSCs.

Multipotent hematopoietic stem cells (HSCs) are progenitors of all types of hematopoietic cells, and the efficient isolation and propagation of HSCs will significantly enhance our ability to manage many human disorders with bone marrow transplantation, stem cell transplantation and gene therapy. We employed "Signal Sequence Trap (SST)" method with yeast invertase to clone proteins on the surface of or secreted by stromal cells that enhance or inhibit the propagation of HSC’s in culture. AFT024, a mouse fetal liver stromal cell line that maintains stem cell activity in long-term culture, was subjected to SST analysis. We identified more than 60 signal sequences or transmembrane domain containing genes expressed by AFT024 cells. We compared their expression levels between AFT024 cells and BFC012 cells, a mouse fetal liver stromal cell line that was developed in the same way as for AFT024 cells but could not support HSC in long-term culture. Pleiotrophin, T16, Sca-1, deltalike and cytokine receptor like-1(CLF-1) are expressed significantly higher in AFT024 cells than in BFC012 cells. We recently employed Affymatrix genechip technology to study the interaction of HSCs and their microenvironment. In genechip experiments, Sca-1, deltalike, pleiotrophin and CLF-1 are among the most differentially expressed genes between AFT024 and BFC012 cells, while T16 was not represented on the chip. In addition, osteopontin, pigment epithelium-derived factor, proliferins, activin subunit, CXC chemokines GRO1 and LIX are more abundant in AFT024 cells than in BFC012 cells. Genechip technology was also applied to bone marrow stromal cell lines, including MS5, S17 and OP9 cells. Two murine multipotent hematopoietic cell lines, FDCP.mix and EML cells, were also analyzed. Data from these experiments are presented. / Singapore-MIT Alliance (SMA)

This thesis explores the capabilities of cationic polymers modified with lipids of different carbon chain length to deliver DNA molecules to primary cells and transformed cell lines. Our studies focus on two different polymers: polyethylenimine (PEI) and poly(L-lysine) (PLL). Firstly, PEI and PLL were conjugated to palmitic acid (C16). The delivery of plasmid DNA to rat bone marrow stromal cells (rat-BMSC) was evaluated by using a Green Fluorescent Protein gene expressing plasmid (pEGFP-N2) as a reporter system. The rationale for lipid substitution is to give the polymer an amphiphilic character so as to improve the transfection efficiency of native polymers by improving the DNA/polymer translocation through the phospholipid-rich cell membranes. In the case of PLL-C16, transfection efficiency was significantly increased (5 fold) as compared to native PLL, and it was significantly higher than commercially available cationic lipids (LipofectamineTM 2000 and FugeneTM). We further explore the use of other lipids with variable chain lengths (carbon chain length ranging from 8 to 18 saturated and unsaturated) in order to identify other candidates to enhance the gene delivery properties of the PLL. Lipid-modified PLL of high molecular weight (25 vs. 4 kDa) was found to be more effective in delivering plasmid DNA in rat-BMSC. We noted that C14-, C16- and C18-substituted PLL gave the most effective DNA delivery. Moreover, a correlation between the extent of lipid substitution and the plasmid DNA delivery efficiency was found Additionally, transgene expression by BMSC significantly increased when amphiphilic PLLs were used as compared to native PLL. The modified polymers were able to transfect the cells up to 7 days, after which the expression decreased. Encouraged by the successful transgene expression agents obtained by modifying low molecular weight PEI with the same series of lipids described above, we explored the possibility of modifying low molecular weight PEI (2 kDa) with longer lipids; saturated fatty acid (C22), trans fat (C18:1T) and essential fatty acids (C22:1, C22:6 and C18:3). Transfection efficiency proved to be cell dependent. Only the transformed 293T cells were able to express GFP compared to human-derived BMSC. The highest transfection efficiency was found with highly unsaturated lipid-substituted PEI (C18:3 and C22:6) and were able to increase transgene expression overtime (6 days). Furthermore, internalization studies indicated that effective transfection of these carries do not follow any known endocytosis pathway instead the DNA/carrier penetrates the plasma membrane directly. / Pharmaceutical Sciences

The islets of Langerhans are small and delicate spheroid organs scattered in the pancreas responsible for insulin production. Transplantation of isolated islets is a beneficial therapy for patients with a severe form of type 1 diabetes. The islets, which normally are richly vascularized in the pancreas, are completely disconnected from the vascular support by the enzymatic digestion during the isolation procedure. Islet viability is affected throughout all steps in this process, from donor death and isolation of islets to culturing and the transplantation process itself. In this thesis a novel strategy to promote islet survival and to re-establish islet vasculature is presented. We show endogenous expression of 51 different genes related to inflammation in cultured islets. Among these genes high expression of MCP-1, MIF, VEGF, thymosin b-10 and IL-8, IL-1β, IL-5R-a, IFN-γ antagonist were found in all donors during the 5- and the 2-day cultures, respectively. Protein expression of these genes can stimulate inflammatory immune responses but also promote tissue repair by attracting curative cells such as endothelial cells (EC) leading to re-establishment of the vasculature. We have established a novel technique by formation of composite islets using EC and mesenchymal stem cells (MSC). EC adhered on the surface of the islets and created a potential blood tolerant surface. The EC-islets showed a degree of protection from the detrimental effects of instant blood-mediated inflammatory reaction (IBMIR) with the major components of IBMIR being decreased in in vitro assays. We combined MSC to the EC-islets with success. The MSC were found to have proliferative effect on EC and the combination of these two cell types on the islets further increased the EC covered surface compared to EC-islets. The EC-MSC-islets in co-culture formed vessel-like structures both into the islets and out to the surrounding matrix. The MSC enhanced the exogenous EC to form vessel-like network in the EC-MSC-islets indicating vascular support by the MSC. The novel strategy and conditions presented herein could alleviate problems related to survival of the islets by promoting revascularization. This would open up a new era in islet transplantation and allow more patients to benefit from this therapy. / Clinical immunology, islet group

Bone marrow (BM)-derived mesenchymal stromal cells (MSCs) can be induced to express cardiac-specific markers by embryonic cardiomyocytes in vitro. To determine whether this phenomenon occurs in vivo, we have developed a cell transplantation system using zebrafish embryonic recipients. We were unable to isolate expandable zebrafish kidney stromal (ZKS) cells from the kidney, the human BM equivalent; hence, we analyzed the established ZKS1 cell line. We found that ZKS1 expresses stromal genes, but also expresses hematopoietic genes not normally expressed by MSCs. Furthermore, we were unable to differentiate ZKS1 cells into adipocytes, osteoblasts or cardiomyocytes in vitro. We created a transgenic ZKS1(CMV:eGFP) cell line which, after transplantation into zebrafish blastulae, was observed within the host heart, among other tissues. Finally, pT2/S2tnnt2-GM2 and pT2/S2tnnt2-DsRed transposons were generated to mark ZKS1 cardiac differentiation. The zebrafish model established here will be useful for studying the molecular mechanisms of exogenous MSC cardiac differentiation in vivo.

Bone marrow (BM)-derived mesenchymal stromal cells (MSCs) can be induced to express cardiac-specific markers by embryonic cardiomyocytes in vitro. To determine whether this phenomenon occurs in vivo, we have developed a cell transplantation system using zebrafish embryonic recipients. We were unable to isolate expandable zebrafish kidney stromal (ZKS) cells from the kidney, the human BM equivalent; hence, we analyzed the established ZKS1 cell line. We found that ZKS1 expresses stromal genes, but also expresses hematopoietic genes not normally expressed by MSCs. Furthermore, we were unable to differentiate ZKS1 cells into adipocytes, osteoblasts or cardiomyocytes in vitro. We created a transgenic ZKS1(CMV:eGFP) cell line which, after transplantation into zebrafish blastulae, was observed within the host heart, among other tissues. Finally, pT2/S2tnnt2-GM2 and pT2/S2tnnt2-DsRed transposons were generated to mark ZKS1 cardiac differentiation. The zebrafish model established here will be useful for studying the molecular mechanisms of exogenous MSC cardiac differentiation in vivo.

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