Final publication is available at http://www.sciencedirect.com/science/article/pii/S0006291X15310664 / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19598号 / 医博第4105号 / 新制||医||1014(附属図書館) / 32634 / 京都大学大学院医学研究科医学専攻 / (主査)教授 三森 経世, 教授 開 祐司, 教授 妻木 範行 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Introduction It is well known that resident adipose stem/stromal cells (ASCs) are a heterogeneous population of multipotent cells characterized by (a) their ability to adhere to plastic; (b) immunophenotypic expression of certain cell surface markers, while lacking others; and (c) the capacity to differentiate into cells of mesodermal origin including osteocytes, chondrocytes and adipocytes. Adipose derived stromal cells offer great therapeutic potential in multiple medical fields, including, orthopedics, cardiology, oncology and degenerative diseases, to name a few. Combining different disciplines of medicine and engineering, organ and tissue repair can be achieved through tissue engineering and regenerative medicine. Adipose derived stromal cells (ASCs) can be utilized as biological vehicles for vector-based gene delivery systems, since they home to sites of inflammation and infection in vivo. In order to reach the long-term aim of clinical translation of cell-based therapy, preclinical safety and efficacy need to be shown in animal models. This has motivated the development of standardized isolation, characterization and differentiation operating procedures as well as an in vivo tracking system for ASCs and lentiviral vector transduction for a vector-based gene delivery system. Methodology Human ASCs were isolated from lipoaspirate, expanded in culture, immunophenotyped using flow cytometery and induced to differentiate into adipogenic, osteogenic and chondrogenic lineages. Tri-lineage differentiation was confirmed by microscopy. The ASCs were then transduced with green fluorescent protein (GFP)-expressing lentiviral vectors in vitro. The effect of the GFP lentiviral vector on ASCs was investigated by studying ASC immunophenotypic expression of surface markers as well as their capacity to differentiate into osteocytes, chondrocytes and adipocytes. Results The isolated and expanded cell population, from harvested lipoaspirate adhered to recommended ASC identity criteria. The heterogeneity of ASCs was confirmed by the presence of sub-populations. Transduction efficiency in ASC cultures of approximately 80% was observed after introducing a total of 300 μl of concentrated lentiviral vector suspension per 4.8 x 104 cells. No immunophenotypic differences were observed between GFP positive and GFP negative cultures. Flow cytometric analysis revealed a progressive increase in GFP expression following in vitro expansion of transduced ASCs. Both non-transduced and transduced cultures successfully differentiated into osteocytes, chondrocytes and adipocytes. Conclusion The isolated and expanded cell population conformed to the recommended characterization criteria. Heterogeneity was demonstrated with the identification of immunophenotypic sub-populations and semi-quantification of adipogenesis was performed. ASCs were efficiently transduced using the GFP lentiviral vectors produced in our facility. In addition, transduced ASCs maintained adherence to plastic, ASC immunophenotype and were able to differentiate successfully into cells of the three lineages of mesodermal origin. This optimized GFP-ASC transduction technique offers a feasible tracking system as well as a vector-based gene delivery system for future preclinical studies. / Dissertation (MSc)--University of Pretoria, 2015. / tm2015 / Immunology / MSc / Unrestricted

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21018号 / 医博第4364号 / 新制||医||1028(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 小川 誠司, 教授 柳田 素子, 教授 江藤 浩之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Les cellules stromales/souches mésenchymateuses de la gelée de Wharton humaines (CSM-WJ) représentent une source abondante et intéressante de cellules souches pour des applications en ingénierie cellulaire et tissulaire. Leur origine fœtale leur confère des caractéristiques spécifiques par rapport aux cellules stromales/souches mésenchymateuses isolées à partir de moelle osseuse humaine (CSM-MO). Tout d'abord, le but de ce travail est d'optimiser les conditions de culture des CSM-GW pour leur utilisation clinique ultérieure. Nous nous concentrons sur l'influence de la concentration en oxygène lors de l'expansion en monocouche de P1 à P7 sur plusieurs paramètres permettant de caractériser les CSM. Les résultats obtenus sont comparés à ceux obtenus avec les CSM-MO. Notre travail a montré des différences entre les deux sources cellulaires en termes de prolifération et de différenciation adipocytaire. D’après nos résultats, l'hypoxie, au cours de l'expansion, est un paramètre important à prendre en compte en ce qui concerne la prolifération et le potentiel de différenciation chondrocytaire. L'influence des facteurs obstétricaux sur les caractéristiques des CSM-GW est également explorée. Cette étude se situant également dans le cadre de l’ingénierie tissulaire du cartilage, la seconde phase du projet consiste à induire la différenciation des cellules en chondrocytes en ensemençant ces dernières dans un biomatériau à base d’alginate et d’acide hyaluronique, et sur une cinétique de 28 jours. Les résultats obtenus sont comparés à ceux obtenus avec les CSM-MO. Après 4 semaines de culture, les CSM-GW sont capables de s'adapter à leur environnement et d’exprimer des gènes et des protéines matriciels spécifiques du cartilage tels que le collagène de type 2, qui se trouve plus exprimé après différenciation à partir des CSM-GW qu’à partir de CSM-MO / Mesenchymal Stromal/Stem Cells from human Wharton’s jelly (WJ-MSC) are an abundant and interesting source of stem cells for applications in cell and tissue engineering. Their fetal origin confers specific characteristics compared to Mesenchymal Stromal/Stem Cells isolated from human bone marrow (BM-MSC). First, the aim of this work is to optimize WJ-MSC culture conditions for their subsequent clinical use. We focus on the influence of oxygen concentration during monolayer expansion on several parameters to characterize MSC. The results are compared to those obtained with BM-MSC. Our work distinguishes WJ-MSC from BM-MSC in terms of proliferation and adipogenic differentiation. Considering our results, hypoxia during cell expansion is an important parameter to take into account regarding proliferation potential but also chondrogenic differentiation potential. The influence of obstetric factors on WJ-MSC characteristics is also explored. In cartilage tissue engineering context, the second phase of the project is to induce cell differentiation into chondrocytes by seeding them in Alginate/Hyaluronic Acid hydrogel scaffold, and during 28 days. The results obtained are compared to those obtained with BM-MSC. After 4 weeks of culture, WJ-MSC are able to adapt to their environment and express specific cartilage-Related genes and matrix proteins such as type 2 collagen, which is found more expressed after differentiation fromWJ-MSC, than from BM-MSC

The acute respiratory distress syndrome (ARDS) and its less severe form, acute lung injury (ALI), are among the leading causes of morbidity and mortality in critically ill patients. Commonly induced by conditions associated with severe pulmonary inflammation, ALI results in disruption of the lung alveolar-capillary membrane barrier and resultant pulmonary edema associated with a proteinaceous alveolar exudate. Sepsis is another frequent and often fatal clinical condition for patients in the intensive care unit. It is characterized by a combination of infection and systemic inflammatory response syndrome (SIRS). Current effective treatment strategies for both ALI/ARDS and sepsis are lacking. We first examined the potential therapeutic role of mesenchymal stromal cells (MSCs) alone or together with the vasculoprotective factor, angiopoietin-1 (ANGPT1), for treatment of experimental ALI in mice. MSCs significantly reduced LPS (lipopolysaccharide)-induced pulmonary inflammation, as reflected by cell counts in bronchoalveolar lavage (BAL) fluid and pro-inflammatory cytokine levels in both BAL fluid and lung parenchymal homogenates. More importantly, administration of MSCs transfected with human ANGPT1 plasmid (MSCs-pANGPT1) completely reversed LPS-induced permeability in the lung (i.e., ALI). A follow-up study showed that MSCs remained effective in rescuing mice with LPS-induced ALI; however, the additional benefit from ANGPT1 was no longer observed. To further evaluate MSC-based therapy in a more clinically relevant model of acute injury, the cecal-ligation-and-puncture (CLP) model for sepsis was employed. Our results demonstrated that MSCs can reduce both systemic and pulmonary inflammation, as well as renal and liver dysfunction/injury, as reflected by plasma urea and bilirubin levels, in septic mice. Most notably, MSCs reduced sepsis-associated mortality from 45% to 24%. Our data demonstrate the feasibility and effectiveness of MSC- and MSC-based gene therapy for experimental ALI and sepsis, and provide the basis for the development of an innovative approach for the prevention and treatment of clinical ALI/ARDS and sepsis.

The acute respiratory distress syndrome (ARDS) and its less severe form, acute lung injury (ALI), are among the leading causes of morbidity and mortality in critically ill patients. Commonly induced by conditions associated with severe pulmonary inflammation, ALI results in disruption of the lung alveolar-capillary membrane barrier and resultant pulmonary edema associated with a proteinaceous alveolar exudate. Sepsis is another frequent and often fatal clinical condition for patients in the intensive care unit. It is characterized by a combination of infection and systemic inflammatory response syndrome (SIRS). Current effective treatment strategies for both ALI/ARDS and sepsis are lacking. We first examined the potential therapeutic role of mesenchymal stromal cells (MSCs) alone or together with the vasculoprotective factor, angiopoietin-1 (ANGPT1), for treatment of experimental ALI in mice. MSCs significantly reduced LPS (lipopolysaccharide)-induced pulmonary inflammation, as reflected by cell counts in bronchoalveolar lavage (BAL) fluid and pro-inflammatory cytokine levels in both BAL fluid and lung parenchymal homogenates. More importantly, administration of MSCs transfected with human ANGPT1 plasmid (MSCs-pANGPT1) completely reversed LPS-induced permeability in the lung (i.e., ALI). A follow-up study showed that MSCs remained effective in rescuing mice with LPS-induced ALI; however, the additional benefit from ANGPT1 was no longer observed. To further evaluate MSC-based therapy in a more clinically relevant model of acute injury, the cecal-ligation-and-puncture (CLP) model for sepsis was employed. Our results demonstrated that MSCs can reduce both systemic and pulmonary inflammation, as well as renal and liver dysfunction/injury, as reflected by plasma urea and bilirubin levels, in septic mice. Most notably, MSCs reduced sepsis-associated mortality from 45% to 24%. Our data demonstrate the feasibility and effectiveness of MSC- and MSC-based gene therapy for experimental ALI and sepsis, and provide the basis for the development of an innovative approach for the prevention and treatment of clinical ALI/ARDS and sepsis.

No description available.

La transplantation rénale, thérapie de choix de l'insuffisance rénale chronique terminale, est limitée par une pénurie d'organes. Les greffons issus de donneurs décédés par arrêt cardiaque (DDAC) peuvent contribuer à pallier à cette pénurie au prix de stratégies thérapeutiques visant à améliorer l'issue de la transplantation. Les cellules souches mésenchymateuses (MSC) de l'organisme adulte ont des propriétés de sécrétion, d'immunomodulation et de différenciation intéressantes dans ce contexte.L'objectif de ce travail est d'évaluer, dans un modèle pré-clinique porcin de DDAC, le potentiel thérapeutique de deux populations cellulaires d'intérêt : les MSC issues du liquide amniotique (AFSC) et les cellules de la fraction vasculaire stromale du tissu adipeux (SVF). Les AFSC porcines injectées dans l'artère rénale 7 jours post-greffe, en raison de leur sensibilité à une séquence d'hypoxie réoxygénation (HR) in vitro, accélèrent la reprise de fonction et réduisent l'extension des lésions chroniques du greffon et sont détectées dans le rein 24h après injection. La SVF porcine, phénotypiquement proche de celle de l'Homme, est moins sensible à cette séquence d'HR et peut être injectée dans l'artère du greffon à sa reperfusion sans perturbation du flux sanguin rénal, avec une rétention des cellules dans le rein 24h post injection.Ce travail met en évidence le rôle bénéfique des AFSC dans la réparation des lésions ischémie-reperfusion des greffons issus des DDAC, ainsi que la faisabilité de l'injection de la SVF dans l'artère rénale après transplantation, et ouvre des pistes pour l'optimisation les protocoles d'administration de produits de thérapie cellulaire en transplantation. / Kidney transplantation is the best therapeutic option for end stage chronic kidney failure, but is limited by transplant shortage. Use of transplants from deceased after cardiac death donors (DCD) could represent an additional graft source, but there is a need for developing new therapeutic approaches like cell therapy to increase their recovery. Mesenchymal stem cells (MSC) potentially extracted from many adult tissues have interesting paracrine, immune-modulating, and differentiation properties in this context. This work aims to assess, in a preclinical porcine model DCD donor, the therapeutic potential of two cell populations of interest: amniotic fluid derived MSC (AFSC) and cells from stromal vascular fraction of adipose tissue (SVF). Delayed injection of AFSC 7 days following kidney transplantation because of their sensitivity to a specific Hypoxia Reoxygenation (HR) sequence in vitro, accelerates graft function recovery and limits chronic injuries to the transplanted organ. Cells are detectable into the transplanted kidney 24h after injection. Porcine SVF is phenotypically similar to human. Injected in renal artery simultaneously with organ reperfusion because of its resistance to the HR sequence, porcine SVF does not disturb renal blood flow and allow cell-retention within the organ 24h after injection. This work highlights the protective effect of AFSC against ischemia reperfusion lesions in grafts from DCD donors and the feasibility of SVF injection directly into the renal artery of the graft following kidney transplantation in DCD conditions. Moreover it opens new lines for optimizing injection protocols of cellular products in kidney transplantation.

Dans le cas de larges pertes de substance osseuse, l’ingénierie tissulaire représente une alternative intéressante aux greffes. Cette technique consiste à associer des cellules à des biomatériaux dans le but de réparer le tissu. L'objectif de ce travail est l'étude de l'amélioration du potentiel ostéogénique des cellules souches mésenchymateuses issues du tissue adipeux humain (ASC), afin d’augmenter la formation de matrice osseuse en territoire ischémique. Nous avons montré que le fucoïdane, un polysaccharide d’origine marine, était capable d’améliorer la différenciation ostéogénique des ASC in vitro. Cependant, la combinaison de ces cellules avec des biomatériaux (granules de biphosphate de calcium) ne suffit pas à permettre une formation osseuse dans un modèle de pousse osseuse en site ectopique chez la souris. Afin d’augmenter l’angiogenèse, essentielle dans la réparation osseuse, nous avons associé les ASC à des cellules progénitrices endothéliales (appelées ECFC), dans ce modèle. Cette association ne permet d’améliorer que faiblement la formation osseuse. Nos études in vitro d'association de CPE et d'ASC ont montré que ces cellules en coculture étaient capables de synthétiser un grand nombre de cytokines impliquées dans les différenciations ostéogénique et angiogénique, telles que le transforming growth factor (TGFß1), l’insulin like growth factor (IGF-1) ou encore le vascular endothelial growth factor (VEGF). Dans nos conditions de culture, le surnageant de l’association des ECFC avec des ASC induit, par rapport au surnageant des ASCs seules, une inhibition de la différenciation ostéogénique dont le mécanisme reste à identifier.L’ensemble de nos données démontre le potentiel du fucoïdane dans l’ingénierie tissulaire osseuse et que les ASC seules ne sont pas capables de former de matrice osseuse. / In the case of large bone defects, tissue engineering represents an attractive alternative to transplantation. Tissue engineering is a combination of cells with biomaterials in order to repair tissue. The aim of this work was the study of the improvement of the osteogenic potential of mesenchymal stromal/stem cells derived from human adipose tissue (ASC) in the order to increase the formation of bone matrix in the ischemic territory. We have shown that fucoidan, a marine polysaccharide, was able to improve the osteogenic differentiation of ASC in vitro. However, the combination of these cells with biomaterials (biphasic calcium phosphate particles) is not enough to have bone formation in an ectopic bone growth model in mice. To promote angiogenesis, a crucial step in bone repair, we associated ASC with endothelial progenitor cells (called ECFC), in our model. This association promotes only lightly the bone formation. Our in vitro coculture studies of ECFC with ASC showed that the cells in coculture were able to synthesize several cytokines involved in angiogenic and osteogenic differentiation, such as transforming growth factor (TGF-ß1), insulin like growth factor (IGF-1) or vascular endothelial growth factor (VEGF). However, ASC in coculture did not express the receptors of these cytokines. In our culture conditions, the supernatant of the association of ECFC + ASC induces, compare to ASC alone, an inhibition of osteogenic differentiation which mechanism has to be identified.Our data show the potential of fucoidan in bone tissue engineering and that ASC alone did not promote bone matrix formation.