CD4+CD25+Foxp3+ T regulatory (Treg) cells are critical in maintaining self tolerance and promoting the acceptance of allogeneic tissue/organ grafts. To be widely applied in clinical settings, there needs to be a readily available source of Treg cells, a requirement that is better met if non-histocompatible donor cells could be utilized in adoptive therapy. Therefore, to investigate the therapeutic potential of fully allogeneic Treg cells to control autoimmune disease or allograft rejection, we utilized IL-2R beta-deficient mice that exhibit rapid lethal autoimmunity due to low production of an ineffective population of Treg cells. We show that adoptive transfer of MHC-mismatched Treg cells into IL-2R beta-/- mice resulted in life-long engraftment of the donor cells, which exhibited skewed reactivity toward host alloantigens, and prevented autoimmunity. When such animals received skin grafts, they exhibited tolerance to those grafts that expressed MHC molecules from which the donor Treg cells were derived. Collectively, these data provide proof-of-principle that effective engraftment by allogeneic Treg cells controls autoimmunity and leads to favorable conditions for long-term acceptance of allografts. Current data indicates that CD4+CD25+Foxp3+ Treg cells exhibit a broad TCR repertoire. However, the relationship between this diversity and capacity to control a similarly diverse population of potentially autoreactive T cells remains to be defined. To investigate this issue, we assessed the TCR repertoire of chimeric donor Treg cells in IL-2R beta-/- mice that were adoptively treated with a diverse polyclonal Treg inoculums. We demonstrate that autoimmune disease was fully prevented by engrafted donor Treg cells in spite of a TCR repertoire that is less diverse than the input cells. However, in settings where the input TCR repertoire is limited by utilizing donor Treg cells that express a single TCR beta chain, control of disease was hampered, correlating with a limited TCR alpha repertoire within the engrafting donor Treg cells. Collectively, these findings suggest that for adoptive therapy, a diverse TCR repertoire of input Treg cell inoculums is an essential requirement for effective control of polyclonal autoreactive T cells but perturbations in the repertoire that results in significant limitation to this diversity may compromise Treg cell efficacy at fully keeping autoaggressive cells in check.

付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム / 京都大学 / 新制・課程博士 / 博士(医科学) / 甲第23116号 / 医科博第127号 / 新制||医科||8(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 藤田 恭之, 教授 渡邊 直樹, 教授 岩田 想 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

<p>This project is undergoing the patent application, so it is confidential and should not be disclosed. Further questions can be asked by contacting Dr. Jeroen Goos, whose contact information was shown in the supervisor section.</p>

While physical rehabilitation facilitates some recovery, it is uncommon for patients to recover completely from stroke. Cell based therapies derived from stem cells have produced promising results in enhancing recovery in pre-clinical studies, but the mechanism is not yet completely understood. We previously evaluated human umbilical tissue-derived cells (hUTC) in our non-human primate model of cortical injury, limited to the hand area of primary motor cortex. hUTC treatment, injected intravenously 24 hours after injury, resulted in significantly greater recovery of fine motor function compared to treatment with vehicle. Based on these striking findings, in the current study, we investigated the hypothesis that hUTC treatment leads to functional recovery through reducing cytotoxic responses and enhancing neurorestorative processes following cortical injury. Brain sections were assessed using histological techniques to quantify perilesional oxidative damage, hemosiderin accumulation, microglial activation, Betz cell number, synaptic density, and astrocytic complexity. Brain sections outside of the primary area of injury were also assessed for microglial activation in white matter pathways, cell activation through c-Fos in premotor cortices, and neurogenesis in neurogenic niches. Finally, blood samples from throughout the recovery period and CSF samples from 16 weeks after injury were analyzed for BDNF levels. In the perilesional area, hUTC treatment was associated with lower oxidative damage and hemosiderin accumulation, but not with a difference in microglial activation. hUTC also resulted in a trend toward higher astrocyte complexity and synaptic density in the lesion area, but no difference in ipsilesional Betz cell number. Further, hUTC treatment led to more microglia in white matter pathways, higher c-Fos activation in ventral premotor cortex, and a trend toward higher neurogenesis in the hippocampus. Finally, BDNF levels were higher in blood with hUTC treatment one week after injury, but there was no change beyond one week in blood serum or in CSF, when compared with vehicle. Taken together, these results suggest that hUTC treatment modulates immune responses, limits perilesional damage and cell death, enables neuroplasticity and reorganization, and enhances acute neurotrophic factor secretion. While many cell therapies are currently undergoing clinical trials, this study advances our understanding of the mechanism of cell based therapies.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19561号 / 医博第4068号 / 新制||医||1013(附属図書館) / 32597 / 京都大学大学院医学研究科医学専攻 / (主査)教授 吉村 長久, 教授 戸口田 淳也, 教授 高橋 淳 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Orientador: Rogerio Martins Amorim / Resumo: Estudos com células estromais mesenquimais multipotentes (MSCs) estão em crescente progresso devido às suas propriedades imunomoduladoras, antiinflamatórias, antiapoptóticas e de regeneração tecidual, tornando essa modalidade de terapia celular promissora no tratamento de diversas doenças. Devido à limitada capacidade regenerativa do sistema nervoso central (CNS), causando sequelas funcionais, as MSCs estão sendo investigadas como uma alternativa terapêutica para condições neurológicas inflamatórias, vasculares, traumáticas e degenerativas em diversas espécies animais. A Mieloencefalite protozoária equina (EPM) causada por ambos os protozoários do filo Apicomplexa, Sarcocystis neurona e Neospora hughesi, permanece como uma importante doença neurológica dos equinos nas Américas, embora a maioria dos casos seja devida à infecção por S. neurona. A aplicação da proteômica com sua gama de ferramentas na clínica de equinos pode contribuir significativamente para o entendimento de processos patológicos e facilitar a descoberta de novos alvos terapêuticos ou marcadores diagnósticos. Neste contexto, os objetivos deste estudo foram avaliar o perfil proteômico do líquido cefalorraquidiano (CSF) antes e após múltiplos transplantes intratecal de MSCs em equinos hígidos e o perfil proteômico do CSF de equinos cronicamente afetados pela EPM. Doze cavalos adultos clinicamente saudáveis foram divididos aleatoriamente em três grupos experimentais: grupo DPBS (DPBS ou control; n = 4) onde a sol... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Multipotent mesenchymal stromal cell (MSCs) studies are under increasing progress because of their immunomodulatory, anti-inflammatory, antiapoptotic and tissue regeneration properties, making this modality of cell therapy promising in the treatment of various diseases. Due to the limited regenerative capacity of the central nervous system (CNS), causing functional sequelae, MSCs are being investigated as a therapeutic alternative for inflammatory, vascular, traumatic and degenerative neurological conditions in various animal species. Equine protozoal myeloencephalitis (EPM) caused by both protozoa of the Apicomplexa phylum, Sarcocystis neurona and Neospora hughesi, remains an important neurological disease in horses in the Americas, although most cases are due to S. neurona infection. The application of proteomics with its range of tools in the equine clinic can contribute significantly to the understanding of pathological processes and facilitate the discovery of new therapeutic targets or diagnostic markers. In this context, the objectives of this study were to evaluate the proteomic profiling of cerebrospinal fluid (CSF) before and after multiple intrathecal transplantations of MSCs in healthy horses and the CSF proteomic profiling of horses chronically affected by EPM. Twelve clinically healthy adult horses were randomly divided into three experimental groups: DPBS (DPBS or control; n = 4), in which intrathecal "transplants" with Dulbecco's phosphate buffered saline (DPB... (Complete abstract click electronic access below) / Doutor

No description available.

Human mesenchymal stem cells (hMSCs) have been identified as a promising cell-based therapy candidate to treat a number of unmet clinical indications, however, in vitro expansion will be required to increase the available number of cells and meet this demand. Scalable manufacturing processes, amenable to closed, single-use and automated technology, must therefore be developed in order to produce safe, effective and affordable hMSC therapies. To address this challenge, a controlled serum-free end-to-end microcarrier process has been developed for hMSCs, which is amenable to large-scale manufacture and therefore increasing economies of scale. Preliminary studies in monolayer culture assessed the level of variability in growth between five hMSC donors, which was found to have a variance of 25.3 % after 30 days in culture. This variance was subsequently reduced to 4.5% by the development of a serum-free monolayer culture process with the maintenance of critical hMSC characteristics and an increased number of population doublings. In order to transfer this into a scalable system, the serum and serum-free expansion processes were transferred into suspension by the addition of plastic microcarriers in 100 mL spinner flasks without control of pH or dissolved oxygen (DO). This achieved a maximum cell density of 0.08 ± 0.01 · 106 cells.mL-1 in FBS-based medium, 0.12 ± 0.01 · 106 cells.mL-1 in HPL-based medium and 0.27 ± 0.03 · 106 cells.mL-1 in serum free medium after six days. In order to drive consistency and yield into the manufacturing process, a process control system was developed for the FBS-based microcarrier expansion process in a 100 mL DASbox bioreactor platform to control DO, pH, impeller rate and temperature. Reduced impeller rates and DO concentrations were found to be beneficial, with a final cell density of 0.11 ± 0.02 · 106 cells.mL-1 and improved post-harvest outgrowth and colony-forming unit (CFU) potential compared to uncontrolled microcarrier and monolayer culture. This controlled bioreactor expansion process was then applied to the previously developed serum-free microcarrier process, eventually achieving a final cell density of 1.04 ± 0.07 · 106 cells.mL-1, whilst retaining key post-harvest hMSC characteristics. Following the controlled serum-free expansion and harvest of hMSCs, a downstream and cryopreservation process was developed to assess the impact of prolonged holding times and subsequent unit-operations on hMSC quality characteristics. This showed that hMSCs are able to maintain key characteristics throughout the entire end-to-end process, demonstrating their potential for commercial scale manufacture.

The intervertebral disc (IVD), situated between adjoining vertebrae, consists of the gelatinous nucleus pulposus (NP) in the centre surrounded by the tougher annulus fibrosus (AF). Its main roles are to distribute loads and to act as joints. With aging, degenerative disc disease (DDD) occurs due to an imbalance in anabolic and catabolic events in the IVD, which results in a loss of function. Lower back pain (LBP) affects 84% of people at some point in their lifetime and is strongly associated with DDD. Current LBP treatments have limited long term efficacy and are symptomatic rather than curative. Cell-based therapies are regarded to hold great potential for the treatment of DDD as it has been hypothesised that they could regenerate the damaged tissue and alleviate LBP. A number of natural and synthetic biomaterials have been investigated as NP tissue engineering scaffolds with varying results. In this study, a self assembling peptide hydrogel (SAPH) was investigated for its potential as a cell carrier and/or scaffold for NP tissue engineering. SAPHs display the advantages of natural polymer hydrogels such as biocompatibility and biodegradability whilst combining the advantages of synthetic materials such as controlled structural and mechanical propertiesCharacterisation determined that the SAPH nanofibrous architecture had features that were of similar scale to extracellular matrix (ECM) components of the human NP. The mechanical properties of the SAPH could be optimised to closely match the native tissue. The system could shear thin and self-heal making the system ideally suited to delivery via minimally invasive procedure. The three dimensional (3D) culture of bovine NP cells (bNPCs) in the SAPH demonstrated that the NP phenotype could be restored after de-differentiation during monolayer culture. Gene expression results demonstrated that ‘traditional’ and ‘novel’ NP markers were highly expressed throughout in vitro culture. Cell viability was high, cell population remained stable and bNPCs adopted the characteristic rounded morphology of native NPCs. Finally, type II collagen and aggrecan, the main ECM components of the NP, were deposited with increasing production over culture period. Growth differentiation factor 6 (GDF-6) has been identified as the most promising current growth factor for inducing discogenic differentiation from human bone marrow mesenchymal stem cell (h-BMMSCs). After samples were stimulated with GDF-6, gene expression results confirmed that a NP-like phenotype could be induced with high expression of ‘traditional’ and ‘novel’ NP markers. Cell viability was high, cell population remained stable and NP associated ECM components were deposited with cells displaying a rounded morphology. Interestingly, when h-BMMSCs were cultured without GDF-6, it was strongly suggested that spontaneous discogenic differentiation occurred after culture in the SAPHs as ‘traditional’ and ‘novel’ NP markers were highly expressed, morphology was comparable to native NPCs and type II collagen and aggrecan were deposited extracellularly. If these findings were accurate then this is the first study to demonstrate that a NP-like phenotype could be induced from MSCs without use of an exogenous growth factor or a discogenic bioactive motif. Despite exciting and novel results, further work is required to confirm the potential of SAPHs for NP tissue engineering scaffolds.