M.Tech. / Stem cells possess self-renewal capacity, long-term viability, and multilineage potential. Stem cells play important roles in normal physiological and disease processes, they also have great therapeutic potential. However, there have been controversies surrounding stem cells in political, religious and ethical arenas. Although the use of certain stem cells (i.e. embryonic stem cells) and the means by which they are obtained contravene certain basic ethical laws, researchers have developed methods with which to ethically obtain and create stem cell lines. Stem cells can be classified as either: totipotent, pluripotent, multipotent, oligopotent and unipotent (Moore, 2007). Totipotent cells have the ability to differentiate into all cell types of an embryo, including the extra-embryonic and post embryonic tissues and organs. Pluripotent cells have the potential to differentiate into almost all tissues found in an embryo (including germ cells), but are not capable of giving rise to supporting cells and tissues. Multipotent stem cells have progeny of several differentiated cell types - but all within a particular tissue, organ, or physiological system. A good example of multipotent cells, are the haematopoietic stem cells that produce blood cell-restricted progenitors, as well as all cell types and elements, such as platelets, that are normal components of blood. Oligopotent stem cells produce two or more lineages within a specific tissue, such as neural stem cells that are able to produce subsets of neurons in the brain. Unipotent cells self-renew, as well as give rise to a single mature cell type, a prime example being the spermatogonial stem cells, that give rise to spermatozoa (Moore, 2007). Adult human subcutaneous adipose tissue contains cells with multilineage developmental plasticity like marrow-derived mesenchymal stem cells (Strem et al., 2005, Tong et al., 2000). Adipose derived stem cells can be obtained in abundance and can differentiate into osteogenic, adipogenic, myogenic and chondrogenic lineages when treated with appropriate growth factors.

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京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22884号 / 医博第4678号 / 新制||医||1048(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 長船 健二, 教授 羽賀 博典, 教授 椛島 健治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Current treatments for peripheral arterial occlusive disease (PAOD) have limited success, so there is a need to develop more effective treatments. Because patients with native collaterals have a better prognosis, promoting collateral arteriogenesis is a potential PAOD treatment. Additionally, female PAOD patients have a worse response to treatment and a worse prognosis compared to males, which could be due to impaired collateralization. Cell transplantation is a potential treatment option to promote collateral arteriogenesis. Bone marrow derived stem cells are the main cell type that has been investigated, but they have had limited clinical success. Delivering a stem cell type native to the tissue like myogenic stem cells could have improved outcomes. In this study, the lateral spinotrapezius feed artery was ligated in male and female Balb/C mice to induce collateral capillary arteriogenesis, and 7 days post ligation ­­­­arterialized collateral capillary (ACC) number and diameter were determined. There were no differences between sexes, which could be because young, healthy mice were used in this study rather than aged and diseased models. Because we observed no sex differences, we then assessed the effect of myogenic cell transplantation in male mice only. Immediately following ligation of the spinotrapezius feed artery, mice were treated with myogenic cells, thrombin, or vehicle, and 7 days post ligation ACC number and diameter were determined. Thrombin increased ACC number, but myogenic cells had no effect. However, myogenic cells increased ACC diameter, and both myogenic cells and thrombin decreased ACC number in the region of the muscle with the largest collateral, and increased the maximum ACC diameter. Another factor that could affect ACC formation is a pre-existing collateral (PEC), which only some Balb/C mice have, so we also separated mice into PEC and non-PEC groups for analysis. In mice with a PEC, thrombin increased ACC number, and both myogenic cells and thrombin increased ACC diameter. There was a trend toward smaller arterialized capillaries in mice with a PEC, which could be because the majority of the blood flow is redirected through the PEC, so the PEC was the main vessel to remodel. These results are consistent with previous studies that indicated that thrombin augments arteriogenesis as well as increasing V-CAM, and suggest that myogenic cells have a similar effect possibly by secreting arteriogenic factors such as VEGF and MMPs. Because myogenic cells increase arteriogenesis, and macrophages are an essential regulator of arteriogenesis, we also tested the hypothesis that myogenic cells would increase macrophage content. Macrophage number increased with ligation, but there was no difference in macrophage number between any of the treatment groups. The lack of difference in macrophage number could be because the day 7 timepoint was too late, as macrophage content peaks at day 3. Because myoblasts increased arteriogenesis, they also may have increased the number M2 macrophages, which are the main macrophage contributor to arteriogenesis, but we used a general macrophage marker and could not detect an increase in M2 macrophages. In future studies, to determine if there is an increase in M2 macrophages a stain specific to M2 macrophages like CD206 could be added. Additionally, a diabetic Balb/C strain could be used to determine if arteriogenesis is impaired in males compared to females in a diseased model.

Costimulation blockade based on a donor-specific transfusion and anti-CD154 mAb is effective for establishing mixed allogeneic hematopoietic chimerism and inducing transplantation tolerance. Despite its potential, recent evidence suggests that the efficacy of costimulation blockade can be reduced by environmental perturbations such as infection or inflammation that activate toll-like receptors (TLR). TLR agonists prevent costimulation blockade-induced prolongation of solid organ allografts, but their effect on the establishment of hematopoietic chimerism has not been reported. In this dissertation, we hypothesized that TLR activation during costimulation blockade would prevent the establishment of mixed hematopoietic chimerism and shorten skin allograft survival. To test this hypothesis, costimulation blockade-treated mice were co-injected with TLR2 (Pam3Cys), TLR3 (poly I:C), or TLR4 (LPS) agonists and transplanted with allogeneic bone marrow and skin grafts. Supporting our hypothesis, we observed that TLR agonists administered at the time of costimulation blockade prevented the establishment of mixed hematopoietic chimerism and shortened skin allograft survival. To investigate underlying cellular and molecular mechanisms, we first determined that LPS administration during costimulation blockade did not increase production of alloantibodies or activate natural killer cells. Similarly, costimulation blockade-treated mice depleted of CD4+ or CD8+ cells did not become chimeric when co-injected with LPS. In contrast, mice depleted of both CD4+ and CD8+cell subsets were resistant to the effects of LPS. We next observed that alloreactive T cells were activated by TLR agonists in mice treated with costimulation blockade, and this activation correlated with LPS-induced maturation of donor and host alloantigen-presenting cells. In contrast, TLR4-deficient mice treated with costimulation blockade and LPS did not upregulate costimulatory molecules on their APCs, and mixed chimerism and permanent skin allograft survival were readily achieved. We further observed that injection of recombinant IFN-β recapitulated the detrimental effects of LPS, and that LPS-injected mice deficient in the type I IFN receptor were partially protected. Importantly, alloantigen-presenting cells did not upregulate costimulatory molecules in response to LPS, and mixed chimerism and permanent skin allograft survival were readily established in type I IFN receptor and MyD88 double deficient mice treated with costimulation blockade. We conclude that the TLR4 agonist LPS prevents the establishment of mixed hematopoietic chimerism and shortens skin allograft survival in mice treated with costimulation blockade by inducing the production of type 1 IFN and MyD88-dependent factors that upregulate costimulatory molecules on APCs, leading to the generation of activated alloreactive T cells.

The attitudes and experiences of the health care team members involved in facial transplant surgery and patient care were explored in this study, which utilized a qualitative descriptive method. The Specific Aims of the study and the interview questions were guided by “Moore’s Ethical Criteria for Surgical Innovation.” Overall, the participants believed that the risk-benefit ratio of facial transplantation favored proceeding with the procedure in the clinical scenarios with which they had been exposed. The participant’s experience was challenging and rewarding, and they expressed personal fulfillment from the opportunity to be involved in the transformation of another human being’s life. Moreover, the entire effort exhibited highly effective team work which displayed esprit de corps, was guided by superior leadership, and illuminated the importance of the clinical, intellectual, and historical environment of the institution where the procedures took place. These components represent a “surgical innovation cluster,” a proposed framework for guiding surgical innovative efforts which represent major paradigmatic shifts in both scientific effort and social philosophy.

Improved therapeutic strategies for patients with leukemia remain in great demand and beckon better understanding of the mechanisms underlying leukemic treatment resistance and relapse. Accordingly, discoveries in leukemic pathophysiology have been achieved in various animal models. Danio rerio—commonly known as the zebrafish—is a vertebrate organism well suited for the investigation of human leukemia. Zebrafish have a conserved hematopoietic program and unique experimental strengths. Recent technological advances in zebrafish research including efficient transgenesis, precise genome editing, and straightforward transplantation techniques have led to the generation of numerous zebrafish leukemia models. Additionally, improved imaging techniques, combined with the transparency of zebrafish, have revealed exquisite details of leukemic initiation, progression, and regression. Finally, advances in high-throughput drug screening in zebrafish are likely to hasten the discovery of novel anti-leukemic agents. Zebrafish provide a reliable experimental system for leukemic disease research and one in which investigators have accumulated knowledge concerning the genetic underpinnings of leukemic transformation and treatment resistance. Without doubt, zebrafish are rapidly expanding our understanding of disease mechanism and are helping to shape therapeutic strategy for improved patient outcomes.

and Key words This diploma thesis is focused on assessing the potential benefit of HLA epitopes for the prediction of de novo antibody production at kidney transplant recipients. The topic and patient selection criteria were selected in accordance with the 18th International HLA and Immunogenetics workshop (IHIWS), which is taking place in May 2022 in the Netherlands, where our data will also be contributed. Our aims were to compare HLA antigens mismatches (counted as total number of mismatched alleles) defined on the high-resolution level by NGS sequencing, HLA eplets mismatches defined by HLA matchmaker, and amino acid mismatches defined by HLA EMMA in their capacity to predict de novo antibody production and compare these results to other works by different authors from this field. We have identified N= 28 patients who developed de novo antibodies and N= 19 who didn't develop de novo antibodies in 5 years follow up their transplant. These two cohorts were compared based on all three approaches and correlation between number of mismatches and number of patients with de novo antibodies were made using ROC curves. Superiority of eplet mismatches over HLA antigen mismatches (total number of mismatched alleles) defined on high resolution was not detected. The HLA epitopes identified by the HLA...

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20082号 / 医博第4175号 / 新制||医||1018(附属図書館) / 33198 / 京都大学大学院医学研究科医学専攻 / (主査)教授 小池 薫, 教授 湊谷 謙司, 教授 山下 潤 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM