京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13194号 / 論医博第2158号 / 新制||医||1030(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 柳田 素子, 教授 横出 正之, 教授 川口 義弥 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Supplementary information 追加（2019-09-30) / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21344号 / 医博第4402号 / 新制||医||1031(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 柳田 素子, 教授 山下 潤, 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

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In Form einer Zusammenfassung aus zwei Publikationen und bisher unveröffentlichtem Material stellt die vorliegende Arbeit die Frage, welche der Zellfraktionen (HUCB-MNC, CD45+/CD34+ und CD45+/CD133+) innerhalb des heterogenen HUCB die am effektivsten wirksame in Hinblick auf die Neuroprotektion nach einem experimentellen Schlaganfall ist und welche Mechanismen dabei zum Tragen kommen. Für die Untersuchung der molekularen Mechanismen des Zusammenwirkens zwischen den genannten HUCB Fraktionen und neuronalen Zellen bzw. Gewebe, kamen ein selbst etabliertes Zellkulturmodel neuronaler Hypoxie bzw. OGD geschädigte hippocampale Schnittkulturen zur Anwendung. Die zu untersuchenden HUCB Zellfraktionen wurden direkt oder indirekt appliziert und ihr Effekt wurde über drei aufeinanderfolgende Tage hinweg untersucht. Das molekulare Mikromilieu, welches durch die hypoxisch geschädigten neuronalen Zellen produziert wurde, stimulierte alle untersuchten HUCB Fraktionen zur Sekretion neurotrophischer Faktoren und/oder immunologisch aktiver Mediatoren, die die neuronale Apoptose günstig beeinflussten. HUCB-MNC zeigten, sowohl in der direkten als auch in der indirekten Kokultur mit geschädigten neuronalen Zellen eine sehr überzeugende Fähigkeit zur Neuroproduktion. In den direkten Kokulturen reduzierten sie die Apoptose der neuronalen Zellen sogar bis auf das Niveau der Kontrollen. Dies kann auf den deutlichen Anstieg der von den HUCB-MNC produzierten Chemokine CCL5; CCL3 und CXCL10 zurückgeführt werden. Weiterhin war zu beobachten, dass HUCB-MNC aktiv zu geschädigten neuronalen Zellen migrierten und sich, vorzugsweise unter Ausbildung von direkten Zell-Zellkontakten, an Axonen und Somata anlagerten. Überraschenderweise zeigte die CD45+/CD133- Zellfraktion ein ähnliches Potential wie HUCB-MNC. Für diese nahezu stammzellfreie Fraktion konnten in den indirekten Kokulturen hohe Konzentrationen an CCL3 und neuroprotektiven G-CSF nachgewiesen werden, wobei letzteres für die Aufrechterhaltung des neuronalen Phänotyps verantwortlich gemacht werden kann. CD45+/CD133+ Stammzellen, die aus der HUCB-MNC Fraktion isoliert wurden, konnten die neuronale Apoptose in direkten Kokulturen signifikant reduzieren. Die Konzentration an löslichen Faktoren, die von den Stammzellen produziert wurde, lag dabei unterhalb der Nachweisbarkeitsgrenze. Die Ergebnisse aus den Untersuchungen der hippocampalen Schnittkulturen zeigen, dass HUCB-MNC direkt neuroprotektiv wirken. Dies gilt insbesondere, wenn sie direkt und in ausreichender Konzentration (12.5x104 Zellen pro Schnitt) appliziert werden. In Kokulturen mit der CD45+/CD34+ Stammzellfraktion fand sich eine verringerte Sekretion an Nervenwachstumsfaktor und damit verbunden eine geringere Anzahl degenerierter Pyramidenzellen. In Kokulturen in welchen die stammzellfreie CD45+/CD34- Fraktion verwendet wurde, trat dieser Effekt nicht auf. Die Resultate, die in den beiden hier verwendeten in vitro Modellen gefunden wurden, legen nahe, dass der Einsatz von HUCB-MNC eine stabile Neuroprotektion hervorruft. Im Vergleich der verwendeten Modelle lieferten die Applikationen von verschiedenen Stammzellfraktion keine einheitlichen Ergebnisse. Damit wird eine starke Systemabhängigkeit induziert. Speziell im Hinblick auf den klinischen Einsatz scheint es keinen deutlich überlegenen Vorteil durch die Verwendung reiner, aus der HUCB-MNC Fraktion gewonnener Stammzellfraktionen zu geben, der den Aufwand rechtfertigt eine zahlenmäßig so geringe Zellfraktion aus der HUCB-MNC Fraktion zu separieren.

Acute damage to the heart, as in the case of myocardial infarction (MI), triggers a robust inflammatory response to the sterile injury and requires a complex and highly organized wound healing processes for survival. Cortical bone stem cell (CBSC) therapy has been shown to attenuate the decline in cardiac function associated with MI in both mouse and swine models. However, the cellular changes brought about by CBSC treatment and their relationship to inflammation and the wound healing process are unknown. We observed that CBSCs secrete paracrine factors known to have immunomodulatory properties, most notably Macrophage Colony Stimulating Factor (M-CSF) and Transforming Growth Factor-b, but not IL-4. Macrophages treated with CBSC medium containing these factors polarized to a hybrid M2a/M2c phenotype characterized by increased CD206 expression but not CD206 and CD163 co-expression, increased efferocytic ability, increased IL-10, TGF-b and IL-1RA secretion, and increased mitochondrial respiration in the absence of IL-4. Media from these macrophages increased proliferation and decreased a-Smooth Muscle Actin expression in fibroblasts in vitro. In addition, CBSC therapy increased macrophages, CD4+ T-cells, and fibroblasts while decreasing myocyte, macrophage, and total apoptosis in an in vivo swine model of MI. From these data, we conclude that CBSCs are modulating the immune response to MI in favor of an anti-inflammatory reparative response, ultimately reducing cell death and altering fibroblast populations resulting in smaller scar and preserved cardiac geometry and function. / Biomedical Sciences

Despite the remarkable success of adoptive T cell therapy in the treatment of melanoma and hematological malignancies, therapeutic capacity in a broad range of solid tumours is impaired due to immunosuppressive events that render tumour-specific T cells unable to persist and kill transformed cells. To address some of the limitations of ACT in solid tumours, our laboratory has developed a therapeutic modality utilizing oncolytic virus, which expresses a tumour-associated antigen, known as an oncolytic viral vaccine (OVV), in combination with tumour specific central memory T cells. With this therapeutic approach (ACT), we can achieve robust in vivo expansion of transferred cells resulting in the complete and durable tumour regression in multiple solid murine tumour models. However, we demonstrate that the curative potential is lost when the tumour stage and burden increase as expanded transferred cells differentiate to a dysfunctional state resulting in the progressive decline in the tumour-specific CD8+ T cell response. Thus, we believe that restoring the T cell response in late-stage tumours will lead to enhanced curative potential of ACT in late-stage tumours. We have previously shown that HDACi, MS-275, can enhance the therapeutic capacity of a T cell-based therapy in an aggressive brain tumour model. In addition, concomitant delivery of MS-275 with ACT ensures durable cures through immunomodulatory mechanisms. Strikingly, concomitant delivery of MS-275, a class 1 histone deacetylase inhibitor (HDACi), with ACT in late-stage tumours completely restores the transferred T cell response to similar levels observed in early-stage tumours resulting in the complete regression of advance-stage tumours. Furthermore, MS-275 enhanced the proliferative capacity and tumour-specific cytotoxic function of transferred cells, independently of tumour stage, type and mouse strain. Interestingly, we did not observe a complete reversal of T cell dysfunction, but rather observed that MS-275 conferred unique properties to T cells as the expression of some markers typically associated with T cell dysfunction was enhanced in addition to persistence and proliferation capacity. Moreover, concomitant delivery of MS-275 also restored the therapeutic capacity of endogenously primed tumour-specific CD8+ T cells expanded by an OVV in late-stage tumours, demonstrating the potential for general use for MS-275 in T cell-based therapies. Our data suggests the use of HDACi may potentiate T cell-based immunotherapies to overcome tumour-mediated T cell dysfunction in advanced stage solid tumours. / Thesis / Master of Science in Medical Sciences (MSMS)

Through the years, cancer therapies have progressed rapidly, pouring out novel treatments such as gene therapy, small molecule therapies and immunotherapy. One such immunotherapy, adoptive cell transfer (ACT), augmented through the addition of a chimeric antigen receptor (CAR), has proven success in treatment of hematological malignancies. Additionally, oncolytic viruses (OV) and OV-based (OVV) therapies, have shown promising results in both clinical and pre-clinical studies. In most instances, when applied as a monotherapy, the aforementioned treatment methods are incapable of inducing complete tumour remission. The Wan lab has developed an approach combining ACT with OVV therapies that dramatically increase therapeutic benefit resulting in complete regression of well-established solid tumours. Despite promising results, certain tumours can still escape this combination therapy through antigen loss resulting in antigen negative relapse (ANR). To further augment the therapy, the addition of a secondary receptor (CAR) provides the ACT multiple avenues of attack to prevent ANR. In this dissertation, we define culture conditions that promote strong expression of the CAR alongside confirmation of function in an in vitro setting. Following, it is demonstrated that OVV boosted dual-targeting T cells carry strong T cell activity by measure of cytokine release in vivo. Despite promising T cell activity data, dual-specific T cells are unable to improve tumour control and survival once relapse occurs. The failure to control relapse remains unclear however evidence points towards lack of T cell persistence, poor CAR function in vivo and a lack of endogenous T cell response leading to compounding effects that prevent dual-targeting T cells from preventing ANR. Although dual specific therapies have shown poor efficacy in preventing ANR, further study must be completed to identify areas of improvement – such as persistence, as the potential for success in using dual-targeting T cells coupled with OVVs still lies untapped. / Thesis / Master of Science (MSc)

Peripheral arterial occlusive disease (PAOD) is characterized by buildup of atherosclerotic plaque in peripheral arteries that leads to an occlusion that can interrupt the supply of blood to the peripheral tissue, causing downstream tissue ischemia/hypoxia. PAOD is estimated to affect over 200 million patients worldwide. Current surgical revascularization treatments can be effective in about half of the patient population, leading to a significant number of patients with no treatment options beyond pharmacological intervention and lifestyle modification. The decrease in blood flow downstream of the occlusion leads to increased blood pressure gradient in the microvasculature, specifically in vessels that connect arterial trees (known as collaterals), which will structurally enlarge and increase blood flow to the downstream ischemic/hypoxic tissue. Targeting this process, known as arteriogenesis, can provide a potential treatment option for patients suffering from PAOD by redirecting blood flow around an occluded artery and therefore supplying hypoxic tissue with blood. In order to enhance this process, cellular transplantation has been used but the current cell types explored have not been successful in enhancing arteriogenesis. Myoblasts, proliferative muscle progenitor cells, mediate muscle regeneration, and promote angiogenesis (the growth of new capillaries to supply hypoxic tissue). Preliminary data indicates that myoblasts also promote arteriogenesis in obese mice, making them an attractive therapeutic candidate. However, the methods used in the preliminary studies limited our ability to confirm those findings and characterize the cell therapy candidate. Specifically, we lacked a reproducible and optimized method to isolate myogenic cells and characterize these cells during in vitro culture and after in vivo transplantation. Therefore, the 1st Aim of this study was to optimize the isolation to obtain the highest number possible of satellite cell-yielding myofibers by modification of enzymatic and mechanical digestion of extensor digitorum longus muscle. Modifications to this methodology increased myofiber yield by more than 150%. The 2nd Aim was to optimize the expansion of satellite cell-derived myoblasts by modification of culture media supplements to promote cell expansion while minimizing maturation. bFGF and SB 203580 supplementation improved cell proliferation and prevented myogenic cell maturation during 7-days of in vitro culture. The 3rd Aim was to develop a process for evaluating the quantity and identity of isolated myogenic cells before and after transplantation. This was achieved by implementing an immunofluorescent transcription factor labeling protocol to determine cell identity and a live/dead cell viability assay to determine cell viability and quantity. All 3 aims were integrated into a proof-of-concept pilot study on a hindlimb ischemic BALB/c mouse model. While myoblast transplantation failed to increase collateral arteriogenesis in this model, the process developed in this project provides a reproducible framework for future studies on myoblast-enhanced arteriogenesis. Further research on the effects of myoblast transplantation on arteriogenesis may facilitate the development of new therapies that improve the prognosis of patients with PAOD.

For a lay summary of the thesis presented in a 1-minute video format, visit the following link: https://www.youtube.com/watch?v=VhLzt_tEz-s / It is projected that, by 2030, 8% of all adults in the world will have diabetes mellitus and treatment will account for 10% of the total healthcare budget in many countries. Polymeric biomaterial research has led to the design of robust polymer hydrogel capsules to develop curative cell-based therapies for chronic disorders such as diabetes mellitus. Encapsulation of insulin-producing beta cells within synthetic, semi-permeable polymer hydrogels can avoid host immune rejection including fibrotic responses, and thus holds the promise of a long-term curative treatment of this disease. There is a paucity of literature regarding methods available for standardized in vitro screening of synthetic polymer hydrogel capsules to predict host responses in vivo. Thus, the focus of this thesis was to design in vitro assays able to screen for subsequent in vivo fibrotic responses. Two dimensional (‘2D’) (cell attachment to thin film hydrogel coatings) and three dimensional (‘3D’) (cell attachment and protein adsorption to hydrogel capsules) in vitro experiments were designed and tested in an iterative process to assess fibrotic responses to a diverse group of polymer hydrogels. Cell attachment assays included fibroblast (NIH 3T3) and macrophage (RAW 264.7) cell lines, and protein adsorption assays included proteins used to model fibrosis including fibrinogen and lysozyme. For some formulations, in vitro assays were compared with in vivo data on pericapsular cellular overgrowth (PCO) after being implanted into mice. A binomial logistic regression model was designed and validated to assess whether the ‘3D’ in vitro assays correlated with in vivo PCO responses. It was found that the RAW 264.7 cell attachment assay was significantly correlated with PCO outcomes in vivo, demonstrating for the first time a simple, cost-effective, and rapid in vitro cell-based approach to screen and select capsules with lower fibrotic potential to be further tested in animals. / Thesis / Master of Health Sciences (MSc) / In North America, one in eleven adults, or about 415 million people, have diabetes. It is projected that by 2030, around 8% of the world population will be diagnosed with this disease. A common form of treatment is through the frequent injection of insulin, but this is costly, requires multiple daily interventions, and cannot prevent regular excursions from the ideal blood glucose range. Cell-based therapies have a lot of promise in treating several chronic diseases including diabetes. Donor and stem-cell derived islets can be implanted into patients with type 1 diabetes and have been shown to function for over a year, albeit at the price of systematic immune suppression. Alternatively, cells that produce insulin can be placed inside immune-evasive capsules and implanted, potentially providing continuous blood glucose regulation without the need for daily insulin injections. However, this novel form of treatment is limited by the encapsulated cells’ survival once implanted. Cell survival can be affected by the body’s response to a foreign body (the capsule), causing deposition of protein or cells on the capsule surface which can limit the oxygen supply to cells in the capsule and the ability of insulin to leave the capsule in a timely fashion. The goal of this project is to develop assays to screen new capsule formulations. This can advance research by using capsules more readily accepted by the body, leading to a more promising and long-term treatment of diabetes.

CAR T-cellbehandling är en immunterapi som har visat lovande resultat vid behandling av cancer. Trots det riktade immunsvaret som kan uppnås, betonar komplexiteten i tillverkningsprocessen och behandlingsproceduren det utrymme somm finns för förbättringar. Omprogrammerade T-celler har illustrerat en hög persistens hos patienter, som utsätter dem för risken för systemisk toxicitet. In-vivo transienta CAR T-celler som använder självförstärkande mRNA leverade genom affinitetsproteinbelagda LNP, föreslås som ett standardiserat alternativ som möjligör dosering av terapin vid behov.  Med hjälp av fagdisplay utfördes ett urval av affibody molekyler mot de tre immunonkologiska målproteinerna CD5, CD8 och CD19, i fyra cykler. Monoklonal fag-ELISA och DNA-sekvensering identifierade sju förmodade kandidater mot CD5, en förmodad kandidat mot CD8 och tre mot CD19. SPR analys visade specifik binding från CD5 kandidaterna, medan binding till målprotein inte kunde påvisas för CD8- och CD19 kandidaterna. De identifierade CD5-bindarna kan konjugeras till LNP för T-cell inriktad leverans av själv-amplififerande mRNA, med genetisk kod för en valfri CAR. / Chimeric antigen receptor (CAR) T-cell therapy is an immunotherapy which has shown promising results in treating patients suffering from oncological malignancies. Despite the targeted immune response that can be achieved, elaborate manufacturing and procedure processes emphasise room for improvement. Engineered T-cells have illustrated a high persistence in patients, exposing them to the risk of systemic toxicity. In-vivo transient CAR T-cells using self-amplifying mRNA by delivery through affinity protein coated lipid nanoparticles (LNP) is proposed as a standardised and reversible alternative, allowing for dosing when needed. Using phage display technology, selection of affibody molecules toward the three immune oncology proteins CD5, CD8 and CD19 was performed in four cycles. Monoclonal phage enzyme-linked immunosorbent assay (ELISA) and DNA sequencing identified seven putative candidates toward CD5, one putative candidate was isolated toward CD8, and three toward CD19. Surface plasmon resonance analysis (SPR) showed specific target binding of the CD5 candidate binders, while target binding could not be demonstrated for the CD8 and CD19 candidates. The identified CD5 binders could be conjugated to LNP for T-cell targeted delivery of self-amplifying mRNA encoding any CAR of interest.