Human umbilical cord perivascular cells (HUCPVCs) derived from regions surrounding the umbilical cord vessels represent an attractive cell source for cellular therapies, given their proliferative potential and the accessibility of donor material compared with human bone marrow-derived mesenchymal stem cells (hBM-MSCs). However, these cells remain poorly characterised. Using flow cytometry, HUCPVCs were shown to express conventional MSC markers CD29, CD44, CD73, CD90, CD105, CD146, CD166 and integrins alpha1 to -5, alphaV, alphaVβ3, alphaVβ5, β1 and β3, but not CD14, CD34, CD45, STRO-1 or integrin alphaVβ6. HUCPVC marker profiles were consistent between three donors and at different passage numbers. Immunostaining for smooth muscle cell (SMC) markers; alpha-SMA, SM22alpha and smoothelin revealed that HUCPVCs shared expression of these markers with SMCs. However, in comparison with SMCs, HUCPVCs deposited more extensive fibronectin-rich matrices. When compared with hBM-MSCs, HUCPVCs differentiated along adipogenic and osteogenic lineages more slowly and did not progress to terminal phenotypes. mRNA expression of recently identified mesenchymal progenitor cell markers, ROR2, EPHA2, PLXNA2, CDH13 and CD9, was confirmed in HUCPVCs from two donors. In addition, all these markers (except EPHA2) were detected in the umbilical cord vessel wall cells of three donors, confirming their expression in both cultured HUCPVCs and cells of the primary tissue. To determine the roles of these markers in HUCPVCs, they were depleted individually using siRNA. Knockdown (KD) efficiencies of 90-97% were achieved. CD9 KD cells appeared elongated compared to cells treated with control siRNA, and these cells along with ROR2, EPHA2 and PLXNA2 KD cells exhibited larger cell areas than controls. All KD cells also showed decreased proliferative potential by day 6 compared with control siRNA or lipofectamine treated cells. A decrease in total β1 integrins was detected in the CD9 KD cells. Up-regulation of ROR2 and PLXNA2 mRNA expression was detected in HUCPVCs from two donors, when they underwent osteogenic differentiation. ROR2 and PLXNA2 knockdown resulted in increases in PLXNA2 and ROR2 mRNAs respectively, when cells were cultured in osteogenic medium compared with basal conditions. In addition, each individual knockdown revealed that the KD cells showed trends in increasing RUNX2 mRNA expression after 13-16 days in osteogenic medium. These data suggest that ROR2 and PLXNA2 may co-operate in promoting an osteogenic phenotype. Culturing HUCPVCs on non-mineralised BVSMC-derived matrices had very little impact on their differentiation status. In contrast, when HUCPVCs were cultured on mineralised BVSMC-derived matrices in osteogenic medium, their ability to further deposit mineralised matrix was enhanced by 7 days; no accompanying changes in RUNX2, ROR2 or PLXNA2 mRNA expression were detected. Taken together, early up-regulation of RUNX2, ROR2 and PLXNA2 appears to be important in driving osteogenic differentiation in HUPCVCs, whilst subsequent down-regulation of these markers may be required for mineralisation to occur. HUCPVCs express ROR2, PLXNA2, CDH13 and CD9 in vitro and in situ; these markers have distinct roles in regulating cell proliferation, shape and differentiation which may be regulated via changes in β1 integrins. It is not known why HUCPVCs might differentiate along adipogenic and osteogenic lineages more incompletely than hBM-MSCs. Further comparative characterisation of HUCPVCs and hBM-MSCs is a prerequisite for exploiting their vast clinical potential.

Alzheimer's disease (AD) is the most prevalent form of progressive dementia and is characterized by the accumulation of amyloid beta (Aß) peptide in the brain and in the cerebral vessels forming cerebral amyloid angiopathy (CAA). As previously reported, an active immunization strategy of mice with Aß1-42 peptide results in decreased Th1 and increased Th2 cytokine responses as well as an effectively clearance of CNS Aß. This approach has also yielded favorable results for many patients, unfortunately, a small percentage of these study participants developed severe aseptic meningoencephalitis likely secondary to CNS invasion of activated T-cells. We have previously demonstrated that disruption of CD40-40L pathway reduces Aß plaque load, promotes Th2 response, and rescues from cognitive impairments. However, direct blockage of the CD40 pathway by passive vaccination with anti-CD40L antibody leads to immunosupression. Therefore, in its current form this therapeutic strategy poses an unacceptable risk to the recipient of treatment, aged individual. For those reasons, the identification and characterization of alternative modulators/inhibitors of CD40 signaling may be necessary for the development of safe and effective AD immunotherapy. This proposal introduces novel immunomodulatory therapies that are based on previous vaccination strategies or cell based therapies across medial field. We showed that transcutaneous vaccination can both be efficacious and safe, thus clearly demonstrating that the right combination of the antigens, adjuvants, and the routes of administration are crucial for the right vaccine. Furthermore, we demonstrated that the effects of current Aß vaccine strategies could be enhanced by a simultaneous blockade of CD40-40L signaling. As an alternative approach, we explored the possibility of cell-based therapies and showed that human umbilical cord blood cells, which are currently used as a treatment for systemic lupus erythematosus and leukemia, and currently investigated against stroke, amyotropic lateral sclerosis, age-related macular degeneration, multiple sclerosis, and Parkinson's disease, and showed that not just they improved the AD like pathology in transgenic animals but altered both the brain and peripheral inflammation levels. Lastly, we discussed the involvement of microglia, one of the key players in both AD pathogenesis and Aß clearance and suggesed that microglia in actuality has a continuum of physiological activation states that contribute to proinflammation, antiinflammation, and phagocytosis.

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

No description available.

The objective of these experiments is to explore the effect of LIPUS on the ultraexpansion and osteogenic differentiation of harvested passage-4 HUCPV-SCs. HUCPV-SCs were divided into two groups: a treatment group that received LIPUS for 10 minutes for 1, 7, and 14 days and a control group that received a sham treatment utilizing both basic and osteogenic media. The results in basic media and osteogenic media demonstrated nonsignificant differences in cell count, ALP, DNA content, and CD90. Statistically significant expression of OSP and PCNA was observed on day 14 in LIPUS treated group. Nucleostemin expression in the LIPUS-treated group was insignificant on days 1 and 7. However, a selective increase in osteogenic markers was obtained on day 7 for ALP and OCN and on day 14 for OPN. Future experiments are required to explore the effects of different application times and/or techniques of LIPUS on the behaviour of HUCPV-SCs. / Medical Science

Cellular mechanisms involved in neurodegeneration and neuroprotection are continuing to be explored, and this paper focuses on some novel discoveries that give further insight into these processes. Oligodendrocytes and activated astroglia are likely generators of the pro-inflammatory cytokines, such as the tumor necrosis factor family and interleukin family, and these glial support cells express adhesion receptors (e.g., VCAM) and release intercellular adhesion molecules (ICAM) that have a major role in neuronal apoptosis. Even brief exposure to some substances, in ontogeny and sometimes in adulthood, can have lasting effects on behaviors because of their prominent toxicity (e.g., NMDA receptor antagonists) or because they sensitize receptors (e.g., dopamine D2 agonists), possibly permanently, and thereby alter behavior for the lifespan. Cell cycle genes which may be derived from microglia, are the most-recent entry into the neuroprotection schema. Neuroprotection afforded by some common substances (e.g., melatonin) and uncommon substances [e.g., nicotine, green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG), trolox], ordinarily thought to be simple radical scavengers, now are thought to invoke previously unsuspected cellular mechanisms in the process of neuroprotection. Although Alzheimer's disease (AD) has features of a continuous spectrum of neural and functional decline, in vivo PET imaging and and functional magnetic resonance imaging, indicate that AD can be staged into an early phase treatable by inhibitors of β and γ secretase; and a late phase which may be more amenable to treatment by drugs that prevent or reverse tau phosphorylation. Neural transplantation, thought to be the last hope for neurally injured patients (e.g., Parkinsonians), may be displaced by non-neural tissue transplants (e.g., human umbilical cord blood; Sertoli cells) which seem to provide similar neurotrophic support and improved behavior-without posing the major ethical dilemma of removing tissue from aborted fetuses. The objective of this paper is to invite added research into the newly discovered (or postulated) novel mechanisms; and to stimulate discovery of additional mechanisms attending neurodegeneration and neuroprotection.

Der ischämische Schlaganfall ist global eine der bedeutendsten Volkskrankheiten. Die derzeit verfügbaren kurativen Therapieoptionen werden vorrangig durch ein enges therapeutisches Zeitfenster limitiert. Ziel der aktuellen Schlaganfallforschung ist die Entwicklung von über dieses Zeitfenster hinaus wirksamen Therapien. Ein vielversprechender neuer Ansatz ist die experimentelle Behandlung mit humanen Nabelschnurblutzellen. Diese Arbeit erforscht das therapeutische Zeitfenster für die systemische Therapie des ischämischen Schlaganfalls mittels mononukleärer Nabelschnurblutzellen (hUCB MNC) in spontanhypertensiven Ratten nach permanentem Verschluss der Arteria cerebri media (pMCAO). Hierzu wurden die Therapiezeitpunkte 4, 24, 72, 120 Stunden und 14 Tage nach experimentellem Schlaganfall in einem komplexen Studiendesign inklusive neurofunktioneller Tests, magnetresonanztomographischer und immunhistochemischer Verfahren untersucht. In vitro wurde der Einfluss kokultivierter hUCB MNC auf Nekrose und Apoptose in neuralem Gewebe unter Sauerstoff-Glukose-Deprivation betrachtet. Die Studie ergab eine verbesserte funktionelle Rekonvaleszenz und eine geringere Ausprägung von Atrophie und Astroglianarbe bei Therapie innerhalb eines 72- Stunden-Zeitfensters. In vitro wurde eine signifikante Reduktion von Nekrose und Apoptose durch kokultivierte hUCB MNC beobachtet. Eine histologische Relokalisierung der intravenös applizierten Zellen war in keiner Therapiegruppe möglich. Die Integration der hUCB MNC ins Hirnparenchym stellt somit keine conditio sine qua non für die funktionelle Erholung nach Schlaganfall dar. Trotz des beobachteten erweiterten Zeitfensters ist die Translation dieses Therapieansatzes in die klinische Realität kritisch zu diskutieren, da weiterführende Studien unserer Arbeitsgruppe eine limitierte Wirksamkeit unter sehr praxisnahen Bedingungen (z.B. Einsatz kryokonservierter hUCB MNC) gezeigt haben. / Experimental treatment strategies using human umbilical cord blood mononuclear cells (hUCB MNCs) represent a promising option for alternative stroke therapies. An important point for clinical translation of such treatment approaches is knowledge on the therapeutic time window. Although expected to be wider than for thrombolysis, the exact time window for hUCB MNC therapy is not known. Our study aimed to determine the time window of intravenous hUCB MNC administration after middle cerebral artery occlusion (MCAO). Male spontaneously hypertensive rats underwent MCAO and were randomly assigned to hUCB MNC administration at 4h, 24h, 72h, 120h or 14d. Influence of cell treatment was observed by magnetic resonance imaging on days 1, 8 and 29 following MCAO and by assessment of functional neurological recovery. On day 30, brains were screened for glial scar development and presence of hUCB MNCs. Further, influence of hUCB MNCs on necrosis and apoptosis in post-ischemic neural tissue was investigated in hippocampal slices cultures. Transplantation within a 72h time window resulted in an early improvement of functional recovery, paralleled by a reduction of brain atrophy and diminished glial scarring. Cell transplantation 120h post MCAO only induced minor functional recovery without changes in the brain atrophy rate and glial reactivity. Later transplantation (14d) did not show any benefit. No evidence for intracerebrally localized hUCB MNCs was found in any treatment group. In vitro hUCB MNCs were able to significantly reduce post-ischemic neural necrosis and apoptosis. Our results for the first time indicate a time window of therapeutic hUCB MNC application of at least 72 hours. The time window is limited, but wider than compared to conventional pharmacological approaches. The data furthermore confirms that differentiation and integration of administered cells is not a prerequisite for poststroke functional improvement and lesion size reduction.

Beim Tissue Engineering werden verschiedene Bereiche der Medizin, der Biologie und der Technik miteinander kombiniert. Das Ziel ist es, ein vitales und funktionales Ersatzgewebe herzustellen, das aus körpereigenen Zellen besteht. Dabei werden körpereigene Zellen auf ein resorbierbares Gerüst transplantiert und in vitro konditioniert, um ein vitales Ersatzgewebe zu schaffen, dass stabil genug ist, um implantiert werden zu können. Dieses Konstrukt hätte die Fähigkeit, sich in das umgebende Gewebe zu integrieren und wie gesundes Gewebe zu entwickeln (z.B. mitwachsen). In dieser Arbeit konnten vaskuläre Zellen aus körpereigenen Nabelschnurgefäßen als neue Zellquelle evaluiert werden. Ein großer Vorteil der Nabelschnur gegenüber den Fragmenten der Aorta ascendens und der Vena saphena magna ist, dass dieses Gewebe nach der Geburt überflüssig ist und verworfen wird. Damit wäre kein weiterer belastender Eingriff bei den Patienten erforderlich. Durch die Kryokonservierung dieser Zellquelle wäre somit auch eine Haltbarkeit möglich gemacht, die eine Verwendung dieser Zellquelle auch bei älteren Patienten zu jedem Zeitpunkt möglich macht. Diese mit kryokonservierten vaskulären Nabelschnurzellen hergestellten Herzklappen waren vital und zeigten unter dynamischen Konditionierungsbedingungen in einem dafür hergestellten Bioreaktor im Vergleich zu den statisch belassenen Klappen eine höhere extrazelluläre Matrix Produktion. Eine Verwendung der kryokonservierten vaskulären Zellen aus humanen Nabelschnurgefäßen ist somit für das Tissue Engineering von kardiovaskulären Strukturen wie das der Herzklappe möglich gemacht worden. / Tissue engineering of heart valves has been developed to overcome the problems of bioprosthetical, mechanical heart valves or homografts by fabricating a viable heart valve substitute from autologous cells with the ability to grow, repair and remodel. This work was focusing of the impact of cryopreserved human umbilical cord cells for fabrication of tissue engineered heart valves for patients diagnosed prenatally with congenital heart lesions potentially enabling heart valve replacement in the early years of life. By using biodegradable polymer matrices as a scaffold for tissue development until cells produce their own matrix autologous cryopreserved human umbilical cord cells are seeded onto the polymer scaffold, forming viable tissue while the polymer degrades. By using a biomimetic flow culture systems these cells shows perfect abilities of creating an in vitro tissue formation. However these cells suggest the potential benefit of establishing autologous human cell banks for pediatric patients diagnosed intrauterinely with congenital defects.

A lesão medular traumática é uma patologia incapacitante, ainda sem tratamento eficaz. As terapias celulares representam uma nova estratégia para o tratamento destas lesões. As células-tronco adultas são fontes potenciais para o transplante celular com o objetivo de minimizar a lesão e promover a recuperação de tecidos lesados, como a medula espinhal. O objetivo desta tese foi avaliar a eficácia do transplante de células-tronco adultas na recuperação funcional e regeneração da lesão medular traumática em modelo experimental de lesão medular contusa em ratas fêmeas Wistar. Os principais objetivos foram: a) comparar os efeitos do transplante da fração mononuclear de sangue de cordão umbilical humano e de células-tronco mesenquimais dos vasos da parede do cordão umbilical humano; b) determinar a janela terapêutica deste tipo de intervenção, comparando os implantes de células- tronco realizados 1 hora, 24 horas e 9 dias após a lesão; c) demonstrar a possível diferenciação das células-tronco implantadas, bem como sua integração no tecido lesado. Os resultados obtidos demonstraram que o transplante de células foi mais eficaz para a recuperação funcional da lesão medular em ratas Wistar quando realizado pela via de administração local 1h após a lesão, quando comparado com a administração na cisterna magna e a aplicação 9 dias a lesão. O tratamento com a fração de células mononucleares ou com as células-tronco mesenquimais do sangue do cordão umbilical 24h após a lesão, não apresentou resultado funcional significativo.Observou-se a neuroproteção do tecido medular quando foi realizado o transplante de células-tronco mesenquimais 1h após a lesão medular. As células humanas transplantadas migraram e sobreviveram no local da lesão quando administradas na cisterna magna ou quando administradas diretamente no local da lesão, porém não se diferenciaram em células gliais ou neurônios. Concluímos que o transplante de células-tronco adultas promoveu a recuperação funcional após a lesão medular contusa, principalmente quando realizado 1h após a lesão diretamente no local da lesão. Apesar das células transplantadas sobreviverem na área da lesão, não foi evidenciada diferenciação celular. / Spinal cord injury is a debilitating disease and yet no effective treatment is available. In this framework cell therapy represents a new strategy to treat this condition. Adult stem cells are potential sources for cell transplantation in order to minimize injury and promote the recovery of damaged tissues, such as the spinal cord. The purpose of this Thesis was to evaluate the action of adult stem cells in the regeneration and functional recovery of spinal cord injury in experimental contusion spinal cord injury in female Wistar rats. Main goals were: a) to compare the effects of transplantation of the mononuclear cells of human umbilical cord blood and mesenchymal stem cells of the vessel wall of human umbilical cord; b) to determine the therapeutic window of this type of intervention, comparing the stem cell implants performed 1 hour, 24 hours and 9 days after injury; c) to demonstrate the possible differentiation of cells implanted, as well as their integration into the damaged tissue. Results reported demonstrate that the transplantation of stem cells was more effective for functional recovery of spinal cord injury when performed into the site of the lesion 1 h after injury, as compared with administration in the cisterna magna 9 days after injury. Treatment with mononuclear cells and mesenchymal cells from umbilical cord blood 24 hours after injury, not showed functional outcome. Neuroprotection was observed when mesenchymal stem cells were transplanted 1 hour after spinal cord injury. The transplanted human cells survived and migrated to the site of injury either when administered in the cisterna magna or directly onto the injury site, but did not differentiated into glial cells or neurons. It is suggested that the transplantation of adult stem cells promotes functional recovery after spinal cord injury when performed 1 hour after injury directly at the injury site, however differentiation of transplanted cells was not detected.

Gegenstand dieser Arbeit war die Synthese von thermisch schaltbaren Kammcopolymeren auf Basis von N-(Isopropylacrylamid) (NiPAAm) und Polyethylenglykolmakromonomeren (PEGMA). Die intensive Charakterisierung der aus diesen Copolymeren hergestellten Schichten und deren Anwendung als Zellkultursubstrate war ein weiteres Forschungsziel dieser Arbeit. Die mit Hilfe der neuartigen Schichten erhaltenen Zellkultursubstrate wurden anhand verschiedener adhärenter Zelllinien erfolgreich getestet. Alle getesten Zelltypen (Mausfibroblasten, humane Endothelzellen der Nabelschnurvene und humane korneale Endothelzellen) proliferierten auf den angebotenen Zellkultursubstraten bei 37°C und konnten durch senken der Temperatur geerntet werden.