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Stem cell therapy is emerging as a possible method for treating many diseases and disorders, such as cardiovascular disease. In particular, stem cells may be able to revive the dead tissue caused by acute myocardial infarction (heart attack). Adipose-derived stem cells were labelled with 18F-fluorodeoxyglucose (FDG) and superparamagnetic iron oxide (SPIO) particles, for imaging with positron emission tomography (PET) and magnetic resonance imaging (MRI), respectively, and injected into several rats following induced myocardial infarction. Stem cell retention in the heart was investigated following three injection sites; two within the heart (intramyocardial and left intraventricular), and one easily accessible vein (tail vein). The PET and MR images were registered and the initial distributions analyzed using region of interest (ROI) analysis, to determine which injection method would result in the highest stem cell retention in the infarcted heart. The ROI results determined that the intramyocardial injection had the highest % injected dose (%ID) in the heart with 14 +/- 4%, followed by left intraventricular and tail vein with %IDs of 3.6 +/- 0.8% and 1.2 +/- 0.6%, respectively. The results indicate that stem cell delivery via intramyocardial injection should be utilized for optimal retention in the heart.

Stem cell therapy is emerging as a possible method for treating many diseases and disorders, such as cardiovascular disease. In particular, stem cells may be able to revive the dead tissue caused by acute myocardial infarction (heart attack). Adipose-derived stem cells were labelled with 18F-fluorodeoxyglucose (FDG) and superparamagnetic iron oxide (SPIO) particles, for imaging with positron emission tomography (PET) and magnetic resonance imaging (MRI), respectively, and injected into several rats following induced myocardial infarction. Stem cell retention in the heart was investigated following three injection sites; two within the heart (intramyocardial and left intraventricular), and one easily accessible vein (tail vein). The PET and MR images were registered and the initial distributions analyzed using region of interest (ROI) analysis, to determine which injection method would result in the highest stem cell retention in the infarcted heart. The ROI results determined that the intramyocardial injection had the highest % injected dose (%ID) in the heart with 14 +/- 4%, followed by left intraventricular and tail vein with %IDs of 3.6 +/- 0.8% and 1.2 +/- 0.6%, respectively. The results indicate that stem cell delivery via intramyocardial injection should be utilized for optimal retention in the heart.

Prostate cancer is one of the most common cancers affecting one of every six men in United States. It is increasingly appreciated that tumor or cancer stem cells are the cells responsible for initiating tumor formation and therefore should be targeted for eradication in cancer treatment. But the mechanism involved in the acquisition of unlimited self-renewal and tumor initiation by cancer stem cells is unknown. Nanog, along with Oct3/4 and Sox-2, constitute the core transcriptional circuitry for the maintenance of stemness in embryonic stem cells. Herein we report that Nanog expression was detected at mRNA and protein levels in prostate cancer cells. The Nanog-expressing LNCaP-T and DU145 cells were enriched by infection with lentiviruses expressing GFP under the control of Nanog promoter. The Nanog-enriched prostate cancer cells had stronger expressions of stem and progenitor cell surface markers, including CD44 and CD133, when compared with those in the control group. Colony formation assay found that the Nanog-enriched LNCaP-T and DU145 cells formed more holoclones and prosta-spheres, which contained more self-renewing cells, than the control cells did. On the other hand, knockdown of Nanog in DU145 or LNCaP-T cells, via shRNAs, reduced their ability to form holoclones. Instead, most clones derived were meroclone and paraclones as result of increased differentiation and senescence due to knockdown of Nanog. When injected into mice, Nanog-enriched DU145 cells were found to possess increased tumorigenic potentials when compared to the vector controls. On the other hand, LNCaP-T cells with Nanog knocked down did not form tumors, while the vector controls readily formed tumors. Taken together, our data suggest an essential role for Nanog in the self-renewal and tumor initiation of prostate cancer cells. Chemotherapy is the major salvage therapeutic modality available for the patients with advanced cancers. However, drug resistance by some prostate cancer cells is a major barrier to efficacious chemotherapy. It has been increasingly appreciated that cancer stem cells are responsible for resistance to chemo- or radio-therapy, in addition to tumor initiation. However, the mechanisms involved remain unknown. In this study, we examined whether Nanog plays an essential role of Nanog in resistance to chemotherapy. In the surviving fractions of prostate cancer cells, we found increased levels of Nanog protein when compared to the cells treated with solvent control. To determine the role of Nanog in resistance of prostate cancer cells, we marked and enriched Nanog-expressing prostate cancer DU145 and LNCaP-T cells using a reporter gene under control of 2.5 kb hNanog1 promoter. When compared to the control, the prospectively enriched Nanog-expressing cells presented increased resistance to Taxol, vinblastine, and doxorubicin. Profiling of genes in drug resistance and metabolism revealed a marked increase in the mRNA level of ATP-binding cassette (ABC) efflux transporters B1 and G2 in tumor cells enriched with endogenous Nanog expression. The increased expression of ABCB1 and ABCG2 at protein levels in Nanog expressing cells was confirmed by Western blot and immunocytochemistry. Inhibition of ABCB1 activities sensitized Nanog expressing cells toward Taxol and vinblastine, and to less extent, doxorubicin. Blocking of ABCG2 activity sensitized Nanog expressing cells toward doxorubicin, but not Taxol and vinblastine. In addition, the tumor cells enriched with Nanog expression showed reduced apoptosis in response to Taxol treatment. Interestingly, Nanog-enriched prostate carcinoma cells displayed aberrantly activated â-catenin signaling, which is potentially associated with their increased chemo-resistant ability as well as the increased acquisition of epithelial to mesenchymal transition. In summary, Nanog is expressed in prostate cancer cells, especially in those positive for stem/progenitor markers. Enrichment of Nanog expressing cells led to enrichment of tumor cells with increased tumor initiating ability and increased resistance toward chemotherapy. Knockdown of Nanog reduces tumor initiating ability of prostate cancer cells and further sensitizes them toward chemotherapy. The gain-of-function and loss-of-function studies suggest an essential role of Nanog for prostate cancer cells to initiate tumor formation and resist chemotherapy.

Bioactive lipids have emerged as prominent regulators of neural stem and progenitor cell (NPC) function under both physiological and pathological conditions. However, how lipid metabolism is regulated, and its role in modulation of NPC function remains unknown. In this regard, my study defines a novel epigenetic pathway that regulates lipid metabolism to determine NPC proliferation versus differentiation. Specifically, I show that activation of an atypical protein kinase C (aPKC)-mediated Ser436 phosphorylation of CREB binding protein (CBP) by aging, metformin stimulation and continued passaging in vitro, represses expression of monoacylglycerol lipase (Mgll) to promote neuronal differentiation of adult NPCs. Mgll, a lipase that hydrolyzes the endocannabinoid 2-arachidonoyl glycerol (2-AG) to produce arachidonic acid (ARA), is thus a key regulator of two critical bioactive lipid signaling pathways in the brain and a potential modulator of NPC function. I observed elevated Mgll levels, concomitant with neuronal differentiation deficits in both the lateral ventricle sub-ventricular zone (SVZ) and the hippocampal subgranular zone (SGZ) NPCs of phospho-null CBPS436A mice, that lack a functional aPKC-CBP pathway. Genetic knockdown of Mgll or inhibition of Mgll activity rescued these neuronal differentiation deficits. In addition, I found that CBPS436A SVZ NPCs exhibit enhanced proliferation at the expense of differentiation as an outcome of increased Mgll levels in culture. Interestingly, I also observed that SVZ NPCs from an Alzheimer’s disease (AD) model, the 3xTg mice, closely resemble CBPS436A NPC behaviour in culture. 3xTg NPCs exhibit attenuation of the aPKC-CBP pathway, which is associated with elevated Mgll expression and increased NPC proliferation at the expense of neuronal differentiation. Reactivation of the aPKC-CBP mediated-Mgll repression in 3xTg AD NPCs mitigates their differentiation deficits. These findings implicate Mgll as a critical switch that regulates NPC function by altering bioactive lipid signaling (2-AG versus ARA). They demonstrate that the aPKC-CBP mediated Mgll repression is essential for normal NPC function, and that when perturbed in AD, it causes impaired NPC function to generate fewer neurons, contributing to AD predisposition.

Polydopamine (PDA), a unique bioinspired polymer, has been a subject of interest in fields including orthopedic biomaterials and antibacterial surfaces. Its osteogenic effects and ability to control surface traits through precise variables (e.g., pH, temperature) have led to its use as a coating in the enhancement of a wide range of materials, including metals and ceramics. In this Thesis, two studies were carried out to better understand the capability and mechanism of PDA-mediated bioactivity. In the first study, we investigated whether PDA coatings can further enhance the bioactivity of nanoporous Titanium (NPTi). While physicochemical traits were in line with literature, PDA was effective in enhancing cell proliferation, beyond NPTi, as early as 8 hours with enhancement in cell spreading and focal adhesion prevalence as early as 1 hour. No changes in adsorptive capacity were found, suggesting a serum-independent component (SIC) of the surface. The second study was focused on (1) determining how treatment parameters influence the physiochemical makeup of PDA surfaces, (2) assessing how PDA surfaces influence stem cell behavior and (3) confirming and investigating the SIC of PDA effect. Results confirm that there is indeed a SIC of PDA coatings with enhancement in cell spreading that improves with the increased size and density of PDA particles. Our findings show that the SIC works in concert with circulating sera to elicit the bioactive effects of PDA. The novel rPDA surface, obtained by adding rotation during the coating deposition, is also shown to elevate bioactivity during normal culturing, beyond classical coatings, with ongoing work suggesting enhancement in the osteogenic differentiation of hMSCs. Taken together, this work has demonstrated novel aspects underlying the potential and mechanism of action for the bioactivity of PDA, ultimately providing new evidence supporting the use of PDA as a biomedical material.

Cardiovascular diseases contribute a large amount of morbidity and mortality in developing and developed countries all around the world. In conditions such as the myocardial infarction, a significant amount of cardiomyocytes die leading to an impaired function of the heart. One promising method for replacing these cardiomyocytes would be with the use of cardiomyocytes derived from embryonic stem cells. However, a large number of cardiomyocytes and a highly efficient method for obtaining cardiomyocytes are needed. Using the principles of small molecule treatment to induce differentiation in a serum-free based differentiation protocol, I have demonstrated that the induction of canonical Wnt signalling via CHIR 99021, and subsequent addition of bone morphogenetic protein 4 was best able to induce cardiomyogenesis in mouse embryonic stem cells. While improvements in efficiency are still required, the manipulation of the Wnt and BMP4 signalling pathways hold great promise in improving cardiomyogenesis in mESCs.

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第14264号 / 工博第3015号 / 新制||工||1448(附属図書館) / 26591 / UT51-2008-T24 / 京都大学大学院工学研究科高分子化学専攻 / (主査)教授 岩田 博夫, 教授 伊藤 紳三郎, 教授 大嶋 正裕 / 学位規則第4条第1項該当

Encapsulating cells within semi-permeable hydrogel material has been shown to boost the therapeutic effectiveness of stem cell therapy in certain applications. Cell encapsulation promotes high retention and engraftment rates, and protects against attack from the immune system of the host, as these are challenges often seen in utilizing stem cells in suspension alone. Leveraging droplet-based microfluidics has yielded a platform capable of producing monodispersed microcapsules embedded with cells at high throughput, typically achieved by mixing an aqueous hydrogel solution that contains cells with an immiscible liquid (oil) in a flow focusing geometry. However, encapsulation using microfluidics results in randomized generation of empty and cell-laden microcapsules, following Poisson statistics, raising the need to institute a successful sorting mechanism, thereby increasing occupancy and ultimately purifying the desired sample. In this thesis we propose a sorting strategy by combining two conceptual mechanisms of electrophoresis (EP) and deterministic lateral displacement (DLD). Different varieties of microcapsules were characterized for EP and DLD respectively. Leveraging these differences was used in a device combining both of the concepts towards sorting of empty and cell-laden microcapsules.

Cell membranes are made up of a complex structure of lipids and proteins that diffuse laterally giving rise to what we call membrane fluidity. During cellular development, such as neuronal differentiation, cell membranes undergo dramatic structural changes induced by proteins such as ARC and Cofilin among others in the case of synaptic modification. In this study we used the generalized polarization (GP) property of fluorescent probe Laurdan using two-photon microscopy to determine membrane fluidity as a function of time and for various cell lines. A low GP value corresponds to a higher fluidity and a higher GP value is associated with a more rigid membrane. Four different cell lines were monitored such as hN2, NIH3T3, HEK293 and L6 cells. As expected, NIH3T3 cells have more rigid membrane at earlier stages of their development. On the other hand neurons tend to have the highest membrane fluidity early in their development emphasizing its correlation with plasticity and the need for this malleability during differentiation. This study sheds light on the involvement of membrane fluidity during neuronal differentiation and development of other cell lines.