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Transplanted embryonic stem (ES) cells following myocardial infarction (MI) contribute to limited cardiac repair and regeneration with improved function. Therefore novel strategies are still needed to enhance the efficacy by which ES cells differentiate into cardiac cell types and inhibit adverse remodeling in the infarcted myocardium. Our studies evaluate whether genetic manipulation of transplanted ES cells employing miR- 1, a pro-cardiac microRNA, and TIMP-1, an anti-apoptotic and anti-fibrotic protein, will enhance cardiac myocyte differentiation, inhibit native cardiac apoptosis, and reduce fibrosis in the infarcted myocardium. Furthermore, we assess levels of associated pro- (caspase-3, PTEN) and anti-(Akt) apoptotic proteins as well as a pro-fibrotic protein (MMP-9) in the post-MI and cell transplanted heart. microRNAs (miRs) have emerged as critical regulators of various physiological processes including development, differentiation, metabolism, and death. Indeed, miR- 1 plays an integral role in early cardiac development in Drosophila and mice as well as mediates differentiation of cardiac myocytes in vitro. To that end, we generated ES cells overexpressing miR-1 (miR-1-ES cells), transplanted them into the infarcted myocardium, and evaluated their impact on cardiac myocyte differentiation, myocardial repair, and left ventricular dysfunction post-MI. We provide evidence demonstrating enhanced cardiac myocyte commitment of transplanted miR-1-ES cells in the mouse infarcted heart as compared to ES cell and culture media transplanted hearts. Assessment of apoptosis revealed overexpression of miR-1 in transplanted ES cells protected host myocardium from MI-induced apoptosis through activation of p-Akt and inhibition of caspase-3, PTEN, and superoxide anion production. A significant reduction iv in interstitial and vascular fibrosis was quantified in miR-1-ES and ES cell transplanted groups compared with control MI. However, no statistical significance between miR-1- ES cell and ES cell groups was observed. Finally mice receiving miR-1-ES cell transplantation post-MI had significantly improved heart function compared with respective controls. Our data suggests miR-1 drives cardiac myocyte differentiation from transplanted ES cells and inhibits apoptosis post-MI ultimately giving rise to enhanced cardiac repair, regeneration, and function. Next, we assessed the role of miR-1-ES cells in a chronic model of MI as research has shown that apoptosis occurs not only hours but months following ischemia. 4 weeks following transplantation into the infarcted myocardium, we provide evidence demonstrating reduced cardiac apoptosis in miR-1-ES cell transplanted hearts compared to respective controls. Moreover, we show significant elevation of p-Akt levels and diminished PTEN levels in hearts transplanted with miR-1-ES cells as determined by enzyme-linked immunoassays. Finally, using echocardiography, we reveal mice receiving miR-1-ES cell transplantation post-MI had significantly improved cardiac function compared with animals transplanted with ES cell and culture media. Our data suggests that miR-1, when overexpressed in transplanted ES cells, has the capacity to inhibit apoptosis long term while attenuating contractility loss. In addition to enhancing cardiac-specific donor cell differentiation, improving the efficacy by which stem cells promote cell survival and repair in the host myocardium is imperative in the pursuit of refining and optimizing stem cell therapy. To that end, we overexpressed TIMP-1, an endogenous inhibitor of apoptosis and fibrosis, in ES cells (TIMP-1-ES cells), transplanted them into infarcted myocardium, and evaluated their v impact on adverse cardiac remodeling. Immunofluorescence, TUNEL staining, caspase-3 activity, ELISAs, histology, and echocardiography were used to assess apoptosis, fibrosis, and heart function. Hearts transplanted with TIMP-1-ES cells demonstrated a reduction in apoptosis as well as an increase in p-Akt activity compared with ES cells or culture media controls. Interstitial and vascular fibrosis was significantly decreased in the TIMP-1-ES cell group compared to controls. Furthermore, MMP-9, a key pro-fibrotic protein, was significantly reduced following TIMP-1-ES cell transplantation. Echocardiography data showed fractional shortening and ejection fraction were significantly improved in the TIMP-1-ES cell group compared with respective controls. Our data suggest that transplanted ES cells overexpressing TIMP- 1 attenuate adverse myocardial remodeling and improve cardiac function compared with ES cells. Overall, our data suggest that genetic manipulation of ES cells following transplantation in the infarcted heart enhances cardiac myocyte differentiation, inhibits apoptosis and fibrosis as well as improves cardiac function.

Alzheimer's disease (AD), marked by a serious and progressive decline in cognitive abilities, is a severely debilitating disease that is becoming an increasing concern with our aging population. Defects in neurogenesis have been shown to exist in AD and aggravate the neuropathology and cognitive deficits associated with the disease. In this study, I aimed to characterize the cellular and molecular defects of neurogenesis in the triple transgenic mouse model of AD (3xTG). To do so, I first performed a detailed immunohistochemistry characterization using neurogenic markers that were quantified and analyzed in the hippocampus of control and 3xTG mice. This analysis not only revealed an overall decrease in the pool of neural stem and progenitor cells (NSPCs) in 3xTG brains, but also defects in proliferation, differentiation and a loss within the neuroblast, immature neuron and mature neuron populations. Subsequent immunohistochemistry analysis of two molecular targets, Hopx and LPAR1, involved in NSC maintenance and proliferation respectively, revealed their dysregulation in 3xTG brains, providing some indication of molecular defects underlying this loss. The neurosphere assay was next employed to assess cell-autonomous defects and fewer neurospheres were formed from cultured 3xTG NSPCs, suggesting a defect in NSPC pool expansion that is intrinsic to 3xTG NSPC function. Molecular characterization of these cultured NSPCs via qPCR revealed the upregulation of mitochondrial and fatty acid oxidation genes in 3xTG NSPCs, suggesting not only a dysregulation of metabolic functions, but also an acclimation to oxidative stress conditions. Interestingly, 3xTG NSPCs formed larger and more neurospheres when grown in galactose medium - which is used to simulate oxidative stress - relative to the control, confirming an adaptative response to oxidative stress conditions. Further characterization of these cellular defects and underlying molecular mechanisms can reveal novel therapeutic strategies for AD.

Quiescent adult stem cells are important for both tissue maintenance and for responding to stress. C. elegans provides an ideal context to dissect pathways involved in the maintenance of long-term cellular quiescence. In favorable environmental conditions, larvae develop continuously into reproductive adults. In adverse environmental conditions, larvae can undergo an alternative life history and enter dauer diapause, a long-lived state with characteristics that promote survival and dispersal. Animals that enter dauer can survive for months, many times the normal life span of an animal that develops continuously. Entry into dauer interrupts the development of the vulva and is associated with a reprogramming-like event that ensures that Vulval Precursor Cells (VPCs) remain multipotent and quiescent until conditions improve. In this work, I aim to understand how the pathways that regulate dauer entry dictate cellular outcomes that oppose VPC fate acquisition and cell division.VPC specification is initiated when an inductive EGF signal from the somatic gonad activates EGFR-Ras-ERK signaling in the nearest VPC, P6.p. Using an ERK activity biosensor, we found that EGFR signal transduction is activated in P6.p prior to dauer entry. However, during the molt into dauer, EGFR signal transduction itself is downregulated and ERK activity remains low in P6.p throughout dauer. To understand how the VPCs are maintained as multipotent precursors in dauer larvae, we investigated the level at which negative regulation of EGFR signaling occurs. We found that dauer VPCs are desensitized to both endogenous and ectopic expression of EGF despite the presence and correct localization of the EGFR. A constitutively active allele of Ras, but not the EGFR, was sufficient to increase ERK activity in the VPCs. This suggests that during dauer, regulation of EGFR signal transduction occurs at or above the level of Ras. We conclude that EGFR signaling is opposed within the VPCs themselves at the level of membrane associated events. Entry into dauer is regulated by Insulin (IIS), TGF-β and Nuclear Hormone Receptor (NHR) signaling pathways. To understand how these pathways might act to block VPC specification and cell division, we characterized mutants acting in either the IIS or NHR pathway that show inappropriate VPC developmental progression in dauer larvae. We found that the phosphatase DAF-18/PTEN, a modulator of IIS, is required to maintain VPC quiescence during dauer. We created an endogenously floxed daf-18 allele and used tissue-specific Cre recombinase drivers to determine the cellular focus of DAF-18/PTEN in regulating VPCs. Our data is consistent with DAF-18/PTEN acting nonautonomously to prevent VPC division and to maintain competence in dauer. DAF-16/FOXO, the major downstream effector of IIS, and DIN-1S/SHARP, which acts in NHR signaling, were previously implicated in regulation of the VPCs during dauer. We looked at null mutants over time in dauer life history and found that each transcription factor opposes VPC division during distinct stages in dauer development. While DIN-1S/SHARP appears to be required to maintain quiescence at the end of the L2d-dauer molt, DAF-16/FOXO is required to maintain quiescence in dauer itself. This suggests that regulation of the VPCs during dauer life history is dynamic and occurs in phases with each stage having distinct regulatory mechanisms, which is like what has been described for dauer exit. Our research provides insights into robust protective mechanisms that maintain multipotency and quiescence over long periods of time. While the pathways required to enact the dauer program are well defined, the downstream consequences of these pathways on individual or groups of cells are less understood. Future work will aim to link dauer regulating pathways to the downregulation of EGFR signaling in the VPCs.

Zinkoxid-Nanopartikel (ZnO-NP) finden in vielen Produkten des täglichen Verbrauchs Verwendung. Daten über die toxikologischen Eigenschaften von ZnO-NP werden kontrovers diskutiert. Die menschliche Haut ist in Bezug auf die ZnO-NP Exposition das wichtigste Kontakt-Organ. Intakte Haut stellt eine suffiziente Barriere gegenüber NP dar. Bei defekter Haut ist ein Kontakt zu den proliferierenden Stammzellen möglich, sodass diese als wichtiges toxikologische Ziel für NP darstellen. Das Ziel dieser Dissertation war die Bewertung der genotoxischen und zytotoxischen Effekte an humanen mesenchymalen Stammzellen (hMSC) durch niedrig dosierte ZnO-NP nach 24 stündiger Exposition, repetitiven Expositionen und im Langzeitversuch bis zu 6 Wochen. Zytotoxische Wirkungen von ZnO-NP wurden mit 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid-Test (MTT) gemessen. Darüber hinaus wurde die Genotoxizität durch den Comet-Assay bewertet. Zur Langzeitbeobachtung bis zu 6 Wochen wurde die Transmissionselektronenmikroskopie (TEM) verwendet. Zytotoxizität nach 24-stündiger ZnO-NP-Exposition war ab einer Konzentration von 50 µg/ml nachweisbar. Genotoxizität konnten bereits bei Konzentrationen von 1 und 10 µg/ml ZnO-NP beschrieben werden. Wiederholte Exposition verstärkte die Zyto-, aber nicht die Genotoxizität. Eine intrazelluläre NP-Akkumulation mit Penetration der Zellorganelle wurde bei einer Exposition bis zu 6 Wochen beobachtet. Die Ergebnisse deuten auf zytotoxische und genotoxisches Effekte von ZnO-NP hin. Bereits geringe Dosen von ZnO-NP können bei wiederholter Exposition toxische Wirkungen hervorrufen sowie eine langfristige Zellakkumulation. Diese Daten sollten bei der Verwendung von ZnO-NP an geschädigter Haut berücksichtigt werden. / Zinc oxide nanoparticles (ZnO-NP) are widely used in many products of daily consumption. Data on the toxicological properties of the ZnO-NP used are discussed controversially. Human skin is the most important organ in terms of ZnO-NP exposure. Intact skin has been shown to provide an adequate barrier against NPs, while defective skin allows NP contact with proliferating cells. Among proliferating cells, stem cells are the main toxicological target for NPs. Therefore, the aim of this dissertation was to evaluate the genotoxic and cytotoxic effects of human mesenchymal stem cells (hMSC) by low-dose ZnO-NP after 24 hours of exposure, repetitive exposures and in long-term experiments up to 6 weeks. Cytotoxic effects of ZnO-NP were measured with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test (MTT). In addition, genotoxicity was assessed by the comet assay. Transmission electron microscopy (TEM) was used for long-term observation after 6 exposure periods. The results of the study show that ZnO-NP has a cytotoxic effect starting at high concentrations of 50 µg/mL and could demonstrate genotoxic effects in hMSC exposed to 1 and 10 µg/ml ZnO-NP. Repeated exposure enhanced cytotoxicity but not genotoxicity. Intracellular NP accumulation with penetration of the cell organelles was observed at exposure up to 6 weeks. The results indicate the cytotoxic and genotoxic potential of ZnO-NP. Even small doses of ZnO-NP can cause toxic effects with repeated exposure and long-term cell accumulation. These data should be considered when using ZnO-NP on damaged skin.

京都大学 / 新制・論文博士 / 博士(医科学) / 乙第13518号 / 論医科博第10号 / 新制||医科||10(附属図書館) / (主査)教授 金子 新, 教授 松田 秀一, 教授 山中 伸弥 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Cryopreservation is currently the only method allowing for the long-term preservation of hematopoietic stem and progenitor cell (HSPC) grafts until their use. However, cryoinjuries reduce cell viability and potency of HSPC. New cryoprotectant (CPA) solutions have recently emerged that have not yet been investigated that may improve the cryopreservation of HSPCs. The overarching hypothesis of the work described in this thesis, is that different CPAs have diverse impact on the key biochemical processes essential for HSPC homeostasis which influences post thaw cell viability and potency. To test this hypothesis, 4 CPAs were extensively characterized for their cryoprotective properties on cord blood (CB) HSPCs in comparison to DMSO control. CryoProtectPure (CPP) supported similar post thaw cell viability and engraftment as DMSO control, whereas pentaisomaltose (PIM) and cryonovo (CN) failed as CPAs for HSPCs. Subsequently, the impact of CPAs on key biological pathways was explored to identify potential biochemical pathways implicated in HSPC cryopreservation. The impact of CPAs on cell membrane integrity, oxidative phosphorylation, glycolysis, and autophagy was examined. CPP and DMSO had varying impact on glycolytic and mitochondrial respiratory activities of HSPCs post-thaw, whereas both CPAs as well as PIM and CN had negligible impact on cell membrane parameters prefreeze. Cryopreservation and thawing strongly induced autophagy in HSPCs. Importantly, early inhibition of autophagy with 3-Methyladenine (3-MA) reduced the recovery of functional CB HSPCs post thaw. Together, my findings provide new insights regarding the biological processes impacted by CPAs and cryopreservation of HSPCs and identify potential targets to improve cryopreservation of HSC grafts.

Long undecoded transcript isoforms (LUTIs) are a class of non-canonical mRNAs that repress gene expression by a combined mechanism of transcriptional and translational interference. Although this mechanism has been shown to be widespread in yeast, its prevalence in mammals has not been established. Using human embryonic stem cells (hESCs) differentiated into endoderm, mesoderm, and ectoderm lineages and further differentiation into polyhormonal cells, cardiomyocytes, and motor neurons, respectively, we obtained a comprehensive dataset through mRNA-seq, ribosome profiling, and quantitative mass spectrometry measurements. Our analysis revealed that LUTI-based regulation is context-dependent, with a total of 271 genes identified in ectoderm to motor neuron differentiation, 69 genes in mesoderm to cardiomyocyte differentiation, and 99 genes in endoderm to polyhormonal cell differentiation. Translational repression of LUTI candidates was found to be primarily dependent on upstream open reading frames (uORFs), while LUTI-based transcriptional repression displayed variability. This study enhances our understanding of gene expression and regulation during mammalian development and highlights the potential significance of LUTI-based regulation in the development of specific cell types or tissues. The findings lay the groundwork for further exploration into the role of LUTI- based regulation in other mammalian developmental programs and its potential implications for therapeutic targets in developmental disorders and diseases.

Cerebral palsy (CP), depending on disease severity, can result in diminished quality of life not only from decreased function but from societal stigmatization. Coordinating various care provider appointments, expense of short-term treatments, difficulty navigating transportation, and relying on caregivers can complicate patient and family lives. The costs of CP are substantial on the healthcare system, with one managed Medicaid database averaged across 15 U.S states finding the average annual Medicaid costs for children with CP to be 15 times higher than children without CP and averaged to $22, 383 United States dollar (USD) compared to $1,358 USD respectively (Pulgar et al., 2019). Cost effective treatment and effective prevention strategies are increasingly warranted for the CP population. Due to the varying manifestations associated with CP, a standardized treatment for this condition is challenging. Current treatments may enhance quality of life and temporarily reduce pain or discomfort, but they do not cure CP. While perinatal prevention strategies potentially provide the greatest chance to prevent CP from occurring and should be the focus of health care policy, financial barriers remain especially with strained health care budgets. While cure remains elusive, focus on treatments and prevention strategies to limit disease impact is paramount. In the last decade some attention has turned to the use of stem cell treatments in children and adolescents with CP to provide more impactful outcomes with earlier intervention potentially limiting the devastating musculoskeletal effects seen with severe disease. While primarily results from clinical trials both nationally and internationally suggest stem cell treatment increases gross motor function in children and adolescents, questions remain whether these treatments provide clinically meaningful improvement compared to traditional therapies. The goal of this thesis is to discuss the current pharmaceuticals and non- pharmaceutical treatments with rehabilitative therapies that are historically used to reduce severity of secondary manifestations associated with CP in children. Human stem cell clinical trials for CP will be reviewed to assess efficacy and risks as this treatment is translated into clinical practice for children. In addition to stem cell treatment, public health practices of prevention during prenatal visits will be reviewed as it is an encouraging method to reduce preterm births which are a risk factor for CP development.

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