Rozprawy doktorskie na temat „Neurospheres”

HIF-1 (Hypoxia Inducible Factor-1) is an oxygen-regulated transcription factor that mediates the intracellular response to hypoxia in human cells, targeting specific genes that promote cell survival by inducing processes such as angiogenesis and glycolysis. The HIF-1 signalling pathway has been of considerable interest due to its role in mammalian development and in particular several pathologies such as ischemia and cancer. Low cellular oxygen levels are found in cancer due to the rapid proliferation of tumour cells distant to blood vessels (forming a hypoxic core) and the typically irregular vasculature is unable to properly perfuse the tumour. In the Centre for Cell Imaging at the University of Liverpool, time lapsed imaging in an oxygen-controlled environment has captured the transient dynamics of the oxygen regulated subunit of HIF-1, HIF-1α, and revealed heterogeneity between individual cells. The essential characteristics of this data are modelled with a system of differential equations describing the feedback inhibition between HIF-1α and the pathway’s effective oxygen sensors. This novel model was formulated by employing a minimalist approach initially, allowing us to use the rich variety of single-cell data to determine the structure of the feedback loop between two key pathway components. Once the central regulatory motif was identified, the model was expanded to include more complexity, including experimental measurements for model parameter values and additional system components. Oxygen plays an especially important role in the early stages of tumour formation. Measurements of oxygen within cells have been recorded in cultured spheres of neuroblastoma cells at different atmospheric conditions. This data is representative of the early development stages of an avascular tumour and a spatial oxygen-diffusion model was coupled with the single-cell HIF-1α model to describe the dynamics of HIF-1α across a developing tumour. This coupled model is used to study HIF-1α dynamics in the context of various oxygen-dependent cellular functions such as cell-cycle progression and apoptosis by integrating with modifications of published mathematical models.

The formation of neurospheres was important in demonstrating that neurogenesis in the adult brain may be fuelled by a stem cell population. The olfactory mucosa is another site of neurogenesis which, in humans, has been observed to contain a stem cell population through the formation of neurospheres (Murrell et al., 2005). Stem cells can be defined as cells capable of self-renewal and multipotency. The aim of this study was to investigate the potential of rat olfactory stem cells growing as neurospheres. The hypothesis is that the rat olfactory mucosa contains a 'true' stem cell population that can be cultured as neurospheres and that will demonstrate multipotency by differentiating into 'non-olfactory' cell types and possess the capacity for self-renewal, if provided with the appropriate environmental niche. Here it was found that adult rat olfactory mucosa is capable of generating neurospheres when cultured in EGF and bFGF. Evidence of self-renewal was provided by the formation of six generations of neurospheres, the formation of neurospheres from single cells and the expression of markers associated with self-renewal by neurosphere cells. The multipotency of olfactory neurosphere cells was demonstrated through manipulation of the stem cell niche. In defined culture conditions, extracellular matrix molecules and growth factors were able to induce the differentiation of neurosphere cells down the dopaminergic lineage pathway. When co-cultured with differentiating cells, neonatal myoblasts and 3T3-L1 cells, olfactory neurosphere cells were able to differentiate and incorporate into a skeletal muscle myotube and differentiate into adipocytes, respectively. In conclusion it was found that the adult rat olfactory mucosa is capable of generating neurospheres. When presented with an appropriate niche neurosphere cells are able to self-renew and demonstrate multipotency.

The formation of neurospheres was important in demonstrating that neurogenesis in the adult brain may be fuelled by a stem cell population. The olfactory mucosa is another site of neurogenesis which, in humans, has been observed to contain a stem cell population through the formation of neurospheres (Murrell et al., 2005). Stem cells can be defined as cells capable of self-renewal and multipotency. The aim of this study was to investigate the potential of rat olfactory stem cells growing as neurospheres. The hypothesis is that the rat olfactory mucosa contains a 'true' stem cell population that can be cultured as neurospheres and that will demonstrate multipotency by differentiating into 'non-olfactory' cell types and possess the capacity for self-renewal, if provided with the appropriate environmental niche. Here it was found that adult rat olfactory mucosa is capable of generating neurospheres when cultured in EGF and bFGF. Evidence of self-renewal was provided by the formation of six generations of neurospheres, the formation of neurospheres from single cells and the expression of markers associated with self-renewal by neurosphere cells. The multipotency of olfactory neurosphere cells was demonstrated through manipulation of the stem cell niche. In defined culture conditions, extracellular matrix molecules and growth factors were able to induce the differentiation of neurosphere cells down the dopaminergic lineage pathway. When co-cultured with differentiating cells, neonatal myoblasts and 3T3-L1 cells, olfactory neurosphere cells were able to differentiate and incorporate into a skeletal muscle myotube and differentiate into adipocytes, respectively. In conclusion it was found that the adult rat olfactory mucosa is capable of generating neurospheres. When presented with an appropriate niche neurosphere cells are able to self-renew and demonstrate multipotency.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Biomedical Sciences
Faculty of Science
Full Text

Thesis (Ph.D.)--University of Florida, 2005.
Typescript. Title from title page of source document. Document formatted into pages; contains 97 pages. Includes Vita. Includes bibliographical references.

The transcription factor FOXP3, a master regulator of Treg cells has been proposed to function as a tumor suppressor in breast and prostate cancer. In the present study we provide evidence that FOXP3 is expressed in normal brain but strongly down-regulated in both primary and recurrent glioblastoma (GB) specimens and in corresponding cell lines growing in culture in the presence of mitogenic factors (mostly Epidermal Growth Factor - EGF and b-Fibroblast Growth Factor – bFGF) as neurospheres (NS). We also found that FOXP3 expression was higher in low-grade gliomas than in GB. Neurosphere generation, a feature present in 58% of the GB that we examined, correlated with lower expression of FOXP3 and shorter patient survival. Our main result refers to the contribution of FOXP3 expression in affecting proliferation and migration in vitro and in vivo. FOXP3 was silenced in one GB-NS expressing measurable levels of the gene. Intracranial injection of these GB-NS cells in nude mouse brains increased significantly tumor development and aggressiveness. Deriving gliomas showed a total absence of FOXP3 expression associated with a significant increase in proliferation and migration. Conversely, FOXP3 over-expression impaired GB-NS migration and proliferation in vitro. We also demonstrated using ChIP that FOXP3 is a transcriptional regulator of p21 and c-MYC supporting the idea that dysregulated expression of these factors is a major mechanism of tumorigenesis driven by the loss of FOXP3 expression in gliomas. These findings support the assertion that FOXP3 exhibits tumor suppressor activity in glioblastomas.

Le glioblastome représente le sous-type de tumeur cérébrale le plus fréquent et le plus agressif. Malgré une meilleure compréhension de la maladie ainsi que l'émergence de nouvelles cibles, voire de nouveaux outils thérapeutiques, son pronostic reste inchangé. En effet, l'échec de l'extrapolation des résultats vers la clinique met en exergue la nature complexe de la maladie et la dimension décisive des modèles animaux adéquats et prédictifs dans l'étude des nouvelles thérapies. Et pour cause, un modèle animal idéal doit pouvoir reproduire les caractéristiques histo-pahologiques, génétiques et diagnostics de la pathologie humaine. Il doit avoir également une survie suffisante pour permettre la mise en place et l'évaluation de nouveaux traitements. Au cours de ce travail, nous avons développé un nouveau modèle de tumeur orthotopique chez la souris à partir de cellules de glioblastome humain cultivées en neurosphères. D'une façon similaire aux protocoles cliniques de neuro-imagerie, les techniques classiques d'IRM et les critères radiographiques ont été utilisés afin d'étudier la croissance tumorale de ce modèle ainsi que l'évaluation de sa réponse au traitement à base de temozolomide. Les observations par imagerie ont été complétées et/ou confirmées par examen histologique ainsi que par l'étude du transcriptome. Comme en clinique, ce nouveau modèle orthotopique présente une tumeur invasive et nécrotique, une résistance au temozolomide ainsi que des signatures moléculaires associées aux observations histologiques. De plus, ce modèle tumoral est caractérisé par une dynamique de signalisation promouvant l'invasion, la migration et la résistance à l'apoptose à l'origine de sa survie post-thérapeutique. Ainsi, ce modèle préclinique mime, au plus près, les caractéristiques de la pathologie humaine avec une médiane de survie des animaux de 82 jours, ce qui le rend pertinent pour l'évaluation préclinique des nouvelles stratégies thérapeutiques
Glioblastoma is the most common and aggressive subtype of brain tumors. Despite a better understanding of the disease and also the emergence of new therapeutic targets and strategies, the prognosis of patients remains unchanged. The failure to extrapolate preclinical results to the clinics highlights the complex nature of the disease and the importance of appropriate and predictive animal models for the study of new therapies. A pertinent animal model should be able to reproduce the characteristic of the human pathology in terms of disease development pattern, histological and transcriptomic specification, drug failure as well as diagnostic features. In this work, we developed a novel orthotopic mouse model derived from human glioblastoma spheres. Like in clinics, conventional MRI techniques and radiographic criteria were used to characterize tumor growth and treatment response to temozolomide. MRI findings have been completed and/or confirmed by histological examination and transcriptomic studies. Like clinically encountered tumors, this new orthotopic tumor model presents an infiltrating growth pattern, resistance to temozolomide and a molecular signature associated with histological features. In addition, this tumor model is characterized by a dynamic signaling pathway, which promotes cell invasion and migration as well as resistance to apoptosis and consequently to treatment. Thus, this preclinical model mimics clinical features of human glioblastoma and has a median host survival time of 82 days, which would be relevant in the assessment of preclinical therapies

Introdução: O glioblastoma multiforme é a neoplasia maligna primária mais prevalente do sistema nervoso central. Enquanto apresenta aumento em sua incidência, mantém a perspectiva de sobrevida global aproximada de 14 meses. Objetivos: O presente estudo objetiva avaliar a expressão dos genes EGFR, PTEN, MGMT e IDH1/2 e dos microRNAs miR-181b, miR-145, miR-149 e miR-128a em neuroesferas (NE) e células aderidas (CA), a partir das linhagens celulares (T98G e U343) submetidas ao tratamento com temozolomida (TMZ) e com radiação ionizante (RI) isolados e em associação (TMZ+RI). Material e métodos: As linhagens celulares T98G e U343 foram tratadas com TMZ, RI e TMZ+RI. A verificação da expressão dos genes e miRNAs foi realizada utilizando o método de PCR em tempo real. Resultados: Observamos: a) o aumento na expressão do IDH1 após RI (4-fold) e TMZ+RI (5-fold) nas NE (T98G); o aumento na expressão do IDH2 (20-fold) nas NE (T98G), após TMZ, e do IDH2 (3-fold) nas CA (U343) submetidas à TMZ+RI; b) a redução na expressão do EGFR após TMZ; o aumento dessa expressão nas NE (T98G), após RI (2-fold) e TMZ+RI (3-fold), e a sua diminuição nas CA (U343) submetidas à TMZ e TMZ+RI; c) a redução na expressão PTEN nas NE (T98G), após TMZ e RI, e sua expressão nas NE (3-fold) frente à TMZ+RI; d) aumento na expressão de MGMT nas NE (T98G) nos grupos RI (10-fold) e TMZ+RI (25-fold). Quanto a expressão de miRNAs, foram identificados os seguintes resultados: a) as CA (U343) expressaram: miR-181b após TMZ (60-fold), RI (20-fold) e TMZ+RI (40- fold); e miR-128a após TMZ (500-fold), RI (200-fold) e TMZ+RI (600-fold); b) as NE (T98G) após TMZ+RI expressaram: miR-181b (30-fold), miR-149 (40-fold), miR-145 (300-fold) e miR-128a (40-fold); c) as NE (U343) após RI hiperexpressaram miR-149 e miR-145 (ambos em 4000-fold). Conclusão: A RI apresentou-se como fator independente e determinante para a radiorresistência das NE, entretanto não observamos ação de complementariedade de regulação dos oncomiRs observados sobre a expressão dos genes estudados. Esta é a primeira análise na literatura que correlaciona a expressão de genes e de miRNAs através das intervenções TMZ, RI e TMZ+RI sobre células aderidas e neurosferas.
Introduction: Glioblastoma multiforme is the most prevalent primary malignant neoplasm of the central nervous system. It has increased its incidence, while the overall survival remains over 14 months. Objectives: This study aims to evaluate the expression of the following genes EGFR, PTEN, MGMT and IDH1/2 and microRNAs miR-181b, miR-145, miR-149 and miR-128a in adhered cells (AC) and neurospheres (NS) from cell lines (T98G and U343) which were submitted to temozolomide (TMZ) and ionizing radiation (IR), isolated and associated (TMZ + IR). Methods: The cell lines T98G and U343 were treated with TMZ, IR and TMZ+IR. The analysis of gene expression and miRNAs was performed using the PCR in real time. Results: This study demonstrated: a) an improvement in the expression of IDH1 after IR (4-fold) and TMZ + IR (5-fold) in the NS (T98G); increased expression of IDH2 (20-fold) in the NS (T98G) after TMZ and IDH2 (3-fold) in AC (U343) submitted to TMZ + IR; b) reduction in EGFR expression after TMZ; increase this expression in NS (T98G) after IR (2-fold) and TMZ + IR (3-fold), and a decrease in AC (U343) submitted to TMZ and TMZ + IR; c) reduction in PTEN expression in NS (T98G) after TMZ and IR, and its improvement in NS (3-fold) when compared to TMZ + IR; d) increase in the expression of MGMT in NS (T98G) in IR groups (10-fold) and TMZ + IR (25-fold). It was also identified the expression of miRNAs results as: a) AC (U343) expressed more miR-181b after TMZ (60-fold), IR (20-fold) and TMZ + IR (40-fold); and miR- 128a improved after TMZ (500-fold), IR (200-fold) and TMZ + IR (600-fold); b) NS (T98G) after TMZ + IR expressed: miR-181b (30-fold); miR-149 (40-fold); miR-145 (300-fold) and miR-128a (40-fold); c) NS (U343) after IR huge expressed miR-149 and miR-145 (both in 4000-fold). Conclusion: IR was an independent and determining radio resistance factor in NS. However, we observed no complementarity action of oncomiRs regulation. This is the first study in the literature correlating gene expression, and miRNAs through TMZ, IR and IR + TMZ interventions in AC and NS.

Glioblastoma Multiforme (GBM) is the most common lethal brain tumour associated with dismal survival rate. GBM is considered to be an incurable malignancy as these tumours evade all intricate attempts of therapy and no contemporary chemotherapeutic regimen is effective. Although the existence of cancer stem cells (CSCs) is still debatable, it is widely accepted that GBM has a small population of cells expressing CSC markers (~1%) that are highly resistant to chemo-radiation therapy. Recent evidence indicates that hypoxia induces cancer stem cell (CSC) phenotypes via epithelial-to-mesenchymal transition (EMT) that promote therapeutic resistance in solid tumours. Given that GBMs are extensively hypooxygenated heterogenous tumours, understanding the molecular relationship between hypoxia, biology of CSCs, EMT and chemoresistance would be invaluable for development of drugs that can target CSCs. Evidence suggests that hypoxia inducible factors (HIFs), NF-B and aldehyde dehydrogenase (ALDH) together orchestrate the stemness and chemoresistance in hypoxia induced CSCs. But the insights on the mechanisms still remain obscure. In this study we used an in vitro GBM CSC and hypoxia model along with NF-B-p65 and HIF transfected GBM cell lines to investigate the relationship between HIFs, NF-B activation and ALDH activity and their role in chemoresistance. The findings of this study demonstrated that GBM cells grown as spheres consist of a vast proportion of hypoxic cells with elevated CSC and EMT markers suggesting hypoxia induced EMT. GBM-CSCs are chemoresistant and displayed increased levels of HIFs, NF-B and ALDH activity. It was also observed that stable transfection of GBM cells with NF-B-p65 or HIFs induced CSC and EMT markers indicating their essential role in maintaining CSC phenotypes. The study also highlighted the importance of NF-B and ALDH in driving chemoresistance and the potential role of NF-B as the master regulator of hypoxia induced stemness in GBM cells. In this study, we used Disulfiram (DS), an anti-alcoholism drug, in combination with copper (Cu) to target the hypoxia-NF-B axis and inhibit ALDH activity to reverse chemoresistance in GBM CSCs. We showed that DS/Cu is cytotoxic to GBM cells and completely eradicated the resistant CSC population at low nanomolar levels in vitro. We also demonstrated that DS/Cu effectively inhibited GBM in vivo using newly formulated PLGA-DS nanoparticles. DS is an FDA approved drug with low/no toxicity to normal tissues and can freely pass through the blood brain barrier (BBB). Further study may lead to quick translation of DS into clinical trials.

O glioblastoma multiforme (GBM) é um dos tumores mais letais e a radioterapia permanece como um dos principais tratamentos. Novas estratégias são necessárias para coibir a resistência ao tratamento, como o silenciamento de fatores de transcrição (FTs). Nossa hipótese é a de que FTs associados a listas de genes diferencialmente expressos, os quais foram selecionados para linhagens de GBM irradiadas, ou comparando amostras de GBM à amostras de tecido cerebral, possam fornecer alvos moleculares que aumentariam a morte das células tumorais, quando silenciados. Foram analisadas a proliferação, morte e ciclo celular, além da formação e diferenciação de neuroesferas, utilizando, em quase todas as etapas, a citometria de fluxo. Os FTs HEB e E2F1, cujas funções principais estão relacionadas à neurogênese e proliferação celular, foram selecionados a partir das análises in silico de GBM irradiados ou não, ou de GBMs comparados a amostras de cérebro normal, respectivamente. Esses FTs encontram-se expressos em linhagens U87, astrócitos primários e neuroesferas provenientes das mesmas, analisadas por Western blot. O silenciamento de HEB e E2F1 na linhagem U87, de forma geral, reduziu a proliferação, induziu morte celular e diminuiu a porcentagem de células em G0/ G1, em pelo menos um dos tempos analisados (24, 48 e 72h) em relação ao grupo transfectado com a sequência scrambled. O silenciamento de HEB e E2F1 reduziu o número de neuroesferas quando comparadas às células transfectadas com a sequência scrambled. Possivelmente, a capacidade anti-proliferativa do silenciamento dos FTs HEB e E2F1 observada no cultivo em monocamada da U87, possam atuar na capacidade de formação de neuroesferas e, consequentemente, podem ter um papel na manutenção das células tronco do GBM. O silenciamento não alterou a radiorresistência da U87 cultivada em monocamada, com exceção dos efeitos do silenciamento de E2F1 em 24 h, em que houve radioproteção. A irradiação não reduziu o número de neuroesferas silenciadas para HEB em comparação ao grupo não irradiado, mas reduziu o número de células presentes nas neuroesferas, indicando uma possível atuação de HEB na resposta à irradiação em neuroesferas, fato este nunca antes descrito. O silenciamento de E2F1 não interferiu na resposta das neuroesferas à radiação. A expressão de CD133 avaliada oito dias após a dissociação das células silenciadas para E2F1 e HEB, cultivadas em meio de diferenciação, foram superiores ao do grupo scrambled, indicando uma possível diminuição na diferenciação celular. O silenciamento dos dois FTs não atuou na seleção positiva de CD133+ após a irradiação, como observado no grupo das neuroesferas transfectadas com a sequência scrambled e irradiadas, comparado às não irradiadas. Assim, E2F1 e HEB mostraram-se alvos interessantes no sentido de reduzir a proliferação, tanto em células U87 cultivadas em monocamada quanto em neuroesferas.
Glioblastoma multiforme (GBM) is one of the most lethal tumors, and radiation therapy remains one of the main treatments. New strategies are needed to suppress typical GBM treatment resistance and transcription factors (TFs) silencing seems to be a promising strategy. Our hypothesis is that TFs associated with lists of differentially expressed genes which were selected for irradiated compared to shamirradiated GBM cell lines, or GBM samples compared to brain tissue samples, could provide molecular targets that are supposed to increase tumor cell death when they are silenced. We analyzed proliferation, cell death and cell cycle progression, besides the formation and differentiation of neurospheres, using several analyses by flow cytometry. The TFs HEB and E2F1, whose primary functions are related to neurogenesis and cell proliferation, were selected from in silico analysis of GBM irradiated or sham-irradiated GBMs and GBM samples compared with normal brain samples, respectively. These TFs were found expressed in U87 GBM cell line, and primary astrocytes, as well as in neurospheres derivated from both, as analyzed by Western blot. Silencing of E2F1 and HEB in U87 cells, reduced proliferation, induced cell death and decreased the percentage of cells at G0/G1 (24, 48 or 72h) compared to the scrambled sequence transfected group. HEB and E2F1 silencing reduced the number of neurospheres when compared to cells transfected with scrambled sequence. Possibly, the anti-proliferative ability of silencing of HEB and E2F1 TFs observed in monolayer culture of U87, may act in neurospheres forming capacity and therefore may play a role in the maintenance of GBM stem cells. In our experiments, gene silencing did not alter the radio-resistance of U87 grown in monolayer. Irradiation did not reduce the number of neurospheres silenced for HEB compared to non-irradiated group, but reduced the number of cells present in neurospheres, indicating a possible role of HEB in response to ionizing irradiation in neurospheres, a fact that was not described yet. The silencing of E2F1 in neurospheres did not affect the response to irradiation. The expression of CD133, as assessed at eight days after the dissociation of cells silenced for E2F1 and HEB (cultured in differentiation culture media), was superior compared with the scrambled group, indicating a possible decrease in cell differentiation. The silencing of both TFs did not influence the positive selection of CD133 after irradiation, as observed in the group of neurospheres transfected with scrambled sequence, and irradiated compared to nonirradiated. Thus, E2F1 and HEB proved to be interesting targets for decreasing proliferation in both U87 cells grown as monolayer or neurospheres.

Cerebral cortex is essential to exert the highest functions in mammals. Understanding the developmental processes that underlie the generation and functionality of cortical cell populations represents a mayor issue in neurobiology. DYRK1A is a dosage-sensitive gene involved in neurodevelopment with putative roles in corticogenesis. In the present study, we show that the transcriptome of the cerebral cortex of newborn mice lacking one functional copy of Dyrk1a (Dyrk1a+/-) is substantially altered and is mostly the consequence of a delayed developmental gene expression program. We also show that neural progenitors of Dyrk1a+/- mice have a reduced capability to self-renew and to differentiate into oligodendroglial cells in vitro. Moreover, we demonstrate that the epigenetic state of S100b and Gfap glial genes in the cortex of Dyrk1a+/- embryos is altered at specific promoter positions before the onset of gliogenesis, suggesting that Dyrk1a+/- neural progenitors may have an increased astrogliogenic and a decreased oligodendrogenic potential in vivo. In accordance, the number of S100b+ oligodendrocytes is reduced in the postnatal cortex of Dyrk1a+/- mutants whereas the number of Gfap+ astrocytes is increased in the adult. Finally, we show that the myelin thickness of Corpus Callosum axons and of axons of the optic and sciatic nerves is significantly decreased in adult Dyrk1a+/- mice, indicating a possible role of Dyrk1a in myelination. Altogether our results indicate that Dyrk1a dose reduction alters the differentiation potential of the embryonic cortical progenitors and suggest a new role of Dyrk1a in glial development and function.
L’escorça cerebral és essencial per dur a terme les funcions més complexes en els mamífers. Entendre el procés de desenvolupament involucrat en la generació i el funcionament de les poblacions cel·lulars de l’escorça representa un dels principals camps d’investigació de la neurobiologia. DYRK1A és un gen sensible a dosis involucrat en el neurodesenvolupament i molt possiblement en la corticogènesis. En aquest estudi demostrem que la reducció de la dosi gènica de Dyrk1a en ratolí (Dyrk1a+/-) provoca una alteració substancial del transcriptoma de l’escorça cerebral neonatal que es principalment la conseqüencia de un retard en el l’expresió seqüencial dels gens durant el desenvolupament. També hem demostrat que els progenitors neuronals dels ratolins Dyrk1a+/- tenen una menor capacitat d’autorenovació i diferenciació en cèl·lules oligodendroglials in vitro. El estat epigenetic dels gens glials S100b i Gfap es troba alterat en l’escorça d’embrions a posicions especifiques del promotor abans de l’inici de la gliogènesis, suggerint que els progenitors neuronals de ratolins Dyrk1a+/- podrien tenir un potencial astrogliogènic incrementat i un potencial oligodendrogènic disminuït in vivo. En concordança, l’escorça de ratolins Dyrk1a+/- neonatals presenta un major número de progenitors oligodendroglials S100b+, mentres que el número d’astròcits Gfap+ es troba incrementat en ratolins Dyrk1a+/- adults. Finalment, hem demostrat que la vaina de mielina dels axons del cos callos i del nervi òptic i ciàtic es troba significativament reduïda en els ratolins Dyrk1a+/- adults, fet que indica una posible implicació de Dyrk1a en mielinització. En conjunt els nostres resultats indiquen que la baixada de dosi de Dyrk1a altera el potencial de diferenciació dels progenitors embrionaris de l'escorça i suggereix un nou paper de Dyrk1a en el desenvolupament i en la funcionalitat glial.

My thesis research began with a project in which we were trying to determine the function of embryonic stem cell (ESC)-specific miRNAs. Using luciferase constructs containing miRNA binding sites, luciferase expression was inhibited by endogenous miRNAs in ESCs, and by exogenous miRNAs in HeLa cells. Inhibition of luciferase expression by miRNAs was inhibited in HeLa cells using 2’O-methyl-oligonucleotides. In ESCs, 2’O-methyl-oligonucleotides were only effective in partially inhibiting miR290 function. Partial inhibition of miR290 did not result in any obvious phenotypic changes in mESCs. Later studies using 2’O-methyl-oligonucleotides in ESCs were also unsuccessful. The function of ESC-specific miRNAs has since been studied by re-introducing miRNAs into Dicer -/- cells which cannot make miRNAs. These studies have shown that ESC-specific miRNAs are involved in de novo DNA methylation, self-renewal, and cell-cycle regulation. Newly diagnosed glioblastoma (GBM) patients rarely survive more than two years even after surgery, radiotherapy, and chemotherapy using temozolomide (TMZ) or 1,3-bis(2-chloroethy)-1-nitrosourea (BCNU). Eventual regrowth of the tumor indicates that some tumor cells are resistant to therapy. GBM neurosphere-initiating cells (NICs) are thought to be similar to tumor-initiating cells in vivo, and will form invasive tumors in mice, making neurosphere cultures a good model system for studying GBMs. To test whether GBM NICs were resistant to chemotherapy, we used a neurosphere formation assay to measure the number of proliferating NICs in the presence of TMZ or BCNU. The concentrations of chemotherapy drugs required to inhibit neurosphere formation were much less than those required to inhibit bulk cell proliferation or to induce cell death in our neurosphere cultures. For some cultures, there was a robust recovery of neurosphere formation after chemotherapy treatment which appeared to be DNA damage independent. Some of the cultures that showed significant recovery of neurosphere formation underwent reversible cell cycle arrest, possibly reducing chemotoxicity in these cultures. Collectively, these results indicate that GBM neurosphere cultures can regrow after being treated with clinically relevant doses of chemotherapy drugs. Chemotherapy-treated neurosphere cultures remained viable, and formed tumors when injected into mice. Our experiments show that these in vitro assays may be useful in predicting in vivo responses to chemotherapeutic agents.

Parkinson’s disease (PD) is the second largest and progressive neurological disorder caused by selective loss of dopaminergic neurons in substantia nigra pars compacta. Familial PD can be attributed to mutations in different genes. Genetic, environmental and unknown factors may interplay and underlie idiopathic forms of PD. Research on PD is confronted by the long preclinical phase of disease, clinical and genetic heterogeneity and incomplete understanding of disease pathogenesis. It is, therefore, critical to understand the molecular basis of disease, and identify biological biomarkers to diagnose before the onset of irreversible loss of neurons. The current project focusses on identifying mutations in known PD genes to comprehend the underlying molecular mechanisms through genetic and functional studies, and identifying a panel of small molecules which can be used as “probes” to differentiate PD mutations and/or PDs and controls through multidimensional screening. All the experiments described in this project were carried out on human olfactory neurosphere derived (ONS) cells which are primary, unmodified cells previously used to model PD and to identify biologically active small molecules. First, ONS cells from control and sporadic PD patients were screened for copy number variation in known PD genes by MLPA (multiplex ligation dependent probe amplification) which identified heterozygous deletions in the ONS cells of two sporadic PD patients i.e. 2704 (PARK2 ex 02), and 2509 (PARK2 ex 5-7). The later exhibited heterozygous deletions in the adjacent exons spanning introns 4 and 7. Whole exome sequencing for this sample did not identify additional disease causing mutations in any putative Parkinsonism-related gene. Alternative splice variant analysis of PARK2 identified four novel alternative splice variants (SV ex 4-7, SV ex 5-7, SVex 5-8 and SVex 3-9) in 2509 (PARK2 ex 5-7) and another PD ONS cell line. Qualitative analysis of PARK2 splice variants showed more alternative variants in the disease group than in the controls. Treatment of ONS cells with mitochondrial toxins, 50 nM rotenone and 10 uM CCCP also showed changes in splicing patterns of PARK2 and SNCA. Overall, this may suggest that splicing of PARK2 and SNCA can be exacerbated on exposure to mitochondrial stressors which can be quantified further by qPCR. Only the full length transcript was observed for DJ1 in the samples tested. PD ONS cells, exposed to 10uM of the mitochondrial uncoupler CCCP, showed increased susceptibility to cell death, as determined by number of cells surviving treatment compared to control ONS. The SNCA full length transcript level was significantly higher in the LRRK2 G2019S containing cell lines on exposure to 50nM rotenone compared to the controls and idiopathic PDs. LRRK2 containing cell lines manifested an overexpression of SYT-11, increased LysoTracker and increased LC3b intensities; this may suggest autophagosomal and lysosomal impairments that may be linked to the ATP13A2/SYT11 pathway. SYT-11 along with ATP13A2, a lysosomal type 5 P-type ATPase, regulates lysosmal function, the autophagy pathway and alpha-synuclein clearance. The altered expression of PGC-1α may suggest impaired mitochondrial biogenesis in some PD cell lines. Interestingly, cell lines carrying mutations in different regions of PARK2 showed different responses to the toxins which may be due to different pathways involved. Seven ONS cell lines carrying mutations in different PD genes (LRRK2 G2019S, LRRK2 R1441, PINK1 G411S, PARK2 ex 5-7, PARK2 x02, KCNJ15) were prepared for high content screening of cellular, nuclear, mitochondrial, lysosomal, endosomal and autophagosomal analysis. The statistical suitability of cytological profiling was determined by subjecting previous collected data-sets to combined analyses. We observed a classification accuracy of approximately 90% using the vehicle (DMSO) treated profiles alone from PDs and controls. The lysosomal, mitochondrial and LC3b parameters were found to be the most important variables for discrimination between disease and control cell lines. Future experiments may involve the profiling of early onset cases or by challenging the ONS cells with different toxins to identify different aetiological subtypes. In the next phase, small molecules which showed some biological activity in one patient cell line were studied in an attempt to identify probes which help to differentiate between PD and control cell lines. This could also lead to the identification of cytological signatures corresponding to the mutations they harbour, or the specific cellular pathways effected, in particular. A probe panel of small molecules was identified by comparing PD mutant ONS and controls, which showed disease or mutation specific cytological profiles. Some small molecules identified in this study have been known previously for their neuroprotective effect e.g. curcumin, Dihydroergocristine mesylate, Iso-tetrandrine, radicicol, berbamine and oxyacanthine sulfate. Little functional evidence is available to ascertain their mechanism of action. Moreover, cytological profiling can be expanded to include other cellular organelles e.g. endoplasmic reticulum and Golgi complex and the results obtained in this study can be validated by using large number of samples. In conclusion, this study identified novel PARK2 splice variants in ONS cells, the functional evidence of which can help to comprehend how alternative splice variants can lead to neurodegeneration in PD. The functional evidence on genetic forms of PD may propose mitochondrial and endo-lysosomal impairments which require further investigation. Cytological profiling also suggests that ONS cells from PD patients reflect subtle morphological differences in various cellular organelles which can be pronounced on treatment with stressors or with small molecules which can be used as biomarkers for PD.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Environment and Sc
Science, Environment, Engineering and Technology
Full Text

Neuroblastoma (NB) is one of the most common extracranial solid tumors of childhood and it accounts for 15% of cancer-related deaths. NB is an embryonal malignancy arising during fetal or early postnatal life from neural crest-derived sympathetic cells. It is commonly found in the adrenal medulla or along the sympathetic chain. Neuroblastoma shows heterogeneous clinical phenotypes with different rate of aggressiveness and responsiveness to therapies. The broad spectrum of clinical manifestations ranges from spontaneous regression, maturation into a benign ganglioneuroma or, in the worst cases, into an aggressive and metastatic disease. Epidermal growth factor receptor (EGFR) is a protein involved in cellular growth and invasiveness, is found frequently overexpressed or aberrantly activated in human neuroblastoma cells, and its inhibition or decreased phosphorylation causes tumor growth suppression and apoptosis in neuroblastomas. Cathepsin D is a ubiquitous soluble aspartic endopeptidase found in acidic intracellular compartments. CD accomplishes bulk protein degradation and mediates the activation of hormones, as well as the inactivation of mature growth factors through extensive lysosomal degradation. In the first original article published, we found that CD reduced the growth of neuroblastoma cells by downregulating EGFR/MAPK signaling and confers better prognosis in neuroblastoma patients. In the second research article, we demonstrated a dual role of cathepsin D in 2D and in 3D growth of neuroblastoma cells in response to EGF. CD upregulation is necessary to guarantee the survival and proliferation of NB cells grown in suspension (as 3D neurospheres) while it is necessary to downregulate its expression for allowing adherence and anchorage-dependent growth of the tumor cells. To our knowledge, this is the first evidence for such a role of CD in neuroblastomas. Another important novelty of the present work is the use of mixed clones expressing CD at different level to mimic cancer heterogeneity arising during tumor evolution. The differential expression and role of CD in the two culture systems, let to hypothesize that cathepsin D could be regulated via epigenetic mechanisms. The bioinformatic analysis performed in this work, identified 38 miRNAs that downregulate CD in human neuroblastomas, and these miRNAs are associated with biological processes involved in cell migration, cell motility, cell growth and EGFR signaling, among others. Collectively, we have uncovered a novel role of CD in the metastatic spreading of tumors. This finding may have a translational relevance, and we propose CD as a possible biomarker for metastatic neuroblastomas and for the stratification of patients in a view of personalized medicine. Modulators of CD synthesis and activity could be a novel therapeutic strategy to ameliorate patients’ outcome. The third part of my work deals with the role of CD in the chaperone-mediated autophagy of annexin A2, which results in attenuation of the NB cell malignant phenotype.

Parkinson’s disease (PD) is the second most common neurodegenerative disease, affecting over five million patients worldwide. Like Alzheimer’s disease (AD), it mostly affects the elderly and causes considerable disability and suffering. Unfortunately, the molecular mechanism of PD is still poorly understood, and there are no drugs available to treat the disease. Our overall aim was to identify natural products to probe PD by phenotypic assay using human olfactory neurosphere-derived (hONS) cells from PD patients. The research presented in this thesis exemplifies the importance of natural products as chemical probes for further investigation of PD as well as lead compounds for future PD-drug development. The thesis begins with an introduction of PD and the chemotherapeutics for PD. It also covers a review on the natural origin anti-PD compounds and the analysis of their physicochemical properties using Lipinski’s rule of five. As part of a research program aiming to identify anti-PD chemical probes, a high throughput screening assay was developed to screen 4224 fractions. Twenty fractions were confirmed to display neuroprotective effects of the PD cells against rotenone. Seven prioritized fractions, representing one Australian marine sponge Jaspis splendens (subject 1) and two Australian terrestrial plants Gloriosa superba (subject 2) and Alangium villosum (subject 3), were selected for large scale extraction and isolation. The results were presented in Chapter 2 to 5.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Natural Sciences
Science, Environment, Engineering and Technology
Full Text

The high composition of fruits and vegetables in the daily diet is associated with cognitive well-being, especially in the elderly population. The phytonutrients are shown to have effects as antioxidants that neutralize oxidative stressors and can interact with molecular pathways to signal neuron survival. Adult hippocampal neurogenesis is a dynamic lifelong process of generating functional newborn neurons in the granular layer of the dentate gyrus from adult precursor cells. This process contributes to brain plasticity and plays a role in learning and memory. External stimuli such as environmental enrichment and physical activity are known to positively regulate this process. However, the role of nutrition and whether nutritional compounds have pro-neurogenic effects on adult hippocampal precursor cells are still elusive. In this study, I investigated the impact of dietary compounds in apples, a significant source of phytonutrients in our food, on adult hippocampal neurogenesis. I demonstrated that quercetin, the most abundant polyphenol in apple, induces cell cycle exit and differentiation of adult hippocampal precursor cells in monolayer culture. Furthermore, this compound also increases the number of surviving cells upon differentiation in vitro, through the activation of endogenous antioxidants in the Nrf2-Keap1 pathway and the prosurvival Akt pathway. Quercetin supplementation in vivo is also shown to significantly increase the number of surviving cells and new neurons in the dentate gyrus. To search for other potential active compounds in apple, I performed bioassay-guided fractionation whereby the flesh extract from apples of the Pinova cultivar was subjected to liquid- and solid phase separation and the active fraction was determined using primary neurosphere assays using cells derived from adult mouse dentate gyrus. Using mass spectometry, we revealed that the active compounds in the apple flesh extract are dihydroxybenzoate glycosides, which are non-flavonoid benzoic acid derivatives. I also confirmed that the isomers of these compounds; 2,3- and 3,5 dihydroxybenzoic acids significantly increase the number of neurospheres. Interestingly, 3,5 dihdroxybenzoic acid is an agonist of lactate receptor hydroxycarboxylic acid receptor 1 (HCAR1), with an even higher affinity than lactate. This receptor is suggested to mediate neurotrophic actions such as increasing production and release of BDNF. I also demonstrated for the first time that this receptor is presence in adult hippocampal precursor cells. To observe whether customary fruits or fruit-related products consumption affects adult hippocampal neurogenesis, I performed an experiment giving apple juice supplementation ad libitum to mice. I did not find a significant increase in net neurogenesis or the performance in the Morris water maze after apple juice supplementation. This is likely due to the low concentration of active compounds in apple juice failing to reach an effective concentration in the body. I conclude that apples provide potential proneurogenic compounds that can influence adult hippocampal neurogenesis through the activation of endogenous antioxidant mechanisms and molecular pathways for cell survival. Further studies are necessary to investigate the role of HCAR1 activation on adult hippocampal neurogenesis, which is a potential new mechanism to explain the health benefits of fruit and vegetable consumption
Eine Ernährung die täglich reich an Obst und Gemüse ist, hat insbesondere bei älteren Menschen einen positiven Einfluss auf kognitive Fähigkeiten. Pflanzeninhaltsstoffe wirken als natürliche Antioxidantien, indem sie oxidative Stressoren neutralisieren. Weiterhin beeinflussen pflanzliche Nährstoffe molekulare Signalwege welche beim Überleben von Neuronen eine Rolle spielen. Die adulte hippocampale Neurogenese ist ein dynamischer, lebenslanger Prozess, bei dem aus Vorläuferzellen funktionelle neue Neuronen in der Körnerzellschicht des Gyrus dentatus gebildet werden. Dieser Prozess trägt zur Plastizität des Gehirns bei und spielt eine bedeutende Rolle beim Lernen und für das Gedächtnis. Externe Stimuli wie zum Beispiel eine reizreiche Umgebung und körperliche Aktivität wirken als positive Regulatoren und begünstigen die adulte hippocampale Neurogenese. Welche Rolle die Ernährung dabei spielt und ob Nahrungsbestandteile einen proneurogenen Effekt auf adulte hippocampale Vorläuferzellen haben ist kaum bekannt. In diesem Projekt habe ich den Effekt von Nahrungsbestandteilen aus Äpfeln, welche eine bedeutende Quelle von pflanzlichen Nährstoffen in unserer Ernährung darstellen, auf die adulte hippocampale Neurogenese untersucht. Ich habe gezeigt, dass Querzetin, das am reichlichsten in Äpfeln enthaltende Polyphenol, in der Monolayer-Zellkultur den Austritt aus dem Zellzyklus induziert und die Differenzierung von adulten hippocampalen Vorläuferzellen fördert. Des Weiteren steigert Querzetin nach der Differenzierung in vitro die Anzahl an überlebenden Zellen. Dies geschieht durch die Aktivierung von endogenen Antioxidantien des Nrf2-Keap1-Signalweges und des für das Überleben von Zellen förderlichen Akt-Signalweges. Die Verabreichung von Querzetin in vivo als Nahrungsergänzungsmittel führte ebenfalls zu einem signifikanten Anstieg der Anzahl an überlebenden Zellen und neu gebildeten Nervenzellen im Gyrus dentatus. Um weitere potentiell aktive Wirkstoffe von Äpfeln zu bestimmen, habe ich eine Bioassay-ausgerichtete Fraktionierung durchgeführt, wobei der Fruchtfleischextrakt von Äpfeln der Sorte Pinova einer Fest-/ Flüssig-Separation unterzogen wurde. Die aktive Fraktion wurde anhand der primären Neurosphäre-Assay-Methode mit Zellen aus dem Gyrus dentatus adulter Mäuse ermittelt. Mittels spektrometrischer Analyse habe ich gezeigt, dass die aktiven Wirkstoffe im Fruchtfleischextrakt von Äpfeln zur Gruppe der Dihydroxybenzol-Glykosiden gehören, welche den nicht-flavonoiden Benzoesäure-Derivaten zuzuordnen sind. Im in vitro Neurosphäre-Assay habe ich zudem gezeigt, dass die Isomere dieser Wirkstoffe, die 2,3- und die 3,5-Dihydroxybenzoesäuren, die Anzahl der Neurosphären signifikant erhöhen. Interessanterweise ist die 3,5-Dihydroxybenzoesäure ein Agonist des Laktatrezeptors Hydroxycarboxylic acid receptor 1 (HCAR1) und weist sogar eine noch höhere Affinität als Laktat auf. Es wird suggeriert, dass dieser Rezeptor neurotrophische Wirkungen vermittelt, wie zum Beispiel eine erhöhte Produktion von BDNF und dessen Ausschüttung. Zudem habe ich das Vorkommen dieses Reporters erstmalig bei adulten hippocampalen Vorläuferzellen nachgewiesen. Um zu untersuchen, ob der Konsum handelsüblicher Obstprodukte die adulte hippocampale Neurogenese beeinflusst, habe ich Mäusen Apfelsaft ad libitum verabreicht. Nach der Gabe von Apfelsaft sah ich keinen signifikanten Anstieg der Gesamtneurogenese und keine Verbesserung der Leistungsfähigkeit im Morris-Wasserlabyrinth-Test. Dies ist bedingt durch eine zu geringe Konzentration der aktiven Wirkstoffe im Apfelsaft wodurch die wirksame Konzentration im Körper nicht erreicht wird. Ich schlussfolgere, dass in Äpfeln potentielle pro-neurogene Inhaltsstoffe enthalten sind, welche die adulte hippocampale Neurogenese beeinflussen. Dies wird insbesondere durch die Aktivierung endogener antioxidativer Mechanismen und molekularer Signalwege vermittelt, die für das Überleben von Zellen von Bedeutung sind. Weitere Studien sind nötig, um zu bestimmen wie sich die Aktivierung von HCAR1 auf die adulte hippocampale Neurogenese auswirkt. Dies stellt einen potentiellen neuen Wirkmechanismus dar, welcher die gesundheitlichen Vorteile von Obst- und Gemüsekonsum belegt

碩士
國立陽明大學
生醫光電研究所
102
Abstract To observe and culture the 3-D biological sample is always big challenge in biological experimints.3-D biological sample is closer to real condition then 2-D. In our study, we used SPIM(selective plane illumination microscopy) which is a type of light sheet microscope to research the growth of neurospheres. neurospheres is a 3-D functional biological sample which are aggregated by a lot of single neural stem cells,and neurospheres have ability of self-renew,proliferationand also will differentiated to neuron,astrocytes and oligodendrocytes. SPIM provide a low phototoxity,fast image acquisitionand 3-D reconstruction by scanning Z axis. Because neural stem cells were cultured to a 3-D functional neurospheres,the proliferation of neorosphere is the basic of cell culture in 3-D environment.The feature of light sheet microscopy is that illumination light will in the vertical direction with detection objective,so we developed a microfluidics device which can provide a micro environment for neurospheres.In the end, we observed the growth of cell in the 3 Days in the device and took the cell counting,demonstrated the relationship between the single neural stem cell and neurospheres. The thickness of beam waist is a key parameter of SPIM,we improve the illumination section by thinning the thickness of beam waist,promote the z resolution of system.The SPIM will be applied in the smaller scale biological sample.

博士
國防醫學院
生命科學研究所
107
Abstract At present, the treatment of peripheral nerve injury has become more and more common. In addition to autologous nerve transplantation, there is no other kind of therapy available for the nerve defect of more than 3 cm. Acellular dermal matrix (ADM) has been widely used in the study of burns and scalds, and its extension and plasticity may be made into suitable nerve channels under different forms of nerve defects. Neural stem cells can now be used for peripheral nerve repair, but it is not easy to obtain. Adipose-derived stem cells (ADSC) are well-acquired, proliferative, and can differentiate into cells of different germ layers. It is assumed that ADSCs can be induced into a large number of neural stem cells and preserved, and neural stem cells can be decellularized. Survival on the tissue may help in the development of neural tissue engineering. The purpose of this dissertation is to use the ADM to incorporate neural stem cells (NSC) induced by ADSCs to establish neural tissue engineering. The focus of this study is that ADSCs can be induced into NSCs via growth factors, which can solve the source and quantity problems of NSCs. It was later confirmed that NSCs cultured on both ADM and ASN survived, and NSCs that survived on ADM also maintained markers of neural stem cells, and in animal experiments, ADM combined with NSC demonstrated improved ability to repair peripheral nerve defects. Importantly, in addition to providing new alternatives to peripheral nerve repair, ADM can be used to meet different forms of peripheral nerve defects in the future. Therefore, the exfoliation and plasticity of the ADM may be utilized and NSCs may be combined to treat different forms of nerve damage.

Huntington Disease (HD) is an autosomal dominant disorder characterized by motor, cognitive and behavioral features caused by a CAG expansion in the HTT (huntingtin) gene beyond 35 repeats. Since the discovery of the HTT mutation 24 years ago, more than 15,000 papers have been published on HD. However, both the role of the huntingtin (wtHTT) in healthy individuals and the molecular mechanisms by which the mutated huntingtin causes the disease remain unclear. The discovery of induced pluripotent stem cell (iPSC) technology offer the possibility to generate patient-specific iPS cells and to enable the development of in vitro HD models that more accurately reflect the human disease. The disease-specific iPSCs can be differentiated into relevant cell-types affected in HD, holding a great potential for disease modeling and drug screening. In the present study, we have obtained dermal fibroblasts from 16 HD patients and 6 healthy controls. Fibroblasts from a young adult healthy control, a young subject with 43 CAG repeats, in an early stage of the disease and two JHD (Juvenile Huntington disease) patients were reprogrammed into induced pluripotent stem cells. All iPS clones that show an uniform flat morphology are characterized for their stemness and pluripotency, both in vitro through embryoid bodies formation and in vivo through teratoma formation assay. iPS cells from a JHD (85CAG) and healthy control, were differentiated into neurospheres of neural precursors (NPCs) by a new protocol optimized for differentiation of iPSCs derived embryoid bodies expressing all the three germ layers (ectoderm, mesoderm and endoderm) in neurospheres of Neural Stem Cells (NSCs). Differentiation assay confirmed that they possessed the potentiality, to differentiate into subtypes of neuronal and glial cells. Gene expression profiling was performed on iPSC HD-derived neurospheres using GeneChip Human Transcriptome Array 2.0. iPSC HD-derived neurospheres exhibit an impaired brain development processes when compared with iPSC-derived neurospheres from healthy individuals. This study aimed to produce a valid model of the Huntington disease. This novel in vitro model will permit us to have a closer view to neuronal development networks by morphologically and physiological studies on the cell types obtained from the neuropheres of NPC.

碩士
國防醫學院
生物及解剖學研究所
95
The nontransmembrane protein tyrosine phosphatase SHP-2 is widely distribution and implicates in cell growth and development. The previous study demonstrated that SHP-2 could express in neural precursor and glial cells. However, little know about SHP-2 subcellular localization in CNS precursor cells and differential cells including glial cells and neuron. This study was aimed to investigate the subcellular localization of SHP-2 in CNS precursor cells and terminal differentiation cells and to determine the role of SHP-2 on neurosphere cell development. We use immunocytochemistry and Western blot analysis to identify the expression of SHP-2 in neurosphere cells. We found phosphorylated SHP-2 expressed in focal adhesions of neural precursor cells, astrocytes and neurons. On activation, SHP-2 might recruit to focal adhesions, suggesting involvement of neurosphere cell adhesion and motility. We used protein tyrosine phosphatase inhibitor IV to block SHP-2 function to observe the role of SHP-2 involved in neurosphere cell spreading and migration. The results showed that neurosphere cell migration was decreased and the morphology of neuron and astrocyte was also changed. Taken together, SHP-2 might translocation to focal adhesions in response to integrin-mediated neural precursor cell adhesion, and thereby facilitate regulation of focal adhesion assembly/disassembly, cytoskeletal reorganization and cell migration by SHP-2.

碩士
國立成功大學
細胞生物與解剖學研究所
105
Adipose-derived stem cells (ASCs) were an ideal cell source for stem cell therapy can promote nerve regeneration after injury. The fibroblast growth factor 9 (FGF9) can alter the proliferation and cell-fate decision in stem cells. In my project, we aim to investigate the role of FGF9 during neurosphere formation of ASCs. We used chitosan- coated dish to culture ASCs to induce sphere formation with or without addition of FGF9. The expression of gene was detected by PCR and the protein expression level was observed by Western blot and IF staining. Besides, we investigated the involvement of potential signaling pathway of FGF9 signaling during the sphere formation using specific inhibitors or shRNA. The results showed the spheres formed by ASCs containing both neuronal and glial lineage cells. Addition of FGF9 induced spheres differentiation into Schwann lineage cells. Then, the activation of Akt, ERK signal pathway was decreased after forming the sphere and the inactivation of signal pathway was necessary for sphere formation. However, the phosphorylation of Akt pathway was transiently preserved by FGF9. Furthermore, the involvement of Akt signaling was confirmed by blockage of Akt signaling. The FGFR2 and 3 blocking antibody and shFGFRs was used to block FGFRs to investigate the major FGFR of FGF9 induced Schwann cell differentiation. The expression of Schwann cell markers induced by FGF9 was decreased when knockdown FGFR2. The result suggested that FGF9 could modulate NLC differentiation by activating Akt signaling via binding with FGFR2 during sphere formation. The finding of current study may show the possibility of differentiating ASCs into desired neural lineage by modulating the level of FGF9 during sphere formation.

碩士
國立臺灣大學
生醫電子與資訊學研究所
100
The purpose of this research is to investigate the infrared irradiation effect on the cell growth and cell physiology of neonatal mice brain cell expression. The plasmonic thermal emitter with broad bandwidth (3~5 μm) and specific emission waveband were designed and used to irradiate the cells for 7 days in order to identify the specific wavelength that induces the cell expression the most significant. Infrared emitters used in this study are fabricated based on the theory of surface plasmon. The purpose of the second experiment is to understand the effect of narrow band infrared irradiation with 3.5, 4.0, and 4.5 μm wavelengths on cell growth features. The heat is generated by inputting electric current to the molybdenum film on silicon substrate. The infrared source can be achieved by heating the triple layer structure which consists of a SiO2 layer between two Ag films on the molybdenum, it is called metal/dielectric/metal (MIM) structure. The top Ag layer is perforated by periodic holes array, and the emission wavelength can be altered and selected by changing the lattice constant and diameter of the holes arrays. By metabolomics analysis, it is found that the mitochondria in the cell tend to follow the glycolysis pathway which can be benefit to the cell growth. It is observed that after illumination by PTE with peak wavelength at 4.0μm, the cell exhibits the largest response and cell number increased up to 45%. The experiments indicate that the cell number can be enhanced and tend to form neurospheres after infrared illumination. The study shows that IR illumination can improve the cell growth and points to new possibilities for future research.

博士
國立成功大學
臨床醫學研究所
103
Peripheral nerve injuries are a significant source of disability. Although adherence to well-established basic principles of evaluation and repair can optimize results, clinical outcomes of peripheral nerve injuries are often suboptimal. The stem cell therapy of neural progenitor cell (NPC) from adipose-derived stem cell (ASC) may bring hope to peripheral nerve injury, especially in the clinical reality of tissue shortage. Transdifferentiation can be induced from ASC to NPC by adding numerous neurotrophic factors, or by mechanical microenvironment such as chitosan surface coating. To our knowledge, chitosan has widely been used for drug release and 3-dimentional scaffold for tissue engineering. And owning to the characteristics of biodegradable and biocompatible in nature, it also applies as nerve conduit to guide nerve regeneration. In author’s previous study, transdifferentiation of ASC into NPC can be achieved by using chitosan surface coating as the 3-D scaffold. Under the consideration of slow nature of nerve regeneration, continued maintenance of the microenvironment for the NPC seems possible to achieve by combined use of the chitosan conduit with induced NPC from ASC. The purpose of this study is to use the in vivo rat model of sciatic nerve transection to survey the therapeutic benefit of chitosan coated conduit containing with induced NPC from ASC. For the analysis of functional recoveries, we will focus on the improvement of the walking track analysis, relative gastrocnemius muscle weight measurement, and the ratio of regenerated myelinated axons. For the exploration of underlying mechanism, we will try to investigate the survival of functional cells after treatment, including the inflammatory profile.

Lo studio in vitro dei progenitori neurali si basa sul concetto che, delle condizioni di coltura cellulare, siano capaci di emulare un ambiente che mantiene uno stato non-differenziato e di autorinnovamento cellulare. Ciò porta alla crescità di neurosfere. Sonic hedgehog (SHH) rappresenta una via di segnalazione chiave sia nel contesto della morfogenesi che nella proliferazione cellulare a livello del sistema nervoso centrale. In particolare, SHH e coinvolto nella segnalazione mitogenica post-natale dei progenitori granulari del cereveletto (GCP). Oltre ai mitogeni communemente usati (EGF e bFGF), dimostriamo che sia il trattamento con SAG, un agonista della via di SHH, che l’attivazione genetica della via SHH tramite delezione del gene PTCH1, portano alla crescità di neurosfere, a partire da espianti di cereveletto all’età di 7 giorni postnatale (p7). Oltremodo, SAG puo indurre la formazione di neurosfere, chiamate sfere SAG dipendente murine (mSS), solo partendo da tessuto cerebellare p7 e non dalla zona sub-ventricolare (SVZ), generalmente nota come fonte di cellule che danno origine alle neurosfere. Inoltre, le colture mSS esprimono ZIC1, ATOH1 e NESTIN, suggerendone l’identità di granuli cerebellari. Notevolmente, rispetto al noto comportamento delle cellule GCP, le cellule mSS possono essere mantenute in colture per tempi indefiniti, come misurato attraverso il saggio di clonogenicità, applicato per un periodo di 10 settimane. Nel contesto dell autorinnovamento confermiamo inoltre l’espressione dei geni POU3f2, POU5f1, NANOG e SOX2 nelle cellule mSS, che sono comunemente associati ai progenitori neuronali. Rilevantemente, le cellule mSS, per essere clonogeniche e per mantenere l’attività della via SHH, misurata in termini di espressione di GLI1, PTCH1 e NMYC, rimangono dipendenti dal SAG. Oltre a mantenere la capacita di autorinnovamento, le colture mSS mantengono anche la capacità di differenziamento. Il differenziamento in vitro porta alla formazione di cellule con morfologia tipica delle cellule granulari del cereveletto che dimostrano positività per tubulina beta-3, misurato sia per western blot che per immunofluorescenza. Inoltre, durante il differenziamento le cellule mSS aumentano l’espressione di GABRA6, un marcatore del differenziamento delle cellule GCP. Questo lavoro e la dimostrazione che si può confezionare su misura un protocollo di coltura cellulare capace di espandere cellule progenitrici da particolari zone del sistema nervoso centrale. Questo permette di estendere il concetto di neurosfera a cellule che non sono state considerate tali fino ad ora. In particolare, estendiamo questo concetto alle cellule GCP rivelando la loro capacità di estensivo autorinnovamento in vitro.
The in vitro study of neural progenitors is based around the idea that defined culture conditions can emulate an environment which maintains an undifferentiated state and self-renewal capability of explanted progenitor cells, culminating in the growth of neurospheres. SHH signaling is a key signaling pathway with roles in morphogenesis and cell proliferation in the central nervous system, in particular, involved in mitogenic signaling within the context of postnatal cerebellar granule cell expansion. We demonstrate that in addition to commonly used mitogens such as EGF and bFGF, the SHH pathway agonist SAG, as well as genetic activation of SHH signaling by PTCH1 deletion, can lead to the growth of neurospheres from postnatal day 7 (p7) cerebellar explants. Interestingly, SAG derived cultures, termed murine SAG dependent spheres (mSS), can be generated solely from the p7 cerebellum and not from the sub-ventricular zone, a confirmed source of neurospheres derived by growth factors. Further, mSS cultures expressed ZIC1, ATOH1 and NESTIN, indicating that their identity is of the cerebellar granule cell progenitor (GCP) lineage. Strikingly, mSS cells can be maintained indefinitely in culture, as demonstrated by extensive clonogenic capability, assayed over a period of ten weeks. In the context of self-renewal, we assay gene expression of POU3f2, POU5f1, NANOG, and SOX2, genes associated with neural progenitors, which we find expressed in mSS cells. Importantly, mSS cultures are continuously dependent on SAG for their clonogenic potential and SHH pathway activation, assayed by expression of GLI1, PTCH1 and NMYC. In addition to the aforementioned extensive self-renewal capability mSS neurosphere cultures also maintain the ability to differentiate. In vitro differentiation leads to formation of cells with typical granule cell morphology and which are positive for beta3-tubulin, as assayed by western blot and immunofluorescence. Additionally, differentiated mSS cells display up-regulation of the cerebellar granule expressed gene GABRA6. Our work demonstrates that by applying culture conditions which are tailored towards biological characteristics of specific regions of the central nervous system the paradigm of the neurosphere can be expanded to include lineages not previously studied in this way. In particular, we apply this principle to unmask the property of cells from the GCP lineage as having extensive self-renewal capability in vitro.

À la fin du 19e siècle, Dr. Ramón y Cajal, un pionnier scientifique, a découvert les éléments cellulaires individuels, appelés neurones, composant le système nerveux. Il a également remarqué la complexité de ce système et a mentionné l’impossibilité de ces nouveaux neurones à être intégrés dans le système nerveux adulte. Une de ses citations reconnues : “Dans les centres adultes, les chemins nerveux sont fixes, terminés, immuables. Tout doit mourir, rien ne peut être régénérer” est représentative du dogme de l’époque (Ramón y Cajal 1928). D’importantes études effectuées dans les années 1960-1970 suggèrent un point de vue différent. Il a été démontré que les nouveaux neurones peuvent être générés à l’âge adulte, mais cette découverte a créé un scepticisme omniprésent au sein de la communauté scientifique. Il a fallu 30 ans pour que le concept de neurogenèse adulte soit largement accepté. Cette découverte, en plus de nombreuses avancées techniques, a ouvert la porte à de nouvelles cibles thérapeutiques potentielles pour les maladies neurodégénératives. Les cellules souches neurales (CSNs) adultes résident principalement dans deux niches du cerveau : la zone sous-ventriculaire des ventricules latéraux et le gyrus dentelé de l’hippocampe. En condition physiologique, le niveau de neurogenèse est relativement élevé dans la zone sous-ventriculaire contrairement à l’hippocampe où certaines étapes sont limitantes. En revanche, la moelle épinière est plutôt définie comme un environnement en quiescence. Une des principales questions qui a été soulevée suite à ces découvertes est : comment peut-on activer les CSNs adultes afin d’augmenter les niveaux de neurogenèse ? Dans l’hippocampe, la capacité de l’environnement enrichi (incluant la stimulation cognitive, l’exercice et les interactions sociales) à promouvoir la neurogenèse hippocampale a déjà été démontrée. La plasticité de cette région est importante, car elle peut jouer un rôle clé dans la récupération de déficits au niveau de la mémoire et l’apprentissage. Dans la moelle épinière, des études effectuées in vitro ont démontré que les cellules épendymaires situées autour du canal central ont des capacités d’auto-renouvellement et de multipotence (neurones, astrocytes, oligodendrocytes). Il est intéressant de noter qu’in vivo, suite à une lésion de la moelle épinière, les cellules épendymaires sont activées, peuvent s’auto-renouveller, mais peuvent seulement ii donner naissance à des cellules de type gliale (astrocytes et oligodendrocytes). Cette nouvelle fonction post-lésion démontre que la plasticité est encore possible dans un environnement en quiescence et peut être exploité afin de développer des stratégies de réparation endogènes dans la moelle épinière. Les CSNs adultes jouent un rôle important dans le maintien des fonctions physiologiques du cerveau sain et dans la réparation neuronale suite à une lésion. Cependant, il y a peu de données sur les mécanismes qui permettent l'activation des CSNs en quiescence permettant de maintenir ces fonctions. L'objectif général est d'élucider les mécanismes sous-jacents à l'activation des CSNs dans le système nerveux central adulte. Pour répondre à cet objectif, nous avons mis en place deux approches complémentaires chez les souris adultes : 1) L'activation des CSNs hippocampales par l'environnement enrichi (EE) et 2) l'activation des CSNs de la moelle épinière par la neuroinflammation suite à une lésion. De plus, 3) afin d’obtenir plus d’information sur les mécanismes moléculaires de ces modèles, nous utiliserons des approches transcriptomiques afin d’ouvrir de nouvelles perspectives. Le premier projet consiste à établir de nouveaux mécanismes cellulaires et moléculaires à travers lesquels l’environnement enrichi module la plasticité du cerveau adulte. Nous avons tout d’abord évalué la contribution de chacune des composantes de l’environnement enrichi à la neurogenèse hippocampale (Chapitre II). L’exercice volontaire promeut la neurogenèse, tandis que le contexte social augmente l’activation neuronale. Par la suite, nous avons déterminé l’effet de ces composantes sur les performances comportementales et sur le transcriptome à l’aide d’un labyrinthe radial à huit bras afin d’évaluer la mémoire spatiale et un test de reconnaissante d’objets nouveaux ainsi qu’un RNA-Seq, respectivement (Chapitre III). Les coureurs ont démontré une mémoire spatiale de rappel à court-terme plus forte, tandis que les souris exposées aux interactions sociales ont eu une plus grande flexibilité cognitive à abandonner leurs anciens souvenirs. Étonnamment, l’analyse du RNA-Seq a permis d’identifier des différences claires dans l’expression des transcripts entre les coureurs de courte et longue distance, en plus des souris sociales (dans l’environnement complexe). iii Le second projet consiste à découvrir comment les cellules épendymaires acquièrent les propriétés des CSNs in vitro ou la multipotence suite aux lésions in vivo (Chapitre IV). Une analyse du RNA-Seq a révélé que le transforming growth factor-β1 (TGF-β1) agit comme un régulateur, en amont des changements significatifs suite à une lésion de la moelle épinière. Nous avons alors confirmé la présence de cette cytokine suite à la lésion et caractérisé son rôle sur la prolifération, différentiation, et survie des cellules initiatrices de neurosphères de la moelle épinière. Nos résultats suggèrent que TGF-β1 régule l’acquisition et l’expression des propriétés de cellules souches sur les cellules épendymaires provenant de la moelle épinière.
At the end of the 19th century, Dr. Ramón y Cajal, a scientific pioneer, discovered that the nervous system was composed of individual cellular elements, later called neurons. He also noticed the complexity of this system and mentioned the impossibility of new neurons to be integrated into the adult nervous system. One of his famous quotes: “In adult centers the nerve paths are something fixed, ended, immutable. Everything may die, nothing may be regenerated” is representative of the prevalent dogma at the time (Ramón y Cajal 1928). Key studies conducted in the 1960-1970s suggested a different point of view. It was demonstrated that new neurons could be born during adulthood, but this discovery created an omnipresent skepticism in the scientific community. It took 30 years for the concept of adult neurogenesis to become widely accepted. This discovery, along with more advanced techniques, opened doors to potential therapeutic avenues for neurodegenerative diseases. Adult neural stem cells (NSCs) reside mainly in two niches in the brain: the subventricular zone of the lateral ventricles and the dentate gyrus of the hippocampus. Under normal conditions, neurogenesis level is relatively high in the SVZ whereas some steps are rate-limiting in the hippocampus. In contrast, the spinal cord is rather defined as a quiescent environment. One of the main questions that arose from these discoveries is: how do you activate adult NSCs in order to increase neurogenesis levels? In the hippocampus, environmental enrichment (including cognitive stimulation, exercise and social interactions) has been shown to promote hippocampal neurogenesis. The plasticity potential of this region is important as it could play a crucial role in rescuing learning and memory deficits. In the spinal cord, studies conducted in vitro demonstrated that ependymal cells found around the central canal have self-renewal and multipotency capacities (neurons, astrocytes, oligodendrocytes). Interestingly, it turns out that in vivo, following a spinal cord lesion, ependymal cells become activated, can self-replicate, but can only give rise to glia cell fate (astrocytes and oligodendrocytes). This new post-injury function shows that plasticity can still occur in a quiescent environment and could be exploited to develop endogenous spinal cord repair strategies. v As mentioned above, NSCs play important roles in normal brain function and neural repair following injury. However, little information is known about how a quiescent NSC becomes activated in order to perform these functions. The general objective of this project was to investigate the mechanisms underlying activation of neural stem cells in the adult central nervous system. My specific aims were to address this question using adult mice in two complementary models: 1) activation of hippocampal NSCs by environmental enrichment, and 2) activation of spinal cord NSCs by injury-induced neuroinflammation. Moreover, 3) to gain new insights into the molecular mechanisms of these models, we will perform transcriptomics studies to open new lines of investigation. The first project is expected to provide us with new insights into the basic cellular and molecular mechanisms through which environmental enrichment modulates adult brain plasticity. We first evaluated the contribution of individual environmental enrichment components to hippocampal neurogenesis (Chapter II). Voluntary exercise promotes neurogenesis, whereas a social context increases neuronal activation. We then determined the effect of these components on behavioural performances and transcriptome using an eight-arm radial maze to assess spatial memory, novel object recognition, and RNA-Seq, respectively (Chapter III). Runners show stronger spatial short-term memory recall, whereas mice exposed to social interactions had a better cognitive flexibility to abandon old memory. Surprisingly, RNA-Seq analysis indicated clear differences in the expression of modified transcripts between low runners and high runners, as well as for social interacting mice (within the complex environment). The second project consists of discovering how ependymal cells acquire NSC properties in vitro or multipotentiality following lesions in vivo. A RNA-Seq analysis revealed that the transforming growth factor-β1 (TGF-β1) acts as an upstream regulator of significant changes following spinal cord injury (Chapter IV). We therefore confirmed the presence of this cytokine after lesion and investigated its role on proliferation, differentiation, and survival of neurosphere-initiating cells from the spinal cord. Our results suggest that TGF-β1 regulates the acquisition and expression of stem cell properties of spinal cord-derived ependymal cells.

La neurogenèse persiste à l’âge adulte dans deux régions du système nerveux central (SNC) des mammifères : la zone sous-ventriculaire (SVZ) du cerveau antérieur et la zone sous-granulaire (SGZ) de l’hippocampe. Cette neurogenèse est possible grâce à la capacité de prolifération des cellules souches présentes dans les niches de la SVZ et la SGZ, mais en vieillissant, le cerveau subit une diminution dramatique du nombre de cellules souches neurales adultes (CSNa), une diminution de la prolifération cellulaire et une altération des niches de neurogenèse. Cependant, une importante question reste sans réponse : comment la perte tardive des CSNa est temporellement reliée aux changements de l’activité de prolifération et de la structure de la principale niche de neurogenèse (la SVZ)? Afin d’avoir un aperçu sur les événements initiaux, nous avons examiné les changements des CSNa et de leur niche dans la SVZ entre le jeune âge et l’âge moyen. La niche de la SVZ des souris d’âge moyen (12 mois) subit une réduction de l’expression des marqueurs de plusieurs sous-populations de précurseurs neuraux en comparaison avec les souris jeunes adultes (2 mois). Anatomiquement, cela est associé avec des anomalies cytologiques, incluant une atrophie générale de la SVZ, une perte de la couche de cellules sousépendymaires par endroit et l’accumulation de gouttelettes lipidiques de grande taille dans l’épendyme. Fonctionnellement, ces changements sont corrélés avec une diminution de l’activité de la SVZ et une réduction du nombre de nouveaux neurones arrivant aux bulbes olfactifs. Pour déterminer si les CSNa de la SVZ ont subi des changements visibles, nous avons évalué les paramètres clés des CSNa in vivo et in vitro. La culture cellulaire montre qu’un nombre équivalent de CSNa ayant la capacité de former des neurosphères peut être isolé du cerveau du jeune adulte et d’âge moyen. Cependant, à l’âge moyen, les précurseurs neuraux semblent moins sensibles aux facteurs de croissance durant leur différenciation in vitro. Les CSNa donnent des signes de latence in vivo puisque leur capacité d’incorporation et de rétention du BrdU diminue. Ensemble, ces données démontrent que, tôt dans le processus du vieillissement, les CSNa et leur niche dans la SVZ subissent des changements significatifs, et suggèrent que la perte de CSNa liée au vieillissement est secondaire à ces événements.
Neurogenesis persists throughout the adulthood in two regions of the mammalian central nervous system (SNC): the sub-ventricular zone (SVZ) of the forebrain and the sub-granular zone (SGZ) of the hippocampus. Neurogenesis is possible due to the proliferation capacity of stem cells present within both the SVZ and SGZ niches, but with aging, the forebrain undergoes a drastic reduction in its number of adult neural stem cells (aNSCs), a decrease of cell proliferation and an alteration of the neurogenic niches. However, a key unresolved question remains: how the onset of aNSC loss is temporally related to changes of proliferating activity and to structural alterations within the principal stem cell niche (the SVZ)? To gain insights into the initial events leading to aging-associated aNSC loss, we investigated the changes occurring to aNSCs and the SVZ niche between young adulthood and middle-age. The SVZ niche of middle-aged mice (12-months-old) was found to display reduced expression of markers for multiple neural precursor sub-populations when compared to young adult mice (2-months-old). Anatomically, this was associated with significant cytological aberrations, including an overall atrophy of the SVZ, loss of sub-ependymal cells, and accumulation of large lipid droplets within the ependyma. Functionally, these changes correlated with diminished SVZ activity and reduced number of newly born neurons reaching the principal target tissue: the olfactory bulbs. To determine whether changes were evident at the level of the SVZ stem cells, we evaluated key in vitro and in vivo parameters of aNSCs. Tissue culture experiments showed that equal numbers of neurosphere-forming aNSCs could be isolated from young adult and middle-aged forebrains. However, at middle-age, neural precursors seemed to be less sensitive to growth factors during their in vitro differentiation and displayed signs of increased quiescence in vivo. Collectively, these findings demonstrate that, with early aging, aNCS and their SVZ niche go through significant changes, and suggest that aging-associated aNSC loss is secondary to these events.