Browsing by Subject "Neural stem cells"

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Open Access
Alterations in the molecular properties of neural stem cells from aged brains and brain tumors
(2017-06) Burhan, Özge Pelin
It is known that new neuron formation in the brain continues throughout the life of an organism. In the adult human brain, it was proven that neurogenesis in the hippocampus is higher than expected, almost 700 new neurons are formed in a day. The formation of new neurons is supported by the stem cell subpopulation in the brain. With learning and the formation of new memories, the neuron production increases. However, changes in the cognitive abilities with advancing age are thought to be caused by the functional and molecular alterations in the stem cell populations. Molecular changes in neural stem cells throughout aging were found to be deterrents of the increased risk of cancer with age, such as tumor suppressor mechanisms. However, the activation and overlap of tumor suppressing mechanisms result in senescence in stem cells that have accumulated oncogenic mutations, which causes the stem cell pool exhaustion. It is thought that cancer cells acquire stem cell-like properties in order to have the unlimited proliferation and self-renewal properties, which are characteristics of both healthy and cancer stem cells. Neural cancer stem cells have the ability to produce glial and neural cells, like normal stem cells. The cancer stem cell subpopulations are implicated in the growth of tumor tissues. Hence, it is important to identify and characterize cancer stem cells and make a distinction between cancer and non-cancer stem cells. In this project, this issue was addressed by studying the marker expressions of brain tumor tissues obtained from humans, which confirmed that the cancer cells do express stem cell and progenitor cell markers, such as Sox2 and Vimentin. The presence of mature neurons was also established by the mature neuronal marker NeuN. In order to determine whether these stem cells may be different in young and old subjects, a study was also carried out in young and old zebrafish neural stem cells in order to identify the expression differences between the groups. The presence of proliferating stem cells and differentiated cells were identified in cell culture. This analysis of neural stem cells in old and young zebrafish revealed 18 differentially-expressed genes. The results indicated a higher differentiation rate in old zebrafish stem cells, which may be due to the increased loss of neural cells in the old zebrafish brain. The development of markers that could be widely used for the diagnosis of cancer and the identification of cell types is important. For reliable diagnosis and identification of cancer cells, multiple cellular markers are used. Hence the distinction of cell types based on light scattering differences would speed up the process of diagnosis, and the elimination of marker used for the distinction of cell types would be beneficial. The final project mentioned in this thesis involves the analysis of C6 (rat glioma) cell line for scattering properties and cell cycle arrest. A general method for definition of a scatter data interval for C6 cells in different stages was developed and can be applied to other cell types and diseases. These studies show that the proliferation and stem cell markers’ expressions differ between cancer and healthy stem cells, and the expression of neuroprotective genes is differentially upregulated in old zebrafish neural stem cells compared to the young. This data could contribute to the knowledge on normal and cancer stem cell expression differences, as well as how age affects the expression, and supply information required for the development of a cancer stem cell identification and targeting methods.
Open Access
Extracellular matrix mimetic peptide scaffolds for neural stem cell culture and differentiation
(Humana Press, 2014) Mammadov, Busra; Güler, Mustafa O.; Tekinay, Ayşe B.
Self-assembled peptide nanofibers form three-dimensional networks that are quite similar to fibrous extracellular matrix (ECM) in their physical structure. By incorporating short peptide sequences derived from ECM proteins, these nanofibers provide bioactive platforms for cell culture studies. This protocol provides information about preparation and characterization of self-assembled peptide nanofiber scaffolds, culturing of neural stem cells (NSCs) on these scaffolds, and analysis of cell behavior. As cell behavior analyses, viability and proliferation of NSCs as well as investigation of differentiation by immunocytochemistry, qRT-PCR, western blot, and morphological analysis on ECM mimetic peptide nanofiber scaffolds are described.
Open Access
Impact of the inflammatory process in the aging brain: evidence from in Vitro and Ex Vivo models
(2023-08) Aktürk, Serena Sevdiye
Aging is a complex and dynamic process that is characterized by a gradual decline over time in the physiological integrity of organisms. Several cellular mechanisms contribute to aging, including telomere shortening, damage accumulation in DNA, disabled macroautophagy, mitochondrial dysfunction, and cellular senescence. These processes, consecutively, lead to impaired cellular function, declined tissue repair, and stem cell exhaustion and are seen in the development of neurodegenerative disorders and healthy aging. One of the hallmarks of brain aging is the altered chronic inflammatory status of the brain. The over-activation and polarization of microglia, increased secretion of pro-inflammatory cytokines and reactive oxygen species, inflammasome activation, and the upregulation of the NF- κB signaling pathway are among the markers of neuroinflammation. This mechanism's anticipated effects include dysregulated nutrition sensing via the mTOR (mammalian target of rapamycin) pathway, decreased neurogenesis, and synaptic integrity over time. Another element that contributes to vulnerability to inflammation is genetic predisposition. Hence, additional research endeavors are required to investigate the influence of dietary interventions and therapeutic modalities targeting inflammation on genetic pathways. Thus, this study aimed to understand how inflammation can be triggered on different models, investigate potential inflammation-related biomarkers with meta-analysis and observe the effect of inflammation for both zebrafish primary brain cells and murine microglial cells. We conducted short-term copper sulfate treatments on both models for this objective. Moreover, to examine the effects of intermittent fasting, an mTOR downregulator, and high-fat diet, an inflammation inducer, on the brain of zebrafish at the molecular level by primary cell culture method. Finally, we applied rapamycin+DMSO treatment to primary cells to assess the possibility of reversing the progression of inflammation. The results showed that copper sulfate is an efficient oxidative stress-induced inflammatory reagent for zebrafish; however, it did not cause a direct inflammatory response in murine microglial cells. For zebrafish, in the copper sulfate+DMSO treated group, age affected Nrf2a mRNA, altering oxidative stress in old animals. Regardless of diet and treatment group, inflammation markers were higher in old animals, which underscores the association between aging and chronic inflammation. Elevated Lc3b levels in young and old animals captured that high copper concentrations can trigger autophagy. Results for neurogenesis markers revealed that overfeeding or acute inflammation could contribute to compromised neurogenesis in advanced stages of life. On the contrary, the enhanced neurogenesis potential of intermittent fasting in old animals was revealed. In conclusion, this study has demonstrated that the modulation of neuroinflammatory responses, as well as oxidative stress, neurogenesis, and autophagy, occurs in an age-related manner. Moreover, dietary or pharmaceutical interventions could yield comprehensive outcomes in perceiving the brain's neuroinflammatory profile during aging.
Open Access
The impact of dietary intake on Sox2 protein levels and adult neurogenesis in the aging zebrafish brain
(2024-07) Gülden, Seçil
Aging in humans is described as the gradual decline in physiological functions due to alterations in complex biological processes. As the lifespan increases and the population shifts towards older age, health problems associated with age become a worldwide problem. Thus, understanding the molecular and cellular changes that contribute to the aging process is essential to ensure healthy aging. Nine hallmarks that describe these changes were identified, which are also observed in the aging brain, leading to structural and cognitive deficiencies. Stem cell exhaustion is one of these hallmarks that explains a loss of stem cell function due to brain aging. A transcription factor, Sox2 is one of the main responsible proteins ensuring the stem cell maintenance through regulation of self-renewal and differentiation. Age-related changes in neural stem cells (NSCs) mediated by Sox2 can lead to a decline in new neuron formation. Neurogenesis is proven to continue in the defined neurogenic niches in the mammalian brain throughout adulthood and is considered to have a crucial role in the healthy functioning of the brain during aging. The regulation of the NSCs in neurogenic niches is achieved through diverse intrinsic and extrinsic factors that are affected by age-related changes. Dietary interventions are thought to modulate these factors and improve the age-related decline. Previous studies show that dietary restriction through lowering the calorie intake improves age-related impairment on neurogenesis, while high-calorie intake has a negative effect. Zebrafish, a small teleost fish, is a highly suitable model organism for investigations on neurogenesis with respect to aging due to exhibiting gradual decline with age, similar to humans and having a high capacity of neurogenesis in a widespread area. Hence, in the first part of the study, the influence of diet on age-associated changes in the brain was observed by following two short-term opposing dietary interventions. Using zebrafish as a model organism, dietary restriction (DR), over-feeding (OF), and ad libitum (AL) diets were included. These interventions demonstrated that short-term DR, compared to the AL, downregulated Sox2, as evidenced by western blot analysis. This might indicate a decreased self-renewal properties and activation of differentiation of NSCs. Short-term OF, on the other hand, did not change its expression levels. Moreover, aging did not alter the Sox2 expression levels. Further correlational and multivariate analyses were performed combining Sox2 with a proliferation marker proliferating cell nuclear antigen (PCNA) and neuronal lineage markers doublecortin-like kinase 1a (DCAMKL1) and ELAV-like neuron-specific RNA binding protein 3 (HuC). The analysis demonstrated a positive relationship between Sox2 and PCNA indicating their similar pattern in the aging process. Also, DCAMKL1 was shown to have a positive relationship with PCNA and a negative relationship with HuC. The multivariate analyses of all datasets exhibited age-specific effects of OF and DR by decreasing the survival of new neurons in old animals. Also, OF reinforced the commitment to neuronal fate with old age. The second part demonstrated a recombinant protein purification approach using immobilized-metal affinity chromatography to purify the His-tagged Sox2 protein. The recombinant Sox2 protein was successfully purified, and concentration values were measured. Taken together, these findings show that although older age led to changing dynamics in the neuronal lineage in the zebrafish brain, Sox2 managed to show a persistent expression. Furthermore, the short-term DR was shown to change the Sox2 expression levels, indicating the importance of calorie intake in alterations in neural stem cell properties. In conclusion, this study makes contributions to understanding cellular and biochemical changes occurring in the neurogenic niches of the aging brain and the altered neurogenic capacity due to dietary interventions.