Cellular Therapy and Stem Cell Production Application and Research Centre, ESTEM, Eskisehir Osmangazi University, Eskisehir, Turkey.
Department of Stem Cell, Institute of Health Sciences, Eskisehir Osmangazi University, Eskisehir, Turkey.
IUBMB Life. 2024 Sep;76(9):697-711. doi: 10.1002/iub.2822. Epub 2024 Apr 25.
The aim of this study was to develop an alternative treatment method for neurodegenerative diseases with dopaminergic neuron loss such as Parkinson's disease by differentiating cells obtained from human olfactory mucosa-derived neural stem cells (hOM-NSCs) with neurotrophic agents in vitro. hOM-NSCs were isolated and subjected to immunophenotypic and MTT analyses. These hOM-NSCs were then cultured in a 3D environment to form neurospheres. The neurospheres were subjected to immunophenotypic analysis and neuronal differentiation assays. Furthermore, hOM-NSCs were differentiated into dopaminergic neuron-like cells in vitro. After differentiation, the dopaminergic neuron-like cells were subjected to immunophenotypic (TH, MAP2) and genotypic (DAT, PITX3, NURR1, TH) characterization. Flow cytometric analysis showed that NSCs were positive for cell surface markers (CD56, CD133). Immunofluorescence analysis showed that NSCs were positive for markers with neuronal and glial cell characteristics (SOX2, NESTIN, TUBB3, GFAP and NG2). Immunofluorescence analysis after differentiation of hOM-NSCs into dopaminergic neuron-like cells in vitro showed that they were positive for a protein specific for dopaminergic neurons (TH). qRT-PCR analysis showed that the expression of dopaminergic neuron-specific genes (DAT, TH, PITX3, NURR1) was significantly increased. It was concluded that hOM-NSCs may be a source of neural stem cells that can be used for cell replacement therapies in neurodegenerative diseases such as Parkinson's disease, are resistant to cell culture, can differentiate into neuronal and glial lineage, are easy to obtain and are cost effective.
本研究旨在通过体外使用神经营养因子分化人嗅黏膜源性神经干细胞（hOM-NSCs），为多巴胺能神经元丢失的神经退行性疾病（如帕金森病）开发替代治疗方法。分离并进行免疫表型和 MTT 分析 hOM-NSCs。然后将这些 hOM-NSCs 在 3D 环境中培养以形成神经球。对神经球进行免疫表型分析和神经元分化测定。此外，还将 hOM-NSCs 体外分化为多巴胺能神经元样细胞。分化后，将多巴胺能神经元样细胞进行免疫表型（TH、MAP2）和基因型（DAT、PITX3、NURR1、TH）鉴定。流式细胞术分析显示 NSCs 呈细胞表面标志物（CD56、CD133）阳性。免疫荧光分析显示 NSCs 呈神经元和神经胶质细胞特征标志物（SOX2、NESTIN、TUBB3、GFAP 和 NG2）阳性。体外将 hOM-NSCs 分化为多巴胺能神经元样细胞后进行免疫荧光分析显示，其对多巴胺能神经元特异性蛋白（TH）呈阳性。qRT-PCR 分析显示多巴胺能神经元特异性基因（DAT、TH、PITX3、NURR1）的表达显著增加。结论：hOM-NSCs 可能是神经干细胞的来源，可用于帕金森病等神经退行性疾病的细胞替代治疗，对细胞培养具有抗性，可分化为神经元和神经胶质谱系，易于获得且具有成本效益。
