Girard Stéphane D, Devéze Arnaud, Nivet Emmanuel, Gepner Bruno, Roman François S, Féron François
NICN, Aix Marseille University.
J Vis Exp. 2011 Aug 22(54):2762. doi: 10.3791/2762.
The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell(1). Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)(2-4) or neurodegenerative (e.g. Parkinson, Alzheimer) diseases(4,5). Olfactory mucosa can be used for either comparative molecular studies(4,6) or in vitro experiments on neurogenesis(3,7). The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria(8-10). We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)(11). Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease(4). We and others have also shown that OE-MSCs are promising candidates for cell therapy, after a spinal cord trauma(12,13), a cochlear damage(14) or in an animal models of Parkinson's disease(15) or amnesia(16). In this study, we present methods to biopsy olfactory mucosa in rats and humans. After collection, the lamina propria is enzymatically separated from the epithelium and stem cells are purified using an enzymatic or a non-enzymatic method. Purified olfactory stem cells can then be either grown in large numbers and banked in liquid nitrogen or induced to form spheres or differentiated into neural cells. These stem cells can also be used for comparative omics (genomic, transcriptomic, epigenomic, proteomic) studies.
位于鼻腔内的嗅黏膜负责检测气味。它也是唯一暴露于外部环境且每个个体都易于获取的神经组织。因此，对于任何旨在识别病理性大脑中的分子异常或分离成体干细胞用于细胞治疗的人来说，这种组织都很独特。脑部疾病中的分子异常通常使用死后收集的神经组织样本进行研究。然而，这种材料有许多局限性。相比之下，嗅黏膜易于获取，并且可以安全地进行活检而不会导致任何嗅觉丧失(1)。因此，嗅黏膜为成年人提供了一扇“开放的窗口”，通过它可以研究发育性疾病（如自闭症、精神分裂症）(2 - 4)或神经退行性疾病（如帕金森病、阿尔茨海默病）(4,5)。嗅黏膜可用于比较分子研究(4,6)或神经发生的体外实验(3,7)。嗅上皮也是一种神经组织，每天都会产生新的神经元以替代那些因污染、细菌或病毒感染而受损的神经元。这种持续的神经发生由祖细胞维持，但也由存在于黏膜两个部分（即神经上皮和下方的固有层）中的干细胞维持(8 - 10)。我们最近开发了一种方法来纯化位于固有层中的成体干细胞，并且在证明它们与骨髓间充质干细胞（BM - MSC）密切相关后，我们将它们命名为嗅外间充质干细胞（OE - MSC）(11)。有趣的是，与BM - MSC相比，OE - MSC显示出高增殖率、高克隆形成能力以及分化为神经细胞的倾向。我们利用这些特性进行了专门研究，以揭示精神分裂症和帕金森病中的新候选基因(4)。我们和其他人还表明，在脊髓损伤(12,13)、耳蜗损伤(14)或帕金森病(15)或失忆症(16)的动物模型中，OE - MSC是细胞治疗的有前景的候选者。在本研究中，我们展示了在大鼠和人类中获取嗅黏膜活检样本的方法。收集后，使用酶解法将固有层与上皮分离，并使用酶法或非酶法纯化干细胞。然后，纯化后的嗅干细胞可以大量培养并保存在液氮中，或者诱导形成球体或分化为神经细胞。这些干细胞还可用于比较组学（基因组学、转录组学、表观基因组学、蛋白质组学）研究。
