Center of Cellular Technologies, Institute of Cytology of the Russian Academy of Science, 194064 St. Petersburg, Russia.
Laboratory of Molecular Neurodegeneration, Peter the Great St. Petersburg Polytechnic University, 195251 St. Petersburg, Russia.
Int J Mol Sci. 2023 Apr 5;24(7):6799. doi: 10.3390/ijms24076799.
The modeling of neuropathology on induced neurons obtained by cell reprogramming technologies can fill a gap between clinical trials and studies on model organisms for the development of treatment strategies for neurodegenerative diseases. Patient-specific models based on patients' cells play an important role in such studies. There are two ways to obtain induced neuronal cells. One is based on induced pluripotent stem cells. The other is based on direct reprogramming, which allows us to obtain mature neuronal cells from adult somatic cells, such as dermal fibroblasts. Moreover, the latter method makes it possible to better preserve the age-related aspects of neuropathology, which is valuable for diseases that occur with age. However, direct methods of reprogramming have a significant drawback associated with low cell viability during procedures. Furthermore, the number of reprogrammable neurons available for morphological and functional studies is limited by the initial number of somatic cells. In this article, we propose modifications of a previously developed direct reprogramming method, based on the combination of microRNA and transcription factors, which allowed us to obtain a population of functionally active induced striatal neurons (iSNs) with a high efficiency. We also overcame the problem of the presence of multinucleated neurons associated with the cellular division of starting fibroblasts. Synchronization cells in the G1 phase increased the homogeneity of the fibroblast population, increased the survival rate of induced neurons, and eliminated the presence of multinucleated cells at the end of the reprogramming procedure. We have demonstrated that iSNs are functionally active and able to form synaptic connections in co-cultures with mouse cortical neurons. The proposed modifications can also be used to obtain a population of other induced neuronal types, such as motor and dopaminergic ones, by selecting transcription factors that determine differentiation into a region-specific neuron.
利用细胞重编程技术获得的诱导神经元进行神经病理学建模，可以填补临床试验和模式生物研究之间的空白，从而为神经退行性疾病的治疗策略开发提供帮助。基于患者细胞的患者特异性模型在这类研究中发挥着重要作用。获得诱导神经元细胞有两种方法。一种是基于诱导多能干细胞，另一种是基于直接重编程，它可以使我们从成体细胞（如皮肤成纤维细胞）中获得成熟的神经元细胞。此外，后一种方法可以更好地保留与年龄相关的神经病理学方面的特征，这对于与年龄相关的疾病非常有价值。然而，直接重编程方法有一个显著的缺点，即在操作过程中细胞存活率较低。此外，用于形态和功能研究的可重编程神经元的数量受到起始体细胞数量的限制。在本文中，我们提出了对以前开发的直接重编程方法的修改，该方法基于 microRNA 和转录因子的组合，使我们能够高效获得具有高功能活性的诱导纹状体神经元（iSN）群体。我们还克服了与起始成纤维细胞细胞分裂相关的多核神经元存在的问题。使细胞同步进入 G1 期增加了成纤维细胞群体的均一性，提高了诱导神经元的存活率，并在重编程过程结束时消除了多核细胞的存在。我们已经证明 iSN 是具有功能活性的，并且能够在与小鼠皮质神经元的共培养物中形成突触连接。通过选择决定分化为特定区域神经元的转录因子，所提出的修改也可用于获得其他类型的诱导神经元群体，如运动神经元和多巴胺能神经元。
