Bellon Alfredo, Hasoglu Tuna, Peterson Mallory, Gao Katherine, Chen Michael, Blandin Elisabeta, Cortez-Resendiz Alonso, Clawson Gary A, Hong Liyi Elliot
Department of Psychiatry and Behavioral Health, Penn State Hershey Medical Center, Hershey, PA 17033, USA.
Department of Pharmacology, Penn State Hershey Medical Center, Hershey, PA 17033, USA.
Brain Sci. 2021 Oct 20;11(11):1372. doi: 10.3390/brainsci11111372.
Deficits in neuronal structure are consistently associated with neurodevelopmental illnesses such as autism and schizophrenia. Nonetheless, the inability to access neurons from clinical patients has limited the study of early neurostructural changes directly in patients' cells. This obstacle has been circumvented by differentiating stem cells into neurons, although the most used methodologies are time consuming. Therefore, we recently developed a relatively rapid (~20 days) protocol for transdifferentiating human circulating monocytes into neuronal-like cells. These monocyte-derived-neuronal-like cells (MDNCs) express several genes and proteins considered neuronal markers, such as MAP-2 and PSD-95. In addition, these cells conduct electrical activity. We have also previously shown that the structure of MDNCs is comparable with that of human developing neurons (HDNs) after 5 days in culture. Moreover, the neurostructure of MDNCs responds similarly to that of HDNs when exposed to colchicine and dopamine. In this manuscript, we expanded our characterization of MDNCs to include the expression of 12 neuronal genes, including tau. Following, we compared three different tracing approaches (two semi-automated and one automated) that enable tracing using photographs of live cells. This comparison is imperative for determining which neurite tracing method is more efficient in extracting neurostructural data from MDNCs and thus allowing researchers to take advantage of the faster yield provided by these neuronal-like cells. Surprisingly, it was one of the semi-automated methods that was the fastest, consisting of tracing only the longest primary and the longest secondary neurite. This tracing technique also detected more structural deficits. The only automated method tested, Volocity, detected MDNCs but failed to trace the entire neuritic length. Other advantages and disadvantages of the three tracing approaches are also presented and discussed.
神经元结构缺陷一直与自闭症和精神分裂症等神经发育疾病相关。然而，无法从临床患者获取神经元限制了直接在患者细胞中研究早期神经结构变化。尽管最常用的方法耗时，但通过将干细胞分化为神经元已规避了这一障碍。因此，我们最近开发了一种相对快速（约20天）的方案，用于将人类循环单核细胞转分化为神经元样细胞。这些单核细胞衍生的神经元样细胞（MDNCs）表达几种被视为神经元标志物的基因和蛋白质，如微管相关蛋白2（MAP-2）和突触后密度蛋白95（PSD-95）。此外，这些细胞具有电活动。我们之前还表明，培养5天后MDNCs的结构与人类发育中的神经元（HDNs）相当。而且，当暴露于秋水仙碱和多巴胺时，MDNCs的神经结构与HDNs的反应相似。在本论文中，我们扩展了对MDNCs的表征，包括12种神经元基因的表达，其中包括tau蛋白。接下来，我们比较了三种不同的追踪方法（两种半自动方法和一种自动方法），这些方法能够使用活细胞照片进行追踪。这种比较对于确定哪种神经突追踪方法在从MDNCs中提取神经结构数据方面更有效，从而使研究人员能够利用这些神经元样细胞更快的产出至关重要。令人惊讶的是，最快的方法之一是一种半自动方法，该方法仅追踪最长的初级神经突和最长的次级神经突。这种追踪技术还检测到更多的结构缺陷。所测试的唯一自动方法Volocity能够检测到MDNCs，但未能追踪到整个神经突长度。还介绍并讨论了这三种追踪方法的其他优缺点。
