School of Life Sciences, Bengbu Medical University, Bengbu 233030, Anhui Province, China.
Anhui Engineering Research Center for Neural Regeneration Technology and Medical New Materials, Bengbu Medical University, Bengbu 233030, Anhui Province, China.
Zhejiang Da Xue Xue Bao Yi Xue Ban. 2024 Aug 25;53(4):498-508. doi: 10.3724/zdxbyxb-2024-0007.
To establish a methodological system for reprogramming rat embryonic fibroblasts (REF) into chemically induced neurons (ciNCs) via small molecule compounds to provide safe and effective donor cells for treatment of neurodegenerative diseases.
Based on the method established by PEI Gang's research group to directly reprogram human fibroblasts into neurons, the induction medium and maturation medium was optimized by replacing the coating solution, mitigating oxidative stress injury, adding neurogenic protective factors, adjusting the concentration of trichothecenes, performing small-molecule removal experiments, and carrying out immunofluorescence and Western blotting on cells at different stages of induction to validate the effect of induction.
When the original protocol was used for induction, the cell survival rate was (34.24±2.77)%. After replacing the coating solution gelatin with matrigel, the cell survival rate increased to (45.41±4.27)%; after adding melatonin, the cell survival rate increased to (67.95±5.61)% and (23.43±1.42)% were transformed into neural-like cells; after adding the small molecule P7C3-A20, the cell survival rate was further increased to (76.27±1.41)%, and (39.72±4.75)% of the cells were transformed into neural-like cells. When the concentration of trichothecene was increased to 30 μmol/L, the proportion of neural-like cells reached (55.79±1.90)%; after the removal of SP600125, (86.96±2.15)% of the cells survived, and the rate of neural-like cell production increased to (63.43±1.60)%. With the optimized protocol, REF could be successfully induced into ciNC through the neural precursor cell stage, in which the neural precursor cells were able to highly express the neural precursor cell markers SRY-related HMG-box gene 2 (Sox2) and paired box 6 (Pax6) as well as neuron-specific marker tubulin 1 (Tuj1), while the expression of fiber-associated protein vimentin was reduced. After two weeks of induction of neural precursor cells in a maturation medium, most cells displayed neuronal-like cell morphology. The induced ciNCs were able to highly express the mature neuronal surface markers Tuj1 and microtubule-associated protein 2 (MAP2), while the expression of vimentin was reduced.
The small molecule combinations optimized in this study can reprogram REF to ciNCs under normoxic conditions.
建立一种通过小分子化合物将大鼠胚胎成纤维细胞（REF）重编程为化学诱导神经元（ciNC）的方法学体系，为治疗神经退行性疾病提供安全有效的供体细胞。
基于 PEI Gang 研究小组建立的直接将人成纤维细胞重编程为神经元的方法，通过更换包被溶液、减轻氧化应激损伤、添加神经保护因子、调整 Trichothecenes 浓度、进行小分子去除实验，并对不同诱导阶段的细胞进行免疫荧光和 Western blot 验证诱导效果，对诱导培养基和成熟培养基进行优化。
当使用原始方案进行诱导时，细胞存活率为（34.24±2.77）%。将包被溶液明胶替换为基质胶后，细胞存活率提高至（45.41±4.27）%；添加褪黑素后，细胞存活率进一步提高至（67.95±5.61）%，并转化为神经样细胞（23.43±1.42）%；添加小分子 P7C3-A20 后，细胞存活率提高至（76.27±1.41）%，并转化为神经样细胞（39.72±4.75）%。当 Trichothecenes 浓度增加到 30 μmol/L 时，神经样细胞的比例达到（55.79±1.90）%；去除 SP600125 后，（86.96±2.15）%的细胞存活，神经样细胞的产生率提高至（63.43±1.60）%。采用优化方案，REF 可通过神经前体细胞阶段成功诱导为 ciNC，其中神经前体细胞能够高表达神经前体细胞标志物 SRY 相关 HMG 盒基因 2（Sox2）和配对盒 6（Pax6）以及神经元特异性标志物微管蛋白 1（Tuj1），同时减少纤维相关蛋白波形蛋白的表达。在成熟培养基中诱导神经前体细胞两周后，大多数细胞呈现神经元样细胞形态。诱导的 ciNC 能够高表达成熟神经元表面标志物 Tuj1 和微管相关蛋白 2（MAP2），同时减少波形蛋白的表达。
本研究优化的小分子组合可在常氧条件下将 REF 重编程为 ciNC。
