Wang Yuan-Yuan, Sun Ting-Ting, Yang Pan, Xu Jia-Jia, Liang Yu, Wu Fan, Ma Cai-Yun, Wang Chun-Jing, Liu Chang-Qing, Guo Yu
School of Life Sciences, Bengbu Medical College, Bengbu 233000, China.
School of Clinical Medicine, Bengbu Medical College, Bengbu 233000, China.
Sichuan Da Xue Xue Bao Yi Xue Ban. 2022 Sep;53(5):790-797. doi: 10.12182/20220960501.
To explore for a protocol for reprogramming rat embryonic fibroblasts (REFs) under hypoxic conditions (5% O ) to form chemically induced rat neural progenitor cells (ciRNPCs).
The reprogramming of REFs into ciNPCs was done in two stages. The first stage involved chemical induction to generate intermediate cells. The REFs were cultured in KSR medium containing valproic acid, CHIR99021, and RepSox (VCR) and 10000 U/mL leukemia inhibitory factor (LIF) for 15 days, under a physiological hypoxic condition. The formation of dense cell colonies, i.e., intermediate cells, were observed. The second stage involved the specific induction of ciRNPCs. The induced intermediate cells were digested with trypsin, seeded on a low adhesion plate, and cultured under normoxic condition to form ciRNPCs neurospheres. Then, after CM-DiI cell-labeling, the ciRNPCs were stereotactically transplanted into the substantia nigra (SN) of rats. The survival, migration, and differentiation of ciRNPCs in the host brain were examined with immunofluorescence assays.
After induction under hypoxic condition for 5 to 10 days, a clear trend of cell aggregation was observed. Compact cell colonies were observed in REFs treated with VCR for 15 days under a hypoxic condition. Approximately 30 colonies emerged from 1×10 cells, and most colonies were positive for AP staining. Moreover, when these cells were cultured further in suspension, free-floating neurospheres formed and stained positive for neural progenitor cell (NPC) markers, including Nestin, Sox2 and Pax6. These ciRNPCs could differentiate into glial cells and neurons, and express neurite marker Tuj1 and astrocyte marker GFAP. Eight weeks after transplantation, the cells could differentiate into GFAP and Tuj1 cells in the rat brain.
Our study demonstrates that VCR, a small molecule compound, can directly induce, under a hypoxic condition, the reprogramming of REFs to form ciRNPCs with the potential to be induced for differentiation into glial cells and neurons and , laying the foundation for transplanting ciRNPCs to treat neurodegenerative diseases.
探索一种在低氧条件（5%氧气）下重编程大鼠胚胎成纤维细胞（REFs）以形成化学诱导的大鼠神经祖细胞（ciRNPCs）的方案。
将REFs重编程为ciNPCs分两个阶段进行。第一阶段涉及化学诱导以产生中间细胞。将REFs在含有丙戊酸、CHIR99021和RepSox（VCR）以及10000 U/mL白血病抑制因子（LIF）的KSR培养基中，在生理性低氧条件下培养15天。观察到致密细胞集落即中间细胞的形成。第二阶段涉及ciRNPCs的特异性诱导。将诱导的中间细胞用胰蛋白酶消化，接种在低粘附板上，并在常氧条件下培养以形成ciRNPCs神经球。然后，在进行CM-DiI细胞标记后，将ciRNPCs立体定向移植到大鼠黑质（SN）中。用免疫荧光测定法检测ciRNPCs在宿主脑中的存活、迁移和分化情况。
在低氧条件下诱导5至10天后，观察到明显的细胞聚集趋势。在低氧条件下用VCR处理15天的REFs中观察到紧密的细胞集落。从1×10个细胞中大约出现30个集落，并且大多数集落对碱性磷酸酶（AP）染色呈阳性。此外，当这些细胞进一步悬浮培养时，形成了自由漂浮的神经球，并且对神经祖细胞（NPC）标志物包括巢蛋白（Nestin）、性别决定区Y框蛋白2（Sox2）和配对盒基因6（Pax6）染色呈阳性。这些ciRNPCs可以分化为神经胶质细胞和神经元，并表达神经突标志物βIII微管蛋白（Tuj1）和星形胶质细胞标志物胶质纤维酸性蛋白（GFAP）。移植八周后，这些细胞可以在大鼠脑中分化为GFAP和Tuj1细胞。
我们的研究表明，小分子化合物VCR可以在低氧条件下直接诱导REFs重编程以形成具有分化为神经胶质细胞和神经元潜力的ciRNPCs，为移植ciRNPCs治疗神经退行性疾病奠定了基础。
