低氧条件下小分子化合物VCR组合诱导大鼠成纤维细胞重编程神经前体细胞的实验研究

王圆圆; 孙婷婷; 杨盼; 徐佳佳; 梁宇; 吴凡; 马彩云; 王春景; 刘长青; 郭俣

doi:10.12182/20220960501

低氧条件下小分子化合物VCR组合诱导大鼠成纤维细胞重编程神经前体细胞的实验研究

VCR, a Small Molecule Compound, Induces Reprogramming of Rat Fibroblasts into Neural Progenitor Cells under Hypoxic Condition

摘要

摘要:
目的探讨大鼠胚胎成纤维细胞（rat embryoic fibroblasts, REFs）在低氧条件下（5%O₂）重编程为化学诱导大鼠神经前体细胞（chemically induced rat neural progenitor cells, ciRNPCs）的方法体系。
方法分两阶段将REFs重编程为ciRNPCs，第一阶段是化学诱导产生中间态细胞，REFs在低氧条件下采用含有小分子化合物的组合VCR（valproic acid、CHIR99021和 RepSox）和10000 U/mL白血病抑制因子（leukemia inhibitory factor, LIF）的KSR培养基培养15 d，观察到致密细胞集落即中间态细胞形成；第二阶段是特异性诱导ciRNPCs，用胰酶消化中间态细胞，接种至低黏附板，在常氧条件下可形成ciRNPCs神经球。CM-DiI标记后，定向移植至大鼠黑质-纹状体，通过免疫荧光检测ciRNPCs在宿主脑内存活、迁移及分化状况。
结果低氧诱导5～10 d时已观察到明显细胞聚集趋势，15 d时已形成聚集紧密的克隆，1×10⁵个细胞中大约产生30个克隆，同时大部分细胞克隆碱性磷酸酶染色呈阳性，消化重铺后2 d即可观察到有神经胚球形成，并可表达神经前体细胞（neural progenitor cells, NPCs）表面特征性抗原（Nestin、Sox2和Pax6），而且具有向神经胶质细胞和神经元分化的能力，分别表达GFAP和Tuj1特异性标志物。移植8周后，大鼠脑内可分化为GFAP⁺和Tuj1⁺细胞。
结论小分子化合物VCR组合在低氧条件下可直接诱导REFs重编程为ciRNPCs，并具备体内与体外诱导分化为神经胶质细胞和神经元的潜能，为ciRNPCs移植治疗神经损伤疾病奠定基础。

Abstract:
Objective To explore for a protocol for reprogramming rat embryonic fibroblasts (REFs) under hypoxic conditions (5% O₂) to form chemically induced rat neural progenitor cells (ciRNPCs).
Methods 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.
Results 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 1×10⁵ 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.
Conclusion 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 in vivo and in vitro, laying the foundation for transplanting ciRNPCs to treat neurodegenerative diseases.

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