南方医科大学学报

南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (3): 360-367.doi: 10.12122/j.issn.1673-4254.2023.03.04

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小分子化合物组合将人胚胎成纤维细胞体外编程为神经前体细胞

杨 盼，王圆圆，高群伟，刘 阳，王 翼，郭 俣，刘长青，刘高峰   

  1. 蚌埠医学院生命科学学院，检验医学院，安徽 蚌埠 233000
  • 出版日期:2023-03-20 发布日期:2023-03-20

Chemical reprogramming of human embryonic fibroblasts into neural progenitor cells in vitro

YANG Pan, WANG Yuanyuan, GAO Qunwei, LIU Yang, WANG Yi, GUO Yu, LIU Changqing, LIU Gaofeng   

  1. Bengbu Medical College School of Life Sciences, School of Laboratory Medicine , Bengbu 233000, China
  • Online:2023-03-20 Published:2023-03-20

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摘要： 目的 通过小分子化合物组合将人胚胎成纤维细胞（HEFs）重编程为化学诱导神经前体细胞（ciNPCs）。方法 HEFs重编程为ciNPCs涉及两个阶段，第1阶段是小分子组合诱导阶段，常氧条件下，将HEFs在含有小分子化合物组合VCR（VPA，CHIR99021，Repsox）的KSR培养基中培养15 d，HEFs形态发生变化，形成致密细胞集落。第2阶段是特异性诱导ciNPCs，将形成的细胞集落胰酶消化后，低粘附板中悬浮培养，可以形成ciNPCs神经球。采用CM-DiI染料标记P3代ciNPCs，并将其移植于6-OHDA法制作的帕金森大鼠模型右脑内侧前脑束（MFB）区，检测ciNPCs在PD大鼠脑内微环境中的存活、迁移以及分化状况。结果 VCR组合诱导10 d细胞开始出现明显的聚集趋势，15 d时，形成致密的细胞集落。单层培养1×105/孔中大约形成40个克隆，且AP染色呈阳性。将细胞集落消化后，低粘附板中悬浮培养培养2 d，可见大量神经球形成，即为第1代ciNPCs（P1代）。ciNPC高表达神经前体细胞（NPCs）特异性标记物（Nestin、Pax6和Sox2），经体外神经特异性诱导分化，ciNPCs表达神经元特异性标记物Tuj1和星形胶质细胞特异性标记物GFAP。而且，P3代ciNPCs移植PD大鼠脑内4 周后，可以分化为Tuj1+、GFAP+、TH+和GABA+细胞。结论 VCR可以将HEFs重编程为ciNPCs，而无需引入外源性基因，为神经科学研究和神经退行性疾病的治疗提供有利的供体材料。

关键词: 小分子化合物组合；细胞重编程；人胚胎成纤维细胞；神经前体细胞

Abstract: Objective To establish a protocol for reprogramming human embryonic fibroblasts (HEFs) into chemically induced neural progenitor cells (ciNPCs). Methods In the two-staged reprogramming of HEFs, the intermediate compact cell colonies were first chemically induced in KSR medium containing small-molecule compounds (VCR) for 15 days in normoxia, followed by the lineage-specific induction stage, in which the compact cell colonies were digested with 0.25% trypsin and the cells were cultured in low adhesion plates. After formation of a large number of free-floating neurospheres 2 days later, the ciNPCs were labeled with CM- DiI and transplanted into rat models of Parkinson's disease (PD)to observe the survival, migration and differentiation of the cells in PD brain. Results After induction with VCR for 10 days under normoxic condition, compact cell colonies occurred in HEF cultures (approximately 40 colonies in each well containing 1×105 HEFs), and most of the colonies expressed high levels of alkaline phosphatase. A large number of free-floating neurospheres formed 2 days after passage and were defined as P1 ciNPCs. These ciNPCs exhibited typical neurosphere-like structures and expressed NPC-specific markers (nestin, Sox2, and Pax6). Under neuronal or glial differentiation condition, the ciNPCs expressed the neuron-specific marker Tuj1 and the astrocyte-specific marker GFAP. These ciNPCs could differentiate into Tuj1+, GFAP+, TH+ and GABA+ cells 4 weeks after transplantation into the brain of PD rats. Conclusion HEFs can be directly reprogrammed into ciNPCs using small-molecule compounds without the need of introducing exogenous genes. This success may provide a solution to the shortage of donor materials for neuroscience research and treatment of neurodegenerative diseases.

Key words: small molecule compounds; cell reprogramming; human embryonic fibroblasts; neural progenitor cells