运动神经元特异性荧光报告系统的建立

Development of a motor neurons-specific fluorescence reporter system

摘要:

人类诱导多能干细胞 (Human induced pluripotent stem cells，hiPSCs) 具有向人体多种类型细胞分化的潜能，其定向分化的运动神经元 (Motor neurons，MNs) 是众多运动神经元疾病的重要体外模型之一。为简化MNs的鉴定方法，通过慢病毒载体将MNs特异性启动子HB9及其控制下的红色荧光蛋白 (Red fluorescent protein，RFP) 基因转入hiPSCs分化而来的神经干细胞 (Human neural stem cells，hNSCs)，经抗生素筛选，获得稳定的阳性细胞株hNSCs-HB9-RFP-Puro。而后，阳性细胞株感染过表达MNs定向分化转录因子的慢病毒LV-Ngn2-Sox11-GFP和LV-Isl1-Lhx3-Hygro，诱导MNs定向分化。诱导分化所得的细胞展现出神经元样结构，并在特异性启动子HB9的作用下表达RFP；同时也表达神经元相关标记物β微管蛋白 (β-tubulin) 和乙酰胆碱转移酶 (Choline acetyltransferase，ChAT)，鉴定为成熟的MNs。该荧光报告系统为MNs的定向分化及鉴定提供了一个更加直观的方法，有效促进了MNs在疾病模型和药物筛选等领域的广泛应用。

关键词:运动神经元，诱导多能干细胞，细胞分化，神经干细胞

Abstract:

Human induced pluripotent stem cells (hiPSCs) have the potential to differentiate into multiple cell types. Motor neurons (MNs) differentiated from hiPSCs are important models of many motor neuron diseases. To simplify the identification of MNs, lentivirus vectors were used to transfer MNs-specific promoter HB9 and red fluorescent protein (RFP) gene into hiPSCs-derived human neural stem cells (hNSCs). Stable positive cells hNSCs-HB9-RFP-Puro were obtained after antibiotic selection. Subsequently, the positive cell line was infected with lentiviruses LV-Ngn2-Sox11-GFP and LV-Isl1-Lhx3-Hygro, which overexpressed the MNs differentiation transcription factor, and differentiated to MNs directly. Differentiated mature MNs showed neuron-like structure, expressed RFP and neuron-related markers β-tubulin and choline acetyltransferase (ChAT) under the control of the MNs-specific promoter HB9. The fluorescence reporter system provides a visual method for directed differentiation and identification of MNs, and may promote the applications of MNs in disease models and drug screening.

Key words:motor neuron, induced pluripotent stem cells, cell differentiation, neural stem cells