Effect of fibronectin on differentiation of human neural stem cells into oligodendrocyte precursor cells

doi:10.12307/2025.693

Abstract

Abstract: BACKGROUND: Oligodendrocyte precursor cells are seed cells for the treatment of white matter damage diseases. Establishing an efficient and stable in vitro differentiation method is an important prerequisite for clinical translational research.
OBJECTIVE: To investigate the effect of fibronectin on biological characteristics such as proliferation, migration, and differentiation of oligodendrocyte precursor cells derived from human neural stem cells.
METHODS: Human neural stem cells cultured in suspension were digested into single cells using Accutase. The expression of specific markers Nestin, Sox2, Vimentin, CD133, and Musashi was detected by flow cytometry. The single cells of human neural stem cells were resuspended in oligodendrocyte precursor cell medium and seeded in six-well plates coated with different concentrations of fibronectin (0, 1, 2.5, 5, and 10 μg/mL). Accutase digestion was performed after 7 days of culture. Cells were counted by trypan staining. Fibronectin-coated group with the strongest amplification ability and the oligodendrocyte precursor cells without fibronectin-coated group were selected for further tests. The migration ability of the two groups of cells was detected by Transwell. Flow cytometry was used to detect the expression of Olig2, Sox10, and PDGFR-α. Oligodendrocyte precursor cells were induced to differentiate into oligodendrocytes for 3 weeks, and the expression of Galc in differentiated cells was detected by immunofluorescence staining.
RESULTS AND CONCLUSION: (1) Human neural stem cells grew in suspension spheres. Flow cytometry showed that human neural stem cells highly expressed Nestin, Sox2, Vimentin, CD133, and Musashi. (2) The cell bodies of oligodendrocyte precursor cells induced by human neural stem cells were round or oval, with strong refractive nature and bipolar or tertiary protrusions. Compared with the 0 μg/mL fibronectin coating group, there was a significant difference in the amplification ability of oligodendrocyte precursor cells in the 2.5, 5, and 10 μg/mL fibronectin coating groups (P < 0.05). The amplification ability of oligodendrocyte precursor cells was the strongest when the fibronectin concentration was 10 μg/mL. (3) Flow cytometry results showed that the oligodendrocyte precursor cell markers Olig2, Sox10, and PDGFR-α were highly expressed in the 0 and 10 μg/mL fibronectin coating groups, and there was no significant difference between the two groups (P > 0.05). (4) Transwell chamber assay results showed that compared with the 0 μg/mL fibronectin-coated group, the migration ability of oligodendrocyte precursor cells in the 10 μg/mL fibronectin-coated group was increased (P < 0.01). (5) After 3 weeks of differentiation into oligodendrocytes, oligodendrocyte precursor cells showed complex morphology with multiple branches, grids or membrane sheets. Immunofluorescence staining results showed that there was no statistical difference in the Galc positive rate of oligodendrocytes between the two groups (P > 0.05). These findings indicate that when the concentration of fibronectin coated well plate is 10 μg/mL, the proliferation and migration of oligodendrocyte precursor cells are the strongest, but it does not affect the expression of oligodendrocyte precursor cells-specific markers Olig2, Sox10, and PDGFR-α and their differentiation into oligodendrocytes.

Key words: fibronectin, neural stem cell, oligodendrocyte precursor cell, oligodendrocyte, cell proliferation, cell differentiation, engineered stem cell, engineered cell

CLC Number:

Wang Zhaoyan, Wang Qian, Liu Weipeng, Yang Hui, Luan Zuo, Qu Suqing. Effect of fibronectin on differentiation of human neural stem cells into oligodendrocyte precursor cells[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(31): 6661-6666.

Figures/Tables 1

2.1 神经干细胞形态及鉴定结果神经干细胞在培养瓶内呈悬浮球状生长，折光性好，神经球周边有较为致密的毛刺。流式细胞术检测结果显示神经干细胞表达特异性标志物Nestin、Musashi、Sox2、Vimentin、CD133，见图1。 2.2 纤维连接蛋白促进少突胶质前体细胞的增殖接种于6孔板中的少突胶质前体细胞胞体呈圆形或卵圆形，折光性强，有双极或三级突起结构，随着纤维连接蛋白质量浓度的增加，镜下可见贴壁细胞数量增多。对照组细胞增殖较慢，细胞贴壁不牢固，视野中可见较多细胞聚集成团现象。Accutase消化各组细胞，锥虫蓝染色计数结果显示，与对照组比较，1 μg/mL纤维连接蛋白组细胞数量无统计学差异，2.5，5，10 μg/mL纤维连接蛋白组细胞数量均有统计学差异，随着纤维连接蛋白质量浓度的增加细胞扩增能力增强，当纤维连接蛋白质量浓度为10 μg/mL时细胞增殖能力最强，见图2。 2.3 纤维连接蛋白不影响少突胶质前体细胞标志物的表达流式检测结果显示，10 μg/mL纤维连接蛋白包被组与对照组获得的少突胶质前体细胞均高表达PDGFR-α、Olig2、Sox10，两组少突胶质前体细胞标志物的表达无统计学差异，说明培养过程中是否使用纤维连接蛋白包被孔板并不会影响少突胶质前体细胞特异性标志物的表达，见图3。 2.4 纤维连接蛋白促进少突胶质前体细胞的迁移 Transwell小室检测少突胶质前体细胞的迁移能力，DAPI染色结果显示，与对照组比较，10 μg/mL纤维连接蛋白组少突胶质前体细胞迁移能力增加(P < 0.01)，见图4。 2.5 少突胶质前体细胞分化为少突胶质细胞及鉴定结果少突胶质前体细胞向少突胶质细胞分化3周，10 μg/mL纤维连接蛋白组与对照组少突胶质前体细胞均可以分化为具有复杂分支或呈膜片状的细胞。免疫荧光染色结果显示Galc染色阳性，见图5。"

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