小分子化合物组合改善多能干细胞向脑类器官定向分化的效率

Small molecule compounds improve the targeteddifferentiation efficiency of cerebral organoids from pluripotent stem cells

目的优化多能干细胞(PSCs)定向分化为脑类器官的条件,提高人脑类器官的分化效率。方法利用人胚胎干细胞(hESCs)系H9向人脑类器官诱导分化体系,在早期脑类器官分化到神经祖细胞的发育阶段添加小分子化合物组合,通过形态学观察不同分化阶段脑类器官中的神经祖细胞形成的效率、细胞凋亡的情况以及分化成神经元的效率,通过RT-qPCR检测标志性基因的表达量,综合评估添加的小分子化合物组合对脑类器官形成的影响。结果在脑类器官神经祖细胞发育的关键阶段(1 d~14 d),培养基中依次添加多索啡(dorsomorphine)、A83-01、GSK-3β抑制剂CHIR99021和SMAD抑制剂SB-431542,可使神经祖细胞阶段的相关标志性基因的表达量显著提高,促进特异性神经管样结构形成,脑类器官中心区域的细胞凋亡减少。结论通过使用上述4种小分子化合物组合,可明显提高早期脑类器官的形成效率,减少脑类器官中的细胞凋亡、促进神经元形成,减少培养过程中的组织结构异质性。

Objective To optimize the conditions and improve the efficiency for the differentiation of pluripotent stem cells(PSCs)into cerebral organoids.Methods Based on the induction differentiation system for inducing human embryonic stem cells(hESCs)H9 towards cerebral organoids,a combination of small molecule compounds was added to the developmental stage of neural progenitor cells in the early stage of cerebral organoid differentiation,and the efficiency of neural progenitor cell formation,apoptosis and differentiation into neurons in cerebral organoids in the differentiation stage were observed through morphology,the expression of marker genes was detected by RT-qPCR and the effect of small molecule compound combination on cerebral organoids was comprehensively evaluated.Results At the critical stage of neural progenitor cell development(1 d-14 d)of cerebral organoids,dorsomorphine,A83-01,GSK-3βinhibitor CHIR99021 and SMAD inhibitor SB-431542 were successively added to the medium.It could significantly increase the expression of marker genes in the neural progenitor cell stage,promote the formation of specific neural tube-like structures and reduce apoptosis in the central region of cerebral organoids.Conclusions By using the combination of four small molecule compounds,the formation efficiency in early cerebral organoids can be significantly improved,apoptosis in cerebral organoids can be reduced,neuronal formation can be promoted,and tissue structure heterogeneity in the culture process can be reduced.