Department of Chemistry and Biochemistry, Florida State University, 102 Varsity Way, Tallahassee, FL, 32306, USA.
Department of Chemical and Biomedical Engineering, FAMU-FSU College of Engineering, Florida State University, 2525 Pottsdamer St., Tallahassee, FL, 32310, USA.
Sci Rep. 2022 Jul 27;12(1):12880. doi: 10.1038/s41598-022-16970-1.
Human cerebellum consists of high density and complexity of neurons. Thus, it is challenging to differentiate cerebellar-like organoids with similar cellular markers and function to the human brain. Our previous study showed that the combination of retinoic acid (RA), Wingless/integrated (Wnt) activator, and Sonic Hedgehog (SHH) activator promotes cerebellar differentiation from human induced pluripotent stem cells (hiPSCs). This study examined phenotypic, metabolic, and biogenesis in early cerebellar development. Cerebellum spheroids were differentiated from human iPSK3 cells. During day 7-14, RA and Wnt activator CHIR99021 were used and SHH activator purmorphamine (PMR) was added later to promote ventralization. Gene expression for early cerebellar layer markers, metabolism, and extracellular vesicle (EV) biogenesis were characterized. Zinc-induced neurotoxicity was investigated as a proof-of-concept of neurotoxicity study. Flow cytometry results showed that there was no significant difference in NEPH3, PTF1A, OLIG2, and MATH1 protein expression between RCP (RA-CHIR-PMR) versus the control condition. However, the expression of cerebellar genes for the molecular layer (BHLE22), the granule cell layer (GABRB2, PAX6, TMEM266, KCNIP4), the Bergmann glial cells (QK1, DAO), and the Purkinje cell layer (ARHGEF33, KIT, MX1, MYH10, PPP1R17, SCGN) was significantly higher in the RCP condition than the control. The shift in metabolic pathways toward glycolysis was observed for RCP condition. The EV biogenesis marker expression was retained. Mild zinc-induced neurotoxicity may exist when zinc exposure exceeds 1.0 µM. RCP treatment can promote specific cerebellar-like differentiation from hiPSCs indicated by gene expression of early cerebellar markers and regionally enriched genes. The higher cerebellar marker expression is accompanied by the elevated glycolysis with the retained EV biogenesis. This study should advance the understanding of biomarkers during early cerebellar development for cerebellum organoid engineering and neurotoxicity study.
人类小脑由高密度和复杂性的神经元组成。因此，区分具有类似细胞标记物和功能的类小脑器官与人类大脑具有挑战性。我们之前的研究表明，维甲酸 (RA)、Wingless/integrated (Wnt) 激活剂和 Sonic Hedgehog (SHH) 激活剂的组合可促进人类诱导多能干细胞 (hiPSC) 向小脑分化。本研究检查了早期小脑发育中的表型、代谢和生物发生。从小脑球体分化出人 iPSK3 细胞。在第 7-14 天期间，使用 RA 和 Wnt 激活剂 CHIR99021，随后添加 SHH 激活剂 purmorphamine (PMR) 以促进腹侧化。表征了早期小脑层标记物、代谢和细胞外囊泡 (EV) 生物发生的基因表达。锌诱导的神经毒性作为神经毒性研究的概念验证进行了研究。流式细胞术结果表明，在 RCP (RA-CHIR-PMR) 与对照条件之间，NEPH3、PTF1A、OLIG2 和 MATH1 蛋白表达没有显著差异。然而，分子层的小脑基因表达（BHLE22）、颗粒细胞层（GABRB2、PAX6、TMEM266、KCNIP4）、Bergmann 神经胶质细胞（QK1、DAO）和浦肯野细胞层（ARHGEF33、KIT、MX1、MYH10、PPP1R17、SCGN）在 RCP 条件下的表达明显高于对照。观察到 RCP 条件下代谢途径向糖酵解的转变。EV 生物发生标记物的表达得到保留。当锌暴露超过 1.0 µM 时，可能存在轻微的锌诱导神经毒性。RCP 处理可促进 hiPSC 中特定的类小脑分化，这表明早期小脑标记物和区域丰富基因的表达。更高的小脑标记物表达伴随着糖酵解的升高和 EV 生物发生的保留。这项研究应该有助于深入了解小脑器官发生工程和神经毒性研究中早期小脑发育过程中的生物标志物。
