Phenotypic, metabolic, and biogenesis properties of human stem cell-derived cerebellar spheroids.

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.