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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.

 

Brain spheroids or organoids derived from human pluripotent stem cells (hiPSCs) are still not capable of completely recapitulating in vivo human brain tissue, and one of the limitations is lack of microglia. To add built-in immune function, coculture of the dorsal forebrain spheroids with isogenic microglia-like cells (D-MG) was performed in our study. The three-dimensional D-MG spheroids were analyzed for their transcriptome and compared with isogenic microglia-like cells (MG). Cortical spheroids containing microglia-like cells displayed different metabolic programming, which may affect the associated phenotype. The expression of genes related to glycolysis and hypoxia signaling was increased in cocultured D-MG spheroids, indicating the metabolic shift to aerobic glycolysis, which is in favor of M1 polarization of microglia-like cells. In addition, the metabolic pathways and the signaling pathways involved in cell proliferation, cell death, PIK3/AKT/mTOR signaling, eukaryotic initiation factor 2 pathway, and Wnt and Notch pathways were analyzed. The results demonstrate the activation of mTOR and p53 signaling, increased expression of Notch ligands, and the repression of NF- κ B and canonical Wnt pathways, as well as the lower expression of cell cycle genes in the cocultured D-MG spheroids. This analysis indicates that physiological 3-D microenvironment may reshape the immunity of in vitro cortical spheroids and better recapitulate in vivo brain tissue function for disease modeling and drug screening.