(486a) Deriving Cerebellum-like Organoids from Induced Pluripotent Stem Cells

Thien Hua, Julie Bejoy, Liqing Song, Zhe Wang, Yan Li*, Qing-Xiang Amy Sang*

Department of Chemical and Biomedical Engineering, FAMU-FSU College of Engineering, Florida State University, Tallahassee, Florida, USA

Abstract:

Human induced pluripotent stem cells (iPSCs) have high differentiation potential into three germ layers. Approaches have been made to explore their ability to generate organoids similar to human tissues for understanding organ development. However, there has yet been a successful model for the cerebellum. Most of the protocols involve costly chemicals including growth factors, neurotrophic factors, and signaling proteins that result with low yield of interested cells. One breakthrough is using fibroblast growth factor (FGF) 2, insulin, and transforming growth factor β inhibitor in the first week of human PSC aggregate formation followed by treating with FGF19 and stromal cell-derived factor 1A (SDF1A) in week 3 and 5 respectively. FGF2 and insulin promote the expression of FGF8 and Wnt1 for dorsal-caudal patterning similar to the isthmic organizer; whereas, FGF19 promotes caudalized patterning with the neural rosette formation. In addition, to mimic the cerebellum, SDF1A promotes self-organization to form the three layers including the molecular layer (NEPH3⁺/PTF1A⁺), the Purkinje cell layer (OLIG2⁺), and the granule cell layer (MATH1⁺). However, the number of progenitors of the Purkinje cells and the granule cells is low. Different morphogens can be used to modulate signal transduction pathways that are important for brain development and can be utilized to improve the differentiation. In this study, we focus on the effects of the wingless (WNT) pathway, the retinoic acid (RA) pathway, and the sonic hedgehog (SHH) pathway on the development of cerebellar spheroids from human iPSCs. WNT and RA pathway was respectively induced by CHIR99021 (CHIR) and RA on week 2; whereas the SHH pathway was induced by purmophamine (PMR) during week 5 of culture. Different combinations of the morphogens (RA/CHIR, RA/PMR, CHIR/PMR, and RA/CHIR/PMR) were utilized, and the spheroids were characterized for each cerebellum marker. Of all the combinations, RA/CHIR/PMR promoted both the Purkinje cell layer and the granule cell layer differentiation. Our results can enhance the understanding of the important role of different molecular pathways in the early stage of cerebellar spheroid differentiation.