Derivation of Cortical Spheroids from Human Induced Pluripotent Stem Cells in a Suspension Bioreactor.

Abstract

Human induced pluripotent stem cells (hiPSCs) emerge as a promising source to construct human brain-like tissues, spheroids, or organoids in vitro for disease modeling and drug screening. A suspension bioreactor can be used to generate large size of brain organoids from hiPSCs through enhanced diffusion, but the influence of a dynamic bioreactor culture environment on neural tissue patterning from hiPSCs has not been well understood. The objective of this study is to assess the influence of a suspension bioreactor culture on cortical spheroid (i.e., forebrain-like aggregates) formation from hiPSCs. Single undifferentiated hiPSK3 cells or preformed embryoid bodies were inoculated into the bioreactor. Aggregate size distribution, neural marker expression (e.g., Nestin, PAX6, β-tubulin III, and MAP-2), and cortical tissue patterning markers (e.g., TBR1, BRN2, SATB2, and vGlut1) were evaluated with static control. Bioreactor culture was found to promote the expression of TBR1, a deep cortical layer VI marker, and temporally affect SATB2, a superficial cortical layer II-IV marker that appears later according to inside-out cortical tissue development. Prolonged culture after 70 days showed layer-specific cortical structure in the spheroids. Differential expression of matrix metalloproteinase-2 and -3 was also observed for bioreactor and static culture. The altered expression of cortical markers by a suspension bioreactor indicates the importance of culture environment on cortical tissue development from hiPSCs.