Improved Protocol for Reproducible Human Cortical Organoids Reveals Early Alterations in Metabolism with MAPT Mutations
Human pluripotent stem cell (hPSC)-derived cortical organoids are powerful models but are often limited by low efficiency, variability, and stress-related artifacts. To address these challenges, we developed a scalable organoid platform with end-to-end quality control (QC) metrics spanning manufacturing and single-cell RNA-sequencing (scRNA-seq), developed using eight MAPT mutation isogenic line sets relevant to frontotemporal dementia (FTD-tau). Using a 96 slit-well format, we achieved [~]100% production efficiency across 64 lines. Controlled-release FGF2 enhanced iPSC pluripotency and reduced mesendodermal contaminants, while optimized SB431542 dosing enhanced cortical patterning across lines with variable TGFBR1/ALK5 expression. The resulting organoids displayed transcriptomic profiles and low-stress signatures closely aligned with the developing human cortex. Applying a cortical organoid scRNA-seq index (CortiCOSI), we identified early dysregulation of phosphatase regulators (PPP2CA, ANP32A) and the prefoldin subunit PFDN6 in MAPT V337M excitatory neurons before tau hyperphosphorylation and oligomerization. This platform improves scalability, reproducibility, and mechanistic insight in cortical organoid studies.