Summary
Cerebral cortical-enriched organoids derived from human pluripotent stem cells (hPSCs) are valuable models for studying neurodevelopment, disease mechanisms, and therapeutic development. However, recognized limitations include the high variability of organoids across hPSC donor lines and experimental replicates. We report a 96-slitwell method for efficient, scalable, reproducible cortical organoid production. When hPSCs were cultured with controlled-release FGF2 and an SB431542 concentration appropriate for their TGFBR1/ALK5 expression level, organoid cortical patterning and reproducibility were significantly improved. Well-patterned organoids included 16 neuronal and glial subtypes by single cell RNA sequencing (scRNA-seq), frequent neural progenitor rosettes and robust BCL11B+ and TBR1+ deep layer cortical neurons at 2 months by immunohistochemistry. In contrast, poorly-patterned organoids contain mesendoderm-related cells, identifiable by negative QC markers including COL1A2. Using this improved protocol, we demonstrate increased sensitivity to study the impact of different MAPT mutations from patients with frontotemporal dementia (FTD), revealing early changes in key metabolic pathways.
Competing Interest Statement
ST is president of StemCultures; TB, ST, SL, patent pending related to FGF2DISCs. AMG serves on the SAB/SRB for Genentech and Muna Therapeutics. SJH serves on the SAB of or is a member of the scientific advisory board of Proximity Therapeutics, Psy Therapeutics, Frequency Therapeutics, Souvien Therapeutics, Vesigen Therapeutics. Sensorium Therapeutics, 4M Therapeutics, Ilios Therapeutics, and Entheos Labs, none of whom were involved in the present study. S.J.H. has also received speaking or consulting fees from Amgen, AstraZeneca, Biogen, Merck, Regenacy Pharmaceuticals, Syros Pharmaceuticals, Juvenescence Life, as well as sponsored research or gift funding from AstraZeneca, JW Pharmaceuticals, Lexicon Pharmaceuticals, Vesigen Therapeutics, Compass Pathways, Atai Life Sciences, and Stealth Biotherapeutics. The funders had no role in the design or content of this article, or the decision to submit this review for publication. The other authors declare no conflicts.