Improved Protocol for Reproducible Human Cortical Organoids Reveals Early Alterations in Metabolism withMAPTMutations

Abstract

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 theirTGFBR1/ALK5expression 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 includingCOL1A2. Using this improved protocol, we demonstrate increased sensitivity to study the impact of differentMAPTmutations from patients with frontotemporal dementia (FTD), revealing early changes in key metabolic pathways.

Fully Human Bifunctional Intrabodies Achieve Graded Reduction of Intracellular Tau and Rescue Survival ofMAPTMutation iPSC-derived Neurons

“…To do this we used cerebral cortical patterned organoids generated from iPSCs using our established methods ( Supplemental Figure 2 ). 47, 48 By 4 months, the resulting organoids include the major cortical deep and upper layer neurons, subsets of inhibitory neurons, astrocytes, and oligodendrocyte progenitor cells.…”

“…3D cortical organoid generation followed our prior published methods. 47, 48 Human iPSC lines and MAPT mutation lines ( Table 1 ) developed from patient samples and knock-in approaches 95 were cultured on Matrigel matrix (Corning Cat # 356231) coated 6-well plates (Corning #3506) with mTeSR (StemCell Technologies Cat # 85851). iPSC lines were cultured using FGF2Discs (Stem Cultures Cat # DSC500-48) and fed with mTeSR three times a week.…”

Advanced Cellular Models for Rare Disease Study: Exploring Neural, Muscle and Skeletal Organoids

“…FTD is included in the spectrum of neurodegenerative tauopathies [120]. The impact of different mutations in the MAPT gene encoding for the Tau protein was studied in cerebral organoids derived from patients with FTD, demonstrating significant differences in the expression of several genes, including the ceramide synthetase genes CERS4, CERS5 and CERS6, and PINI, TBK1, FUS and ELAV4 [78,121]. In particular, using human cerebral organoids, it was shown that the MAPTp.V3337M mutation induces Tau phosphorylation, glutaminergic dysfunction and a loss of glutamatergic neurons [78].…”