Reduced mechanical shear is associated with early cortical differentiation and GDNF-GFRA1/RET signalling in human brain organoids.

Early cortical differentiation in human brain organoids is a critical step of corticogenesis, but remains poorly coordinated under conventional culture conditions. In a standard orbital shaker system, mechanical shear and variability in extracellular matrix support, can disrupt neuroepithelial organisation, leading to inconsistent early cortical differentiation. Here, we investigated how ClinoStar-associated low-shear culture conditions affect early corticogenic marker expression in brain organoids and examined GFRA1/RET-related signaling as a candidate pathway associated with these culture-related effects. Using a low-shear ClinoStar bioreactor, we observed improved neuroepithelial-like expansion, enhanced tissue organisation, and promoted early cortical differentiation during the neuroepithelial expansion stage. Compared with conventional conditions, low-mechanical shear in ClinoStar was associated with increased proliferation capacity and supported the number of TBR2+ intermediate progenitors and TBR1+ early cortical neurons. Transcriptomic analysis revealed enrichment of neuronal differentiation and neuroactive ligand-receptor interaction pathways, with increased upregulation of GDNF signalling components under reduced mechanical shear. Functional experiments showed that GDNF supplementation increased TBR2+ intermediate progenitor-associated and TBR1+ early neuronal marker-related populations in brain organoids, suggesting a positive effect on early corticogenic marker expression. Consistent with this, GFRA1/RET levels were elevated in organoids in the ClinoStar culture with low-mechanical shear stress, and manipulation of GFRA1 affected early cortical differentiation outcomes. These findings suggest that ClinoStar low-shear culture promoted early corticogenic marker expression, potentially associated with GDNF-GFRA1/RET signalling. Collectively, this study provides a refined culture framework for improving the reproducibility and structural organisation of human brain organoid models of early cortical differentiation.