Expression profiling by high throughput sequencing
Summary
Here, we used agarose microwells to control embryos body (EB) shapes and generated whole brain human cerebral organoids (hCO) from these EBs to investigate the effects of early spatial regulation on transcriptomic changes during brain region specification using bulk RNA sequencing. Our results showed that gross morphological and transcriptional changes related to hCO differentiation were similar between 96-well and microwell hCOs, indicating that the microwell growth environment did not significantly affect the overall neural differentiation or viability of the hCOs. However, investigating individual developmental time points and comparing across 96-well and microwell conditions more closely revealed notable differences in genes associated with pluripotency, mesoderm, MGE, retina and cortical development, and epithelial and mesenchymal fates, implying that the microwell growth environment may bias lineage commitment in neurodevelopment. Furthermore, we found that the direction and intensity of this transcriptional bias was associated with the starting EB shapes. The butterfly shape showed the most noticeable differences, followed closely by the peanut shape. Among other notable differences, round microwells exhibited a potential delay in the kinetics of neurodevelopment, and our results also demonstrated that non-spherical shapes may favor the development of MGE-associated brain regions and cell types over cortical regions.
Overall design
Investigation of the affects of four different microwell shapes on the development of whole brain human cerebral organoids at three different time points.
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