"Introduction: Mitochondrial dynamics and metabolic alterations play a pivotal role in neuron maintenance and differentiation during early human neurodevelopment. Brain organoids (BO) derived from human induced pluripotent stem cells (hiPSC) provide a unique model to study developmental stage-specific sensitivity of mitochondrial dynamics to different microenvironmental cues. Our previous experiments performed on hiPSC-derived neuronal cultures showed that physiological normoxia (5% O2) impacts neural to glial cell fate by increasing expression of the astrocytic markers and lowering expression of the neuronal markers. In this work, we used a brain organoid model from hiPSCs grown in two different oxygen conditions – 5 and 21% O2 - to decipher the influence of mitochondrial dynamics on neural cell fate.
Methods: BO were generated from hiPSCs and cultured either in 21% (control) or 5% O2 (physiological normoxia). Then, 11-day neurospheres (11D-N), 44-days (44D-BO) and 4-month brain organoids (4M-BO) were collected. BO metabolism was evaluated by monitoring the ATP levels and through Alamar Blue assay. Changes in expression for markers specific to neural stem cells (Nestin, Pax6), neuronal cells (MAP2, bTubIII), glial cells (GFAP), proliferation (Ki-67), mitochondria (MAB1273) were determined by immunofluorescence labelling. To determine the effect of low oxygen on 44D-BO at the genomic level, RNA-seq experiment was performed.
To assess changes in mitochondrial networks, organoid sections were immunostained with anti-MAB1273, a mitochondrial surface protein. Confocal and structured illumination microscopy images were acquired on a Zeiss LSM 780. Image processing and quantitative analysis of the mitochondrial morphology were determined using ZEN software, FIJI plugins (Mitochondrial Analyzer plugin, MorpholibJ plugins and 3D Manager) and ad hoc Matlab routines.
Results: Our mitochondria analysis framework revealed changes in mitochondrial network parameters throughout brain organoid development and show that physiological normoxia affects key parameters of mitochondrial morphology in a developmental stage-specific manner. The most noticeable effect of low oxygen conditions on mitochondrial shape, connectivity and size predictors was observed at the stage of 44-day brain organoids therefore most of the analyses were performed at this stage. Metabolic assays showed a lower rate of metabolism, which is accompanied by a noticeably smaller diameter of 44D-BO in 5% O2 compared to 21% O2 controls. Furthermore, 3D analysis of segmented mitochondrial objects from the super-resolution microscopy images revealed significant alterations in mitochondrial volume, surface area, equivalent diameter, sphericity in the cortical zone of BO grown in 5% O2 with respect to 21 % O2 -cultured ones. Transcriptomic analysis revealed upregulation of genes involved in: HIF-1 signaling pathway, glycolysis/gluconeogenesis, central carbon metabolism and pyruvate metabolism in 44D-BO grown in 5% O2. Our results suggest that in physiological normoxia, glycolysis prevails over oxidative phosphorylation (Oxphos). This is accompanied with decreased expression of neuronal markers (βtubIII, MAP2) and increased level of glial marker (GFAP) in brain organoids grown in low oxygen conditions compared to controls, confirming the influence of low oxygen on neural to glial cell fate transition.
In summary, this study shows that oxygen conditions influence neural fate by inducing changes in glycolysis/Oxphos ratio and mitochondrial dynamics in a stage-specific manner during brain organoid development."