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ICE Krakow

ICE Krakow

ul. Marii Konopnickiej 17 30-302 Kraków


Metin, Erkan (Stem Cell Bioengineering Department of Mossakowski Medical Research Centre Polish Academy of Sciences)


"Introduction: The brain organoids derived from human-induced Pluripotent Stem Cells (iPSC) provide a tool to model and study human brain development and its pathology. Up to date reports regarding the role of mitochondrial biogenesis (MB) in cell-type specification during in vitro human cortical development are not available. By inducing mitochondrial biogenesis at different developmental stages of cortical organoid - Embryonic Body (EB), Neurosphere (NS), and Cortical Organoid (CO), we aim to elucidate its role in neurodevelopment.
The main objective of this study was to determine the effect of MB induction on cell fate commitment specifically in the Embryonic Body structures, resembling the earliest developmental stage of human cortical development.
Methods: In this study brain region-specific cortical organoids were derived from human iPSCs with EBs as the earliest stage of development. EBs were maintained in Essential 6 media for 13 days, to induce spontaneous differentiation and expression of 3-germ layers markers. MB was induced by alpha-7 nicotinic acetylcholine receptor (α7nAChR) agonist, PNU-282987 for seven days. The toxicity of different doses of PNU-282987 (5, 10, 20, 50, 100 µM) was evaluated by AlamarBlue and CellTiter-3D Glo viability assays and an apoptotic assay using CellEvent™ caspase-3/7 green detection reagent and Hoechst. The level of mitochondria biogenesis was determined by the ratio of mtDNA copy number to genomic DNA, ND1/HBB and ND5/SERPINA1 via qPCR. The presence of 3-germ layers markers – FOXA2 for endoderm, α-SMA for mesoderm as well as Pax6 and Nestin for ectoderm were evaluated using immunofluorescence labeling and RT-PCR analysis for the expression of markers at cellular and genomic level respectively. Proliferation was evaluated by calculating the number of Ki67-positive cells after 7-day induction of MB.
Results: The cell viability assays using resazurin and luciferin indicating live cells as well as apoptotic assay using Cas3/7 detection reagent with Hoechst, indicating the percentage of apoptotic cells, revealed that all tested concentrations of PNU-282987 had no toxic effect on the EBs formation. Furthermore, the measurement of the ratio of mtDNA to genomic DNA revealed that while most of the used concentrations resulted in increased mtDNA copy number, the concentration of 10 µM induced MB more effectively in statistically significant manner. The early, Embryonic Body stage of cortical organoid development treated with 10 µM PNU-282987 showed significant increase in cell proliferation, as revealed by the calculation of Ki67 positive cells. Lastly, changes in expression of ectodermal markers- Pax6 and Nestin as compared to endodermal and mesodermal markers at genomic level, revealed that in 3D Embryonic Bodies, MB induction drives cell fate commitment into ectoderm layer. Quantitative evaluation of the fluorescence intensity of the ectodermal markers at cellular level supports the notion of the influence of MB induction in cell fate specification during the early stage of human cortical organoid.
Conclusions: Induction of Mitochondrial Biogenesis in EB stage of cortical organoid development guides cell fate specification into neuroectodermal lineage. Mimicking human early neurodevelopment with 3D cortical organoid model provides a useful framework to study the role of mitochondrial biogenesis in developmental stage-dependent cell fate specification."

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