Associate Professor North Carolina State University, United States
Introduction:: Organoids are in vitro 3D models capable of recapitulating complex tissue architectures. They are typically derived from embryonic and induced pluripotent stem cells and are scalable and renewable. The differentiation of stem cells into defined cell types and spatial patterning within the organoids is controlled and directed using morphogens. Organoids mimicking tissues like the liver, brain, heart, and many more have been developed. However, the in vitro growth conditions impose limitations on these models. Heterogeneity between and within organoids, incomplete differentiation, and non-physiological cell types are often observed. Previous work hints at the role of metabolism in organoid development. Substrates in metabolic pathways have been shown to impact growth and play a role in epigenetic modifications, thus governing cell fate. Our aim is to investigate the influence of glucose metabolism on germ-layer and lineage specification in human stem cell-derived organoids.
Materials and Methods:: Stem Cell culture and organoid generation: H9 and H1 stem cells were maintained in 6-well tissue culture dishes (Fisher Scientific Corning Costar) coated with 0.5 g/cm2 Vitronectin (VTN-N) (Thermo Fisher Scientific) in E8 medium (Stemcell Technologies) and passaged using standard protocols. Organoids were generated by seeding a single-cell suspension of stem cells at 10,000 cells/well in a 96-well ultra-low attachment plate (S-bio). The media was changed every 2-3 days and the morphology was observed every 3 days. Immunofluorescence study: Organoids were fixed using 4% paraformaldehyde and embedded in a sucrose gelatin solution. Prior to analysis, they were cryosectioned into 16μm slices using cryoStat (ThermoFisher). The following primary antibodies were used: SOX2 (Goat, Invitrogen), TUJ1 (Rabbit, Sigma), ZO1 (mouse, BD biosciences), AQP1 (rabbit, Abcam), and TTR (mouse, R&D Systems). TUJ1 ( Secondary antibodies used were donkey Alexa Fluor 488, 594, and 647 conjugates (Invitrogen). Images were taken by using Nikon A1R confocal microscope (Nikon Instruments).
Results, Conclusions, and Discussions:: Our studies with H1-derived cortical organoids showed that organoids grown in low glucose conditions form large Choroid Plexus (ChP) cysts within two weeks of culture whereas the organoids grown in high-glucose conditions are limited to cortical cell types. Organoids in low glucose conditions lacked the neural rosettes, that are intrinsic to cortical organoids and were positive for expression of ChP markers TTR, AQP1, and ZO-1 by immunofluorescence analysis. In addition, morphological differences in neuroepithelium development were observed in low vs high glucose conditions for H9-derived cortical organoids. These findings highlight the importance of glucose metabolism in lineage specification and suggest that nutrient concentrations are important knobs controlling organoid development. This study will contribute to the understanding of the interdependence between metabolism and regulation of cell fate, specifically differentiation. It has the potential to uncover molecular components and mechanisms that convert metabolic changes to differentiation patterns and help inform strategies for generating improved in vitro human organoid models with higher fidelity and reproducibility. Furthermore, this can lead to advanced models for developmental diseases and reveal new therapeutic avenues.
