GSK3ß inhibitor CHIR 99021 modulates cerebral organoid development through dose-dependent regulation of apoptosis, proliferation, differentiation and migration
Cerebral organoids generated from human pluripotent stem cells (hiPSCs) are unique in their ability to recapitulate human-specific neurodevelopmental events. They are capable of modeling the human brain and its cell composition, including human-specific progenitor cell types; ordered laminar compartments; and both cell-specific transcriptional signatures and the broader telencephalic transcriptional landscape. The serine/threonine kinase, GSK3β, plays a critical role in neurodevelopment, controlling processes as varied as neurogenesis, morphological changes, polarization, and migration. In the generation of cerebral organoids, inhibition of GSK3β at low doses has been used to increase organoid size and decrease necrotic core. However, little is known of the effects of GSK3β inhibition on organoid development.
Here, they demonstrate that while low dose of GSK3β inhibitor CHIR 99021 increases organoid size, higher dose actually reduces organoid size; with the highest dose arresting organoid growth. To examine the mechanisms that may contribute to the phenotypic size differences observed in these treatment groups, they show that low dose of CHIR 99021 increases cell survival, neural progenitor cell proliferation and neuronal migration. A higher dose, however, decreases not only apoptosis but also proliferation, and arrests neural differentiation, enriching the pool of neuroepithelial cells, and decreasing the pools of early neuronal progenitors and neurons. These results reveal new mechanisms of the pleiotropic effects of GSK3β during organoid development, providing essential information for the improvement of organoid production and ultimately shedding light on the mechanisms of embryonic brain development.
Glycogen synthase kinase 3 beta (GSK3β) is a serine/threonine kinase, which has an extended 13 amino acid loop by the catalytic domain. During neurodevelopment, GSK3β controls the level, binding, and localization of transcription factors (such as CREB, Nfat, Neurogenin 2, SMAD1, and β-catenin) as well as the activity of cytoskeletal proteins involved in migration and axonal growth and guidance. Altogether, GSK3β coordinates a wide range of signaling processes guiding neurodevelopmental events that include neurogenesis; structural changes and cell advancement; polarization and orientation; and migration. The canonical Wnt/β-catenin pathway is the best characterized pathway mediated by GSK3β. The absence of Wnt facilitates GSK3β-mediated phosphorylation of β-catenin, which leads to its ubiquitination and proteasome-mediated degradation. In the presence of Wnt, or inhibition of GSK3β, the consequent reduction of β-catenin phosphorylation and degradation allows β-catenin to accumulate in the cytoplasm, associated with its translocation to the nucleus and the transcriptional regulation of Wnt target genes. Inhibition of GSK3β stabilizes β-catenin and in turn activates the Wnt pathway, independently of Wnt ligand-receptor interactions.
Understanding human brain development with little access to fetal tissues remains a major challenge in neuroscience. In vitro cultures of cerebral organoids derived from human pluripotent stem cells have therefore raised the promise of effectively studying and modeling human brain development. However, protocols for brain organoid production are limited by their partial knowledge of the mechanisms of brain development. In the past decade the field has developed both self-organizing protocols and more recent directed protocols for the production of brain organoids encompassing broad and specific regional identity, respectively; however many limitations remain, including difficulties in the maintenance of healthy cells throughout long-term cultures, and efficient and reliable determination of cell fate and identity. Inhibition of GSK3β by CHIR 99021 to promote cell survival or trigger regionalization in brain organoids has been suggested in several recent studies; however its multiple effects on organoid development are not well characterized. Here, they demonstrate that the inhibition of GSK3β using high and low dosages of CHIR 99021 during neuronal differentiation of hiPSC-derived organoids elicits differential effects on their growth and cellular phenotypes. Understanding how the GSK3β inhibition affects cerebral organoid production not only helps improve cerebral organoid protocols but also reveals human neurodevelopmental processes1.
GSK3β inhibition by CHIR 99021 during neuronal differentiation affects neurodevelopmental signaling pathways and organoid growth. A. Schematic of organoid production protocol. Dual SMAD inhibition was performed during the neural induction step. GSK3β inhibitor CHIR 99021 (1μM, 10μM or 50μM), or vehicle DMSO, was added to the culture media throughout neuronal differentiation (day 14 and onward). B. Representative organoids at day 35 of organoid development; organoids treated with 50μM CHIR 99021 failed to grow. C. Quantification of organoid size. CHIR 99021 treatment had a dose-dependent effect on organoid size.
A. Representative images of immunostained organoids. The apoptosis marker cleaved caspase 3 (c-Cas3) and proliferation marker KI67 were immunolabeled. B. Quantification of the c-Cas3 or KI67 positive areas normalized by the DAPI positive area for each organoid.
A. Western Blot bands and B. quantification of neuroepithelium marker E-cadherin (E-Cad), neural progenitor cells (NPCs) marker SOX2, radial glia marker BLBP, intermediate progenitor marker TBR2, and neuronal markers TUJ1 and DCX, relative to DMSO control.
A. Representative images of immune-stained organoids for detection of NPC marker PAX6, neuronal markers DCX and TUJ1, intermediate progenitor marker TBR2, and B. neuroepithelial marker E-cadherin. C. Quantification of the cell type marker positive areas normalized by the DAPI positive area for each organoid, and relative to DMSO organoids
A. Representative images of GFP expressing cells 7 days after injection and electroporation of the GFP expression plasmid, with immunostaining of proliferative cell marker KI67 and neuronal marker DCX. B. Quantification of the percentage of neurons (DCX+, KI67- cells) (DMSO: 91.33% ± 8.083, n = 3; 1μM: 93.33% ± 5.508, n = 3; p-value = 0.7411, unpaired t-test) C. GFP positive area, distance and maximum distance (in gray) from injection ventricle were measured. D. Quantification of cell distance from injected ventricle.
Delepine, C., Pham, V. A., Tsang, H. W. S. & Sur, M. (2021). GSK3ß inhibitor CHIR 99021 modulates cerebral organoid development through dose-dependent regulation of apoptosis, proliferation, differentiation and migration. PLOS ONE, 16 (5), s. e0251173. doi:10.1371/journal.pone.0251173