It is currently challenging to adequately model the growth and migration of glioblastoma using two-dimensional (2D) in vitro culture systems as they quickly lose the original, patient-specific identity and heterogeneity. However, with the advent of three-dimensional (3D) cell cultures and human-induced pluripotent stem cell (iPSC)-derived cerebral organoids (COs), studies demonstrate that the glioblastoma-CO (GLICO) coculture model helps to preserve the phenotype of the patient-specific tissue. Here, we aimed to set up such a model using mature COs and develop a pipeline for subsequent analysis of cocultured glioblastoma. Our data demonstrate that the growth and migration of the glioblastoma cell line within the mature COs are significantly increased in the presence of extracellular matrix proteins, shortening the time needed for glioblastoma to initiate migration. We also describe in detail the method for the visualization and quantification of these migrating cells within the GLICO model. Lastly, we show that this coculture model (and the human brain-like microenvironment) can significantly transform the gene expression profile of the established U87 glioblastoma cell line into proneural and classical glioblastoma cell types.
- Klíčová slova
- GLICO, cerebral organoids, glioblastoma, induced pluripotent stem cells,
- MeSH
- buněčné kultury metody MeSH
- buněčné linie MeSH
- glioblastom * genetika metabolismus MeSH
- lidé MeSH
- mozek MeSH
- nádorové mikroprostředí MeSH
- organoidy metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
MicroRNAs (miRNAs), a class of small, noncoding RNA molecules represent important regulators of gene expression. Recent reports have implicated their role in the cell specification process acting as "fine-tuners" to ensure the precise gene expression at the specific stage of cell differentiation. Here, we used retinal organoids differentiated from human pluripotent stem cells (hPSCs) as a model to closely investigate the role of a sensory organ-specific and evolutionary conserved miR-183/96/182 cluster. Using a miRNA tough decoy approach, we inhibited the miR-183/96/182 cluster in hPSCs. Inhibition of the miRNA cluster resulted in an increased expansion of neuroepithelium leading to abnormal "bulged" neural retina in organoids, associated with upregulation of neural-specific and retinal-specific genes. Importantly, we identified PAX6, a well-known essential gene in neuroectoderm specification, as a target of the miR-183/96/182 cluster members. Taken together, the miR-183/96/182 cluster not only represents an important regulator of PAX6 expression, but it also plays a crucial role in retinal tissue morphogenesis.
- Klíčová slova
- microRNAs, organoids, pluripotent stem cells, retina,
- Publikační typ
- časopisecké články MeSH
