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DOI: 10.1055/s-0039-1698203
Establishment of a 3-dimensional Low-grade Glioma Model in Induced Pluripotent Stem-cell-derived Brain Organoids
Publication History
Publication Date:
11 September 2019 (online)
Objective: Preclinical models to investigate the most common pediatric brain tumor entity, low-grade glioma (LGG), are still scarce. We sought to establish a human-induced pluripotent stem-cell-derived (hiPSC) brain organoid harboring low-grade glioma.
Methods: We generated and cultivated HiPSC-derived brain organoids using the STEMdiff™ Cerebral Organoid Kit (Lancaster et al. Nature 2013), LGG tumor spheres derived from pilocytic astrocytoma cells DKFZ-BT66 and high-grade glioma (HGG) tumor spheres derived from U-87 cells. Next, co-cultures of brain organoids with either LGG or HGG tumor spheres were analyzed using immunohistochemistry for MIB1, SOX2, Nestin, GFAP, S-100, TP53, Synaptophysin, OLIG2, NeuN, Vimentin, AE1/3, RFP and H&E after 30 and 45 days.
Results: Brain organoids displaying neuronal rosettes stained positively for SOX2 and MIB1, indicating immaturity and high proliferation. Radial structures within rosettes expressed Nestin and MAP2. Nestin is transiently expressed during neuro- and gliogenesis. Differentiating cells surrounding the rosettes labeled positively for MAP2. Plexus-like structures were also detected. The nearby localized cells were characterized by co-expression of Nestin and GFAP. GFAP as glial marker indicates astrocytic and ependymal like differentiation. Nestin/GFAP co-expressing cells as well as randomly detected pigmented areas were also positive for Vimentin. In addition, 3-layered cortical structures reminding mature 6-layered human cortex were detected which may represent intermediate stages of brain development. The number of MAP2 expressing mature neurons increased from day 30 to day 45. Other proteins such as S-100, TP53, Synaptophysin, OLIG2, NeuN and AE1/3 were only expressed focally. In tumor co-cultured brain organoids, we observed a partial or complete fusion of both tissues within the first three days. In co-cultured LGG red fluorescent protein (RFP) expressing LGG cells migrated into the inner organoid without destruction of the organoid architecture. Areas representing cortical-like structures, neuronal rosettes and grouped cells with similar expression patterns were equally found in co-cultured LGG. In contrast, in co-cultured HGG U-87 cells massively spread around the organoids showing a highly proliferating and invading biology. U-87 cells only expressed MIB1 and no mature glial markers.
Conclusions: Cultivating hiPSC-derived brain organoids with human LGG tumor spheres points to the first 3-dimensional animal free tumor model that allows studying LGG disease.