Neuropediatrics 2019; 50(S 02): S1-S55
DOI: 10.1055/s-0039-1698203
Poster Presentations
Poster Are a GNP Varia 1/CNS Tumours
Georg Thieme Verlag KG Stuttgart · New York

Establishment of a 3-dimensional Low-grade Glioma Model in Induced Pluripotent Stem-cell-derived Brain Organoids

Annabelle Bahr
1   Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Department of Pediatric Oncology and Hematology, Berlin, Germany
,
Simon Michèle
1   Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Department of Pediatric Oncology and Hematology, Berlin, Germany
,
David Capper
2   Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Institute of Neuropathology, Berlin, Germany
,
Olaf Witt
3   Heidelberg University Hospital, Hopp Children’s Center Heidelberg (KiTZ), Heidelberg, Germany, Clinical Cooperation Unit Pediatric Oncology, German Cancer Research Center (DKFZ) and German Consortium for Translational Cancer Research (DKTK), Heidelberg, Germany, KiTZ Clinical Trial Unit, Heidelberg, Germany
,
Till Milde
3   Heidelberg University Hospital, Hopp Children’s Center Heidelberg (KiTZ), Heidelberg, Germany, Clinical Cooperation Unit Pediatric Oncology, German Cancer Research Center (DKFZ) and German Consortium for Translational Cancer Research (DKTK), Heidelberg, Germany, KiTZ Clinical Trial Unit, Heidelberg, Germany
,
Florian Selt
3   Heidelberg University Hospital, Hopp Children’s Center Heidelberg (KiTZ), Heidelberg, Germany, Clinical Cooperation Unit Pediatric Oncology, German Cancer Research Center (DKFZ) and German Consortium for Translational Cancer Research (DKTK), Heidelberg, Germany, KiTZ Clinical Trial Unit, Heidelberg, Germany
,
Juliane Buhl
4   Heidelberg University, Hopp Children’s Cancer Center Heidelberg (KiTZ), Heidelberg, Germany, Faculty of Biosciences, Heidelberg University, Heidelberg, Germany, Clinical Cooperation Unit Pediatric Oncology, German Cancer Research Center (DKFZ) and German Consortium for Translat, Heidelberg, Germany
,
Harald Stachelscheid
5   Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, BIH Stem Cell Core Facility, Berlin-Brandenburg Center for Regenerative Therapies, Berlin, Germany
,
Pablo Hernáiz Driever
1   Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Department of Pediatric Oncology and Hematology, Berlin, Germany
› Author Affiliations
Further Information

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.