{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":["Hendrikus Ariese"],"organism":["Homo sapiens"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-15147"],"description":["Diffuse midline glioma (DMG) is a lethal pediatric brain cancer with limited treatment options. We developed a human brainstem organoid model to study H3.3K27M-driven DMG, enabling formation of patient-like tumors with molecular heterogeneity. To characterize this model, we performed a time-course transcriptomic analysis of healthy organoid development. We then introduced a DMG-inducing plasmid cocktail via electroporation and profiled the resulting tumors using single-cell RNA sequencing. Tumors were treated with GD2 CAR T cells, which were subsequently analyzed by single-cell RNA sequencing to capture treatment responses. Finally, we assessed the role of brain-resident myeloid cells by performing sort-seq on PMP-DMGO cocultures with or without CAR T cell treatment. This platform supports extended in vitro experimentation and reveals immune dynamics relevant for therapy development."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Sample Collection - DMGOs treated with GD2 CAR T cells were dissociated 14 or 35 days after initial T cell addition with the Neural Tissue Dissociation Kit (P) (Miltenyi Biotec, Cat. #130-092-628), as described above for preparation of single cell RNA and tracker seq libraries. Dissociated cells were washed and stained in FC buffer with CD3-APC (1:80; BD Biosciences, clone SK7) and LIVE/DEAD Fixable Near-IR Dead Cell Stain (1:1000; Thermo Fisher) for 30 min at 4˚C. CD3+ T cells and GFP+ tumour cells were sorted on a CytoFLEX SRT Benchtop Cell Sorter (Beckman Coulter) and immediately processed for scRNA-seq.","Library Construction - Sequencing libraries were prepared using Single Cell 3’ Reagent Kits v4 (10X Genomics) and then converted using the NovaSeq 6000 S2 Reagent Kit (Illumina).","Sequencing - Sequencing of single cell libraries was performed using an Illumina NovaSeq 6000 instrument using a SP or S2 flow cell.","Sample Collection - We performed single cell dissociation using the Neural Tissue Dissociation Kit (P) (Miltenyi Biotec, Cat. #130-092-628), adjusted for DMGOs. After cutting the organoids into smaller pieces and adding the enzyme mixes, the samples were incubated on an orbital shaker at 37°C and resuspended in regular intervals with a P1000 until a single cell suspension was reached, which was verified under the microscope using trypan blue to check for cell death. After dissociation, the single cell suspension was washed twice with PBS +/+ (magnesium/calcium+ 3% FBS). Next, DAPI was used as a viability dye (DAPI 1:5000). Stained single cells suspensions were filtered using a 40 µm Flowmi cell strainer and sorted by FACS on DAPI exclusion and enriched for GFP expression for tumour cells.","Library Construction - Library generation was provided by Leiden Genome Technology Center (Leiden, The Netherlands)","Library Construction - Library generation was provided by Single Cell Discoveries (Utrecht, Netherlands)","Sample Collection - Multiple organoids from the same batch and age were pooled. For timepoints after Matrigel embedding, Cell Recovery Solution (Corning, 354253) was used to dissolve the Matrigel. Organoids were incubated in this solution at 4°C for 15 minutes, halved, and washed in HBSS without Ca²⁺ and Mg²⁺. A single cell supsension was obtained using the Neural Tissue Dissociation Kit (Miltenyi Biotec, 130-092-628) according manufactures protocol.","Nucleic Acid Extraction - Nuclei acid extraction was provided by Leiden Genome Technology Center (Leiden, The Netherlands)","Nucleic Acid Extraction - Sequencing samples were thawed by warming the cryovials in a 37°C water bath until only a small clump of ice remained. The contents were then transferred to 10 mL of pre-warmed DMEM containing 10% FBS and centrifuged to pellet the cells. The cells were washed twice with PBS containing 5% BSA and filtered through a 40 µm Flowmi cell strainer to remove debris and aggregates. Approximately 30,000 dissociated cells per sample/timepoint were loaded into separate channels of a single Chromium controller (10x Genomics) chip. Consequently, gel bead-in-emulsions (GEMS) were generated using 10X Genomics Single Cell 3’ Reagent Kit v4.","Sequencing - Sequencing was provided by Single Cell Discoveries (Utrecht, Netherlands), on NovaSeq X Plus.","Nucleic Acid Extraction - Nuclei acid extraction was provided by Single Cell Discoveries (Utrecht, Netherlands)","Sample Collection - BrOs and DMGOs containing microglia and optionally treated with GD2 CAR T cells were dissociated 21 days after initial microglia incorporation with the Neural Tissue Dissociation Kit (P) (Miltenyi Biotec, Cat. #130-092-628), as described above for preparation of single cell RNA and tracker seq libraries. Dissociated cells, control PMPs and unexposed GD2 CAR T cells were washed and stained in FC buffer with CD3-BV421 (1:100; BD Biosciences, clone SK7) and LIVE/DEAD Fixable Near-IR Dead Cell Stain (1:1000; Thermo Fisher) for 30 min at 4˚C. CD3+ T cells and mScarlet+ microglia/ PMP were sorted into 386-well plates containing well-specific barcoded primers (Single Cell Discoveries), one cell per well, on a Sony SH800s (Sony Biotechnology). Plates containing sorted cells were immediately snap frozen on dry ice and processed for SORT-seq by Single Cell Discoveries."],"figure_sub":["Organization","MINSEQE Score","Assays and Data","MAGE-TAB Files"],"omics_type":["Metabolomics","Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["CytoFLEX SRT Benchtop Cell Sorter","Sony SH800S Cell Sorter","Illumina NovaSeq 6000","NextSeq 500"],"study_type":["RNA-seq of coding RNA from single cells"],"species":["Homo sapiens"],"pubmed_title":["De novo H3.3K27M-altered Diffuse Midline Glioma in human brainstem organoids to dissect GD2 CAR T cell function"],"pubmed_authors":["Hendrikus Ariese","Anne Rios","Nils Bessler, Amber K.L. Wezenaar, Hendrikus C.R. Ariese, Celina Honhoff, Ellen J. Wehrens, Noëlle Dommann, Cristian Ruiz Moreno, Thijs van den Broek, Raphaël V.U. Collot, Farid F. Keramati, Mieke Roosen, Sam de Blank, Esmée van Vliet, Mario Barrera Román, Lucrezia C.D.E. Gatti, Ali Ertürk, Jürgen Kuball, Zsolt Sebestyén, Marcel Kool, Sara Patrizi, Evelina Miele, Annette Künkele, Mariëtte E.G. Kranendonk, Annelisa M. Cornel, Stefan Nierkens, Christian Mayer, Henk Stunnenberg, Anna Alemany,  Maria Alieva and Anne C. Rios"],"additional_accession":[]},"is_claimable":false,"name":"Transcriptomic profiling of BrO, DMGO and GD2 CAR T cells","description":"Diffuse midline glioma (DMG) is a lethal pediatric brain cancer with limited treatment options. We developed a human brainstem organoid model to study H3.3K27M-driven DMG, enabling formation of patient-like tumors with molecular heterogeneity. To characterize this model, we performed a time-course transcriptomic analysis of healthy organoid development. We then introduced a DMG-inducing plasmid cocktail via electroporation and profiled the resulting tumors using single-cell RNA sequencing. Tumors were treated with GD2 CAR T cells, which were subsequently analyzed by single-cell RNA sequencing to capture treatment responses. Finally, we assessed the role of brain-resident myeloid cells by performing sort-seq on PMP-DMGO cocultures with or without CAR T cell treatment. This platform supports extended in vitro experimentation and reveals immune dynamics relevant for therapy development.","dates":{"release":"2025-08-28T00:00:00Z","modification":"2025-09-04T13:48:38.279Z","creation":"2025-05-19T13:15:44.338Z"},"accession":"E-MTAB-15147","cross_references":{"ENA":["ERP172720"],"Biostudies":["E-MTAB-15559"],"EFO":["EFO_0002944","EFO_0004170","EFO_0005684","EFO_0005518","EFO_0004184"]}}