biostudies-arrayexpressAkanksha JainHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15057Here we have provided scRNAseq datasets of unguided human brain organoids. The organoids were sequenced as a 1) Timecourse, 2) With different extracellular matrix treatments, 3) chemical treatment and 4) from organoids ere generated with a gene knockout cell line. Brain organoids enable the mechanistic study of human brain development, and provide opportunities to explore self-organization in unconstrained developmental systems. We have established long-term, live light sheet microscopy on unguided brain organoids generated from fluorescently labeled human induced pluripotent stem cells, which enables tracking of tissue morphology, cell behaviors, and subcellular features over weeks of organoid development. Based on imaging and single-cell transcriptome modalities, we find that lumenal expansion and cell morphotype composition within the developing neuroepithelium are associated with modulation of gene expression programs involving extracellular matrix (ECM) pathway regulators and mechanosensing. We show that an extrinsically provided matrix enhances lumen expansion as well as telencephalon formation, and unguided organoids grown in the absence of an extrinsic matrix have altered morphologies with increased neural crest and caudalized tissue identity. Matrix induced regional guidance and lumen morphogenesis are linked to the WNT and Hippo (YAP1) signaling pathways, including spatially restricted induction of the Wnt Ligand Secretion Mediator (WLS) that marks the earliest emergence of non-telencephalic brain regions. Altogether, our work provides a new inroad into studying human brain morphodynamics, and supports a view that matrix-linked mechanosensing dynamics play a central role during brain regionalization.biostudies-arrayexpressSample Collection - For all experiments, single-cell suspensions were generated by dissociation of the organoids with a papain-based neural dissociation kit (Miltenyi Biotec, 130-092-628). Briefly, organoids were washed three times with HBSS without Ca2+ and Mg2+ (STEMCELL Technologies, 37250). Prewarmed papain solution (1-2 ml) was added to the organoids and incubated for 15 min at 37°C. The tissue pieces were triturated 5–10 times with 1000 µl wide-bore and then P1000 pipette tips. The tissue pieces were incubated twice for 10 min at 37 °C with additional trituration steps in between and after with P200 and P1000 pipette tips. Cells were filtered consecutively with a 30 or 40µm filter, centrifuged at 300g for 5 min and resuspended in cold PBS. The viability and cell count for the single cell suspensions were assessed using a Trypan Blue assay on the automated cell counter Countess (Thermo Fisher Scientific).Sequencing - 10x Genomics 3' scRNAseq libraries were sequenced on a NovaSeq 6000 in paired end mode with 28/8/0/91 cyclesSample Treatment - For the experiments with YAP activator (Fig. 6e,f, Extended Fig. 10h-l), organoids were generated using the unlabelled WTC-11 parent iPSC line. For the scRNAseq (Fig. 6e,f) organoids were cultured as described above with addition of 1:1000 DMSO in neural induction medium to control organoids on day 5 and 10µM Py-60 (MedChem Express, HY-141644) to organoids on day 5 or day 7. Media with fresh inhibitor or control media were exchanged every other day till dissociation and sequencing on day 10. For the scRNAseq (Extended Figure 9c-f; Extended Figure 10c-g), EBs were generated from 500 cells in mTSR+ (1:200 Rocki and 1:200 Pen-strep), per well of a 96 well plate and centrifuged at 200g for 5 min to generate embryoid bodies (EBs). Fresh mTSR+ with 1:200 ROCKi and 1:200 Pen-strep was exchanged on day 2 and day 4. Fresh Neural induction media was supplied on day 6 and exchanged every other day, followed by differentiation media - VitA on day 10 and differentiation media + VitA on Day 15. Organoids were given 2% matrigel, or 10µM Py-60 with 2% matrigel or no matrix on day 10. All media compositions were exchanged on day 13 followed by dissociation and sequencing on day 16.Nucleic Acid Extraction - For scRNA-seq, cells were resuspended to a final concentration after counting and viability checking that enabled targeting 8,000 cells and, in case the cell numbers were not sufficient, all cells were loaded. The scRNA-seq libraries were generated using the Chromium Single Cell 3′ V3 Library & Gel Bead Kit. Single-cell encapsulation and library preparation were performed according to the manufacturer’s protocol.Library Construction - Single-cell encapsulation and library preparation were performed according to the manufacturer’s protocol.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - normalization.method = \"LogNormalize\", scale.factor = 10000UnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000Brain organoids enable the mechanistic study of human brain development, and provide opportunities to explore self-organization in unconstrained developmental systems. Here, we establish long-term, live light sheet microscopy on unguided brain organoids generated from fluorescently labeled human induced pluripotent stem cells, which enables tracking of tissue morphology, cell behaviors, and subcellular features over weeks of organoid development. We provide a novel dual-channel, multi-mosaic and multi-protein labeling strategy combined with a computational demultiplexing approach to enable simultaneous quantification of distinct subcellular features during organoid development. We track Actin, Tubulin, plasma membrane, nucleus, and nuclear envelope dynamics, and quantify cell morphometric and alignment changes during tissue state transitions including neuroepithelial induction, maturation, lumenization, and brain regionalization. Based on imaging and single-cell transcriptome modalities, we find that lumenal expansion and cell morphotype composition within the developing neuroepithelium are associated with modulation of gene expression programs involving extracellular matrix (ECM) pathway regulators and mechanosensing. We show that an extrinsically provided matrix enhances lumen expansion as well as telencephalon formation, and unguided organoids grown in the absence of an extrinsic matrix have altered morphologies with increased neural crest and caudalized tissue identity. Matrixinduced regional guidance and lumen morphogenesis are linked to the WNT and Hippo (YAP1) signaling pathways, including spatially restricted induction of the Wnt Ligand Secretion Mediator (WLS) that marks the earliest emergence of nontelencephalic brain regions. Altogether, our work provides a new inroad into studying human brain morphodynamics, and supports a view that matrix-linked mechanosensing dynamics play a central role during brain regionalization.RNA-seq of coding RNA from single cellsHomo sapiensMorphodynamics of human early brain organoid developmentZhisong HeAkanksha JainBarbara TreutleinfalsescRNAseq datasets of human brain organoids generated for the manuscript "Morphodynamics of human early brain organoidsHere we have provided scRNAseq datasets of unguided human brain organoids. The organoids were sequenced as a 1) Timecourse, 2) With different extracellular matrix treatments, 3) chemical treatment and 4) from organoids ere generated with a gene knockout cell line. Brain organoids enable the mechanistic study of human brain development, and provide opportunities to explore self-organization in unconstrained developmental systems. We have established long-term, live light sheet microscopy on unguided brain organoids generated from fluorescently labeled human induced pluripotent stem cells, which enables tracking of tissue morphology, cell behaviors, and subcellular features over weeks of organoid development. Based on imaging and single-cell transcriptome modalities, we find that lumenal expansion and cell morphotype composition within the developing neuroepithelium are associated with modulation of gene expression programs involving extracellular matrix (ECM) pathway regulators and mechanosensing. We show that an extrinsically provided matrix enhances lumen expansion as well as telencephalon formation, and unguided organoids grown in the absence of an extrinsic matrix have altered morphologies with increased neural crest and caudalized tissue identity. Matrix induced regional guidance and lumen morphogenesis are linked to the WNT and Hippo (YAP1) signaling pathways, including spatially restricted induction of the Wnt Ligand Secretion Mediator (WLS) that marks the earliest emergence of non-telencephalic brain regions. Altogether, our work provides a new inroad into studying human brain morphodynamics, and supports a view that matrix-linked mechanosensing dynamics play a central role during brain regionalization.2025-04-30T00:00:00Z2025-04-17T15:26:40.539Z2025-04-17T15:26:40.539ZE-MTAB-15057ERP171779EFO_0002944EFO_0004170EFO_0005684EFO_0005518EFO_0003816EFO_0004184EFO_000396910.1101/2023.08.21.553827