Information accessibility snRNA-seq information are offered in Gene Expression Omnibus (GEO) under the accession 19459007 GSE185472 19459008 The following public datasets were utilized for snRNA-seq analysis: Allen Brain Institute human adult snRNA-seq information from numerous cortical locations ( https://portal.brain-map.org/atlases-and-data/rnaseq/human-multiple-cortical-areas-smart-seq 19459008; accessed October 2022), snRNA-seq information from broad temporal protection from fetal to the adult years phases of the Brodmann location 8, 9, 10 and 46 prefrontal cortex areas (GEO accession GSE168408 19459008 and snRNA-seq from 8-month-old cortical organoid transplants (GEO accession GSE190815. For single-nucleus analysis, we utilized hg19 human referral genome v1.2.0 and mm10 mouse recommendation genome v1.2.0 offered by 10x Genomics. The series and gene files utilized to construct the referrals can be accomplished at 19459012 ftp:// ftp.ensembl.org/pub/grch37/release-84/fasta/homo_sapiens/dna/ 19459008 and ftp:// ftp.ensembl.org/pub/grch37/release-84/gtf/homo_sapiens/ 19459008 (for human hg19 genome); 19459014 ftp:// ftp.ensembl.org/pub/release-84/fasta/mus_musculus/dna/ 19459008 and 19459015 ftp:// ftp.ensembl.org/pub/release-84/gtf/mus_musculus/ (for mouse mm10 genome). All other raw information utilized for outlining in the figures are offered as source information. 19459016 Source information are offered with this paper. Referrals Liddelow, S. A. et al. Neurotoxic reactive astrocytes are caused by triggered microglia. Nature 541 19459028, 481– 487 (2017 ). Post 19459030 ADS 19459008 19459031 CAS 19459032 PubMed 19459033 PubMed Central Google Scholar 19459008 19659007 Qian, X., Song, H. & & Ming, G. L. Brain organoids: advances, applications and difficulties. Advancement 146 , dev166074 (2019 ). 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WebGestalt 2019: gene set analysis toolkit with revamped UIs and APIs. 19459025 Nucleic Acids Res. 19659188 47 19459028, W199– W205 (2019 ). Post CAS 19459008 PubMed Central 19459008 Google Scholar 19459008 Download referrals 19659191 Recognitions We thank A. Mansour (Salk Institute) for offering the pCSC-CAG-GFP lentiviral identified H9 ESC line, N. Hah for technical help with snRNA-seq and R. Garg for support with EM division. We likewise thank M.L. Gage for editorial remarks and A. Cao and T. Bartol for their assist with 3DEM visualization. This work was supported by the American Heart Association and the Paul G. Allen Frontiers Group Grant (19PABHI34610000/TEAM LEADER: Fred H. Gage/2019), JPB Foundation, Annette C. Merle-Smith, Lynn and Edward Streim, the Milky Way Foundation, the Ray and Dagmar Dolby Family Fund and NIH (R37 AG072502-03, P30 AG062429-05, P30 AG068635-04, R01 AG070154-04, AG056306-07 and P01 AG051449-08). This work was likewise supported by the NGS Core Facility, the GT3 Core Facility, the Razavi Newman Integrative Genomics and Bioinformatics Core Facility and the Waitt Advanced Biophotonics Core Facility of the Salk Institute with financing from NIH– NCI (CCSG, P30 014195), the Chapman Foundation, NINDS R24 Core Grant, NEI and the Waitt Foundation. M.W. is supported by a Young Investigator Grant from the Brain & & Behavior Research Foundation (BBRFNARSAD) and a Pioneer Fund Postdoctoral Scholar Award from the Salk Institute. Figures 19459458 2a 19459008, 2h , 19459459 4a 19459008, 5a , 6a and 19459461 6d 19459008 were developed with 19459462 BioRender.com 19459008 19659193 Author info 19659194 Author notes Uri Manor 19659196 Present address: Division of Biological Sciences, University of California, San Diego, La Jolla, CA, USA 19659197 These authors contributed similarly: Meiyan Wang, Lei Zhang. Authors and Affiliations Lab of Genetics, The Salk Institute for Biological Studies, La Jolla, CA, USA Meiyan Wang, Lei Zhang, Iryna S. Gallina, Lynne L. Xu, Christina K. Lim, Sarah Fernandes, Monisha D. Saxena, Shashank Coorapati, Sarah L. Parylak & & Fred H. Gage Waitt Advanced Biophotonics Core, The Salk Institute for Biological Studies, La Jolla, CA, USA Sammy Weiser Novak, Leonardo R. Andrade & & Uri Manor 19659203 Integrative Genomics and Bioinformatics Core, The Salk Institute for Biological Studies, La Jolla, CA, USA Jingting Yu, Maxim N. Shokhirev & & April E. Williams Department of Biological Sciences, University of California, San Diego, La Jolla, CA, USA 19659206 Christina K. Lim & & Sarah Fernandes 19659207 Next Generation Sequencing Core, The Salk Institute for Biological Studies, La Jolla, CA, USA 19659208 Cristian Quintero & & Elsa Molina 19659209 Contributions M.W., L.Z. and F.H.G. developed the research study and composed the paper. M.W., L.Z., C.K.L. and S.F. carried out cell culture and organoid distinction. M.W., L.Z., C.K.L. and I.S.G. carried out surgical treatments. S.L.P. helped in surgical treatments. M.W. and I.S.G. carried out snRNA-seq and scRNA-seq. M.W., C.Q. and E.M. carried out NanoString GeoMx DSP. M.W., M.N.S., J.Y. and A.E.W. carried out bioinformatics analyses. M.W. and L.Z. carried out imaging analysis with support from L.L.X., C.K.L., S.C. and M.D.S. L.Z., S.W.N. and L.R.A. carried out sample processing for SEM. S.W.N. and L.R.A. carried out electron tiny image analysis under the guidance of M.W., L.Z. and U.M. F.H.G. offered financing. 19659211 Corresponding author 19659212 Correspondence to Fred H. Gage Principles statements 19659214 Completing interests 19659215 The authors state no contending interests. Peer evaluation 19659217 Peer evaluation details 19659218 Nature Biotechnology 19459026 thanks the confidential customers for their contribution to the peer evaluation of this work. 19659219 Extra info 19659220 Publisher’s note 19459028 Springer Nature stays neutral with regard to jurisdictional claims in released maps and institutional associations. 19659221 Prolonged information 19659222 Extended Data Fig. 1 Rapid astrogliogenesis in glia-enriched cortical organoids. a Test bright-field pictures of hiPSCs/hESCs nests and glia-enriched cortical organoids at days 0, 14, 30 and 60. Day 0 describes embryoid bodies (EB). Scale bars, 890 μm. 19459027 b Quantitative PCR analysis of the expression levels of NFIA and SOX2 in three-week-old organoids cultured under differing conditions (Hues6: 1, n= 5; 2, n= 5; 3, n= 5; 4, n= 5; 5, n= 4. iPSC822: 1, n= 5; 2, n= 5; 3, n= 5; 4, n= 5; 5, n= 4). Each dot represents a swimming pool of 3 organoids. Bars, suggest ± s.d. Two-sided t-test, ns, not considerable, * p < 0.05, *** p < 0.001, **** p < 0.0001. 19459027 c Immunostaining of two-month-old glia-enriched cortical organoids: stem cells (SOX2, magenta), astrocytes (GFAP, green). Scale bar, 100 μm. d Immunostaining of two-month-old glia-enriched cortical organoids: intermediate progenitor cells (EOMES, green), cortical excitatory nerve cells (CTIP2, magenta; SATB2, gray), stem cells (SOX2, green), nerve cells (NeuN, magenta), astrocytes (GFAP, gray), glia (HOPX, green; S100B, magenta; GFAP, gray). Scale bars, 20 μm. 19459027 e 19459028 Immunostaining of three-month-old glia-enriched cortical organoids distinguished in 2% FBS (leading left panel), 2% SATO (leading best panel; serum-free condition, SATO part reported in ref. 30 , 10% FBS (bottom left panel) and 10% SATO (bottom ideal panel). Astrocytes (GFAP, green). Scale bars, 100 μm. 19459027 f Metrology of the GFAP fluorescence strength in glia-enriched cortical organoids distinguished in 2% FBS, 2% SATO, 10% FBS and 10% SATO conditions (Hues6: 10% FBS, n= 3; 10% SATO, n= 6; 2% FBS, n= 4; 2% SATO, n= 6. iPSC822: 10% FBS, n= 6; 10% SATO, n= 8; 2% FBS, n= 5; 2% SATO, n= 6). Each dot represents one organoid. Bars, imply ± s.e.m. Two-sided t-test, *** p < 0.001, **** p < 0.0001. 19659226 Source information Extended Data Fig. 2 snRNA-seq of 10-week-old glia-enriched cortical organoids. a UMAP plot of snRNA-seq information from 10-week-old glia-enriched cortical organoids. The portion of each significant cell types is revealed. APC, astrocyte progenitor cell; Ast, astrocyte; IPC, intermediate progenitor cell; In, repressive nerve cell; Cortical Ex, cortical excitatory nerve cell; Ex1 and Ex2, excitatory nerve cell. 19459027 b 19459028 Expression of picked marker genes utilized in cell type recognition. The violin plot reveals the circulation of stabilized expression in nuclei in each cluster. Scale: stabilized checked out counts. 19459027 c Heatmap plot reveals the expression of the leading 10 function genes determined in each cluster. d Dot plot reveals the expression of astrocyte gene modules in each significant cell type. 19459027 e 19459028 Dot plot of the enrichR combined rating for the leading enriched GO terms for astrocyte gene modules M12 and M14. 19459027 f WGCNA dendrogram of Cortical Ex gene modules. g 19459028 Dot plot reveals the expression of Cortical Ex gene modules in each significant cell type. 19459027 h 19459028 UMAP plots of module center gene expression rating for Cortical Ex gene modules M1-- 4. i Co-expression plots of the leading 25 module genes for Cortical Ex gene modules M1-- 4. 19459027 j Dot plot of the enrichR combined rating for the leading enriched GO terms for cortical excitatory nerve cell gene modules M1-- 4. Extended Data Fig. 3 Maturation of astrocytes in glia-enriched cortical organoids. 19659230 a 19459028 Immunostaining of five-month-old glia-enriched cortical organoids: GFAP:: GFP AAV-labeled astrocytes (GFP, green), presynaptic blisters (SV2, gray), postsynaptic density (PSD95, magenta). Inset, a bigger view of astrocyte procedure and synapses. Scale bars, 20 μm and 2 μm (inset). b 19459028 Immunostaining of five-month-old glia-enriched cortical organoids: GFAP:: GFP AAV-labeled astrocytes (GFP, green), glutamate transporter (EAAT2, magenta). Scale bar, 20 μm. 19459027 c 19459028 Immunostaining of five-month-old glia-enriched cortical organoids: GFAP:: GFP AAV-labeled astrocytes (GFP, green) and matricellular protein (HEVIN, magenta). Scale bar, 5 μm. 19459027 d Immunostaining of five-month-old glia-enriched cortical organoids: GFAP:: GFP AAV-labeled astrocytes (GFP, green), inward-rectifier potassium channel (Kir4.1, magenta), postsynaptic density protein (PSD95, cyan). Scale bar, 2 μm. e 19459028 Immunostaining of five-month-old glia-enriched cortical organoids: astrocytes (hGFAP, green), Connexin 43 (CX43, magenta). Inset, a bigger view of astrocyte procedures and expression of Connexin 43. Scale bars, 20 μm and 2 μm (inset). 19459027 f 19459028 Metrology of glutamate uptake of neural progenitor cells (NPCs, n= 8) and astrocytes cleansed from five-month-old glia-enriched cortical organoids stemmed from Hues6 (n= 8), iPSC822 (n= 8) and H9 (n= 7) stem cell lines. Each dot represents one independent experiment. Bars, imply ± s.e.m. 19659231 Source information 19659232 Extended Data Fig. 4 Formation of anatomically specified morphological subclasses of human astrocytes in engrafted glia-enriched cortical organoids. 19659233 a Immunostaining of glia-enriched cortical organoid transplants: human nuclear antigen (HuNu, Cyan), astrocytes (GFAP, green), nerve cells (NeuN, magenta). Scale bar, 20 μm. 19459027 b 19459028 Metrology of the portion of NeuN 19459290 + 19459291 HuNu 19459290 + 19459291 cells amongst NeuN + 19459291 cells (left) or huGFAP + GFAP + cells amongst GFAP 19459290 + 19459291 cells (right) in the transplants. n= 4 transplants. Bars, indicate ± s.e.m. c Immunostaining of the center (left) and border (right) of glia-enriched cortical organoid transplants: human astrocytes (hGFAP, green), astrocytes (GFAP, magenta). Scale bars, 50 μm. d Immunostaining of the center (left) and border (right) of glia-enriched cortical organoid transplants: oligodendrocyte progenitor cells (PDGFRα, green), human nuclear antigen (HuNu, magenta). Inset, bigger views of human oligodendrocyte progenitor cells. Scale bars, 50 μm and 10 μm (inset). 19459027 e 19459028 Immunostaining of human interlaminar, protoplasmic and fibrous astrocytes in glia-enriched cortical organoid transplants: human astrocytes (hGFAP, green), white matter (myelin standard protein or MBP, gray). Scale bars, 20 μm. 19459027 f 19459028 Immunostaining of glia-enriched cortical organoid transplants: human cells (GFP, green), astrocytes (S100B, gray; CD44, magenta). Scale bar, 100 μm. 19459027 g 19459028 Immunostaining of the top, center and bottom areas of the transplants: astrocytes (CD44, magenta), white matter (myelin fundamental protein or MBP, gray). Scale bars, 20 μm. 19659234 Source information 19659235 Extended Data Fig. 5 Spatial transcriptome profiling of layer-specific subclasses of human astrocytes in engrafted glia-enriched cortical organoids. 19659236 a Immunofluorescent staining images on the left revealing hGFAP in yellow, SOX9 in magenta and CYTO-13 in blue, in addition to the cell division of SOX9 19459290 + 19459291/ CYTO-13 19459290 + 19459291 Cells portrayed in magenta within a chosen ROI on the. Scale bars, 100 μm. b Box plots portraying the stabilized expression levels of picked genes in each group (pial= 10 ROIs, cortex= 16 ROIs, WM= 12 ROIs). Centerline, mean; box limitations, upper and lower quartiles; hairs, 1.5 × interquartile variety; points, outliers. 19459027 c Bubble plots portraying leading considerable terms determined from GSEA utilizing a weighted Kolmogorov-- Smirnov test. NES, stabilized enrichment rating. Considerable limits set at adjusted p worth < 0.1. 19659237 Extended Data Fig. 6 Close association of human protoplasmic astrocytes with synapses and host vasculature in engrafted glia-enriched cortical organoids. 19659238 a Immunostaining of transplants: human astrocytes (hGFAP, green), glutamate transporters (EAAT2, magenta). Scale bar, 20 μm. 19459027 b 19459028 Immunostaining of transplants: human astrocytes (hGFAP, green), matricellular protein (HEVIN, gray), postsynaptic density (PSD95, magenta). Scale bar, 20 μm. 19459027 c Electron micrograph of a multi-synaptic bouton in an eight-month-old transplant. Arrowheads: synapses. Scale bar, 1 μm. 19459027 d 3D restoration of serial area electron microscopic lense pictures of synaptic structures in an eight-month-old transplant. Presynaptic bouton (orange), postsynaptic density (magenta), dendritic spinal column (blue). Scale bar, 1 μm. 19459027 e Electron micrograph of a synapse within the transplant. Left, presynaptic bouton (orange), postsynaptic density (PSD, magenta), dendritic spinal column (blue) and astrocytic procedures (AP, green). Initial image. Scale bar, 1 μm. f Immunostaining of transplants at 2 (left panel) or 7 months (ideal panel) post-transplantation: capillary (Ly6C, cyan). Scale bars, 100 μm. 19459027 g 19459028 Ly6C + 19459291 location (left) or vessel size (right) in transplants at 2 months (n= 4 transplants) or 7 months (n= 4 transplants) post-transplantation versus contralateral mouse cortex (Ms brain; n= 8 mice). Bars, indicate ± s.e.m. Two-sided t-test, ** p=0.001, **** p < 0.0001. hImmunostaining of transplants: human nuclear antigen (HuNu, green), pericytes (NG2, magenta, suggested by arrowhead in the left image), monocytes (Ly6C, gray, shown by arrowhead in the center image), endothelial cells (CD31, magenta). Scale bars, 10 μm. iImmunostaining of transplants (left) or host brains (right): human nuclear antigen (HuNu, green), microglia (IBA1, magenta). Inset: microglia. Scale bars, 50 μm and 10 μm (insets). jImmunostaining of transplants: human astrocytes (hGFAP, green), inward-rectifier potassium channel (Kir4.1, magenta), capillary (Ly6C, cyan). Scale bar, 20 μm. kImmunostaining of transplants: human astrocytes (hGFAP, green), glucose transporter (Glut1, magenta) and capillary (Ly6C, cyan). Scale bar, 20 μm. lConfocal pictures of the transplant utilized in EM research studies: capillary (DiI, magenta), transplant (GFP, green). Scale bar, 1 mm. m3D restoration of series SEM images: tight junction (yellow) and basement membrane (blue). Scale bar, 1 μm.
Morphological diversification and functional maturation of human astrocytes in glia-enriched cortical organoid transplanted in mouse brain
