Morphological diversification and helpful maturation of human astrocytes in glia-enriched cortical organoid transplanted in mouse brain

Morphological diversification and helpful maturation of human astrocytes in glia-enriched cortical organoid transplanted in mouse brain

   February 29, 2024  Posted in Health 110 Views
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Files availability

snRNA-seq data are readily available in Gene Expression Omnibus (GEO) below the accession GSE185472. The following public datasets were passe for snRNA-seq prognosis: Allen Mind Institute human adult snRNA-seq data from extra than one cortical areas (https://portal.brain-scheme.org/atlases-and-data/rnaseq/human-extra than one-cortical-areas-trim-seq; accessed October 2022), snRNA-seq data from broad temporal coverage from fetal to adulthood stages of the Brodmann standing 8, 9, 10 and 46 prefrontal cortex regions (GEO accession GSE168408) and snRNA-seq from 8-month-passe cortical organoid transplants (GEO accession GSE190815). For single-nucleus prognosis, we passe hg19 human reference genome v1.2.0 and mm10 mouse reference genome v1.2.0 supplied by 10x Genomics. The sequences and gene files passe to originate the references is at possibility of be accomplished at ftp://ftp.ensembl.org/pub/grch37/originate-84/fasta/homo_sapiens/dna/ and ftp://ftp.ensembl.org/pub/grch37/originate-84/gtf/homo_sapiens/ (for human hg19 genome); ftp://ftp.ensembl.org/pub/originate-84/fasta/mus_musculus/dna/ and ftp://ftp.ensembl.org/pub/originate-84/gtf/mus_musculus/ (for mouse mm10 genome). All other raw data passe for plotting within the figures are supplied as source data. Offer data are supplied with this paper.

References

  1. Liddelow, S. A. et al. Neurotoxic reactive astrocytes are triggered by activated microglia. Nature 541, 481–487 (2017).

    Article 
    ADS 
    CAS 
    PubMed 
    PubMed Central 

    Google Student 

  2. Qian, X., Music, H. & Ming, G. L. Mind organoids: advances, applications and challenges. Construction 146, dev166074 (2019).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Student 

  3. Wang, M., Zhang, L. & Gage, F. H. Modeling neuropsychiatric disorders utilizing human triggered pluripotent stem cells. Protein Cell 11, forty five–59 (2020).

    Article 
    CAS 
    PubMed 

    Google Student 

  4. Lancaster, M. A. et al. Cerebral organoids mannequin human brain construction and microcephaly. Nature 501, 373–379 (2013).

    Article 
    ADS 
    CAS 
    PubMed 

    Google Student 

  5. Kim, J., Koo, B. Good enough. & Knoblich, J. A. Human organoids: mannequin programs for human biology and treatment. Nat. Rev. Mol. Cell Biol. 21, 571–584 (2020).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Student 

  6. Velasco, S. et al. Particular particular person brain organoids reproducibly invent cell diversity of the human cerebral cortex. Nature 570, 523–527 (2019).

    Article 
    ADS 
    CAS 
    PubMed 
    PubMed Central 

    Google Student 

  7. Sloan, S. A. et al. Human astrocyte maturation captured in 3D cerebral cortical spheroids derived from pluripotent stem cells. Neuron 95, 779–790 (2017).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Student 

  8. Qian, X. et al. Sliced human cortical organoids for modeling determined cortical layer formation. Cell Stem Cell 26, 766–781 (2020).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Student 

  9. Molofsky, A. V. et al. Astrocytes and illness: a neurodevelopmental perspective. Genes Dev. 26, 891–907 (2012).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Student 

  10. Tchieu, J. et al. NFIA is a gliogenic switch enabling instant derivation of helpful human astrocytes from pluripotent stem cells. Nat. Biotechnol. 37, 267–275 (2019).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Student 

  11. Canals, I. et al. Rapidly and ambiance pleasant induction of helpful astrocytes from human pluripotent stem cells. Nat. Programs 15, 693–696 (2018).

    Article 
    CAS 
    PubMed 

    Google Student 

  12. Santos, R. et al. Differentiation of inflammation-responsive astrocytes from glial progenitors generated from human triggered pluripotent stem cells. Stem Cell Experiences 8, 1757–1769 (2017).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Student 

  13. Barbar, L. et al. CD49f is a singular marker of helpful and reactive human iPSC-derived astrocytes. Neuron 107, 436–453 (2020).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Student 

  14. Zhang, J. & Liu, Q. Cholesterol metabolism and homeostasis within the brain. Protein Cell 6, 254–264 (2015).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Student 

  15. Daneman, R. & Prat, A. The blood-brain barrier. Cold Spring Harb. Perspect. Biol. 7, a020412 (2015).

    Article 
    PubMed 
    PubMed Central 

    Google Student 

  16. Mansour, A. A. et al. An in vivo mannequin of helpful and vascularized human brain organoids. Nat. Biotechnol. 36, 432–441 (2018).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Student 

  17. Bao, Z. et al. Human cerebral organoid implantation alleviated the neurological deficits of irritating brain harm in mice. Oxid. Med. Cell. Longev. 2021, 6338722 (2021).

    Article 
    PubMed 
    PubMed Central 

    Google Student 

  18. Daviaud, N., Friedel, R. H. & Zou, H. Vascularization and engraftment of transplanted human cerebral organoids in mouse cortex. eNeuro 5, ENEURO.0219-18.2018 (2018).

    Article 
    PubMed 
    PubMed Central 

    Google Student 

  19. Kitahara, T. et al. Axonal extensions along corticospinal tracts from transplanted human cerebral organoids. Stem Cell Experiences 15, 467–481 (2020).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Student 

  20. Shi, Y. et al. Vascularized human cortical organoids (vOrganoids) mannequin cortical construction in vivo. PLoS Biol. 18, e3000705 (2020).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Student 

  21. Revah, O. et al. Maturation and circuit integration of transplanted human cortical organoids. Nature 610, 319–326 (2022).

    Article 
    ADS 
    CAS 
    PubMed 
    PubMed Central 

    Google Student 

  22. Jgamadze, D. et al. Structural and helpful integration of human forebrain organoids with the injured adult rat visible scheme. Cell Stem Cell 30, 137–152 (2023).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Student 

  23. Qian, X. et al. Generation of human brain situation-notify organoids utilizing a miniaturized spinning bioreactor. Nat. Protoc. 13, 565–580 (2018).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Student 

  24. Qian, X. et al. Mind-situation-notify organoids utilizing mini-bioreactors for modeling ZIKV publicity. Cell 165, 1238–1254 (2016).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Student 

  25. Langfelder, P. & Horvath, S. WGCNA: an R equipment for weighted correlation network prognosis. BMC Bioinformatics 9, 559 (2008).

    Article 
    PubMed 
    PubMed Central 

    Google Student 

  26. Morabito, S. et al. Single-nucleus chromatin accessibility and transcriptomic characterization of Alzheimer’s illness. Nat. Genet. Fifty three, 1143–1155 (2021).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Student 

  27. Allen, N. J. & Eroglu, C. Cell biology of astrocyte-synapse interactions. Neuron 96, 697–708 (2017).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Student 

  28. Stogsdill, J. A. et al. Astrocytic neuroligins management astrocyte morphogenesis and synaptogenesis. Nature 551, 192–197 (2017).

    Article 
    ADS 
    CAS 
    PubMed 
    PubMed Central 

    Google Student 

  29. Oberheim, N. A. et al. Uniquely hominid substances of adult human astrocytes. J. Neurosci. 29, 3276–3287 (2009).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Student 

  30. Zhang, Y. et al. Purification and characterization of progenitor and ragged human astrocytes unearths transcriptional and helpful differences with mouse. Neuron 89, 37–Fifty three (2016).

    Article 
    CAS 
    PubMed 

    Google Student 

  31. Oberheim, N. A., Wang, X., Goldman, S. & Nedergaard, M. Astrocytic complexity distinguishes the human brain. Traits Neurosci. 29, 547–553 (2006).

    Article 
    CAS 
    PubMed 

    Google Student 

  32. Merritt, C. R. et al. Multiplex digital spatial profiling of proteins and RNA in fixed tissue. Nat. Biotechnol. 38, 586–599 (2020).

    Article 
    CAS 
    PubMed 

    Google Student 

  33. Li, Y. et al. Advise labeling and visualization of blood vessels with lipophilic carbocyanine dye DiI. Nat. Protoc. 3, 1703–1708 (2008).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Student 

  34. Herring, C. A. et al. Human prefrontal cortex gene regulatory dynamics from gestation to adulthood at single-cell decision. Cell 185, 4428–4447 (2022).

    Article 
    CAS 
    PubMed 

    Google Student 

  35. Cao, J. et al. Primarily the most titillating-cell transcriptional panorama of mammalian organogenesis. Nature 566, 496–502 (2019).

    Article 
    ADS 
    CAS 
    PubMed 
    PubMed Central 

    Google Student 

  36. Sofroniew, M. V. & Vinters, H. V. Astrocytes: biology and pathology. Acta Neuropathol. 119, 7–35 (2010).

    Article 
    PubMed 

    Google Student 

  37. Zamanian, J. L. et al. Genomic prognosis of reactive astrogliosis. J. Neurosci. 32, 6391–6410 (2012).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Student 

  38. Anderson, M. A. et al. Astrocyte scar formation aids central anxious scheme axon regeneration. Nature 532, 195–200 (2016).

    Article 
    ADS 
    CAS 
    PubMed 
    PubMed Central 

    Google Student 

  39. Tarrago, M. G. et al. A potent and notify CD38 inhibitor ameliorates age-associated metabolic dysfunction by reversing tissue NAD+ decline. Cell Metab. 27, 1081–1095 e1010 (2018).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Student 

  40. Sprenger, H. G. & Langer, T. The nice and the harmful of mitochondrial breakups. Traits Cell Biol. 29, 888–900 (2019).

    Article 
    CAS 
    PubMed 

    Google Student 

  41. Krencik, R. & Zhang, S. C. Directed differentiation of helpful astroglial subtypes from human pluripotent stem cells. Nat. Protoc. 6, 1710–1717 (2011).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Student 

  42. Palm, T. et al. Rapidly and tough technology of lengthy-term self-renewing human neural stem cells with the skill to generate ragged astroglia. Sci. Win. 5, 16321 (2015).

    Article 
    ADS 
    CAS 
    PubMed 
    PubMed Central 

    Google Student 

  43. Han, X. et al. Forebrain engraftment by human glial progenitor cells enhances synaptic plasticity and learning in adult mice. Cell Stem Cell 12, 342–353 (2013).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Student 

  44. Windrem, M. S. et al. A aggressive advantage by neonatally engrafted human glial progenitors yields mice whose brains are chimeric for human glia. J. Neurosci. 34, 16153–16161 (2014).

    Article 
    PubMed 
    PubMed Central 

    Google Student 

  45. Mariani, J. N., Zou, L. & Goldman, S. A. Human glial chimeric mice to account for the aim of glial pathology in human illness. Programs Mol. Biol. 1936, 311–331 (2019).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Student 

  46. Zeisel, A. et al. Mind construction. Cell types within the mouse cortex and hippocampus printed by single-cell RNA-seq. Science 347, 1138–1142 (2015).

    Article 
    ADS 
    CAS 
    PubMed 

    Google Student 

  47. Bayraktar, O. A. et al. Astrocyte layers within the mammalian cerebral cortex printed by a single-cell in situ transcriptomic scheme. Nat. Neurosci. 23, 500–509 (2020).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Student 

  48. Hodge, R. D. et al. Conserved cell types with divergent substances in human versus mouse cortex. Nature 573, 61–68 (2019).

    Article 
    ADS 
    CAS 
    PubMed 
    PubMed Central 

    Google Student 

  49. Jorstad, N. L. et al. Transcriptomic cytoarchitecture unearths suggestions of human neocortex organization. Science 382, eadf6812 (2023).

    Article 
    CAS 
    PubMed 

    Google Student 

  50. Burda, J. E. et al. Divergent transcriptional legislation of astrocyte reactivity all over disorders. Nature 606, 557–564 (2022).

    Article 
    ADS 
    CAS 
    PubMed 
    PubMed Central 

    Google Student 

  51. Cowan, C. A. et al. Derivation of embryonic stem-cell lines from human blastocysts. Fresh Engl. J. Med. 350, 1353–1356 (2004).

    Article 
    CAS 
    PubMed 

    Google Student 

  52. Thomson, J. A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998).

    Article 
    ADS 
    CAS 
    PubMed 

    Google Student 

  53. Mertens, J. et al. Differential responses to lithium in hyperexcitable neurons from patients with bipolar disorder. Nature 527, 95–Ninety nine (2015).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Student 

  54. Goncalves, J. T. et al. In vivo imaging of dendritic pruning in dentate granule cells. Nat. Neurosci. 19, 788–791 (2016).

    Article 
    MathSciNet 
    CAS 
    PubMed 
    PubMed Central 

    Google Student 

  55. Longair, M. H., Baker, D. A. & Armstrong, J. D. Simple Neurite Tracer: originate source scheme for reconstruction, visualization and prognosis of neuronal processes. Bioinformatics 27, 2453–2454 (2011).

    Article 
    CAS 
    PubMed 

    Google Student 

  56. Kuwajima, M., Mendenhall, J. M. & Harris, Good enough. M. Huge-quantity reconstruction of brain tissue from high-decision serial portion photos received by SEM-primarily based scanning transmission electron microscopy. Programs Mol. Biol. 950, 253–273 (2013).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Student 

  57. Deerinck, T. J., Bushong, E. A., Thor, A. & Ellisman, M. H. NCMIR programs for 3D EM: a brand unique protocol for preparation of biological specimens for serial block face scanning electron microscopy. Microscopy 1, 6–8 (2010).

    Google Student 

  58. Horstmann, H., Korber, C., Satzler, Good enough., Aydin, D. & Kuner, T. Serial portion scanning electron microscopy (S3EM) on silicon wafers for ultra-structural quantity imaging of cells and tissues. PLoS ONE 7, e35172 (2012).

    Article 
    ADS 
    CAS 
    PubMed 
    PubMed Central 

    Google Student 

  59. Hao, Y. et al. Integrated prognosis of multimodal single-cell data. Cell 184, 3573–3587 e3529 (2021).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Student 

  60. Conway, J. R., Lex, A. & Gehlenborg, N. UpSetR: an R equipment for the visualization of intersecting devices and their properties. Bioinformatics 33, 2938–2940 (2017).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Student 

  61. Kuleshov, M. V. et al. Enrichr: a comprehensive gene space enrichment prognosis web server 2016 update. Nucleic Acids Res. 44, W90–W97 (2016).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Student 

  62. Liao, Y., Wang, J., Jaehnig, E. J., Shi, Z. & Zhang, B. WebGestalt 2019: gene space prognosis toolkit with revamped Usaand APIs. Nucleic Acids Res. 47, W199–W205 (2019).

    Article 
    CAS 
    PubMed 
    PubMed Central 

    Google Student 

Gather references

Acknowledgements

We thank A. Mansour (Salk Institute) for offering the pCSC-CAG-GFP lentiviral labeled H9 ESC line, N. Hah for technical assistance with snRNA-seq and R. Garg for assistance with EM segmentation. We also thank M.L. Gage for editorial comments and A. Cao and T. Bartol for their aid with 3DEM visualization. This work became once supported by the American Heart Affiliation and the Paul G. Allen Frontiers Crew Grant (19PABHI34610000/TEAM LEADER: Fred H. Gage/2019), JPB Basis, Annette C. Merle-Smith, Lynn and Edward Streim, the Milky Contrivance Basis, 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 became once also supported by the NGS Core Facility, the GT3 Core Facility, the Razavi Newman Integrative Genomics and Bioinformatics Core Facility and the Waitt Evolved Biophotonics Core Facility of the Salk Institute with funding from NIH–NCI (CCSG, P30 014195), the Chapman Basis, NINDS R24 Core Grant, NEI and the Waitt Basis. M.W. is supported by a Younger Investigator Grant from the Mind & Habits Compare Basis (BBRFNARSAD) and a Pioneer Fund Postdoctoral Student Award from the Salk Institute. Figures 2a, 2h, 4a, 5a, 6a and 6d were created with BioRender.com.

Author data

Author notes

  1. Uri Manor

    Expose take care of: Division of Biological Sciences, College of California, San Diego, La Jolla, CA, USA

  2. These authors contributed equally: Meiyan Wang, Lei Zhang.

Authors and Affiliations

  1. Laboratory of Genetics, The Salk Institute for Biological Compare, La Jolla, CA, USA

    Meiyan Wang, Lei Zhang, Iryna S. Gallina, Lynne L. Xu, Christina Good enough. Lim, Sarah Fernandes, Monisha D. Saxena, Shashank Coorapati, Sarah L. Parylak & Fred H. Gage

  2. Waitt Evolved Biophotonics Core, The Salk Institute for Biological Compare, La Jolla, CA, USA

    Sammy Weiser Novak, Leonardo R. Andrade & Uri Manor

  3. Integrative Genomics and Bioinformatics Core, The Salk Institute for Biological Compare, La Jolla, CA, USA

    Jingting Yu, Maxim N. Shokhirev & April E. Williams

  4. Division of Biological Sciences, College of California, San Diego, La Jolla, CA, USA

    Christina Good enough. Lim & Sarah Fernandes

  5. Subsequent Generation Sequencing Core, The Salk Institute for Biological Compare, La Jolla, CA, USA

    Cristian Quintero & Elsa Molina

Contributions

M.W., L.Z. and F.H.G. conceived the gaze and wrote the paper. M.W., L.Z., C.Good enough.L. and S.F. conducted cell tradition and organoid differentiation. M.W., L.Z., C.Good enough.L. and I.S.G. conducted surgeries. S.L.P. assisted in surgeries. M.W. and I.S.G. conducted snRNA-seq and scRNA-seq. M.W., C.Q. and E.M. conducted NanoString GeoMx DSP. M.W., M.N.S., J.Y. and A.E.W. conducted bioinformatics analyses. M.W. and L.Z. conducted imaging prognosis with assistance from L.L.X., C.Good enough.L., S.C. and M.D.S. L.Z., S.W.N. and L.R.A. conducted sample processing for SEM. S.W.N. and L.R.A. conducted electron cramped image prognosis below the supervision of M.W., L.Z. and U.M. F.H.G. supplied funding.

Corresponding creator

Correspondence to
Fred H. Gage.

Ethics declarations

Competing pursuits

The authors boom no competing pursuits.

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Nature Biotechnology thanks the anonymous reviewers for their contribution to the ask overview of this work.

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Prolonged data

Prolonged Files Fig. 1 Rapidly astrogliogenesis in glia-enriched cortical organoids.

a. Pattern shiny-discipline photos of hiPSCs/hESCs colonies and glia-enriched cortical organoids at days 0, 14, 30 and 60. Day 0 refers to embryoid our bodies (EB). Scale bars, 890 μm. b. Quantitative PCR prognosis of the expression ranges of NFIA and SOX2 in three-week-passe organoids cultured below varying 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). Every dot represents a pool of three organoids. Bars, mean ± s.d. Two-sided t-test, ns, no longer vital, *p < 0.05, ***p < 0.001, ****p < 0.0001. c. Immunostaining of two-month-passe glia-enriched cortical organoids: stem cells (SOX2, magenta), astrocytes (GFAP, green). Scale bar, 100 μm. d. Immunostaining of two-month-passe glia-enriched cortical organoids: intermediate progenitor cells (EOMES, green), cortical excitatory neurons (CTIP2, magenta; SATB2, grey), stem cells (SOX2, green), neurons (NeuN, magenta), astrocytes (GFAP, grey), glia (HOPX, green; S100B, magenta; GFAP, grey). Scale bars, 20 μm. e. Immunostaining of three-month-passe glia-enriched cortical organoids differentiated in 2% FBS (top left panel), 2% SATO (top graceful panel; serum-free condition, SATO factor reported in ref. 30), 10% FBS (backside left panel) and 10% SATO (backside graceful panel). Astrocytes (GFAP, green). Scale bars, 100 μm. f. Quantification of the GFAP fluorescence depth in glia-enriched cortical organoids differentiated 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). Every dot represents one organoid. Bars, mean ± s.e.m. Two-sided t-test, ***p < 0.001, ****p < 0.0001.

Offer data

Prolonged Files Fig. 2 snRNA-seq of 10-week-passe glia-enriched cortical organoids.

a. UMAP put of snRNA-seq data from 10-week-passe glia-enriched cortical organoids. The proportion of each and every fundamental cell types is confirmed. APC, astrocyte progenitor cell; Ast, astrocyte; IPC, intermediate progenitor cell; In, inhibitory neuron; Cortical Ex, cortical excitatory neuron; Ex1 and Ex2, excitatory neuron. b. Expression of selected marker genes passe in cell form identification. The violin put reveals the distribution of normalized expression in nuclei in each and every cluster. Scale: normalized be taught counts. c. Heatmap put reveals the expression of the tip 10 purpose genes known in each and every cluster. d. Dot put reveals the expression of astrocyte gene modules in each and every fundamental cell form. e. Dot put of the enrichR blended ranking for the tip enriched GO phrases for astrocyte gene modules M12 and M14. f. WGCNA dendrogram of Cortical Ex gene modules. g. Dot put reveals the expression of Cortical Ex gene modules in each and every fundamental cell form. h. UMAP plots of module hub gene expression ranking for Cortical Ex gene modules M1–4. i. Co-expression plots of the tip 25 module genes for Cortical Ex gene modules M1–4. j. Dot put of the enrichR blended ranking for the tip enriched GO phrases for cortical excitatory neuron gene modules M1–4.

Prolonged Files Fig. 3 Maturation of astrocytes in glia-enriched cortical organoids.

a. Immunostaining of 5-month-passe glia-enriched cortical organoids: GFAP::GFP AAV-labeled astrocytes (GFP, green), presynaptic vesicles (SV2, grey), postsynaptic density (PSD95, magenta). Inset, an enlarged glimpse of astrocyte task and synapses. Scale bars, 20 μm and a pair of μm (inset). b. Immunostaining of 5-month-passe glia-enriched cortical organoids: GFAP::GFP AAV-labeled astrocytes (GFP, green), glutamate transporter (EAAT2, magenta). Scale bar, 20 μm. c. Immunostaining of 5-month-passe glia-enriched cortical organoids: GFAP::GFP AAV-labeled astrocytes (GFP, green) and matricellular protein (HEVIN, magenta). Scale bar, 5 μm. d. Immunostaining of 5-month-passe 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. Immunostaining of 5-month-passe glia-enriched cortical organoids: astrocytes (hGFAP, green), Connexin 43 (CX43, magenta). Inset, an enlarged glimpse of astrocyte processes and expression of Connexin 43. Scale bars, 20 μm and a pair of μm (inset). f. Quantification of glutamate uptake of neural progenitor cells (NPCs, n = 8) and astrocytes purified from five-month-passe glia-enriched cortical organoids derived from Hues6 (n = 8), iPSC822 (n = 8) and H9 (n = 7) stem cell lines. Every dot represents one honest experiment. Bars, mean ± s.e.m.

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Prolonged Files Fig. 4 Formation of anatomically outlined morphological subclasses of human astrocytes in engrafted glia-enriched cortical organoids.

a. Immunostaining of glia-enriched cortical organoid transplants: human nuclear antigen (HuNu, Cyan), astrocytes (GFAP, green), neurons (NeuN, magenta). Scale bar, 20 μm. b. Quantification of the proportion of NeuN+ HuNu+ cells amongst NeuN+ cells (left) or huGFAP+ GFAP+ cells amongst GFAP+ cells (graceful) within the transplants. n = 4 transplants. Bars, mean ± s.e.m. c. Immunostaining of the guts (left) and border (graceful) of glia-enriched cortical organoid transplants: human astrocytes (hGFAP, green), astrocytes (GFAP, magenta). Scale bars, 50 μm. d. Immunostaining of the guts (left) and border (graceful) of glia-enriched cortical organoid transplants: oligodendrocyte progenitor cells (PDGFRα, green), human nuclear antigen (HuNu, magenta). Inset, enlarged views of human oligodendrocyte progenitor cells. Scale bars, 50 μm and 10 μm (inset). e. Immunostaining of human interlaminar, protoplasmic and fibrous astrocytes in glia-enriched cortical organoid transplants: human astrocytes (hGFAP, green), white subject (myelin standard protein or MBP, grey). Scale bars, 20 μm. f. Immunostaining of glia-enriched cortical organoid transplants: human cells (GFP, green), astrocytes (S100B, grey; CD44, magenta). Scale bar, 100 μm. g. Immunostaining of the tip, heart and backside regions of the transplants: astrocytes (CD44, magenta), white subject (myelin standard protein or MBP, grey). Scale bars, 20 μm.

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Prolonged Files Fig. 5 Spatial transcriptome profiling of layer-notify subclasses of human astrocytes in engrafted glia-enriched cortical organoids.

a. Immunofluorescent staining photos on the left exhibiting hGFAP in yellow, SOX9 in magenta and CYTO-13 in blue, along with the cell segmentation of SOX9+/CYTO-13+ cells depicted in magenta internal a particular ROI on the calm. Scale bars, 100 μm. b. Field plots depicting the normalized expression ranges of selected genes in each and every neighborhood (pial = 10 ROIs, cortex = 16 ROIs, WM = 12 ROIs). Centerline, median; field limits, larger and lower quartiles; whiskers, 1.5× interquartile differ; points, outliers. c. Bubble plots depicting top vital phrases known from GSEA utilizing a weighted Kolmogorov–Smirnov test. NES, normalized enrichment ranking. Significant thresholds space at adjusted p mark < 0.1.

Prolonged Files Fig. 6 End association of human protoplasmic astrocytes with synapses and host vasculature in engrafted glia-enriched cortical organoids.

a. Immunostaining of transplants: human astrocytes (hGFAP, green), glutamate transporters (EAAT2, magenta). Scale bar, 20 μm. b. Immunostaining of transplants: human astrocytes (hGFAP, green), matricellular protein (HEVIN, grey), postsynaptic density (PSD95, magenta). Scale bar, 20 μm. c. Electron micrograph of a multi-synaptic bouton in an eight-month-passe transplant. Arrowheads: synapses. Scale bar, 1 μm. d. 3D reconstruction of serial portion electron microscope photos of synaptic constructions in an eight-month-passe transplant. Presynaptic bouton (orange), postsynaptic density (magenta), dendritic spine (blue). Scale bar, 1 μm. e. Electron micrograph of a synapse internal the transplant. Left, presynaptic bouton (orange), postsynaptic density (PSD, magenta), dendritic spine (blue) and astrocytic processes (AP, green). Just appropriate, fashioned image. Scale bar, 1 μm. f. Immunostaining of transplants at 2 (left panel) or 7 months (graceful panel) post-transplantation: blood vessels (Ly6C, cyan). Scale bars, 100 μm. g. Ly6C+ standing (left) or vessel diameter (graceful) 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, mean ± s.e.m. Two-sided t-test, **p = 0.001, ****p < 0.0001. h. Immunostaining of transplants: human nuclear antigen (HuNu, green), pericytes (NG2, magenta, indicated by arrowhead within the left image), monocytes (Ly6C, grey, indicated by arrowhead within the guts image), endothelial cells (CD31, magenta). Scale bars, 10 μm. i. Immunostaining of transplants (left) or host brains (graceful): human nuclear antigen (HuNu, green), microglia (IBA1, magenta). Inset: microglia. Scale bars, 50 μm and 10 μm (insets). j. Immunostaining of transplants: human astrocytes (hGFAP, green), inward-rectifier potassium channel (Kir4.1, magenta), blood vessels (Ly6C, cyan). Scale bar, 20 μm. k. Immunostaining of transplants: human astrocytes (hGFAP, green), glucose transporter (Glut1, magenta) and blood vessels (Ly6C, cyan). Scale bar, 20 μm. l. Confocal photos of the transplant passe in EM stories: blood vessels (DiI, magenta), transplant (GFP, green). Scale bar, 1 mm. m. 3D reconstruction of series SEM photos: tight junction (yellow) and basement membrane (blue). Scale bar, 1 μm.

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Prolonged Files Fig. 7 Transcriptomic prognosis of built-in snRNA-seq datasets.

a. Heatmap put reveals the expression ranges of the tip 10 purpose genes known in each and every cluster. b. Dot put reveals the usual expression of selected marker genes in nuclei in each and every cluster. c. Heatmap depicting the pairwise transcriptional cluster correlation of cell clusters utilizing top 50 marker genes from each and every cluster in built-in organoid datasets with cell clusters in snRNA-seq datasets of extra than one human cortical areas from Allen Mind Procedure. Immature, immature excitatory neuron; UL, larger layer cortical excitatory neuron; DL, deep-layer cortical excitatory neuron; In, inhibitory neuron; APC, astrocyte progenitor cell; Ast, astrocyte; OPC, oligodendrocyte progenitor cell; unknown, undetermined cell. d. Ridge put reveals the distributions of predicted age for nuclei derived from snRNA-seq data received from prefrontal cortex (PFC) samples starting from 22 weeks of gestation (ga) to 40 years of age (ref. 34). e. UMAP put of snRNA-seq data from PFC samples (ref. 34) coloured by fundamental cell types. f. UMAP put of snRNA-seq data from PFC samples (ref. 34) with each and every nucleus coloured by its donor age. g. UMAP plots of snRNA-seq data from 5-month-passe organoids (5m_Org), 5-month-passe transplants (5m_T), 6-month-passe transplants (6m_T) and 8-month-passe transplants (8m_T), projected on to the reference dataset, with each and every nucleus coloured by the expected age. h. Ridge put reveals the distributions of predicted age for UL nuclei from 5m_Org, 5m_T, 6m_T and 8m_T. Wilcoxon test (two-sided, ****, p < 0.0001; reference neighborhood, 5m_Org).

Prolonged Files Fig. 8 WGCNA and pseudotime analyses of built-in snRNA-seq datasets.

a. WGCNA dendrogram of gene modules comprised of astrocytes all over numerous time points. b. Dot put reveals the expression ranges of astrocyte gene modules all over fundamental cell form. c. WGCNA dendrogram of gene modules comprised of immature and UL excitatory neurons all over numerous time points. d. Dot put reveals the expression ranges of neuron gene modules in each and every fundamental cell form. e. Co-expression put of the tip 25 hub genes (left) and UMAP put (graceful) of module hub gene expression ranking for neuron gene modules M1. f. Violin put of harmonized module ranking of neuron gene module M1 in UL excitatory neurons all over numerous time points. Centerline, median; field limits, larger and lower quartiles; whiskers, 1.5× interquartile differ; points, outliers. Wilcoxon test (two-sided, ****, p < 0.0001; reference neighborhood, 5m_Org). g. UMAP dimensionality discount displays the pseudotime trajectories of neurons from the built-in snRNA-seq. Every cell is coloured by its pseudotime trajectory task. Pseudotime prognosis separated by time point. One-sided Kolmogorov–Smirnov test (****, p < 2.2e-16; reference neighborhood, 5m_Org).

Prolonged Files Fig. 9 Transcriptome profiling unearths instant activation of pro-inflammatory pathways in a subpopulation of astrocytes in vivo.

a. Drift cytometry sorting technique for glial cells. b. UMAP put (left) and violin put (graceful) reveals the expression of selected cell form marker genes. c. UMAP plots prove the expression of selected genes. d. Violin put reveals the expression of selected genes in astrocyte clusters 1–4. Ast, astrocyte. e. Bar put of the enrichR blended ranking for gene ontology phrases of the tip 100 very much upregulated (top) and downregulated (backside) genes following TNFα therapy in cluster 2 astrocytes. f. GSEA evaluating TNFα-treated and saline-treated cluster 2 astrocytes. GO phrases are confirmed. FDR, wrong discovery rate; NES, normalized enrichment ranking.

Prolonged Files Fig. 10 CD38 mediates inflammation-triggered metabolic and mitochondrial stresses in human astrocytes.

a. Immunostaining of in vitro glia-enriched cortical organoids at day 0, day 1 and day 2 post-TNFα therapy: astrocytes (hCD38, green; CD44 cyan), chemokines (CXCL10, magenta). Scale bars, 50 μm. b. Quantitative PCR analyses of selected genes in organoids (CTRL = 4; TNFα1d = 4; TNFα2d = 6). Every dot represents one organoid. Bars, mean ± s.d. c. Drift cytometry sorting technique for GFAP::tdTomato+ cells. d. Quantitative PCR analyses of selected genes in sorted astrocytes (CTRL = 3 honest experiments; TNFα1d = 3 honest experiments). Every dot represents one honest experiment. Bars, mean ± s.d. Two-sided t-test, *p = 0.013555, ***p = 0.000948.

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Supplementary data

Supplementary Files

Supplementary Figs. 1 and a pair of.

Reporting Summary

Supplementary Video 1

Three-dimensional EM reconstruction of astrocytic endfeet construction.

Supplementary Tables

Supplementary Table 1: 10-week-passe glia-enriched cortical organoid cell form cluster marker genes. Supplementary Table 2: Co-expression gene modules constructed utilizing astrocytes in 10-week-passe glia-enriched cortical organoids. Supplementary Table 3: Co-expression gene modules constructed utilizing cortical excitatory neurons in 10-week-passe glia-enriched cortical organoids. Supplementary Table 4: Differential gene expression all over layer-notify astrocyte subclasses known by GeoMx DSP. Supplementary Table 5: Differential gene expression all over layer-notify astrocyte subclasses known by GeoMx DSP. Supplementary Table 6: Differential gene expression all over layer-notify astrocyte subclasses known by GeoMx DSP. Supplementary Table 7: Integrated glia-enriched cortical organoid and transplant cell form cluster marker genes. Supplementary Table 8: Co-expression gene modules constructed utilizing astrocytes in built-in glia-enriched cortical organoids and transplants. Supplementary Table 9: Co-expression gene modules constructed utilizing immature and ragged UL cortical excitatory neurons in built-in glia-enriched cortical organoids and transplants. Supplementary Table 10: Cell cluster marker genes from 8-month-passe transplants treated with saline or TNFα. Supplementary Table 11: Primers passe for quantitative PCR assay.

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Wang, M., Zhang, L., Novak, S.W. et al. Morphological diversification and helpful maturation of human astrocytes in glia-enriched cortical organoid transplanted in mouse brain.
Nat Biotechnol (2024). https://doi.org/10.1038/s41587-024-02157-8

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