Injury and therapy in a human spinal cord organoid

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Injury and therapy in a human spinal cord organoid

Data availability

The materials used in this study are available upon request. The scRNA-seq, omics data will be deposited under GEO accession numbers GSE267252 and GSE267067. The bulk RNA-seq, omics data will be deposited under GEO accession number GSE297701. Source data are provided with this paper.

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Acknowledgements

Research funding was provided by the Center for Regenerative Nanomedicine at Northwestern University and a gift from the John Potocsnak Family for spinal cord injury research. Peptide amphiphile synthesis was performed at the Peptide Synthesis Core Facility of the Center for Regenerative Nanomedicine at Northwestern University. The Peptide Synthesis Core is supported from the Soft and Hybrid Nanotechnology Experimental (SHyNE) Resource (NSF ECCS-2025633). We are grateful to M. Karver, S. Biswas, E. Testa, J. Grzybek and T. Lusis for their assistance and advice on peptide synthesis. Analytical bioNanoTechnology Equipment Core (ANTEC) (RRID:SCR_023706) Facility of the Center for Regenerative Nanomedicine at Northwestern University is supported by the SHyNE Resource. We thank A. Kolot, H. Sai and K. Lee for general instrumentation service, and M. Seniw for the preparation of scientific illustrations at ANTEC. We thank X. Wang for service in bulk and single-cell RNA sequencing work supported by the Northwestern University NUSeq Core Facility. We thank C. Arvanitis, W. Liu and F. Korobova for the assistance in imaging work performed at the university core facility, Center for Advanced Microscopy generously supported by NCI CCSG P30 CA060553 awarded to the Robert H. Lurie Comprehensive Cancer Center. This work made use of the EPIC facility of Northwestern University’s NUANCE Center, which has received support from the SHyNE Resource (NSF ECCS-2025633), the IIN and Northwestern’s MRSEC programme (NSF DMR-2308691). We acknowledge the Robert H. Lurie Comprehensive Cancer Center of Northwestern University for the use of the Flow Cytometry Core Facility. The Lurie Cancer Center is supported in part by an NCI Cancer Center Support Grant number P30 CA060553. We appreciate G. Oliver (Northwestern University) for research instruments. We thank H. Sakaguchi (RIKEN), E. Kiskinis (Northwestern University), J. A. Ortega (University of Barcelona), Z. Alvarez (University of Barcelona) and J. Miska (Northwestern University) for technical advice and discussion. We would like to thank Northwestern undergraduate researchers, R. Gil, N. Coppisetti, T. Yang, J. McCubbin, N. Bruha and E. Lee for helping with stem cell organoid engineering projects.

Author information

Authors and Affiliations

  1. Center for Regenerative Nanomedicine, Northwestern University, Chicago, IL, USA

    Nozomu Takata, Zhiwei Li, Feng Chen, Nicholas A. Sather, Oscar A. Carballo-Molina, Cassandre Jamroz, Cara S. Smith, Yang Yang, Suitu Wang, Liam C. Palmer & Samuel I. Stupp

  2. Department of Medicine, Northwestern University, Chicago, IL, USA

    Nozomu Takata & Samuel I. Stupp

  3. Department of Biomedical Engineering, McCormick School of Engineering, Northwestern University, Evanston, IL, USA

    Anna Metlushko, Madison E. Strong, Cara S. Smith & Samuel I. Stupp

  4. Department of Chemistry, Northwestern University, Evanston, IL, USA

    Xinyi Lin, Liam C. Palmer & Samuel I. Stupp

  5. Center for Genetic Medicine, Northwestern University, Chicago, IL, USA

    Matthew J. Schipma, Ching M. Wai & Neha Joshi

  6. Medical Scientist Training Program, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA

    Jack Kolberg-Edelbrock

  7. Department of Materials Science and Engineering, Northwestern University, Evanston, IL, USA

    Jack Kolberg-Edelbrock, Kyle J. Gray & Samuel I. Stupp

Authors

  1. Nozomu Takata
  2. Zhiwei Li
  3. Anna Metlushko
  4. Feng Chen
  5. Nicholas A. Sather
  6. Xinyi Lin
  7. Matthew J. Schipma
  8. Oscar A. Carballo-Molina
  9. Cassandre Jamroz
  10. Madison E. Strong
  11. Cara S. Smith
  12. Yang Yang
  13. Ching M. Wai
  14. Neha Joshi
  15. Jack Kolberg-Edelbrock
  16. Kyle J. Gray
  17. Suitu Wang
  18. Liam C. Palmer
  19. Samuel I. Stupp

Contributions

S.I.S. supervised the project and secured funding for the research. N.T. and S.I.S. designed the study. N.T., L.C.P. and S.I.S. wrote the paper. N.A.S. and L.C.P. provided critical intellectual and technical advice. N.T. performed most of the experiments. N.T. and Z.L. performed the microglia and immune-competent organoid culture. N.T., A.M., Z.L., F.C., C.J. and M.E.S. performed the human pluripotent stem cell culture and spinal cord organoid production. N.T., A.M. and X.L. performed the PA preparation and evaluation. N.T. performed the PA treatment in healthy and injured spinal cord organoids. N.T., Z.L., F.C., C.J., M.E.S. and S.W. performed the immunohistological analysis. N.A.S. and K.J.G. performed the SEM preparation and imaging. N.T., C.S.S., F.C. O.A.C.-M. and J.K.-E. prepared the device materials and performed the contusion injury experiments. N.T. and Y.Y. generated the polar histogram. N.T., A.M. and M.E.S. purified the RNA and performed the qPCR analysis. N.T. and C.M.W. performed the preparation for RNA-seq and scRNA-seq. N.T., M.J.S. and N.J. performed the bioinformatic analysis and assisted with results interpretation. All authors helped to edit and approved the final version of the paper.

Corresponding author

Correspondence to Samuel I. Stupp.

Ethics declarations

Competing interests

N.T., N.A.S., Z.L. and S.I.S. are co-inventors on a patent application related to this work filed by Northwestern University (US patent application number 18/735,858, filed on 6 June 2024). The other authors declare no competing interests.

Peer review

Peer review information

Nature Biomedical Engineering thanks Shi Yan Ng and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Additional information

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Extended data

Extended Data Fig. 1 Inflammatory response after contusion injury of spinal cord organoids and addition of microglia.

(a) Schematic illustration of microglia assembly pathway 2. (b) Representative bright-field micrographs of mature microglia. (c) Schematic illustration of a protocol for the assembly 2. Mature microglia were assembled in freshly injured organoids (24-week) in the spinal cord maturation medium supplemented with IL-34 and mCSF. The organoids in 96 wells were then agitated at 55 rpm (revolutions per minute) using orbital shaking for 2 days before transder to 24 well dish. (d) Representative fluorescence micrographs of CSPG (green) and IBA-1 (magenta) and DAPI (blue). The square indicates high magnification image. (e) Schematic illustration showing the regions outlined by the red or black square, which indicates injured (inj.) or uninjured (uninj.) areas, respectively. Representative fluorescence micrographs of IBA1 (red) and GFAP (green). (f) Representative fluorescence micrographs of TUJ-1 (green) and DAPI (blue) in conditions with E2 PA or IKVAV PA2 (DAPI, blue). (g) Bar graphs of relative TUJ-1 intensity compared to uninjured area. Data are presented as mean values +/- s.e.m. of independently repeated experiments (n = 3; two-sided unpaired t-test). (h) Fluorescence micrographs of neurofilament (yellow) specific for axon represented in injured organoid surface treated with E2 PA or IKVAV PA2 (DAPI, blue). (i) Representative fluorescence micrographs of CSPG (red) treated with E2 PA or IKVAV PA2 (DAPI, blue). (j) Bar graph of relative CSPG intensity in injured versus uninjured areas. Data are presented as mean values +/- s.e.m. of independently repeated experiments (n = 3; two-sided unpaired t-test). Scale bars: 200 μm (d, f, h), 100 μm (b, e, i).

Source data

Supplementary information

Source data

Source Data Figs. 1–5, Extended Data Fig. 1

Figure 1. (g, o) Unprocessed absorbance intensities. Figure 2. (e) Intensities from fluorescence microscopy (‘w’ represents the condition after washing with culture medium); (o) average lengths of neurites measured by fluorescence microscopy; (p) average radial distribution of neurites relative to normal of organoid surface. Figure 3. (c, n) Average fluorescence intensities in injured regions relative to uninjured regions; (e, p) average lengths of neurites measured by fluorescence microscopy. Figure 4. (i) % of CD11b+/CD45+ cells measured by fluorescence-activated cell sorting; (j, k) normalized expression of C1QA and C3 relative to housekeeping genes; (m) average lengths of neurites measured by fluorescence microscopy; (q, s) average fluorescence intensities in injured regions relative to uninjured regions. Figure 5. Differential expression of genes detected by RNA sequencing. Extended Data Fig. 1. (g, j) Average fluorescence intensities in injured regions relative to uninjured regions.

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Takata, N., Li, Z., Metlushko, A. et al. Injury and therapy in a human spinal cord organoid. Nat. Biomed. Eng (2026). https://doi.org/10.1038/s41551-025-01606-2

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