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Three-dimensional brain-like microenvironments facilitate the direct reprogramming of fibroblasts into therapeutic neurons

Nature Biomedical Engineering volume 2, pages 522–539 (2018)Cite this article

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Abstract

Biophysical cues can improve the direct reprogramming of fibroblasts into neurons that can be used for therapeutic purposes. However, the effects of a three-dimensional (3D) environment on direct neuronal reprogramming remain unexplored. Here, we show that brain extracellular matrix (BEM) decellularized from human brain tissue facilitates the plasmid-transfection-based direct conversion of primary mouse embryonic fibroblasts into induced neuronal (iN) cells. We first show that two-dimensional (2D) surfaces modified with BEM significantly increase the generation efficiency of iN cells and enhance neuronal transdifferentiation and maturation. Moreover, in an animal model of ischaemic stroke, iN cells generated on the BEM substrates and transplanted into the brain led to significant improvements in locomotive behaviours. We also show that compared with the 2D BEM substrates, 3D BEM hydrogels recapitulating brain-like microenvironments further promote neuronal conversion and potentiate the functional recovery of the animals. Our findings suggest that 3D microenvironments can boost nonviral direct reprogramming for the generation of therapeutic neuronal cells.

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Fig. 1: Preparation of human decellularized BEM and characterization of BEM-based 2D coatings and 3D hydrogels.
Fig. 2: Nonviral direct reprogramming of mouse fibroblasts into iN cells on human decellularized BEM.
Fig. 3: Functional maturation of iN cells on decellularized BEM at day 30.
Fig. 4: Transplantation of iN cells generated on BEM improves functional recovery of hypoxic–ischaemic brain injured mice.
Fig. 5: 3D BEM boosts nonviral direct neuronal reprogramming.
Fig. 6: The effect of contractility inhibition on YAP translocation and direct neuronal reprogramming in 2D and 3D conditions.
Fig. 7: Improvement of direct neuronal reprogramming by 3D brain-like microenvironments.
Fig. 8: Proposed mechanistic model of 3D BEM-induced direct neuronal reprogramming of PMEFs.

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Acknowledgements

This work was supported by grants (2018M3A9H1021382, 2017R1A2B3005994 and 2014R1A2A11052042) from the National Research Foundation (NRF) of Korea funded by the Ministry of Science and ICT (MSIT), Republic of Korea. This work was supported by the Institute for Basic Science (IBS-R026-D1). It was also supported in part by a grant (HI14C1588) from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI) funded by the Ministry of Health and Welfare, Republic of Korea.

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  1. These authors contributed equally: Yoonhee Jin, Jung Seung Lee.

Authors and Affiliations

  1. Department of Biotechnology, Yonsei University, Seoul, Republic of Korea

    Yoonhee Jin, Jung Seung Lee, Jin Kim, Sungjin Min, Gyeong-Eon Chang, Ann-Na Cho, Eunji Cheong & Seung-Woo Cho

  2. Department and Research Institute of Rehabilitation Medicine, Yonsei University College of Medicine, Seoul, Republic of Korea

    Soohyun Wi, Ji Hea Yu & Sung-Rae Cho

  3. Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, Republic of Korea

    Soohyun Wi, Yeeun Choi, Sung-Rae Cho & Hyong-Pyo Kim

  4. Department of Environmental Medical Biology, Yonsei University College of Medicine, Seoul, Republic of Korea

    Yeeun Choi & Hyong-Pyo Kim

  5. Department of Chemical Engineering, Soongsil University, Seoul, Republic of Korea

    Da-Hee Ahn & Yun-Gon Kim

  6. Division of Biological Sciences, Sookmyung Women’s University, Seoul, Republic of Korea

    Yonghwan Kim

  7. Division of Pediatric Neurosurgery, Severance Children’s Hospital, Department of Neurosurgery, Yonsei University College of Medicine, Seoul, Republic of Korea

    Dong Seok Kim

  8. Division of Pediatric Neurology, Department of Pediatrics, Yonsei University College of Medicine, Seoul, Republic of Korea

    Hyun Woo Kim, Zhejiu Quan & Hoon-Chul Kang

  9. Center for Nanomedicine, Institute for Basic Science (IBS), Seoul, Republic of Korea

    Seung-Woo Cho

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Contributions

Y.J. and J.S.L. contributed equally to this work. Y.J. and J.S.L. designed, led the experiments, performed data analysis and wrote the paper. J.K. fabricated the microfluidic devices and S.M. supported cell culture work and data analyses. S.W., J.H.Y., and S.-R.C. performed the animal experiments and advised on the data analyses. G.-E.C. and E.C. supported the electrophysiological evaluation of cells and data analyses. A.-N.C. cultured and provided the human stem cells. D.-H.A. and Y.-G.K. helped with the proteomic analyses of BEM. Y.C. and H.-P.K. conducted the ChIP assay and advised on the results. Y.K. provided materials for transfection. D.S.K. H.W.K. and Z.Q. helped with the human brain tissue preparation for the fabrication of the BEM. H.-C.K. and S.W.C. designed and supervised the experiments, and wrote the paper.

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Correspondence to Hoon-Chul Kang or Seung-Woo Cho.

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Supplementary methods, references, figures, tables and video captions.

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Supplementary Video 1

Real-time calcium imaging of Fluo-4-AM-treated induced neuronal cells, cultured on a brain extracellular matrix substrate, during glutamate stimulation at day 30.

Supplementary Video 2

Tuj1 and MAP2 co-immunostaining of induced neuronal cells, cultured in 3D brain extracellular matrix within a microfluidic channel, on day 14.

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Jin, Y., Lee, J.S., Kim, J. et al. Three-dimensional brain-like microenvironments facilitate the direct reprogramming of fibroblasts into therapeutic neurons. Nat Biomed Eng 2, 522–539 (2018). https://doi.org/10.1038/s41551-018-0260-8

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