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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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.
The authors declare no competing interests.
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Supplementary methods, references, figures, tables and video captions.
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.
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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