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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Xu, J., Du, Y. & Deng, H. Direct lineage reprogramming: strategies, mechanisms, and applications. Cell Stem Cell 16, 119–134 (2015).
Sancho-Martinez, I., Baek, S. H. & Izpisua Belmonte, J. C. Lineage conversion methodologies meet the reprogramming toolbox. Nat. Cell Biol. 14, 892–899 (2012).
Heinrich, C., Spagnoli, F. M. & Berninger, B. In vivo reprogramming for tissue repair. Nat. Cell Biol. 17, 204–211 (2015).
Vierbuchen, T. et al. Direct conversion of fibroblasts to functional neurons by defined factors. Nature 463, 1035–1041 (2010).
Caiazzo, M. et al. Direct conversion of fibroblasts into functional astrocytes by defined transcription factors. Stem Cell Rep. 4, 25–36 (2015).
Kim, J. et al. Direct reprogramming of mouse fibroblasts to neural progenitors. Proc. Natl Acad. Sci. USA 108, 7838–7843 (2011).
Kim, J. et al. Functional integration of dopaminergic neurons directly converted from mouse fibroblasts. Cell Stem Cell 9, 413–419 (2011).
Han, D. W. et al. Direct reprogramming of fibroblasts into neural stem cells by defined factors. Cell Stem Cell 10, 465–472 (2012).
Pfisterer, U. et al. Direct conversion of human fibroblasts to dopaminergic neurons. Proc. Natl Acad. Sci. USA 108, 10343–10348 (2011).
Lujan, E. et al. Direct conversion of mouse fibroblasts to self-renewing, tripotent neural precursor cells. Proc. Natl Acad. Sci. USA 109, 2527–2532 (2012).
Xue, Y. et al. Direct conversion of fibroblasts to neurons by reprogramming PTB-regulated microRNA circuits. Cell 152, 82–96 (2013).
Yoo, A. S. et al. MicroRNA-mediated conversion of human fibroblasts to neurons. Nature 476, 228–231 (2011).
Thier, M. et al. Direct conversion of fibroblasts into stably expandable neural stem cells. Cell Stem Cell 10, 473–479 (2012).
Ambasudhan, R. et al. Direct reprogramming of adult human fibroblasts to functional neurons under defined conditions. Cell Stem Cell 9, 113–118 (2011).
Liu, M.-L. et al. Small molecules enable neurogenin 2 to efficiently convert human fibroblasts into cholinergic neurons. Nat. Commun. 4, 2183 (2013).
Son, E. Y. et al. Conversion of mouse and human fibroblasts into functional spinal motor neurons. Cell Stem Cell 9, 205–218 (2011).
Najm, F. J. et al. Transcription factor-mediated reprogramming of fibroblasts to expandable, myelinogenic oligodendrocyte progenitor cells. Nat. Biotechnol. 31, 426–433 (2013).
Yang, N. et al. Generation of oligodendroglial cells by direct lineage conversion. Nat. Biotechnol. 31, 434–439 (2013).
Li, X. et al. Small-molecule-driven direct reprogramming of mouse fibroblasts into functional neurons. Cell Stem Cell 17, 195–203 (2015).
Hu, W. et al. Direct conversion of normal and Alzheimer’s disease human fibroblasts into neuronal cells by small molecules. Cell Stem Cell 17, 204–212 (2015).
Thomas, C. E., Ehrhardt, A. & Kay, M. A. Progress and problems with the use of viral vectors for gene therapy. Nat. Rev. Genet. 4, 346–358 (2003).
Cesana, D. et al. Uncovering and dissecting the genotoxicity of self-inactivating lentiviral vectors in vivo. Mol. Ther. 22, 774–785 (2014).
Chandler, R. J. et al. Vector design influences hepatic genotoxicity after adeno-associated virus gene therapy. J. Clin. Invest. 125, 870–880 (2015).
Montserrat, N. et al. Simple generation of human induced pluripotent stem cells using poly-β-amino esters as the non-viral gene delivery system. J. Biol. Chem. 286, 12417–12428 (2011).
Narsinh, K. H. et al. Generation of adult human induced pluripotent stem cells using nonviral minicircle DNA vectors. Nat. Protoc. 6, 78–88 (2010).
Gonzalez, F. et al. Generation of mouse-induced pluripotent stem cells by transient expression of a single nonviral polycistronic vector. Proc. Natl Acad. Sci. USA 106, 8918–8922 (2009).
Wang, H., Luo, X. & Leighton, J. Extracellular matrix and integrins in embryonic stem cell differentiation. Biochem. Insights 8, 15–21 (2015).
Rozario, T. & DeSimone, D. W. The extracellular matrix in development and morphogenesis: a dynamic view. Dev. Biol. 341, 126–140 (2010).
Solozobova, V., Wyvekens, N. & Pruszak, J. Lessons from the embryonic neural stem cell niche for neural lineage differentiation of pluripotent stem cells. Stem Cell Rev. 8, 813–829 (2012).
Li, Y., Liu, M., Yan, Y. & Yang, S.-T. Neural differentiation from pluripotent stem cells: the role of natural and synthetic extracellular matrix. World J. Stem Cells 6, 11–23 (2014).
Sun, Y. et al. Hippo/YAP-mediated rigidity-dependent motor neuron differentiation of human pluripotent stem cells. Nat. Mater. 13, 599–604 (2014).
Caiazzo, M. et al. Defined three-dimensional microenvironments boost induction of pluripotency. Nat. Mater. 15, 344–352 (2016).
Broccoli, V., Colasante, G., Sessa, A. & Rubio, A. Histone modifications controlling native and induced neural stem cell identity. Curr. Opin. Genet. Dev. 34, 95–101 (2015).
Wapinski, OrlyL. et al. Hierarchical mechanisms for direct reprogramming of fibroblasts to neurons. Cell 155, 621–635 (2013).
DeQuach, J. A., Yuan, S. H., Goldstein, L. S. & Christman, K. L. Decellularized porcine brain matrix for cell culture and tissue engineering scaffolds. Tissue Eng. Part A 17, 2583–2592 (2011).
Zhang, Y. et al. Tissue-specific extracellular matrix coatings for the promotion of cell proliferation and maintenance of cell phenotype. Biomaterials 30, 4021–4028 (2009).
Lee, J. S. et al. Liver extracellular matrix providing dual functions of two-dimensional substrate coating and three-dimensional injectable hydrogel platform for liver tissue engineering. Biomacromolecules 15, 206–218 (2013).
Seal, A. et al. Mechanical properties of very thin cover slip glass disk. Bull. Mater. Sci. 24, 151–155 (2001).
Sood, D. et al. Fetal brain extracellular matrix boosts neuronal network formation in 3D bioengineered model of cortical brain tissue. ACS Biomater. Sci. Eng. 2, 131–140 (2015).
Medberry, C. J. et al. Hydrogels derived from central nervous system extracellular matrix. Biomaterials 34, 1033–1040 (2013).
Yang, F. et al. Genetic engineering of human stem cells for enhanced angiogenesis using biodegradable polymeric nanoparticles. Proc. Natl Acad. Sci. USA 107, 3317–3322 (2010).
Mangraviti, A. et al. Polymeric nanoparticles for nonviral gene therapy extend brain tumor survival in vivo. ACS Nano 9, 1236–1249 (2015).
Green, J. J., Langer, R. & Anderson, D. G. A combinatorial polymer library approach yields insight into nonviral gene delivery. Acc. Chem. Res. 41, 749–759 (2008).
Dupont, S. et al. Role of YAP/TAZ in mechanotransduction. Nature 474, 179–183 (2011).
Lian, I. et al. The role of YAP transcription coactivator in regulating stem cell self-renewal and differentiation. Genes Dev. 24, 1106–1118 (2010).
Gjorevski, N. et al. Designer matrices for intestinal stem cell and organoid culture. Nature 539, 560–564 (2016).
Musah, S. et al. Substratum-induced differentiation of human pluripotent stem cells reveals the coactivator YAP is a potent regulator of neuronal specification. Proc. Natl Acad. Sci. USA 111, 13805–13810 (2014).
Zhang, H. et al. Negative regulation of Yap during neuronal differentiation. Dev. Biol. 361, 103–115 (2012).
Halder, G., Dupont, S. & Piccolo, S. Transduction of mechanical and cytoskeletal cues by YAP and TAZ. Nat. Rev. Mol. Cell Biol. 13, 591–600 (2012).
Iwafuchi-Doi, M. & Zaret, K. S. Pioneer transcription factors in cell reprogramming. Genes Dev. 28, 2679–2692 (2014).
Wu, H.-W., Lin, C.-C. & Lee, G.-B. Stem cells in microfluidics. Biomicrofluidics 5, 013401 (2011).
Luni, C. et al. High-efficiency cellular reprogramming with microfluidics. Nat. Methods 13, 446–452 (2016).
Balestrini, J. L. et al. Comparative biology of decellularized lung matrix: implications of species mismatch in regenerative medicine. Biomaterials 102, 220–230 (2016).
Song, K. et al. Heart repair by reprogramming non-myocytes with cardiac transcription factors. Nature 485, 599–604 (2012).
Qian, L. et al. In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes. Nature 485, 593–598 (2012).
Gattazzo, F., Urciuolo, A. & Bonaldo, P. Extracellular matrix: a dynamic microenvironment for stem cell niche. Biochim. Biophys. Acta 1840, 2506–2519 (2014).
Watt, F. M. & Huck, W. T. S. Role of the extracellular matrix in regulating stem cell fate. Nat. Rev. Mol. Cell Biol. 14, 467–473 (2013).
Downing, T. L. et al. Biophysical regulation of epigenetic state and cell reprogramming. Nat. Mater. 12, 1154–1162 (2013).
Petersen, G. F. et al. Direct conversion of equine adipose-derived stem cells into induced neuronal cells is enhanced in three-dimensional culture. Cell Reprogram. 17, 419–426 (2015).
Velve-Casquillas, G., Le Berre, M., Piel, M. & Tran, P. T. Microfluidic tools for cell biological research. Nano Today 5, 28–47 (2010).
Ruoslahti, E. Brain extracellular matrix. Glycobiology 6, 489–492 (1996).
Ranga, A. et al. 3D niche microarrays for systems-level analyses of cell fate. Nat. Commun. 5, 4324 (2014).
Nguyen, E. H. et al. Versatile synthetic alternatives to Matrigel for vascular toxicity screening and stem cell expansion. Nat. Biomed. Eng. 1, 0096 (2017).
Hargus, G. et al. Tenascin-R promotes neuronal differentiation of embryonic stem cells and recruitment of host-derived neural precursor cells after excitotoxic lesion of the mouse striatum. Stem Cells 26, 1973–1984 (2008).
Wu, Y. et al. Versican V1 isoform induces neuronal differentiation and promotes neurite outgrowth. Mol. Biol. Cell 15, 2093–2104 (2004).
Pang, Z. P. et al. Induction of human neuronal cells by defined transcription factors. Nature 476, 220–223 (2011).
Kulangara, K. et al. The effect of substrate topography on direct reprogramming of fibroblasts to induced neurons. Biomaterials 35, 5327–5336 (2014).
Chanda, S. et al. Generation of induced neuronal cells by the single reprogramming factor ASCL1. Stem Cell Rep. 3, 282–296 (2014).
Fusaki, N. et al. Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proc. Jpn. Acad. Ser. B Phys. Biol. Sci. 85, 348–362 (2009).
Giorgetti, A. et al. Generation of induced pluripotent stem cells from human cord blood using OCT4 and SOX2. Cell Stem Cell 5, 353–357 (2009).
Seki, T. et al. Generation of induced pluripotent stem cells from human terminally differentiated circulating T cells. Cell Stem Cell 7, 11–14 (2010).
Lu, J. et al. Generation of integration-free and region-specific neural progenitors from primate fibroblasts. Cell Rep. 3, 1580–1591 (2013).
Cheng, X., Putz, K. W., Wood, C. D. & Brinson, L. C. Characterization of local elastic modulus in confined polymer films via AFM indentation. Macromol. Rapid Commun. 36, 391–397 (2015).
Jin, Y. et al. Triboelectric nanogenerator accelerates highly efficient nonviral direct conversion and in vivo reprogramming of fibroblasts to functional neuronal cells. Adv. Mater. 28, 7365–7374 (2016).
Hilgenberg, L. G., & Smith, M. A. Preparation of dissociated mouse cortical neuron cultures.J. Vis. Exp. 10, e562 (2007).
Wu, H. Y. et al. Critical role of calpain-mediated cleavage of calcineurin in excitotoxic neurodegeneration. J. Biol. Chem. 279, 4929–4940 (2004).
Yang, K. et al. Polydopamine-mediated surface modification of scaffold materials for human neural stem cell engineering. Biomaterials 33, 6952–6964 (2012).
Seo, H. I. et al. Thermo-responsive polymeric nanoparticles for enhancing neuronal differentiation of human induced pluripotent stem cells. Nanomedicine 11, 1861–1869 (2015).
Chambers, S. M. et al. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat. Biotechnol. 27, 275–280 (2009).
Shi, Y., Kirwan, P. & Livesey, F. J. Directed differentiation of human pluripotent stem cells to cerebral cortex neurons and neural networks. Nat. Protoc. 7, 1836–1846 (2012).
Han, S. et al. Three-dimensional extracellular matrix-mediated neural stem cell differentiation in a microfluidic device. Lab Chip 12, 2305–2308 (2012).
Yu, J. H. et al. Time-dependent effect of combination therapy with erythropoietin and granulocyte colony-stimulating factor in a mouse model of hypoxic–ischemic brain injury. Neurosci. Bull. 30, 107–117 (2014).
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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