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

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.

References

  1. Xu, J., Du, Y. & Deng, H. Direct lineage reprogramming: strategies, mechanisms, and applications. Cell Stem Cell 16, 119–134 (2015).

    Article  PubMed  CAS  Google Scholar 

  2. Sancho-Martinez, I., Baek, S. H. & Izpisua Belmonte, J. C. Lineage conversion methodologies meet the reprogramming toolbox. Nat. Cell Biol. 14, 892–899 (2012).

    Article  PubMed  CAS  Google Scholar 

  3. Heinrich, C., Spagnoli, F. M. & Berninger, B. In vivo reprogramming for tissue repair. Nat. Cell Biol. 17, 204–211 (2015).

    Article  PubMed  CAS  Google Scholar 

  4. Vierbuchen, T. et al. Direct conversion of fibroblasts to functional neurons by defined factors. Nature 463, 1035–1041 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  5. Caiazzo, M. et al. Direct conversion of fibroblasts into functional astrocytes by defined transcription factors. Stem Cell Rep. 4, 25–36 (2015).

    Article  CAS  Google Scholar 

  6. Kim, J. et al. Direct reprogramming of mouse fibroblasts to neural progenitors. Proc. Natl Acad. Sci. USA 108, 7838–7843 (2011).

    Article  PubMed  Google Scholar 

  7. Kim, J. et al. Functional integration of dopaminergic neurons directly converted from mouse fibroblasts. Cell Stem Cell 9, 413–419 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  8. Han, D. W. et al. Direct reprogramming of fibroblasts into neural stem cells by defined factors. Cell Stem Cell 10, 465–472 (2012).

    Article  PubMed  CAS  Google Scholar 

  9. Pfisterer, U. et al. Direct conversion of human fibroblasts to dopaminergic neurons. Proc. Natl Acad. Sci. USA 108, 10343–10348 (2011).

    Article  PubMed  Google Scholar 

  10. 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).

    Article  PubMed  Google Scholar 

  11. Xue, Y. et al. Direct conversion of fibroblasts to neurons by reprogramming PTB-regulated microRNA circuits. Cell 152, 82–96 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Yoo, A. S. et al. MicroRNA-mediated conversion of human fibroblasts to neurons. Nature 476, 228–231 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  13. Thier, M. et al. Direct conversion of fibroblasts into stably expandable neural stem cells. Cell Stem Cell 10, 473–479 (2012).

    Article  PubMed  CAS  Google Scholar 

  14. Ambasudhan, R. et al. Direct reprogramming of adult human fibroblasts to functional neurons under defined conditions. Cell Stem Cell 9, 113–118 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  15. Liu, M.-L. et al. Small molecules enable neurogenin 2 to efficiently convert human fibroblasts into cholinergic neurons. Nat. Commun. 4, 2183 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  16. Son, E. Y. et al. Conversion of mouse and human fibroblasts into functional spinal motor neurons. Cell Stem Cell 9, 205–218 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. Najm, F. J. et al. Transcription factor-mediated reprogramming of fibroblasts to expandable, myelinogenic oligodendrocyte progenitor cells. Nat. Biotechnol. 31, 426–433 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Yang, N. et al. Generation of oligodendroglial cells by direct lineage conversion. Nat. Biotechnol. 31, 434–439 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  19. Li, X. et al. Small-molecule-driven direct reprogramming of mouse fibroblasts into functional neurons. Cell Stem Cell 17, 195–203 (2015).

    Article  PubMed  CAS  Google Scholar 

  20. 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).

    Article  PubMed  CAS  Google Scholar 

  21. 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).

    Article  PubMed  CAS  Google Scholar 

  22. Cesana, D. et al. Uncovering and dissecting the genotoxicity of self-inactivating lentiviral vectors in vivo. Mol. Ther. 22, 774–785 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  23. Chandler, R. J. et al. Vector design influences hepatic genotoxicity after adeno-associated virus gene therapy. J. Clin. Invest. 125, 870–880 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  24. 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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  25. Narsinh, K. H. et al. Generation of adult human induced pluripotent stem cells using nonviral minicircle DNA vectors. Nat. Protoc. 6, 78–88 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  26. 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).

    Article  PubMed  Google Scholar 

  27. Wang, H., Luo, X. & Leighton, J. Extracellular matrix and integrins in embryonic stem cell differentiation. Biochem. Insights 8, 15–21 (2015).

    PubMed  PubMed Central  Google Scholar 

  28. Rozario, T. & DeSimone, D. W. The extracellular matrix in development and morphogenesis: a dynamic view. Dev. Biol. 341, 126–140 (2010).

    Article  PubMed  CAS  Google Scholar 

  29. 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).

    Article  PubMed  PubMed Central  Google Scholar 

  30. 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).

    Article  PubMed  PubMed Central  Google Scholar 

  31. Sun, Y. et al. Hippo/YAP-mediated rigidity-dependent motor neuron differentiation of human pluripotent stem cells. Nat. Mater. 13, 599–604 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  32. Caiazzo, M. et al. Defined three-dimensional microenvironments boost induction of pluripotency. Nat. Mater. 15, 344–352 (2016).

    Article  PubMed  CAS  Google Scholar 

  33. 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).

    Article  PubMed  CAS  Google Scholar 

  34. Wapinski, OrlyL. et al. Hierarchical mechanisms for direct reprogramming of fibroblasts to neurons. Cell 155, 621–635 (2013).

    Article  PubMed  CAS  Google Scholar 

  35. 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).

    Article  PubMed  PubMed Central  Google Scholar 

  36. 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).

    Article  PubMed  CAS  Google Scholar 

  37. 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).

    Article  PubMed  CAS  Google Scholar 

  38. Seal, A. et al. Mechanical properties of very thin cover slip glass disk. Bull. Mater. Sci. 24, 151–155 (2001).

    Article  CAS  Google Scholar 

  39. 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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  40. Medberry, C. J. et al. Hydrogels derived from central nervous system extracellular matrix. Biomaterials 34, 1033–1040 (2013).

    Article  PubMed  CAS  Google Scholar 

  41. 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).

    Article  PubMed  Google Scholar 

  42. Mangraviti, A. et al. Polymeric nanoparticles for nonviral gene therapy extend brain tumor survival in vivo. ACS Nano 9, 1236–1249 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  43. 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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  44. Dupont, S. et al. Role of YAP/TAZ in mechanotransduction. Nature 474, 179–183 (2011).

    Article  PubMed  CAS  Google Scholar 

  45. Lian, I. et al. The role of YAP transcription coactivator in regulating stem cell self-renewal and differentiation. Genes Dev. 24, 1106–1118 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  46. Gjorevski, N. et al. Designer matrices for intestinal stem cell and organoid culture. Nature 539, 560–564 (2016).

    Article  PubMed  CAS  Google Scholar 

  47. 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).

    Article  PubMed  CAS  Google Scholar 

  48. Zhang, H. et al. Negative regulation of Yap during neuronal differentiation. Dev. Biol. 361, 103–115 (2012).

    Article  PubMed  CAS  Google Scholar 

  49. 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).

    Article  PubMed  CAS  Google Scholar 

  50. Iwafuchi-Doi, M. & Zaret, K. S. Pioneer transcription factors in cell reprogramming. Genes Dev. 28, 2679–2692 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  51. Wu, H.-W., Lin, C.-C. & Lee, G.-B. Stem cells in microfluidics. Biomicrofluidics 5, 013401 (2011).

    Article  PubMed Central  CAS  Google Scholar 

  52. Luni, C. et al. High-efficiency cellular reprogramming with microfluidics. Nat. Methods 13, 446–452 (2016).

    Article  PubMed  CAS  Google Scholar 

  53. Balestrini, J. L. et al. Comparative biology of decellularized lung matrix: implications of species mismatch in regenerative medicine. Biomaterials 102, 220–230 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  54. Song, K. et al. Heart repair by reprogramming non-myocytes with cardiac transcription factors. Nature 485, 599–604 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  55. Qian, L. et al. In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes. Nature 485, 593–598 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  56. Gattazzo, F., Urciuolo, A. & Bonaldo, P. Extracellular matrix: a dynamic microenvironment for stem cell niche. Biochim. Biophys. Acta 1840, 2506–2519 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  57. 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).

    Article  PubMed  CAS  Google Scholar 

  58. Downing, T. L. et al. Biophysical regulation of epigenetic state and cell reprogramming. Nat. Mater. 12, 1154–1162 (2013).

    Article  PubMed  CAS  Google Scholar 

  59. 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).

    Article  PubMed  CAS  Google Scholar 

  60. Velve-Casquillas, G., Le Berre, M., Piel, M. & Tran, P. T. Microfluidic tools for cell biological research. Nano Today 5, 28–47 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  61. Ruoslahti, E. Brain extracellular matrix. Glycobiology 6, 489–492 (1996).

    Article  PubMed  CAS  Google Scholar 

  62. Ranga, A. et al. 3D niche microarrays for systems-level analyses of cell fate. Nat. Commun. 5, 4324 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  63. Nguyen, E. H. et al. Versatile synthetic alternatives to Matrigel for vascular toxicity screening and stem cell expansion. Nat. Biomed. Eng. 1, 0096 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  64. 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).

    Article  PubMed  CAS  Google Scholar 

  65. Wu, Y. et al. Versican V1 isoform induces neuronal differentiation and promotes neurite outgrowth. Mol. Biol. Cell 15, 2093–2104 (2004).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  66. Pang, Z. P. et al. Induction of human neuronal cells by defined transcription factors. Nature 476, 220–223 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  67. Kulangara, K. et al. The effect of substrate topography on direct reprogramming of fibroblasts to induced neurons. Biomaterials 35, 5327–5336 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  68. Chanda, S. et al. Generation of induced neuronal cells by the single reprogramming factor ASCL1. Stem Cell Rep. 3, 282–296 (2014).

    Article  CAS  Google Scholar 

  69. 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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  70. 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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  71. Seki, T. et al. Generation of induced pluripotent stem cells from human terminally differentiated circulating T cells. Cell Stem Cell 7, 11–14 (2010).

    Article  PubMed  CAS  Google Scholar 

  72. Lu, J. et al. Generation of integration-free and region-specific neural progenitors from primate fibroblasts. Cell Rep. 3, 1580–1591 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  73. 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).

    Article  PubMed  CAS  Google Scholar 

  74. 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).

    Article  PubMed  CAS  Google Scholar 

  75. Hilgenberg, L. G., & Smith, M. A. Preparation of dissociated mouse cortical neuron cultures.J. Vis. Exp. 10, e562 (2007).

    Google Scholar 

  76. Wu, H. Y. et al. Critical role of calpain-mediated cleavage of calcineurin in excitotoxic neurodegeneration. J. Biol. Chem. 279, 4929–4940 (2004).

    Article  PubMed  CAS  Google Scholar 

  77. Yang, K. et al. Polydopamine-mediated surface modification of scaffold materials for human neural stem cell engineering. Biomaterials 33, 6952–6964 (2012).

    Article  PubMed  CAS  Google Scholar 

  78. Seo, H. I. et al. Thermo-responsive polymeric nanoparticles for enhancing neuronal differentiation of human induced pluripotent stem cells. Nanomedicine 11, 1861–1869 (2015).

    Article  PubMed  CAS  Google Scholar 

  79. 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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  80. 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).

    Article  PubMed  CAS  Google Scholar 

  81. Han, S. et al. Three-dimensional extracellular matrix-mediated neural stem cell differentiation in a microfluidic device. Lab Chip 12, 2305–2308 (2012).

    Article  PubMed  CAS  Google Scholar 

  82. 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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

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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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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 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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