Topical tissue nano-transfection mediates non-viral stroma reprogramming and rescue

Abstract

Although cellular therapies represent a promising strategy for a number of conditions, current approaches face major translational hurdles, including limited cell sources and the need for cumbersome pre-processing steps (for example, isolation, induced pluripotency)1,2,3,4,5,6. In vivo cell reprogramming has the potential to enable more-effective cell-based therapies by using readily available cell sources (for example, fibroblasts) and circumventing the need for ex vivo pre-processing7,8. Existing reprogramming methodologies, however, are fraught with caveats, including a heavy reliance on viral transfection9,10. Moreover, capsid size constraints and/or the stochastic nature of status quo approaches (viral and non-viral) pose additional limitations, thus highlighting the need for safer and more deterministic in vivo reprogramming methods11,12. Here, we report a novel yet simple-to-implement non-viral approach to topically reprogram tissues through a nanochannelled device validated with well-established and newly developed reprogramming models of induced neurons and endothelium, respectively. We demonstrate the simplicity and utility of this approach by rescuing necrotizing tissues and whole limbs using two murine models of injury-induced ischaemia.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: TNT mediates enhanced reprogramming factor delivery and propagation beyond the transfection boundary.
Figure 2: EFF TNT leads to increased vascularization and rescue of skin tissue under ischaemic conditions.
Figure 3: EFF TNT rescues whole limbs from necrotizing ischaemia.

References

  1. 1

    Rosova, I., Dao, M., Capoccia, B., Link, D. & Nolta, J. A. Hypoxic preconditioning results in increased motility and improved therapeutic potential of human mesenchymal stem cells. Stem Cells 26, 2173–2182 (2008).

    CAS  Article  Google Scholar 

  2. 2

    Kinoshita, M. et al. Long-term clinical outcome after intramuscular transplantation of granulocyte colony stimulating factor-mobilized CD34 positive cells in patients with critical limb ischemia. Atherosclerosis 224, 440–445 (2012).

    CAS  Article  Google Scholar 

  3. 3

    Losordo, D. W. & Dimmeler, S. Therapeutic angiogenesis and vasculogenesis for ischemic disease. Part II: cell-based therapies. Circulation 109, 2692–2697 (2004).

    Article  Google Scholar 

  4. 4

    Lee, A. S., Tang, C., Rao, M. S., Weissman, I. L . & Wu, J. C. Tumorigenicity as a clinical hurdle for pluripotent stem cell therapies. Nat. Med. 19, 998–1004 (2013).

    CAS  Article  Google Scholar 

  5. 5

    Cunningham, J. J., Ulbright, T. M., Pera, M. F. & Looijenga, L. H. Lessons from human teratomas to guide development of safe stem cell therapies. Nat. Biotechnol. 30, 849–857 (2012).

    CAS  Article  Google Scholar 

  6. 6

    Leduc, P. R. et al. Towards an in vivo biologically inspired nanofactory. Nat. Nanotech. 2, 3–7 (2007).

    CAS  Article  Google Scholar 

  7. 7

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

    Article  Google Scholar 

  8. 8

    Karagiannis, P. & Yamanaka, S. The fate of cell reprogramming. Nat. Methods 11, 1006–1008 (2014).

    CAS  Article  Google Scholar 

  9. 9

    Grande, A. et al. Environmental impact on direct neuronal reprogramming in vivo in the adult brain. Nat. Commun. 4, 2373 (2013).

    Article  Google Scholar 

  10. 10

    Morita, R. et al. ETS transcription factor ETV2 directly converts human fibroblasts into functional endothelial cells. Proc. Natl Acad. Sci. USA 112, 160–165 (2015).

    CAS  Article  Google Scholar 

  11. 11

    Gallego-Perez, D. et al. Deterministic transfection drives efficient nonviral reprogramming and uncovers reprogramming barriers. Nanomedicine 12, 399–409 (2016).

    CAS  Article  Google Scholar 

  12. 12

    Marx, V. Cell biology: delivering tough cargo into cells. Nat. Methods 13, 37–40 (2016).

    CAS  Article  Google Scholar 

  13. 13

    Boukany, P. E. et al. Nanochannel electroporation delivers precise amounts of biomolecules into living cells. Nat. Nanotech. 6, 747–754 (2011).

    CAS  Article  Google Scholar 

  14. 14

    Sen, C. K. & Ghatak, S. miRNA control of tissue repair and regeneration. Am. J. Pathol. 185, 2629–2640 (2015).

    CAS  Article  Google Scholar 

  15. 15

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

    CAS  Article  Google Scholar 

  16. 16

    Valadi, H. et al. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat. Cell Biol. 9, 654–659 (2007).

    CAS  Article  Google Scholar 

  17. 17

    Davis, D. M. & Sowinski, S. Membrane nanotubes: dynamic long-distance connections between animal cells. Nat. Rev. Mol. Cell Biol. 9, 431–436 (2008).

    CAS  Article  Google Scholar 

  18. 18

    Khanna, S. et al. Loss of miR-29b following acute ischemic stroke contributes to neural cell death and infarct size. J. Cereb. Blood Flow Metab. 33, 1197–1206 (2013).

    CAS  Article  Google Scholar 

  19. 19

    Venugopal, V. & Sundaresan, V. B. Polypyrrole-based amperometric cation sensor with tunable sensitivity. J. Intel. Mat. Syst. Str. 27, 1702–1709 (2016).

    CAS  Article  Google Scholar 

  20. 20

    Venugopal, V., Venkatesh, V., Northcutt, R. G., Maddox, J. & Sundaresan, V. B. Nanoscale polypyrrole sensors for near-field electrochemical measurements. Sensors Actuat. B Chem. 242, 1193–1200 (2017).

    CAS  Article  Google Scholar 

  21. 21

    Hunt, D. P. et al. A highly enriched niche of precursor cells with neuronal and glial potential within the hair follicle dermal papilla of adult skin. Stem Cells 26, 163–172 (2008).

    CAS  Article  Google Scholar 

  22. 22

    Higgins, C. A . et al. Reprogramming of human hair follicle dermal papilla cells into induced pluripotent stem cells. J. Invest. Dermatol. 132, 1725–1727 (2012).

    CAS  Article  Google Scholar 

  23. 23

    Helisch, A. et al. Impact of mouse strain differences in innate hindlimb collateral vasculature. Arterioscler. Thromb. Vasc. Biol. 26, 520–526 (2006).

    CAS  Article  Google Scholar 

  24. 24

    Chalothorn, D., Clayton, J. A., Zhang, H., Pomp, D. & Faber, J. E. Collateral density, remodeling, and VEGF-A expression differ widely between mouse strains. Physiol. Genomics 30, 179–191 (2007).

    CAS  Article  Google Scholar 

  25. 25

    Anokye-Danso, F. et al. Highly efficient miRNA-mediated reprogramming of mouse and human somatic cells to pluripotency. Cell Stem Cell 8, 376–388 (2011).

    CAS  Article  Google Scholar 

  26. 26

    Limbourg, A. et al. Evaluation of postnatal arteriogenesis and angiogenesis in a mouse model of hind-limb ischemia. Nat. Protoc. 4, 1737–1746 (2009).

    CAS  Article  Google Scholar 

  27. 27

    Fiedler, G. B. et al. Localized semi-LASER dynamic 31P magnetic resonance spectroscopy of the soleus during and following exercise at 7 T. MAGMA 28, 493–501 (2015).

    Article  Google Scholar 

  28. 28

    Gnyawali, S. C. et al. High-frequency high-resolution echocardiography: first evidence on non-invasive repeated measure of myocardial strain, contractility, and mitral regurgitation in the ischemia-reperfused murine heart. J. Vis. Exp. 2010, e1781 (2010).

    Google Scholar 

  29. 29

    Roy, S . et al. Transcriptome-wide analysis of blood vessels laser captured from human skin and chronic wound-edge tissue. Proc. Natl Acad. Sci. USA 104, 14472–14477 (2007).

    CAS  Article  Google Scholar 

  30. 30

    Rink, C. et al. Oxygen-sensitive outcomes and gene expression in acute ischemic stroke. J. Cereb. Blood Flow Metab. 30, 1275–1287 (2010).

    CAS  Article  Google Scholar 

  31. 31

    Roy, S., Khanna, S., Rink, C., Biswas, S. & Sen, C. K. Characterization of the acute temporal changes in excisional murine cutaneous wound inflammation by screening of the wound-edge transcriptome. Physiol. Genomics 34, 162–184 (2008).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

Funding for C.K.S. was partly provided by NIGMS/NINR (R01GM07718507, R01GM10801402, R01NR01567601, R01NR01389804 and R01NS42617) and a philanthropic gift from Leslie and Abigail Wexner. Funding for L.J.L. was partly provided by NIBIB (R21EB017539), NSF (NSEC EEC-0914790) and the National Center for the Advancing Translational Sciences (UL1TR001070). Funding for D.G.-P., S.K. and C.R. was partly provided by NINDS (R21NS099869). Additional funding for D.G.-P. and S.K. was provided in part by the NIDDK Diabetic Complications Consortium (DiaComp, www.diacomp.org), grant DK076169 (U24DK076169). J.J.O., V.B.S., S.K., C.R. and S.R. acknowledge financial support from NIH (R01HL132355, R21EB017539, R01NS099869 and R01DK076566) and NSF (1325114), respectively. The authors thank T. Wilgus and B. Wulff (Department of Pathology, The Ohio State University) for providing the Col1A1 mice used in this study. Fsp1-Cre mice were a gift from A. Deb (University of California, Los Angeles). Etv2 and Fli1 plasmids were donated by A. Ferdous (Department of Internal Medicine, UT Southwestern). Foxc2 plasmid was donated by T. Kume (Department of Medicine-Cardiology and Pharmacology, Northwestern University-FCVRI, Chicago). X. Wang (The Ohio State University) provided support with plasmid design and preparation. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center for Advancing Translational Sciences, National Science Foundation or the National Institutes of Health. This work was sponsored by and represents activity of The Ohio State University Center for Regenerative Medicine and Cell Based Therapies (regenerativemedicine.osu.edu) and Nanoscale Engineering Center for Affordable Nanoengineering of Polymeric Biomedical Devices.

Author information

Affiliations

Authors

Contributions

TNT platform design, implementation and optimization (for different applications) was performed by D.G.-P., L.J.L., D.P., S.G. and C.K.S. TNT chip fabrication, transductions, cell/tissue imaging, transgenic mouse experiments and histology were performed by D.G.-P., D.P., S.G., N.H.-C., V.M., S.G., L.C., M.S., E.S., A.S., J.M., P.B., W.L., J.J.O., L.J.L. and C.K.S. Electrophysiological activity measurements were conducted by T.Z., R.G.N., V.B.S. and J.J.O., with support from S.G. and N.H.-C. Global gene expression analyses were conducted by S.R., S.K., K.S. and C.K.S. TNT chip simulations were conducted by W.-C.L., J.S., L.C., D.G.-P. and L.J.L. S.R. oversaw and participated in the LCM work. Stroke recovery experiments were conducted by D.G.-P., V.M., A.S., R.S., M.H., S.K., C.R. and C.K.S. The manuscript was written by D.G.-P., L.J.L., D.P., S.G. and C.K.S. C.K.S. and L.J.L. jointly supervised this work.

Corresponding authors

Correspondence to L. James Lee or Chandan K. Sen.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary Information (PDF 10863 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Gallego-Perez, D., Pal, D., Ghatak, S. et al. Topical tissue nano-transfection mediates non-viral stroma reprogramming and rescue. Nature Nanotech 12, 974–979 (2017). https://doi.org/10.1038/nnano.2017.134

Download citation

Further reading