Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Engineering pulmonary vasculature in decellularized rat and human lungs

Abstract

Bioengineered lungs produced from patient-derived cells may one day provide an alternative to donor lungs for transplantation therapy. Here we report the regeneration of functional pulmonary vasculature by repopulating the vascular compartment of decellularized rat and human lung scaffolds with human cells, including endothelial and perivascular cells derived from induced pluripotent stem cells. We describe improved methods for delivering cells into the lung scaffold and for maturing newly formed endothelium through co-seeding of endothelial and perivascular cells and a two-phase culture protocol. Using these methods we achieved 75% endothelial coverage in the rat lung scaffold relative to that of native lung. The regenerated endothelium showed reduced vascular resistance and improved barrier function over the course of in vitro culture and remained patent for 3 days after orthotopic transplantation in rats. Finally, we scaled our approach to the human lung lobe and achieved efficient cell delivery, maintenance of cell viability and establishment of perfusable vascular lumens.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Endothelial delivery, vascular maturation and functional assessment of regenerated lungs during in vitro culture.
Figure 2: Endothelial and perivascular cell differentiation from hiPSCs.
Figure 3: Pulmonary vascular regeneration with hiPSC-ECs and hiPSC-PCs.
Figure 4: Human lung regeneration using hiPSC-ECs and hiPSC-PCs.

Similar content being viewed by others

Zixuan Zhao, Xinyi Chen, … Hanry Yu

References

  1. Ott, H.C. et al. Regeneration and orthotopic transplantation of a bioartificial lung. Nat. Med. 16, 927–933 (2010).

    Article  CAS  Google Scholar 

  2. Petersen, T.H. et al. Tissue-engineered lungs for in vivo implantation. Science 329, 538–541 (2010).

    Article  CAS  Google Scholar 

  3. Herbert, S.P. & Stainier, D.Y.R. Molecular control of endothelial cell behaviour during blood vessel morphogenesis. Nat. Rev. Mol. Cell Biol. 12, 551–564 (2011).

    Article  CAS  Google Scholar 

  4. Strilicć, B. et al. The molecular basis of vascular lumen formation in the developing mouse aorta. Dev. Cell 17, 505–515 (2009).

    Article  Google Scholar 

  5. Au, P., Tam, J., Fukumura, D. & Jain, R.K. Bone marrow-derived mesenchymal stem cells facilitate engineering of long-lasting functional vasculature. Blood 111, 4551–4558 (2008).

    Article  CAS  Google Scholar 

  6. Bayless, K.J., Salazar, R. & Davis, G.E. RGD-dependent vacuolation and lumen formation observed during endothelial cell morphogenesis in three-dimensional fibrin matrices involves the αvβ3 and α5β1 integrins. Am. J. Pathol. 156, 1673–1683 (2000).

    Article  CAS  Google Scholar 

  7. Kim, S.H. et al. Antagonism of VEGF-A–induced increase in vascular permeability by an integrin α3β1-Shp-1-cAMP/PKA pathway. Blood 120, 4892–4902 (2012).

    Article  CAS  Google Scholar 

  8. Förster, C. et al. Occludin as direct target for glucocorticoid-induced improvement of blood–brain barrier properties in a murine in vitro system. J. Physiol. (Lond.) 565, 475–486 (2005).

    Article  Google Scholar 

  9. Rock, J.R. et al. Multiple stromal populations contribute to pulmonary fibrosis without evidence for epithelial to mesenchymal transition. Proc. Natl. Acad. Sci. USA 108, E1475–E1483 (2011).

    Article  CAS  Google Scholar 

  10. Gavard, J. et al. A role for a CXCR2/phosphatidylinositol 3-kinase γ signaling axis in acute and chronic vascular permeability. Mol. Cell. Biol. 29, 2469–2480 (2009).

    Article  CAS  Google Scholar 

  11. Carter, E.P., Ölveczky, B.P., Matthay, M.A. & Verkman, A.S. High microvascular endothelial water permeability in mouse lung measured by a pleural surface fluorescence method. Biophys. J. 74, 2121–2128 (1998).

    Article  CAS  Google Scholar 

  12. Baptista, P.M. et al. The use of whole organ decellularization for the generation of a vascularized liver organoid. Hepatology 53, 604–617 (2011).

    Article  CAS  Google Scholar 

  13. Matthay, M.A., Folkesson, H.G. & Clerici, C. Lung epithelial fluid transport and the resolution of pulmonary edema. Physiol Rev. 82, 569–600 (2002).

    Article  CAS  Google Scholar 

  14. Mackersie, R.C., Christensen, J. & Lewis, F.R. The role of pulmonary lymphatics in the clearance of hydrostatic pulmonary edema. J. Surg. Res. 43, 495–504 (1987).

    Article  CAS  Google Scholar 

  15. Melero-Martin, J.M. et al. In vivo vasculogenic potential of human blood-derived endothelial progenitor cells. Blood 109, 4761–4768 (2007).

    Article  CAS  Google Scholar 

  16. James, D. & et al. Expansion and maintenance of human embryonic stem cell-derived endothelial cells by TGFbeta inhibition is Id1 dependent. Nat. Biotechnol. 28, 161–166 (2010).

    Article  CAS  Google Scholar 

  17. Stone, K., Mercer, R., Freeman, B., Chang, L. & Crapo, J. Distribution of lung cell numbers and volumes between alveolar and nonalveolar tissue. Am. Rev. Respir. Dis. 146, 454–456 (1992).

    Article  CAS  Google Scholar 

  18. Crapo, J., Barry, B., Gehr, P., Bachofen, M. & Weibel, E. Cell number and cell characteristics of the normal human lung. Am. Rev. Respir. Dis. 126, 332–337 (1982).

    CAS  PubMed  Google Scholar 

  19. Crisan, M. et al. A perivascular origin for mesenchymal stem cells in multiple human organs. Cell Stem Cell 3, 301–313 (2008).

    Article  CAS  Google Scholar 

  20. Meyrick, B. & Reid, L. The effect of continued hypoxia on rat pulmonary arterial circulation. An ultrastructural study. Lab. Invest. 38, 188–200 (1978).

    CAS  PubMed  Google Scholar 

  21. Booth, A.J. et al. Acellular normal and fibrotic human lung matrices as a culture system for in vitro investigation. Am. J. Respir. Crit. Care Med. 186, 866–876 (2012).

    Article  CAS  Google Scholar 

  22. Nichols, J.E. et al. Production and assessment of decellularized pig and human lung scaffolds. Tissue Eng. Part A 19, 2045–2062 (2013).

    Article  CAS  Google Scholar 

  23. Gilpin, S.E. et al. Perfusion decellularization of human and porcine lungs: bringing the matrix to clinical scale. J. Heart Lung Transplant. 33, 298–308 (2014).

    Article  Google Scholar 

  24. O′Neill, J.D. et al. Decellularization of human and porcine lung tissues for pulmonary tissue engineering. Ann. Thorac. Surg. 96, 1046–1055 (2013).

    Article  Google Scholar 

  25. Price, A.P. et al. Automated decellularization of intact, human-sized lungs for tissue engineering. Tissue Eng. Part C Methods 21, 94–103 (2015).

    Article  CAS  Google Scholar 

  26. Wagner, D.E. et al. Comparative decellularization and recellularization of normal versus emphysematous human lungs. Biomaterials 35, 3281–3297 (2014).

    Article  CAS  Google Scholar 

  27. Bonvillain, R.W. et al. Nonhuman primate lung decellularization and recellularization using a specialized large-organ bioreactor. J. Vis. Exp. e50825 (2013).

  28. Ott, H.C. et al. Perfusion-decellularized matrix: using nature's platform to engineer a bioartificial heart. Nat. Med. 14, 213–221 (2008).

    Article  CAS  Google Scholar 

  29. Song, J.J. et al. Regeneration and experimental orthotopic transplantation of a bioengineered kidney. Nat. Med. 19, 646–651 (2013).

    Article  CAS  Google Scholar 

  30. Uygun, B.E. et al. Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix. Nat. Med. 16, 814–820 (2010).

    Article  CAS  Google Scholar 

  31. Salvagiotto, G. et al. A defined, feeder-free, serum-free system to generate in vitro hematopoietic progenitors and differentiated blood cells from hESCs and hiPSCs. PLoS ONE 6, e17829 (2011).

    Article  CAS  Google Scholar 

  32. Sahara, M. et al. Manipulation of a VEGF-Notch signaling circuit drives formation of functional vascular endothelial progenitors from human pluripotent stem cells. Cell Res. 24, 820–841 (2014).

    Article  CAS  Google Scholar 

  33. Prasain, N. et al. Differentiation of human pluripotent stem cells to cells similar to cord-blood endothelial colony-forming cells. Nat. Biotechnol. 32, 1151–1157 (2014).

    Article  CAS  Google Scholar 

  34. Huang, S.X.L. et al. Efficient generation of lung and airway epithelial cells from human pluripotent stem cells. Nat. Biotechnol. 32, 84–91 (2014).

    Article  CAS  Google Scholar 

  35. Longmire, T.A. et al. Efficient derivation of purified lung and thyroid progenitors from embryonic stem cells. Cell Stem Cell 10, 398–411 (2012).

    Article  CAS  Google Scholar 

  36. Mou, H. et al. Generation of multipotent lung and airway progenitors from mouse ESCs and patient-specific cystic fibrosis iPSCs. Cell Stem Cell 10, 385–397 (2012).

    Article  CAS  Google Scholar 

  37. Wong, A.P. et al. Directed differentiation of human pluripotent stem cells into mature airway epithelia expressing functional CFTR protein. Nat. Biotechnol. 30, 876–882 (2012).

    Article  CAS  Google Scholar 

  38. Ghaedi, M. et al. Human iPS cell–derived alveolar epithelium repopulates lung extracellular matrix. J. Clin. Invest. 123, 4950–4962 (2013).

    Article  CAS  Google Scholar 

  39. Ren, X. et al. Ex vivo non-invasive assessment of cell viability and proliferation in bio-engineered whole organ constructs. Biomaterials 52, 103–112 (2015).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This study was supported by the United Therapeutics Corporation and the National Institutes of Health (NIH) Director's New Innovator Award (DP2-OD008749-01). The authors thank the HSCI/MGH Flow Cytometry Core Facility for analysis and purification of hiPSC differentiation, the MGH Center for Skeletal Research Core (NIH P30 AR066261) for histological processing and MGH Wellman Center for Photomedicine for transmission electron microscopy.

Author information

Authors and Affiliations

Authors

Contributions

H.C.O. conceived, designed and oversaw all of the studies, collection of results, interpretation of the data and writing of the manuscript and was responsible for the primary undertaking, completion and supervision of all experiments. X.R. designed the studies, conducted cell culture, animal surgeries, decellularization, whole-organ culture, histological and functional analysis, and prepared the manuscript. P.T.M. assisted in animal surgeries, histological analysis and figure preparation. S.E.G. and L.F.T. conducted human lung decellularization, and assisted in human lung recellularization. T.O. and L.X. performed orthotopic lung transplantation. T.W. performed perivascular cell characterization and labeling. F.E.M. designed and constructed the lentiviral vector. R.G. assisted in histological analysis. D.T.S. and D.J.M. provided input on experimental design and edited the manuscript.

Ethics declarations

Competing interests

H.C.O. is founder and stockholder of IVIVA Medical Inc. This relationship did not affect the content or conclusions contained in this manuscript.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–13 (PDF 38301 kb)

Supplementary Table 1 (PDF 152 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ren, X., Moser, P., Gilpin, S. et al. Engineering pulmonary vasculature in decellularized rat and human lungs. Nat Biotechnol 33, 1097–1102 (2015). https://doi.org/10.1038/nbt.3354

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt.3354

This article is cited by

Search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research