Research ArticleLUNG DISEASE

Functional vascularized lung grafts for lung bioengineering

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Science Advances  30 Aug 2017:
Vol. 3, no. 8, e1700521
DOI: 10.1126/sciadv.1700521
  • Fig. 1 Experimental setup of single lung airway de-epithelialization.

    (A) EVLP circuit and circuit elements. (B) Double lung cannulation method enabling ventilation of one lung (native) and airway de-epithelialization of contralateral lung (de-epith). R, right; L, left. (C and D) Schematic of experimental approach demonstrating (C) native airway, interstitium, and adjacent pulmonary vasculature and (D) denuded airway basement membrane following de-epithelialization with intact interstitium and preservation of vascular viability and function. (E) Components of vascular perfusate (see Materials and Methods for the complete list of components) and airway de-epithelialization solution.

  • Fig. 2 Removal of pulmonary epithelium in conducting and respiratory zones.

    (A to D) Hematoxylin and eosin (H&E) staining of conducting zone in native and de-epithelialized lungs with intact pseudostratified epithelium (arrowheads) in the native lung (A and C) and denuded basement membrane (arrowheads) following the removal of respiratory epithelium in the de-epithelialized lung (B and D). Vessel is indicated by asterisk. (E and F) Immunofluorescence staining confirming the removal of bronchial epithelial cells by decreased EpCAM in de-epithelialized lung, with preservation of vessels indicated by vWF. (G and H) H&E staining of respiratory zone in native and de-epithelialized lungs. (I to L) Immunofluorescence staining confirmed the removal of alveolar type I cells by decreased Aq5, alveolar type II cells by decreased SPC, with preservation of vessels indicated by vWF in de-epithelialized lungs (J and L). (M and N) Western blot of CD31 and EpCAM from native and de-epithelialized lungs (asterisk indicates nonspecific band) (M). Quantification data indicate that de-epithelialized lungs contained five times less EpCAM than native (n = 3, values normalized to levels in native lung; error bars represent means ± SD of experimental values) (N). (O and P) TEM of native (O) and de-epithelialized (P) lungs showing intact microvessels and endothelial cell nuclei (asterisks) but absent alveolar cells (arrowheads) in de-epithelialized lungs.

  • Fig. 3 Preservation of lung structure and ECM.

    (A) Immunofluorescence staining demonstrating preservation of collagen IV, elastin, fibronectin, and laminin in de-epithelialized lung. (B to I) H&E staining (B and C) and special staining of native and de-epithelialized lung: trichrome (D and E), Alcian blue (F and G), and van Gieson (H and I). (J) Quantification of sGAGs (n = 3; P = 0.15), HOP (n = 3; P = 0.71), elastin (n = 3; P = 0.26), and DNA (native, n = 5; de-epithelialized, n = 5; decellularized, n = 4; *P < 0.05 and **P < 0.0001). (K) Scanning electron microscopy of native and de-epithelialized lungs. (L) Morphometry and stereology of native and de-epithelialized lungs: airspace volume (n = 3; P = 0.27), septal volume fraction (n = 3; P = 0.31), surface density (n = 3; P = 0.27), and septal thickness (n = 3; P = 0.98). Data shown were analyzed by Student’s t test. Error bars represent means ± SD of experimental values.

  • Fig. 4 Bronchial structure, viability, and function.

    Preservation of bronchial structure of native and de-epithelialized lungs (A to F). Pentachrome stain (A and B). SMA immunofluorescence staining (C and D). Airways are indicated by arrows, and vessels are indicated by stars. Scanning electron microscopy of airway casts (E and F). (G and H) Airway responsiveness during intravascular administration of methacholine measured by pressure-volume loops. Lung compliance in native (G) (0.190 ± 0.046 ml/cmH2O; after methacholine, 0.048 ± 0.007 ml/cmH2O; reduction of ~73.48 ± 8.704%) and de-epithelialized (H) (0.065 ± 0.036ml/cmH2O; after methacholine, 0.031 ± 0.004 ml/cmH2O; reduction of 45.28 ± 18.71%) lungs. Values are expressed as means ± SD.

  • Fig. 5 Vascular preservation, viability, and function.

    (A) Immunofluorescence staining demonstrating the preservation of endothelial cells (vWF and CD31), vascular smooth muscle (SMA), pericytes (NG2), and tight and gap junction proteins (ZO-1 and Cx43) of the vascular bed following de-epithelialization. (B) Viability of endothelial cells by capture of acetylated LDL (aLDL) and loss of alveolar type I cells (Aq5) after de-epithelialization. (C) Nonapoptotic endothelial cells following de-epithelialization shown by TUNEL and vWF costaining; apoptotic cells (negative for vWF) in de-epithelialized lungs are indicated by arrowheads. (D) Intravascular administration of endothelin-1 and treprostinil demonstrating the preservation of vasoresponsiveness in native and de-epithelialized lungs (n = 3; Student’s t test; error bars represent means ± SD of experimental values). (E) Integrity of the pulmonary vascular bed shown by quantification of fluorescein isothiocyanate (FITC)–conjugated dextran recovered from the pulmonary venous drainage in native lung, following 4 and 8 mM CHAPS treatment (n = 3 for each group; *P = 0.01 and **P < 0.01, Student’s t test; error bars represent means ± SD of experimental values). (F) H&E histologic comparison of native lung and de-epithelialization by 4 and 8 mM CHAPS treatment. (G to I) Transpleural imaging to visualize pulmonary microvasculature. (G) Imaging setup with water-dipping lens. Images are captured during vascular perfusion of 0.2-μm fluorescent microspheres in (H) native and (I) de-epithelialized lungs (see movies S3 and S4). Blood vessels are indicated by asterisks.

  • Fig. 6 Cell delivery, attachment, and viability in de-epithelialized lung.

    (A) Bioreactor schematic. R, reservoir of perfusate, cell culture medium; green circles, human SAECs or hiPSC-derived lung-specified epithelial cells delivered into de-epithelialized lung. (B and C) Delivery and attachment of SAECs in de-epithelialized lungs. Global distribution demonstrated with transpleural imaging of NIR-labeled cells (B) (dotted line indicates pleura). CFSE-labeled cells are distributed in the respiratory zone (C). (D to G) Lung section and histologic analysis of CFSE-labeled cells. H&E staining of native lung demonstrating intact pseudostratified respiratory epithelium (arrowhead) and vasculature (asterisk) (D) and of repopulated lung with intact native vasculature (asterisk) (E). CFSE-labeled SAECs attachment in the alveoli (F and G). (H to J) Immunostaining of native (H) and recellularized lungs with CFSE-labeled SAECs (I) with the epithelial marker Pankeratin. Inset: Higher-magnification images. Quantification of human and rat epithelial cells in recellularized lungs (n = 3; percentage of normalized to total Pankeratin-positive epithelial cells) (J). (K) Metabolic activity of native and recellularized lungs by resazurin assay (n = 3; P = 0.87, Student’s t test; error bars represent means ± SD of experimental values). OD, optical density. (L) Dynamic lung compliance measured by pressure-volume loops of native, de-epithelialized, and recellularized lungs (n = 3; native, 0.066 ± 0.0022 ml/cmH2O; de-epithelialized, 0.0169 ± 0.0021 ml/cmH2O; recellularized, 0.0257 ± 0.0015 ml/cmH2O; compliance decreased by ~74.20 ± 3.872% after de-epithelialization and increased by ~53.38 ± 20.83% after recellularization). Values are expressed as means ± SD. (M and N) Ki67 and caspase-3 (Cas 3) staining (indicated by filled arrowheads) demonstrating viable CFSE-labeled SAECs following 24 hours of culture in the bioreactor. Endogenous cells are indicated by the empty arrowhead. (O to T) Day 40 to 45 hiPSC-derived lung-specified epithelial cells, which were differentiated as described in Materials and Methods, were delivered into and attached in the de-epithelialized lung (O and P), expressing the human alveolar type I cell marker HT1-56 (Q and R) and the alveolar type II cell marker HT2-280 (S and T). (R) and (T) show higher magnification of boxed areas.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/8/e1700521/DC1

    table S1. Primary antibodies.

    table S2. Secondary antibodies.

    fig. S1. Lung de-epithelialization.

    fig. S2. Optimization of de-epithelialization solution in rodent lungs on EVLP.

    fig. S3. Efficiency of de-epithelialization.

    fig. S4. Preservation of lung structure and ECM.

    fig. S5. Dynamic lung compliance.

    fig. S6. Vascular viability and function after lung de-epithelialization.

    fig. S7. Cell attachment and viability in de-epithelialized lung.

    fig. S8. Schematic of ex vivo lung regeneration.

    fig. S9. Outline of stereologic analysis.

    movie S1. HFOV before de-epithelialization on EVLP.

    movie S2. HFOV during de-epithelialization on EVLP.

    movie S3. Native lung perfusion with microspheres.

    movie S4. De-epithelialized lung perfusion with microspheres.

  • Supplementary Materials

    This PDF file includes:

    • table S1. Primary antibodies.
    • table S2. Secondary antibodies.
    • fig. S1. Lung de-epithelialization.
    • fig. S2. Optimization of de-epithelialization solution in rodent lungs on EVLP.
    • fig. S3. Efficiency of de-epithelialization.
    • fig. S4. Preservation of lung structure and ECM.
    • fig. S5. Dynamic lung compliance.
    • fig. S6. Vascular viability and function after lung de-epithelialization.
    • fig. S7. Cell attachment and viability in de-epithelialized lung.
    • fig. S8. Schematic of ex vivo lung regeneration.
    • fig. S9. Outline of stereologic analysis.

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    Other Supplementary Material for this manuscript includes the following:

    • movie S1 (.mp4 format). HFOV before de-epithelialization on EVLP.
    • movie S2 (.mp4 format). HFOV during de-epithelialization on EVLP.
    • movie S3 (.mov format). Native lung perfusion with microspheres.
    • movie S4 (.mov format). De-epithelialized lung perfusion with microspheres.

    Files in this Data Supplement: