Production and transplantation of bioengineered lung into a large-animal model by Joan E. Nichols, Saverio La Francesca, Jean A. Niles, Stephanie P. Vega, Lissenya B. Argueta, Luba Frank, David C. Christiani, Richard B. Pyles, Blanca E. Himes, Ruyang Zhang, Su Li, Jason Sakamoto, Jessica Rhudy, Greg Hendricks, Filippo Begarani, Xuewu Liu, Igor Patrikeev, Rahul Pal, Emiliya Usheva, Grace Vargas, Aaron Miller, Lee Woodson, Adam Wacher, Maria Grimaldo, Daniil Weaver, Ron Mlcak, and Joaquin Cortiella Sci Transl Med Volume 10(452):eaao3926 August 1, 2018 Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

Fig. 1 Study overview. Study overview. (A) Left lung scaffolds were produced from whole acellular pig lungs. (B) Catheters were placed into the trachea (TR), pulmonary artery (Pa), and pulmonary vein (Pv) and (C and D) were positioned in the chamber to permit visualization of catheters. (E) Diagram of the fluidic system shows the microfluidic and pumping system. OS, oxygen sensor. (F) Photograph of the system outlined in (E) (arrow points to oxygenator). (G and H) BEL on culture day 30. The BEL in (G) and (H) was produced using the scaffold in images (A) to (D). (H) Photograph of BEL being prepared for transplantation. Scale bar, 20 cm. (I) BEL in surgical suite and (J) after the trachea-to-trachea anastomosis. n = 6 BELs were created. Joan E. Nichols et al., Sci Transl Med 2018;10:eaao3926 Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

Fig. 2 Gross assessment of BEL and native lung. Gross assessment of BEL and native lung. (A to C) Bronchoscopy images of (A) BEL before and after transplant and of (B) the area above the anastomosis site showing left main stem bronchus, and (C) BEL trachea-to-trachea anastomosis (black arrow). (D and E) CT angiograms of the thorax of pig 1, 2 weeks after transplant. Native lung (NL) and BEL. (D) Collateral circulation in BEL is highlighted (white arrows), and aerated regions appear black in this colorized image. (E) Collateral vessels formed in BEL after transplantation (black arrows). (F) Gross image of BEL [left lung (LL)] from pig 1 after transplant. Black arrow indicates anastomosis site. (G and H) Micro-CTs of open airway in nonventilated (G) native lung and (H) BEL of pig 1. (I to N) BEL of pig 4, 1 month after transplant. (I) CT angiogram of the thorax in the arterial phase, axial image showing BEL in the left thoracic cavity (red dots denote edges of BEL). The pulmonary artery (Pa), aorta (Ao), right ventricle (RV), and left ventricle (LV) are shown, as well as the left side (L) of the animal in this coronal image. (J) Coronal x-ray image showing BEL in the left hemithorax (red dots denote edges of BEL). (K) Axial CT image of both native lung and BEL. The heart (Ht) and left side of the animal are noted. (L) Coronal and (M) axial images of MRI angiography showing peripheral enhancement outlining the left BEL, indicating capillary vascularization. (M) MRI image showing full expansion of both right native lung and left BEL. “R” denotes the right side of the animal and “L” the left side. (L and M) White arrows point to large collateral vessels in BEL. (N) Gross image of the BEL after necropsy showing native lung and the smaller BEL. Joan E. Nichols et al., Sci Transl Med 2018;10:eaao3926 Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

Fig. 3 Genes expressed in BEL. Genes expressed in BEL. RNA sequence analysis of GE in BEL. FC > 1 indicates that GE value of BEL was greater than value of native lung, FC = 1 indicates that GE value of engineered lung was equal to value of native lung, and FC < 1 indicates that GE value of engineered lung was less than the value of native lung. (A) Heat map of the top 1000 genes (ranked by P values) from samples removed from three different regions of the BEL and native lung. (B) Table summarizing the number of genes in BEL exhibiting FC between 0 and 0.5, between 0.5 and 2, or >2 FCs in expression compared to native lung for tissue sets 1 to 3. (C) Table of BEL genes of interest related to angiogenesis with FC > 1 as compared to native lung. Joan E. Nichols et al., Sci Transl Med 2018;10:eaao3926 Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

Fig. 4 Vascular tissue development in BELs. Vascular tissue development in BELs. (A) Transmission electron microscopy (TEM) of BEL on day 30 of bioreactor culture demonstrating capillaries (ca; black arrows) without red blood cells. (B) Hematoxylin and eosin (H&E) image of BEL at 2 weeks after transplant and (C) TEM of red blood cell–filled collateral capillaries (black arrows). (D) Cross-sectional H&E image of collateral blood vessels in BEL 2 weeks after transplant. (E to W) BEL harvested 1 month after transplant. (E) Cross section of CFSE-labeled (green) vessel in BEL. (F and G) Blood vessels within BEL formed from CFSE-labeled (green) primary lung–derived vascular cells. (G) Overlay of CD31+ (red) staining with CFSE+ (green). (H) 4′,6-Diamidino-2-phenylindole (DAPI; blue, nuclei) staining control lung for (I). (I) Junction of collateral vessel outside of the BEL. VE-cadherin+ (VE-Cad; red) endothelial cells and CFSE-labeled primary lung–derived vascular cells (white arrow) were found where collateral vessels joined with the BEL vasculature. (J to S) Cross sections of BEL blood vessels. (J, L, N, P, and R) DAPI staining controls and sections stained for (K and M) CD31+ (red), (O) ERG+ (red), (Q) eNOS+ (red), and (S) ACE+ (red) cells, all of which are indicators of endothelial cell function in the BEL. (T and V) DAPI control and representative image showing LYVE-1+ (green) lymphatic cells at (U) 2 weeks and (W) 1 month after transplant. Joan E. Nichols et al., Sci Transl Med 2018;10:eaao3926 Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

Fig. 5 Lung tissue development in BELs. Lung tissue development in BELs. (A) Scanning electron microscopy (SEM) of acellular scaffold. (B) Methylene blue–stained thin section of BEL before transplantation highlighting nonaerated (black arrows) and aerated spaces. (C) SEM of alveoli of BELs before transplant. White arrows indicate nonaerated areas. (D) TEM image of AEC II (inset, lamellar body) before transplant. (E to K) Evaluation of pig 4 BEL at 1 month after transplant. (E) SEM of BEL after transplant demonstrating increased aerated regions due to normal breathing. A small compressed area remains (white arrow). (F) TEM image of AEC II (inset, lamellar body) after transplant. (G) TEM of BEL containing AEC I pneumocytes (arrow). (H) DAPI-stained control and (I) P-SPC+ (red) AEC II. (J) DAPI-stained control and (K) AQP5+ (green) AEC I cells. (L to O) Mean counts of total number of cells and number of AEC I cells in native lungs or BELs for pigs that survived for (L) 10 hours, (M) 2 weeks, (N) 1 month, or (O) 2 months. Student’s t test was used to compare total number of cells and total numbers of AEC I in native lung and BEL. Analysis of variance (ANOVA) was used to assess statistical significance in the comparison of (L) to (M), (N), and (O) (*P < 0.001) and (M) and (N) to (O) (***P < 0.0001). Joan E. Nichols et al., Sci Transl Med 2018;10:eaao3926 Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

Fig. 6 Pig 5 tissue development. Pig 5 tissue development. Pig 5 developed a partial airway occlusion after transplantation. Bronchoscopy images of (A) carina, (B) bronchial occlusion (arrow) and open airway, and (C) image of bronchial occlusion alone (black arrow) in BEL. (D) Anastomosis site of BEL in recipient’s trachea. (E) Chest x-ray of the nonaerated BEL, seen as the dense homogeneous opacity, projecting over the mediastinum. Extensive compensatory hyperinflation of the native lung occurred. (F) CT image of chest in venous phase through the left hemithorax showing collapsed BEL containing multiple small intercostal vessels (arrows). (G, I, and L) DAPI-stained controls and BEL in (G and H) aerated and (J to N) nonaerated regions containing P-SPC+ (red) and TUNEL+ (green) AEC II. (H) P-SPC+ (red) cells with inset of enlarged image. (J and K) BEL in nonaerated region cells stained for P-SPC+ (red) and TUNEL+ (green), a marker indicative of cells undergoing apoptosis. (L) DAPI-stained control and (M and N) FSP-1+ (red) fibroblasts in nonaerated regions. Five randomly selected areas from 10 different sections of tissue immunostained were examined for TUNEL+ or FSP-1+ cells in native lung and in aerated or nonaerated sections of BEL. (O) Averaged number of cells, number of P-SPC+ AEC II, TUNEL+ AEC II, and FSP-1+ cells ± SD are shown for native lung and aerated and nonaerated BEL. Data were analyzed using ANOVA. *P < 0.05, **P < 0.005, ***P < 0.0005. NS, not significant. Joan E. Nichols et al., Sci Transl Med 2018;10:eaao3926 Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

Fig. 7 BEL immune response. BEL immune response. BAL was performed on BEL and native lungs of all animals. (A and B) Examination of IL-8, 1L-1β, IL-6, IL-10, IL-12p70, IL-2, IL-4, and interferon-γ (IFN-γ) concentrations in BAL of (A) native lungs and BELs before transplant. (B) Polar plot showing cytokine concentrations for native lungs and overlay of data from BALs evaluated at 10 hours, 2 weeks, 1 month, or 2 months after transplantation. The polar plot highlights each animal’s immune response. (A to D) BALs performed on native lung at the time of the pneumonectomy and on BEL after euthanasia. (C) Number of CD8+ cells isolated from BALs. (D) Percentage of CD4+, CD8+ T lymphocytes, perforin-positive cells, and CD20+ B lymphocytes are shown. (E and G) DAPI-stained controls and (F and H) representative images of CD8+ T lymphocytes (green) in tissues of (F) native lung or (H) BEL. (I) Averaged CD8+ lymphocyte counts for tissue sections from pigs 1, 2, 4, and 5 native lungs and BELs. Analyses to compare native lung to BEL per pig were done using Student’s t test. Joan E. Nichols et al., Sci Transl Med 2018;10:eaao3926 Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

Fig. 8 Analysis of BEL microbiome. Analysis of BEL microbiome. (A) Representative SEM image of the native lung of pig 1 demonstrating the normal microbiome. (B) SEM image of sterile BELs before transplantation. (C) SEM of BEL 2 weeks after transplantation showing reduced microbial colonization of BEL. The composition of each microbiome was evaluated for (D) tracheal and (E) lung samples from native lung and BELs of pigs that survived for 10 hours, 2 weeks, or 1 month and are shown as proportional bar charts (average of at least two independent evaluations per sample). Data for native lung and BELs are labeled at the top of each bar. Joan E. Nichols et al., Sci Transl Med 2018;10:eaao3926 Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works