SC I ENCE TRANS LAT IONAL MED I C I N E | R E S EARCH ART I C L E

T I S SUE ENG INEER ING

1Division of Infectious Diseases, Department of Internal Medicine, University ofTexas Medical Branch (UTMB), Galveston, TX 77555, USA. 2Biostage Inc., Holliston,MA 01746, USA. 3Department of Microbiology and Immunology, UTMB, Galveston,TX 77555, USA. 4Weill Cornell Medical College, New York, NY 10065, USA. 5Depart-ment of Radiology, UTMB, Galveston, TX 77555, USA. 6Pulmonary Division, Depart-ment of Medicine, Massachusetts General Hospital, Boston, MA 02114, USA.7Environmental, Occupational Medicine, Epidemiology Department, TH Chan Schoolof Public Health, Harvard University, Boston, MA 02115, USA. 8Galveston NationalLaboratory, Assay Development Core, UTMB, Galveston, TX 77555, USA. 9Departmentof Biostatistics and Epidemiology, University of Pennsylvania, Philadelphia, PA 19104,USA. 10Houston Methodist Hospital Research Institute, Houston, TX 77030, USA.11Radiology Division of Cell Biology, University of Massachusetts Medical School,Worchester, MA 01605, USA. 12Center for Biomedical Engineering, UTMB, Galveston,TX 77555, USA. 13University of Texas Southwestern Medical School, Dallas, TX75390, USA. 14Department of Anesthesiology, UTMB, Galveston, TX 77555, USA.15Shriners Hospital for Children, Galveston, TX 77550, USA.*Corresponding author. Email: jnichols@utmb.edu (J.E.N.); jocortie@utmb.edu (J.C.)

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Production and transplantation of bioengineered lunginto a large-animal modelJoan E. Nichols1*, Saverio La Francesca2, Jean A. Niles1, Stephanie P. Vega3, Lissenya B. Argueta4,Luba Frank5, David C. Christiani6,7, Richard B. Pyles8, Blanca E. Himes9, Ruyang Zhang7, Su Li7,Jason Sakamoto10, Jessica Rhudy10, Greg Hendricks11, Filippo Begarani10, Xuewu Liu10,Igor Patrikeev12, Rahul Pal12, Emiliya Usheva13, Grace Vargas12, Aaron Miller8, Lee Woodson14,Adam Wacher14, Maria Grimaldo1, Daniil Weaver1, Ron Mlcak15, Joaquin Cortiella14*

The inability to produce perfusable microvasculature networks capable of supporting tissue survival and ofwithstanding physiological pressures without leakage is a fundamental problem facing the field of tissueengineering. Microvasculature is critically important for production of bioengineered lung (BEL), which requiressystemic circulation to support tissue survival and coordination of circulatory and respiratory systems to ensureproper gas exchange. To advance our understanding of vascularization after bioengineered organ trans-plantation, we produced and transplanted BEL without creation of a pulmonary artery anastomosis in aporcine model. A single pneumonectomy, performed 1 month before BEL implantation, provided the sourceof autologous cells used to bioengineer the organ on an acellular lung scaffold. During 30 days of bioreactorculture, we facilitated systemic vessel development using growth factor–loaded microparticles. We evaluatedrecipient survival, autograft (BEL) vascular and parenchymal tissue development, graft rejection, and micro-biome reestablishment in autografted animals 10 hours, 2 weeks, 1 month, and 2 months after transplant.BEL became well vascularized as early as 2 weeks after transplant, and formation of alveolar tissue wasobserved in all animals (n = 4). There was no indication of transplant rejection. BEL continued to developafter transplant and did not require addition of exogenous growth factors to drive cell proliferation or lungand vascular tissue development. The sterile BEL was seeded and colonized by the bacterial community ofthe native lung.

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INTRODUCTIONWhole bioengineered lungs (BELs) produced on acellular lung scaf-folds have been transplanted in small animal models, but lungs faileddue to intravascular coagulation and defects in endothelial barrierfunction leading to pulmonary edema (1, 2). No approach has allowedfor long-term survival of BELs after transplantation.

Awork examining passive diffusion of gas into the lung suggests thatnonvascularized lung can survive for periods of time without vascularsupport (3), such as ligation of the pulmonary artery (4). Here, wefocused on development of the bronchial systemic circulation in non-immunosuppressed pigs to support BEL growth and survival aftertransplantation. We performed a pilot study to establish feasibility ofBEL transplantation, with an airway anastomosis but without a vascular(pulmonary) anastomosis. We relied on the development of collateralsystemic circulation to support tissue survival (5, 6). BELs were createdusing autologous cells isolated from a left lung pneumonectomy for n =

6 pigs. Four pigs received implanted BELs 30 days after pneumonecto-my, whereas two animals were euthanized before receiving a BEL. Thisapproach allowed the opportunity to enhance our understanding ofpulmonary vascular development, initiate examination of the BEL tran-scriptome, evaluate BEL tissue development after transplant, examineBEL immune response, evaluate acute and chronic rejection, and exam-ine reestablishment of the microbiome within the BEL.

RESULTSDecellularizationPorcine acellular lung scaffolds were produced, as described previously(7, 8), with one modification. A dextrose pretreatment step was addedbefore decellularization of whole lungs. This was done to enhance pro-tein stability (9), reducing collagen loss during decellularization.Established multiphoton microscopy (MPM) and second harmonicgeneration methods (7, 8) demonstrated that collagen fibers were lessdamaged (fig. S1A compared to fig. S1B), and significantly more col-lagen (P < 0.002) was retained in scaffolds using dextrose-SDS decel-lularization (fig. S1C). Bronchoscopy was performed on all scaffoldsbefore recellularization (movie S1). Table S1 lists all of the abbrevia-tions used in the manuscript.

Supplementation of scaffoldIn past studies, acellular lung scaffolds were supplementedwith platelet-rich plasma (PRP)–loaded pluronic F-127 hydrogel (BASF) (7, 8) beforeinstallation of cells. The ability of hydrogels and nanoparticles to targetdelivery in support of vascular tissue development has been previouslydemonstrated (10, 11). We combined microparticle (MP) delivery ofvascular endothelial growth factor (VEGF) with hydrogel delivery of

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PRP, fibroblast growth factor 2 (FGF2), and keratinocyte growth factor(KGF). Discoidal porous silicon MPs (12) with 30- or 60-nm poresdelivered VEGF to vascular portions of acellular scaffolds. Images of1-mmVEGF-MPs showMP shape and structure (fig. S1, D and E). Suit-ability of growth factors was determined by measuring attachment ofprimary lung–derived vascular cells to 3 × 3 × 0.5–cm pieces of acellularblood vessel scaffold pretreated with media, VEGF-MP, a mixture ofVEGF-MP and FGF2 hydrogel, or FGF2 hydrogel (fig. S1, F to I). Useof VEGF-MP, FGF2-loaded hydrogel, or VEGF-MP mixed with FGF2hydrogel enhanced cell attachment (fig. S1J) beyond media or FGF2 hy-drogel alone, and VEGF-MP and FGF2 hydrogel provided best cell at-tachment. Use ofMPs with different pore sizes allowed for staged releaseof VEGF (fig. S1K). Hydrogels loaded with FGF2 or KGF released at asteady rate over time (fig. S1L). In whole acellular lung scaffolds, VEGF-MP (fig. S1, M and N) and FGF2 hydrogel (fig. S1O) were depositedwithin the small vessels and capillaries of the scaffold. Tracheal deliveryof KGF hydrogel was also used to support cell attachment (fig. S1P).

Mesenchymal stem cells (MSCs) support angiogenesis, produce im-munomodulatory factors, promote lung repair (13), regulate macro-phage function (14), and combined with M2 macrophages contributeto tissue regeneration (15). To study the effects ofMSC andM2 cells onlung tissue development, we added porcine MSCs, unpolarized macro-phages, M1 or M2 macrophage subsets, mononuclear leukocytes(MNLs), or lipopolysaccharide (LPS)–stimulated MNLs or culturesupernatants from these cell types to primary lung cells seeded onto3 × 3 × 0.5–cm pieces of acellular lung scaffold. Increased cell attach-ment (fig. S2A) and proliferation, measured by Ki67 staining, occurredwhen primary lung cells were cultured on KGF hydrogel–pretreatedscaffolds (fig. S2B) or with addition of MSC supernatant, MSC, M2 cellsupernatant, orM2 cells to primary lung cultures, justifying use of thesesupplements in the production of whole BEL.

Production of BEL for transplantationProcedures for recellularization of whole acellular pediatric scaffoldswith adult lung–derived cells (7) were modified for use in this study.Changes included installation of VEGF-MP and FGF2 hydrogel intothe pulmonary artery of whole acellular scaffolds 2 hours beforeprimary vascular cell installation and addition of KGFhydrogel 2 hoursbefore primary lung cell installation.

The primary lung cell preparation included aquaporin-5–positive(AQP5+) alveolar epithelial type I (AEC I) cells, prosurfactant proteinC–positive (P-SPC+) AEC II, smooth muscle actin–positive (SMA+)cells, and fibroblast-specific protein 1–positive (FSP-1+) fibroblasts (fig.S3, A to I). Cells in the primary lung–derived vascular cell preparationcontained CD31+ and vascular endothelial cadherin–positive (VE-CAD+)cellswith SMA+andFSP-1+ cells (fig. S3, J toR). Primary tracheal-bronchialcells were pan-cytokeratin–positive (Ck+), Ck-18+, epithelial celladhesion molecule–positive (Ep-CAM+) cells, with few Clara cellprotein-10+ (CC10) or FSP-1+ cells included (fig. S3S to CC). Cell in-stallation information (table S2) and numbers of autologous cellsinstalled into lung scaffold (table S3) are provided. Primary lung–derived vascular cells were installed into the pulmonary artery andprimary lung, and primary tracheal-bronchial cells were installed intothe trachea; MSC supernatant, MSCs, M2 macrophage supernatant,andM2 cells were added during BEL culture (tables S2 and S3). Oxygenconcentrations were uniform for media alone or media and scaffoldcultures over 30 days. There was a slow decrease in oxygen concen-tration over the 30-day BEL culture period, as oxygenwas consumed bythe cells of the BEL (fig. S4). In a subset of scaffolds, carboxyfluorescein

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succinimidyl ester (CFSE)–labeled primary lung cells were installed intoscaffolds, and a Spectrum in vivo imaging system (IVIS) was used toexamine cell dispersal on pieces of acellular scaffolds (fig. S5, A andB) or whole-lung scaffolds (fig. S5, C to F).

An overview of BEL production is shown in Fig. 1 (A to H). The leftlungs removed fromdonor pigs (Fig. 1A) were used to produce left lungscaffolds for this study (Fig. 1B). The pulmonary artery, pulmonaryvein, and trachea of the scaffold were cannulated as described (Fig. 1,B to D) (7).

Transplantation and outcomesSix nonimmunosuppressed pigs were slated to receive a BEL transplant,with two pigs euthanized before BEL transplantation because of surgicalcomplications related to the left lung pneumonectomy. Four animals re-ceived autologous BELs 30 days after a left pneumonectomy and wereeuthanized at 10 hours (pig 2), 2 weeks (pig 1), 1 month (pig 4), and2 months (pig 5) after transplantation (fig. S6A). After surgery, pig 5developed a partial airway occlusion that reduced lung expansion. Pulseoximetry remained at 100% throughout the testing period. All pulmo-nary function measurements showed a trend toward return to baselinevalues, suggesting that transplanted lungs had normal pressures andvolumes (fig. S6, B to D). Bronchoscopy of BELs was performed before(Fig. 2A and movie S2) and after transplantation (Fig. 2B, fig. S2C, andmovie S3). Small blood vessels were seen mid-trachea and at the anas-tomosis site (Fig. 2, B and C) in animals that survived for longer than10 hours. Computed tomography (CT) angiograms of the thorax ofpig 1 (survived 2weeks) comparing BEL andnative lung (Fig. 2, D andE)depict the development of collateral blood circulation in BEL by 2 weeksafter transplant. Figure 2F is a gross image of this BEL. Micro-CTs ofnative lung and BEL demonstrated that both BEL and native lung con-tained open airways and comparable tissue density (Fig. 2, G and H).

In pig 4 (survived 1 month), a CT angiogram of the thorax in thearterial phase, axial image, shows aerated portions of the BEL (Fig. 2I).The aorta, pulmonary artery, right ventricle, and left ventricle are noted.A coronal image of this animal shows the BEL in the left hemithorax(Fig. 2J). Hyperinflation of the right lung resulted in herniation of thenative lung into the inferior left hemithorax. This contributed to therestricted expansion of the BEL (Fig. 2J), although the transplanted leftlung became aerated during breathing (Fig. 2K). Coronal and axialimages of magnetic resonance imaging (MRI) angiography displaythe peripheral enhancement outlining the left BEL due to capillary re-vascularization (Fig. 2, L andM). A large intercostal vessel arising fromthe aorta with branches extending toward the BEL is noted on the axialimage (Fig. 2M, arrow). The gross image of the BEL from pig 4, afternecropsy, shows the smaller size of the left BEL compared to the rightnative lung (Fig. 2N).

BEL transcriptome profileWe initiated the examination of BEL gene expression (GE) profiles at1 month after transplant (pig 4) to determine whether angiogenesis ortissue development was still in progress and to identify key time pointsfor examination of the BEL transcriptome in later studies. We tested4128 genes isolated from BEL or native lung samples isolated fromthe same animal, setting the GE of native lung as reference andcalculating fold changes (FCs) of GE for BEL. Here, FC was defined as

FC ¼ ðGEEngineered þ 1ÞðGENative þ 1Þ

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Although there were variations in levels of GE in BEL compared tonative lung, the types of genes expressed were similar (Fig. 3A). Com-pared with native lung, an average of 11.79% of the genes were down-regulated (0 < FC ≤ 0.5), and 15.93% were up-regulated (FC ≥ 2) inBEL (Fig. 3B). Themajority of genes (72.28%, 0.5 < FC < 2) remained atthe same expression level as found in native lung (Fig. 3B). We per-formed a paired Student’s t test between BEL and native lung GE withlog2 transformation. Genes with FC ≥ 2 or FC ≤ 0.5 (P < 0.05) were

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defined as potential differentially ex-pressed genes (Fig. 3, A to C, and tablesS4 to S5).

Angiogenesis-related genes that wereup-regulated in the BEL at 1 month aftertransplant includedMAPK14 (FC=5.00),TGFB2 (FC = 5.00), PDGFC (FC = 3.00),VCAM1 (FC = 3.00), VEGFD (FC =3.00), HEY 1 (FC = 2.50), SRY-Box-9(SOX-9; FC = 3.00), PDGFRA (FC =2.50), SHH (FC = 2.25), SRY-Box-15(SOX-15; FC = 2.00), FGFR1 (FC =2.00), SELP (FC = 2.00), Wnt10B (FC =2.00), ETV2 (FC = 2.00), and ICAM1(FC = 2.00; Fig. 3C). Other up-regulatedgenes included KDR/VEGF2R (FC =1.55), CXCL12/SDF-1 (FC = 1.33), NRP1(FC = 1.28), SRY-Box-4 (SOX-4; FC =1.25), and CXCR4 (FC = 1.17; Fig. 3C),as well as ITG2AB/CD41, ETS1, TGFB1,HEY2, PROX1, VEGFC, PECAM1,NOS2, NOS1, and SELE (table S4). Innormal vascular development, one of themajor signaling pathways is Notch (16).Although there was increased expressionof downstreamNotch target ligands in theBEL, Hey1 (FC = 3.00) and HeyL (FC =2.50), this expression was not as robustas would have been expected if produc-tion of the BELwas purely a developmen-tal process (17). Genes expressed in theBEL also included lung lineage geneNKX2-1 (FC = 1.40) and AEC I cell–associated genes AQP5 (FC = 2.00),SCNN1G (FC = 2.00), CAV-1, andRAGE/Ager or AEC II–associated genesSFTPC, SFTPB, SFTPD, and SFTPA1(table S5). Other lung epithelial cell–associated genes expressed in the BELincluded KRT19, MUC20, MUC13,MUC15, TP63, MUC1, and KRT5 (tableS4). Genes normally expressed by neuro-endocrine cells (CHGA, ENO2, andFOXF2), Clara cells (SCGb3A2), or musclecells (ACTG2,ACTA1,ACTB, andACTA2;table S5) were also expressed.

The gene profile of the BEL was simi-lar to that of the native lung, although theBEL exhibited distinct expression pro-files. Despite this study’s limitations dueto the small sample size, the information

generated provides an importantGE data set to build from in the future.

BEL vascular and lymphatic developmentPretransplant capillaries in BEL contained no red blood cells (Fig. 4A).Posttransplantation collateral circulation developed in pig 1 within2 weeks (Fig. 4B) and developed in all animals that survived 2 weeks orlonger (Fig. 2E). BEL microvasculature appeared normal (Fig. 4, C andD), and, on the basis of CFSE labeling of primary vascular cells, vessels

Fig. 1. Study overview. (A) Left lung scaffolds were produced from whole acellular pig lungs. (B) Catheters were placedinto the trachea (TR), pulmonary artery (Pa), and pulmonary vein (Pv) and (C and D) were positioned in the chamber topermit visualization of catheters. (E) Diagram of the fluidic system shows themicrofluidic and pumping system. OS, oxygensensor. (F) Photographof the systemoutlined in (E) (arrowpoints to oxygenator). (G andH) 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.

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in the BELs were formed from installed cells (Fig. 4, E to G), although fewCFSE-labeled cells were found at the junction point between the BEL cir-culation and the animals’ normal vasculature (Fig. 4I, with Fig. 4H as the

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control). Pigs were spontaneously breathing 21% oxy-gen beforeBELharvest. The average partial pressure ofoxygen (pO2) in the BEL pulmonary artery of thesepigs was 123 ± 10 mmHg, indicating it was receivingoxygenated blood and not venous blood from the col-lateral circulation. The lack of an oxygen gradient atthe alveolus capillary junction prevented gas exchange,as has been documented in past studies (4, 6).

Vessels in BELs expressed CD31 (Fig. 4, K andM; J and L are controls) and angiogenesis markersincluding transcription factor early growth responseprotein 1 (ERG1) (Fig. 4O, with Fig. 4N as the con-trol), endothelium nitric oxide synthase (eNOS; Fig.4Q, with Fig. 4P as the control), and angiotensin-converting enzyme (ACE; Fig. 4S, with Fig. 4R asthe control), which contributes to vascular muscletone and blood flow (18). Lymphatic vessel endothe-lial receptor 1–positive (LYVE-1+) areas were seen at2 weeks (Fig. 4U, with Fig. 4T as the control), and by1 month after transplant, lymphatic vessels werefound throughout the BEL (Fig. 4W, with Fig. 4Vas the control), suggesting reestablishment of pul-monary lymphatics.

BEL tissue developmentAcellular distal lung scaffold lacks structure (Fig. 5A).After recellularization, on day 30 of bioreactorculture, the BEL contained well-developed alveolarareas (Fig. 5, B and C), although nonaerated regions(Fig. 5, B andC, arrows)were evident. PSP-C+AEC II(Fig. 5D) was the predominant cell type beforetransplant. After transplantation, normal breath-ing enhanced aeration of the BEL, although occa-sional nonaerated areas were present (Fig. 5E).PSP-C+ AEC II (Fig. 5, F and I, with Fig. 5H asthe control) and AQP5+ AEC I were present inall animals (Fig. 5, G and K, with Fig. 5J as thecontrol). Compared to the animal that survived for10 hours (Fig. 5L), the total number of cells increasedin BEL (Fig. 5M) of animals that survived for1month (Fig. 5N) and 2months (Fig. 5O). The totalnumber of AEC I also increased in pigs that sur-vived for 2 weeks and 1 month but not in pig 5, inwhich a partial occlusion (Fig. 5O) reduced aerationand stretch of tissues.

Acellular trachea scaffold (fig. S7A) was sup-plemented with FGF2 hydrogel to support trache-al cell attachment (fig. S7, B and C). FGF2hydrogel provided for better cell attachment thanmedia or hydrogel alone (fig. S7D). Two weeks af-ter transplant, cells were dispersed (fig. S7, E andF) and lacked cell-to-cell contacts. By 2 months,cells in the BEL trachea had reestablished cell-cellcontacts and developed intercellular junctions(fig. S7, G to J) in most areas.

Ki67+, proliferating cells were found in

bronchioles (fig. S8B, with fig. S8A as the control) and lungs (fig.S8D, with fig. S8C as the control) of all BELs. Higher numbers ofKi67+ cells were found in animals that survived for 2 weeks and

Fig. 2. Gross assessment of BEL and native lung. (A to C) Bronchoscopy images of (A) BEL before andafter 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 weeksafter 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 trans-plantation (black arrows). (F) Gross image of BEL [left lung (LL)] from pig 1 after transplant. Black arrowindicates 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 arterialphase, axial image showing BEL in the left thoracic cavity (red dots denote edges of BEL). The pulmo-nary 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 (reddots denote edges of BEL). (K) Axial CT image of both native lung and BEL. The heart (Ht) and left side ofthe animal are noted. (L) Coronal and (M) axial images of MRI angiography showing peripheral enhance-ment outlining the left BEL, indicating capillary vascularization. (M) MRI image showing full expansion ofboth right native lung and left BEL. “R” denotes the right side of the animal and “L” the left side. (L andM) White arrows point to large collateral vessels in BEL. (N) Gross image of the BEL after necropsyshowing native lung and the smaller BEL.

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1 month after transplantation (fig. S8E). Key changes after trans-plantation included continued angiogenesis and development ofthe epithelial lining of the trachea, bronchi, and bronchioles inBEL. Although alveoli and bronchioles were not well developedat 10 hours (fig. S8, F to H) or 2 weeks (fig. S8, I to K), continueddevelopment occurred in animals that survived for 1 month (fig.S8, L to N) or 2 months (fig. S8, O to Q). BEL alveoli and bronchiwere indistinguishable from native lung except within nonaeratedregions in pig 5.

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Ck-18+ cells (fig. S9B, with fig. S9A asthe control) and CC10+ Clara cells werepresent in developing bronchioles (fig.S9D, with fig. S9C as the control) as weremucin-producing cells (fig. S9E) includ-ingmucin 5a (MUC5a), a proteinmarkerof developing airway epithelium (fig. S9G,with fig. S9F as the control), and MUC1(fig. S9I, with fig. S9H as the control).Low numbers of lung progenitor cellphenotypes, such as CK5+/ P63+ cells(19), were found (fig. S9K, with fig. S9Jas the control) in BELs.

Pig 5 developed an occlusion of thefirst branch of the main stem bronchusof the BEL after transplantation. Bothpassageways at the point of the carinawere open, as shown by bronchoscopy,2 months after transplantation (Fig. 6A),but the left bifurcation of the lung wasoccluded (Fig. 6, B to D). Chest x-raywas performed due to breath soundsin the left chest cavity. The left lung ap-peared small, dense, and partially aerated(Fig. 6, D and E), although CT imagesindicated the presence of multiple inter-costal vessels (Fig. 6F). P-SPC+ AEC II(Fig. 6H, with Fig. 6G as the control)were found in compressed, nonaeratedareas, and many of these cells were un-dergoingapoptosis [terminal deoxynucleo-tidyl transferase–mediated deoxyuridinetriphosphate nick end labeling–positive(TUNEL+)] (Fig. 6, J, K, and O, withFig. 6I as the control). More FSP-1+ fi-broblasts were found in nonaeratedversus aerated regions of the lung orcompared to native lung (Fig. 6, M toO, with Fig. 6L as the control). Asexpected, native lung contained morecells and more P-SPC+ cells than BEL(Fig. 6O).

Immune response of BELContamination of long-term bioengi-neered tissue cultures is a commonproblem. There is also increased sus-ceptibility to infection of pulmonarygrafts after transplantation, due to con-tact with microbial contaminants dur-

ing breathing (20, 21). As a preventative antimicrobial strategy, theimmune systems of BELs were reconstituted. Autologous MNLswere added on day 11 of bioreactor culture and autologous serum,alveolar macrophages, and MNLs on day 30 before transplantation.

Cytokine analysis was performed on bonchioalveolar lavage (BAL)fluid isolated from native lung and BEL. Before transplant, there werelow concentrations of proinflammatory cytokines in native lungs andBELs (Fig. 7A). Pig 2, euthanized 10 hours after transplant, had a mea-surable proinflammatory response due to an undiagnosed infection at

Fig. 3. Genes expressed in BEL. RNA sequence analysis of GE in BEL. FC > 1 indicates that GE value of BEL wasgreater than value of native lung, FC = 1 indicates that GE value of engineered lung was equal to value of nativelung, and FC < 1 indicates that GE value of engineered lung was less than the value of native lung. (A) Heat map ofthe top 1000 genes (ranked by P values) from samples removed from three different regions of the BEL and nativelung. (B) Table summarizing the number of genes in BEL exhibiting FC between 0 and 0.5, between 0.5 and 2, or >2FCs in expression compared to native lung for tissue sets 1 to 3. (C) Table of BEL genes of interest related to angi-ogenesis with FC > 1 as compared to native lung.

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the time of euthanasia (Fig. 7B). Cytokineconcentrations decreased as survival timeof animals increased (Fig. 7B). There wasno indication of a T cell response afterBEL transplantation as demonstrated bylow concentrations of interleukin-2(IL-2) or IL-12p70 and lack of increasein CD8+ cell numbers in BAL (Fig. 7C).Numbers of CD4+, CD8+, perforin-containing cells, andCD20+- or immuno-globulin G–expressing B lymphocyteswere not significantly different in BELcompared to native lung (Fig. 7D). Therewas also no difference in location of CD8+

lymphocytes in airways of native lung(Fig. 7F, with Fig. 7E as the control) andBELs (Fig. 7H,with Fig. 7G as the control)or number of CD8+ cells in tissues (Fig.7I). These data indicate that the autologousBELs were well tolerated, with no infil-tration of leukocytes into tissues or up-regulation of T cell responses indicativeof graft dysfunction or rejection.

BEL microbiome developmentTransplantation of the sterile BELsprovided an opportunity to observe theestablishment of the pulmonary micro-biome communities in the respiratorytree (21). Native lung contains a well-developed microbiome (Fig. 8A), butBELs are sterile before transplant (Fig.8B), and no organisms were found inBEL until after transplantation (Fig. 8C).Evaluation of the established microbiomeover a time course helped us address theBEL from the perspective of the bacterialcommunity. We completed initial next-generation sequencing of samples fromfour pigs housed in our facility to identifythe core microbiome of the respiratorytree. The resulting data were consistentwith the limited published microbiomedata for pig lung (22) and identified themost common genera or species presentin these laboratory animals. Optimizedquantitative polymerase chain reaction(qPCR) targets and assays were thenestablished to quantify the commonbacteria and selected minor species asso-ciated with pathogenic infections to eval-uate the seeding of BEL (table S8). Onebacterial target was based on identifica-tion of 16S sequences that did not alignwith sequences in the SILVA rRNAdatabase. Specifically, IOLA (infectiousorganism lurking in airways) (22, 23)16S was seen in one of the transplantedand two of the control animals studied.

Fig. 4. Vascular tissue development in BELs. (A) Transmission electron microscopy (TEM) of BEL on day 30 ofbioreactor 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 (blackarrows). (D) Cross-sectional H&E image of collateral blood vessels in BEL 2 weeks after transplant. (E to W) BELharvested 1 month after transplant. (E) Cross section of CFSE-labeled (green) vessel in BEL. (F and G) Blood vesselswithin BEL formed from CFSE-labeled (green) primary lung–derived vascular cells. (G) Overlay of CD31+ (red) stain-ing with CFSE+ (green). (H) 4′,6-Diamidino-2-phenylindole (DAPI; blue, nuclei) staining control lung for (I). (I) Junctionof 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 theBEL. (T and V) DAPI control and representative image showing LYVE-1+ (green) lymphatic cells at (U) 2 weeks and (W)1 month after transplant.

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Fig. 5. Lung tissuedevelopment in BELs. (A) Scanning electronmicroscopy (SEM) of acellular scaffold. (B) Methyleneblue–stained thin section of BEL before transplantationhighlighting nonaerated (black arrows) and aerated spaces. (C) SEMof 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 tonormal 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 toO) Mean counts of total number of cells and numberof AEC I cells in native lungs or BELs for pigs that survived for (L) 10 hours, (M) 2 weeks, (N) 1month, or (O) 2months. Student’s t test was used to compare total number of cellsand total numbers of AEC I in native lung and BEL. Analysis of variance (ANOVA)was used to assess statistical significance in the comparisonof (L) to (M), (N), and (O) (*P<0.001)and (M) and (N) to (O) (***P < 0.0001).

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This resultwas confirmed through cloning and sequencing of additionalgenomic fragments using published PCR primers (23).

The composition of eachmicrobiomewas evaluated for tracheal andlung samples from each of three pigs and is shown as proportional barcharts (average of at least two independent evaluations per sample) inFig. 8. Tracheal and lung colonization occurred within 10 hours of thetransplant; however, the profile of these communities appeared to beless stabilized, with more bacterial targets detected in the trachea ofthe transplant relative to the normal lung. Respiratory problems forcedearly euthanasia of this animal. The qPCR detected extremely highamounts of Mycoplasma flocculare in the bioengineered trachea andlung communities, suggesting that this organismmay have contributedto the signs of clinical disease that warranted early euthanasia. The2-week BEL tissue showed slight but not significant differences inproportions ofMoraxella species and Staphylococcus species in thetrachea (P > 0.05). The paired native lung and BEL samples for the

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2- and 1-month transplants also showed similar bacterial communitieswith nearly identical representation and proportions. There were somenotable differences in the 1-month tissues, including theM. flocculare(24) observed in the 10-hour samples.

DISCUSSIONTo date, regenerative laboratories have attempted to engineer only a fewwhole organs. This endeavor requires engineering not only the organbut also vascular tissues to maintain a healthy organ with full function-ality. We concentrated our initial efforts on developing the micro-vasculature and systemic support in the BEL and found that collateralsystemic circulation developed in all animals that survived 2 weeks orlonger. Because BEL was supplied with oxygenated rather thandeoxygenated blood, wewere unable to assess gas exchange due to a lackof an oxygen gradient at the alveolar capillary junction.

Fig. 6. Pig 5 tissue development. Pig 5 developed a partial airway occlusion after transplantation. Bronchoscopy images of (A) carina, (B) bronchial occlusion (arrow) andopen 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, seenas the dense homogeneous opacity, projecting over the mediastinum. Extensive compensatory hyperinflation of the native lung occurred. (F) CT image of chest in venousphase 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 nonaeratedregion 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) fibroblastsin 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 inaerated 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 aeratedand nonaerated BEL. Data were analyzed using ANOVA. *P < 0.05, **P < 0.005, ***P < 0.0005. NS, not significant.

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GE related to angiogenesis and lung tissue development indicatedthat tissue development was still in progress 1 month after transplanta-tion. Histological examination of tissues indicated that collateral circu-lation developed in all animals as early as 2 weeks after transplant.Histological evaluation showed progression in lung and airway epithe-lial cell development with an associated increase in overall cell numbersand AEC I in animals that survived from 10 hours to 2 months. Cellsassociated with lung-specific lineages were found in all animals at alltime points examined, although there were few Clara cells in the devel-oping airways of animals due to the lack of primary Clara cells in theprimary tracheal bronchial cell preparation.

One obvious limitation of this study is the small sample size relatedto genome analysis. However, this finding was supported by other

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FrfoluB12(IBa(Btonoa1tepa(Antho(CisclypB(EcpCinoCfoplutotou

methods of analysis and indicated that genes related to angiogenesis andlung cell lineages remained elevated in BEL 2 months after transplan-tation. Another limitation is the need to continue survival of animalsbeyond 2 months with subsequent evaluation of the ability of theseanimals to survive relying only on oxygen provided by their BEL alone.

Acute lung rejection, characterized by perivascular and sub-endothelial mononuclear infiltrates or by lymphocytic bronchitis andbronchiolitis, was not seen inBELs.Wedid not see a significant increasein the presence of proinflammatory cytokines in tissues isolated fromBELs, except in pig 2 (10-hour survival). This animal was later shown tohave high numbers of M. flocculare, a swine pathogen. We saw noindication of primary graft rejection in animals that survived for10 hours, 2 weeks, 1 month, or 2 months based on BAL evaluations

ig. 7. BEL immuneesponse. BAL was per-rmed on BEL and nativengs of all animals. (A and) Examination of IL-8, 1L-b, IL-6, IL-10, IL-12p70, IL-, IL-4, and interferon-gFN-g) concentrations inAL of (A) native lungsndBELs before transplant.) Polar plot showing cy-kine concentrations forative lungs and overlayf data from BALs evalu-ted at 10 hours, 2 weeks,month, or 2 months af-r transplantation. Theolar plot highlights eachnimal’s immune response.toD)BALsperformedonative lung at the time ofe pneumonectomy andn BEL after euthanasia.) Number of CD8+ cellsolated fromBALs. (D) Per-entage of CD4+, CD8+ Tmphocytes, perforin-ositive cells, and CD20+

lymphocytes are shown.and G) DAPI-stained

ontrols and (F and H) re-resentative images ofD8+ T lymphocytes (green)tissues of (F) native lungr (H) BEL. (I) AveragedD8+ lymphocyte countsr tissue sections fromigs 1, 2, 4, and 5 nativengs and BELs. Analysescompare native lungBEL per pig were done

sing Student’s t test.

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or histopathology. No marked structural abnormalities were found inBEL tissues in pigs 1, 2, or 4. Pig 5, however, developed a partial airwayocclusion after surgery and showed some underdeveloped lung areas.Representative images indicated that aerated regions of the lungdisplayed normal lung architecture.

Our study provided the opportunity to examine the reestablishmentof the microbiome in a sterile BEL after transplantation. Recent reportshighlight a role for lung microbiota in control of lung injury and re-modeling after transplantation (25) and development of bronchiolitisobliterans syndrome, which impacts long-term survival (26). The steriletissues appear to have been seeded via the trachea, as noted from theresults of the animal that survived for 10 hours; however, more workwill be required to confirm this route of colonization. The distinct bac-terial communities we observed were consistent with other reports forthe swine lung (21) and were consistently reproduced in BEL. Theseevaluations also led to the observation of an infectious organism presentin airways, suggesting that this organismmay be of pathogenic concernin swine; moreover, IOLA had previously been reported only in thehuman respiratory tract in association with clinical disease (23).

In conclusion, we have shown that the nanoparticle and growthfactor hydrogel modification of acellular scaffolds was essential to thesuccess of this study and that continued vascular development occurs

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in animals after transplantation of BELs. These resultsalso support the utility of the platform used to produceand transplant BEL for the general study of BEL develop-ment, including the transcriptome, vascular tissue devel-opment, immune response related to rejection, andmicrobiome formation. This platform would also allowexamination of the influence of the microbiome onBEL survival and function in future studies. Together,these findings represent a significant advance in ourunderstanding of the production of bioengineered tissuesfor transplantation. Future studies should concentrate onprocedures to allow continued maturation of the BEL invivo and establishment of vascular flow via the pulmo-nary artery and pulmonary vein.

MATERIALS AND METHODSStudy designThe objective of this studywas to explore development ofthe systemic circulation after transplanting BELs into alarge-animal (pig) model with tracheal anastomosis butwithout reattachment of the pulmonary circulation. Weused a three-dimensional model of porcine lung tissue toselect methods of growth factor delivery and scaffoldsupplements that enhanced vascular and lung tissue de-velopment. BELs were created from autologous primarylung, and vascular cells were isolated from a left lungpneumonectomy performed 30 days before BEL trans-plantation. Porcine lungs for acellular scaffold produc-tion were obtained as discarded surgical materials attheUniversity of TexasMedical Branch (UTMB) or wereobtained following the Institutional Animal Care andUse Committee (IACUC)–approved protocols at theTexas Methodist Hospital Research Institute. Animalhandling and surgical procedures for obtaining porcineperipheral blood or BEL transplantation were performedaccording to protocols approved by the IACUC of

UTMB at Galveston and were compliant with guidelines of the Amer-ican Association for the Accreditation of Laboratory Animal Care.Animals were not immunosuppressed in this study. Replicate numbersof each experiment are included in the figure captions. Tissues from n =6BELs before transplant and n= 4BELs after successful transplantationwere randomized before examination. Histology analysis and cellcounts were performed by trained individuals who were blinded tothe study. One animal (pig 2) was euthanized early due to respiratorycomplications at 10 hours. Pig 5 developed an airway occlusion aftersurgery, limiting BEL development, and samples from this animal werenot used for microbiome analysis. Animals that survived for 2 weeks,1 month, and 2 months demonstrated development of collateral sys-temic circulation, BEL survival, and tissue development after transplan-tation. Antibodies used in this study are listed in tables S6 and S7.

Statistical analysisAll viability, genomic, histology, imaging, and microbiome analysesdata for each pig compared each animal’s BEL to its native lung.For cell phenotype analysis, 10,000 cells were collected for each flowcytometry sample examined. For specified data comparisons, a pairedsamples Student’s t test was used to compare means. For other datasets, ANOVA was used as noted. Statistical analyses for these data

Fig. 8. Analysis of BEL microbiome. (A) Representative SEM image of the native lung of pig 1 de-monstrating the normal microbiome. (B) SEM image of sterile BELs before transplantation. (C) SEM ofBEL 2 weeks after transplantation showing reduced microbial colonization of BEL. The compositionof each microbiome was evaluated for (D) tracheal and (E) lung samples from native lung and BELsof 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 arelabeled at the top of each bar.

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were performed using GraphPad Prism v7.0.04. Mean values and SDsare reported. Mean differences in the values were considered significantwhen P < 0.05. For microbiome analysis, mathematical analyses wereperformed using Excel (Microsoft Corp.). Graphing was competedusing Excel or GraphPad InSTAT software (version 2003).

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SUPPLEMENTARY MATERIALSwww.sciencetranslationalmedicine.org/cgi/content/full/10/452/eaao3926/DC1Materials and MethodsFig. S1. Scaffold production and modification.Fig. S2. BEL culture supplements.Fig. S3. Cell phenotypes installed in BEL.Fig. S4. Bioreactor culture BEL pO2 measurements.Fig. S5. IVIS imaging to estimate cell dispersal.Fig. S6. Information regarding study animals.Fig. S7. Evaluation of tracheal development.Fig. S8. BEL tissue development.Fig. S9. Cell phenotypes in BEL.Table S1. Abbreviations used in the manuscript.Table S2. Cell installation information.Table S3. Number of cells Installed in scaffolds.Table S4. RNA sequence data: Angiogenesis.Table S5. RNA sequence data: Cell lineage.Table S6. Antibodies used for histochemical cell phenotype analysis.Table S7. Antibodies used for flow cytometry analysis.Table S8. Microbiome primers used in this study.Movie S1. Bronchoscopy of acellular pig lung scaffold.Movie S2. Bronchoscopy of BEL before transplant.Movie S3. Bronchoscopy of BEL after transplant.References (27–44)

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Acknowledgments: We thank the UTMB Animal Resource Center and our veterinary staffC. Klages, D. Brining, and D. Deyo for help with support of our animals. We thank A. Duarte,D. Christiani, and J. Leduc for reading and suggesting edits for the manuscript. We thankJ. Barral for his help regarding use of osmolytes to protect proteins from denaturation. Wethank M. Riddle, E. Suarez, and C. Bryant for their help with this study. We thankM. Susman and C. Holubar for editorial assistance. Funding: This work was supported in partby NIH U18 Grant (grant no. U18TR000560-01). J. Leduc provided funds to support BELproduction and transplantation. Additional funding for production of nanoparticles wasprovided from startup funds provided to J.S. from Houston Methodist Research Institute ofHouston. Author contributions: J.E.N. managed the BEL production team and data analysis

Nichols et al., Sci. Transl. Med. 10, eaao3926 (2018) 1 August 2018

and prepared the manuscript. S.L.F. performed surgeries including transplantation of theBEL and was involved in the preparation of the manuscript. S.P.V., J.E.N., J.A.N., and L.B.A. wereinvolved in the production of BEL, data preparation, data analysis, and preparation of themanuscript. L.F. did the MRI and CT analyses of animals. J.S., X.L., and J.R. provided MPor produced porcine lung scaffolds. G.H. managed TEM and SEM. G.V. and R.P. did MPM. D.C.C.,R.Z., B.E.H., and S.L. did the genomic analysis. R.B.P. and A.M. performed the microbiomeanalysis. L.W. performed bronchoscopic evaluations. F.B. developed the dextrosedecellularization protocol. A.W. produced anesthesia protocol and provided anesthesiato animals. E.U., M.G., and D.W. did the histological and histopathological analyses. I.P.performed micro-CTs and IVIS. R.M. examined the respiratory function testing. J.C.managed the clinical transplant team and contributed to data analysis and preparationof the manuscript. Competing interests: J.S., J.E.N., and J.A.N. are inventors listed onPatent Cooperation Treaty (PCT) International Application No. PCT/US2016/057977 for useof dextrose in production of whole-lung scaffolds for BEL production. J.E.N., J.C., S.L.F., J.A.N.,and J.S. are inventors on a U.S. provisional patent application #62/659,321 that is beingsubmitted by UTMB at Galveston that covers the process of producing scaffolds for use in theproduction of BELs, use of nanoparticles to facilitate this process, and the method oftransplantation of BEL into a large-animal model. S.L.F. worked for the Methodist ResearchInstitute at the beginning of this project and now works for Biostage. J.S. worked for theMethodist Research Institute at the beginning of this project and now works for NanoMedicalSystems Inc. Data and materials availability: All data associated with this study arepresent in the paper or the Supplementary Materials.

Submitted 15 July 2017Accepted 19 June 2018Published 1 August 201810.1126/scitranslmed.aao3926

Citation: J. E. Nichols, S. La Francesca, J. A. Niles, S. P. Vega, L. B. Argueta, L. Frank,D. C. Christiani, R. B. Pyles, B. E. Himes, R. Zhang, S. Li, J. Sakamoto, J. Rhudy, G. Hendricks,F. Begarani, X. Liu, I. Patrikeev, R. Pal, E. Usheva, G. Vargas, A. Miller, L. Woodson, A. Wacher,M. Grimaldo, D. Weaver, R. Mlcak, J. Cortiella, Production and transplantation of bioengineeredlung into a large-animal model. Sci. Transl. Med. 10, eaao3926 (2018).

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Production and transplantation of bioengineered lung into a large-animal model

Adam Wacher, Maria Grimaldo, Daniil Weaver, Ron Mlcak and Joaquin CortiellaFilippo Begarani, Xuewu Liu, Igor Patrikeev, Rahul Pal, Emiliya Usheva, Grace Vargas, Aaron Miller, Lee Woodson,

Hendricks,Christiani, Richard B. Pyles, Blanca E. Himes, Ruyang Zhang, Su Li, Jason Sakamoto, Jessica Rhudy, Greg Joan E. Nichols, Saverio La Francesca, Jean A. Niles, Stephanie P. Vega, Lissenya B. Argueta, Luba Frank, David C.

DOI: 10.1126/scitranslmed.aao3926, eaao3926.10Sci Transl Med

lungs closer to the realm of clinical possibility.This work represents a considerable advance in the lung tissue engineering field and brings tissue-engineered lung-like microbiomes. One pig had no respiratory symptoms when euthanized a full 2 months after transplant.within 2 weeks after transplantation. The transplanted bioengineered lungs became aerated and developed native

seeded bioengineered lungs showed vascular perfusion via collateral circulation−lung scaffolds. Autologous cell model. Nanoparticle and hydrogel delivery of growth factors promoted cell adhesion to whole decellularized pig

of vascular perfusion, recellularization, and engraftment of tissue-engineered lungs in a clinically relevant pig . tackled the challengeset alwith a unique architecture that must maintain compliance during respiration. Nichols

Lungs are complex organs to engineer: They contain multiple specialized cell types in extracellular matrixNew life for lungs

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REFERENCES

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