WO2024112691A1 - In vivo lung de-epithelialization - Google Patents

Abstract

Methods and compositions for selectively de-epithelializing a portion of a lung in vivo or ex vivo, without removing lung microvasculature. The so-treated lung portion can be subsequently re- epithelialized endogenously, using stem cells or using genetically engineered cells.

Description

IN VIVO LUNG DE-EPITHELIALIZATION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 63/426,959, filed November 21, 2022 and U.S. Provisional Application No. 63/531,943, filed August 10, 2023, the contents of each of which are hereby incorporated by reference.

STATEMENT OF GOVERNMENTAL INTEREST

[0002] This invention was made with government support under grant HL120046 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

[0003] The disclosures of all publications, patents, patent application publications and books referred to in this application are hereby incorporated by reference in their entirety into the subject application to more fully describe the art to which the subject invention pertains.

[0004] Nearly 25 million people suffer from lung disease in the United States alone, with a staggering -400,000 patients dying each year. Lung transplantation, the only definitive treatment for these patients, remains constrained by the severe shortage of donor organs, with only one out of three donor lung meeting transplantable criteria.

[0005] Tissue engineering aims at replacing or regenerating human tissue with the final goal of restoring normal function, through an integrated use of cells, signaling molecules, and scaffolds. To provide the main function of lung, i.e., gas exchange, lung bioengineering has been focusing in utilizing biological scaffolds, which maintain the structural, biomechanical, and biochemical properties of the native organ and therefore can guide cells to reconstitute the physiological function of the organ. These features of biological scaffolds have turned interest to lung decellularization, where all cellular material is removed from the lung and only the native scaffold, made of extra-cellular matrix (ECM), is left behind. Of course, whole decellularization does not come without pitfalls. In fact, harsh decellularization can damage ECM’s microstructure and ultrastructure, making recellularization difficult and incomplete.

[0006] Two groups pioneered lung decellularization to obtain a scaffold for seeding primary epithelial into the airways and endothelial cells into the vascular compartment, to enable restoration of gas exchange (Ott et al. 2010; Petersen et al. 2010). However, these bioengineered lungs failed after only a few hours upon transplant, due to the incomplete regeneration of vasculature that remained leaky and resulted in alveolar edema and thrombosis (Petersen et al. 2010; 2011; 2012; Ott et al. 2010; Song et al. 2011). Typically, fully decellularized lungs are used as scaffolds for seeding of epithelial and endothelial cells (Wallis et al. 2012; O’Neill et al. 2013; Ott et al. 2010; Petersen et al. 2010; Wagner et al. 2014). But because human lungs contain more than a billion cells, complete decellularization and recellularization may be impractical at the clinical scale. Unlike some other tissues (e.g., blood vessels and bones), lungs cannot be grown using cells on synthetic scaffolds, due to the structural and biological complexity of the parenchyma and vasculature and the need for many different cell types to reconstruct such a complex organ. Lung regeneration using a completely decellularized lung repopulated with epithelial and vascular cells remains slow and incomplete, due in large part to the fact that the lung contains more than 40 different cell types (Colby TV, Leslie KO, and Yousem SA 2007; Franks et al. 2008; Beers and Morrisey 2011; Wagner et al. 2013).

SUMMARY OF THE INVENTION

[0007] The cellular and structural complexity of the lung makes it a challenge to use as a biological scaffold for lung bioengineering. Herein a method of gently treating lungs, either inside or outside of the body, to selectively remove only a predetermined portion of the lung epithelial tissues, leaving other critical cellular components intact, such as the lung microvasculature, is described. The method allows, among other uses, treatment of lung diseases that damage epithelia and treatment of lungs obtained from donors in order to improve the rate of donor lungs that meet transplantable criteria.

[0008] Disclosed herein is a method for selective de-epithelialization of a portion of a lung in a subject comprising administering an amount of a composition comprising a zwitterionic detergent to a portion of a lung epithelial surface in vivo effective to de-epitheli alize a portion of a lung without effecting endothelial removal in the portion of the lung, thereby selectively de- epithelizing a portion of the lung in vivo.

[0009] A method for treating a lung disease in a subject comprising selectively de- epithelializing a portion of the lung affected by lung disease in vivo so as to thereby permit the de-epithelialized portion of the lung to subsequently, endogenously, re-epithelialize, wherein the selectively de-epithelializing comprises administering an amount of a composition comprising a zwitterionic detergent to a portion of a lung epithelial surface in vivo effective to selectively de- epithelialize of a portion of the lung.

[0010] A method for treating a lung disease in vivo in a subject comprising: a) selectively de-epithelializing a portion of the lung affected by lung disease in vivo, and b) subsequently administering an amount of (i) stem cells or (ii) genetically-engineered stem cells to the de-epithelialized portion of the lung so as to treat the lung disease, wherein the selectively de-epithelializing comprises administering an amount of a composition comprising a zwitterionic detergent to a portion of a lung epithelial surface in vivo effective to selectively de-epithelialize of a portion of the lung.

[0011] A method for treating a lung ex vivo comprising: a) selectively de-epithelializing a portion of the lung which has been removed from a subject, by administering an amount of a composition comprising a zwitterionic detergent to a portion of a lung epithelial surface effective to selectively de-epithelialize of a portion of the lung; and b) optionally, administering a medication to the lung ex vivo prior to, during, or after step a). [0012] A composition for selective de-epithelialization of a lung comprising: an amount of a zwitterionic detergent; an amount of a salt; and an amount of a chelating agent.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 : Regional lung de-epithelialization in vivo. Schematics of regional lung de- epithelialization in Sprague-Dawley rats using a single lung catheter

[0014] FIG. 2: Protocol for regional lung de-epithelialization in vivo. Scheme of the main steps for the procedure of regional de-epithelialization.

[0015] FIG. 3A-3C: Setting for regional lung de-epithelialization in vivo. (3A) Engineered cannula with a tracheal plug. (3B) Experimental setting shows a intubated animal connected to the ventilation during the procedure. (3C) Blue dye was added to the CHAPS solution to confirm de-epithelialization region in the left lung.

[0016] FIG. 4A-4B: (4A) Vitals are monitored to keep animals stable. SpCh and heart rate decrease after every intratracheal injection of any fluid (CHAPS or PBS) but they recover completely by end of procedure. (4B) Hematological, renal and hepatic profiles, comparing pre- and post- treatments, show no alterations.

[0017] FIGS. 5A-5D: Regional de-epithelialization characterization. 5A. Schematic representation of the targeted region. Loss of epithelial markers (ProSPC for ATII and Aq5 for ATI cells) and preservation of endothelial markers (CD31) in the de-epithelialized area (outlined by the dotted white line), compared to the surrounding and contralateral untreated lung regions. Representative red squares are shown at higher magnification (lower panels). 5B. Lung injury score of de-epithelialized region compared to the contralateral, untreated lung. 5C. Scores for individual features of the LIS. In both c and d, data are reported as mean+SEM and analyzed by paired t-test [n=3 animals per condition, 60 ROI (30 from left lung, 30 from right lung) per animal; ****, p<0.0001], 5D. H&E, Pankeratin (Pank) and CD31 (endothelial cells) immunohistochemistry showing only removal of epithelial layers in the targeted region. Representative black squares are shownl 1 A, 1 IB at higher magnification (lower panels).

[0018] FIG. 6: Rat lung geometry plots. On the left, biologarithmic plot of lung volume (VL) against body weight (W). On the right, biologarithmic plot of alveolar surface area (Sa) and long volume (Burn, Dbaly and Weibel, 1974).

[0019] FIG. 7: Normal spirogram and subdivisions of lung volume during a respiratory cycle.

[0020] FIG. 8: Endogenous re-epithelialization of the lung seen at day 5 after partial, selective de-epithealization. By day 10 full re-epithelialization by endogenous progenitors occurred. [0021] FIGS. 9A-9G: Endogenous re-epithelialization. 9A. EdU incorporation in the de- epithelialized region and in the contralateral lung. Arrowheads: EdU incorporation in AT2 cells. 9B-9C. Immunofluorescent staining for epithelial cells (AQ5, ProSPC), and endothelial cells (CD31) in treated regions 5 (9B) and 10 (9C) days post de-epithelialization. 9D. Lung injury score of de-epithelialized region evaluated 2, 5, and 10 days post-treatment and compared to an untreated animal. Mean+SEM, one-way ANOVA, n=3 animals per condition, 60 ROI (30 from left lung, 30 from right lung) per animal; *, p=0.0287; ****, p<0.0001. 9E. Scores for the most pronouncedly different individual features of the LIS. Mean+SEM, one-way ANOVA, n=3 animals per condition, 60 ROI (30 from left lung, 30 from right lung) per animal; ****, p<0.0001. f-g. Pankeratin (Pank) and CD31 (endothelial cells) immunohistochemistry 5 days (9F) and 10 days (9G) after de-epithelialization. Representative black squares in lower panels.

[0022] FIGS. 10A-10E: Engraftment of DLEPs after de-epithelialization and irradiation. 10A. Schematic representation of experimental approach. 10B. Annotations (red squares) of the location of human cells based on hMit staining, using de-epithelialized, non-engrafted lung as background control. 10C. RNA in situ hybridization (RNAscope) for human P2-microglobulin (hB2M), and IF for hMit, hKRT5, hTP63, ProSPB and SCGB3A2 in representative engrafted area from left lung. The left upper panel shows a region without human cells as a negative control for RNAscope. 10D. Human gDNA content in sections of lung regions as represented in the schematic on the right. Aggregated data from equal numbers of experiments with ESC- and iPSC-derived DLEPs. 10E. Percent engraftment in the most highly engrafted section of panel d for each experiment. Mean+SEM, unpaired Student’s t-test (n=6 per condition; *, p=0.02).

[0023] FIGS. 11A-11C: Repair of lung injury. 11 A. H&E staining of injured (De-Epi + IR) and engrafted (De-Epi + IR + DLEPs) lower left lobes. 11B. Representative higher magnification images. 11C. Lung injury scores (LIS) (IR: irradiation, UN: normal control lung, 800 blindedly evaluated fields in total, 30 ROI per tile scan, one-way ANOVA; *, p=0.0383; ****, p<0.0001).

DETAILED DESCRIPTION

[0024] A method for selective de-epithelialization of a portion of a lung in a subject comprising administering an amount of a composition comprising a zwitterionic detergent to a portion of a lung epithelial surface in vivo effective to de-epithelialize a portion of a lung without effecting endothelial removal in the portion of the lung, thereby selectively de-epithelizing a portion of the lung in vivo.

[0025] A method for treating a lung disease in a subject comprising selectively de-epithelializing a portion of the lung affected by lung disease in vivo so as to thereby permit the de-epithelialized portion of the lung to subsequently, endogenously, re-epithelialize, wherein the selectively de- epithelializing comprises administering an amount of a composition comprising a zwitterionic detergent to a portion of a lung epithelial surface in vivo effective to selectively de-epithelialize of a portion of the lung. In embodiments, re-epithelialization occurs from endogenous lung progenitors.

[0026] A method for treating a lung disease in vivo in a subject comprising: a) selectively de-epithelializing a portion of the lung affected by lung disease in vivo, and subsequently administering an amount of (i) stem cells or (ii) genetically-engineered stem cells to the de-epithelialized portion of the lung so as to treat the lung disease, b) wherein the selectively de-epithelializing comprises administering an amount of a composition comprising a zwitterionic detergent to a portion of a lung epithelial surface in vivo effective to selectively de-epithelialize of a portion of the lung.

[0027] In embodiments, the (i) stem cells, or (ii) stem cells which are genetically-engineered, are autologous to the subject being treated. In embodiments, the (i) stem cells, or (ii) stem cells which are genetically-engineered, are allogeneic to the subject being treated. In embodiments, the subject has a genetic disease affecting lung function and the stem cells which are genetically- engineered are administered. In embodiments, the stem cells are genetically-engineered to correct the genetic sequence associated with the genetic disease affecting lung function. In embodiments, the genetically-engineered stem cells correct a genetic defect of an Alveolar Type II (ATII) cell. In In embodiments, the genetically-engineered stem cells correct a genetic mutation in one or more of SFTPB, SFTPC, ABCA3, SFTPA2). In embodiments, the stem cells are, or are derived from, human embryonic stem cells or human pluripotent stem cells.

[0028] In embodiments, the stem cells are embryonic stem cells. In embodiments, the genetically-engineered stem cells are autologous induced pluripotent stem cells (iPSC). In embodiments, the genetically-engineered stem cells are allogeneic HLA haploidentical iPSC. Examples or human pluripotent stem cells, including lung progenitor cells, and how to make them can be found in, for example, in US 11739299 B2, which is hereby incorporated by reference in its entirety. In embodiments the cells are human stem cells.

[0029] In embodiments, the lung microvasculature in the de-epithelialized portion of the lung remains functional. Prior methods damage endothelial cells, and/or microvasculature, in the lungs, since they do not selectively de-epithelialize the lung, rather, they non-selectively decellularize the lung. The present methods can overcome these limitations of the prior art.

[0030] In embodiments, the methods further comprise irradiating the portion of the lung subsequent to selectively de-epithelializing the portion of the lung. In embodiments, irradiating the portion of the lung subsequent to selectively de-epithelializing the portion of the lung is conducted prior to endogenous re-epithelialization or prior to subsequently administering an amount of (i) stem cells or (ii) genetically-engineered stem cells to the de-epithelialized portion of the lung so as to treat the lung disease. In embodiments, the methods further comprise administering to the portion of the lung an inhibitor of endogenous endothelium production either prior to or during selectively de-epithelializing the portion of the lung. In embodiments, the methods further comprise administering to the portion of the lung an inhibitor of endogenous epithelium production either prior to or during selectively de-epithelializing the portion of the lung. In embodiments, the methods further comprise administering to the portion of the lung an inhibitor of endogenous endothelium production subsequent to selectively de-epithelializing the portion of the lung. In embodiments, administering to the portion of the lung an inhibitor of endogenous endothelium production is conducted prior to endogenous re-epithelialization or prior to subsequently administering an amount of (i) stem cells or (ii) genetically-engineered stem cells to the de-epithelialized portion of the lung so as to treat the lung disease. In embodiments, an inhibitor of endogenous endothelium production comprises a cyclin-dependent kinase 4 and 6 (Cdk4/6) inhibitor. In embodiments, the Cdk4/6 inhibitor inhibits progression of one or more lung cells through the Gl-to-S cell cycle checkpoint. In embodiments, the Cdk4/6 inhibitor is a small molecule Cdk4/6 inhibitor. In embodiments, the Cdk4/6 inhibitor comprises abemaciclib, palbociclib or ribociclib.

[0031] In embodiments, no more than 25% of the lung surface is de-epithelialized.

[0032] In embodiments, no more than 20% of the lung surface is de-epithelialized.

[0033] In embodiments, the amount of a zwitterionic detergent is delivered via a cannula.

[0034] In embodiments, the cannula is introduced into the lung via the trachea. [0035] In embodiments, the portion of the lung to be selectively de-epithelialized is prior- identified by a computer tomography (CT) scan and/or via a bronchoscope. In embodiments, the area of lung to be de-epithelialized is monitored during the procedure using bronchoscopy.

[0036] In embodiments, placement within the lung of a delivery device to administer the amount of a zwitterionic detergent is assisted using an inflatable bronchoscope balloon.

[0037] In embodiments, at least two amounts of a zwitterionic detergent are administered to the portion of the lung epithelial surface in vivo, and wherein the administration of the two amounts is separated in time.

[0038] In embodiments, the time between administrations is from 15 to 45 minutes.

[0039] In embodiments, the lung is ventilated for a period of time immediately subsequent to the administration of the amount of the zwitterionic detergent.

[0040] In embodiments, the lung is ventilated for a period of time immediately subsequent to each administration of the amount of the zwitterionic detergent.

[0041] In embodiments, each period of time of ventilation is, independently, from 15 to 45 minutes.

[0042] In embodiments, a bronchoalveolar lavage is performed at least on the portion of the lung to which the amount of zwitterionic detergent has been administered, subsequent to it being ventilated.

[0043] In embodiments, a bronchoalveolar lavage is performed at least on the portion of the lung subsequent to the lung being ventilated after a second of the two amounts of a zwitterionic detergent has been administered. In embodiments, the lavage comprises phosphate-buffered saline.

[0044] In embodiments, the lung of the subject being selectively de-epithelialized is affected by a lung disease that compromises lung epithelia. Such diseases include those that negatively impact function of type II alveolar epithelial cells (e.g., idiopathic pulmonary fibrosis).

[0045] In embodiments, the subject has emphysema, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, acute respiratory distress syndrome (ARDS), cystic fibrosis, or interstitial lung disease.

[0046] In embodiments involving a subject, the methods can further comprise subject can be administered an immunosuppressive medication. Immunosuppressive medications are known in the art, for example, tacrolimus, cyclosporin etc. [0047] A method for treating a lung ex vivo comprising: a) selectively de-epithelializing a portion of the lung which has been removed from a subject, by administering an amount of a composition comprising a zwitterionic detergent to a portion of a lung epithelial surface effective to selectively de-epitheli alize of a portion of the lung; and b) optionally, prior to, during, or after step a) administering a medication to the lung ex vivo.

[0048] In embodiments, the method further comprises removing the lung from the subject prior to step a).

[0049] In embodiments, the subject has a lung infection, further comprising administering an antibiotic and/or antiviral medication to the lung ex vivo.

[0050] In embodiments, the method further comprises treating the de-epithelialized lungs so as to effect re-epithelization ex vivo. In embodiments, the de-epithelialized are treated with (i) stem cells, or (ii) stem cells which are genetically-engineered, effective to re-epithelialize the portion of the lung. In embodiments, the (i) stem cells, or (ii) stem cells which are genetically- engineered, are allogeneic to the subject being treated. In embodiments, the subject from whom the lung is removed has a genetic disease affecting lung function and the stem cells which are genetically-engineered are used to re-epithelialize the lung portion. In embodiments, the stem cells are genetically-engineered to correct the genetic sequence associated with the genetic disease affecting lung function. In embodiments, the genetically-engineered stem cells correct a genetic defect of an Alveolar Type II (ATII) cell. In In embodiments, the genetically-engineered stem cells correct a genetic mutation in one or more of SFTPB, SFTPC, ABCA3, SFTPA2). In embodiments, the stem cells are, or are derived from, human embryonic stem cells or human pluripotent stem cells.

[0051] In embodiments, the methods further comprise irradiating the portion of the lung subsequent to selectively de-epithelializing the portion of the lung. In embodiments, irradiating the portion of the lung subsequent to selectively de-epithelializing the portion of the lung is conducted prior to endogenous re-epithelialization or prior to subsequently administering an amount of (i) stem cells or (ii) genetically-engineered stem cells to the de-epithelialized portion of the lung so as to treat the lung disease. In embodiments, the methods further comprise administering to the portion of the lung an inhibitor of endogenous endothelium production either prior to or during selectively de-epithelializing the portion of the lung. In embodiments, the methods further comprise administering to the portion of the lung an inhibitor of endogenous endothelium production subsequent to selectively de-epithelializing the portion of the lung. In embodiments, administering to the portion of the lung an inhibitor of endogenous endothelium production is conducted prior to endogenous re-epithelialization or prior to subsequently administering an amount of (i) stem cells or (ii) genetically-engineered stem cells to the de- epithelialized portion of the lung so as to treat the lung disease. In embodiments, an inhibitor of endogenous endothelium production comprises a cyclin-dependent kinase 4 and 6 (Cdk4/6) inhibitor. In embodiments, the Cdk4/6 inhibitor inhibits progression of one or more lung cells through the Gl-to-S cell cycle checkpoint. In embodiments, the Cdk4/6 inhibitor is a small molecule Cdk4/6 inhibitor. In embodiments, the Cdk4/6 inhibitor comprises abemaciclib, palbociclib or riboci clib.

[0052] In embodiments, no more than 25% of the lung surface is de-epithelialized.

[0053] In embodiments, the method further comprises comprising transplanting the so-treated lung (a) back into the subject from which the lung was removed or (b) into a non-donor subject.

[0054] In embodiments, the zwitterionic detergent comprises (3-((3-cholamidopropyl) dimethylammonio)-l -propanesulfonate) (CHAPS).

[0055] In embodiments, the composition comprising the zwitterionic detergent comprises a minimum amount of zwitterionic detergent effective to achieve micellar formation.

[0056] In embodiments, the composition comprising the zwitterionic detergent comprises 4 - 5 mM zwitterionic detergent.

[0057] In embodiments, the composition comprising the zwitterionic detergent comprises an amount of a salt.

[0058] In embodiments, the salt is NaCl.

[0059] In embodiments, the salt is 0.25 - 0.75 mM.

[0060] In embodiments, the salt is 0.45 - 0.55 mM.

[0061] In embodiments, the amount of salt does not effect decellularization of non-epithelial tissue in the lung.

[0062] In embodiments, the composition comprising the zwitterionic detergent comprises a chelating agent.

[0063] In embodiments, the chelating agent is EDTA. [0064] In embodiments, the composition has a pH of 7.8 - 8.2.

[0065] In embodiments, the composition has a pH of 8.0.

[0066] In embodiments, the amount of zwitterionic detergent does not result in hepatic damage in the subject.

[0067] In embodiments, the amount of zwitterionic detergent does not result in hematologic damage in the subject.

[0068] A composition for selective de-epithelialization of a lung comprising: an amount of a zwitterionic detergent; an amount of a salt; and an amount of a chelating agent.

[0069] In embodiments, the zwitterionic detergent comprises (3-((3-cholamidopropyl) dimethylammonio)-l -propanesulfonate) (CHAPS).

[0070] In embodiments, the composition comprises an amount of zwitterionic detergent effective to achieve micellar formation in a lung.

[0071] In embodiments, the composition comprises 4 - 5 mM zwitterionic detergent.

[0072] In embodiments, the salt is NaCl.

[0073] In embodiments, the salt is 0.25 - 0.75 mM.

[0074] In embodiments, the salt is 0.45 - 0.55 mM.

[0075] In embodiments, the amount of salt does not effect decellularization of non-epithelial tissue in a human lung.

[0076] In embodiments, the chelating agent is EDTA.

[0077] In embodiments, the composition has a pH of 7.8 - 8.2.

[0078] In embodiments, the composition has a pH of 8.0.

[0079] In embodiments, the composition comprises deionized water.

[0080] In embodiments, the subject is a human subject.

[0081] In embodiments, the subject is 60 years or older. In embodiments, the subject is immunocompromised.

[0082] Non-limiting examples of lung epithelial disease that can be treated by the methods and compositions disclosed herein include, cystic fibrosis; emphysema; chronic obstructive pulmonary disease (COPD); interstitial lung diseases including pulmonary fibrosis, idiopathic pulmonary fibrosis (IPF), Hermansky-Pudlak Syndrome (HPS), hypersensitivity pneumonitis, sarcoidosis, asbestosis, autoimmune-mediated interstitial lung disease; pulmonary hypertension; lung cancer; acute lung injury (adult respiratory distress syndrome); respiratory distress syndrome of prematurity, chronic lung disease of prematurity (bronchopulmonary dysplasia); congenital surfactant deficiencies, including surfactant protein B deficiency, surfactant protein C deficiency, ABCA3 deficiency; ciliopathies; congenital diaphragmatic hernia; pulmonary alveolar proteinosis; Nieman-Pick disease; surfactant proteins defects (SFPTA1, SFPTA2, SFPTB, SFTPC); and pulmonary hypoplasia. Lung epithelia damages by lung cancer can also be treated by the methods and compositions described herein.

[0083] Selectively de-epithelializing a portion of a lung means that epithelial cells are removed in a greater preponderance than other cell types present in the portion of the lung, for example, endothelial cells. For example, the removal of epithelial cells is greater than 75% of their total presence in the lung portion at the start of treatment whereas the removal of endothelial cells is 10% or less of their total presence in the lung portion at the start of treatment. For example, the removal of epithelial cells is greater than 90% of their total presence in the lung portion whereas the removal of endothelial cells is 10% or less. In embodiments, the selectively de-epithelializing a portion of a lung means a functional microvasculature remains present in the portion of the lung after the treatment. In embodiments, the selectively de-epithelializing a portion of a lung means the extracellular matrix, or ECM, is substantially maintained: (e.g., biochemical moieties, adhesion molecules, matrix peptides) and the interstitial and support cells (e.g., fibroblasts, pericytes, endothelial, mesothelial, and lymphatic cells).

[0084] In embodiments the administered stem cells or genetically engineered stem cells are from human pluripotent stem cell-derived expandable cell lines shares one or more features of human airway secretory and/or basal cells.

[0085] “And/or” as used herein, for example, with option A and/or option B, encompasses the separate embodiments of (i) option A, (ii) option B, and (iii) option A plus option B.

[0086] All combinations of the various elements described herein are within the scope of the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0087] Definitions: The terms used in this specification generally have their ordinary meanings in the art, within the context of this invention and the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the methods of the invention and how to use them. Moreover, it will be appreciated that the same thing can be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein, nor is any special significance to be placed upon whether or not a term is elaborated or discussed herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of the other synonyms. The use of examples anywhere in the specification, including examples of any terms discussed herein, is illustrative only, and in no way limits the scope and meaning of the invention or any exemplified term. Likewise, the invention is not limited to its preferred embodiments.

[0088] The term “subject” as used in this application means a mammal. Mammals include canines, felines, rodents, bovine, equines, porcines, ovines, and primates including humans. Thus, the invention can be used in human medicine or also in veterinary medicine, e.g., to treat companion animals, farm animals, laboratory animals in zoological parks, and animals in the wild. The invention is particularly desirable for human medical applications. In a preferred embodiment the subject is a human.

[0089] The terms “treat”, “treatment” of a disease or condition, and the like refer to slowing down, relieving, ameliorating or alleviating at least one of the symptoms of the disease.

[0090] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system, i.e., the degree of precision required for a particular purpose, such as a pharmaceutical formulation. For example, “about” can mean within 1 or more than 1 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” meaning within an acceptable error range for the particular value should be assumed.

Results

[0091] Lung epithelium is not only the main target of lung diseases, congenital and acquired, but also the most critical component involved in lung repair and functional recovery. There are several examples demonstrating the critical role of epithelium in the initiation of parenchymal lung disease, including monogenic disease such as Hemansky-Pudlack syndrome, Nieman-Pick disease, surfactant proteins defects (SFPTA1, SFPTA2, SFPTB, SFTPC,), and mutations in the ABC subfamily 3 (ABCA3) (Beers and Morrisey 2011). These diseases are characterized by dysfunctional ATII cells displaying fibrotic phenotype. In mice, targeted ablation of ATII cells results in extensive lung fibrosis (Sisson et al. 2010; Kim et al. 2018). Expression of mutant forms of SFTPC associated with human respiratory disease show ATII cell injury, such as endoplasmic reticulum (ER) stress and apoptosis (Korfei et al. 2008; Lawson et al. 2011; Maitra et al. 2010; Mulugeta et al. 2007; Katzen et al. 2019). SFTPC and ABCA3 mutations are associated with idiopathic interstitial pneumonia shown to induce apoptosis of epithelial cells in vitro and in vivo (Wert et al. 2009; Glasser et al. 2003; Bullard et al. 2005; Rindler et al. 2017). Epithelial injury is also a central finding in the lungs of patients with the acute respiratory distress syndrome (ARDS) patients.

[0092] Extensive epithelial damage is often involved in the loss of the epithelial-mesenchymal homeostasis, with rearrangement of ECM and lung architecture leading into fibrosis. Reconstitution of functional epithelium (re-epithelialization) is crucial for preventing pathological lung remodeling and for recovering the most important lung function: gas exchange. Hypothetically, regeneration with healthy epithelial cells could promote local proliferation of the remaining undamaged epithelium, activated by the local lung progenitor or use of exogenous stem cells (Beers and Morrisey 2011).

[0093] Given that the vascular component of the lung is critical for the supply of nutrients and oxygen, much effort has been invested into finding the best strategies to repair lung epithelium while keeping the vascular network intact and functional. The derivation and use of vascularized lung scaffolds has been pioneered by our group (Dorrello et al. 2017).

[0094] Our de-epithelialization approach has been designed to overcome three major limitations of the whole lung decellularization: (i) the challenging hurdle to properly recellularize an organ consisting of 40 distinct cell types (Colby TV, Leslie KO, and Yousem SA 2007; Franks et al. 2008; Beers and Morrisey 2011; Wagner et al. 2013); (ii) the lack of functional vascular network with the relative high risk of thrombogenicity, pulmonary edema and hemorrhage within the lung graft (Ott et al. 2010; Petersen et al. 2010; 2011; 2012; Song et al. 2011; Ren et al. 2015); (iii) the paucity of all supporting cells (interstitial and vascular) that through delivery of growth factors and signaling molecules could foster appropriate lung epithelial regeneration.

[0095] To enable replacement of the epithelium in specific local areas of the lung (rather than in the entire lung) while maintaining intact vascular perfusion and whole body support our group developed a methodology for targeted de-epithelialization in vivo (Fig. 1).

[0096] De-epithelialization in vivo has not been demonstrated before; we have established in fact both the feasibility and efficacy of de-epithelialization in vivo.

[0097] The de-epithelialization solution volume was selected to target 17-20% of the alveolar surface in the left lung. According to the geometry of the rat left lung (Lai and Hildebrandt 1978; Barre et al. 2014a), volume of de-epithelialization solution (DE) was chosen as follows (see below for details):

[0098] DE volume uL) = 6.327 * 1V^O'⁷(I L g body weight)

[0099] An effective de-epithelialization solution containing 4 mM CHAPS (Sigma-Aldrich), 0.5 M NaCl, and 25 mM EDTA in deionized water. Each dose was divided equally into two, with 30 minutes between each instillation. 30 minutes after the second instillation, in case of animals with a weight higher than 230g, the workstand was lowered to its horizontal position. At a 15-minute interval, bronchoalveolar lavage (BAL) was performed twice (1 mL of PBS each) via a 3-mL syringe (Fig. 2). 6mM and 8mM CHAPS were not effective for the procedure since they caused decellularization beyond just epithelial cells.

[00100] To reach the lower side of left lower lung, we engineered in lab a long cannula that allows to reach the lower side of one lung (left was chosen for simplicity of the approach since it is a single lobe) (Fig. 3A). The cannula is confirmed on in the left lung using a flexible bronchoscope. The cannula has also a plastic plug at proximal extremity (Fig. 3B). The tracheal plug was used the cannula to create a temporary seal at the entrance of the trachea and allowed proper airways washing. The procedure set up with surgical thermostable pad, ventilator, pulse oximetry, heart rate and end tidal CO2 monitoring are shown in Fig. 3B. After the procedure, the animals were extubated to room air, returned to their cage, and sacrificed at 48 hours to evaluate the efficacy of de-epithelialization in vivo. Blue dye was used to show macroscopically the area treated (Fig. 3C). The rats were kept on the ventilator to recover and returned to their cages. This methodology allowed us to treat only a discrete region of lung (the lower side of left lung, in our case) -as hypothetically applicable to patients - while using the contralateral lung as control in the same animal. After the single lung de-epithelialization treatment, the animals were extubated to room air, returned to their cage, and sacrificed at 48 hours to evaluate the efficacy of de- epithelialization in vivo.

[00101] Heart rate, oxygen saturation, and temperature were monitored during the intervention with overall stable trend and improvement at the end of the treatment (Fig. 4A). To evaluate the toxicity of de-epithelialization solution, we analyzed the hematological profile and organ function parameters of the animals before and after the procedure: no abnormal changes noted (Fig. 4B). We had a mortality of 5%, and none of the animals showed any external signs and symptoms of respiratory distress at the 48-hour mark.

[00102] Validation of in vivo de-epithelialization

[00103] Lungs harvested 48hrs post de-epithelization were stained for the alveolar epithelial markers, aquaporin 5 (AQ5, expressed in ATI cells), pro-surfactant protein C (proSPC, expressed in AT2 cells) and CD31, an endothelial cell marker. Compared to the untreated right lung within the same animal, there was a considerable loss of ATI and AT2 cells in the treated region (Fig. 5A). Likely because of gravity and the positioning of the canula, the de- epithelialized regions were mainly localized to the dorsal part of the lower left lobe and had a similar spatial and volumetric distribution across all the experimental animals (not shown). Hematoxylin and eosin (H&E) staining showed patency of the alveoli in the targeted region (Fig. 5A). However, neutrophils in alveolar and interstitial space, inflammatory infiltrates, hyaline membranes, and thickening of septa were detected area across the treated left lower lobe (Fig. 5B,5C). These changes were quantified by assessing the lung injury score (LIS) according to ATS guidelines. [44,48,49] LIS was significantly elevated in the lower left lung (treated) compared to the right lung (untreated) within the same animal (Fig. 5B, 5C). We did observe a lower amount of proteinaceous debris in the de-epithelialized lungs compared to untreated lungs (Fig. 5C). However, during the LIS evaluation, the abundant presence of hyaline membranes in de-epithelialized lungs most likely obscured the underlying proteinaceous debris.

[00104] Despite these changes, expression of the endothelial marker, CD31, was maintained, indicating mostly selective removal of the epithelium (Fig. 5A). Regional de- epithelialization was further confirmed by immunohistochemistry (IHC) using antibodies against the epithelial marker Pankeratin (PANK) and CD31, respectively showing lack of PANK but maintenance of CD31 staining in the targeted region (Fig. 5D). [00105] EdU labeling 48hrs after de-epithelialization showed extensive proliferation in the epithelium of airways and the alveoli, both in the targeted region and in the surrounding areas of the lower left lung (Fig. 9A). Outside of the de-epithelialized region, proliferating cells included proSPC+ (AT2) cells indicating a regional regenerative response of the lung to the local injury (Fig. 9A). After 5 days, partial recovery of alveolar epithelium as evidenced by increased expression of AQP5 and proSPC had occurred in the treated regions (Fig. 9B). At dlO, the alveolar epithelium appeared fully reconstituted (Fig. 9C). Assessment of LIS (Fig. 9D, 9E) and IHC for PANK confirmed these findings (Fig. 9F).

[00106] Engraftment of DLEPs.

[00107] Next, de-epithelialization was applied followed by an immune suppression regimen (oral mycophenolate, intramuscular (IM) methylprednisolone, and subcutaneous (sq) tacrolimus). 48hrs post-de-epithelialization we administered 107 DLEPs intrabronchially targeting the lower region of left lung. After 10 days, patches of human cells were found scattered throughout the lower left lobe as judged by staining for human mitochondria (hMit). Co-staining with proSPC or with RAGE indicated ATI and AT2 development from the engrafted cells (Fig. 10A-10C). No contribution to large or small airways was observed however. [00108] To improve engraftment, we blocked the endogenous repair with 12Gy local irradiation of entire lung field 24hr prior to cell delivery. This intervention blocked EdU incorporation after administration of CHAPS. Transplantation of DLEPs at day 2 after de- epithelialization combined with irradiation at day 1 (Fig. 10A) resulted in extensive areas containing human alveolar epithelium detected by staining for hMit (Fig. 10B). RNAscope50 for human |32-microglobulin (B2M) confirmed patchy locoregional replacement of rat alveoli by human cells that corresponded to the staining for hMit (Fig. 10C). Engraftment was observed in occasional airways, as identified by staining for hKRT5, hTP63 and hMit. Markers of mature cells were not found at this 10-day time point. While specifically targeting airways might be challenging, the data do demonstrate airway potential of DLEPs in vivo. In addition, dense aggregates of human cells were observed as judged by staining for hMit and RNAscope for B2M in localized areas that appeared histologically more severely damaged (alveolar collapse, thickened septa, infiltrate). These structures stained with human-specific anti-KRT5 antibodies, while human p63 was detected at the periphery (Fig. 10C, right panels). These cell clusters are therefore highly reminiscent of the KRT5-pods identified in the distal lungs of mice after severe influenza. [12-14] Furthermore, the human engrafted ‘KRT5-pods’ expressed SCGB3A2, proSPC, and proSPB (Fig. 11C, lower right panels), markers co-expressed in recently identified distal lung progenitors in human lungs that are more abundant after injury. [22,23]

[00109] To estimate the number of engrafted human cells, genomic DNA (gDNA) was extracted from 10 150pm thick sections from right and left lungs according to the schematic in Fig. 10D and subjected to qPCR for human AluYb8.51 Compared to a standard curve of human cells alone and mixed with rat lung cells, we estimated the engraftment of human cells using the section with the highest engraftment in each individual animal. (Fig. 10D). In de- epithelialized/transplanted region, we found -0.6% human cells per section (Fig. 10E). With irradiation, however, average engraftment was -10%. Fig. 10D also shows that in occasional rats, the right lung was injured and engrafted as well, most likely due to spillover or to anatomical variation in the airways of these outbred rats, which could lead inadvertent targeting of the right lung. We conclude that DLEPs can engraft in rat lungs after appropriate conditioning and immunosuppression and that three patterns of engraftment were observed: KRT5-pods in severely damage areas, integrated replacement of alveolar epithelium, and, occasionally, airways.

[00110] Injury repair by DLEPs.

[00111] Low-magnification images of HE-stained sections showed that, whereas after 10 days no residual injury was detected in CHAPS-treated lungs (Fig. 9C-9F), the combination of both CHAPS and irradiation induced severe damage (Fig. 11 A, 1 IB). The lower left lobes of rats engrafted with cells, however, showed a remarkable attenuation of lung damage, although some localized injury was still observed (Fig. 11 A, 1 IB). To quantify injury and the effect DLEPs on injury repair, we assessed the lung injury score (LIS) according to ATS guidelines. [44,48,49] In recipients of ESC and iPSC-derived lines there was a significant reduction in LIS that, importantly, quantitatively approached that of uninjured lungs especially for ESC-DLEPs, where there was no difference (Fig. 1 IB). The variance however was higher, as some localized areas remained abnormal. These are the areas containing the KRT5+ pods. The most strikingly elevated feature of the LIS in the conditioned lower left lobe was septal thickening, an early sign of interstitial lung disease. [52] This feature, in particular, was reduced to near uninjured levels in all recipients. These data indicate that DLEP engraftment promotes repair, and therefore unequivocally showing functionality. [00112] Further explanation of the design of the formula for calculate the dose of CHAPS- based solution is as follows, though other zwitterionic detergents can be used. To make the result of the de-epithelialization comparable among animals with different sizes of lungs, a dose formula for CHAPS solution was established. Based on published data of rat lung geometry (Lai and Hildebrandt 1978) (Barre et al. 2014b), a formula (eq. 1) was generated to calculate the amount of CHAPS necessary to remove the epithelium only from -20% of the alveolar surface of one lung, in agreement to our overall goal of “targeted de-epithelialization” of only injured regions of lung.

[00113] Firstly, the relationship between the body weight (W) of rats and their total lung capacity (TLC) is found.

TLC (mL) = 0.222 VIZ ^{0 7} (VIZ, g body weight)

[00114] From the relationship between the lung volume (VL) and the body weight in the rat (Burri, Dbaly, and Weibel 1974) (Fig. 6 left):

[00115] V_L - 0.176 VP^{0 70}

[00116] and from the rapport between the alveolar surface area (S_a) and the lung volume [Fig. 6 right]:

[00117] S_a = 1113.1 P_L ^{0 71}

[00118] it was possible to calculate the ratio between two different surface areas by:

[00120] From the data collected from fixed lungs of CFN-COBS-strain rat by saline displacement (Lai and Hildebrandt 1978), the definition of total tissue volume of lungs (TTV) has been adapted in order to find the equation to TLC in live Sprague-Dawley rats.

[00121] TLC [ml] = 0.222 W^{0 7}[g]

[00122] Then, the total capacity of the left lobe was calculated, knowing the percentage it takes up in the whole lung. Using data from X-ray computed microtomography from several rats (Barre et al. 2014b), mathematical models can be built to estimate the volume of each lobe. Table 1 shows the amount of volume specific for each lobe compared to the whole lung of one of the first treated rat. Table 1 : Relationship between the Whole lung volume and relatively lobes

[00123] The air space in the left lobe takes up approximately 35.4% of the TLC.

[00124] Finally, the dose to cover a reasonable (for treating enough epithelium while maintaining the rat able to breath) amount of alveolar surface, was defined. Since the residual volume (RV) [Fig. 7] stays constant throughout the respiratory cycle, the dose volume was based on the RV of the left lobe, which equals to 10% of its total capacity volume. Considering the possible uneven distribution of liquid, once injected in the lobe, 8% instead of 10% is considered with a security factor of 20%.

[00125] It is possible to estimate the alveolar surface area of this specific volume as (8%)^0,71 = 17% of the total alveolar surface area in the left lobe, according to the equation developed above.

[00126] The final de-epithelializing dose formula (eq. 2) obtained was

[00127] Dose(uL) = 6.327 * VT^{0 7}^)

[00128] Regional de-epithelialization in vivo can serve as a physiologic scaffold for cell therapy by: (?) enabling the delivery of oxygen, nutrients, growth factors, and signaling molecules, (//') providing biophysical and mechanical signals via perfusion (flow, shear) and ventilation (strain), and (iii) maintaining the ECM (biochemical moieties, adhesion molecules, matrix peptides) and the interstitial and support cells (fibroblasts, pericytes, endothelial, mesothelial, and lymphatic cells). Lungs de-epithelialized by the present methods have been successfully repopulated with human embryonic pluripotent stem cells in a xenotransplant model (data not shown). Lungs de-epithelialized by the present methods, ex vivo, have also been observed to shown to repopulation by endogenous epithelial cells.

[00129] DISCUSSION

[00130] We report here that distal lung endothelial progenitor cells (DLEPs) engraft distal lung of rats conditioned with regional de-epithelialization followed by irradiation and promote extensive repair of lung damage inflicted by the conditioning regimen. These findings validate this model for both the preclinical development of cell therapy for lung disease and the study of cell types and mechanisms involved in human lung regeneration.

[00131] That a cell population that contains subsets sharing expression signatures with BCs or secretory cells in airway engrafts in the distal lung may seem surprising. However, a common thread in the sometimes conflicting reports on cell types involved in lung regeneration and repair in mice and humans is that subsets of cells with BC-like and secretory-like phenotypes may be interconvertible or overlapping to some extent and participate in distal lung regeneration. [8, 9, 13, 13-15, 18, 22, 23] The cells we generated, which contain a BC-like population and a population similar to Notch-induced UPK3A+ variant secretory-like population identified in the mouse, [16, 17, 41] therefore fit this paradigm. This notion is further supported by the observation that Upka3+ contribute to alveolar regeneration after bleomycin injury in the mouse. [17] The finding that DLEP-derived structures similar to KRT5-pods[12-14] are present in the most damaged areas, and co-express SCGB3A2, proSPC, and proSPB fits this paradigm as well. The DLEPs that we generated from lung organoids are therefore likely equivalent to the precursors of KRT5-pods observed in injured mouse lungs. [12-14] Expansion of KRT5+ cells in the distal lung, though not in structures resembling KRT5-pods in the mouse, has also been demonstrated after severe lung injury in humans. [53] Interestingly, and in contrast to mouse KRT5-pods, these cells co-express SFTPC, similar to the human KRT5+ cells engrafted in injured rat lungs in our study. [53] We note that injury repair was almost complete, and therefore much more extensive than actual alveolar engraftment. This maybe be consistent with the notion that KRT5+ cells arising after severe lung injury may primarily fulfill a supportive role, either structural and/or mediated by paracrine factors, in lung repair.[3, 14,54]

[00132] Airway repopulation was rare and only observed in two recipients. The reasons are unclear, as the DLEPs show gene-expression signatures associated with airways. It is possible that physical forces caused by spontaneous breathing and mechanical ventilation drive both the detergent solution and the cells to the distal lung, such that injury and cell attachment occur predominantly in the alveoli. We also could not reproducibly engraft NSG mice. The reasons could also be technical, however. It was not feasible to specifically target a single lobe in mouse using an intratracheal canula while ventilating the animal under anesthesia. Therefore, insufficient distal injury was achieved. However, bleomycin, naphthalene, and polidocanol followed by intratracheal administration of cells between 1 and 10 days after injury did not result in engraftment either. Multiple explanations could exist for this discrepancy between mouse and rat. It is possible that the strategies we could use in the mouse model do not create conditions that are sufficiently permissive for engraftment. Human cells may also not respond properly to the regenerative and homing cues in mouse. Another possibility is that engraftment requires lymphocytes that are absent in NSG mice. A final possibility is that the size of human cells, in particular of developing ATI cells, is not compatible with the architecture and size of mouse alveoli.

[00133] Our observations may also constitute a pre-clinical model for cell therapy for lung disease.55 Cell therapy could be an alternative for lung transplantation in at least some diseases where lung transplantation is currently the only curative standard of care. Cystic fibrosis (CF) refractory to pharmacological modulator therapy and primary ciliary dyskinesia are monogenic airway disease that could be targeted by cell therapy. [56] Congenital, lethal surfactant deficiency is another potential target for cell therapy. [57-59] IPF is primarily caused by acquired or genetic AT2 dysfunction.60 At least 10% of IPF cases are inherited. Mutations among others in the surfactant proteins SFTPA and SFTPC, in some Hermansky-Pudlak Syndrome genes, and in telomerase cause familial IPF. [60-63] It could be envisaged that in highly penetrant and early- onset forms of IPF, replacing AT2 cells before the onset of fibrosis might prevent disease. The fact that the engrafted DLEPs prevented septal thickening, which can be an early sign of interstitial lung disease, [52] is compelling in this respect.

[00134] Implementation of cell therapy for lung disease requires both appropriate engrafting cells than can be expanded at clinical scale, and a safe strategy to condition the recipient lungs, although it is possible that a milder injury might allow engraftment in diseased lungs. In the mouse, different types of injury, including bleomycin, toxic gases, and naphthalene target distinct regions and cell types of the lung, but cannot be used clinically because of unacceptable toxicity. [64, 65] Two groups pioneered ex vivo lung decellularization, prior to seeding primary epithelial into the airways and endothelial cells into the vascular compartment followed by transplantation. However, these bioengineered lungs failed only a few hours after transplant due to alveolar edema and thrombosis. [66,67] Complete decellul arization and recellularization in vivo or even ex vivo is likely neither feasible nor necessary. Sequential regional, mild detergent-mediated de-epithelialization, as we demonstrate here, might provide an avenue to develop CT for lung diseases.

[00135] Animals, anesthesia and euthanasia

[00136] Sprague-Dawley (SD) rats weighing 230-250 and 180-220 g were used in the de-epithelialization optimization and characterization experiments and the transplant studies, respectively. All animal work was approved by the Columbia University Institutional Animal Care and Use Committee (IACUC), complying with the National Research Council Guide for the Care and Use of Laboratory Animals (eighth edition).

[00137] Prior to experiments, rats are anesthetized with isoflurane vapor (3-5%) and an intraperitoneal (IP) injection of ketamine (80-95 mg/kg) and xylazine (5-10 mg/kg). The animals were then positioned upright on the rodent workstand (Hallowell EMC) and endotracheally intubated with a modified cannula (JoVet #J0458B). The cannula had a tracheal plug (cut from a 200 mL pipette tip) that fitted it tightly. Throughout the experiment, animals were ventilated with 1% isoflurane on a small animal ventilator (Harvard Apparatus). Ventilation parameters were automatically set by the machine according to the weight.

[00138] Euthanasia was performed with carbon dioxide (CO2) inhalation, followed by bilateral thoracotomy. The aorta was then cut, and the pulmonary vessels were perfused by phosphate-buffered saline (PBS) via the right ventricle. The static pressure was maintained at 13 cm H2O above the heart. OCT compound (Sakura) was diluted in PBS (8 OCT:2 PBS in volume) and injected into the trachea. The injection volume was equivalent to 60% of the animal’s total lung capacity (TLC, calculation see below). A total of six tissue samples were taken from the lung and embedded in the undiluted OCT compound for frozen sectioning.

[00139] In vivo de-epithelialization (DE)

[00140] A dosage was selected to let the injected fluid cover 17% of the alveolar surface in the left lung (derivation not shown):

Dose(uL) = 6.3 * W°-⁷(W, g body weight) [00141] The DE solution contains 4 mM CHAPS (Sigma-Aldrich), 0.5 M NaCl, and 25 mM EDTA in deionized water. Each dose was divided equally into two, with 30 minutes between each instillation. 30 minutes after the second instillation, the work stand was lowered to its horizontal position. At a 15-minute interval, bronchoalveolar lavage (BAL) was performed twice (1 mL of PBS each) via a 3-mL syringe. The tracheal plug was pushed down the cannula to create a temporary seal at the entrance of the trachea. The plug was quickly retrieved after each lavage to ensure ventilation. The BAL fluid was collected for further analysis. The rats were kept on the ventilator to recover and returned to their cages.

[00142] Monitoring and blood analysis

[00143] Heart rate, oxygen saturation, and rectal temperature were monitored during each experiment using a pulse oximeter (Nonin) and a digital thermometer, respectively. Tail vein blood was collected one day before and 48 hours after DE experiments and delivered to Columbia University Comparative Pathology Laboratory (CPL) for pathologic analysis.

[00144] EdU (5-ethynyl-2'-deoxyuridine) incorporation

[00145] To label proliferating cells, 50 mg/kg body weight of EdU was intraperitoneally administered 3 hours before euthanasia. EdU was purchased from Click Chemistry Tools, and the EdU staining kit was purchased from ThermoFisher (#C10338). The staining was done according to the kit manufacturer’s protocol.

[00146] Histology and immunofluorescence (IF) staining

[00147] Frozen sections (5-pm thick) and H&E staining/imaging were provided by the Molecular Pathology Shared Resource (MPSR) facility at Herbert Irving Comprehensive Cancer Center (HICCC), New York. Upon returning to the room temperature, the sections were submerged in 4% paraformaldehyde for 10 minutes, washed with PBS for 5 minutes, and permeabilized in 0.25% Triton X-100/PBS solution for 20 min. After one hour of blocking in 10% donkey serum/PBS solution, EdU staining was performed. Subsequently, the primary antibodies were added to the sections diluted in 5% donkey serum/PBS solution. The slides were then incubated overnight in dark at 4°C. The next day, following three 10-minute washes in 0.025% Triton X-100/PBS solution, the sections were introduced to secondary antibodies (in 5% donkey serum/PBS) for a one-hour binding in the dark. At last, DAPI staining (1 : 1000 in PBS) was performed before the slides were mounted and sealed with nail polish. A list of antibodies can be found in supplementary material. IF images were taken and processed using a Leica DMi8 system.

[00148] hPSC maintenance

[00149] RUES 2 (Rockfeller University Embryonic Stem Cell Line 2, passage 20-27), Sendai Virus human dermal fibroblast iPSC lines (from healthy fibroblasts, purchased from Mount Sinai Stem Cell Core Facility, passage 17-23) were cultured on mouse embryonic fibroblasts (GlobalStem) plated at 17,000-20,000 cells/cm². hPSC maintenance media consisted of DMEM/F12 (Cellgro) 20% Knockout Serum Replacement (Gibco), 0.1 mM [3- mercaptoethanol (Sigma- Aldrich), 1% GlutaMax (Gibco), 1% non-essential amino acids (Gibco), 0.2% primocin (InvivoGen) and 20 ng/ml FGF-2 (R&D Systems). Media were changed daily, and cells were passaged every 4 to 5 days using Accutase/EDTA (Innovative Cell Technologies) and plated 10,000-12,000 cells/cm². Cells were maintained in an undifferentiated state in a humidified 5% CO2 atmosphere at 37°C. The cells are tested for Mycoplasma contamination by PCR every 6 months. Karyotype was performed every 6 months.

[00150] Generation of hPSC-derived lung organoids

[00151] The hPSC-derived human lung organoids were generated as described. [37] Briefly, MEFs were depleted by passaging 5-7 million hPSCs onto Matrigel coated 10-cm dish. Cells were maintained in hPSC media in a humidified 5% CO2 atmosphere at 37°C. After 24 hours, cells were detached with 0.05% Trypsin/EDTA and distributed to the 6-well low attachment plate containing primitive streak/embryoid body media (10 uM Y-27632, 3 ng/ml BMP4) to allow embryoid body formation. Embryoid bodies were fed every day with fresh endoderm induction media (10 uM Y-27632, 0.5 ng/ml BMP4, 2.5 ng/ml FGF2 and 100 ng/ml ActivinA) and maintained in a humidified 5% CCh/5% O2 atmosphere at 37°C. Endoderm yield efficiency was determined by dissociating embryoid bodies and evaluating CXCR4 and c-KIT co-expression by flow cytometry on day 4.3 from endoderm induction. Cells used in all experiments had > 90% endoderm yield and were plated on 0.2% fibronectin-coated wells at a density of 80,000 cells/cm². Cells were incubated in Anteriorization media-1 (100 ng/ml Noggin and lOuM SB431542) for 24 hours, followed by Anteriorization media-2 (10 uM SB431542 and 1 uM IWP2) for another 24 hours. At the end of anterior foregut endoderm induction, cells were switched to Ventralization/Branching media (3 uM CHIR99021, 10 ng/ml FGF10, 10 ng/ml rhKGF, 10 ng/ml BMP4 and 50 nM all-trans Retinoic acid) for 48 hours and three-dimensional clump formation was observed. The adherent clumps were detached by gentle pipetting and transferred to the low-attachment plate, where they folded into lung bud organoids as early as dl0-dl2 (LBOs). Branching media was changed every other day until d20-d25 and LBOs were embedded in 100% Matrigel in 24-well trans-well inserts. Branching media was added after Matrigel solidified and changed every 2-3 days to facilitate proper growth into lung organoids. Culture of embedded organoids can be kept for more than 6 months.

[00152] Generation of hEPC -derived Distal Lung Epithelial Progenitors

[00153] Matrigel embedded lung organoids can be used for generating DEEP when they reach d42 and up of the development. Lung organoids were released from Matrigel by incubating with dispase (lU/ml) for 30-45 minutes in normoxic incubator. Organoids were transferred and washed in the 15 ml conical tube with wash media to neutralize protease, then centrifuged at 200 g for 5 minutes. Pellet was dissociated into small cell clump to single cell with of 0.05% Trypsin/EDTA in normoxic incubator for 10-12 minutes with occasional pipetting with P1000. Dissociated cells were neutralized with wash media, then centrifuged at 400 g for 4 minutes. Dissociated cells were seeded on the Mitomycin C treated 3T3-J2 feeders (20,000 cells/cm²) and cultured with DLEP media (DMEM: Ham’ s-F 12=2:1, 6% FBS, 250 ng/ml amphotericin B, 25 ng/ml hydrocortisone, 5 ug/ml recombinant human insulin, 8 ug/ml cholera toxin, 5 uM Y-27632 Rock Inhibitor). DLEP media was changed every other day. DLEP cells were passaged every week and could be kept more than 20 passages with normal karyotype.

[00154] RNAscope

[00155] RNAscope staining were performed according to the manufacturer’s instruction using the following probe and reagents: Beta-microglobulin (B2M)-C2 (Cat No. 1211661-C2) with RNAscope 2.5 HD Duplex Detection Ki (Chromogenic). Briefly, tissue was freshly harvested and embedded into OCT and further stored at the -80. Tissues were sectioned in 5um and fixed with ice cold 4% paraformaldehyde at 4°C for 15 min. Slides were dehydrated with EtOH and air dry completely. A hydrophobic barrier was drawn around the tissue with ImmEdge Pen (Vector Labs, catalog no. H-4000). Endogenous peroxidase activity was blocked with hydrogen peroxide for 10 min at room temperature (RT). Slides were applied with RNAscope Protease IV for 15-30 min at RT and then proceeded to run the RNAscope assay. We hybridized the probes, applied RNAscope signal amplifiers and labeled probes accordingly. The image was taken with Leica AT2 bright field whole slide scanning system with 40x magnification. [00156] Genomic qPCR

[00157] Genomic DNA was extracted from 200-250um cryopreserved rat lung tissue using the Zymo Quick-DNA MicroPrep, according to the manufacturer’s instructions. Tissues samples were first washed with water to remove residual OCT before lysing with Genomic Lysis Buffer (Zymo D3021). DNA concentration was assessed by absorbance in a spectrophotometer (Thermo Scientific NanoDrop2000c). Human and Rat DNA were verified by qPCR assays using human AluYb8 and rat aryl hydrocarbon receptor gene. Serial diluted human genomic DNA, from 0.01 pg-1 Ong, were mixed with lOOng of rat genomic DNA for standard curve at each set of qPCR. PCR was performed on QuantStudio 5 Real-Time PCR System. qPCR was performed with denaturation at 95°C for 10 minutes, followed by 40 cycles of amplification of 95°C for 15 seconds, 62°C for 5 seconds and 72°C for 15 seconds.

[00158] Quantification of xenotransplantation human cells in Rat lung

[00159] Human AluYb8 qPCR was performed in lOOng gDNA extracted from each section of the rat lung. Human gDNA quantity was calculated from raw Cq value based on the standard curve generated each batch. Human cell engraftment percentage was calculated on the quantity of human gDNA within lOOng rat tissue per section.

[00160] Statistical analysis

[00161] All the applied statistical tests are reported in the legends of the respective figures.

[00162] The foregoing merely illustrates the principles of the disclosure. Various modifications and alterations to the described embodiments will be apparent to those skilled in the art in view of the teachings herein. It will thus be appreciated that those skilled in the art will be able to devise numerous systems, arrangements, and procedures which, although not explicitly shown or described herein, embody the principles of the disclosure and can be thus within the spirit and scope of the disclosure. Various different exemplary embodiments can be used together with one another, as well as interchangeably therewith, as should be understood by those having ordinary skill in the art. In addition, certain terms used in the present disclosure, including the specification, drawings and claims thereof, can be used synonymously in certain instances, including, but not limited to, for example, data and information. It should be understood that, while these words, and/or other words that can be synonymous to one another, can be used synonymously herein, that there can be instances when such words can be intended to not be used synonymously. Further, to the extent that the prior art knowledge has not been explicitly incorporated by reference herein above, it is explicitly incorporated herein in its entirety. All publications referenced are incorporated herein by reference in their entireties.

[00163] REFERENCES

[00164] 1. Bellani, G. et al. Epidemiology, Patterns of Care, and Mortality for

Patients With Acute Respiratory Distress Syndrome in Intensive Care Units in 50 Countries. JAMA 315, 788 (2016).

[00165] 2. Lederer, D. J. & Martinez, F. J. Idiopathic Pulmonary Fibrosis. N. Engl. J.

Med. 378, 1811-1823 (2018).

[00166] 3. Basil, M. C. et al. The Cellular and Physiological Basis for Lung Repair and Regeneration: Past, Present, and Future. Cell Stem Cell 26, 482-502 (2020).

[00167] 4. Barkauskas, C. E. et al. Type 2 alveolar cells are stem cells in adult lung.

J. Clin. Invest. 123, 3025-3036 (2013).

[00168] 5. Nabhan, A. N., Brownfield, D. G., Harbuiy, P. B., Krasnow, M. A. &

Desai, T. J. Single-cell Wnt signaling niches maintain sternness of alveolar type 2 cells. Science 359, 1118-1123 (2018).

[00169] 6. Zacharias, W. J. et al. Regeneration of the lung alveolus by an evolutionarily conserved epithelial progenitor. Nature 555, 251-255 (2018).

[00170] 7. Jain, R. et al. Plasticity of Hopx(+) type I alveolar cells to regenerate type

II cells in the lung. Nat. Commun. 6, 6727 (2015).

[00171] 8. Kobayashi, Y. et al. Persistence of a regeneration-associated, transitional alveolar epithelial cell state in pulmonary fibrosis. Nat. Cell Biol. 22, 934-946 (2020).

[00172] 9 Strunz, M. et al. Alveolar regeneration through a Krt8+ transitional stem cell state that persists in human lung fibrosis. Nat. Commun. 11, 3559 (2020).

[00173] 10. Kim, C. F. B. et al. Identification of bronchioalveolar stem cells in normal lung and lung cancer. Cell 121, 823-835 (2005). [00174] 11 Liu, Q. et al. Lung regeneration by multipotent stem cells residing at the bronchioalveolar-duct junction. Nat. Genet. 51, 728-738 (2019).

[00175] 12. Kumar, P. A. et al. Distal airway stem cells yield alveoli in vitro and during lung regeneration following H1N1 influenza infection. Cell 147, 525-538 (2011).

[00176] 13. Zuo, W. et al. p63(+)Krt5(+) distal airway stem cells are essential for lung regeneration. Nature 517, 616-620 (2015).

[00177] 14. Vaughan, A. E. et al. Lineage-negative progenitors mobilize to regenerate lung epithelium after major injury. Nature 517, 621-625 (2015).

[00178] 15. Kathiriya, J. J., Brumwell, A. N., Jackson, J. R., Tang, X. & Chapman, H.

A. Distinct Airway Epithelial Stem Cells Hide among Club Cells but Mobilize to Promote Alveolar Regeneration. Cell Stem Cell 26, 346-358. e4 (2020).

[00179] 16. Guha, A. et al. Analysis of Notch signaling-dependent gene expression in developing airways reveals diversity of Clara cells. PloS One 9, e88848 (2014).

[00180] 17. Guha, A., Deshpande, A., Jain, A., Sebastiani, P. & Cardoso, W. V.

Uroplakin 3a+ Cells Are a Distinctive Population of Epithelial Progenitors that Contribute to Airway Maintenance and Post-injury Repair. Cell Rep. 19, 246-254 (2017).

[00181] 18. Choi, J. et al. Release of Notch activity coordinated by IL-ip signalling confers differentiation plasticity of airway progenitors via Fosl2 during alveolar regeneration. Nat. Cell Biol. 23, 953-966 (2021).

[00182] 19. Choi, J. et al. Inflammatory Signals Induce AT2 Cell-Derived Damage-

Associated Transient Progenitors that Mediate Alveolar Regeneration. Cell Stem Cell 27, 366- 382. e7 (2020).

[00183] 20. Rawlins, E. L. et al. The role of Scgblal+ Clara cells in the long-term maintenance and repair of lung airway, but not alveolar, epithelium. Cell Stem Cell 4, 525-534 (2009). [00184] 21. Habermann, A. C. el al. Single-cell RNA sequencing reveals profibrotic roles of distinct epithelial and mesenchymal lineages in pulmonary fibrosis. Sci. Adv. 6, eabal972 (2020).

[00185] 22. Kadur Lakshminarasimha Murthy, P. et al. Human distal lung maps and lineage hierarchies reveal a bipotent progenitor. Nature 604, 111-119 (2022).

[00186] 23. Basil, M. C. et al. Human distal airways contain a multipotent secretory cell that can regenerate alveoli. Nature 604, 120-126 (2022).

[00187] 24. Melms, J. C. et al. A molecular single-cell lung atlas of lethal COVID-19.

Nature 595, 114-119 (2021).

[00188] 25. Nichane, M. et al. Isolation and 3D expansion of multipotent Sox9+ mouse lung progenitors. Nat. Methods 14, 1205-1212 (2017).

[00189] 26. Weiner, A. I. et al. Mesenchyme-free expansion and transplantation of adult alveolar progenitor cells: steps toward cell-based regenerative therapies. NPJ Regen. Med. 4, 17 (2019).

[00190] 27. Louie, S. M. et al. Progenitor potential of lung epithelial organoid cells in a transplantation model. Cell Rep. 39, 110662 (2022).

[00191] 28. Rosen, C. et al. Preconditioning allows engraftment of mouse and human embryonic lung cells, enabling lung repair in mice. Nat. Med. 21, 869-879 (2015).

[00192] 29. Nikolic, M. Z. et al. Human embryonic lung epithelial tips are multipotent progenitors that can be expanded in vitro as long-term self-renewing organoids. eLife 6, e26575 (2017).

[00193] 30. Miller, A. J. et al. In Vitro Induction and In Vivo Engraftment of Lung

Bud Tip Progenitor Cells Derived from Human Pluripotent Stem Cells. Stem Cell Rep. 10, 101— 119 (2018).

[00194] 31. Dye, B. R. et al. A bioengineered niche promotes in vivo engraftment and maturation of pluripotent stem cell derived human lung organoids. eLife 5, el 9732 (2016). [00195] 32. Rosen, C. et al. Preconditioning allows engraftment of mouse and human embryonic lung cells, enabling lung repair in mice. Nat. Med. 21, 869-879 (2015).

[00196] 33. Green, M. D. et al. Generation of anterior foregut endoderm from human embryonic and induced pluripotent stem cells. Nat Biotechnol 29, 267-72 (2011).

[00197] 34. Huang, S. X. et al. Efficient generation of lung and airway epithelial cells from human pluripotent stem cells. Nat Biotechnol 32, 84-91 (2014).

[00198] 35. Chen, Y. W. et al. A three-dimensional model of human lung development and disease from pluripotent stem cells. Nat Cell Biol 19, 542-549 (2017).

[00199] 36. D’ Amour, K. A. et al. Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat. Biotechnol. 23, 1534-1541 (2005).

[00200] 37. Matkovic Leko, I. et al. A distal lung organoid model to study interstitial lung disease, viral infection and human lung development. Nat. Protoc. (2023) doi : 10.1038/s41596-023-00827-6.

[00201] 38. Butler, C. R. et al. Rapid Expansion of Human Epithelial Stem Cells

Suitable for Airway Tissue Engineering. Am. J. Respir. Crit. Care Med. 194, 156-168 (2016).

[00202] 39. Sun, X. et al. A census of the lung: CellCards from LungMAP. Dev. Cell

57, 112-145. e2 (2022).

[00203] 40. Montoro, D. T. et al. A revised airway epithelial hierarchy includes

CFTR-expressing ionocytes. Nature 560, 319-324 (2018).

[00204] 41. Guha, A. et al. Neuroepithelial body microenvironment is a niche for a distinct subset of Clara-like precursors in the developing airways. Proc. Natl. Acad. Sei. U. S. A. 109, 12592-12597 (2012).

[00205] 42. Rock, J. R. et al. Notch-dependent differentiation of adult airway basal stem cells. Cell Stem Cell 8, 639-648 (2011).

[00206] 43. Tsao, P.-N. et al. Notch signaling controls the balance of ciliated and secretory cell fates in developing airways. Dev. Camb. Engl. 136, 2297-2307 (2009). [00207] 44. Dorrello, N. V. et al. Functional vascularized lung grafts for lung bioengineering. Sci. Adv. 3, el700521 (2017).

[00208] 45. Burri, P. H., Dbaly, J. & Weibel, E. R. The postnatal growth of the rat lung. I. Morphometry. Anal. Rec. 178, 711-730 (1974).

[00209] 46. Lai, Y. L. & Hildebrandt, J. Respiratory mechanics in the anesthetized rat.

J. Appl. Physiol. 45, 255-260 (1978).

[00210] 47. Barre, S. F., Haberthiir, D., Stampanoni, M. & Schittny, J. C. Efficient estimation of the total number of acini in adult rat lung. Physiol. Rep. 2, el 2063 (2014).

[00211] 48. Kulkami, H. S. el al. Update on the Features and Measurements of

Experimental Acute Lung Injury in Animals: An Official American Thoracic Society Workshop Report. Am. J. Respir. Cell Mol. Biol. 66, el -el 4 (2022).

[00212] 49. Toth, A. et al. Inflammatory blockade prevents injury to the developing pulmonary gas exchange surface in preterm primates. Sci. Transl. Med. 14, eabl8574 (2022).

[00213] 50. Wang, F. et al. RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues. J. Mol. Diagn. JMD 14, 22-29 (2012).

[00214] 51. Prigent, J. et al. Human Progenitor Cell Quantification after

Xenotransplantation in Rat and Mouse Models by a Sensitive qPCR Assay. Cell Transplant. 24, 1639-1652 (2015).

[00215] 52. Renzoni, E. A., Poletti, V. & Mackintosh, J. A. Disease pathology in fibrotic interstitial lung disease: is it all about usual interstitial pneumonia? Lancet Lond. Engl. 398, 1437-1449 (2021).

[00216] 53. Kathiriya, J. J. et al. Human alveolar type 2 epithelium transdifferentiates into metaplastic KRT5+ basal cells. Nat. Cell Biol. 24, 10-23 (2022).

[00217] 54. Kanegai, C. M. et al. Persistent Pathology in Influenza-Infected Mouse

Lungs. Am. J. Respir. Cell Mol. Biol. 55, 613-615 (2016). [00218] 55. Bos, S., Vos, R., Van Raemdonck, D. E. & Verleden, G. M. Survival in adult lung transplantation: where are we in 2020? Curr. Opin. Organ Transplant. 25, 268-273 (2020).

[00219] 56. Jia, S. & Taylor-Cousar, J. L. Cystic Fibrosis Modulator Therapies. Annu.

Rev. Med. 74, 413-426 (2023).

[00220] 57. Cunningham, S., Jaffe, A. & Young, L. R. Children’s interstitial and diffuse lung disease. Lancet Child Adolesc. Health 3, 568-577 (2019).

[00221] 58. Deterding, R. R. et al. Approaching Clinical Trials in Childhood

Interstitial Lung Disease and Pediatric Pulmonary Fibrosis. Am. J. Respir. Crit. Care Med. 200, 1219-1227 (2019).

[00222] 59. Nogee, L. M. Genetic causes of surfactant protein abnormalities. Curr.

Opin. Pediatr. 31, 330-339 (2019).

[00223] 60. Lederer, D. J. & Martinez, F. J. Idiopathic Pulmonary Fibrosis. N Engl J

Med 378, 1811-1823 (2018).

[00224] 61. Yokoyama, T. & Gochuico, B. R. Hermansky-Pudlak syndrome pulmonary fibrosis: a rare inherited interstitial lung disease. Eur. Respir. Rev. Off. J. Eur. Respir. Soc. 30, 200193 (2021).

[00225] 62. Alder, J. K. & Armanios, M. Telomere-mediated lung disease. Physiol.

Rev. 102, 1703-1720 (2022).

[00226] 63. Katzen, J. & Beers, M. F. Contributions of alveolar epithelial cell quality control to pulmonary fibrosis. J. Clin. Invest. 130, 5088-5099 (2020).

[00227] 64. Matute-Bello, G., Frevert, C. W. & Martin, T. R. Animal models of acute lung injury. Am. J. Physiol. Lung Cell. Mol. Physiol. 295, L379-399 (2008).

[00228] 65. Pannu, A. K. & Singla, V. Naphthalene Toxicity in Clinical Practice.

Curr. Drug Metab. 21, 63-66 (2020).

[00229] 66. Ott, H. C. et al. Regeneration and orthotopic transplantation of a bioartificial lung. Nat. Med. 16, 927-933 (2010). [00230] 67. Petersen, T. H. et al. Tissue-engineered lungs for in vivo implantation.

Science 329, 538-541 (2010).

Claims

1. A method for selective de-epithelialization of a portion of a lung in a subject comprising administering an amount of a composition comprising a zwitterionic detergent to a portion of a lung epithelial surface in vivo effective to de-epithelialize a portion of a lung without effecting endothelial removal in the portion of the lung, thereby selectively de- epithelizing a portion of the lung in vivo.

2. A method for treating a lung disease in a subject comprising selectively de-epithelializing a portion of the lung affected by lung disease in vivo so as to thereby permit the de- epithelialized portion of the lung to subsequently, endogenously, re-epithelialize, wherein the selectively de-epithelializing comprises administering an amount of a composition comprising a zwitterionic detergent to a portion of a lung epithelial surface in vivo effective to selectively de-epithelialize of a portion of the lung.

3. A method for treating a lung disease in vivo in a subject comprising: a) selectively de-epithelializing a portion of the lung affected by lung disease in vivo, and b) subsequently administering an amount of (i) stem cells or (ii) genetically-engineered stem cells to the de-epithelialized portion of the lung so as to treat the lung disease, wherein the selectively de-epithelializing comprises administering an amount of a composition comprising a zwitterionic detergent to a portion of a lung epithelial surface in vivo effective to selectively de-epithelialize of a portion of the lung.

4. The method of Claim 3, wherein the (i) stem cells, or (ii) stem cells which are genetically-engineered, are autologous to the subject being treated.

5. The method of any of Claims 1-4, wherein lung microvasculature in the de-epithelialized portion of the lung remains functional. The method of any of Claims 1 -5, wherein no more than 25% of the lung surface is de- epithelialized. The method of any of Claims 1-6, further comprising irradiating the portion of the lung subsequent to selectively de-epithelializing the portion of the lung, or administering to the portion of the lung an inhibitor of endogenous endothelium production and/or an inhibitor of endogenous epithelium production prior to or subsequent to selectively de- epithelializing the portion of the lung. The method of any of Claims 1-7, wherein the amount of a zwitterionic detergent is delivered via a cannula. The method of any of Claims 3-8, wherein the stem cells are embryonic stem cells, or the genetically-engineered stem cells are autologous induced pluripotent stem cells (iPSC) or allogeneic HLA haploidentical iPSC. The method of any of Claims 1-9, wherein the portion of the lung to be selectively de- epithelialized is prior-identified by a computer tomography (CT) scan and/or via a bronchoscope. The method of any of Claims 1-10, wherein placement within the lung of a delivery device to administer the amount of a zwitterionic detergent is assisted using an inflatable bronchoscope balloon. The method of any of Claims 1-11, wherein at least two amounts of a zwitterionic detergent are administered to the portion of the lung epithelial surface in vivo, and wherein the administration of the two amounts is separated in time. The method of Claim 12, wherein the time between administrations is from 15 to 45 minutes. The method of any of Claims 1-11 , wherein the lung is ventilated for a period of time immediately subsequent to the administration of the amount of the zwitterionic detergent. The method of Claims 12 or 13, wherein the lung is ventilated for a period of time immediately subsequent to each administration of the amount of the zwitterionic detergent. The method of Claim 14 or 15, wherein each period of time of ventilation is, independently, from 15 to 45 minutes. The method of Claims 14 or 16, wherein a bronchoalveolar lavage is performed at least on the portion of the lung to which the amount of zwitterionic detergent has been administered, subsequent to it being ventilated. The method of Claim 15 or 16, wherein a bronchoalveolar lavage is performed at least on the portion of the lung subsequent to the lung being ventilated after a second of the two amounts of a zwitterionic detergent has been administered. The method of any of Claims 17 or 18, wherein the bronchoalveolar lavage comprises phosphate-buffered saline. The method of any of Claims 1-19, wherein the lung of the subject being selectively de- epithelialized is affected by a lung disease that compromises lung epithelia. The method of any of Claims 7-20, wherein irradiating the portion of the lung, or administering to the portion of the lung an inhibitor of endogenous endothelium production is conducted prior to endogenous re-epithelialization or prior to subsequently administering an amount of (i) stem cells or (ii) genetically-engineered stem cells to the de-epithelialized portion of the lung. A method for treating a lung ex vivo comprising: a) selectively de-epithelializing a portion of the lung which has been removed from a subject, by administering an amount of a composition comprising a zwitterionic detergent to a portion of a lung epithelial surface effective to selectively de- epithelialize of a portion of the lung; and b) optionally, administering a medication to the lung ex vivo prior to, during, or after step a). The method of Claim 22, further comprising removing the lung from the subject prior to step a). The method of Claim 22 or 23, wherein the subject has a lung infection, further comprising administering an antibiotic and/or antiviral medication to the lung ex vivo. The method of any of Claims 22-24, further comprising treating the de-epithelialized lungs so as to effect re-epithelization ex vivo. The method of any of Claims 22-25, further comprising transplanting the so-treated lung (a) back into the subject from which the lung was removed or (b) into a non-donor subject. The method of any of Claims 1-26, wherein the zwitterionic detergent comprises (3 -((3 - cholamidopropyl) dimethylammonio)-l -propanesulfonate) (CHAPS). The method of any of Claims 1-27, wherein the composition comprising the zwitterionic detergent comprises a minimum amount of zwitterionic detergent effective to achieve micellar formation. The method of any of Claims 1-28, wherein the composition comprising the zwitterionic detergent comprises 4 - 5 mM zwitterionic detergent. The method of any of Claims 1-29, wherein the composition comprising the zwitterionic detergent comprises an amount of a salt. The method of Claim 30, wherein the salt is NaCl. The method of Claim 30 or 31, wherein the salt is 0.25 - 0.75 mM or 0.45 to 0.55mM. The method of any of Claims 22-32, further comprising administering to the portion of the lung an inhibitor of endogenous endothelium production prior to or subsequent to selectively de-epitheli alizing the portion of the lung. The method of any of Claims 30-33, wherein the amount of salt does not effect decellularization of non- epitheli al tissue in the lung. The method of any of Claims 1-34, wherein the composition comprising the zwitterionic detergent comprises a chelating agent. The method of Claim 35, wherein the chelating agent is EDTA. The method of any of Claims 1-35, wherein the composition has a pH of 7.8 - 8.2. The method of any of Claims 1-37, wherein the composition has a pH of 8.0. The method of any of Claims 1-21 or 27-38, wherein the amount of zwitterionic detergent does not result in hepatic damage in the subject. The method of any of Claims 1-21 or 27-39, wherein the amount of zwitterionic detergent does not result in hematologic damage in the subject. The method of any of Claims 33-40, further comprising administering to the portion of the lung an inhibitor of endogenous endothelium production subsequent to selectively de- epithelializing the portion of the lung prior to endogenous re-epithelialization or prior to subsequently administering an amount of (i) stem cells or (ii) genetically-engineered stem cells to the de-epithelialized portion of the lung. A composition for selective de-epithelialization of a lung comprising: an amount of a zwitterionic detergent; an amount of a salt; and an amount of a chelating agent. The composition of Claim 42, wherein the zwitterionic detergent comprises (3 -((3 - cholamidopropyl) dimethylammonio)-l-propanesulfonate) (CHAPS). The composition of Claim 42, wherein the composition comprises an amount of zwitterionic detergent effective to achieve micellar formation in a lung. The composition of any of Claims 42 to 44, wherein the composition comprises 4 - 5 mM zwitterionic detergent. The composition of any of Claims 42 to 45, wherein the salt is NaCl. The composition of any of Claims 42 to 46, wherein the salt is 0.25 - 0.75 mM. The composition of Claim 47, wherein the salt is 0.45 - 0.55 mM. The composition of any of Claims 42 to 48, wherein the amount of salt does not effect decellularization of non-epithelial tissue in a human lung. The composition of any of Claims 42 to 49, wherein the chelating agent is EDTA. The composition of any of Claims 42 to 50, wherein the composition has a pH of 7.8 -

8.2. The composition of Claim 51, wherein the composition has a pH of 8.0. The composition of any of Claims 42 to 52, comprising deionized water.