Multipotent lung progenitor cells for lung regeneration

Abstract

Multipotent lung progenitor cells for lung regeneration are provided.Accordingly, a method for culturing and expanding the isolated lung cell population, a method for confirming the suitability of the isolated lung cell population for administration to a subject in need thereof, and a method for generating the isolated lung cell population are provided, comprising selecting a cell population that is double positive for the expression of epithelial cell markers and endothelial cell markers.Also provided are the isolated lung cell population, pharmaceutical compositions comprising the same, and uses thereof.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Application No. 63 / 287,147, filed December 8, 2021, the contents of which are incorporated by reference in their entirety into this specification.

[0002] FIELD AND BACKGROUND OF THEINVENTION The present invention, in some embodiments thereof, relates to lung progenitor cells, and more particularly, but not exclusively, to methods of generating lung progenitor cells and the use of lung progenitor cells in therapeutic applications. [Background technology]

[0003] End-stage respiratory diseases are one of the leading causes of death worldwide, causing over 5.5 million deaths each year (World Health Organization data for 2020). Currently, the only curative treatment for these conditions is replacing the damaged organ through a lung transplant. Due to a shortage of suitable organs, many patients die while waiting for a transplant, making lung diseases a prime candidate for stem cell therapy.

[0004] Various cell populations have been shown to exhibit regenerative potential, including BM-derived cells (1), lung-derived p63+ cells (2), LNEP (lineage-negative epithelial progenitor cells) (3), and mouse and human sox9+ cells (4), (5). Recently, fetal lung progenitor cells have been proposed as an attractive source for transplantation in mice, provided that endogenous lung progenitor cells are purged from the recipient's lung stem cell niche by suitable conditioning. Thus, in a procedure similar to bone marrow transplantation (BMT), single-cell suspensions of mouse or human fetal / fetal lung cells harvested during the tubular phase of gestation (20–22 weeks in humans, E15–E16 in mice) and IV-injected into recipient mice after conditioning with naphthalene and 6 Gy TBI resulted in significant lung chimerism within the alveolar and bronchiolar lineages. This chimerism was associated with a marked improvement in lung function (6). More recently, remarkable lung chimerism has been extended to transplantation of single-cell suspensions from adult mouse lung donors (7)(8), with approximately three-fold higher cell doses required to achieve levels of chimerism similar to those seen after fetal / fetal cell transplantation (6).

[0005] Further background art includes the following:

[0006] WO 2013 / 084190 discloses a pharmaceutical composition comprising as an active ingredient a suspension of isolated cell population from mammalian fetal / fetal lung tissue, the fetal / fetal lung tissue being at a developmental stage corresponding to the developmental stage of human lung organ / tissue at a gestational age selected from the range of about 20 weeks to about 22 weeks of gestation.

[0007] WO 2016 / 203477 discloses a method for pre-treating a subject in need of transplantation of progenitor cells in suspension of a tissue of interest.

[0008] WO 2017 / 203520 discloses a method for treating a lung disorder or injury, comprising administering to a subject an effective amount of non-syngeneic lung tissue cells in a suspension containing hematopoietic progenitor cells (HPCs) or supplemented with HPCs, the effective amount being sufficient to achieve tolerance to the lung tissue cells without a long-term immunosuppressive regimen. Summary of the Invention

[0009] According to an aspect of some embodiments of the present invention there is provided a method of expanding an isolated lung cell population in culture, comprising the steps of: (a) dissociating lung tissue to obtain an isolated lung cell population; and (b) growing the isolated lung cell population in a medium containing a factor that promotes the proliferation of endothelial cells, a factor that promotes the proliferation of epithelial cells, and a factor that prevents differentiation, to grow a cell population that is double positive for the expression of epithelial cell markers and endothelial cell markers; whereby the isolated lung cell population is expanded in culture.

[0010] According to an aspect of some embodiments of the present invention there is provided a method of confirming suitability of an isolated lung cell population for administration to a subject in need thereof, comprising the steps of: (a) dissociating lung tissue to obtain an isolated lung cell population; (b) expanding the isolated lung cell population in culture; and (c) determining the expression of epithelial and endothelial cell markers on the isolated lung cell population during and / or after culture; Including, proliferation of the cell population double positive for expression of epithelial cell markers and endothelial cell markers above a predetermined threshold indicates that the isolated lung cell population is suitable for administration to the subject; and a lack of proliferation or proliferation below a predetermined threshold of the cell population double positive for expression of epithelial cell markers and endothelial cell markers indicates that the isolated lung cell population is unsuitable for administration to the subject; Thereby, methods are provided for confirming the suitability of an isolated lung cell population for administration to a subject.

[0011] According to an aspect of some embodiments of the present invention, there is provided a method of generating an isolated lung cell population, the method comprising: (a) dissociating lung tissue to obtain an isolated lung cell population; and (b) contacting the isolated lung cell population with at least one reagent capable of binding to an epithelial cell marker and an endothelial cell marker to select a cell population that is double positive for expression of an epithelial cell marker and an endothelial cell marker, thereby generating the isolated lung cell population.

[0012] According to some embodiments of the invention, the method further comprises expanding the lung cells in culture after step (b).

[0013] According to some embodiments of the invention, the culture medium comprises factors that promote endothelial cell proliferation, factors that promote epithelial cell proliferation, and factors that prevent differentiation. According to some embodiments of the invention, the method further comprises determining expression of epithelial and endothelial cell markers on the lung cells during and / or after culture.

[0014] According to some embodiments of the present invention, proliferation of the cell population double positive for expression of epithelial cell markers and endothelial cell markers above a predetermined threshold indicates that the isolated lung cell population is suitable for administration to a subject in need thereof, and no proliferation or proliferation below a predetermined threshold indicates that the isolated lung cell population is unsuitable for administration to a subject.

[0015] According to some embodiments of the invention, the factor that promotes endothelial cell proliferation is selected from the group consisting of vascular endothelial growth factor (VEGF), FGF, FGF2, IL-8, and BMP4.

[0016] According to some embodiments of the invention, the factor that promotes endothelial cell proliferation comprises vascular endothelial growth factor (VEGF).

[0017] According to some embodiments of the invention, the factor that promotes epithelial cell proliferation is selected from the group consisting of epidermal growth factor (EGF), noggin, and R-spondin.

[0018] According to some embodiments of the invention, the factor that promotes epithelial cell proliferation comprises epidermal growth factor (EGF).

[0019] According to some embodiments of the invention, the factor that prevents differentiation is selected from the group consisting of a ROCK inhibitor, a GSK3b inhibitor, and an ALK5 inhibitor.

[0020] According to some embodiments of the invention, the factor that prevents differentiation comprises a ROCK inhibitor.

[0021] According to an aspect of some embodiments of the present invention there is provided an isolated lung cell population comprising at least 40% CD326+CD31+ cells.

[0022] According to an aspect of some embodiments of the present invention there is provided an isolated lung cell population obtained according to the method of some embodiments of the present invention.

[0023] According to an aspect of some embodiments of the present invention there is provided a pharmaceutical composition comprising as an active ingredient the isolated lung cell population of some embodiments of the present invention and a pharma- ceutically acceptable carrier.

[0024] According to an aspect of some embodiments of the present invention, there is provided a method of regenerating pulmonary epithelial and / or endothelial tissue in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an isolated lung cell population of some embodiments of the present invention, thereby regenerating pulmonary epithelial and / or endothelial tissue.

[0025] According to an aspect of some embodiments of the present invention, there is provided a method of treating a pulmonary disorder or lung injury in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an isolated lung cell population of some embodiments of the present invention, thereby treating the pulmonary disorder or lung injury.

[0026] According to an aspect of some embodiments of the present invention, there is provided a therapeutically effective amount of the isolated lung cell population of some embodiments of the present invention for use in treating a pulmonary disorder or lung injury in a subject in need thereof.

[0027] According to one aspect of some embodiments of the present invention, there is provided a kit for isolating lung cells characterized by double positive expression of epithelial cell markers and endothelial cell markers, the kit comprising: (I) at least one reagent capable of binding to (i) CD31 or CD144, and (ii) CD326, CD324, CD24, aquaporin 5 (AQP-5), podoplanin (PDPN), or receptor for advanced glycation end products (RAGE); and (II) instructions for use.

[0028] According to one aspect of some embodiments of the present invention, there is provided a cell bank comprising: (i) a plurality of isolated lung cell populations in suspension, wherein the lung cells are characterized as double positive for expression of epithelial cell markers and endothelial cell markers, and the plurality of isolated lung cell populations are HLA typed to form an allogeneic cell bank, each individually disposed in separate containers; and (ii) a catalog comprising information regarding the HLA-typed cells of the plurality of isolated lung cell populations.

[0029] According to some embodiments of the invention, the epithelial cell markers include CD326, CD324, CD24, aquaporin 5 (AQP-5), podoplanin (PDPN), or receptor for advanced glycation end products (RAGE).

[0030] According to some embodiments of the invention, the endothelial cell marker comprises CD31 or CD144 (VE-cadherin).

[0031] According to some embodiments of the present invention, the cell population that is double positive for the expression of epithelial cell markers and endothelial cell markers is CD326 + CD31 + Includes signature.

[0032] According to some embodiments of the present invention, the cell population that is double positive for expression of epithelial cell markers and endothelial cell markers is CD324 + CD31 + Includes signature.

[0033] According to some embodiments of the present invention, the cell population that is double positive for the expression of epithelial cell markers and endothelial cell markers is CD326 + CD144 + Includes signature.

[0034] According to some embodiments of the present invention, the cell population that is double positive for expression of epithelial cell markers and endothelial cell markers is CD324 + CD144 + Includes signature.

[0035] According to some embodiments of the invention, the method further comprises depleting CD45 expressing cells.

[0036] According to some embodiments of the invention, CD45 expressing cells are depleted by contacting the isolated lung cell population with an agent capable of binding to CD45 and selecting for a cell population that is negative for expression of CD45.

[0037] According to some embodiments of the invention, the method further comprises depleting T cells.

[0038] According to some embodiments of the invention, the method further comprises expanding the lung cells in culture after step (b).

[0039] According to some embodiments of the invention, the at least one reagent capable of binding is an antibody.

[0040] According to some embodiments of the invention, the antibody is a monospecific antibody.

[0041] According to some embodiments of the invention, the antibody is a bispecific antibody.

[0042] According to some embodiments of the invention, the dissociating is by enzymatic digestion.

[0043] According to some embodiments of the invention, the method is performed ex vivo.

[0044] According to some embodiments of the invention, the lung tissue is fetal / fetal lung tissue.

[0045] According to some embodiments of the invention, the lung tissue is adult lung tissue.

[0046] According to some embodiments of the invention, the lung tissue is human lung tissue.

[0047] According to some embodiments of the invention, the lung tissue is from a cadaveric donor.

[0048] According to some embodiments of the invention, the lung tissue is from a living donor.

[0049] According to some embodiments of the invention, the lung cells are capable of regenerating lung epithelial tissue.

[0050] According to some embodiments of the invention, the lung cells are capable of regenerating pulmonary endothelial tissue.

[0051] According to some embodiments of the invention, the cells are in suspension.

[0052] According to some embodiments of the invention, the cells are embedded or attached to the scaffold.

[0053] According to some embodiments of the invention, the pharmaceutical composition further comprises hematopoietic progenitor cells (HPCs) as an active ingredient.

[0054] According to some embodiments of the invention, the HPCs comprise T cell depleted immature hematopoietic cells.

[0055] According to some embodiments of the invention, the method further comprises, prior to administration, administering to the subject a reagent capable of inducing injury to lung tissue, which injury results in proliferation of resident stem cells in the lung tissue.

[0056] According to some embodiments of the invention, the method further comprises pretreating the subject under a sublethal, lethal, or supralethal pretreatment protocol prior to administration.

[0057] According to some embodiments of the invention, the method further comprises administering to the subject an effective amount of hematopoietic progenitor cells (HPCs).

[0058] According to some embodiments of the invention, the method further comprises treating the subject with an immunosuppressant after administration.

[0059] According to some embodiments of the present invention, the isolated lung cell population for use further comprises the use of an agent capable of inducing injury to lung tissue, which injury results in proliferation of resident stem cells within the lung tissue.

[0060] According to some embodiments of the invention, the isolated lung cell population for use further comprises a sublethal, lethal, or supralethal conditioning protocol.

[0061] According to some embodiments of the invention, the isolated lung cell population for use further comprises the use of an effective amount of hematopoietic progenitor cells (HPCs).

[0062] According to some embodiments of the invention, the isolated lung cell population for use further comprises use of an immunosuppressant.

[0063] According to some embodiments of the invention, the agent capable of inducing damage to lung tissue is selected from the group consisting of chemotherapeutic agents, immunosuppressants, amiodarone, beta blockers, ACE inhibitors, nitrofurantoin, procainamide, quinidine, tocainide, and minoxidil.

[0064] According to some embodiments of the present invention, the agent capable of inducing damage to lung tissue comprises naphthalene.

[0065] According to some embodiments of the invention, the conditioning protocol includes reduced intensity conditioning (RIC).

[0066] According to some embodiments of the invention, the conditioning protocol includes at least one of total body irradiation (TBI), partial body irradiation, chemotherapy, and / or antibody immunotherapy.

[0067] According to some embodiments of the invention, the antibody immunotherapy comprises T cell debulking.

[0068] According to some embodiments of the invention, the antibody immunotherapy comprises an antithymocyte globulin (ATG) antibody, alemtuzumab, muromonab-CD3, or a combination thereof.

[0069] According to some embodiments of the invention, the TBI comprises a single or fractionated dose of radiation in the range of 1-10 Gy.

[0070] According to some embodiments of the invention, HPCs are derived from lung tissue and / or CD34 + Contains cells.

[0071] According to some embodiments of the invention, HPCs are derived from bone marrow CD34 + Cell or mobilized peripheral blood CD34 + Contains cells.

[0072] According to some embodiments of the invention, the HPCs comprise T cell depleted immature hematopoietic cells.

[0073] According to some embodiments of the invention, the isolated lung cell population and the HPCs are obtained from the same donor.

[0074] According to some embodiments of the invention, the isolated lung cell population and the hematopoietic progenitor cells (HPCs) are present in separate formulations.

[0075] According to some embodiments of the invention, the isolated lung cell population and the HPCs are present in the same formulation.

[0076] According to some embodiments of the invention, the isolated lung cell population and / or HPCs are formulated for intravenous or intratracheal routes of administration.

[0077] According to some embodiments of the invention, the immunosuppressant comprises cyclophosphamide, busulfan, fludarabine, tacrolimus, cyclosporine, mycophenolate mofetil, azathioprine, everolimus, sirolimus, glucocorticoids, or combinations thereof.

[0078] According to some embodiments of the invention, the subject is a human subject.

[0079] According to some embodiments of the invention, the isolated lung cell population is non-syngeneic to the subject.

[0080] According to some embodiments of the invention, the lung disorder or injury comprises chronic inflammation of the lungs.

[0081] According to some embodiments of the invention, the lung disorder or injury is selected from the group consisting of cystic fibrosis, emphysema, asbestosis, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, idiopathic pulmonary fibrosis, pulmonary hypertension, lung cancer, sarcoidosis, acute lung injury (adult respiratory distress syndrome), respiratory distress syndrome of prematurity, chronic lung disease of prematurity (bronchopulmonary dysplasia), surfactant protein B deficiency, congenital diaphragmatic hernia, pulmonary alveolar proteinosis, pulmonary hypoplasia, and asthma.

[0082] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used to practice or test embodiments of the present invention, exemplary methods and / or materials are described below. In case of conflict, the present patent specification, including definitions, will control. In addition, the materials, methods, and examples are merely illustrative and are not necessarily intended to be limiting.

[0083] Some embodiments of the present invention are described herein, by way of example only, with reference to the accompanying drawings. With specific reference now made in detail to the drawings, it is stressed that the particulars shown are by way of example only and for the purpose of illustratively discussing embodiments of the present invention. In this regard, reading the description in conjunction with the drawings will make apparent to those skilled in the art how embodiments of the present invention may be practiced.

[0084] The drawings are as follows: [Brief description of the drawings]

[0085] [Figure 1-1]Figure 1A-F shows multilineage engraftment of mTom donor-derived cells in recipient lungs, as assessed by sc-RNA seq. Figure 1A shows the cell sorting design of host and donor lung cells derived from chimeric lungs. Three chimeric lungs were first verified by fluorescence microscopy to exhibit significant chimerism, pooled, enzymatically dissociated, gated on single CD45- live cells, and then FACS-sorted into donor and recipient compartments based on Td-Tomato expression. Figure 1B shows FACS analysis of chimeric lungs transplanted with Td-Tomato cells and control non-transplanted lungs. Figure 1C-D shows transcriptome analysis of FACS-sorted donor (Figure 1C) and recipient (Figure 1D)-derived compartments 6 months after transplantation. Monocle3 UMAP and proportional plots of clusters show analysis of donor (n=6081) and recipient cells (n=3210) and clarify that the major epithelial and endothelial clusters in the donor-derived compartment are equivalent to those defined in the recipient-derived compartment. Figure 1E-F show heatmaps identifying functionally distinct gCap and aCap endothelial cells in the donor (Figure 1E) and recipient (Figure 1F) compartments, respectively. [Figure 1-2] Same as above [Figure 1-3] Same as above [Figure 1-4] Same as above [Figure 2-1]Figure 2A-E show the transplantation of fetal lung cells from Confetti donors. Figure 2A shows the experimental scheme used to implant E16 fetal lung cells after tamoxifen-induced CRE recombination into immunodeficient RAG recipients pretreated with naphthalene and 6 Gy TBI. Figure 2B shows FACS analysis of E16 R26R-Confetti fetal lungs showing the percentage of Cre-recombined cells (n=5 embryos). Figure 2C shows two-photon microscopy images of E16 fetal lung tissue illustrating single monochromatic cells in R26R-Confetti mice after TMX induction and before transplantation (from n=3 embryos). Figure 2D shows a monochromatic patch 6 weeks after transplantation of E16 fetal lung cells from R26R-Confetti donors into RAG2- / - recipient mice. Whole mounts of chimeric lungs were analyzed by low-magnification fluorescence microscopy (scale bar=200 μm). Figure 2E shows a typical analysis of the same chimeric lung by two-photon microscopy, showing monochromatic patches expressing different fluorescent tags (scale bar = 50 μm). Results are representative of three independent experiments, with n = 3 mice in each experiment. [Figure 2-2] Same as above [Figure 3-1]Figure 3A-G shows lung chimerism analysis 8 weeks after transplantation of adult R26R-Confetti lung cells. Figure 3A is a schematic representation of the experimental procedure. The left image in Figure 3B is a two-photon microscopy image of an adult lung before transplantation, revealing a single cell expressing one of the four tag colors after Cre recombination (n=3). The right image in Figure 3B is a confocal image of an adult R26R-Confetti lung slice, showing the bronchial and alveolar parts of the lung tissue, as well as the diffuse and random localization of fluorescent cells within the donor lung. Results are representative of R26R-Confetti mice (n=6) from two independent experiments. Figure 3C-D shows FACS analysis of adult R26R-Confetti lungs after administration of two doses of TMX, showing the percentage of Cre-recombined cells carrying the different fluorescent tags (n=5). Figure 3E-F shows a clearing procedure resulting in transparent chimeric lungs, which were analyzed by light sheet microscopy. A total of n=2 chimeric lungs were evaluated by LSM. Scale bars are 60 μm in FIG. 3E and 40 μm in FIG. 3F. FIG. 3G shows typical monochromatic patches, each of which exhibits a distinct color, in a whole-mount chimeric lung. A total of n=4 chimeric lungs were evaluated by two-photon microscopy. Scale bars=50 μm. [Figure 3-2] Same as above [Figure 4-1]Figure 4A-H show donor-derived lung patches after transplantation of different lung cell subpopulations. Figure 4A shows the gating strategy for FACS sorting of CD45- lung cells into four subpopulations including CD326+CD31- cells, CD326+CD31+ cells, CD326-CD31+ cells, and CD326-CD31- cells. Figure 4B shows visualization of double positive CD45-CD326+CD31+ lung cells by Imagestream analysis. Figure 4C shows a schematic representation of the transplantation experiments. Figure 4D shows the percentage of each subpopulation sorted from the CD45- non-hematopoietic lung cell population in 16 experiments. Figure 4E shows the percentage of chimeric mice exhibiting donor-derived patches out of the total number of mice that received transplants (n=16 experiments). Figure 4F shows donor-derived lung patches 6 weeks after transplantation of 0.3–0.5 × 106 sorted double-positive CD326+CD31+ cells or single-positive CD31+ endothelial cells (red) from an nTnG donor mixed with 0.5 × 106 unsorted cells (green) from a GFP+ donor. Whole-mount confocal images shown for each group are representative of n=16 experiments (at least n=10 mice in each group), scale bar=500 μm. Of all mice that underwent transplantation (n=98), 70% were transplanted with td-tomato sorted+unsorted GFP cells and 30% were transplanted with td-tomato FACS-sorted cells only. Figure 4G–H show representative images of whole-mount lungs from mice transplanted with 0.5 × 106 nTnG CD326+CD31+ cells or CD31+ cells without co-transplantation of GFP+unsorted lung cells. Donor-derived red patches express nuclear td-tomato. Low (scale bar = 500 μm) and high magnification (scale bar = 100 μm) images are representative of each group of mice (n = 5). [Figure 4-2] Same as above [Figure 5-1]Figure 5A-K shows the different cellular composition of donor-derived patches after transplantation of sorted CD326+CD31+ lung cell subpopulations versus donor-derived patches after transplantation of sorted CD326-CD31+ lung cell subpopulations. Figure 5A-D shows staining of a typical lung patch derived from sorted nTnG CD326-CD31+ lung cells (red). Figure 5A-B shows staining for the endothelial marker ERG (nuclei, green), scale bar = 100 μm (Figure 5A) and 10 μm (Figure 5B). Figure 5C shows staining for the endothelial nuclear marker SOX17 (cyan) in a donor-derived nTnG positive patch after transplantation of CD326-CD31+ cells. Stained markers are indicated above the images, scale bar = 10 μm. FIG. 5D shows staining for cell surface endothelial marker CD31 and epithelial marker HOPX in donor-derived patches formed after transplantation of CD326-CD31+ cells. Left: double staining for CD31 and SOX17, right: triple staining for nTnG+ (red), CD31 (blue), and HOPX (green). Donor-derived endothelial CD31+ cells are present in close proximity to recipient HOPX+AT1 cells (indicated by arrowheads), whereas donor-derived AT1 cells are undetectable, scale bar = 10 μm. FIG. 5E-F show staining of a typical lung patch derived from sorted nTnG CD326+CD31+ lung progenitor cells, showing donor-derived epithelial and endothelial cells. FIG. 5E, Donor-derived AT1 epithelial cells (arrowheads) and endothelial cells (arrows) are shown. Left panel: double staining for nTnG (red) and SOX17 (cyan); center panel: single staining for SOX17; right panel: triple staining for nTnG (red), SOX17 (cyan), and HOPX (green); scale bar=15 μm. FIG. 5F, high magnification of typical staining for HOPX showing donor AT1 cells (red, indicated by arrowheads) and host AT1 cells (indicated by arrowheads). FIG. 5G, high magnification of triple staining of CD326+CD31+ derived patches demonstrating the close presence of donor-derived AT1 cells (red) and endothelial CD31+ cells. Arrows indicate donor-derived AT1 cells and arrowheads indicate host-derived AT1 cells. In FIG. 5F and G, scale bar=10 μm. Images are representative of n=3 mice.FIG. 5H shows staining of a typical lung patch derived from sorted nTnG CD326+CD31+ lung cells, showing donor-derived epithelial AT1 cells (AQP-5+, purple) and endothelial cells (SOX17+, green). Alveolar AT1 cells (arrowheads) and endothelial cells (arrows) are depicted. Left panel: double staining for nTnG (red) and SOX17 (green); middle panel: double staining for nTnG (red) and AQP-5 (purple); right panel: triple staining for nTnG, SOX17, and AQP-5; Scale bar=20 μm. Images are representative of n=3 mice. FIG. 5I shows staining of a typical donor-derived patch for CD31 (green) and single molecule RNA FISH probe for SPC (cyan), showing endothelial and AT2 cells; Scale bar=20 μm. Figure 5J-K shows a graphical summary of the quantitative differences between the composition of patches derived from transplantation of CD326+CD31+ cells and those derived from transplantation of CD326-CD31+ cells. Figure 5J is a graph of donor-derived nuclei / patch showing greater patch size derived from CD326+CD31+ cells compared to CD326-CD31+ cells, p=0.035, Student's t-test, n=20 patches were evaluated from each group of mice (n=3). Figure 5K is a graph of the absolute number of donor-derived epithelial and endothelial cells per patch after transplantation from CD326+CD31+ cells or CD326-CD31+ cells. A and B represent epithelial cells derived from transplanted CD326-CD31+ and CD326+CD31+ populations, respectively (p=0.0001, Student's t-test); C and D represent endothelial cells derived from transplanted CD326-CD31+ and CD326+CD31+ populations, respectively (p=0.04, Student's t-test); n=10 patches from 2 mice per group were evaluated. [Figure 5-2] Same as above [Figure 6-1]Figure 6A-J demonstrates differential transgene expression in double positive CD326+CD31+ patch forming lung cell progenitors. CD326+CD31+ lung cells from transgenic reporter mice expressing GFP under the Shh promoter (Figure 6A,C,F) or VE-cadherin promoter (Figure 6B,C,G) were analyzed for expression of these markers by FACS and Imagestream analysis (n=6 mice). Cells from these mice express td tomato and are red, but express GFP and turn green when the transgene is expressed. Figure 6A-B shows representative dot plots demonstrating GFP expression of gated CD326+CD31+ double positive cells. Figure 6C shows the percentage of CD326+CD31+ double positive cells compared to CD326+VEcad+ cells in VE-cadherin Cre mTmG mice or CD31+Shh+ cells in Shh Cre mTmG mice. The figures show the results of individual transgenic mice (n=6 for each genotype). Figure 6D left shows the culture of FACS-purified CD326+VEcad- (mT) and CD326+VEcad+ (mG) lung cell populations from VEcad mTmG mice under 3D conditions (see Figure 14A for FACS sorting scheme). CD326+VEcad- cells that do not express VEcad result in mT+ red organoids that do not express GFP, whereas double-positive CD326+VEcad+ cells result in the generation of green organoids that express both GFP and various epithelial markers (e.g., CK, AQP-5, and SPC (magenta)). Figure 6D right shows the absolute number of organoids per well observed when 5x105 CD326+VEcad- or CD326+VEcad+ FACS-sorted lung cells were seeded. The top panel of Figure 6E shows an organoid expressing GFP (green, from VE-cadherin expressing cells) and the epithelial marker cytokeratin (magenta). The bottom left panel of Figure 6E shows an organoid expressing GFP (green, from VE-cadherin expressing cells) and the epithelial marker AQP-5 (magenta) for alveolar AT1 pneumocytes.The lower right panel of Figure 6E shows organoids expressing GFP (green, from VE-cadherin expressing cells) and the epithelial marker SPC (magenta) for alveolar AT2 lung cells. Figure 6F-G shows Imagestream analysis of CD326+CD31+ lung cell progenitors from Shh Cre nTnG or VEcad Cre nTnG mice, illustrating the expression of Shh and VE-cad in these cells. Figure 6H-J shows FACS analysis of double positive CD326+CD31+ lung cells from Nkx2.1 Cre ER2 mTmG mice (n=3), Ager Cre ER2 mTmG mice (n=5), and Hopx Cre ER2 mTmG mice (n=3), clarifying the percentages of Nkx2.1+, Ager+, and Hopx+ cells. [Figure 6-2] Same as above [Figure 6-3] Same as above [Figure 6-4] Same as above [Figure 7]Figure 7A-E show staining of regenerating patches within chimeric lungs for epithelial and endothelial markers. Figure 7A shows a donor-derived bronchial patch demonstrating extensive engraftment of donor cells in the bronchi. Donor cells stain positive for membranous Ecad (blue) and nuclear Nkx-2.1 (cyan), nuclei (yellow) (scale bar = 20 μm). Figure 7B shows a donor-derived bronchoalveolar patch stained with anti-CD31 (blue) and anti-cytokeratin (cyan) antibodies demonstrating chimerism in the epithelial and endothelial compartments (scale bar = 50 μm). Figure 7C shows staining of a donor-derived bronchial patch with anti-CD31 (blue) and anti-CC-10 (cyan) antibodies as well as nuclei (gray) (scale bar = 50 μm) demonstrating engraftment in the secretory cell compartment. Images shown are representative of n = 3 mice. FIG. 7D shows staining with anti-AQP-5 antibody (green) demonstrating the presence of donor-derived mTom AT1 cells (red) within the patch (nuclei (blue), scale bar=5 μm). FIG. 7E shows staining of an alveolar patch with an SPC probe (smFISH) (green) and anti-CD31 antibody (cyan) demonstrating the presence of AT2 cells and endothelial cells within the patch (nuclei (grey), scale bar=10 μm). [Figure 8-1] Figure 8A-E shows transplantation of adult R26R-Confetti BM from donors induced to express Cre recombination with tamoxifen. Figure 8A is a schematic representation of a spleen colony assay using adult BM from an R26R-Confetti donor. Figure 8B-C shows FACS analysis demonstrating expression of the fluorescent tag in LSK+ BM progenitor cells prior to BM transplantation. BM was analyzed from adult R26R-Confetti mice (n=8) in two independent experiments. Figure 8D shows the formation of monochromatic splenic colonies 9 days after transplantation of BM from an R26R-Confetti donor into lethally irradiated mice. Figure 8E shows peripheral blood chimerism, demonstrating the presence of fluorescent clones in the blood compartment that originated from transplanted Cr recombination cells. Results shown in Figure 8D-E are representative of two independent experiments, with n=7 mice in each experiment. [Figure 8-2] Same as above [Figure 9-1] 9A-E show transplantation of E16 R26R-Confetti livers into mice pretreated with NA+6Gy. 9A shows the experimental workflow. 9B shows FACS analysis of E16 confetti fetal livers 4 days after Tmx administration, demonstrating the expression of four fluorescent tags within the Sca1+Ckit+ hematopoietic progenitor population. 9C shows monochromatic splenic colonies generated by fetal liver cells transplanted into mice pretreated with NA+6Gy TBI. Scale bar=500 μm. 9D shows FACS analysis of peripheral blood from chimeric mice 2 months after transplantation, demonstrating the persistence of monochromatic clones within the blood compartment. 9E shows representative lung two-photon images of mice pretreated with Na+6GY TBI and transplanted with E16 fetal liver cells, demonstrating the presence of isolated fluorescent cells and the absence of monochromatic donor-derived patches, confirming the unique ability of lung cells to mediate lung regeneration. Scale bar = 50 μm. Results are representative of mice (n = 10) transplanted in two independent experiments. [Figure 9-2] Same as above [Figure 10] Figures 10A-B show the appearance of cleared chimeric lung samples prior to evaluation by LSM. Staining of the cleared chimeric lungs is shown in Figures 3E-F. [Figure 11] Figure 11A-B show staining of the lungs of chimeric mice transplanted with FACS-sorted CD326+CD31+ cells from mTmG mice. Figure 11A shows staining of the regenerated patch with anti-CD31 (blue) and anti-Nkx2.1 (cyan) antibodies, demonstrating the presence of epithelial and endothelial cells within the regenerated patch (scale bar = 50 μm). Figure 11B shows staining of the sample shown in Figure 11A with anti-AQP-5 (cyan) and anti-CD31 (blue) antibodies, demonstrating the presence of donor-derived AT1 cells (indicated by arrows) and endothelial cells (indicated by arrowheads) in the engrafted area (scale bar = 10 μm). [Figure 12]Figure 12A-D demonstrates long-term chimerism 9 months after transplantation in mice transplanted with CD326+CD31+nTnG FACS-sorted cells. Figure 12A shows whole-mount lung tissue assessed by confocal microscopy, scale bar = 500 μm. Figure 12B shows low-magnification confocal images of chimeric lung slices stained with anti-Lyve-1 (blue) and anti-HOPX (green) antibodies, demonstrating extensive engraftment of donor-derived cells in different compartments of the lung, scale bar = 200 μm. Figure 12C is a confocal image of chimeric lung stained with anti-CD31 (blue) showing endothelial cells with nT nuclei (indicated by arrowheads) and anti-SPC (green) showing AT2 cells with nT nuclei (indicated by arrows), scale bar = 20 μm. FIG. 12D shows staining of chimeric lungs with anti-Hopx antibody, showing donor-derived AT1 cells (indicated by arrows), Scale bar=20 μm. [Figure 13-1] Figure 13A-E show FACS analysis of the lungs of transgenic mice. Figure 13A shows a typical dot plot representing double positive mG-Shh+CD31+ cells in the lungs of Shh Cre mTmG mice (n=6). Figure 13B shows a typical dot plot representing double positive CD326+mG-VEcad+ cells in the lungs of VE cad Cre mTmG mice (n=6). Figure 13C shows a typical dot plot representing double positive mG-Nkx2.1+CD31+ cells in the lungs of Nkx2.Cre ER2 mTmG mice (n=5). Figure 13D shows a typical dot plot representing double positive mG-Ager+CD31+ cells in the lungs of Ager Cre ER2 mTmG lungs (n=5). FIG. 13E shows a representative dot plot representing double positive mG-Hopx+CD31+ cells in the lungs of Hopx Cre ER2 mTmG lungs (n=3). [Figure 13-2] Same as above [Figure 14-1]Figure 14A-D shows the staining for epithelial markers of organoids grown from FACS-purified CD326+VEcad- harvested from the lungs of VEcad mTmG mice. Figure 14A shows the gating strategy for purifying single-positive CD326+VE cad mG- and double-positive CD326+VE cad mG+ cells from VEcad mTmG mice. CD45-TER119-Sytox-CD326+ single live cells were further gated according to the expression of VE-cad mG to purify CD326+VE-cad mG- and CD326+VE cad mG+ lung cell subpopulations. The isolated cells were used to generate lung organoids as described in the methods below. Organoids were stained with anti-CK (Figure 14B) (cyan, scale bar = 20 μm), anti-AQP-5 (Figure 14C) (cyan, scale bar = 10 μm), and anti-SPC (Figure 14D) (cyan, scale bar = 7 μm). Nuclei were stained with DAPI (blue). Images are representative of two independent experiments. [Figure 14-2] Same as above [Figure 15-1] Figures 15A-C demonstrate that different culture media have different effects on the proliferation of lung cells dually expressing endothelial and epithelial markers. The top panel of Figure 15A shows FACS analysis demonstrating the levels of double positive CD326+CD31+ cells at various culture time points when incubated in conditioned medium (CM) from mouse fibroblasts supplemented with EGF and low concentration of ROCK inhibitor (5 μm) (labeled "original medium"). The bottom panel of Figure 15A shows FACS analysis demonstrating the levels of double positive CD326+CD31+ cells at various culture time points when incubated in CM supplemented with EGF, VEGF, and high concentration of ROCK inhibitor (20 μm) (labeled "CM+Epi+Endo+High RICK-I"). Figure 15B shows the total cell counts at the indicated days in the two cultures described in Figure 15A. FIG. 15C shows the number of CD326+CD31+ double positive cells on the indicated days for the two cultures described in FIG. 15A. [Figure 15-2] Same as above DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0086] The present invention, in some embodiments thereof, relates to lung progenitor cells, and more particularly, but not exclusively, to methods of generating lung progenitor cells and the use of lung progenitor cells in therapeutic applications.

[0087] The principles and operation of the present invention may be better understood with reference to the drawings and accompanying descriptions.

[0088] Before describing at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or illustrated by the examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.

[0089] Currently, the only curative treatment for end-stage respiratory disease is replacing the damaged organ via lung transplantation. A shortage of suitable organs means many patients die while waiting for a transplant, making lung disease a prime candidate for stem cell therapy.

[0090] It has been shown that different cell populations, e.g. BM- or lung-derived cells, exhibit lung regenerative potential and result in significant lung host-donor chimerism. Notably, the majority of donor-derived patches exhibit different lineages of lung compartments, including epithelial and endothelial cells. We investigated the possibility that each donor patch originates from a single progenitor cell after lung cell transplantation. To that end, as shown in the Examples section below (see Example 1), fetal or adult lung cells from Rosa26-Confetti mice (9) carrying a multicolor Cre reporter system were transplanted into recipients pretreated with naphthalene and TBI. This four-color Cre recombination system provides preparations of fetal or adult lung cells in which each cell expresses only one randomly determined color. Thus, the chance that each cell within a doublet of transplanted cell populations will be the same color is significantly reduced. Notably, immunohistochemistry, confocal microscopy, and two-photon and light sheet microscopy demonstrated that all donor-derived lung patches that developed after transplantation were monochromatic, strongly supporting the clonal origin of the donor-derived lung patches observed after transplantation, which is strikingly similar to the splenic colony-forming cells typically identified after bone marrow transplantation. These results conclusively demonstrated a single multipotent lung progenitor cell that can differentiate into various lung cell lineages. In line with the observation that the majority of patches contain both endothelial and epithelial cells, and that each such patch originates from a single progenitor cell, the inventors searched for putative multipotent lung progenitor cells that can differentiate along these two distinct lineages.

[0091] Subsequently, in putting the embodiments of the present invention into practice, the inventors discovered a novel population of lung progenitor cells that dually express endothelial and epithelial markers (see Example 1 in the Examples section below). These lung progenitor cells were obtained from both fetal and adult lung tissues. Furthermore, these lung progenitor cells were capable of differentiating into lung epithelial cells in addition to lung endothelial cells, and as a result, were capable of generating lung endothelial and epithelial tissues after transplantation into a recipient. Taken together, these results demonstrate the use of the novel lung progenitor cells for regeneration of lung organs or lung tissues, such as for treating lung injury or lung disease.

[0092] Thus, according to one aspect of the invention there is provided a method of generating an isolated lung cell population comprising the steps of: (a) dissociating lung tissue to obtain an isolated lung cell population; and (b) contacting the isolated lung cell population with at least one reagent capable of binding to an epithelial cell marker and an endothelial cell marker to select a cell population that is double positive for expression of the epithelial cell marker and the endothelial cell marker; thereby producing an isolated lung cell population.

[0093] Furthermore, the inventors found that different culture conditions have different effects on the proliferation of this novel lung progenitor cell population. Specifically, it was shown that when dissociated lung cells were cultured in a medium containing VEGF, EGF, and a ROCK inhibitor, the cells that double-express endothelial and epithelial markers significantly proliferated, whereas when these cells were cultured in a medium containing only EGF and a ROCK inhibitor, the total number of cells expanded but not the total number of double-positive cells (Example 2 in the Examples section below). Taken together, these results demonstrate the need to culture lung cells under conditions that allow the proliferation of lung progenitor cells that double-express endothelial and epithelial markers, and / or to confirm the proliferation of these progenitor cells before using the cultured cells (e.g., for regenerating lung organs or tissues for treating lung injury or disease).

[0094] Thus, according to one aspect of the invention there is provided a method of expanding an isolated lung cell population in culture, comprising the steps of: (a) dissociating lung tissue to obtain an isolated lung cell population; and (b) growing the isolated lung cell population in a medium containing a factor that promotes the proliferation of endothelial cells, a factor that promotes the proliferation of epithelial cells, and a factor that prevents differentiation, to grow a cell population that is double positive for the expression of epithelial cell markers and endothelial cell markers; whereby the isolated lung cell population is expanded in culture.

[0095] According to a further or alternative aspect of the invention there is provided a method for confirming suitability of an isolated lung cell population for administration to a subject in need thereof, comprising the steps of: (a) dissociating lung tissue to obtain an isolated lung cell population; (b) expanding the isolated lung cell population in culture; and (c) determining the expression of epithelial and endothelial cell markers on the isolated lung cell population during and / or after culture; wherein proliferation of the cell population that is double positive for expression of epithelial cell markers and endothelial cell markers exceeds a predetermined threshold, indicating that the isolated lung cell population is suitable for administration to a subject; and a lack of proliferation of the cell population double positive for expression of epithelial cell markers and endothelial cell markers or proliferation below a predetermined threshold indicates that the isolated lung cell population is unsuitable for administration to the subject; Thereby, methods are provided for confirming the suitability of an isolated lung cell population for administration to a subject.

[0096] According to certain embodiments, the methods disclosed herein are performed ex vivo.

[0097] The term "pulmonary tissue" as used herein refers to lung tissue or organ. The lung tissue of the present invention may be a complete organ or tissue, or a partial organ or tissue. Thus, the lung tissue of some embodiments may include the right lung, the left lung, or both. The lung tissue of some embodiments of the present invention may include one, two, three, four, or five lobes (of either the right or left lung). Furthermore, the lung tissue of some embodiments of the present invention may include one or more lung segments or lung lobules. Furthermore, the lung tissue of some embodiments of the present invention may include any number of bronchi and bronchioles (e.g., bronchial trees) and any number of alveoli or alveolar sacs.

[0098] Depending on the application and available sources, the cells of the invention may be obtained from prenatal organisms, postnatal organisms, adults, or cadaveric donors. Making such decisions is well within the capabilities of one of ordinary skill in the art.

[0099] It will be appreciated that the lung cells of some embodiments of the present invention may be fresh or frozen (eg, cryopreserved) preparations, as further described below.

[0100] According to certain embodiments, the lung tissue is human lung tissue.

[0101] According to certain embodiments, the lung tissue is from a cadaveric donor.

[0102] According to certain embodiments, the lung tissue is from a living donor.

[0103] According to one embodiment, the lung tissue is of adult origin (eg, from a mammalian organism at any post-natal stage).

[0104] According to one embodiment, the lung tissue is of embryonic origin.

[0105] According to one embodiment, the lung tissue is of fetal / fetal origin.

[0106] Thus, the embryo or fetal / fetal organism can be of either human or xenogeneic origin (e.g., porcine) and can be at any gestational age. Making such determinations is within the ability of one of skill in the art.

[0107] A variety of methods can be used to obtain organs or tissues from embryonic or fetal / fetal organisms. Thus, for example, lung tissue can be obtained by removing tissue (e.g., by surgical procedures) from a developing fetal / fetal organism.

[0108] According to one embodiment, pulmonary tissue (i.e., lung tissue) is obtained from a fetal organism at a gestational age corresponding to the tubular stage of human development (e.g., 16-25 weeks gestation). According to one embodiment, the lung tissue ...17 weeks gestation, 16-18 weeks gestation, 16-19 weeks gestation, 16-20 weeks gestation, 16-21 weeks gestation, 16-22 weeks gestation, 16-24 weeks gestation, 17-18 weeks gestation, 17-19 weeks gestation, 17-20 weeks gestation, 17-21 weeks gestation, 17-22 weeks gestation, 17-24 weeks gestation, 18-19 weeks gestation, 18-20 weeks gestation, 18-21 weeks gestation, 18-22 weeks gestation, 18-24 weeks gestation, 19-20 weeks gestation. , 19-21 weeks gestation, 19-22 weeks gestation, 19-23 weeks gestation, 19-24 weeks gestation, 20-21 weeks gestation, 20-22 weeks gestation, 20-23 weeks gestation, 20-24 weeks gestation, 21-22 weeks gestation, 21-23 weeks gestation, 2 Obtained from a fetus / fetus organism at a gestational age corresponding to 1 to 24 weeks, 22 to 23 weeks of gestation, 22 to 24 weeks of gestation, 22 to 25 weeks of gestation, 23 to 24 weeks of gestation, 23 to 25 weeks of gestation, 24 to 25 weeks of gestation, or 25 to 26 weeks of gestation.

[0109] According to certain embodiments, the lung tissue is obtained from a fetal / fetal organism at a gestational age corresponding to 20-22 weeks of human gestation.

[0110] According to certain embodiments, lung tissue is obtained from a fetus / fetal organism at a gestational age corresponding to 21-22 days of human gestation.

[0111] According to certain embodiments, lung tissue is obtained from a fetus / fetal organism at a gestational age corresponding to 20-21 days of human gestation.

[0112] Those skilled in the art will understand that the gestational age of an organism is the period of time that has elapsed following fertilization of the oocyte that gives rise to that organism. The following table provides examples of gestational ages of human and porcine tissues from which fetal / fetal tissues at essentially corresponding developmental stages can be obtained.

[0113] [Table 1]

[0114] Similarly, various methods can be used to obtain lung organs or lung tissue from adult organisms (e.g., live or cadaveric). Thus, for example, lung tissue can be obtained by harvesting tissue from an organ donor by surgical procedures (e.g., laparotomy or laparoscopy). After obtaining the organ / tissue from the adult organism, lung cells and hematopoietic progenitor cells (described in detail below) can be isolated therefrom according to methods known in the art. Such methods depend on the source and lineage of the cells and can include, for example, flow cytometry and cell sorting (e.g., as taught at www(dot)bio-rad(dot)com / en-uk / applications-technologies / isolation-maintenance-stem-cells).

[0115] It will be appreciated that the lung tissue does not need to be intact (i.e., maintaining tissue structure so as to be suitable for whole organ transplantation) to obtain lung cells, but the lung tissue must contain viable cells.

[0116] Additionally, according to certain embodiments, the lung tissue may be obtained from multiple donors. Thus, according to certain embodiments, the lung cells may comprise cells obtained from multiple cell donors.

[0117] The present invention further contemplates obtaining a lung organ / tissue (e.g., fetal / fetal tissue or adult tissue) and then generating an isolated cell population therefrom. The phrase "isolated lung cell population" refers to isolated cells that do not form tissue structures (i.e., are free of connective tissue structures).

[0118] Thus, the lung cells may be contained in a suspension of single cells or in cell aggregates having no more than 5, no more than 10, no more than 50, no more than 100, no more than 200, no more than 300, no more than 400, no more than 500, no more than 1000, no more than 1500, no more than 2000 cells in the aggregates.

[0119] As used herein, the phrase "lung cells in suspension" refers to cells that have been isolated from their native environment (e.g., the human body) and extracted from lung tissue while maintaining viability, but do not maintain tissue structure (i.e., lack vascularized tissue structure) and are not attached to a solid support.

[0120] The cell suspension of the present invention can be obtained by any mechanical or chemical (e.g. enzymatic) means.There are several methods for dissociating cell clusters to form cell suspensions (e.g. single cell suspensions) from primary tissues, adherent cells in culture, and aggregates, including, for example, physical force (mechanical dissociation, e.g. cell scraper, trituration with small-bore pipette, fine needle aspiration, disaggregation by vortexing, and forced filtration through nylon or stainless steel fine mesh), enzyme (enzymatic dissociation, e.g. trypsin, collagenase, Acutase, etc.), or a combination of both.

[0121] According to certain embodiments, the dissociating is by enzymatic digestion.

[0122] Thus, for example, tissues / organs can be enzymatically digested into isolated cells by subjecting the tissue to enzymes such as collagenase type IV (Worthington biochemical corporation, Lakewood, NJ, USA) and / or dispase (a product of Invitrogen Corporation, Grand Island, NY, USA). For example, tissues can be enzymatically digested by mincing with a razor blade in the presence of collagenase, dispase, and CaCl2 at 37° C. for about 1 hour. This method may further comprise removing non-specific debris from the resulting cell suspension, for example by successive filtration through filters (e.g., 70 μm and 40 μm filters), essentially as described in the Examples section below.

[0123] Additionally, tissues can be mechanically dissociated into isolated cells using devices designed to break tissues into size segments. Such devices are available from CellArtis Goteborg, Sweden. Additionally or alternatively, mechanical dissociation can be performed manually using a needle such as a 27g needle (BD Microlance, Drogheda, Ireland) while viewing the tissue / cells under an inverted microscope.

[0124] After enzymatic or mechanical dissociation of the tissue, the dissociated cells are further broken down into small clumps (eg, by pipetting the cells up and down) using, for example, a 200 μl Gilson pipette tip.

[0125] According to certain embodiments, the cell suspension of lung cells comprises viable cells. Cell viability can be monitored using any method known in the art, for example, using a cell viability assay (e.g., MultiTox Multiplex Assay available from Promega), flow cytometry, trypan blue, etc.

[0126] In accordance with the teachings of the present invention, lung tissue and isolated cells obtained therefrom contain cells that express both epithelial and endothelial cell markers.

[0127] According to certain embodiments, these cells expressing both epithelial and endothelial cell markers are precursor cells capable of differentiating into both pulmonary endothelial and epithelial cells. Methods for determining differentiation include in vitro and in vivo (e.g., transplantation) methods well known to those skilled in the art. Non-limiting examples are provided in the Examples section below.

[0128] Thus, according to one embodiment, lung cells are characterized by the expression of epithelial and endothelial cell markers.

[0129] As used herein, the phrase "epithelial cell marker" refers to cell surface proteins characteristic of lung epithelial cells, including, but not limited to, CD326, CD324, CD24, aquaporin 5 (AQP-5), podoplanin (PDPN), and receptor for advanced glycation end products (RAGE, i.e., encoded by the AGER gene).

[0130] As used herein, the phrase "endothelial cell marker" refers to cell surface proteins characteristic of pulmonary endothelial cells. Such markers include, but are not limited to, CD31 and CD144 (VE-cadherin).

[0131] According to certain embodiments, the lung cells have the co-expression signature: CD326 + and CD31 + It is characterized by:

[0132] According to certain embodiments, the lung cells have the co-expression signature: CD324 + and CD31 + It is characterized by:

[0133] According to certain embodiments, the lung cells have the co-expression signature: CD24 + and CD31+ It is characterized by:

[0134] According to certain embodiments, the lung cells have the co-expression signature: AQP-5 + and CD31 + It is characterized by:

[0135] According to certain embodiments, the lung cells have the co-expression signature: PDPN + and CD31 + It is characterized by:

[0136] According to certain embodiments, the lung cells have the co-expression signature: RAGE. + and CD31 + It is characterized by:

[0137] According to certain embodiments, the lung cells have the co-expression signature: CD326 + and CD144 + It is characterized by:

[0138] According to certain embodiments, the lung cells have the co-expression signature: CD324 + and CD144 + It is characterized by:

[0139] According to certain embodiments, the lung cells have the co-expression signature: CD24 + and CD144 + It is characterized by:

[0140] According to certain embodiments, the lung cells have the co-expression signature: AQP-5 + and CD144 + It is characterized by:

[0141] According to certain embodiments, the lung cells have the co-expression signature: PDPN + and CD144 + It is characterized by:

[0142] According to certain embodiments, the lung cells have the co-expression signature: RAGE. + and CD144 + It is characterized by:

[0143] According to certain embodiments, the lung cells are further characterized by expression of at least one of Nkx2.1, CD200, Akap5, Sec14l3, Prdx6, and Clic3.

[0144] According to certain embodiments, the lung cells comprise a heterogeneous cell population (eg, an unseparated cell population) comprising cells that co-express endothelial and epithelial markers.

[0145] According to other particular embodiments, the lung cells comprise a purified cell population. Thus, the cells can be treated to remove specific cell populations therefrom (e.g., removal of subpopulations) or to positively select for a desired population (e.g., a cell population that is double positive for the expression of epithelial and endothelial cell markers). Purification of a particular cell type can be performed by any method known to the skilled artisan, for example, eradication (e.g., killing) with a specific antibody, or affinity-based purification (e.g., by using MACS beads, FACS sorter, and / or capture ELISA label) using a specific antibody that recognizes any particular cell marker (e.g., CD31, CD34, CD41, CD45, CD8, CD8, CD48, CD105, CD150, CD271, CD326, MUCIN-1, PODOPLANIN, etc.). Such methods are described herein and in THE HANDBOOK OF EXPERIMENTAL IMMUNOLOGY, volumes 1-4 (ed. DN Weir) and FLOW CYTOMETRY AND CELL SORTING (ed. A. Radbruch, Springer Verlag, 1992). For example, cells can be sorted, for example, by flow cytometry or FACS. Thus, fluorescence-activated cell sorting (FACS) can be used, which may have various degrees of color channels, small-angle and obtuse-angle light scattering detection channels, and impedance channels. Any ligand-dependent separation technique known in the art can be used in conjunction with both positive and negative separation techniques that rely on physical properties of cells rather than antibody affinity, including, but not limited to, elutriation and density gradient centrifugation. Other methods for cell sorting include, for example, panning and separation using affinity techniques, including techniques that use solid supports such as plates, beads, and columns. Thus, biological samples can be separated by "panning" with antibodies attached to a solid matrix (e.g., a plate). Alternatively, cells can be sorted / isolated by magnetic separation techniques, some of which utilize magnetic beads.A variety of magnetic beads are available from a number of sources, including, for example, Dynal (Norway), Advanced Magnetics (Cambridge, Massachusetts, USA), Immuncon (Philadelphia, USA), Immunotec (Marseille, France), Invitrogen, Stem cell Technologies (USA), and Cellpro (USA). Alternatively, antibodies can be biotinylated or conjugated with digoxigenin and used in conjunction with avidin or anti-digoxigenin coated affinity columns.

[0146] According to one embodiment, different depletion / separation methods can be combined, e.g. magnetic cell sorting can be combined with FACS to improve separation quality or to allow sorting by multiple parameters.

[0147] According to certain embodiments, such selection is carried out by contacting with an agent capable of binding to the desired marker. Such an agent may for example be an antibody. The antibody may be monospecific or at least bispecific.

[0148] According to certain embodiments, the lung cells comprise a purified cell population that expresses both epithelial and endothelial markers.

[0149] According to certain embodiments, the selection is performed by contacting the isolated lung cell population with at least one reagent capable of binding to an epithelial cell marker and an endothelial cell marker, and selecting a cell population that is double positive for expression of the epithelial cell marker and the endothelial cell marker.

[0150] According to certain embodiments, the at least one reagent is a single reagent. In such cases, the agent has specificity for both endothelial and epithelial markers.

[0151] According to certain embodiments, the at least one reagent comprises at least two reagents, in such case at least one of the agents has specificity for an endothelial marker and at least one of the agents has specificity for an epithelial marker.

[0152] According to certain embodiments, at least one reagent is an antibody.

[0153] According to a particular embodiment, at least one antibody is a monospecific antibody, in which case the contacting is carried out by two distinct antibodies, one with specificity for an endothelial marker and one with specificity for an epithelial marker.

[0154] According to a particular embodiment, the antibody is a bispecific antibody, in which case the contacting may be carried out with one antibody having specificity for both endothelial and epithelial markers.

[0155] According to certain embodiments, at least about 0.1%, 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the lung cells generated by the methods of some embodiments of the invention are characterized by dual expression of epithelial and endothelial cell markers (e.g., CD326 + CD31 + , CD324 + CD31 + , CD326 + CD144 + , or CD324 + CD144 + (It is.)

[0156] According to certain embodiments, about 0.1-10%, 0.1-20%, 0.1-50%, 0.1-100%, 1-10%, 1-20%, 1-50%, 1-100%, 10-20%, 10-50%, 10-100%, such as about 20-30%, for example about 20-40%, such as about 20-60%, for example about 30-50%, such as about 30-70%, for example about 40-50%, such as about 40-80%, for example about 50-60%, such as about 50-70%, for example about 60-80%, for example about 60-90%, for example about 70-90%, such as about 80-100% of the pulmonary cells generated by the methods of some embodiments of the invention are characterized by dual expression of epithelial cell markers and endothelial cell markers (e.g. CD326 + CD31 + , CD324 + CD31 + , CD326 + CD144 + , or CD324 + CD144 + (It is.)

[0157] According to certain embodiments, at least about 0.1%, 1%, 2%, 5%, or 10% of the lung cells generated by the methods of some embodiments of the present invention are characterized by dual expression of epithelial cell markers and endothelial cell markers.

[0158] According to certain embodiments, approximately 0.1-10% or 1-10% of the lung cells generated by the methods of some embodiments of the present invention are characterized by dual expression of epithelial cell markers and endothelial cell markers.

[0159] According to certain embodiments, at least 20%, 30%, 40%, or 50% of the lung cells generated by the methods of some embodiments of the present invention are characterized by dual expression of epithelial cell markers and endothelial cell markers.

[0160] Also provided herein is a kit for isolating lung cells characterized by double positive expression of an epithelial cell marker and an endothelial cell marker, comprising: (i) CD31 or CD144, and (ii) CD326, CD324, CD24, aquaporin 5 (AQP-5), podoplanin (PDPN), or receptor for advanced glycation end products (RAGE) The kit includes at least one reagent capable of binding to the

[0161] According to certain embodiments, the kit further comprises instructions for use.

[0162] According to certain embodiments, the selection is carried out prior to culturing.

[0163] According to certain embodiments, the selection is carried out after or during the culture.

[0164] According to certain embodiments, the selection is performed prior to administration of the cells to a subject in need thereof.

[0165] According to one embodiment, the lung cells are characterized by a lack of expression of white blood cell markers.

[0166] According to certain embodiments, the lung cells are characterized by a lack of expression of CD45.

[0167] Thus, according to a particular embodiment, the method comprises depleting CD45 expressing cells.

[0168] Methods for depleting cells are well known to those skilled in the art and are further described above and below. According to certain embodiments, depletion of CD45-expressing cells is performed by contacting the isolated lung cell population with a reagent capable of binding to CD45 in order to select a cell population negative for expression of CD45.

[0169] According to one embodiment, the lung cells comprise less than 10%, less than 50%, or less than 2% CD45+ cells.

[0170] According to one embodiment, the lung cells are T cell depleted.

[0171] Thus, according to certain embodiments, the method comprises depleting T cells.

[0172] Methods for depleting T cells are well known to those skilled in the art and are further described above and below. According to certain embodiments, depleting T cell expressing cells is performed by contacting the isolated lung cell population with a reagent capable of binding to T cells to select a cell population that is negative for T cells.

[0173] As used herein, the phrase "T cell depleted" refers to a lung cell population that is depleted of T lymphocytes. T cell depleted lung cells are CD3 + cells, CD2 + cells, CD8 + cells, CD4 + T cells, α / β T cells, and / or γ / δ T cells may be depleted.

[0174] According to one embodiment, the T cell depleted lung cells contain less than 10%, less than 50%, or less than 2% T cells.

[0175] According to certain embodiments, the lung cells are characterized by a lack of expression of CD3.

[0176] According to one embodiment, the therapeutically effective amount of T cell depleted lung cells is 50×10 5 Fewer than CD3 + T cells, 40 x 10 5 Fewer than CD3 + T cells, 30 x 10 5 Fewer than CD3 + T cells, 20 x 10 5 Fewer than CD3 + T cells, 15 x 10 5 Fewer than CD3 + T cells, 10 x 10 5 Fewer than CD3 + T cells, 9 x 10 5 Fewer than CD3 + T cells, 8 x 10 5 Fewer than CD3+ T cells, 7 x 10 5 Fewer than CD3 + T cells, 6 x 10 5 Fewer than CD3 + T cells, 5 x 10 5 Fewer than CD3 + T cells, 4 x 10 5 Fewer than CD3 + T cells, 3 x 10 5 Fewer than CD3 + T cells, 2 x 10 5 Fewer than CD3 + T cells, 1 x 10 5 Fewer than CD3 + T cells, or 5 × 10 4 Fewer than CD3 + Includes T cells.

[0177] According to certain embodiments, the lung cells are characterized by a lack of expression of CD2.

[0178] According to certain embodiments, the lung cells are characterized by a lack of expression of CD4.

[0179] According to certain embodiments, the lung cells are characterized by a lack of expression of CD8.

[0180] According to one embodiment, the therapeutically effective amount of T cell depleted lung cells is about 50×10 5 Fewer than CD8 + cells, 25 x 10 5 Fewer than CD8 + cells, 15 x 10 5 Fewer than CD8 + cells, 10x10 5 Fewer than CD8 + cells, 9 x 10 5 Fewer than CD8 + cells, 8 x 10 5 Fewer than CD8 + cells, 7 x 10 5 Fewer than CD8 + cells, 6 x 10 5 Fewer than CD8 + cells, 5 x 10 5Fewer than CD8 + cells, 4 x 10 5 Fewer than CD8 + cells, 3 x 10 5 Fewer than CD8 + cells, 2 x 10 5 Fewer than CD8 + cells, 1 x 10 5 Fewer than CD8 + cells, 9 x 10 4 Fewer than CD8 + cells, 8 x 10 4 Fewer than CD8 + cells, 7 x 10 4 Fewer than CD8 + cells, 6 x 10 4 Fewer than CD8 + cells, 5 x 10 4 Fewer than CD8 + cells, 4 x 10 4 Fewer than CD8 + cells, 3 x 10 4 Fewer than CD8 + cells, 2 x 10 4 Fewer than CD8 + cells, or 1 x 10 4 Fewer than CD8 + Contains cells.

[0181] According to certain embodiments, the lung cells are characterized by a lack of expression of the T cell receptor alpha and beta chains.

[0182] According to certain embodiments, the lung cells are characterized by a lack of expression of T cell receptor gamma and delta chains.

[0183] According to one embodiment, the T cell depleted lung cells are obtained by T cell debulking (TCD), which can be performed using antibodies, including, for example, anti-CD8, anti-CD4, anti-CD3, anti-CD2, anti-TCR alpha / beta, and / or anti-TCR gamma / delta antibodies.

[0184] According to one embodiment, the lung cells are B cell depleted.

[0185] According to one embodiment, the B cell depleted lung cells contain less than 10%, less than 50%, or less than 2% B cells.

[0186] According to one embodiment, the therapeutically effective amount of lung cells is about 50×10 5 < 40 × 10 B cells 5 < 30 × 10 B cells 5 <20 × 10 B cells 5 < 10 x 10 B cells 5 < 9 × 10 B cells 5 <8 x 10 B cells 5 < 7 x 10 B cells 5 < 6 × 10 B cells 5 <5 × 10 B cells 5 <4 x 10 B cells 5 < 3 × 10 B cells 5 <2 x 10 B cells 5 < 1 x 10 B cells, or 1 x 10 5 Contains less than 1 B cell.

[0187] According to one embodiment, depletion of B cells is performed by B cell debulking. B cell debulking can be performed using antibodies, including, for example, anti-CD19 or anti-CD20 antibodies. Alternatively, B cell debulking can be achieved in vivo by injection of anti-CD20 antibodies.

[0188] T cell or B cell debulking can be performed in vitro or in vivo (eg, in the donor prior to obtaining lung tissue from the donor).

[0189] According to certain embodiments, the lung cells comprise a heterogeneous population of cells, including hematopoietic progenitor or precursor cells (HPCs), mesenchymal progenitor cells, epithelial cells, endothelial cells, and the like, in addition to cells that co-express endothelial and epithelial markers.

[0190] According to certain embodiments, the lung cells are immediately used for transplantation.

[0191] According to other particular embodiments, the lung cells are cultured ex vivo.

[0192] As used herein, the term "culturing" or "culture" refers to at least pulmonary cells and culture medium in an ex vivo environment. The culture is maintained under conditions capable of supporting at least pulmonary cell survival. Such conditions include, for example, appropriate temperature (e.g., 37° C.), atmosphere (e.g., O2%, CO2%), pressure, pH, light, media, supplements, etc.

[0193] The culture medium of some embodiments may be a water-based medium, which includes a combination of substances such as salts, nutrients, minerals, vitamins, amino acids, antibiotics, nucleic acids, proteins (e.g., cytokines, growth factors, and hormones), all of which are necessary to maintain lung cells in a viable state. For example, the culture medium may be a synthetic tissue culture medium, such as RPMI-1640 (Life Technologies, Israel), Ko-DMEM (a product of Gibco-Invitrogen Corporation, Grand Island, NY, USA), DMEM / F12 (Biological Industries, Beit Haemek, Israel), Mab ADCB medium (HyClone, Utah, USA), DMEM / F12 (Biological Industries, Biet Haemek, Israel), or a conditioned medium (e.g., from a feeder medium, e.g., iMEF) supplemented with necessary additives. Preferably, all components included in the culture medium of the present invention are substantially pure and tissue culture grade.

[0194] According to certain embodiments, the medium is a conditioned medium.

[0195] "Conditioned medium (CM)" refers to a culture medium supplemented with soluble factors (culture-derived growth factors) produced and secreted by cells (e.g., fibroblasts, e.g., iMEFs) cultured in the medium. As will be understood, conditioned medium is substantially free of cells. Techniques for isolating conditioned medium from cell cultures are well known in the art. Conditioned medium can also be obtained commercially, for example, from R&D Systems (e.g., MEF conditioned medium, catalog number AR005).

[0196] The cultures may be in glass, plastic, or metal containers that can provide a sterile environment for tissue culture. According to certain embodiments, the culture containers include dishes, plates, flasks, bottles, and vials. Culture containers are commercially available from various manufacturers, such as COSTAR®, NUNC®, and FALCON®.

[0197] According to certain embodiments, the culture vessel is a tissue culture plate.

[0198] According to certain embodiments, the culture is maintained under sterile conditions.

[0199] According to certain embodiments, the culture is maintained at 37-38°C.

[0200] According to certain embodiments, the pulmonary cells are cultured under conditions that allow their proliferation, in other words, according to certain embodiments, the pulmonary cells are propagated ex vivo by culture.

[0201] The terms "expanding," "expanded," or "proliferation" refer to an increase in the number of cells in a population by cell division. Methods for assessing proliferation are well known in the art and include, but are not limited to, proliferation assays such as CFSE and BrDU, and determining cell number by direct cell counting and microscopic evaluation.

[0202] According to certain embodiments, the proliferation is at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 40-fold, at least about 80-fold, at least about 120-fold, at least about 140-fold, or greater, over a given time interval (and e.g., compared to non-proliferating cells prior to culturing).

[0203] According to certain embodiments, lung cells are cultured ex vivo to expand a cell population that is double positive for expression of epithelial and endothelial cell markers as described herein.

[0204] According to certain embodiments, such conditions include a culture medium that includes factors that promote endothelial cell proliferation, factors that promote epithelial cell proliferation, and factors that prevent differentiation.

[0205] Endothelial cells are thin, flat cells that cover the inner surface of blood and lymphatic vessels, constituting the endothelium. As used herein, the term "endothelial cells" refers to isolated endothelial cells at any developmental stage from precursor cells to mature differentiated cells. Endothelial cells may express markers typical of the endothelial lineage, including, but not limited to, CD31, CD144 (VE-cadherin), CD54 (I-CAM1), vWF, VCAM, CD106 (V-CAM), and VEGF-R2.

[0206] Epithelial cells are cells that line either the cavities or the surfaces of structures throughout the mammalian body, constituting the epithelium. The basic cell types are squamous, cuboidal, and columnar, and are classified according to their shape. As used herein, the term "epithelial cell" refers to an isolated epithelial cell at any developmental stage from a precursor cell to a mature differentiated cell. Epithelial cells may express markers typical of the epithelial lineage, including cytokeratin, CD326, CD324, CD24, aquaporin 5 (AQP-5), podoplanin (PDPN), advanced glycation end products, HOPX, cytokeratin, Nkx 2.1, SP-A, SP-B, SP-D, Clara cell protein (CC16, CC10), mucin-related antigens: KL-6, 17-Q2, 17-B1.

[0207] As used herein, a "growth-enhancing factor" refers to a biomolecule (eg, amino acid- or nucleic acid-based) or small molecule chemical that promotes growth in culture.

[0208] Factors that promote endothelial cell proliferation are well known in the art. Non-limiting examples that may be used with certain embodiments of the present invention include vascular endothelial growth factor (VEGF), b-FGF, FGF2, IL-8, and BMP4.

[0209] According to certain embodiments, the factor that promotes endothelial cell proliferation comprises VEGF.

[0210] Non-limiting examples of VEGFs that can be used with certain embodiments of the present invention include hVEGF 165, rhVEGF-121, rhVEGF-164, VEGF-c.

[0211] VEGF is commercially available from a number of sources, such as, for example, Stemcell, R&D systems, Peprotech, etc. According to certain embodiments, VEGF is included in a medium, such as, for example, Endo medium, available from Sartorius.

[0212] According to some embodiments of the invention, a factor that promotes endothelial cell proliferation (e.g., VEGF) is provided at a concentration of at least 0.1 ng / ml, at least 0.5 ng / ml, at least 1 ng / ml, at least 5 ng / ml, or at least 10 ng / ml.

[0213] According to certain embodiments, factors that promote endothelial cell proliferation (eg, VEGF) are provided at a concentration of 10 μg / ml or less, 1 μg / ml or less, or 100 ng / ml or less.

[0214] According to certain embodiments, the factor that promotes endothelial cell proliferation (e.g., VEGF) is provided at a concentration of 5 to 100 ng / ml. According to certain embodiments, the factor that promotes endothelial cell proliferation (e.g., VEGF) is provided at a concentration of about 30 ng / ml.

[0215] Factors that promote epithelial cell proliferation are well known in the art. Non-limiting examples that may be used with certain embodiments of the present invention include epidermal growth factor (EGF), noggin, and R-spondin.

[0216] According to certain embodiments, the factor that promotes epithelial cell proliferation comprises EGF (eg, hEGF).

[0217] EGF is commercially available from a number of sources, for example, Stemcell, R&D systems, and Sigma-Aldrich.

[0218] According to some embodiments of the invention, a factor that promotes epithelial cell proliferation (e.g., EGF) is provided at a concentration of at least 0.1 ng / ml, at least 0.5 ng / ml, at least 1 ng / ml, at least 5 ng / ml, or at least 10 ng / ml.

[0219] According to certain embodiments, factors that promote epithelial cell proliferation (eg, EGF) are provided at a concentration of 10 μg / ml or less, 1 μg / ml or less, or 100 ng / ml or less.

[0220] According to certain embodiments, a factor that promotes epithelial cell proliferation (eg, EGF) is provided at a concentration of 5-100 ng / ml.

[0221] According to certain embodiments, a factor that promotes epithelial cell proliferation (eg, EGF) is provided at a concentration of about 30 ng / ml.

[0222] As used herein, "factors that prevent differentiation" refer to biomolecules (e.g., amino acid- or nucleic acid-based) or small molecule chemicals that, alone or in combination with other factors, prevent differentiation of progenitor cells in culture (i.e., maintain the pluripotent state).

[0223] Factors that prevent differentiation are well known in the art. Non-limiting examples that can be used with certain embodiments of the present invention include ROCK inhibitors, GSK3b inhibitors (e.g., CHIR99021), ALK5 inhibitors (e.g., A83-01).

[0224] According to certain embodiments, the factor that prevents differentiation comprises a ROCK inhibitor.

[0225] Many ROCK inhibitors are known in the art and commercially available. Non-limiting examples include Y27632 (TOCRIS, Catalog No. 1254), Blebbistatin (TOCRIS, Catalog No. 1760), and Thiazovivin (Axon Medchem-Axon1535).

[0226] According to some embodiments of the invention, the factor that prevents differentiation (eg, a ROCK inhibitor) is provided at a concentration of at least 0.1 μM, at least 0.5 μM, at least 1 μM, at least 5 μM, or at least 10 μM.

[0227] According to certain embodiments, the factor that prevents differentiation (eg, a ROCK inhibitor) is provided at a concentration of 10 mM or less, 1 mM or less, 100 μM or less.

[0228] According to certain embodiments, the factor that prevents differentiation (eg, a ROCK inhibitor) is provided at a concentration of 5-50 μM.

[0229] According to certain embodiments, the factor that prevents differentiation (eg, a ROCK inhibitor) is provided at a concentration of about 20 μM.

[0230] According to certain embodiments, the culture is carried out until a desired number of viable cells is obtained. The measurement of the number of cells (e.g., viable cells) can be carried out using any method known to those skilled in the art, for example, by counting chamber, FACs analysis, or spectrophotometer.

[0231] According to certain embodiments, the culturing or growing is carried out for at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 72 hours.

[0232] According to certain embodiments, the culturing or growing is carried out for at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 10 days, at least 14 days, at least 21 days, at least 24 days.

[0233] According to a particular embodiment, the culturing or growing is carried out for 20 to 30 days.

[0234] According to particular embodiments, the culturing or growing is carried out for up to 5 weeks or up to 4 weeks.

[0235] According to certain embodiments, the culturing or propagation is carried out at a cell number of at least 1×10 7 This will be continued until it reaches 100.

[0236] According to certain embodiments, culturing or expansion is performed until the cells expressing both epithelial and endothelial markers reach a cell number of at least 50,000, at least 100,000, at least 150,000, at least 200,000.

[0237] According to certain embodiments, the method further comprises determining the expression of epithelial and endothelial cell markers on the lung cells.

[0238] According to a particular embodiment, the decision is made after the selection.

[0239] According to certain embodiments, the determination is made prior to culturing.

[0240] According to certain embodiments, the determination is performed during and / or after the culture.

[0241] Methods for determining expression are well known in the art and include flow cytometry, immunocytochemistry, Western blot, PCR, and the like.

[0242] According to a particular embodiment, the determination of expression is carried out by flow cytometry.

[0243] According to certain embodiments, proliferation of the cell population that is double positive for expression of epithelial cell markers and endothelial cell markers above a predetermined threshold indicates that the isolated lung cell population is suitable for administration to a subject.

[0244] On the other hand, according to certain embodiments, the absence of proliferation or a decrease below a predetermined threshold of the double positive cell population for expression of epithelial and endothelial cell markers indicates that the isolated lung cell population is unsuitable for administration to a subject. Then, according to certain embodiments, if the absence of proliferation or a decrease below a predetermined threshold is detected, the cells are cultured again until proliferation of double positive cells occurs or are discarded.

[0245] According to certain embodiments, such a pre-defined threshold is determined relative to the total number of epithelial / endothelial double positive cells per se.

[0246] Thus, according to certain embodiments, proliferation above a predetermined threshold refers to an increase in the number of epithelial / endothelial double positive cells of at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold or more compared to the number before culture.

[0247] According to certain embodiments, proliferation above a predetermined threshold refers to an increase in the number of epithelial / endothelial double positive cells of at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or more compared to the number before culture.

[0248] According to certain embodiments, proliferation below a predetermined threshold means that the number of epithelial / endothelial double positive cells increases by less than about 1.5-fold, less than about 2-fold, less than about 3-fold, less than about 4-fold, less than about 5-fold, or less than about 10-fold compared to the number before culture.

[0249] According to certain embodiments, proliferation below a predetermined threshold means that the number of epithelial / endothelial double positive cells increases by less than 5%, less than 10%, less than 15%, less than 20%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, less than 80%, less than 90%, or less than 100% compared to the number before culture.

[0250] According to certain embodiments, the determination is made after at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, or at least 72 hours of incubation.

[0251] According to certain embodiments, the determination is made after at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 10 days, at least 14 days, at least 21 days, or at least 24 days of culture.

[0252] According to a particular embodiment, the determination is made after 20 to 30 days of culture.

[0253] According to particular embodiments, the determination is made after up to 5 weeks or up to 4 weeks of culture.

[0254] The lung tissue or cells obtained therefrom of some embodiments of the present invention can be preserved (typically by freezing) under appropriate conditions at any step (e.g., after dissociation, after selection, before, during, or after culture) so that the cells remain viable and functional for use in transplantation. According to one embodiment, the lung cells are preserved as a cryopreserved population. Other preservation methods are described in U.S. Patent Nos. 5,656,498, 5,004,681, 5,192,553, 5,955,257, and 6,461,645. Methods for banking stem cells are described, for example, in U.S. Patent Application Publication No. 2003 / 0215942.

[0255] Thus, according to one aspect of the present invention, (i) a plurality of isolated lung cell populations in suspension, the lung cells being characterized as double positive for expression of epithelial and endothelial cell markers, the plurality of isolated lung cell populations being HLA typed to form an allogeneic cell bank, each individually disposed in a separate container; (ii) a catalog containing information about HLA-typed cells of a plurality of isolated lung cell populations; A cell bank is provided, comprising:

[0256] The present invention, in some embodiments thereof, also contemplates cells obtainable or obtained by the methods disclosed herein.

[0257] Thus, according to one aspect of the invention there is provided an isolated lung cell population obtained according to the method.

[0258] According to certain embodiments, the isolated lung cell population is comprised of cells that express both epithelial and endothelial markers, such as CD326. + CD31 + The cells comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%.

[0259] According to one aspect of the present invention, CD326 + CD31 + An isolated lung cell population comprising at least 40% cells is provided.

[0260] According to certain embodiments, the isolated lung cell population is CD326 + CD31 + It comprises at least 50%, at least 60%, at least 70%, at least 80% of cells.

[0261] According to certain embodiments, the pulmonary cells disclosed herein are capable of regenerating pulmonary epithelial tissue.

[0262] According to certain embodiments, the pulmonary cells disclosed herein are capable of regenerating pulmonary endothelial tissue.

[0263] According to certain embodiments, the cells can be grown in 2D or 3D culture.

[0264] According to certain embodiments, the cells are in suspension.

[0265] According to certain embodiments, the cells are embedded or attached to a scaffold or carrier that allows them to grow in suspension.

[0266] Scaffold materials can include natural (e.g., fibrinogen, fibrin, thrombin, chitosan, collagen, alginate, poly(N-isopropylacrylamide), albumin, collagen, synthetic polyamino acids, prolamines, polysaccharides (e.g., alginate, heparin, and other naturally occurring biodegradable polymers of sugar units)) or synthetic organic polymers (e.g., PLGA, PMMA, PCL, and the like) that can be gelled or polymerized or solidified (e.g., by aggregation, coagulation, hydrophobic interactions, or crosslinking) into two- or three-dimensional structures. Such scaffolds are known in the art and are disclosed, for example, in Florian Weinberger et al. (2017) Circulation Research. 120:1487-1500, Rochkind S et al (2004) Neurol Res. 26(2):161-6, Rochkind S. et al. (2006) Eur Spine J. 15(2):234-45, FSY Wong, ACY Lo (2015) J Stem Cell Res Ther 5:267, and International Patent Application Publication No. 2020 / 245832, the contents of which are incorporated by reference in their entirety.

[0267] The polymers used in the scaffolding composition may be biocompatible, biodegradable, and / or bioerodible and may serve as an attachment substrate for cells. In an exemplary embodiment, the structural scaffolding material is easily fabricated into complex shapes and has suitable stiffness and mechanical strength to maintain a desired shape under in vivo conditions.

[0268] In certain embodiments, structural scaffolding materials can be non-absorbable or non-biodegradable polymers or materials. Such non-absorbable scaffolding materials can be used to manufacture materials designed for long-term or permanent implantation into a host organism.

[0269] Scaffolds can be made by any of a variety of techniques known to those of skill in the art. Salt leaching, porogenization, solid-liquid phase separation (sometimes called freeze-drying), and phase inversion fabrication can all be used to produce porous scaffolds. Fiber pulling and weaving (see, e.g., Vacanti, et al., (1988) Journal of Pediatric Surgery, 23: 3-9) can be used to produce scaffolds with more aligned polymer threads. Those of skill in the art will recognize that standard polymer processing techniques can be utilized to create polymer scaffolds with a variety of porosities and microstructures.

[0270] Scaffolding materials are readily available (usually in the form of a solution) to those skilled in the art (e.g., suppliers such as BDH, UK and Pronova Biomedical Technology, Norway). For a general overview of the selection and preparation of scaffolding materials, see the American National Standards Institute publication No. F2064-00 entitled "Standard Guide for Characterization and Testing of Alginates as Starting Materials Intended for Use in Biomedical and Tissue Engineering Medical Products Applications."

[0271] The present invention, in some embodiments thereof, also contemplates administering the pulmonary cells described herein to a subject.

[0272] The method can be performed using lung cells that are syngeneic or non-syngeneic to the subject, depending on the application.

[0273] As used herein, the term "syngeneic" cells refers to cells that are essentially genetically identical to a subject, or essentially all of the subject's lymphocytes. Examples of syngeneic cells include cells derived from a subject (also referred to in the art as "autologous"), cells derived from a clone of a subject, or cells derived from an identical twin of a subject.

[0274] According to certain embodiments, the lung tissue or cells are non-syngeneic to the subject.

[0275] As used herein, the term "non-syngeneic" cells refers to cells that are not essentially genetically identical to a subject, such as allogeneic or xenogeneic cells, that are not essentially all of the subject's lymphocytes.

[0276] As used herein, the term "allogeneic" refers to cells derived from a donor that is the same species as the subject, but is substantially non-clonal to the subject. Usually, non-inbred, non-zygotic twin mammals of the same species are allogeneic to each other. It will be understood that allogeneic cells can be HLA-identical, partially HLA-identical, or non-HLA-identical (i.e., exhibit one or more different HLA determinants) to the subject.

[0277] As used herein, the term "xenogeneic" refers to cells that substantially express antigens of a different species compared to the species of a substantial proportion of the lymphocytes of the subject. Typically, outbred mammals of different species are xenogeneic to each other.

[0278] The present invention contemplates that the heterologous cells are derived from various species. Thus, according to one embodiment, the lung cells are derived from any mammal. Suitable species sources for the lung cells include major farm animals or livestock and primates. Such animals include, but are not limited to, porcine (e.g., pigs), bovine (e.g., cows), equine (e.g., horses), ovine (e.g., goats, sheep), feline (e.g., Felis domestica), canine (e.g., Canis domestica), rodents (e.g., mice, rats, rabbits, guinea pigs, gerbils, hamsters), and primates (e.g., chimpanzees, rhesus monkeys, macaques, marmosets).

[0279] Lung cells of xenogeneic origin (e.g., porcine origin) are preferably obtained from a source known to be free of zoonotic diseases, such as porcine endogenous retroviruses. Similarly, cells or tissues of human origin are preferably obtained from a source that is substantially free of pathogens.

[0280] According to one embodiment, the lung cells are non-syngeneic to the subject.

[0281] According to one embodiment, the lung cells are allogeneic to the subject.

[0282] According to one embodiment, the lung cells are xenogeneic to the subject.

[0283] According to one embodiment of the invention, the subject is a human and the lung cells are of mammalian origin (eg, allogeneic or xenogeneic).

[0284] According to one embodiment of the invention, the subject is a human and the lung cells are of human origin (eg, syngeneic or non-syngeneic).

[0285] According to one embodiment, the subject is a human and the lung cells are of xenogeneic origin (eg, porcine origin).

[0286] According to one embodiment, the lung cells can be genetically modified prior to transplantation.

[0287] The lung cells of some embodiments of the present invention include progenitor cells that have the capacity to differentiate into epithelial and endothelial cells and can therefore be used to treat lung disorders and / or regenerate lung tissue in subjects in need thereof.

[0288] Thus, according to one aspect of the present invention, there is provided a method of regenerating pulmonary epithelial and / or endothelial tissue in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an isolated lung cell population disclosed herein, thereby regenerating pulmonary epithelial and / or endothelial tissue.

[0289] According to further or alternative aspects of the present invention, there is provided an isolated lung cell population as disclosed herein for use in regenerating pulmonary epithelial and / or endothelial tissue in a subject in need thereof.

[0290] According to further or alternative aspects of the present invention, there is provided a method of treating a pulmonary disorder or lung injury in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the isolated lung cell population disclosed herein, thereby treating the pulmonary disorder or lung injury.

[0291] According to further or alternative aspects of the invention, there is provided an isolated lung cell population as disclosed herein for use in treating a pulmonary disorder or injury in a subject in need thereof.

[0292] As used herein, the term "treating" includes arresting, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating a clinical or cosmetic symptom of a condition, or substantially preventing the appearance of a clinical or cosmetic symptom of a condition.

[0293] As used herein, the term "subject" or "subject in need" refers to a mammal, preferably a human (male or female), of any age, suffering from or predisposed to lung tissue damage or loss as a result of disease, disorder, or injury. Typically, the subject is in need of lung cell or lung tissue transplantation (also referred to herein as recipient) due to a disorder or pathological or undesirable condition, state, or syndrome, or physical, morphological, or physiological abnormality that results in loss of organ functionality and is amenable to treatment by lung cell or lung tissue transplantation.

[0294] According to certain embodiments, the subject is a human subject.

[0295] As used herein, the phrase "pulmonary disorder or injury" refers to any disease, disorder, condition, or any pathological or undesirable condition, state, or syndrome, or any physical, morphological, or physiological abnormality, involving loss or deficiency of lung cells or lung tissue, or loss of function of lung cells or lung tissue.

[0296] Exemplary pulmonary diseases include cystic fibrosis (CF), emphysema, asbestosis, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, idiopathic pulmonary fibrosis, pulmonary hypertension, lung cancer, sarcoidosis, acute lung injury (adult respiratory distress syndrome), respiratory distress syndrome of prematurity, chronic lung disease of prematurity (bronchopulmonary dysplasia), surfactant protein B deficiency, congenital diaphragmatic hernia, pulmonary alveolar proteinosis, pulmonary hypoplasia, pneumonia (e.g., bacterial, viral, or These conditions include, but are not limited to, pulmonary fibrosis, pulmonary fibrosis, nonspecific interstitial pneumonia (including those associated with autoimmune conditions such as lupus, rheumatoid arthritis, or scleroderma), hypersensitivity pneumonitis, idiopathic organizing pneumonia (COP), acute interstitial pneumonia, desquamative interstitial pneumonia, asbestosis, and lung injury (e.g., induced by ischemia / reperfusion pulmonary hypertension or hyperoxic lung injury).

[0297] According to one embodiment, the lung disorder or injury comprises chronic inflammation of the lungs (eg, inflammation that persists for more than two weeks).

[0298] Exemplary chronic inflammatory conditions of the lung include, but are not limited to, chronic airway inflammation, asthma, chronic obstructive pulmonary disease (COPD), lung cancer, cystic fibrosis (CF), granulomatous lung disease, idiopathic pulmonary fibrosis, chronic lung disease of prematurity, radiation pneumonitis, and pulmonary diseases associated with systemic diseases such as scleroderma, lupus, dermatomyositis, sarcoidosis, and adult and neonatal respiratory distress syndrome.

[0299] According to certain embodiments, the pulmonary disorder or injury is selected from the group consisting of cystic fibrosis, emphysema, asbestosis, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, idiopathic pulmonary fibrosis, pulmonary hypertension, lung cancer, sarcoidosis, acute lung injury (adult respiratory distress syndrome), respiratory distress syndrome of prematurity, chronic lung disease of prematurity (bronchopulmonary dysplasia), surfactant protein B deficiency, congenital diaphragmatic hernia, pulmonary alveolar proteinosis, pulmonary hypoplasia, and asthma.

[0300] According to one embodiment, the subject may benefit from a transplant of lung cells or lung tissue.

[0301] According to one embodiment, transplantation of lung cells regenerates structural / functional lung tissue.

[0302] According to one embodiment, transplantation of lung cells generates a chimeric lung (i.e., a lung containing cells derived from genetically distinct sources).

[0303] It will be appreciated that the lung cells of some embodiments of the present invention can regenerate structural / functional lung tissue, including the generation of chimeric lungs. The chimeric lungs include alveoli, bronchial and / or bronchiolar structures, and / or vascular structures. Furthermore, the structural / functional lung tissue of some embodiments includes surfactant synthesis capability detectable by specific cell staining (e.g., Clara cell secretory protein (CCSP), aquaporin-5 (AQP-5), and surfactant protein C (sp-C)), and / or ion transport capability (e.g., as indicated by staining for CFTR (cystic fibrosis transmembrane conductance regulator)). The lung cells of some embodiments of the present invention can further regenerate epithelial, mesenchymal, and / or endothelial tissues (e.g., as indicated by the formation of complete chimeric lung tissues including all these components).

[0304] As used herein, the term "regeneration" refers to the reconstitution of pulmonary epithelial and / or endothelial tissue. Thus, in some embodiments of the present invention, regeneration refers to an increase of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of epithelial and / or endothelial tissue. Regeneration can be assessed using any method known to those skilled in the art, including, for example, X-ray, ultrasound, CT, MRI, histological staining of tissue samples, and the like.

[0305] After transplanting lung cells into a subject according to some embodiments, it is recommended to monitor the growth functionality and immune compatibility of transplanted cells according to any one of various standard techniques in the art, according to standard medical practice.For example, the functionality of regenerated lung tissue can be monitored after transplantation by standard pulmonary function tests, such as by analyzing the functional characteristics of developing grafts, as indicated by surfactant synthesis ability, detectable by staining for surfactant protein C (sp-C), and ion transport ability, as indicated by staining for CFTR (cystic fibrosis transmembrane conductance regulator).

[0306] In order to promote engraftment of the lung cells and to reduce or preferably avoid transplant rejection and / or graft-versus-host disease (GVHD) by at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%, the methods and uses disclosed herein may advantageously further comprise pretreating the subject prior to administration of the lung cells.

[0307] As used herein, the term "conditioning" refers to the preparatory treatment of a subject prior to transplantation.

[0308] According to one embodiment, in order to increase the rate of successful transplantation (e.g., formation of chimerism), the subject is treated with a pretreatment capable of emptying the cellular niche within the lung tissue or organ.

[0309] Thus, the subject is pretreated by administering to the subject a therapeutically effective amount of an agent capable of inducing injury to lung tissue, which injury results in proliferation of resident stem cells in the lung tissue.

[0310] The phrase "injury to lung tissue" refers to localized injury to the lung organ / tissue or a portion thereof.

[0311] The term "resident stem cell proliferation" refers to the induction of cell division of endogenous stem cells resident within lung tissue upon exposure to an agent.

[0312] A variety of pretreatment agents can be used in accordance with the present invention, so long as they induce injury to at least a portion of the lung tissue, resulting in proliferation of resident stem cells within the lung tissue. Thus, for example, the agent can include a chemical, an antibiotic, a therapeutic agent, a toxin, or a medicinal plant or extract thereof.

[0313] The pretreatment protocol can be adjusted taking into account the age and condition (e.g., disease, stage) of the subject, and making such decisions is well within the capabilities of one of ordinary skill in the art, especially in light of the disclosure provided herein.

[0314] Without wishing to be bound by theory, a therapeutically effective amount of pretreatment is an amount of pretreatment agent sufficient to induce local lung tissue damage and proliferation of resident stem cells, but which is not toxic to other organs of the subject being treated (e.g., liver, kidney, heart, etc.) Determining such a therapeutically effective amount is well within the capabilities of one of ordinary skill in the art.

[0315] Exemplary agents that cause pulmonary cytotoxicity include, but are not limited to, chemotherapeutic agents, immunosuppressants, amiodarone, beta blockers, ACE inhibitors, nitrofurantoin, procainamide, quinidine, tocainide, minoxidil, amiodarone, methotrexate, taxanes (e.g., paclitaxel and docetaxel), gemcitabine, bleomycin, mitomycin C, busulfan, cyclophosphamide, chlorambucil, nitrosoureas (e.g., carmustine), and sirolimus.

[0316] Additional agents that cause pulmonary cytotoxicity are listed in Table 2 below [adapted from Collard, www(dot)merckmanuals(dot)com / professional / pulmonary-disorders / interstitial-lung-diseases / drug-induced-pulmonary-disease].

[0317] [Table 2]

[0318] According to certain embodiments, the agent capable of inducing damage to lung tissue is selected from the group consisting of chemotherapeutic agents, immunosuppressants, amiodarone, beta blockers, ACE inhibitors, nitrofurantoin, procainamide, quinidine, tocainide, and minoxidil.

[0319] According to certain embodiments, the agent capable of inducing damage to lung tissue comprises naphthalene.

[0320] According to one embodiment, naphthalene treatment is administered to the subject 1 to 10 days (eg, 7, 6, 5, 4, 3, 2 days, for example 3 days) prior to administration of the lung cells.

[0321] Assessment of lung tissue damage can be performed using any method known in the art, such as pulmonary function tests, chest X-rays, chest CT scans, or PET scans. Determining lung damage is well within the capabilities of those skilled in the art.

[0322] As mentioned above, injury to lung tissue leads to proliferation of resident stem cells within the tissue.

[0323] Assessment of proliferation of resident stem cells (e.g., endogenous stem cells in lung tissue) can be performed using any method known to one of skill in the art, such as, for example, by in vivo imaging of cell proliferation using positron emission tomography (PET) with a PET tracer (e.g., 18F-labeled 2-fluoro-2-deoxy-D-glucose (18FDG) or [18F]3'-deoxy-3-fluorothymidine ((18)FLT)), as taught by, for example, Francis et al, Gut. (2003) 52(11):1602-6 and Fuchs et al., J Nucl Med. (2013) 54(1):151-8.

[0324] Thus, according to one embodiment of the present invention, after administration of an agent capable of inducing damage to the tissue of interest, the subject is exposed to a second pretreatment agent, an agent that eliminates resident stem cells in the tissue. As will be apparent to those skilled in the art of cell biology, sensitivity to radiation is only achieved during the proliferation phase.

[0325] According to another embodiment, an agent for depleting resident stem cells in a tissue (described below) can be administered to a subject without prior pretreatment with an agent that induces damage to the tissue (eg, naphthalene).

[0326] According to one embodiment, the agents that ablate resident stem cells constitute a sublethal, lethal, or supralethal pretreatment protocol.

[0327] According to one embodiment, the conditioning protocol includes reduced intensity conditioning (RIC).

[0328] According to one embodiment, reduced intensity conditioning is performed for up to two weeks (eg, 1-14 days, 1-10 days, or 1-7 days) prior to transplantation of the lung cells.

[0329] According to one embodiment, the conditioning protocol includes total body irradiation (TBI), total lymphoid irradiation (TLI, i.e. exposure to all lymph nodes, thymus, and spleen), partial irradiation, T cell debulking (TCD), chemotherapy, and / or antibody immunotherapy.

[0330] Thus, according to one embodiment, the TBI may be administered in doses ranging from 0.5 to 1 Gy, 0.5 to 1.5 Gy, 0.5 to 2.5 Gy, 0.5 to 5 Gy, 0.5 to 7.5 Gy, 0.5 to 10 Gy, 0.5 to 15 Gy, 0.5 to 20 Gy, 1 to 1.5 Gy, 1 to 2 Gy, 1 to 2.5 Gy, 1 to 3 Gy, 1 to 3.5 Gy, y, 1~4Gy, 1~4.5Gy, 1~1.5Gy, 1~7.5Gy, 1~10Gy, 1~15Gy, 1~12Gy, 2~3Gy, 2~4Gy , 2~5Gy, 2~6Gy, 2~7Gy, 2~8Gy, 2~9Gy, 2~10Gy, 2~15Gy, 2~20Gy, 3~4Gy, 3~5Gy, 3 ~6Gy, 3~7Gy, 3~8Gy, 3~9Gy, 3~10Gy, 3~15Gy, 3~20Gy, 4~5Gy, 4~6Gy, 4~7Gy, 4~ 8Gy, 4~9Gy, 4~10Gy, 4~15Gy, 4~20Gy, 5~6Gy, 5~7Gy, 5~8Gy, 5~9Gy, 5~10Gy, 5~ Includes single or fractionated doses in the ranges of 15 Gy, 5–20 Gy, 6–7 Gy, 6–8 Gy, 6–9 Gy, 6–10 Gy, 6–20 Gy, 7–8 Gy, 7–9 Gy, 7–10 Gy, 7–20 Gy, 8–9 Gy, 8–10 Gy, 10–12 Gy, 10–15 Gy, or 10–20 Gy.

[0331] According to certain embodiments, the TBI comprises a single or fractionated dose of radiation in the range of 1-20 Gy.

[0332] According to certain embodiments, the TBI comprises a single or fractionated dose of radiation in the range of 1-10 Gy.

[0333] According to one embodiment, the TBI treatment is administered to the subject 1-10 days (e.g., 1-3 days) prior to transplantation. According to one embodiment, the subject is pretreated once with TBI 1 or 2 days prior to transplantation.

[0334] According to certain embodiments, the TLI is 0.5-1 Gy, 0.5-1.5 Gy, 0.5-2.5 Gy, 0.5-5 Gy, 0.5-7.5 Gy, 0.5-10 Gy, 0.5-15 Gy, 0.5-20 Gy, 1-1.5 Gy, 1-2 Gy, 1-2.5 Gy, 1-3 Gy, 1-3.5 Gy, 1-4 Gy, 1-4.5 Gy, 1-1.5 Gy, 1-7.5 Gy, 1-10 Gy, 2-3 Gy, 2-4 Gy, 2-5 Gy, 2-6 Gy, 2-7 Gy, 2-8 Gy, 2-9 Gy, 2-10 Gy, 3-4 Gy, 3-5 Gy, 3-6 Gy, 3-7 Gy, 3-8 Gy, 3-9 Gy, 3-10 Gy, 3-4 Gy, 3-5 Gy, 3-6 Gy, 3-7 Gy, 3-8 Gy, 3-10 Gy, 3-10 Gy, 3-20 Gy, 3-20 Gy, 3-20 Gy, 3-30 Gy, 3-40 Gy, 3-50 Gy, 3-60 Gy, 3-7 Gy, 3-80 Gy, 3-100 Gy, 3-200 Gy, 3-200 Gy, 3-200 Gy, 3-300 Gy, 3-400 Gy, 3-500 Gy, 3-600 Gy, 3-700 Gy, 3-800 Gy, 3-100 Gy, 3-200 Gy, Gy, 3-9 Gy, 3-10 Gy, 4-5 Gy, 4-6 Gy, 4-7 Gy, 4-8 Gy, 4-9 Gy, 4-10 Gy, 5-6 Gy, 5-7 Gy, 5-8 Gy, 5-9 Gy, 5-10 Gy, 6-7 Gy, 6-8 Gy, 6-9 Gy, 6-10 Gy, 7-8 Gy, 7-9 Gy, 7-10 Gy, 8-9 Gy, 8-10 Gy, 10-12 Gy, 10-15 Gy, 10-20 Gy, 10-30 Gy, 10-40 Gy, 10-50 Gy, 0.5-20 Gy, 0.5-30 Gy, 0.5-40 Gy, or 0.5-50 Gy.

[0335] According to certain embodiments, TLI comprises a single or fractionated radiation dose in the range of 1-20 Gy.

[0336] According to certain embodiments, TLI comprises a single or fractionated radiation dose in the range of 1-10 Gy.

[0337] According to one embodiment, the TLI treatment is administered to the subject 1-10 days (e.g., 1-3 days) prior to transplantation. According to one embodiment, the subject is pretreated once with TLI 1 or 2 days prior to transplantation.

[0338] According to certain embodiments, the subject may be treated by in vivo T cell debulking with, for example, an anti-CD4 antibody, an anti-CD8 antibody, an anti-CD3 (OKT3) antibody, an anti-CD52 antibody (e.g., CAMPATH), and / or an anti-thymocyte globulin (ATG) antibody (e.g., at a therapeutically effective dose of about 100-500 μg, e.g., 300 μg each, 10, 9, 8, 7, 6, or 5 days prior to transplant).

[0339] According to one embodiment, the conditioning comprises a chemotherapeutic agent. Exemplary chemotherapeutic agents include, but are not limited to, busulfan, myleran, busulfex, fludarabine, melphalan, dimethyl mileran, and thiotepa, and cyclophosphamide. The chemotherapeutic agent may be administered to the subject in a single dose or multiple doses, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses (e.g., daily doses) prior to transplantation. According to one embodiment, the subject is administered a chemotherapeutic agent (e.g., about 30 mg / m2 or more) for 3 to 7 consecutive days, e.g., 5 consecutive days, prior to transplantation (e.g., from day -7 to day -3). 2 Fludarabine (e.g., at a dose of 100 mg / day) is administered.

[0340] According to one embodiment, the conditioning treatment includes antibody immunotherapy. Exemplary antibodies include, but are not limited to, anti-CD52 antibodies (e.g., alemtuzumab, sold under trade names such as Campath, MabCampath, Campath-1H, and Lemtrada), and anti-thymocyte globulin (ATG) agents (e.g., Thymoglobulin (rabbit ATG, rATG, available from Genzyme) and Atgam (horse ATG, eATG, available from Pfizer). Additional antibody immunotherapy may include anti-CD3 (OKT3) agents, anti-CD4 agents, or anti-CD8 agents. According to one embodiment, the antibody is administered to the subject prior to transplantation (e.g., 4-8 days, e.g., 6 days, prior to transplantation) in a single dose or in several doses, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses (e.g., daily doses).

[0341] According to one embodiment, the pretreatment comprises costimulatory blockade. Thus, for example, the pretreatment may comprise transiently administering to the subject at least one T cell costimulatory inhibitor and at least one CD40 ligand inhibitor, and more preferably may further comprise administering to the subject a T cell proliferation inhibitor.

[0342] According to one embodiment, the T cell costimulation inhibitor is CTLA4-Ig, the CD40 ligand inhibitor is an anti-CD40 ligand antibody, and the T cell proliferation inhibitor is rapamycin. Alternatively, the T cell costimulation inhibitor may be an anti-CD40 antibody. Alternatively, the T cell costimulation inhibitor may be an antibody specific for B7-1, B7-2, CD28, anti-LFA-1, and / or anti-LFA3.

[0343] According to certain embodiments, the pretreatment comprises naphthalene treatment (e.g., 10, 9, 8, 7, 6, 5, 4, 3, or 2 days prior to transplantation, e.g., 3 days prior) and TBI treatment (e.g., at a dose of 1-20 Gy, e.g., 6 Gy, e.g., 9, 8, 7, 6, 5, 4, 3, 2, or 1 day prior to transplantation, e.g., 1 day prior).

[0344] According to another particular embodiment, the conditioning comprises a T cell debulking treatment (e.g., with anti-CD8 and / or anti-CD4 antibodies, e.g., 10, 9, 8, 7, 6, 5, 4, 3, or 2 days prior to transplant, e.g., 6 days prior), a naphthalene treatment (e.g., 10, 9, 8, 7, 6, 5, 4, 3, or 2 days prior to transplant, e.g., 3 days prior), and a TBI treatment (e.g., at a dose of 1-20 Gy, e.g., 6 Gy, e.g., 9, 8, 7, 6, 5, 4, 3, 2, or 1 day prior to transplant, e.g., 1 day prior).

[0345] According to another particular embodiment, the conditioning comprises only TBI treatment (e.g., at a dose of 1-20 Gy, e.g., 6 Gy, e.g., 9, 8, 7, 6, 5, 4, 3, 2, or 1 days prior to transplant, e.g., 1 day prior).

[0346] To avoid graft rejection of the lung cells, the subject may be placed on a post-transplant immunosuppressive regimen.

[0347] According to one embodiment, the subject is treated with an immunosuppressive regimen for up to two weeks following administration of the lung cells.

[0348] According to one embodiment, the subject is treated with an immunosuppressive regimen for up to 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days following administration of the lung cells.

[0349] Examples of suitable types of immunosuppressive regimens include administration of immunosuppressive drugs (also called immunosuppressants) and / or immunosuppressive irradiation.

[0350] Ample guidance regarding the selection and administration of appropriate immunosuppressive regimens for transplantation is provided in the art (see, e.g., Kirkpatrick CH. and Rowlands DT Jr., 1992. JAMA. 268, 2952; Higgins RM. et al., 1996. Lancet 348, 1208; Suthanthiran M. and Strom TB., 1996. New Engl. J. Med. 331, 365; Midthun DE. et al. 1997. Mayo Clin Proc. 72, 175; Morrison VA. et al. 1994. Am J Med. 97, 14; Hanto DW., 1995. Annu Rev Med. 46, 381; Sendorowicz AM. et al., 1997. Ann Intern Med. 126, 882; Vincenti F. et al., 1998. New Engl. J. Med. 338, 161; Dantal J. et al. 1998. Lancet 351, 623).

[0351] Examples of immunosuppressants include, but are not limited to, methotrexate, cyclophosphamide, cyclosporine, cyclosporine A, chloroquine, hydroxychloroquine, sulfasalazine (sulphasalazopyrine), gold salts, D-penicillamine, leflunomide, azathioprine, anakinra, infliximab (REMICADE), etanercept, TNF-alpha blockers, biologics targeting inflammatory cytokines, and nonsteroidal anti-inflammatory drugs (NSAIDs). Examples of NSAIDs include, but are not limited to, acetylsalicylic acid, choline magnesium salicylate, diflunisal, magnesium salicylate, salsalate, sodium salicylate, diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, naproxen, nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin, acetaminophen, ibuprofen, Cox-2 inhibitors, tramadol, rapamycin (sirolimus), and rapamycin analogs (CCI-779, RAD001, AP23573, etc.). These agents can be administered individually or in combination.

[0352] According to certain embodiments, the immunosuppressant comprises cyclophosphamide, busulfan, fludarabine, tacrolimus, cyclosporine, mycophenolate mofetil, azathioprine, everolimus, sirolimus, glucocorticoids, or combinations thereof.

[0353] According to one embodiment, the immunosuppressant is cyclophosphamide.

[0354] According to one embodiment, the present invention further contemplates administration of cyclophosphamide pre-transplant (e.g., on days 4, 3, or 2 after transplant, i.e., T-4, -3, or -2), in addition to the post-transplant administration described herein.

[0355] According to certain embodiments, the pulmonary cells administered to the subject comprise an effective amount of hematopoietic progenitor cells (HPCs). Alternatively, or in addition, according to certain embodiments, the pulmonary cells are administered to the subject in combination with an effective amount of hematopoietic progenitor cells (HPCs).

[0356] According to certain embodiments, the HPCs may be administered prior to, simultaneously with, or after administration of the pulmonary cells.

[0357] As used herein, the term "hematopoietic progenitor cells" or "HPCs" refers to a cell preparation that contains immature hematopoietic cells. Such cell preparations can be obtained from biological samples, such as lung tissue (e.g., fetal / fetal or adult tissue), bone marrow (e.g., T cell-depleted bone marrow), mobilized peripheral blood (e.g., CD34 + to increase its concentration), umbilical cord blood (e.g., umbilical cord), fetal / fetal liver, yolk sac, and / or placenta. + cells or other hematopoietic stem cells (e.g., CD131 + Cells) can be used according to some embodiments of the present teachings with or without ex vivo expansion.

[0358] According to certain embodiments, the HPCs comprise lung tissue-derived CD34+ cells.

[0359] According to certain embodiments, the HPCs are bone marrow CD34 + Cell or mobilized peripheral blood CD34 + Contains cells.

[0360] According to certain embodiments, the HPCs comprise T cell depleted immature hematopoietic cells.

[0361] According to certain embodiments, the isolated lung cell population and the HPCs are obtained from the same donor.

[0362] As used herein, the term "effective amount of HPCs" refers to an amount sufficient to achieve tolerance to lung cells without a long-term immunosuppressive regimen.

[0363] As used herein, the term "tolerance" refers to a state in which the responsiveness of a recipient's cells (e.g., the recipient's T cells) when contacted with a donor's cells (e.g., donor HPCs) is reduced compared to the responsiveness of the recipient's cells in the absence of such treatment.

[0364] Tolerance induction allows for the transplantation of cell or tissue grafts (eg, lung cells) with reduced risk of graft rejection or graft-versus-host disease (GVHD).

[0365] An effective amount of HPC is typically about 1×10 per kg of subject body weight. 5 ~10×10 7 Contains cells.

[0366] The cells of some embodiments of the present invention can be administered to an organism by themselves or in a pharmaceutical composition mixed with suitable carriers or excipients.

[0367] As used herein, "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components, such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

[0368] As used herein, the term "active ingredient" refers to the pulmonary cells responsible for the biological effect.

[0369] Thus, according to one aspect of the present invention there is provided a pharmaceutical composition comprising an isolated population of lung cells as an active ingredient and a pharma- ceutically acceptable carrier.

[0370] According to certain embodiments, the pharmaceutical composition further comprises hematopoietic progenitor cells (HPCs) as an active ingredient.

[0371] According to certain embodiments, the isolated lung cell population and the hematopoietic progenitor cells (HPCs) are present in separate formulations.

[0372] According to other specific embodiments, the isolated lung cell population and the HPCs are present in the same formulation.

[0373] Hereinafter, the terms "physiologically acceptable carrier" and "pharmaceutical acceptable carrier", which may be used interchangeably, refer to a carrier or diluent that does not cause significant irritation to an organism and does not interfere with the biological activity and properties of the administered compound. These terms include auxiliary agents.

[0374] As used herein, the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples of excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

[0375] Techniques for formulation and administration of drugs can be found in the latest edition of "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, incorporated herein by reference.

[0376] Suitable routes of administration can include, for example, oral, rectal, mucosal, especially nasal, intestinal, or parenteral delivery (including intramuscular, subcutaneous, and intramedullary injections, as well as intrathecal, direct intraventricular, intracardiac (e.g., into the right or left ventricular cavity, into the common coronary artery), intravenous, intratracheal, intrabronchial, intraalveolar, intraperitoneal, intranasal, or intraocular injections).

[0377] According to one embodiment, administration is by intravenous route.

[0378] Thus, according to certain embodiments, the cells are formulated for intravenous administration.

[0379] According to one embodiment, administration is by the intratracheal route.

[0380] Thus, according to certain embodiments, the cells are formulated for intratracheal administration.

[0381] Alternatively, the administration of lung cells to a subject can be performed by administering the lung cells to various suitable anatomical sites for therapeutic effect. Thus, depending on the application and purpose, the lung cells can be administered to an orthotopic anatomical site (an anatomical site that is normal for the organ or tissue type of the cells) or an ectopic anatomical site (an anatomical site that is abnormal for the organ or tissue type of the cells).

[0382] Thus, depending on the application and purpose, lung cells can be advantageously implanted (e.g., transplanted) under the renal capsule, or into the kidney, testicular fat, subcutaneous tissue, omentum, portal vein, liver, spleen, cardiac cavity, heart, thoracic cavity, lung, pancreas, skin, and / or intraperitoneal space.

[0383] Conventional approaches to deliver drugs to the central nervous system (CNS) include neurosurgical strategies (e.g., intracerebral injection or intraventricular infusion), molecular manipulation of the agent in an attempt to exploit one of the endogenous transport pathways of the BBB (e.g., production of chimeric fusion proteins that include a transport peptide with affinity for an endothelial cell surface molecule in combination with an agent that cannot cross the BBB itself), pharmacological strategies designed to increase the lipid solubility of the agent (e.g., conjugation of a water-soluble agent to a lipid or cholesterol carrier), and temporary disruption of the integrity of the BBB by disruption at high osmolarity (caused by infusion of a mannitol solution into the carotid artery or use of a biologically active agent such as angiotensin peptide). However, each of these strategies has limitations, such as inherent risks associated with invasive surgical procedures, size limitations imposed by limitations inherent to endogenous transport systems, potentially undesirable biological side effects associated with systemic administration of chimeric molecules composed of carrier motifs that may be active outside the CNS, and possible risks of brain damage in brain regions where the BBB is disrupted, making them suboptimal delivery methods.

[0384] Alternatively, the pharmaceutical composition can be administered locally rather than systemically, for example, by injecting the pharmaceutical composition directly into a tissue area (eg, lung tissue) of the patient.

[0385] Pharmaceutical compositions of some embodiments of the present invention can be manufactured by processes well known in the art, e.g., conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0386] Thus, pharmaceutical compositions for use in accordance with some embodiments of the present invention can be formulated in a conventional manner using one or more physiologically acceptable carriers, including excipients and auxiliaries, that facilitate processing of the active ingredient into a pharma- ceutically usable preparation. Appropriate formulations depend on the chosen route of administration.

[0387] For injection, the active ingredients of the pharmaceutical composition can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0388] For oral administration, pharmaceutical compositions can be easily formulated by combining the active compounds with pharma- ceutically acceptable carriers well known in the art. Such carriers allow the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions, and the like for oral ingestion by the patient. Pharmacological preparations for oral use can be made by using solid excipients, optionally grinding the resulting mixture, and processing the mixture of granules after adding suitable auxiliaries as necessary, to obtain tablets or dragee cores. Suitable excipients are in particular fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol, for example cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose, and / or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0389] Dragee cores are provided with suitable coatings.For this purpose, gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions, and concentrated sugar solutions, which may optionally contain suitable organic solvents or solvent mixtures, can be used.Dyes or pigments can be added to tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

[0390] Orally usable pharmaceutical preparations include push-fit capsules made of gelatin, as well as soft sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. Push-fit capsules may contain the active ingredient mixed with a filler such as lactose, a binder such as starch, a lubricant such as talc or magnesium stearate, and optionally mixed with a stabilizer. In soft capsules, the active ingredient may be dissolved or suspended in a suitable liquid, such as fatty oils, liquid paraffin, or liquid polyethylene glycol. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.

[0391] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0392] For administration by nasal inhalation, the active ingredient for use according to some embodiments of the present invention is conveniently delivered in the form of an aerosol spray provided by a pressurized pack or nebulizer using a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane, or carbon dioxide.In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount.Capsules and cartridges (e.g., made of gelatin) for use in a dispenser can be formulated to contain a powder mix of the compound and a suitable powder base, such as lactose or starch.

[0393] The pharmaceutical compositions described herein can be formulated for parenteral administration, for example, by bolus injection or continuous infusion.The preparations for injection can be provided in unit dosage form, for example, in ampoules or in multi-dose containers with optional addition of preservatives.The compositions can be suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulating agents such as suspending agents, stabilizing agents, and / or dispersing agents.

[0394] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Furthermore, suspensions of the active ingredient can be prepared as suitable oil-based or water-based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate, triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the active ingredient to allow the preparation of highly concentrated solutions.

[0395] Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, for example, sterile pyrogen-free water-based solution, before use.

[0396] Pharmaceutical compositions of some embodiments of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, for example, conventional suppository bases such as cocoa butter or other glycerides.

[0397] Pharmaceutical compositions suitable for use in the context of some embodiments of the present invention include compositions in which the active ingredient is contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount refers to an amount of active ingredient (e.g., lung cells) effective to prevent, alleviate, or ameliorate symptoms of a disorder (e.g., a lung disease or condition) or to prolong the survival of the subject being treated.

[0398] Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0399] For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or potency. Such information can be used to more accurately determine useful doses in humans.

[0400] Exemplary animal models that can be used to evaluate a therapeutically effective amount of lung cells include murine models (e.g., mice) in which lung injury is induced, for example, by intraperitoneal injection of naphthalene (e.g., greater than 99% purity) with or without further irradiation (e.g., 40-48 hours after naphthalene administration), as detailed in the Examples section below. For example, immunodeficient mice, such as NOD-SCID mice, can be treated with naphthalene followed by irradiation (e.g., low dose TBI, e.g., 1-3 Gy), prior to administration of lung cells.

[0401] According to certain embodiments, the lung cells are administered at a dose of about 1-10×10 per Kg of subject body weight. 6 , 5~10×10 6 , 1 to 50 × 10 6 , 10~50×10 6 , 10~60×10 6 , 10~70×10 6 , 10~80×10 6 , 10~90×10 6 , 1 to 100 x 10 6 , 5~100×10 6 , 10~100×10 6 , 40~100×10 6 , 50~100×10 6 , 1~200×10 6 , 5~200×10 6 , 10~200×10 6 , 50~200×10 6 , 100~200×10 6 , 1~500×10 6 , 5~500×10 6 , 10~500×10 6 , 100~500×106 、1~1000×10 6 、5~1000×10 6 、10~1000×10 6 、40~1000×10 6 、50~1000×10 6 、100~1000×10 6 、500~1000×10 6 、1~2000×10 6 、5~2000×10 6 、10~2000×10 6 、20~2000×10 6 、30~2000×10 6 、40~2000×10 6 、50~2000×10 6 、60~2000×10 6 、70~2000×10 6 、80~2000×10 6 、90~2000×10 6 、100~2000×10 6 、200~2000×10 6 、300~2000×10 6 、400~2000×10 6 、500~2000×10 6 、600~2000×10 6 、700~2000×10 6 、800~2000×10 6 、900~2000×10 6 、1000~2000×10 6 、1500~2000×10 6 、100~3000×10 6 、200~3000×10 6 、300~3000×10 6 、400~3000×10 6 、500~3000×10 6 、600~3000×10 6 、700~3000×10 6 、800~3000×10 6 、900~3000×10 6 、1000~2000×10 6 、1000~3000×10 6, 2000~3000×10 6 , 500~4000×10 6 , 1000~4000×10 6 , 2000~4000×10 6 , 3000~4000×10 6 A dose range of cells is administered to the subject.

[0402] According to certain embodiments, CD45-depleted lung cells expanded in culture, containing about 1-10% cells double positive for expression of epithelial cell markers and endothelial cell markers, are present at about 1-10×10 per Kg of subject body weight. 6 , 5~10×10 6 , 1 to 50 × 10 6 , 10~50×10 6 , 10~60×10 6 , 10~70×10 6 , 10~80×10 6 , 10~90×10 6 , 1 to 100 x 10 6 , 5~100×10 6 , 10~100×10 6 , 40~100×10 6 , 50~100×10 6 , 1~200×10 6 , 5~200×10 6 , 10~200×10 6 , 50~200×10 6 , 100~200×10 6 , 1~500×10 6 , 5~500×10 6 , 10~500×10 6 , 100~500×10 6 , 1 to 1000 x 10 6 , 5~1000×10 6 , 10~1000×10 6 , 40~1000×10 6 , 50~1000×10 6 , 100~1000×10 6 , 500~1000×10 6 , 1~2000×10 6 , 5~2000×10 6 , 10~2000×10 6, 20~2000×10 6 , 30~2000×10 6 , 40~2000×10 6 , 50~2000×10 6 , 60~2000×10 6 , 70~2000×10 6 , 80~2000×10 6 , 90~2000×10 6 , 100~2000×10 6 , 200~2000×10 6 , 300~2000×10 6 , 400~2000×10 6 , 500~2000×10 6 , 600~2000×10 6 , 700~2000×10 6 , 800~2000×10 6 , 900~2000×10 6 , 1000~2000×10 6 , 1500~2000×10 6 , 100~3000×10 6 , 200~3000×10 6 , 300~3000×10 6 , 400~3000×10 6 , 500~3000×10 6 , 600~3000×10 6 , 700~3000×10 6 , 800~3000×10 6 , 900~3000×10 6 , 1000~2000×10 6 , 1000~3000×10 6 , 2000~3000×10 6 , 500~4000×10 6 , 1000~4000×10 6 , 2000~4000×10 6 , 3000~4000×10 6 A dose range of cells is administered to the subject.

[0403] According to one embodiment of the invention, the lung cells are administered at a concentration of at least about 1×10 per kilogram of the subject's body weight. 6 , 1.5×106 、2×10 6 、2.5×10 6 、3×10 6 、3.5×10 6 、4×10 6 、4.5×10 6 、5×10 6 、5.5×10 6 、6×10 6 、6.5×10 6 、7×10 6 、7.5×10 6 、8×10 6 、8.5×10 6 、9×10 6 、9.5×10 6 、10×10 6 、12.5×10 6 、15×10 6 、20×10 6 、25×10 6 、30×10 6 、35×10 6 、40×10 6 、45×10 6 、50×10 6 、60×10 6 、70×10 6 、80×10 6 、90×10 6 、100×10 6 、110×10 6 、120×10 6 、130×10 6 、140×10 6 、150×10 6 、160×10 6 、170×10 6 、180×10 6 、190×10 6 、200×10 6 、250×10 6 、300×10 6 、320×10 6 、350×10 6 、400×10 6 、450×10 6 、500×10 6 、600×10 6 、700×10 6 、800×10 6, 900×10 6 , 1000×10 6 , 1100×10 6 , 1200×10 6 , 1300×10 6 , 1400×10 6 , 1500×10 6 , or 2000×10 6 A dose of cells is administered to the subject.

[0404] According to certain embodiments, lung cells, including cells expanded in culture and selected or enriched (e.g., at least 20%) for double positive expression of epithelial and endothelial cell markers (either before or after culture), are present at approximately 1×10 6 cells / kg of subject body weight. 6 , 1.5×10 6 , 2×10 6 , 2.5×10 6 , 3×10 6 , 3.5×10 6 , 4×10 6 , 4.5×10 6 , 5×10 6 , 5.5×10 6 , 6×10 6 , 6.5×10 6 , 7×10 6 , 7.5×10 6 , 8×10 6 , 8.5×10 6 , 9×10 6 , 9.5×10 6 , 10×10 6 , 12.5×10 6 , 15×10 6 , 20×10 6 , 25×10 6 , 30×10 6 , 35×10 6 , 40×10 6 , 45×10 6 , 50×10 6 , 60×10 6 , 70×10 6 , 80×10 6 , 90×10 6 , 100×10 6 , 110×10 6, 120×10 6 , 130×10 6 , 140×10 6 , 150×10 6 , 160×10 6 , 170×10 6 , 180×10 6 , 190×10 6 , 200×10 6 , 250×10 6 , 300×10 6 , 320×10 6 , 350×10 6 , 400×10 6 , 450×10 6 , 500×10 6 , 600×10 6 , 700×10 6 , 800×10 6 , 900×10 6 , 1000×10 6 , 1100×10 6 , 1200×10 6 , 1300×10 6 , 1400×10 6 , 1500×10 6 , or 2000×10 6 A dose range of cells is administered to the subject.

[0405] The toxicity and therapeutic efficacy of the active ingredients described herein can be determined in vitro, in cell culture, or in experimental animals by standard pharmaceutical procedures. The data obtained from these in vitro and cell culture assays and animal tests can be used in formulating a dosage range for use in humans. The dosage can vary depending on the dosage form used and the route of administration utilized. The exact formulation, route of administration, and dosage can be selected by the individual physician in consideration of the patient's condition (see, for example, Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.1).

[0406] Dosage and intervals can be adjusted individually to provide a sufficient level of active ingredient to induce or suppress a biological effect (minimal effective concentration, MEC). The MEC varies from preparation to preparation but can be estimated from in vitro data. The dosage required to achieve the MEC depends on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

[0407] Depending on the severity and responsiveness of the condition being treated, dosage may be single or multiple administrations, with the course of treatment lasting from several days to several weeks, or until a cure is effected or a diminution of the disease state is achieved.

[0408] The amount of a composition administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

[0409] The composition of some embodiments of the present invention can be provided in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient, if necessary. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also conform to a notice associated with the container, in a format prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals, which notice indicates that the composition form or is approved by the agency for human or veterinary administration. Such notice may, for example, be that of a label approved by the U.S. Food and Drug Administration for prescription drugs, or that of an approved product insert. The composition comprising the preparation of the present invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in a suitable container, and labeled for the treatment of an indicated condition, as further detailed above.

[0410] To further avoid residual immune responses that may still be present when administering lung cells, several approaches have been developed to reduce the chance of rejection. These include encapsulating non-syngeneic cells in semi-permeable immunoisolation membranes prior to transplantation. Alternatively, cells that do not express xenogeneic surface antigens can be used, such as cells developed in transgenic animals (e.g. pigs).

[0411] Encapsulation techniques are generally classified as microencapsulation, which involves small spherical vesicles, and macroencapsulation, which involves larger flat plate and hollow fiber membranes (Uludag, H. et al. (2000). Technology of mammalian cell encapsulation. Adv Drug Deliv Rev 42, 29-64).

[0412] Methods for preparing microcapsules are known in the art and include, for example, those disclosed in Lu, MZ et al. (2000). Cell encapsulation with alginate and alpha-phenoxycinnamylidene-acetylated poly(allylamine). Biotechnol Bioeng 70, 479-483; Chang, TM and Prakash, S. (2001) Procedures for microencapsulation of enzymes, cells and genetically engineered microorganisms. Mol Biotechnol 17, 249-260; and Lu, MZ, et al. (2000). A novel cell encapsulation method using photosensitive poly(allylamine alpha-cyanocinnamylideneacetate). J Microencapsul 17, 245-521.

[0413] For example, microcapsules have been prepared using modified collagen complexed with a terpolymer shell of 2-hydroxyethylmethylacrylate (HEMA), methacrylic acid (MAA), and methyl methacrylate (MMA), resulting in capsules with a thickness of 2-5 μm. Such microcapsules can be further encapsulated with an additional 2-5 μm terpolymer shell to impart a negatively charged smooth surface and minimize the absorption of plasma proteins (Chia, SM et al. (2002). Multi-layered microcapsules for cell encapsulation. Biomaterials 23, 849-856).

[0414] Other microcapsules are based on the marine polysaccharide alginate (Sambanis, A. (2003). Encapsulated islets in diabetes treatment. Diabetes Thechnol Ther 5, 665-668) or its derivatives. For example, microcapsules can be prepared by polyelectrolyte complexation between the polyanions sodium alginate and sodium cellulose sulfate and the polycation poly(methylene-co-guanidine) hydrochloride in the presence of calcium chloride.

[0415] It is understood that the use of smaller capsules improves cell encapsulation. Thus, for example, reducing capsule size from 1 mm to 400 μm improved the quality control, mechanical stability, diffusion properties, and in vitro activity of encapsulated cells (Canaple, L. et al. (2002). Improving cell encapsulation through size control. J Biomater Sci Polym Ed 13, 783-96). Furthermore, nanoporous biocapsules with well-controlled pore sizes of only 7 nm, tailored surface chemistry, and precise microstructures have been found to successfully immunoisolate the microenvironment for cells (see Williams, D. (1999). Small is beautiful: microparticle and nanoparticle technology in medical devices. Med Device Technol 10, 6-9, and Desai, TA (2002). Microfabrication technology for pancreatic cell encapsulation. Expert Opin Biol Ther 2, 633-646).

[0416] As used herein, the term "about" refers to ±10%.

[0417] The terms "comprises," "comprising," "includes," "including," "having" and conjugations thereof mean "including but not limited to."

[0418] The term "consisting of" means "including and limited to."

[0419] The term "consisting essentially of" means that a composition, method, or structure may include additional components, steps, and / or moieties, but only if the additional components, steps, and / or moieties do not materially alter the basic and novel characteristics of the claimed composition, method, or structure.

[0420] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" can include a plurality of compounds, including mixtures thereof.

[0421] Throughout this application, various embodiments of the invention may be presented in a range format. The description in range format should be understood to be merely for convenience and brevity, and should not be construed as an inflexible limitation of the scope of the invention. Thus, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to have specifically disclosed not only subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., but also individual numerical values ​​within that range, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0422] Whenever a numerical range is given herein, the numerical range is meant to include any recited numbers (fractional or integer) within the given range. The phrases "range between" a first indicated number and a second indicated number and "range from" a first indicated number to a second indicated number are used interchangeably herein and are meant to include the first indicated number and the second indicated number, and all fractional and integer numbers therebetween.

[0423] As used herein, the term "method" refers to manners, means, techniques, and procedures for accomplishing a given task, including, but not limited to, manners, means, techniques, and procedures known to or readily developed by practitioners in the fields of chemistry, pharmacology, biology, biochemistry, and medicine from known manners, means, techniques, and procedures.

[0424] As used herein, the term "treating" includes arresting, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating a clinical or cosmetic symptom of a condition, or substantially preventing the appearance of a clinical or cosmetic symptom of a condition.

[0425] It will be understood that certain features of the invention that are described for clarity in the context of separate embodiments can also be provided in combination in a single embodiment. Conversely, various features of the invention that are described for brevity in the context of a single embodiment can also be provided separately, or in any suitable subcombination, or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments should not be regarded as essential features of those embodiments, unless the embodiment is inoperable without those elements.

[0426] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below, find experimental support in the following examples. EXAMPLES

[0427] Reference is now made to the following examples, which together with the above descriptions illustrate the invention in a non-limiting fashion.

[0428] In general, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological, and recombinant DNA techniques. Such techniques are fully explained in the literature, e.g., "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, RM, ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York. (1998), U.S. Pat. No. 4,666,828, U.S. Pat. No. 4,683,202, U.S. Pat. No. 4,801,531, U.S. Pat. (1994), "Current Protocols in Immunology" Volumes I-III Colligan JE, ed. (1994), Stites et al.(eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", WH Freeman and Co., New York (1980); available immunoassays are widely described in the patent and scientific literature, e.g., U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879, 262, 3,901,654, 3,935,074, 3,984,533, 3,996,345, 4,034,074, 4,098,876, 4,879,219, 5,011,771, and 5,281,521, "Oligonucleotide "Synthesis" Gait, MJ, ed. (1984), "Nucleic Acid Hybridization" Hames, BD, and Higgins SJ, eds. (1985), "Transcription and Translation" Hames, BD, and Higgins SJ, Eds. (1984), "Animal Cell Culture" Freshney, RI, ed. (1986), "Immobilized Cells and Enzymes" IRL Press, (1986), "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol.1-317, Academic Press, "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996), all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout the specification. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All information contained therein is incorporated herein by reference.

[0429] General materials and experimental procedures Mice - Animals were maintained under conditions approved by the Weizmann Institute and MD Anderson Cancer Center Animal Care and Use Committees. All procedures were monitored by the Weizmann Institute and MD Anderson Cancer Center Veterinary Resources Unit and approved by the Institutional Animal Care and Use Committee (IACUC). Mouse strains used included Rag1 - / - (on a C57BL / BJ background), C57BL / 6J (CD45.2) and C57BL / 6-Tg(CAG-EGFP)1Osb / J (Weizmann Institute Animal Breeding Center, Rehovot, Israel), Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo / J ( 39 ), B6.129P2-Gt(ROSA)26Sortm1(CAG-Brainbow2.1)Cle / J ( 9 ), B6.Cg-Shh tm1(EGFP / cre)Cjt / J(40), B6.129-Tg(Cdh5-cre)1Spe / J(21), B6.Cg-Ager tm2.1(cre / ERT2)Blh / J(23), Hopx tm2.1(cre / ERT2)Joe / J(35), B6N.129S6-Gt(ROSA)26Sor tm1(CAG-tdTomato*,-EGFP*)EesMice included 1.1(cre / ERT2)Zjh / J(41), 1.1(cre / ERT2)Zjh / J(22) (Jackson Laboratory, Bar Harbor, USA). All mice were used at 6–12 weeks of age. Mice were maintained in small cages (maximum of 5 animals per cage) and fed sterile diet and acidified water. Randomization: Animals of the same age, sex, and genetic background were randomly assigned to treatment groups. Prespecified exclusion criteria were based on IACUC guidelines and included systemic disease, toxicity, respiratory distress, difficulty eating and drinking, and significant (>15%) weight loss. The majority of animals appeared to be in good health throughout the study period and were therefore included in the appropriate analyses. In all experiments, animals were randomly assigned to treatment groups.

[0430] Naphthalene-induced lung injury - As previously described (6-8), naphthalene (>99% purity, Sigma-Aldrich) was dissolved in corn oil and administered at a dose of 200 mg / kg body weight by intraperitoneal injection 40-48 h before exposure to TBI. In the case of "double" lung injury, naphthalene-treated animals were additionally irradiated in an X-ray irradiator (40-48 h after naphthalene administration). C57BL / 6 mice and Rag1 mice were - / - Mice were irradiated with 6 Gy of TBI.

[0431] Lung cell suspensions - Fetal / embryo or adult lung cell suspensions were obtained by enzymatic digestion as previously described (6-8) with some modifications. Briefly, suspensions were prepared by centrifugation in PBS (Ca + Mg + Dissociation was performed by either mincing the tissue with a razor blade in the presence of 1% collagenase in 100 mM NaCl, 2.4 U / ml Dispase II (Roche Diagnostics, Indianapolis, IN), 1 mg / ml DNAse-I (Roche Diagnostics, Indianapolis, IN) or in phosphate-buffered saline (PBS) in a GentleMACS™ Octo Dissociator with Heaters (Miltenyi Biotec) using the mouse lung dissociation protocol provided by the supplier. +Mg + Lungs were digested by enzymatic digestion of lung tissue in the presence of 1 mg / ml collagenase, 2.4 U / ml dispase II, and 1 mg / ml DNAse-I (Roche Diagnostics) in 100 mL of PBS. Removal of nonspecific debris was performed by sequential filtration through 70 μm and 40 μm filters, or 100 μm, 70 μm, and 40 μm filters. Cells were then washed with PBS containing 2% FCS, 2 mM EDTA, and antibiotics.

[0432] Hepatocyte suspension - Fetal / fetal liver or adult liver was placed on a 40 μm cell strainer, crushed using the flat end of a 5 mL syringe plunger in a 6-well plate, and dispensed into 3–4 mL of ice-cold DPBS. Subsequently, cells were rinsed with ice-cold DPBS and the filtered cell suspension was collected in a 15 μL conical tube. Cells were collected by centrifugation in a refrigerated centrifuge (1,300 rpm, 10 min) and resuspended in 3–5 mL of DPBS before injection. Cells were counted and resuspended using trypan blue and diluted with 10 mL of DPBS. 7 A stock solution of cells / mL was made.

[0433] Bone marrow cell suspension - Bone marrow cells were prepared for transplantation by grinding the long bones of donor mice using a mixer homogenizer (OMNI). Cells were depleted of CD4 and CD8 cells using Miltenyi's magnetic microbead separation protocol according to the manufacturer's instructions.

[0434] Fetal / fetal and adult lung cell transplants – C57BL / 6 mice or Rag1 - / - Recipient mice were pretreated with naphthalene and exposed to 6 Gy TBI 48 hours later. 6 10 x E15–E16 embryonic mouse lung cells or 3–16 x 10 6 Adult mouse lung cells were engrafted into mice by injection into the tail vein 4-8 h after irradiation.

[0435] TMX administration - TMX (Sigma-Aldrich) was prepared as a stock solution of 20 mg / ml in corn oil. To induce Cre recombination in R26R-Confetti adult mice, mice were injected intraperitoneally (IP) with two doses of TMX (5 mg), 5 and 4 days before harvesting bone marrow or lungs. Lungs or BM were harvested 5 days after TMX administration. To induce CRE recombination in Confetti embryos, female mice were treated with a single IP dose of TMX 5 mg at E12. Embryos were harvested at E16, and lung and liver cells from Confetti-positive embryos were used for transplantation experiments.

[0436] Nkx2-1tm1.1(cre / ERT2)Zjh / J mTmG, B6.Cg-Ager tm2.1(cre / ERT2)Blh / J mTmG and Hopx tm2.1(cre / ERT2)Joe To induce Cre recombination in mTmG / J mice, 4 mg of TMX was injected 6 days before harvesting lungs for FACS analysis.

[0437] Spleen colony assay - Five days after TMX administration, bone marrow was harvested from 8-week-old R26R-Confetti mice by flushing the long bones with ice-cold PBS and BM cells were transplanted into lethally irradiated (10 Gy TBI) mice via tail vein injection. Nine days after transplantation, mice were sacrificed and spleens were assessed for the presence of splenic colonies by binocular and fluorescent microscopy.

[0438] Flow cytometric analysis of R26R-Confetti donor cells and transgenic mouse lungs - FACS analysis was performed on a 5-laser LSRII (BD Biosciences) or Fortessa analyzer. E16 fetal / fetal Confetti lung and liver cells, as well as adult Confetti lung and bone marrow cells were analyzed. E16 fetal / fetal and adult lung cells were analyzed for YFP / GFP, RFP, and CFP labeled cells, as well as epithelial (CD326), endothelial (CD31), and hematopoietic (CD45) lineage markers to quantify distinct cell populations within the different fluorescently tagged cells after Cre recombination induced by TMX administration.

[0439] Samples were stained with conjugated antibodies or matching isotype controls according to the manufacturer's instructions. A complete list of anti-CD326, anti-CD31, and anti-CD45 antibodies is provided in Table 3 below. - Sca-1 + c-kit + E16 fetal / fetal liver cells and adult bone marrow cells were analyzed for cell (LSK) populations. Cells were stained with the following antibodies or matching isotype controls: Lineage Panel-Streptavidin followed by staining with Biotin-APC or Biotin-APC-CY7, Sca-1-Brilliant Violet 711, C-kit PE-CY7. A complete list of antibodies used is provided in Table 3 below. Antibodies were purchased from e-Bioscience, BD, and Biolegend.

[0440] Data were analyzed using FlowJo software (version vX.0.7 Tree Star Inc).

[0441] Acquisition of images by TPLSM - A Zeiss LSM 880 upright microscope equipped with a Coherent Chameleon Vision laser was used for the imaging experiments of explanted lung tissue. Images were acquired with a femtosecond pulsed two-photon laser tuned to 940 nm. The microscope was equipped with a filter cube containing a 565 LPXR, which splits the emission light to a PMT detector (with a 579-631 nm filter for tdTomato fluorescence) and a further 505 LPXR mirror, which further splits the emission light to two GaAsp detectors (with 500-550 nm filters for GFP fluorescence). Images were acquired as 100-150 μm Z-stacks with 1-5 μm intervals in each Z-plane. The zoom was set to 0.7 and pictures were acquired with an xy resolution of 512 × 512.

[0442] Tissue clearing - Mice were perfused with a monomer solution containing 4% PFA, 4% acrylamide, 0.0125% bis-acrylamide, and 0.1% azo initiator VA-044. To avoid premature polymerization, the lungs were immediately immersed in the above solution for another 24-48 h with constant shaking at 40 °C. After degassing, the lungs were polymerized for 3 h at 37 °C and washed with 20 mM sodium borate buffer (pH = 9) containing 200 mM SDS. The lungs were then cleared for 4 days using a rotary electrophoretic clearing system (SmartClear II Pro, Life Canvas Technologies, Seoul, Korea). Once the lungs reached a sufficiently cleared state, they were washed with 20 mM sodium borate buffer for 24 h and immersed in index matching solution (EasyIndex, RI = 1.46, Lifecanvas technologies) until imaging.

[0443] Light sheet microscopy - To image large lung volumes, 3D images of cleared lungs were acquired using an ultramicroscope II (LaVision BioTec GmbH, Astastrasse 14, 33617 Bielefeld, Germany) operated with ImspectorPro software (LaVision BioTec). The light sheet was generated by a Superk supercontinuum white light laser (DK-3460 Birkerod, Brocken 84, NKT photonics) with an emission range of 460-800 nm and 1 mW / nm. The excitation filters used to detect red and green patches were 545 / 25 and 470 / 40, respectively. The corresponding emission filters were 595 / 40 and 525 / 50. The microscope was equipped with a single lens configuration, 4X objective (LVMI-Fluor 4X / 0.3, NA: 0.3, WD: 5.6-6.0 mm, RI range: 1.33-1.57). Samples were glued to a sample holder and placed in a 100% quartz imaging chamber (LaVision BioTec) filled with EasyIndex solution (RI = 1.46, lifecanvas technologies) and illuminated from the side. Images were acquired by an Andor Neo sCMOS camera (16-bit, 2150 × 2560, pixel size 1.626 × 1.626 μm, Andor, 277.3 mi Belfast, UK). Z-stacks were acquired in 5 μm increments and two fields of view (3510 × 4160 μm each) were acquired with 20% overlap and stitched together using an Imaris stitcher (BITPLANE, a part of Oxford Instruments, www(dot)bitplane(dot)com).

[0444] To increase the 3D imaging resolution of the red and green engrafted lung patches, the samples were also imaged using a LightSheet Z.1 microscope (Zeiss Ltd, Tegeluddsvaegen 76, 115 28 Stockholm, Sweden) equipped with two sCMOS cameras PCO.Edge, 1920x1920. The red and green patches were illuminated using Zeiss illumination optics LightSheet 10X / 0.2 and the emission was detected using Clr Plan-Neofluar 20x / 1.0 Corr nd=1.45 (Zeiss Ltd.). The edges of the samples were glued to a designated holder and immersed in an imaging chamber filled with EasyIndex solution (Lifecanvas Technologies).

[0445] Imaging was performed using one-sided illumination with two fields of view: 1093.91 × 1093.91 × 296.22 μm and 437.56 × 437.56 × 315.33 μm. The excitation lines for the red and green patches were 561 nm for 1% and 488 nm for 2%, with emission collected from 575-615 nm and 505-545 nm, respectively.

[0446] R26R-Confetti in chimeric lungs + Immunohistological assessment of lesions - Lungs were fixed with 4% PFA solution introduced through the trachea under a constant pressure of 20 cm H2O. Lungs were then immersed in fixative overnight at 4°C. After PFA treatment, lungs were processed, fixed in 30% sucrose, and frozen in Optimal Cutting Temperature (OCT) compound (Tissue-Tek, Sakura Finetek USA, Inc.). Serial step sections of 12 μm thickness were taken along the longitudinal axis of the lobe. Fixation distances between sections were calculated to allow systematic sampling of at least 20 sections throughout the lung. Lung slices were analyzed by fluorescence or confocal microscopy. The actual number of "confetti" patches (defined as a group of 6 or more adjacent cells labeled with the same fluorescent tag (cytoplasmic RFP and YFP, nuclear GFP, and membrane CFP)) was counted per slice.

[0447] Confocal microscopy - 12 μm thin sections of engrafted lungs were imaged using a laser scanning Leica TCS SP8 upright microscope equipped with an acousto-optic beam splitter and acousto-optic tunable filters for wavelength separation (Leica microsystems CMS GmbH, Germany) and two internal HyD detectors with spectral separation. The confocal pinhole was opened to 1 AU (58.6 μm for 580 nm). Images were acquired at two magnifications using an 8 kHz resonant scanner in 1024 × 1024, 8-bit format. Images were acquired using a 20× air objective (HC PL 20x / 0.75W, Leica microsystems) with a field of view of 443.29 μm and pixel size = 0.43 μm. For higher resolution images, a 60X oil objective (HC PL APO 63x / 104 Oil CS2, Leica microsystems) was used, obtaining images with a FOV of 140.73 μm and pixel size of 0.137 μm. To clearly separate the excitation and signal collection from the five different markers (see list for antibody and staining details), a sequential imaging step was applied, with a sequence transition after the acquisition of each Z-stack. In the first sequence, excitation was applied with an Ar laser at 488 nm at 2% (out of 30% lasers) and a HeNe633 laser at 1%, collecting emission at 505-566 nm and 651-708 nm, respectively. In the second sequence, excitation was applied with a DPSS561 laser at 4% and collecting at 574-638 nm. In the third sequence, excitation was applied using the Diode 405 laser at 8% and two emissions were collected: 413-446 nm and 493-521 nm.

[0448] Wide-field microscopy - To image the entire area of ​​the lung tissue sections, a Leica DMi8 inverted microscope equipped with a motorized stage for high-speed imaging was used. Images were taken with a 10X air objective (HC PL FLUOTAR 10x / 0.3 DRY, Leica microsystems) and recorded with a CCD camera (1392 × 1040, 8-bit, Leica DFC7000 GT, monochrome, Leica microsystems).

[0449] Transplantation of sorted CD326+CD31+, CD326+, CD31+ cell populations into mice pretreated with NA+6Gy TBI - Experiments were performed involving transplantation of FACS sorted cell populations from adult mouse lungs. The labeled donor mice used included GFP (C57BL / 6-Tg(CAG-EGFP)1Osb / J), mTmG (Gt(ROSA)26Sor tm4(ACTB-tdTomato,-EGFP)Luo / J), and nTnG(Gt(ROSA)26Sor tm1(CAG-tdTomato*,-EGFP*)Ees The donors included 0.3–0.5 × 10 64-cells from either mTmG or nTnG donors (mTmG mice expressing membrane td-tomato and nTnG mice expressing nuclear td-tomato). Overall, a total of 16 independent transplantation experiments were performed testing the patch-forming activity of sorted cells. Single CD45- live lung cells were FACS sorted into four subpopulations, including CD326+CD31- cells, CD326+CD31+ cells, CD326-CD31+ cells, and CD326-CD31- cells. 6 Sorted cells were collected from 0.5 x 10 mice donor 6 The donor-derived cells were then transplanted into naphthalene-treated, irradiated C57BL mice with or without unseparated lung cells. Lungs from transplanted mice were harvested and assessed for the presence of donor-derived cells 6–24 weeks after transplantation.

[0450] Single molecule fluorescence in situ hybridization (smFISH) - SmFISH was performed as previously described (42). For smFISH, lung tissues were harvested, inflated, fixed in 4% paraformaldehyde for 3 h, incubated overnight with 30% sucrose in 4% paraformaldehyde, and then embedded in OCT and frozen. 6 μm frozen sections were used for hybridization. smFish probes were coupled with Cy5. Probes were purchased from Stellaris® Biosearch™ Technologies.

[0451] Single-cell RNAseq analysis of chimeric lungs - Chimeric lungs were harvested 6 months after transplantation from C57Bl mice transplanted with TdTom single-cell suspensions. Lungs from three chimeric mice were enzymatically treated, dissociated into single cells, pooled, and FACS-sorted for CD45- td-tomato positive and CD45- td-tomato negative cells. Single-cell RNA transcriptome analysis was performed at the MD Anderson core lab using the 10x Genomics platform. Cell Ranger Single Cell Software Suite v3.0.1 (www(dot)support(dot)10xgenomics(dot)com / single-cell-gene-expression / software / overview / welcome) was used to demultiplex samples, align to the mm10 mouse reference genome and transcriptome, and generate a filtered unique molecular identifier (UMI) count matrix for downstream analysis. Data QC, normalization, and integration of the three datasets were performed using the R package Seurat (43–45) v3.1.1. Cells with dataset-specific outliers of high percentage of mitochondria, extremely high or low gene counts, or high RNA content were excluded as potential dead cells or doublets. LogNormalize was used for normalization. Integration was based on 2000 anchor genes by default. The R package monocle3 (46–48) v0.2.0 was used for dimensionality reduction of the integrated dataset using the uniform manifold approximation and projection (UMAP) method (49) and unsupervised clustering of cells was performed using Louvain / Leiden community detection (50). Marker genes for each cluster were identified using Seurat by comparing the gene expression profile of each cluster with that of all other clusters using the Wilcoxon rank sum test.Single-sample gene set variation analysis was performed using the R package GSVA(51) v1.32.0 against the MSigDB(52) hallmark gene set (v7.0) and curated lung gene sets (www(dot)research(dot)cchmc(dot)org / pbge / lunggens / mainportal(dot)html) to calculate gene set enrichment scores per cluster. The resulting clusters were manually curated and / or merged based on the marker genes and gene set analysis results.

[0452] Epithelial Cell Colony Formation Assay - Lung organoids were grown by culturing FACS-sorted cells from VEcad mTmG adult mouse lungs in 50% growth factor reduced (GFR) Matrigel (BD Biosciences) on IBIDI glass-bottom u-slides (Cat. No. 81507). For cell sorting experiments, cells were cultured in phosphate-buffered saline (PBS) (Ca) in a GentleMACS™ Octo Dissociator with Heaters (Miltenyi Biotec) using the mouse lung dissociation protocol provided by the supplier. + Mg + Lung single cell suspensions were prepared by enzymatic digestion of lung tissue in the presence of 1 mg / ml collagenase, 2.4 U / ml dispase, and 1 mg / ml DNAse-I (Roche Diagnostics) in 100 mM NaCl. Single cells were stained with conjugated antibodies against CD45, CD326, and CD31. CD326+mG- and CD326+mG+ cells were purified by FACS, mixed with Matrigel, and cultured at 4–5 × 10 510 μl of cell / Matrigel mixture containing 10 cells was cultured per well. A few hours after the gel solidified, 50 μl of epithelial growth medium was added on top of the cultured cells. The medium was changed every 48–72 hours. The absolute number of epithelial clones was determined after 7–10 days of culture, and in some experiments, the colony formation efficiency was calculated as a percentage (number of seeded cells that gave rise to growing clones divided by the total number of cells seeded in the well) as the number of growing clones per number of cells seeded × 100. All cell cultures were performed at 37°C in a humidified incubator with 5% CO2. After 7–10 days of culture, whole-mount epithelial colonies were stained and analyzed by confocal microscopy (Olympus FV3000).

[0453] Statistical analysis and reproducibility - No sample size calculations were performed. Both female and male mice were used as donors and recipients. Fetal / fetal lungs used for transplantation experiments were harvested on embryonic day (E) 16. Adult lung donors used for transplantation of either unseparated or FACS-purified populations were 6-12 weeks old. All experiments were performed in at least three biological replicates. All data are expressed as mean ± SEM or SD. Statistical analysis was performed using Prizm software. Comparisons were tested using Student's t-test, χ2 distribution test, or one-way analysis of variance (ANOVA). P values ​​<0.05 were considered significant. All graphs were generated using Excel, Prizm, and Adobe Illustrator CC 2018 software.

[0454] Lung cell culture - Conditioned medium (CM) from irradiated mouse embryonic fibroblasts (iMEFs) was collected every 24–48 h. Lungs were freshly harvested from 6–12 week old C57BL / 6 and enzymatically digested into single cell suspensions as described above. CD45+ cells were subsequently depleted by magnetic beads (CD45 microbeads, Miltenyi Biotec, #130-052-301) according to the manufacturer's instructions. 3 × 10 CM were then cultured per 10 ml of CM. 6 cells, or 3 x 10 per well of a 6-well tissue culture plate5 The cells were resuspended in CM supplemented with epidermal growth factor (hEGF, Stemcell, #78006 (20 μg / ml)) and ROCK inhibitor (Y-27632, Tocris, #1254 (5 μm)) or CM supplemented with epidermal growth factor (EGF) (20 μg / ml), ROCK inhibitor (RI) (20 μm) and vascular endothelial growth factor (hVEGF, Stemcell, #78073) and plated in 6-well plates (3 × 10 cells per well). 5 The cells were cultured in 1000 mL of 1000 mM NaCl (100 mM MgCl2) at 37°C in a humidified incubator with 5% CO2. The medium was changed every 48-72 hours. To track cell growth, cells were passaged, counted, and stained with anti-CD31 and anti-CD326 antibodies (see Table 3 below) for FACS analysis every 72-96 hours.

[0455] [Table 3-1] [Table 3-2]

[0456] Example 1 Identification of a novel lung progenitor cell population that dually expresses endothelial and epithelial markers Long-term chimerism in recipient lungs after transplantation of td-tomato-labeled cells In previous studies, pulmonary chimerism was followed up for up to 4 months using immunohistology (IH). Using and combining the results of immunohistology (Figure 7A-E) and single-cell RNA transcriptome analysis (Figure 1A-F), 6-8 month follow-up of chimerism demonstrated donor-derived epithelial clusters (ciliated cells, club cells, AT1 and AT2 cells) as well as endothelial clusters (lymphatic vessels, endothelial progenitor cells, and the recently described (10) gCap-"generic" and aCap-"aerocyte" capillary cells). The resulting clusters were defined based on marker genes and gene set analysis results using the publicly available LGEA (Lung Gene Expression Analysis) web portal. We used well-defined hallmark gene sets for the definition of each cluster.

[0457] Transplantation of lung progenitor cells from fetal / embryo or adult Cag-Cre ER2 “Confetti” mice results in monochromatic patches.

[0458] To reliably establish the single-cell origin of the lung patches observed after transplantation, we performed a series of experiments utilizing the multicolor reporter system “R26R-Confetti” mice (9) as donors. In these mice, the recombination process is independent and stochastic in each cell. Daughter cells produce the same fluorescent protein, but if indeed all cells in each patch originated from a single cell, they should all be the same color. Multiple studies have used this system to demonstrate single-cell-derived clonal behavior in organs, e.g., intestine (9), (11), lung (12), brain (13), kidney, mammary gland, and others (14), (15), (16), (17), (18).

[0459] To validate this approach for lung cell transplantation studies, we first tested it in the context of hematopoietic stem cell transplantation, which is known to result in single-cell-derived splenic colonies (Figures 8A-E).

[0460] We next investigated lung patch formation after transplantation of lung cell suspensions from E16 R26R-Confetti donors into RAG-2 recipients pretreated with naphthalene and 6 Gy TBI (see schematic representation in Figure 2A). Embryos were harvested at E16, tamoxifen (TMX) was administered to pregnant females at E12, and fetal / embryo lungs expressing fluorescent cells were isolated under a fluorescent microscope. Approximately 5-6% of the harvested lungs expressed one of the fluorescent tags (Figure 2B). Two-photon micrographs show typical monochromatic cells before transplantation, with cells each expressing one of the four fluorescent tags (Figure 2C).

[0461] Transplantation of fetal lung cells from tamoxifen-induced donors into naphthalene-treated and irradiated recipient mice gave rise to discrete monochromatic lung patches expressing one of four fluorescent proteins, strongly suggesting that each patch may be derived from a single lung progenitor cell (Figure 2D-E). Similar transplantation of R26R-Confetti fetal liver single-cell suspensions (schematically shown in Figure 9A), which expressed fluorescent tags in hematopoietic LSK+ (lineage-Sca1+c-kit+) stem cells (Figure 9B), and induced hematopoietic splenic colonies (Figure 9C) and hematopoietic blood chimerism (Figure 9D), failed to give rise to donor-derived lung patches (Figure 9E), strongly supporting the lung specificity of the observed patch-forming activity.

[0462] Next, based on recent findings that patch-forming progenitor cells are also present in adult mouse lungs, we applied the same approach to analyze the origin of lung patches after transplantation of adult lung cells. For that purpose, we used a previously described protocol, but because the frequency of patch-forming cells in adult lungs is approximately 3-4-fold lower compared to the frequency found in E16 fetal / embryo lungs (7), and because only a small fraction of cells undergo Cre recombination, we used a higher dose (16 × 10 6) of lung cells were used for transplantation (Figure 3A). As shown in Figure 3B, after TMX treatment, monochromatic GFP, YFP, RFP, and CFP fluorescent cells could be recorded by two-photon microscopy in the adult lung, and as shown by confocal microscopy, these tagged cells were distributed throughout the lung (Figure 3B). Further FACS analysis of R26R-Confetti adult lungs after two doses of TMX allowed us to detect the percentage of Cre-recombinant cells (up to 5%, Figure 3C-D). To assess the clonality of the donor-derived patches 8 weeks after transplantation, confocal, two-photon, and light sheet microscopy were used. As shown in Figure 3E-F and Figure 10A-B, light sheet microscopy of the "cleared" chimeric lungs revealed distinct monochromatic patches.

[0463] Similarly, two-photon snapshots of whole-mount chimeric lungs recorded monochromatic patches expressing either membrane CFP, nuclear GFP, or cytoplasmic RFP or YFP (Figure 3G). Full-depth two-photon microscopy scans of these monochromatic patches in whole-mount lung tissue are presented (data not shown).

[0464] In total, 50 fields collected from 15 chimeric mice, including 12 transplanted with adult lung cells and 3 transplanted with E16 fetal / fetal lung cells, were analyzed, and notably, only single-color donor-derived patches were found to be present. The experimental distribution of single-color clones (n=50) versus two-color clones (n=0) was tested against the theoretical distribution of 25% versus 75% (χ21=150) with a χ2 distribution test, suggesting that it was highly unlikely (p<0.001) that any clone was derived from two cells. This demonstrates that the lung patches observed after transplantation of fetal / fetal or adult lung cells are derived from a single progenitor cell.

[0465] Two distinct patches of pneumocyte progenitors To clarify the identity of the putative "patch-forming" cells in the system, we transplanted FACS-purified cell populations from mouse lungs. For this purpose, we transplanted GFP (C57BL / 6-Tg(CAG-EGFP)1Osb / J) mice, mTmG (Gt(ROSA)26Sor tm4(ACTB-tdTomato,-EGFP)Luo / J) mice, and nTnG (Gt(ROSA)26Sor tm1(CAG-tdTomato*,-EGFP*)Ees We used labeled donor mice, including mTmG mice (mTmG mice express membrane td-tomato, nTnG mice express nuclear td-tomato) and nTmG mice (mTmG mice express membrane td-tomato, nTnG mice express nuclear td-tomato). Unique localization of fluorescent tags to either the membrane or nucleus of sorted lung cells allowed us to track membrane, cytoplasmic, and nuclear epithelial and endothelial markers at the single-cell level within monochromatic patches of chimeric lungs and characterize the cellular composition of the patches after transplantation of sorted lung cell subpopulations. In line with our observations that the majority of patches contain both endothelial and epithelial cells, and that each such patch originates from a single progenitor cell, we searched for putative multipotent lung progenitor cells capable of differentiating along these two distinct lineages. FACS analysis (Figure 4A) and image-stream analysis (Figure 4B) of CD45-negative lung cells revealed a unique subpopulation expressing both the CD326 epithelial and CD31 endothelial markers. We therefore hypothesized that patch-forming cells might be contained within this double-positive subpopulation. To test this hypothesis, we used a transplantation assay for patch-forming cells after sorting the four lung subpopulations obtained by staining for CD326 and CD31, as described in FIG. 4C.

[0466] Patch-forming activity could only be found upon transplantation of CD326+CD31+ double-positive cells (21 of 37 mice) or single-positive CD326-CD31+ cells (28 of 61 mice), whereas no patch-forming activity was found in single-positive CD326+CD31- epithelial cell fractions or double-negative CD326-CD31- fractions (Figure 4F). Sorted cells may require other facilitating cells in the donor cell preparation for initial colonization in the recipient lung. Thus, 0.3–0.5 × 10 64-cells from lungs of mTmG or nTnG mice were transplanted. 6 Sorted cells were collected from 0.5 x 10 6 We attempted to transplant FACS-sorted cells with (Figure 4F) or without (Figure 4G-H) unsorted lung cells. These experiments clearly demonstrated that transplanted FACS-sorted cells were capable of patch formation even in the absence of potential supporting cells from the co-transplanted unsorted GFP+ lung cell preparation (Figure 4G-H).

[0467] Staining of donor-derived patches for alveolar epithelial markers including AQP-5, HOPX, and SPC, or for endothelial markers including CD31, ERG, and SOX17 demonstrated that patches formed after transplantation of double-positive subpopulations contained both endothelial and epithelial cells (Figure 5E-I, Figure 11A-B, Figure 12A-D), whereas patches formed by sorted CD326-CD31+ single-positive cells were composed mainly of endothelial cells (Figure 5A-D). This analysis is strongly supported by the use of nuclear staining, which allows the identification of cell boundaries between adjacent cells. To this end, colocalization of donor-derived nT markers with nuclear ERG / SOX17 or with nuclear expression of HOPX allowed the identification of donor-derived endothelial or AT1 epithelial cells (Figure 5E-G). Similarly, within the patch, AT1 cells stained for AQP-5 could be distinguished from endothelial cells stained for SOX17 (Figure 5H). Staining for CD31 and smRNA FISH probe for SPC similarly allowed the differentiation of AT2 cells from endothelial cells (Fig. 5I). Quantitative analysis of the number of cells composing the patches suggests that the number of cells found after transplantation of CD326+CD31+ lung cells was higher than that after transplantation of CD326-CD31+ cells (Fig. 5J). This analysis revealed differences in the cellular composition between the two types of patches, with colocalization of endothelial and epithelial cells being predominantly found in the patches formed after transplantation of CD326+CD31+ lung cells (Fig. 5K).

[0468] Further characterization of CD326+CD31+ patch-forming lung progenitor cells To further characterize the sorted CD326+CD31+ lung subpopulation, we used transgenic mice expressing GFP under different promoters that are typically activated in epithelial or endothelial cells (e.g., Sonic hedgehog (Shh) and VE-cadherin (cadherin 5)). Shh Cre mTmG mice and Shh Cre nTnG mice (B6.Cg-Shh tm1(EGFP / cre)Cjt / J and Gt(ROSA)26Sortm4(ACTB-tdTomato EGFP)Luo / J mice, and B6N.129S6-Gt(ROSA)26Sor tm1(CAG-tdTomato*,-EGFP*)Ees / J mice, respectively) express GFP in the epithelial lineage (19, 20), whereas VE-cadherinCre mTmG and VE-cadherinCre nTnG mice (B6.129-Tg(Cdh5-cre)1Spe / J mice and Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo / J mice and B6N.129S6-Gt(ROSA)26Sor tm1(CAG-tdTomato*,-EGFP*)Ees Transgenic mice (generated by crossing with VE-cadherinCre / J mice, respectively) express GFP in the endothelial lineage (21). FACS analysis of the lungs of these transgenic mice revealed that double-positive CD326+CD31+ lung progenitors also expressed additional typical endothelial and epithelial markers, namely VE-cadherin (71%) and Shh (74%) (Figure 6A-C). Furthermore, CD326+CD31+ progenitor cells were able to form single-cell-derived patches composed of donor-derived epithelial and endothelial cells, and these cells were able to generate epithelial organoids in vitro. This assay was also used to confirm that VE-cadherin+ cells within the CD326+CD31+ subpopulation purified from the lungs of VE-cadherinCre mTmG mouse donors were indeed able to form epithelial colonies. Thus, organoids generated from FACS-purified double-positive CD326+VEcad mG+ cells displayed GFP+ epithelial cells in contrast to organoids generated from sorted CD326+VEcad mG- lung cells that were GFP-negative and expressed mT (Figure 6D and Figures 14A-D). The epithelial character of the GFP+ organoids was confirmed by staining for additional epithelial markers such as cytokeratin, AQP-5, and SPC (Figure 6E).

[0469] The dual nature of CD326+CD31+ cells was also confirmed by Imagestream analysis of individual cells. Thus, CD326+CD31+ lung cell progenitors from Shh Cre nTnG or VEcad Cre nTnG mice were found to be double positive for Shh and VE cad (Figure 6F-G and Figure 13A-E). Furthermore, analysis of Nkx2.1CreER2 mTmG lungs in which Cre recombination was induced by administration of a single dose of tamoxifen 6 days prior to FACS analysis (22) similarly showed high levels of NKX2.1 (68%) within the CD31+CD326+ lung cell population. Notably, Ager-CreER2 mTmG (23), (24) and Hopx-CreER2 mTmG transgenic mice (25), (26), (27) showed substantial levels of receptor for advanced glycation end products (RAGE) (25.5%) and low expression of Homeobox only protein x (Hopx) (14%). Collectively, these results, based on lineage-specific GFP expression in transgenic mice but not cell surface antibody staining, further support the dual character of CD326+CD31+ lung progenitor cells (Figure 6H–J).

[0470] Consideration It was recently demonstrated that 6–8 weeks after transplantation of mouse or human fetal lung cells into mice pretreated with naphthalene and 6 Gy TBI, numerous donor-derived patches containing epithelial and endothelial cells could be detected throughout the lung (6). Similar findings were subsequently demonstrated after transplantation of adult lung cells, although the concentration of patch-forming cells was 3–4-fold lower compared to that in fetal lungs (7). Using R26R-Confetti donors, in which each fluorescent cell is labeled with one of four possible colors, we now find that, by Cre recombination, each donor-derived lung patch found after transplantation of mouse fetal or adult lung cells is formed by colonization and differentiation of a single multipotent lung progenitor cell. This finding further allowed us to characterize the putative multipotent lung progenitor cells by FACS purification. Thus, using transplantation assays, we found two distinct patch-forming progenitors in the non-hematopoietic CD45-negative lung compartment: CD326+CD31+ and CD326-CD31+ cells, however, only the former were capable of forming patches containing both epithelial and endothelial cells, whereas the latter were primarily associated with the formation of endothelial cells.

[0471] A classic example of such cellular plasticity was demonstrated by Tata et al. (28, 29), who showed that fully mature secretory cells dedifferentiate into basal stem cells with regenerative potential. Another example of cellular plasticity is known as transdifferentiation or transcommitment, where stem cells from one region of the lung can be transformed into stem cells from other lung regions (29). Furthermore, evidence for multipotent progenitor cells capable of developing into endothelial and epithelial lineages has also been described for human breast progenitor cells, which were found to express significant levels of the "Yamanaka" transcription factors (30, 31).

[0472] Notably, the dual character of double-positive CD326+CD31+ progenitor cells was further substantiated by the demonstration that in transgenic mice expressing fluorescent tags under the Shh or VE-cadherin promoter, double-positive CD326+CD31+ cells also express both these typical epithelial and endothelial markers. In addition, using the same approach of tamoxifen-induced Cre recombination, the CD326+CD31+ cell fraction was also found to contain similar levels of NKX2.1 and comparable (but lower) levels of cells expressing Ager and Hopx. The observed expression of NKX2.1 is consistent with previous suggestions that this transcription factor, a hallmark of lung specificity, is expressed in endothelial cells within the developing lung (32). Notably, Ager, a multiligand pattern recognition receptor involved in several disease states, is widely expressed not only in AT1 cells but also in alveolar endothelium (24). Hopx (homeobox-only protein x) is an AT1 marker and a key regulator of cardiac development (33)(34) as well as the biology of hair follicle, intestinal, and hematopoietic stem cells (35)(36)(37)(38).

[0473] In conclusion, our data reveal a novel lung subpopulation, including multipotent CD326+CD31+ lung progenitor cells capable of repairing lung injury. Although various lung progenitor cells have been described previously that are restricted to differentiation along epithelial lineages, this unique progenitor cell exhibits a dual phenotype, expressing well-established epithelial and endothelial markers, and can differentiate into both epithelial or endothelial fates after transplantation into lung-injured mice. This duality is particularly valuable for repairing lung injury, considering that all major lung diseases not only exhibit epithelial injury but also involve endothelial injury.

[0474] Furthermore, the identification of lung progenitor cells may contribute to potential translational research aimed at repairing various lung diseases as well as basic research aimed at improving our understanding of fetal / embryo lung development and the maintenance of steady-state of different cell lineages in the adult lung.

[0475] Example 2 Expansion of pulmonary progenitor cells dually expressing endothelial and epithelial markers in culture. Considering that double positive CD31+CD326+ lung cells represent patch-forming lung progenitor cells, this marker was used to guide an ex vivo expansion strategy. Briefly, lungs were freshly harvested from 6-12 week old C57BL / 6 mice and enzymatically digested into single cell suspensions to deplete CD45+ cells. Cells were subsequently cultured in various media to optimize the conditions most favorable for the expansion of CD31+CD326+ cells.

[0476] After 4–5 passages, we found that culture in medium containing EGF with low doses of ROCK inhibitor expanded the total number of cells but induced differentiation to an epithelial fate without retaining double-positive CD31+CD326+ cells in culture (Figure 15A–C). In contrast, culture in medium containing VEGF, EGF, and high doses of ROCK inhibitor led to a significant expansion of CD31+CD326+ cells (Figure 15A–C).

[0477] It is the intention of the applicant(s) that all publications, patents, and patent applications mentioned herein are incorporated herein by reference in their entirety as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference at the time of reference. In addition, citation or identification of any reference in this application should not be construed as an admission that such reference is available as prior art to the present invention. To the extent section headings are used, they should not be construed as necessarily limiting. In addition, any priority documents of this application are incorporated herein by reference in their entirety.

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Claims

1. 1. A method for expanding an isolated lung cell population in culture, comprising: (a) dissociating lung tissue to obtain an isolated lung cell population; and (b) growing the isolated lung cell population in a medium containing a factor that promotes the proliferation of endothelial cells, a factor that promotes the proliferation of epithelial cells, and a factor that prevents differentiation, to grow a cell population that is double-positive for the expression of epithelial cell markers and endothelial cell markers; whereby said isolated lung cell population is expanded in culture.

2. 1. A method for confirming the suitability of an isolated lung cell population for administration to a subject in need thereof, comprising: (a) dissociating lung tissue to obtain an isolated lung cell population; (b) expanding the isolated lung cell population in culture; and (c) determining the expression of epithelial and endothelial cell markers on the isolated lung cell population during and / or after said culturing; wherein proliferation of the cell population double positive for expression of the epithelial cell marker and the endothelial cell marker above a predetermined threshold indicates that the isolated lung cell population is suitable for administration to the subject; and a lack of proliferation or proliferation below the predetermined threshold of the cell population double positive for expression of the epithelial cell marker and the endothelial cell marker indicates that the isolated lung cell population is unsuitable for administration to the subject; thereby confirming the suitability of the isolated lung cell population for administration to the subject.

3. 1. A method for generating an isolated lung cell population, comprising: (a) dissociating lung tissue to obtain an isolated lung cell population; and (b) contacting the isolated lung cell population with at least one reagent capable of binding to an epithelial cell marker and an endothelial cell marker to select a cell population that is double positive for expression of an epithelial cell marker and an endothelial cell marker; thereby producing said isolated lung cell population.

4. 4. The method of claim 3, further comprising expanding the lung cells in culture after step (b).

5. The method of claim 2 , wherein the culture medium comprises factors that promote endothelial cell proliferation, factors that promote epithelial cell proliferation, and factors that prevent differentiation.

6. 10. The method of claim 1, further comprising determining the expression of said epithelial cell markers and endothelial cell markers on said lung cells during and / or after said culturing.

7. proliferation of the cell population double positive for expression of the epithelial cell marker and the endothelial cell marker above a predetermined threshold indicates that the isolated lung cell population is suitable for administration to a subject in need thereof; and The method of claim 6, wherein the absence of proliferation of the cell population double-positive for expression of the epithelial cell marker and the endothelial cell marker or a proliferation below the predetermined threshold indicates that the isolated lung cell population is unsuitable for administration to the subject.

8. The method according to any one of claims 1 and 5 to 7, wherein the factor that promotes the proliferation of endothelial cells comprises vascular endothelial growth factor (VEGF).

9. The method according to any one of claims 1 and 5 to 7, wherein the factor that promotes the proliferation of epithelial cells is selected from the group consisting of epidermal growth factor (EGF), noggin, and R-spondin.

10. The method according to any one of claims 1 and 5 to 7, wherein the factor that promotes the proliferation of epithelial cells comprises epidermal growth factor (EGF).

11. The method of any one of claims 1 and 5 to 7, wherein the factor that prevents differentiation is selected from the group consisting of a ROCK inhibitor, a GSK3b inhibitor, and an ALK5 inhibitor.

12. The method according to any one of claims 1 and 5 to 7, wherein the factor that prevents differentiation comprises a ROCK inhibitor.

13. The cell population double-positive for the expression of epithelial cell markers and endothelial cell markers is CD326 ＋ CD31 ＋ The method of any one of claims 1 to 7, comprising a signature.

14. An isolated lung cell population obtained according to the method described in any one of claims 1 to 7.

15. 15. A therapeutically effective amount of the isolated lung cell population of claim 14 for use in treating a lung disorder or lung injury in a subject in need thereof.

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