Lung cell transplantation for the treatment of pulmonary fibrosis

Abstract

Provided herein is a lung-forming progenitor cell therapy that does not require preconditioning treatment. Administration of the progenitor cells can replace or replenish a population of host-derived patch-forming cells to reverse or suppress fibrosis, promote healing, and improve lung function. Also disclosed herein are methods for monitoring fibrosis and determining whether a subject is susceptible to progenitor cell therapy.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 441,122, filed January 25, 2023, the entire contents of which are incorporated herein by reference.

[0002] FIELD OF THE INVENTION The present invention relates generally to pulmonary fibrosis, and more particularly to a method of treating a subject with pulmonary fibrosis by transplantation of lung-forming progenitor cells. [Background technology]

[0003] Background information Chronic lung diseases, including chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF), are the most common non-infectious diseases and leading causes of morbidity and mortality, with an estimated prevalence of 13–20 per 100,000 people worldwide. Approximately 100,000 people in the United States are affected, with 30,000–40,000 new cases diagnosed annually. IPF has a poor prognosis, with a median survival time from diagnosis of 3–8 years in adults aged 65 years and older. IPF is characterized by an atypical interstitial pneumonia pattern on high-resolution CT scans, manifesting as basal-predominant reticulation, traction bronchiectasis, and honeycombing. Histologically, these features are represented by heterogeneous paraseptal fibrosis, architectural disarray, and fibroblastic nests. Although the anti-fibrotic drugs pirfenidone and nintedanib can slow the progression of the disease, lung transplantation is often the only treatment that can improve quality of life and survival rate.However, the shortage of lungs suitable for transplantation and the high mortality risk associated with the procedure have led to extensive research into lung stem cell populations, which can potentially provide an alternative source for transplantation and less invasive treatment.

[0004] Various cell populations, including BM-derived cells, lung-derived p63+ cells, LNEP (lineage-negative epithelial progenitor cells), and mouse and human Sox9+ cells, have shown regenerative potential. Recently, fetal and adult lung progenitor cells have been shown to potentially provide an attractive source for transplantation in mice, as long as endogenous lung progenitor cells are eliminated from the lung stem cell niche in the recipient by appropriate conditioning. Following lung injury with naphthalene (NA) or cyclophosphamide (CY), endogenous host lung progenitor cells, which are normally quiescent, exhibit a robust proliferative response, outcompeting donor progenitor cells and preventing engraftment. However, these endogenous proliferating cells are radiosensitive and can be eliminated by subsequent sublethal 6 Gy total body irradiation (TBI). Thus, in a procedure similar to bone marrow transplantation (BMT), single-cell suspensions of mouse or human fetal lung cells harvested at the tubular stage of gestation (20–22 weeks in humans, E15–E16 in mice) and infused intravenously (IV) after conditioning of recipient mice with naphthalene and 6 Gy TBI resulted in remarkable lung chimerism within the alveolar and bronchial lineages. This chimerism is associated with significantly improved lung function. Notably, transplantation of such conditioned fetal or adult lung cells results in extensive donor-derived "patches" with bronchial, alveolar, and endothelial cell lineages. More recently, this approach to inducing lung chimerism has been extended to transplantation of single-cell suspensions of adult mouse lung donors, which requires an approximately three-fold higher cell dose to obtain a similar level of chimerism. Notably, in these studies where donor-derived cells were derived from GFP+ or TdTomato+ donors, the infused lung-forming progenitor cells colonized discrete green or red patches containing multiple cell lineages, including epithelial and endothelial cells. Summary of the Invention

[0005] The present invention is based on the pioneering discovery that preconditioning regimens are not essential for lung-forming progenitor cell therapy. In particular, certain lung pathologies can promote donor cell receptivity to engraftment, enabling progenitor cell therapy without the need for stem cell depletion preconditioning. Based on transplant failure in non-preconditioned mouse models, it was previously believed that preconditioning was required for lung transplantation therapy. However, it has surprisingly been discovered herein that certain lung pathologies obviate the need for preconditioning, allowing for less intensive treatment regimens for subjects with these conditions. This disclosure provides studies in different mouse models using single-cell lung suspensions, suggesting that conditioning is required in normal recipients but not in two models of pulmonary fibrosis. The present invention does not include a preconditioning step, thus providing progenitor cell therapy accessible to a broader population of pulmonary fibrosis patients.

[0006] In one embodiment, the present invention provides a method for treating pulmonary fibrosis in a subject, comprising administering a therapeutically effective amount of lung-forming progenitor cells to a subject having at least about 15% pulmonary fibrosis, thereby treating the pulmonary fibrosis in the subject.

[0007] In one embodiment, prior to administration, the level of fibrosis in the subject's lungs is measured by comparing the volume of fibrosis in the lungs to the total lung volume. In another embodiment, the measurement comprises subjecting the subject to a computed tomography (CT) scan and / or assessing the level of fibrosis via a lung biopsy sample. In some embodiments, assessing the level of fibrosis in the lung biopsy sample comprises subjecting the lung biopsy sample to an Ashcroft test. In some embodiments, the treatment is administered in the absence of a preconditioning treatment prior to administration of the cells. In certain embodiments, the method further comprises dissociating the lung tissue to obtain lung-forming progenitor cells. In certain embodiments, dissociating the lung tissue comprises subjecting the lung tissue to enzymatic digestion. In certain embodiments, the lung tissue is fetal lung tissue or adult lung tissue. In some embodiments, the lung tissue is human lung tissue. In some embodiments, the cells are autologous cells. In some embodiments, the cells are allogeneic cells. In some embodiments, the cells are administered in a cell suspension. In various embodiments, the method further comprises enriching for cells expressing endothelial and / or epithelial markers and / or depleting the cells of cells expressing CD45. In some embodiments, the epithelial marker is selected from CD326, CD324, CD245, aquaporin-5, podoplanin, or receptor for advanced glycation end products. In certain embodiments, the endothelial marker is CD31, CD144, or ERG. ... CD326. + CD31 + , CD324 + CD31 + , CD326 + CD144 + , and / or CD324 + CD144 + In certain embodiments, the method further comprises enriching the cells for cells that are CD326 + CD31 + , CD324 + CD31 + , CD326 + CD144 + , and / or CD324 + CD144 +In certain embodiments, the method further comprises determining that the isolated lung cells comprise a T cell. In certain embodiments, the method further comprises depleting the cells of T cells, depleting the cells of B cells, or a combination thereof. In one embodiment, enriching or depleting the cells comprises contacting the cells with an agent that binds to an epithelial marker, an endothelial marker, or CD45. In another embodiment, the binding agent is an antibody. In some embodiments, the method further comprises expanding the isolated lung cells in culture. In various embodiments, the subject has idiopathic pulmonary fibrosis or chronic obstructive pulmonary disease. In certain embodiments, the cells are administered in one or more doses.

[0008] In some aspects, the method further comprises determining expression of an epithelial marker, an endothelial marker, or a combination thereof in the cells. In some aspects, the epithelial marker is selected from CD326, CD324, CD245, aquaporin-5, podoplanin, or advanced glycation end-product specific receptor. In some aspects, the epithelial marker is CD326. In some aspects, the endothelial marker is CD31, CD144, or ERG. In some aspects, the endothelial marker is CD31. In some aspects, a predetermined percentage of the cells expresses the epithelial marker and the endothelial marker. In some embodiments, the predetermined percentage is at least about 0.1%, at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% of the cells. In some embodiments, the epithelial marker is CD326 and the endothelial marker is CD31. In some embodiments, the cells are expanded for at least about 12 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 72 hours, at least about 120 hours, at least about 180 hours, at least about 240 hours, at least about 300 hours, or at least about 600 hours prior to determining. In some embodiments, if the percentage of cells that do not express epithelial and endothelial markers exceeds a predetermined threshold, the cells are determined to be unsuitable for administration to a subject. In some embodiments, the predetermined threshold is at least about 0.1%, at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% of the cells.

[0009] In further embodiments, the method further comprises administering to the subject an immunosuppressive therapy, hi certain embodiments, the immunosuppressive therapy comprises tacrolimus, everolimus, sirolimus, rapamycin, cyclosporin A, an antilymphocyte globulin antibody, an antithymocyte globulin (ATG) antibody, an anti-CD3 antibody, a steroid, azathioprine, an anti-IL-2Ra receptor antibody, mycophenolic acid, or an anti-CD20 antibody.

[0010] In another embodiment, the present invention provides a method for improving lung function in a subject in need thereof, comprising administering a therapeutically effective amount of lung-forming progenitor cells to a subject having at least about 15% pulmonary fibrosis, thereby improving lung function in the subject.

[0011] In one embodiment, prior to administration, the level of fibrosis in the subject's lungs is measured by comparing the volume of fibrosis in the lungs with the total lung volume. In another embodiment, improving lung function comprises reducing fibrosis and / or preventing or slowing the progression of fibrosis. In some embodiments, improving lung function comprises improving Newtonian resistance of the respiratory system, lung tissue compliance, lung tissue elastance, tissue damping, and / or forced expiratory volume. In certain embodiments, the subject has pulmonary fibrosis or chronic obstructive pulmonary disease. In some embodiments, the treatment is administered in the absence of a preconditioning treatment prior to administration of the cells. In some embodiments, the cells are administered in a cell suspension.

[0012] In another embodiment, the present invention provides a method for identifying a subject with pulmonary fibrosis as a candidate for treatment with lung progenitor cells, comprising: a) measuring the level of fibrosis in the subject's lung; and i) classifying a subject having a level of fibrosis of at least about 15% or more as a likely responder to cell transplantation, thereby identifying the subject as suitable for cell transplantation; or ii) classifying a subject having a level of fibrosis of less than about 0-15% as a likely non-responder to lung cell transplantation, thereby identifying the subject as not suitable for cell transplantation, thereby identifying the subject with pulmonary fibrosis as a candidate for treatment with lung progenitor cells.

[0013] In one embodiment, measuring the level of fibrosis is performed by computed tomography (CT) scanning and / or by assessing the level of fibrosis in a lung biopsy sample. In a further embodiment, the method further comprises administering a cell suspension comprising isolated lung-forming progenitor cells to the subject identified as a likely responder. In various embodiments, administering the cell suspension comprises increasing the number of donor-derived patch-forming cells and / or decreasing the number of host-derived patch-forming cells. In certain embodiments, the donor-derived patch-forming cells express both endothelial and epithelial markers. In some embodiments, the endothelial marker is CD31, CD144, or ERG. In some embodiments, the endothelial marker is CD31. In some embodiments, the epithelial marker is CD326, CD324, CD245, aquaporin-5, podoplanin, or receptor for advanced glycation end products. In some embodiments, the epithelial marker is CD326. In some embodiments, the method further comprises measuring a second level of fibrosis in the subject's lung at a second time point, and (i) maintaining the subject as a unlikely responder if the second level of fibrosis is less than about 0-15%, or (ii) reclassifying the subject as a likely responder if the second level of fibrosis is at least about 15% or greater. In some embodiments, a level of fibrosis of at least about 15% or greater indicates a reduction in the number of endogenous pulmonary stem cells sufficient to ensure engraftment of lung-forming progenitor cells. In certain embodiments, a level of fibrosis of less than about 1-15% indicates an insufficient reduction in the number of endogenous pulmonary stem cells to allow engraftment of lung-forming progenitor cells. [Brief explanation of the drawings]

[0014] [Figure 1A]Figures 1A-1C are a schematic, a set of images, and a plot showing the number of patches forming lung-forming progenitor cells remaining in mice treated with BLM for different periods. This is tested by transplanting different numbers of lung cells from BLM-treated mice into preconditioned recipient mice. Figure 1A is a schematic of the study design. Figure 1B is a graph showing the number of TdTomato- and GFP-positive donor-derived patches after transplantation of different numbers of lung cells from mice treated with BLM for different periods (size scale = 500 µm). Figure 1C is a plot showing a quantitative analysis of the number of donor-derived patches per 2 mm2 lung area after transplantation of different numbers of lung cells from mice treated with BLM for different periods. [Figure 1B] See legend to Figure 1A. [Figure 1C] See legend to Figure 1A. [Figure 2A] Figures 2A-2D are diagrams, images, and plots showing donor-derived patches after transplantation of TdTomato+ lung cells into mice treated with BLM for different periods. Figure 2A is a schematic of the experimental design. Figure 2B is a graph showing the number of donor-derived lung patches per 2 mm2 lung area in mice treated with BLM for different intervals. Figure 2C is a set of images showing typical examples of donor-derived lung patches in large lung areas of mice treated with BLM for different intervals. Figure 2D is a plot showing the percentage of chimeric mice, defined by at least four visible donor-derived patches, each consisting of more than 20 donor cells. [Figure 2B] See legend to Figure 2A. [Figure 2C] See legend to Figure 2A. [Figure 2D] See legend to Figure 2A. [Figure 3A]Figures 3A-3E are a set of images and plots outlining the typical cellular composition of GFP+ donor-derived patches in the lungs of transplanted mice pretreated with BLM for 4 weeks. Figure 3A is a set of images of typical immunohistological staining of HOPX+ AT1 alveolar cells. Figure 3B is a set of images of typical immunohistological staining of LAMP3+ AT2 alveolar cells. Figure 3C is a set of images of typical immunohistological staining of ERG+ endothelial cells. Figure 3D is a set of images of typical immunohistological staining of PDGRa+ mesenchymal cells. Figure 3E is a series of plots of the average percentage of different cell types in donor-derived lung patches, representing a minimum of 40 patches for each staining obtained from 3-5 chimeric mice. [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 3D] See legend to Figure 3A. [Figure 3E] See legend to Figure 3A. [Figure 4A]Figures 4A-4H are a schematic diagram and a set of images and plots showing the attenuation of fibrosis and functional benefit in the BLM mouse model 8 weeks after lung cell transplantation. Figure 4A is a schematic diagram of the experimental design. Figure 4B is a photograph showing typical IHC staining of lung tissue from mice preconditioned with BLM for 4 weeks and transplanted with TdTomato+ and GFP+ C57BL donor-derived lung cells 1 week later. Lung tissue was harvested 8 weeks after transplantation (scale bar = 200 μm). Figure 4C shows photographs showing typical trichrome staining of lungs treated with vehicle (PBS, left), bleomycin (center), or bleomycin along with transplantation of a total of 8 × 10 donor-derived cells (right). All groups were tested for fibrosis simultaneously, i.e., 13 weeks after the start of BLM treatment. Figure 4D is a graph showing an Ashcroft test comparing fibrosis levels in different groups of mice based on Mason trichrome staining. Figure 4E is a graph showing percent lung tissue volume measured by CT. Figure 4F shows graphs showing functional parameters measured by FlexiVent (one-way anova with Dunnett's test was used for statistical analysis; *p<0.03, **p<0.002, ***p<0.0002, ****p<0.0001). Figure 4G is a plot of chimerism level, defined by the average number of patches per 2 mm2 of lung area. Mice showing an average of less than four patches per 2 mm2 of lung tissue (purple) were excluded from further comparison. Figure 4H is a plot of total collagen in paraffin-embedded lung tissue by hydroxyproline assay from one transplant experiment. [Figure 4B] See legend to Figure 4A. [Figure 4C] See legend to Figure 4A. [Figure 4D] See legend to Figure 4A. [Figure 4E] See legend to Figure 4A. [Figure 4F] See legend to Figure 4A. [Figure 4G] See legend to Figure 4A. [Figure 4H] See legend to Figure 4A. [Figure 5A] Figures 5A-5H are a set of diagrams, images, and plots showing donor-derived patches after transplantation of TdTomato+ lung cells into TRF1ko / SPC mice treated with TMX after different time periods. Figure 5A is a schematic diagram of the experimental design showing induction of TRF1 knockout in AT2 cells by TMX treatment three times per week for 6.5 weeks. Mice were then transplanted with lung cells from TdTomato+ donors at different time points during fibrosis progression. Figure 5B is a graph showing the number of donor-derived lung patches per 2-nm2 lung area in mice treated with donor lung cells at different time points after TMX induction. Figure 5C shows a photograph showing a typical example of donor-derived lung patches in a large lung area of ​​a mouse treated with donor lung cells at different time points after TMX induction. Figure 5D is a scheme showing the generation of SPC-Cre TRF1fl / fl mice in which TMX induces Cre recombination, resulting in specific deletion of TRF1 in SPC+ AT2 alveolar cells. Figure 5E is a plot of the number of donor-derived lung patches per 2 mm2 of lung area in mice treated with donor lung cells at different time points after TMX induction. Each dot represents the average number of patches per 2 mm2 of lung area based on at least 10 measurements in an individual mouse. Figure 5F is a plot of the percentage of fibrosis measured by microCT at different time points after the initiation of TMX treatment in the absence of lung cell transplantation. Figure 5G is a plot showing the percentage of mice exhibiting donor chimerism as a function of time. Figure 5H is a set of representative 3D lung images generated by microCT imaging of untreated lung tissue (top) and lung tissue 14 weeks after the initiation of TMX administration (bottom). Tissue volume, representing the fibrotic area, is shown in contrast and calculated for all images. [Figure 5B] See the legend to Figure 5A. [Figure 5C] See the legend to Figure 5A. [Figure 5D] See the legend to Figure 5A. [Figure 5E] See the legend to Figure 5A. [Figure 5F] See the legend to Figure 5A. [Figure 5G]See the legend to Figure 5A. [Figure 5H] See the legend to Figure 5A. [Figure 6A] Figures 6A-6E are a set of plots and images showing the typical cellular composition of GPIF+ donor-derived patches in the lungs of a TRF1ko / SPC mouse model transplanted 14 weeks after TMX induction of TRF1 knockout in AT2 cells. Figure 6A is a set of magnified images of typical immunohistological staining of HOPX+ AT1 alveolar cells (left, 10 µm scale bar), HOPX and GFP staining (second from left, 2 µm scale bar), HOPX and nuclear staining (second from right, 2 µm scale bar), and GFP and nuclear double staining (right, 2 µm scale bar). Figure 6B is a set of magnified images of typical immunohistological staining of LAMP3+ AT2 alveolar cells (left, 10 µm scale bar), LAMP3 and GFP staining (second from left, 2 µm scale bar), LAMP3 and nuclear staining (second from right, 2 µm scale bar), and GFP and nuclear double staining (right, 2 µm scale bar). Figure 6C is a set of magnified images of typical immunohistochemical staining of ERG+ endothelial cells at low magnification (left, 10 μm scale bar), ERG and GFP staining (second from left, 2 μm scale bar), ERG and nuclear staining (second from right, 2 μm scale bar), and GFP and nuclear double staining (right, 2 μm scale bar). Figure 6D is a set of magnified images of typical immunohistochemical staining of PDRGa+ mesenchymal cells at low magnification (left, 10 μm scale bar), PDRGa and GFP staining (second from left, 2 μm scale bar), PDRGa and nuclear staining (second from right, 2 μm scale bar), and GFP and nuclear double staining (right, 2 μm scale bar). Figure 6E is a set of plots showing the prevalence of multiple cell types in donor-derived lung patches. [Figure 6B] See legend to Figure 6A. [Figure 6C] See legend to Figure 6A. [Figure 6D] See legend to Figure 6A. [Figure 6E] See legend to Figure 6A. [Figure 7A] Figures 7A-7I are a set of schematics and plots and images showing fibrosis attenuation and functional benefit in TRF1ko / SPC mice transplanted 14 weeks after the initiation of TMX induction of TRF1 knockout in AT2 cells. Figure 7A shows the experimental design with induction of TRF1 knockout in TRF- / -SPEC-Cre mice and transplantation 7 weeks after TMX induction with a mixture of GFP and TdTomato fluorescent C57BL mice. Figure 7B is a photograph showing typical fibrosis determined by trichrome staining 14 weeks after the initiation of TMX induction. Figure 7C is a graph showing an Ashcroft test demonstrating statistically significant lower levels of fibrosis in transplanted mice compared to non-transplanted mice (P<0.002). Figure 7D shows graphs demonstrating improved lung function in transplanted mice compared with non-transplanted mice, as measured using Flexivent to measure resistance (left, P<0.03), tissue damping (second from left, P<0.002), tissue elastance (second from right, P<0.03), and forced expiratory volume (right). Figure 7E shows a set of images of typical examples of fibrosis determined by trichrome staining 22 weeks after the start of TMX induction (high and low magnifications, respectively; scale bars = 200 μm and 100 μm). Figure 7F shows a plot of total collagen in paraffin-embedded lung tissue, as measured by the hydroxyproline assay. Figure 7G shows an image of fibrosis determined by trichrome staining 22 weeks after the start of TMX induction. Figure 7H shows a plot of chimerism levels, defined by the average number of patches per 2 mm2 lung area. Figure 7I shows a fluorescent image of transplanted lung tissue 8 weeks after transplantation of a 1:1 mixture of TdTomato+ and GFP+ C57BL donor-derived lung cells, 14 weeks after host preconditioning. Scale bar = 200 μm. [Figure 7B] See the legend to Figure 7A. [Figure 7C] See the legend to Figure 7A. [Figure 7D] See the legend to Figure 7A. [Figure 7E] See the legend to Figure 7A. [Figure 7F]See the legend to Figure 7A. [Figure 7G] See the legend to Figure 7A. [Figure 7H] See the legend to Figure 7A. [Figure 7I] See the legend to Figure 7A. [Figure 8] 1 is a diagram showing the conditioning scheme for lung injury in C57BL / 6 mice. [Figure 9A] Figures 9A-9D show the engraftment of embryonic progenitor lung cells following different lung preconditioning regimens. Figure 9A shows fluorescence microscopy images of lungs from C57BL / 6 adult mice pretreated with 6 Gy of TBI and transplanted with GFP+ embryonic progenitor lung cells. Lungs were harvested 8 weeks after transplantation. Figure 9B shows fluorescence microscopy images of lungs from C57BL / 6 adult mice pretreated with naphthalene and transplanted with GFP+ embryonic progenitor lung cells. Lungs were harvested 8 weeks after transplantation. Figure 9C shows fluorescence microscopy images of lungs from C57BL / 6 adult mice pretreated with naphthalene and 6 Gy of TBI and transplanted with GFP+ embryonic progenitor lung cells. Lungs were harvested 8 weeks after transplantation. Figure 9D shows the results of quantitative morphometric analysis of GFP+ patches of transplanted cells per mm3 of lung tissue after conditioning regimens with 6 Gy radiotherapy, naphthalene, or 6 Gy radiotherapy plus naphthalene. [Figure 9B] See the legend to Figure 9A. [Figure 9C] See the legend to Figure 9A. [Figure 9D] See the legend to Figure 9A. [Figure 10A]Figures 10A-10I are plots and images showing the assessment of persistent BLM-induced pulmonary fibrosis. Figure 10A is a set of representative microCT 3D images of lung tissue from mice after treatment with PBS (left) and 8 weeks after completion of BLM treatment (right). Figure 10B is a plot of the percentage of fibrotic tissue calculated based on microCT scans 8 weeks after completion of BLM treatment. Figure 10C is a plot showing quantitative measurement of total collagen in paraffin-embedded lung tissue, measured by hydroxyproline assay. Figure 10D is a set of representative H&E lung staining images 8 weeks after completion of BLM treatment (middle and bottom rows) compared to treatment with PBS (top row). Figure 10E is a set of representative three-color lung staining images 8 weeks after completion of BLM treatment (middle and bottom rows) compared to treatment with PBS (top row). Figure 10F is a set of representative images of alpha-SMA lung staining 8 weeks after completion of BLM treatment (middle and bottom rows) compared to treatment with PBS (top row). Figure 10G is a set of representative images of fibronectin lung staining 8 weeks after completion of BLM treatment (middle and bottom rows) compared to treatment with PBS (top row). Figure 10H is a set of representative images of collagen IV lung staining 8 weeks after completion of BLM treatment (middle and bottom rows) compared to treatment with PBS (top row). Figure 10I is a set of plots of pulmonary function assessments (from left to right, elastance (E), resistance (R), tissue damping (G), tissue elastance (H), and forced expiratory volume (FEV0.1) measured by Flexivent at different time points after completion of BLM treatment. [Figure 10B] See the legend to Figure 10A. [Figure 10C] See the legend to Figure 10A. [Figure 10D] See the legend to Figure 10A. [Figure 10E] See the legend to Figure 10A. [Figure 10F] See the legend to Figure 10A. [Figure 10G] See the legend to Figure 10A. [Figure 10H] See the legend to Figure 10A. [Figure 10I] See the legend to Figure 10A. [Figure 11A] Figures 11A-11C show representative trichrome and alpha-SMA stained images of mouse lungs 8 weeks after completion of BLM treatment. Figures 11A-B show 200 μm (left) and 500 μm (center and right) images of trichrome-stained lung tissue from a BLM-treated mouse. Figure 11C shows a set of 200 μm (left) and 500 μm (center and right) images of alpha-SMA stained lung tissue from a BLM-treated mouse. [Figure 11B] See the legend to Figure 11A. [Figure 11C] See the legend to Figure 11A. [Figure 12] A set of images of a typical example of a donor-derived lung patch in the whole lung of a mouse treated with BLM for 4 weeks and transplanted with 8 million TdTomato+ lung cells. Each panoramic view represents a different transplanted mouse (scale size = 500 µm). [Figure 13A] Figures 13A-13D show images of single cells tracked along the Z axis within donor-derived patches found in the lungs of BLM-treated mice 8 weeks after transplantation of GFP+ lung cells. Chimeric lung tissue was stained for the expression of GFP and different markers and analyzed by confocal microscopy through optical slices z=1 to z=10. Nuclei were tracked by Hoechst staining in GFP+ donor-derived cells. AT1 and AT2 alveolar cells, endothelial cells, and mesenchymal cells are shown by staining for HOPX, LAMP, ERG, and PDGRa, respectively. The left column shows low-magnification images of donor-derived patches (scale bar = 10 μm), and the right column shows higher-magnification images of double-positive cells (scale bar = 3 μm). Figure 13A shows a set of images with HOPX staining. Figure 13B shows a set of images with LAMP3 staining. Figure 13C shows a set of images with ERG staining. Figure 13D shows a set of images with PDGRa staining. [Figure 13B] See the description of Figure 13A. [Figure 13C] See the description of Figure 13A. [Figure 13D] See the description of Figure 13A. [Figure 14] This is a set of images showing panoramic views of donor-derived lung patches in SPC-Cre TRF1fl / fl recipients of TdTomato+ lung cells. Mice were treated with TMX for 7 weeks and then transplanted with 8 million lung cells from a TdTomato+ donor. Chimeric lungs were harvested 8 weeks after transplantation. Scale bar 200 μm, N=8 mice. [Figure 15A] Figures 15A-15D show images of Z-tracking of a single cell within a donor-derived patch found in an SPC-Cre TRF1fl / fl recipient of TdTomato+ lung cells. Chimeric lung tissue was stained for GFP and different marker expression and analyzed by confocal microscopy through optical slices z=1 to z=6. Nuclei were tracked by Hoechst staining in GFP+ donor-derived cells. AT1 and AT2 alveolar cells, endothelial cells, and mesenchymal cells are shown by staining for HOPX, LAMP3, ERG, and PDGRa, respectively. Figure 15A shows a set of images with HOPX staining. Figure 15B shows a set of images with LAMP3 staining. Figure 15C shows a set of images with ERG staining. Figure 15D shows a set of images with PDGRa staining. [Figure 15B] See the legend to Figure 15A. [Figure 15C] See the legend to Figure 15A. [Figure 15D] See the legend to Figure 15A. [Figure 16A]Figures 16A-16B are images of hyaluronic acid staining of lungs before and after transplantation. Figure 16A is a set of images of chimeric lungs taken 8 weeks after transplantation and stained for HA. The top row shows typical staining at 500 μm for mice not induced with BLM (PBS), mice induced with BLM, and mice induced with BLM and transplanted after 4 weeks. The bottom row shows a 200 μm magnification of the boxed area shown in the top row. Figure 16B is a set of images of HA staining for mice treated with TMX for 7 weeks and transplanted with 8 million lung cells from a TdTomato+ donor after an additional 7 weeks. The top row shows typical staining at 500 μm magnification for mice not induced with TMX (PBS), mice induced with TMX, and mice treated with TMX and transplanted after 7 weeks. The bottom row shows a 200 μm magnification of the boxed area shown in the top row. [Figure 16B] See the legend to Figure 16A. [Figure 17A] Figures 17A-17C are sets of images showing the incorporation of donor-derived AT1, AT2, and endothelial cells into the alveoli of BLM-treated mice 2 months after transplantation of lung single-cell suspensions from GFP+ donors. Figure 17A is a set of images stained with Hoechst and HOPX. Figure 17B is a set of images stained with Hoechst and LAMP3. Figure 17C is a set of images stained with Hoechst and ERG. The leftmost image in each of Figures 17A-C contains GFP staining to highlight donor cells. The second-left image in each of Figures 17A-C contains nuclear staining. The second-right image in each of Figures 17A-C contains staining for the lineage markers tested. The rightmost image in each of Figures 17A-C shows colocalization of donor-derived cells. [Figure 17B] See the legend to Figure 17A. [Figure 17C] See the legend to Figure 17A. DETAILED DESCRIPTION OF THE INVENTION

[0015] Detailed Description of the Invention The present invention is based on the pioneering discovery that a preconditioning regimen is not required for lung-forming progenitor cell therapy in subjects with certain lung pathologies.

[0016] Before describing the compositions and methods of the present invention, it is to be understood that this invention is not limited to the particular compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the invention will be limited only in the appended claims.

[0017] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, it will be apparent to persons skilled in the art upon reading this disclosure and so forth that reference to "the method" includes one or more methods and / or steps of the type described herein.

[0018] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0019] As used herein, the term "about" in conjunction with a numerical value is meant to include any additional numerical values ​​reasonably close to the stated numerical value. For example, based on the context, the value may be increased or decreased by 5-10%. For example, a value of about 100 means 90-110 (or any value between 90-110).

[0020] As used in this specification and claims, the terms "comprising," "containing," and "including" are inclusive and open-ended and do not exclude additional, unrecited elements, components, or method steps. Thus, the terms "comprising" and "including" encompass the relatively more restrictive terms "consisting of" and "consisting essentially of."

[0021] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the practice or testing of the present invention, any methods and materials similar or equivalent to those described herein can be used, but it is understood that modifications and variations are within the spirit and scope of the present disclosure. Preferred methods and materials are now described.

[0023] The present invention provides cell transplantation therapies for the treatment of pulmonary fibrosis. Previously, preconditioning regimens were thought to be essential for achieving lung-forming progenitor cell engraftment. However, it has surprisingly been discovered herein that moderate and advanced stages of fibrosis are more susceptible to donor cell engraftment, enabling regenerative progenitor cell transplantation without preconditioning. As further demonstrated herein (e.g., as shown in Example 3), these treatments not only replenish multiple stem cell phenotypes but also improve overall lung health and reduce fibrosis. Thus, the therapies disclosed herein provide an improved treatment option that does not require the induction of injury through preconditioning to promote cell engraftment and fibrosis repair.

[0024] As used herein, the terms "fibrosis" and "fibrosis" refer to levels of fibrotic tissue that exceed those observed in healthy tissue. For example, fibrosis can refer to the deposition of extracellular matrix (ECM) and collagen at levels that reduce elasticity and inhibit normal tissue function. Fibrosis can include excessive accumulation or hyperactivity of fibroblasts in an organ or tissue. Treating fibrosis means reducing the degree of fibrosis (e.g., decreasing collagen and / or ECM levels, or decreasing the Ashcroft score) or slowing the rate of fibrosis in a subject, organ, or tissue.

[0025] As used herein, the term "pulmonary fibrosis" refers to fibrosis of the airways. Examples of pulmonary fibrosis and diseases that may include pulmonary fibrosis include, but are not limited to, cystic fibrosis, emphysema, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis, lung cancer, acute lung injury, respiratory distress syndrome, chronic lung disease, and chronic pulmonary inflammation.

[0026] As used herein, the term "fibroblast" may refer to a connective tissue cell that is active in the deposition of extracellular matrix (ECM) and collagen.

[0027] As used herein, the term "pulmonary tissue" refers to tissue of the respiratory tract. In certain instances, pulmonary tissue refers to tissue of the lower respiratory tract, including tissue of the lungs, trachea, bronchi, bronchioles, and alveoli. Lung tissue can be tissue located within the respiratory tract, tissue obtained from the respiratory tract, or tissue produced from respiratory cells.

[0028] This invention is based on the pioneering discovery that preconditioning regimens are not essential for lung-forming progenitor cell therapies, allowing these therapies to be administered without preconditioning, thereby reducing the intensity of such therapies and expanding the subset of IPF patients who are candidates for this treatment. Currently, the primary option available for the treatment of IPF is lung transplantation, which carries a relatively high risk. Despite the emergence of novel compounds for the treatment of IPF patients, lung transplantation remains the only "curative" option for IPF. The low 5-year median survival rate after transplantation highlights the urgent need for novel therapies.

[0029] The presently disclosed study, which investigated the efficacy of lung stem cell transplantation in two different models of pulmonary fibrosis, demonstrates that lung cell transplantation is histologically, biochemically, radiographically, and physiologically effective. These included three independent transplantation experiments in each model, including at least two transplantation experiments for functional analysis. After transplantation of lung cell suspensions into mice exhibiting moderate levels of pulmonary fibrosis, donor-derived patches occupied a significant volume of the lung in most transplanted mice, exceeding 2 mm. 2 More than 12 donor-derived patches were found per lung area. Immunohistochemical analysis revealed that in both fibrosis models, numerous donor-derived patches expressed AT1 (HOPX +) and AT2 (LAMP-3 +、 SPC +) Alveolar cells and endothelial cells (ERG + ) and mesenchymal cells (PDGRa +Furthermore, functional analyses measuring lung tissue resistance, elastance, and forced expiratory volume demonstrated that this robust chimerism was associated with improved lung function across all parameters. Pathological analysis of the tissue also revealed a significant reduction in fibrotic tissue compared to untreated mice.

[0030] Notably, in both models, the progression of pulmonary fibrosis was associated with a significant depletion of endogenous patch-forming lung progenitor cells in the recipient, demonstrating receptivity to donor cell engraftment without the need for further conditioning. This surprising finding supports a model similar to bone marrow transplantation (BMT), in which stem cell competition for the lung stem cell niche represents a major barrier to achieving lung chimerism after lung stem cell transplantation. The lack of stem cell competition during fibrosis progression in the two mouse models is reminiscent of BMT in patients with severe combined immunodeficiency (SCID). These patients possess genetically defective T cell progenitors that are unable to compete with normal donor-derived progenitor cells and can develop in the recipient thymus after transplantation without the need for host stem cell ablation.

[0031] More broadly, it has been discovered herein that lung-forming progenitor cells can reside in, proliferate within, and repair fibrotic lungs, enabling various therapies for treating fibrosis. Because fibrosis often occurs simultaneously with proinflammatory immune polarization, suppressing tissue repair processes, many fibrotic subjects are unable to undergo the preconditioning treatment required by conventional cell transplantation procedures, and therefore lack options for managing and treating fibrosis. The compositions and methods disclosed herein provide treatment and management options for previously untreatable forms of fibrosis. Furthermore, because many subjects with lung pathology have a reduced ability to recover from preconditioning regimens, the methods disclosed herein can increase the likelihood of a successful treatment outcome.

[0032] The present study demonstrates that a single intravenous infusion of a lung cell suspension can lead to robust regeneration of both donor-derived AT2 and AT1 alveolar cells and endothelial cells. Considering that all major pulmonary fibrotic diseases are associated with not only epithelial damage but also vascular and endothelial damage (leading to poor oxygen exchange in the lung), this multilineage engraftment is particularly valuable and may be advantageous over AT2 cell transplantation for lung function restoration.

[0033] Leveraging these discoveries, in one embodiment, the present invention provides a method for treating pulmonary fibrosis in a subject, comprising administering a therapeutically effective amount of lung-forming progenitor cells to a subject having at least about 15% pulmonary fibrosis, thereby treating the pulmonary fibrosis in the subject.

[0034] As used herein, the term "subject" refers to any individual or patient to whom a disclosed composition is administered or a disclosed method is performed, or to whom biological material (e.g., tissue samples, cells, or biological fluids) is obtained. Generally, a subject is a human, as would be understood by one of skill in the art, although a subject can also be a non-human animal. Thus, other animals, including vertebrates such as rodents (including mice, rats, hamsters, and guinea pigs), cats, dogs, rabbits, farm animals (including cows, horses, goats, sheep, pigs, chickens, etc.), and primates (including monkeys, chimpanzees, orangutans, and gorillas), are also included within the definition of a subject.

[0035] The term "treatment" is used interchangeably herein with the terms "therapeutic method" or "therapy" and refers to 1) therapeutic procedures or treatments that cure, delay, alleviate symptoms, and / or halt progression of a diagnosed pathological condition or disorder (e.g., idiopathic pulmonary fibrosis), and 2) prophylactic / preventative measures. Individuals in need of treatment can include those who already have a particular medical disorder as well as those who may eventually acquire the disorder (i.e., those in need of preventative measures).

[0036] The terms "administration of" and / or "administering" should be understood to mean providing a therapeutically effective amount of a pharmaceutical composition to a subject in need of treatment. The route of administration can be enteral, topical, or parenteral. Thus, routes of administration include, but are not limited to, intradermal, subcutaneous, intravenous, intraperitoneal, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transdermal, transtracheal, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal, oral, sublingual, buccal, rectal, vaginal, nasal, ocular administration, as well as injection, inhalation, and spray. As used herein, the phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration.

[0037] In some aspects, administration comprises intravenous injection, intrapulmonary administration, intratracheal administration, intrabronchial administration, intranasal administration, nebulization, powder inhalation, intrapulmonary injection, intraperitoneal, intrathecal, or pulmonary artery injection. In some cases, administration comprises intravenous or intrapulmonary administration.

[0038] Herein, we found that fibrosis can increase donor cell engraftment receptivity, and as a result, administration of lung-forming progenitor cells can result in chimerism leading to lung repair and fibrosis suppression. In particular, in the BLM model, the optimal time for engraftment was found to be 3–4 weeks after the initiation of BLM treatment (a total of 6–8 BLM administrations), but not in the SPC-Cre TRF1 fl / fl In our model, the optimal time for treatment was found to be 6–7 weeks after the completion of TMX treatment (a total of 12–14 weeks from the initiation of TMX treatment). In contrast, poor lung chimerism was observed at earlier time points in both models. Furthermore, using CT analysis, we demonstrated that a level of fibrosis occupying approximately 15% or more of the lung led to effective engraftment and colonization of the lung with donor-derived patch-forming lung progenitor cells (e.g., as shown in Figures 5E–5G). This level of fibrosis is similar to that found by CT in IPF patients with moderate fibrosis. Such patients, whose disease would otherwise inevitably follow a fatal trajectory, are likely to benefit from the initiation of cell therapy.

[0039] Based on these findings, in some cases, the subject has at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, or at least about 25% pulmonary fibrosis. In some cases, the subject has honeycombing, ground-glass opacity, reticular opacity, predominantly basal reticulation, traction bronchiectasis, or a combination thereof. In some cases, the subject's pulmonary health precludes lung preconditioning (e.g., due to induced damage by chemical treatment or radiation).

[0040] In one embodiment, before administration, the level of fibrosis in the subject's lung is measured by comparing the fibrous volume in the lung with the total lung volume. As used herein, the term "fibrous volume" can refer to the amount of space in the lung occupied by fibrous tissue. When taken as a ratio to the total lung volume, the fibrous volume can indicate the degree of fibrosis in the lung. Therefore, the fibrous volume can be useful for determining the severity of fibrosis in a subject. In some cases, the fibrous volume indicates the volume of the lung with honeycombing, ground-glass opacity, or reticular opacity (as described in detail, for example, in Bartholmai et al. J Thorac Imaging. 2013; 28: 298-307). In some cases, the fibrous volume indicates the volume of fibrotic lung parenchyma (as described in detail, for example, in Radiat Oncol J. 2014; 32 (1): 43-47).

[0041] Fibrosis volume and total lung volume can be determined by numerous techniques well known in the art. In many embodiments, measurements can include subjecting a subject to a computed tomography (CT) scan and / or assessing the level of fibrosis in a lung biopsy sample. As used herein, computed tomography refers to an imaging technique that utilizes X-rays and computational modeling to generate two-dimensional cross-sectional or three-dimensional images of solid structures, such as the lungs. CT is commonly used to monitor lung pathologies, including fibrosis, due to its resolution and non-invasiveness (see, e.g., Nemoto et al. Respiratory Research 2020;21:275). In many lung imaging applications, CT provides millimeter to submillimeter spatial resolution (e.g., about 0.1-5 mm). 3 ), allowing for subtle distinction between fibrous and non-fibrous tissue.

[0042] In some embodiments, assessing the level of fibrosis in a lung biopsy sample includes subjecting the lung biopsy sample to the Ashcroft test. The term "Ashcroft test," used interchangeably herein with "Ashcroft scoring," refers to a method of aggregating fibrosis scores from separate microscopic images to determine the extent of fibrosis in the lung. In one implementation, the Ashcroft test can score tissue from 0 to 8, with 0 indicating normal lung; 1 indicating minimal fibrotic thickening of the alveolar or bronchial walls; 3 indicating moderate thickening of the walls with no obvious damage to the lung structure; 5 indicating advanced fibrosis with definitive damage to the lung structure and the formation of fibrotic bands or small fibrotic masses; 7 indicating severe structural distortion and large fibrotic areas; and 8 indicating complete fibrotic obstruction of the lung field (see, e.g., Ashcroft et al. J Clin Pathol. 1988;41(4):467-470). The Ashcroft test typically utilizes a light microscope, such as a confocal microscope, a bright field microscope, a dark field microscope, or an oblique illumination microscope.

[0043] In some embodiments, the treatment is administered without preconditioning treatment prior to cell administration. Donor cell engraftment and colonization are typically prevented by endogenous stem cell populations. Depleting stem cell populations through preconditioning can provide a window of time during which donor cells can engraft and induce chimerism. Preconditioning regimens often achieve this depletion through cytotoxic conditions or agents that collaterally damage other lung cell types, necessitating subsequent repair and recovery. For example, preconditioning can include the use of cytotoxic agents such as naphthalene, irradiation, ischemia, or a combination thereof. Fibrotic lungs often exhibit impaired repair and immune function and may be unable to recover from such pretreatments, making them resistant to conventional cell therapy. Thus, the compositions and methods of the present invention can provide treatment for otherwise untreatable conditions.

[0044] As used herein, "preconditioning" can refer to a treatment that reduces the density of endogenous stem cells in a target tissue. Examples of preconditioning regimens include total body irradiation (TBI), partial body irradiation, chemotherapy, and chemotoxic treatments (e.g., naphthalene treatment). In many cases, preconditioning regimens can be distinguished from immune cell reduction and immunosuppressive therapies that primarily target immune cells, and can optionally be used in conjunction with the methods disclosed herein to limit graft rejection.

[0045] Thus, in some aspects, the treatment further comprises a treatment to suppress graft rejection. In some aspects, the treatment to suppress graft rejection includes immune cell reduction, immunosuppression, or a combination thereof. The treatment to suppress graft rejection can optionally be provided before, concurrently with, and / or after cell transplantation to enhance chimerism and graft stability.

[0046] In some embodiments, the subject is administered immunosuppressive therapy. The immunosuppressive therapy can be chronic (e.g., similar to the immunosuppressive regimens provided to organ transplant patients) or can occur directly concurrently with or precede administration of the progenitor cells. By way of non-limiting example, immunosuppressive therapy can include administration of an immunomodulatory agent such as tacrolimus, everolimus, sirolimus, rapamycin, cyclosporin A, antilymphocyte globulin antibody, antithymocyte globulin (ATG) antibody, azathioprine, anti-IL-2Ra receptor antibody, mycophenolic acid, or anti-CD20 antibody.

[0047] The progenitor cells can be derived from lung tissue. Fetal and adult lung tissue often contains a complex cellular environment, which can include hematopoietic cells, endothelial cells, epithelial cells, and mesenchymal progenitor cells. After administration, these cells can localize to the lung tissue, proliferate, and differentiate into functional lung tissue. In many cases, the progenitor cells contain multiple cell lineages (e.g., epithelial and endothelial). However, in certain embodiments, the progenitor cells are depleted or enriched for specific cell types.

[0048] Lung-forming progenitor cells can be obtained from a variety of sources. While the primary source of allogeneic lung cell transplantation is currently limited to cadaveric lungs, it is contemplated herein that patch-forming lung progenitors can be cultured to allow for the collection of sufficient cells from a lung biopsy from a suitable donor (e.g., a family member). Furthermore, as disclosed herein, induction of lung chimerism can be achieved in mismatched recipients without chronic immunosuppression, for example, by combining lung cell transplantation with bone marrow transplantation from the same donor. However, given that it can be difficult to collect sufficient numbers of hematopoietic stem cells from cadaveric lungs, this approach may be more feasible using living donors to collect ex vivo expanded lung progenitor cells in conjunction with freshly isolated hematopoietic stem cells.

[0049] In certain aspects, the method includes dissociating lung tissue to obtain lung progenitor cells. Dissociation can liberate lung progenitor cells from the lung tissue, which can increase their suitability for delivery and engraftment. Dissociation can include various mechanical, chemical, and enzymatic means. Prior to dissociation, the lung tissue can optionally be dissected or minced with a razor. After dissociation, reagents and cellular debris can be separated from the lung progenitor cells by, for example, filtration, chromatography, or affinity purification.

[0050] In some embodiments, dissociating the lung tissue comprises subjecting the lung tissue to enzymatic digestion. Enzymatic digestion can be performed using proteolytic enzymes such as trypsin or dispase, collagen-degrading enzymes such as collagenase, ECM-targeting enzymes such as matrix metalloproteinases, or combinations thereof. After enzymatic digestion, the enzymes can be inactivated or separated from the cells, for example, by chromatography or filtration.

[0051] In some embodiments, dissociating the lung tissue comprises mechanical dissociation. In many such cases, mechanical dissociation can break the tissue into fragments of a predetermined size. Mechanical dissociation can include cutting, grinding, scraping, or extrusion through a fine filter. In some cases, mechanical dissociation is performed before enzymatic digestion.

[0052] In certain aspects, the lung tissue is fetal lung tissue or adult lung tissue. In many aspects, the fetal lung tissue or adult lung tissue is derived from the lower respiratory tract, such as a portion of the lung. In some aspects, the lung tissue is human lung tissue.

[0053] The tissues can be autologous (i.e., derived from the subject to whom they are administered) or allogeneic (i.e., derived from and delivered to a separate subject). In some embodiments, the cells are autologous cells. In some embodiments, the cells are allogeneic cells. In some embodiments, the cells are administered in a cell suspension.

[0054] In many cases, the cells contain multiple cell types (e.g., a combination of epithelial, endothelial, and mesenchymal progenitor cells) that are effective in restoring homeostasis and suppressing or reversing fibrosis. Furthermore, as shown herein, coadministration of terminally differentiated lung cells with progenitor cells often does not affect the engraftment or antifibrotic activity of the progenitor cells. As shown in Figures 3A-3E, successful donor-derived patches can contain AT1 pneumocytes (e.g., identified by HOPX staining or flow cytometry), AT2 pneumocytes (e.g., identified by LAMP3 staining or flow cytometry), endothelial cells (e.g., identified by ERG staining or flow cytometry), or mesenchymal cells (e.g., identified by PDGRa staining or flow cytometry). It is contemplated herein that coadministration of one or more of these cells with lung-forming progenitor cells can promote successful engraftment and recovery from IPF.

[0055] For example, lung-forming progenitor cells can be co-administered with about 1%-5% AT1 cells (i.e., AT1 cells comprise a total of 1%-5% of all cells administered to the subject). More commonly, lung-forming progenitor cells can be co-administered with about 1%-10%, about 1%-15%, about 1%-20%, about 1%-25%, about 1%-30%, about 1%-40%, about 1%-50%, about 1%-60%, about 1%-80%, about 5%-10%, about 5%-15%, about 5%-20%, about 5%-25%, about 5%-30%, about 5%-40%, about 5%-50%, about 5%-60%, about 5%-80%, 10%-20%, about 10%-25%, about 10%-30%, about 10%-40%, or about 1%-50%. It may be co-administered with about 10% to 50%, about 10% to 60%, about 10% to 80%, about 15% to 25%, about 15% to 30%, about 15% to 40%, about 15% to 50%, about 15% to 60%, about 15% to 80%, about 20% to 30%, about 20% to 40%, about 20% to 50%, about 20% to 60%, about 20% to 80%, about 30% to 50%, about 30% to 60%, about 30% to 80%, about 40% to 60%, about 40% to 80%, or about 50% to 80% AT1 cells. Similarly, lung-forming progenitor cells are approximately 1%-5%, approximately 1%-10%, approximately 1%-15%, approximately 1%-20%, approximately 1%-25%, approximately 1%-30%, approximately 1%-40%, approximately 1%-50%, approximately 1%-60%, approximately 1%-80%, approximately 5%-10%, approximately 5%-15%, approximately 5%-20%, approximately 5%-25%, approximately 5%-30%, approximately 5%-40%, approximately 5%-50%, approximately 5%-60%, approximately 5%-80%, 10%-20%, approximately 10%-25%, approximately 10%-30%, approximately 10%-40% %, about 10% to 50%, about 10% to 60%, about 10% to 80%, about 15% to 25%, about 15% to 30%, about 15% to 40%, about 15% to 50%, about 15% to 60%, about 15% to 80%, about 20% to 30%, about 20% to 40%, about 20% to 50%, about 20% to 60%, about 20% to 80%, about 30% to 50%, about 30% to 60%, about 30% to 80%, about 40% to 60%, about 40% to 80%, or about 50% to 80% of the AT2 cells.Furthermore, lung-forming progenitor cells are approximately 1% to 5%, approximately 1% to 10%, approximately 1% to 15%, approximately 1% to 20%, approximately 1% to 25%, approximately 1% to 30%, approximately 1% to 40%, approximately 1% to 50%, approximately 1% to 60%, approximately 1% to 80%, approximately 5% to 10%, approximately 5% to 15%, approximately 5% to 20%, approximately 5% to 25%, approximately 5% to 30%, approximately 5% to 40%, approximately 5% to 50%, approximately 5% to 60%, approximately 5% to 80%, 10% to 20%, approximately 10% to 25%, approximately 10% to 30%, approximately 10% to 40% %, about 10% to 50%, about 10% to 60%, about 10% to 80%, about 15% to 25%, about 15% to 30%, about 15% to 40%, about 15% to 50%, about 15% to 60%, about 15% to 80%, about 20% to 30%, about 20% to 40%, about 20% to 50%, about 20% to 60%, about 20% to 80%, about 30% to 50%, about 30% to 60%, about 30% to 80%, about 40% to 60%, about 40% to 80%, or about 50% to 80% endothelial cells. Lung-forming progenitor cells are also expressed in approximately 1% to 5%, approximately 1% to 10%, approximately 1% to 15%, approximately 1% to 20%, approximately 1% to 25%, approximately 1% to 30%, approximately 1% to 40%, approximately 1% to 50%, approximately 1% to 60%, approximately 1% to 80%, approximately 5% to 10%, approximately 5% to 15%, approximately 5% to 20%, approximately 5% to 25%, approximately 5% to 30%, approximately 5% to 40%, approximately 5% to 50%, approximately 5% to 60%, approximately 5% to 80%, 10% to 20%, approximately 10% to 25%, approximately 10% to 30%, approximately 10% to 40% , about 10% to 50%, about 10% to 60%, about 10% to 80%, about 15% to 25%, about 15% to 30%, about 15% to 40%, about 15% to 50%, about 15% to 60%, about 15% to 80%, about 20% to 30%, about 20% to 40%, about 20% to 50%, about 20% to 60%, about 20% to 80%, about 30% to 50%, about 30% to 60%, about 30% to 80%, about 40% to 60%, about 40% to 80%, or about 50% to 80% mesenchymal cells. Lung-forming progenitor cells may be co-administered with a combination of AT1, AT2, endothelial cells, and mesenchymal cells.For example, lung-forming progenitor cells are about 1% to 5%, about 1% to 10%, about 1% to 15%, about 1% to 20%, about 1% to 25%, about 1% to 30%, about 1% to 40%, about 1% to 50%, about 1% to 60%, about 1% to 80%, about 5% to 10%, about 5% to 15%, about 5% to 20%, about 5% to 25%, about 5% to 30%, about 5% to 40%, about 5% to 50%, about 5% to 60%, about 5% to 80%, 10% to 20%, about 10% to 25%, about 10% to 30%, about 10% to 40%, about 10% to 50%, about 1% to 60% The present invention may be co-administered with about 0% to 60%, about 10% to 80%, about 15% to 25%, about 15% to 30%, about 15% to 40%, about 15% to 50%, about 15% to 60%, about 15% to 80%, about 20% to 30%, about 20% to 40%, about 20% to 50%, about 20% to 60%, about 20% to 80%, about 30% to 50%, about 30% to 60%, about 30% to 80%, about 40% to 60%, about 40% to 80%, or about 50% to 80% of cells selected from AT1, AT2, endothelial cells, or mesenchymal cells. Alternatively, instead of lung-forming progenitor cells, the methods disclosed herein may include administering cells selected from AT1, AT2, endothelial, or mesenchymal cells.

[0056] In some embodiments, the lung-forming progenitor cells are CD45 + Hematopoietic cells include, for example, lung-forming progenitor cells, about 1% to 5%, about 1% to 10%, about 1% to 15%, about 1% to 20%, about 1% to 25%, about 1% to 30%, about 1% to 40%, about 1% to 50%, about 1% to 60%, about 1% to 80%, about 5% to 10%, about 5% to 15%, about 5% to 20%, about 5% to 25%, about 5% to 30%, about 5% to 40%, about 5% to 50%, about 5% to 60%, about 5% to 80%, 10% to 20%, about 10% to 25%, about 10% to 30%, about 1% to 5 ... 0% to 40%, about 10% to 50%, about 10% to 60%, about 10% to 80%, about 15% to 25%, about 15% to 30%, about 15% to 40%, about 15% to 50%, about 15% to 60%, about 15% to 80%, about 20% to 30%, about 20% to 40%, about 20% to 50%, about 20% to 60%, about 20% to 80%, about 30% to 50%, about 30% to 60%, about 30% to 80%, about 40% to 60%, about 40% to 80%, or about 50% to 80% CD45 + It may contain hematopoietic cells. +Hematopoietic cells represent about 1% to 5%, about 1% to 10%, about 1% to 15%, about 1% to 20%, about 1% to 25%, about 1% to 30%, about 1% to 40%, about 1% to 50%, about 1% to 60%, about 1% to 80%, about 5% to 10%, about 5% to 15%, about 5% to 20%, about 5% to 25%, about 5% to 30%, about 5% to 40%, about 5% to 50%, about 5% to 60%, about 5% to 80%, 10% to 20%, about 10% to 25%, about 10% to 30% of all cells administered to the subject. , about 10% to 40%, about 10% to 50%, about 10% to 60%, about 10% to 80%, about 15% to 25%, about 15% to 30%, about 15% to 40%, about 15% to 50%, about 15% to 60%, about 15% to 80%, about 20% to 30%, about 20% to 40%, about 20% to 50%, about 20% to 60%, about 20% to 80%, about 30% to 50%, about 30% to 60%, about 30% to 80%, about 40 to 60%, about 40% to 80%, or about 50% to 80%.

[0057] In certain embodiments, the lung-forming progenitor cells include AT2 cells. In particular, it has been demonstrated herein that in IPF patients, asymptomatic epithelial damage is associated with AT2 cell depletion and the presence of a subset of dysfunctional AT2 cells, and furthermore, that AT2 cells in IPF exhibit a profibrotic phenotype that likely activates fibroblast and mesenchymal cell proliferation and ECM deposition. Without being bound by theory, it is contemplated herein that these changes are associated with the failure of AT2 cells to differentiate into AT1 alveolar cells and the disruption of the epithelial-mesenchymal interface. Furthermore, AT2 cell invasion may lead to opportunistic proliferation of fibroblasts, further impairing the gas exchange properties of the lung surface.

[0058] In some cases, the efficacy of progenitor cell therapy can be enhanced by targeted depletion or enrichment. For example, in various embodiments, the methods include enriching cells for cells expressing endothelial and / or epithelial markers and / or depleting cells from cells expressing CD45.

[0059] In some embodiments, the method includes enriching cells for cells expressing an epithelial marker. Epithelial cell populations are often depleted and suppressed in fibrotic lungs. In particular, profibrotic conditions can induce epithelial-mesenchymal transition within the epithelial population, adversely affecting epithelial (e.g., alveolar) structure and potentially suppressing epithelial repair. Administration of epithelial progenitor cells can replenish the lung epithelial population, improving lung function and suppressing fibrosis. In some embodiments, the epithelial marker is selected from CD326, CD324, CD245, aquaporin-5, podoplanin, or receptor for advanced glycation end products. In some embodiments, the epithelial marker is CD326.

[0060] In some embodiments, the method includes enriching cells for cells expressing endothelial markers. Administration of endothelial progenitor cells can restore anti-fibrotic homeostasis in fibrotic lungs. Fibrosis, inflammation, and endothelial damage can induce resident pulmonary endothelial cells to produce pro-fibrotic factors and epithelial repair inhibitors, thereby promoting the progression of fibrosis. Restoring healthy endothelial lung tissue can not only reverse fibrosis but also promote lung repair. In certain embodiments, the endothelial marker is CD31 or CD144.

[0061] In some embodiments, the methods include enriching cells for cells that express at least two markers. In some embodiments, the at least two markers are CD31 + , CD144 + , CD324 + , and CD326 + In some embodiments, the method comprises: + CD31 + , CD324 + CD31 + , CD326 + CD144 + , and / or CD324 + CD144 + and further enriching the cells for cells in which:

[0062] Advantageously, the lung tissue may contain a sufficient density of patch-forming cells so as not to require concentration prior to administration. In some aspects, the method includes determining that the cells include cells that are positive for endothelial and epithelial markers. In some aspects, the method includes determining that the cells are positive for CD326. + CD31 + , CD324 + CD31 + , CD326 + CD144 + , and / or CD324 + CD144 + In some embodiments, the method does not include enrichment.

[0063] In some aspects, the method further includes determining expression of an epithelial marker, an endothelial marker, or a combination thereof in the cells. By way of non-limiting example, the epithelial marker can be selected from CD326, CD324, CD245, aquaporin-5, podoplanin, or advanced glycation end products-specific receptor. In some cases, the epithelial marker is CD326. In some cases, the endothelial marker is CD31, CD144, or ERG. In some cases, the endothelial marker is CD31. In some cases, the epithelial marker is CD326 and the endothelial marker is CD31. In some cases, a predetermined percentage of the cells express both the epithelial marker and the endothelial marker. For example, at least about 0.1%, at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% of the cells may express epithelial and endothelial markers. In some cases, the cells are grown for at least about 12 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 72 hours, at least about 120 hours, at least about 180 hours, at least about 240 hours, at least about 300 hours, at least about 600 hours, or at least about 1200 hours prior to determining. In some cases, the cells are expanded for at least about 12 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 72 hours, at least about 120 hours, at least about 180 hours, at least about 240 hours, at least about 300 hours, at least about 600 hours, or at least about 1200 hours after the determination.

[0064] In some embodiments, if the percentage of cells that do not express epithelial and endothelial markers exceeds a predetermined threshold, the cells are determined to be unsuitable for administration to a subject. The predetermined threshold can be at least about 0.1%, at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% of the cells. In such cases, endothelial and epithelial-positive cells can be enriched from the sample, or a new sample can be obtained and analyzed for endothelial and epithelial-positive cell content.

[0065] In certain embodiments, the method further comprises depleting T cells from the cells, depleting B cells from the cells, or a combination thereof. Depletion of T cells can prevent graft-versus-host disease (GVHD) and common GVHD-associated symptoms, including nausea, ulcers, and skin discoloration. Depletion of T cells and B cells can also limit B cell responses that could otherwise induce Epstein-Barr virus-associated proliferative disorders. In such embodiments, the method further comprises depleting CD19 + , CD20 + , CD45 + , CD4 + , CD8 + , CD127 + , PD-1 + , CD122 + , and / or CD132 + In one aspect, the method may comprise depleting CD45 cells. + This involves depleting the cells.

[0066] In one embodiment, enriching or depleting the cells comprises contacting the cells with an agent that binds to an epithelial marker, an endothelial marker, or a T cell marker. In one embodiment, enriching or depleting the cells comprises contacting the cells with an agent that binds to an epithelial marker, an endothelial marker, or CD45. In another embodiment, the binding agent is an antibody. In some embodiments, the antibody is bound to a substrate.

[0067] In some embodiments, the method further comprises expanding the isolated lung cells in culture. The isolated lung cells can be cultured in a suitable medium, antibiotics, growth factors, and nutrients (e.g., amino acids) conducive to cell proliferation. This proliferation can enrich for specific types of cells, such as epithelial cells, endothelial cells, or mesenchymal progenitor cells.

[0068] In various aspects, the subject has idiopathic pulmonary fibrosis or chronic obstructive pulmonary disease. In some aspects, the idiopathic pulmonary fibrosis or chronic obstructive pulmonary disease comprises honeycombing, ground-glass opacity, reticular opacity, predominantly basal reticulation, traction bronchiectasis, or a combination thereof. In some aspects, the idiopathic pulmonary fibrosis or chronic obstructive pulmonary disease comprises aging.

[0069] In certain embodiments, the cells are administered in one or more doses. In some embodiments, a subject receives a single dose of lung-forming progenitor cells. In some embodiments, a subject receives multiple doses of lung-forming progenitor cells. The doses may be the same or different in form, excipient type, and number and type of lung-forming progenitor cells. In some embodiments, the same dose of lung-forming progenitor cells is administered at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten times. In some embodiments, a subject receives a dose of lung-forming progenitor cells until fibrosis is ameliorated or suppressed. For example, a subject may receive a dose of lung-forming progenitor cells at regular intervals until pulmonary fibrosis falls below a certain threshold. In some embodiments, the method includes repeating the administration every 1 to 400 days. For example, a single dose of lung-forming progenitor cells can be administered to a subject on a daily, weekly, biweekly, monthly, bimonthly, semi-annual, or yearly basis.

[0070] In some embodiments, the subject receives about 5×10 2 ~5×10 7 In some embodiments, the subject receives about 5×10 lung-forming progenitor cells. 2 ~5×105 , about 5×10 3 ~5×10 6 , or approximately 5 × 10 4 ~5×10 7 In an additional embodiment, the composition comprising lung-forming progenitor cells comprises 5 million / kg to 500 million / kg of lung cells. In a further embodiment, CD326 + CD31 + Lung progenitor cells are approximately 0.1%-1%, approximately 0.1%-2%, approximately 0.1%-3%, approximately 0.1%-4%, approximately 0.1%-5%, approximately 0.1%-6%, approximately 0.1%-8%, approximately 0.5%-1%, approximately 0.5%-2%, approximately 0.5%-3%, approximately 0.5%-4%, approximately 0.5%-5%, approximately 0.5%-6%, approximately 0.5%-8%, approximately 1%-2%, approximately 1% ~3%, about 1%-4%, about 1%-5%, about 1%-6%, about 1%-8%, about 2%-3%, about 2%-4%, about 2%-5%, about 2%-6%, about 2%-8%, about 3%-4%, about 3%-5%, about 3%-6%, about 3%-8%, about 4%-5%, about 4%-6%, about 4%-8%, about 5%-6%, about 5%-8%, or about 6%-8% CD326 + CD31 + Contains lung progenitor cells.

[0071] In one embodiment, the present invention provides a method for improving lung function in a subject in need thereof, comprising administering a therapeutically effective amount of pneumonogenic progenitor cells to a subject having at least about 15% pulmonary fibrosis, thereby improving lung function in the subject. As demonstrated herein, in addition to repairing fibrosis, pneumonogenic progenitor cell therapy can improve lung function, including forced expiratory volume and pulmonary resistance, promote wound healing, and replace the population of host-derived patch-forming cells. In many aspects, administration is performed in the absence of a preconditioning treatment prior to administration of the cells. Thus, the present method can provide a gentle alternative to cell therapy treatments that require injury-inducing preconditioning regimens. In some aspects, the pneumonogenic progenitor cells increase the number of donor-derived patch-forming cells. In some aspects, the pneumonogenic progenitor cells decrease the number of host-derived patch-forming cells.

[0072] In one embodiment, prior to administration, the level of fibrosis in the subject's lungs is measured by comparing the volume of fiber in the lungs to the total lung volume. In some embodiments, the fiber volume is parenchymal fiber volume (e.g., as described in detail in Ikezoe et al. American Journal of Respiratory and Critical Care Medicine 2021;204(9):1045-59). In some embodiments, the fiber volume and total lung volume are determined by CT scan and / or lung biopsy analysis.

[0073] In another embodiment, improving pulmonary function includes reducing fibrosis and / or preventing or slowing the progression of fibrosis. In some embodiments, improving pulmonary function includes improving Newtonian resistance of the respiratory system, lung tissue compliance, lung tissue elastance, tissue damping, and / or forced expiratory volume. For example, a subject may demonstrate improved performance in a pulmonary mechanics test.

[0074] In certain embodiments, the subject has pulmonary fibrosis or chronic obstructive pulmonary disease. In some embodiments, the idiopathic pulmonary fibrosis or chronic obstructive pulmonary disease comprises honeycombing, ground-glass opacity, reticular opacity, predominantly basal reticulation, traction bronchiectasis, or a combination thereof. In some cases, the subject has at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20% pulmonary fibrosis, at least about 21% pulmonary fibrosis, at least about 22% pulmonary fibrosis, at least about 23% pulmonary fibrosis, at least about 24% pulmonary fibrosis, or at least about 25% pulmonary fibrosis.

[0075] In some embodiments, the cells are administered in a cell suspension. The cell suspension may comprise a consortium of lung cells, including lung-forming progenitor cells. Alternatively, lung-forming progenitor cells may comprise the majority of the cells in the suspension. For example, in some embodiments, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% of the cells in the cell suspension are lung-forming progenitor cells. In some cases, less than about 20%, less than about 15%, less than about 12%, less than about 10%, less than about 8%, less than about 6%, less than about 4%, less than about 2%, or less than about 1% of the cells in the cell suspension are immune cells. The cell suspension may comprise approximately 5×10 2 ~5×10 7 , about 5×10 2 ~5×10 5 , about 5×10 3 ~5×10 6 , or approximately 5 × 10 4 ~5×10 7 cells, or 5 x 10 7 ~1×10 9 Alternatively, or additionally, the cell suspension may contain about 5 x 10 cells. 2 ~5×10 7 , about 5×10 2 ~5×10 5 , about 5×10 3 ~5×10 6 , or approximately 5 × 10 4 ~5×10 7 The cells may contain lung-forming progenitor cells.

[0076] The cell suspension may contain a therapeutically effective amount of lung-forming progenitor cells. As used herein, the term "effective amount" of an active agent refers to an amount of the active agent that is non-toxic to the subject but sufficient to provide the desired effect (e.g., treatment of skeletal muscle disorders, metabolic disorders, blood disorders, or cancer). This amount may vary from subject to subject depending on the species, age, and physical condition of the subject, the severity of the disease being treated, the specific conjugate, or more specifically, the specific active agent used, its mode of administration, etc. Therefore, it is difficult to generalize the exact "effective amount," but a suitable effective amount can be determined by one skilled in the art.

[0077] The terms "therapeutically effective amount," "effective dose," "therapeutically effective dose," "effective amount," and the like refer to the number of lung-forming progenitor cells that elicit the biological or medical response in a tissue, system, animal, or human that is sought by a researcher, veterinarian, physician, or other clinician. Generally, the response is either an improvement in a patient's symptoms or a desired biological outcome (e.g., prevention or amelioration of fibrosis). Such an amount should be sufficient to inhibit or treat fibrosis and can be determined as described herein.

[0078] In one embodiment, the invention provides a method for identifying a subject with pulmonary fibrosis as a candidate for treatment with lung progenitor cells, comprising: a) measuring the level of fibrosis in the subject's lung; and i) classifying a subject having a level of fibrosis of at least about 15% or more as a likely responder to cell transplantation, thereby identifying the subject as suitable for cell transplantation; or ii) classifying a subject having a level of fibrosis of less than about 0-15% as a likely non-responder to lung cell transplantation, thereby identifying the subject as not suitable for cell transplantation, thereby identifying the subject with pulmonary fibrosis as a candidate for treatment with lung progenitor cells.

[0079] In one embodiment, measuring the level of fibrosis is performed by computed tomography (CT) scan and / or by assessing the level of fibrosis in a lung biopsy sample. In some embodiments, the level of fibrosis in the subject's lung is measured by comparing the volume of fiber in the lung with the total lung volume. In some embodiments, the fiber volume is parenchymal fiber volume. In some embodiments, fibrosis is identified as honeycombing, ground-glass opacity, reticular opacity, and combinations thereof.

[0080] In further aspects, the method further comprises administering a cell suspension comprising isolated lung-forming progenitor cells to a subject identified as a likely responder. In some aspects, administering the cell suspension comprises increasing the number of donor-derived patch-forming cells and / or decreasing the number of host-derived patch-forming cells. For example, in some cases, engraftment of donor cells results in a decrease in the number of host-derived patch-forming cells in the subject. In certain aspects, the donor-derived patch-forming cells express both endothelial and epithelial markers. In some aspects, the endothelial marker is CD31, CD144, or ERG. In some aspects, the endothelial marker is CD31. In some aspects, the epithelial marker is CD326. In some aspects, the epithelial marker is selected from CD326, CD324, CD245, aquaporin-5, podoplanin, or receptor for advanced glycation end products. In some aspects, the epithelial marker is CD326.

[0081] In some embodiments, the method further includes measuring a second level of fibrosis in the subject's lungs at a second time point and (i) maintaining the subject as a unlikely responder if the second level of fibrosis is less than about 0-15%, or (ii) reclassifying the subject as a likely responder if the second level of fibrosis is at least about 15% or greater. In some embodiments, a level of fibrosis of at least about 15% or greater indicates a reduction in the number of endogenous pulmonary stem cells sufficient to ensure engraftment of lung-forming progenitor cells. In some embodiments, after measuring the second level of fibrosis in the subject's lungs (e.g., after the subject is reclassified as a likely responder to lung-forming progenitor cell therapy), the subject is administered a cell suspension containing isolated lung-forming progenitor cells. In certain embodiments, a level of fibrosis of less than about 1-15% indicates an insufficient reduction in the number of endogenous pulmonary stem cells to allow engraftment of lung-forming progenitor cells. In some embodiments, the second time point is 10-1000 days after the first instance of measuring the level of fibrosis in the subject's lungs. In some embodiments, the second time point is 50 to 500 days after the first instance in which the level of fibrosis in the subject's lungs is measured.

[0082] Presented below are examples illustrating lung-forming progenitor cell therapies contemplated for the discussed applications. The following examples are provided to further illustrate embodiments of the present invention, but are not intended to limit the scope of the invention. While they are typical of those that might be used, other procedures, methods, or techniques known to those skilled in the art may alternatively be used. [Example]

[0083] Example 1 Materials and Methods mouse Animals were maintained under conditions approved by the Institutional Animal Care and Use Committee at MD Anderson (Protocol No. 1976, 1819). Mouse strains used included: C57BL / 6, C57BL / 6-TRF1 (B6-129P2-Terf1), and C57BL / 6-TRF1. tm2.1Tdl) and B6-SPC-Cre (B6.129S-Sftpc tm1(cre-ERT2)Blh ), and C57BL / 6-Tg(CAG-EGFP)1Osb / J and B6.129(Cg)-Gt(ROSA)26Sor tm4(ACTB-tdTomato,-EGFP)Luo Donor mice for transplantation experiments included 1 / J mice. These latter mice express GFP or TdTomato, respectively, in all lung cells. All mice were used between 6 and 30 weeks of age. Mice were housed in small cages (maximum of 5 animals per cage) and provided with sterilized food and acidified water. Animals of the same age, sex, and genetic background were randomly assigned to treatment groups. Pre-established exclusion criteria were based on IACUC guidelines and included systemic illness, respiratory distress, refusal to eat or drink, and significant (>15%) weight loss. In BLM experiments, only male mice were used as hosts because male mice develop pulmonary fibrosis to a greater extent than female mice. SPC-Cre TRF1 fl / fl Mice of both sexes were used as hosts in experiments using the model, and a minimum of five mice per group were used in all experiments.

[0084] SPC-Cre TRF1 fl / fl Genetic Model C57BL / 6-TRF1 (B6-129P2-Terf1tm2.1Tdl) mice were received from the Jax lab after cryo-recovery and backcrossed to C57BL / 6 mice for four generations within the lab to ensure a clean C57BL / 6 background. Mice homozygous for the Lox insertion were crossed with transgenic mice expressing the Cre-ERT protein under the control of the surfactant promoter (SFTPC)-B6-SPC-Cre (B6.129S-Sftpctm1(cre-ERT2)Blh). First-generation heterozygous B6-TRFWT / Lox SPC-CreERT mice were backcrossed between them until the B6-TRFWT / Lox SPC-CreERT mouse strain was obtained. To activate the Cre protein, mice were intraperitoneally injected with 50 mg of tamoxifen (TMX, Sigma) per kg, three times a week for 6–7 weeks. Before each administration, TMX was freshly dissolved in corn oil to a concentration of 10 μg / μl.

[0085] Induction of fibrosis by bleomycin Nine to ten week old C57BL / 6 male mice were administered 0.035 U / gr bleomycin (Hikma or Teva) by intraperitoneal injection twice weekly for weeks 1 to 5. Control groups received PBS vehicle. Mice were weighed twice weekly, and mice that lost weight were placed on an ultra-high fat diet. Mice were euthanized if they lost more than 20% of their body weight.

[0086] Preconditioning of host C57BL / 6 mice before transplantation Using an assay previously described by the inventors (Milman et al., Stem Cells Transl Med, 2022;11(2):178-188), we examined the level of patch-forming lung progenitor cells after BLM treatment, based on conditioning of recipient mice with CY and 6 Gy TBI. Three days before transplantation, C57BL / 6 host mice were treated with an intraperitoneal injection of 200 mg / kg CY (Sandoz or Baxter) dissolved in PBS. One day before transplantation, mice were treated with 6 Gy TBI using an Xrad-320 biological X-ray irradiator. On day 0, mice were transplanted with various cell populations, as shown in Figure 1.

[0087] Preparation of single cell lung suspension Single-cell suspensions were obtained from enzymatically treated adult and fetal mouse lungs. Briefly, lung tissue was dissociated by mincing with fine scissors in the presence of 1 mg / ml collagenase, 2.4 U / ml dispase, and 1 mg / ml DNAse I (Roche Diagnostics) diluted in Ca+Mg+ phosphate-buffered saline. Cells were dissociated by GentelMax (Miltenyi Biotec) incubation at 370°C for 30 minutes. Nonspecific debris was removed by sequential filtration through 100 μm and 70 μm filters. Cells were then transferred to PBS (Ca, Mg, Fe, Fe-20) with 2% bovine serum albumin (BSA), antibiotics, and 2 mM anticoagulant citrate dextrose solution A. 2+ and Mg2+ Before intravenous injection, donor cells were filtered again through a 40 μm filter. Host mice were injected with 2–8 × 10 cells of either B6-GFP or B6-TdTomato. 6 Adult lung cells were transplanted. The transplanted cells were introduced by intravenous cell injection (IV) into the tail vein.

[0088] Flow cytometry Cell samples were stained with conjugated antibodies or matching isotype controls according to the antibody manufacturer's instructions. Antibodies were purchased from e-Bioscience and Biolegend. Data were acquired on a BD FACSCanto II or BD LSRFortessa flow cytometer and analyzed using FlowJo software (version 9 or version 10).

[0089] immunostaining Mice were sacrificed at different time points after transplantation. Lungs were inflated to maximum volume with 4% paraformaldehyde (PFA) solution introduced through the trachea under a constant pressure of 20 cm H2O. They were then immersed in fixative overnight at 4°C. The following day, the lungs were divided in half, and one half was preserved in 30% sucrose for an additional 24 hours before being snap-frozen in pre-cooled isopentane with liquid nitrogen in the presence of Optimal Cutting Temperature (OCT) compound (Sakura Finetek USA, Inc. Tissue-Tek.; product code no. 4583). The other half was preserved in 70% ethanol before paraffin embedding. Samples were collected for cryosectioning, inflated in a 1:1 mixture of OCT and PBS, and snap-frozen in pre-cooled isopentane with liquid nitrogen. Frozen samples were cut into 6-12 μm sections and stained. All secondary antibodies were purchased from Jackson Laboratories or Abcam. Stained samples were evaluated using an upright Olympus BX51 fluorescence microscope equipped with ×4, ×10, ×20, and ×40 air objectives and a ×100 oil immersion objective and an Olympus digital camera (DP70). Confocal microscopy was performed on an Olympus 3000FV laser scanning confocal microscope using cell sense software (Olympus). Images were processed, rendered, and reconstructed in 3D with Imaris software (Bitplane AG, Switzerland, www.bitplane.com).

[0090] Trichrome staining and Ashcroft test for assessment of fibrosis The PFA-fixed mid-lower right lung was deparaffinized and stained with Masson's trichrome using a Masson's trichrome staining kit (Thomas Scientific) according to the manufacturer's instructions. More than 20 bright-field images were taken per mouse using 20x magnification. Fields were randomly selected by blindly moving the microscope stage control and photographing the resulting field without further stage adjustment. All imaged fields were at least 80% covered by lung tissue. Sections were selected to ensure that all grades of fibrosis were represented. Image files were name-coded and given to three different observers, blinded to the experimental groups. Observers were provided with a copy of the scoring scheme and a set of examples for different grades. Each observer scored all images from 0 to 8 using specific criteria described in Ashcroft et al., J Clin Pathol., 1988;41(4):467-70 and Hubner et al., Biotechniques, 2008;44(4):507-17. All scores were then pooled and averaged for each field and mouse, followed by averages for each treatment group. Results were also divided into three scoring bins: normal lung tissue scored 0-3, mild damage scored 4-5, and severe fibrotic damage scored 6-8. Results were analyzed using PRISM software. Large field images were captured using ImageXpress Micro Confocal.

[0091] CT scan and calculation of fibrosis area In vivo micro-CT scans were performed using a high-resolution Skyscan 1276 (Bruker BioSpin Corporation, MA) with a 30 μm resolution, 135 msec exposure time, and a 0.5 mm filter, using a 1008 × 672 matrix. Mice were anesthetized with 0.5% oxygen and 1.5% isoflurane. 3D images and fibrosis calculations were performed using a 55 / 255 threshold. Lung volume (LV) and fibrous volume (FV) were measured. Fibrosis percentage (FV / LV) was calculated as the ratio of fibrous volume to total lung volume.

[0092] Quantitative assay of total collagen levels To assess total collagen levels, an assay based on a colorimetric readout of free hydroxyproline was used. Briefly, paraffin-embedded lung samples were weighed, hydrolyzed overnight, and then assessed for collagen content by a color reaction using a QuickZyme kit (QuickZyme, Biosciences, Netherlands) according to the manufacturer's instructions.

[0093] Assessment of pulmonary function Respiratory system mechanics and PV relationships were measured using a FlexiVent device (SCIREQ, Montreal, QC, Canada). Briefly, mice were anesthetized with Avertin, tracheotomized using a 19-gauge metal cannula (Brico), connected to the FlexiVent via an endotracheal cannula, and ventilated at a respiratory rate of 150 breaths / min and a tidal volume of 10 ml / kg against a positive end-expiratory pressure of 3 cm H2O. Force measurements and PV loops were performed using the FlexiVent software.

[0094] Linear single-frequency forced oscillation technique (FOT) was used to assess total respiratory system resistance (R), compliance (C), and elastance (E). Broadband FOT was used to determine Newtonian resistance (Rn), tissue elastance (H), and tissue damping (G). Volume-driven PV loops measured the lungs from functional residual capacity (defined as 3 cm HO) to 40 ml kg. -1 The airway opening pressure was recorded after delivery of each volume increment. The area under the PV curve was calculated using flexiVent software, providing data for quantitative analysis of elastic properties. All measurements of respiratory system mechanics were performed in mice with intact chest walls. Upon completion of measurements, anesthetized animals were killed by cervical dislocation. Results were analyzed using PRISM software.

[0095] Hyaluronic acid (HA) and alpha-SMA double staining Deparaffinized lung sections were blocked with normal horse serum (2.5%, Vector Laboratories) and then stained with biotinylated HA-binding protein (HABP) (Vector Laboratories, US). After washing, sections were stained blue using alkaline phosphatase (AP) enzyme substrate (Vector Blue Kit). Alpha-SMA-stained sections were blocked again, washed, incubated overnight with alpha-SMA (Thermo Fisher, US) primary antibody, and treated with the ImmPact detection kit for alkaline phosphatase (Vector Laboratories) according to the manufacturer's instructions to stain alpha-SMA red.

[0096] statistical analysis Using Prism software, differences between groups were assessed using one-way analysis of variance (ANOVA) and Dunnett's post-hoc test to calculate p-values ​​between three or more different groups, or using an unpaired t-test for comparison of only two groups. For each data set, the mean ± SD or mean ± SEM was calculated and presented in the Results section of the text. A p-value ≤ 0.03 was considered statistically significant.

[0097] Example 2 Time frame for successful engraftment of donor-derived epithelial and endothelial cells in the BLM mouse model In mice treated with NA (naphthalene) or CY (cyclophosphamide), engraftment and colonization of lung-forming progenitors were shown to require subsequent treatment with sublethal 6 Gy total body irradiation (TBI) 2 days later. This pretransplantation conditioning effectively eliminated endogenous lung-forming progenitor cells from their respective niches, thereby reducing stem cell competition between host and donor cells for these niches.

[0098] In this study, we evaluated the therapeutic effect of this transplantation modality on pulmonary fibrosis using two different mouse models: the bleomycin (BLM) and TRF1 / SPC models.

[0099] BLM induces a fibrotic response in mice within a short period (1–3 weeks after administration), with some strain variability, with C57BL / 6 and Balb / c mice exhibiting high and low fibrotic damage, respectively. Females also exhibit higher resistance to BLM, which is associated with the production of X-linked antiprotease damaging proteins. Notably, BLM-induced fibrosis has been suggested to be partially reversible; therefore, its use has been primarily limited to short-term experiments evaluating potential therapeutic agents within 4–5 weeks after completion of BLM administration. Bleomycin is generally administered intratracheally (IT) using a single dose, which produces a predominantly bronchocentric distribution of fibrosis. Alternatively, it may be administered intravenously (IV) or intraperitoneally (IP), both of which primarily induce subpleural scarring of lung tissue, as commonly observed in IPF patients. Chronic IP administration of bleomycin results in more persistent and less reversible fibrosis. Therefore, the protocol used relies on low doses of BLM administered over several weeks to induce more stable pulmonary fibrosis, which makes it possible to evaluate pulmonary stem cell transplantation modalities that require 6–8 weeks to obtain significant levels of donor-derived lung cell chimerism.

[0100] In NA- or CY-treated mice, engraftment and colonization of lung progenitor cells requires a subsequent conditioning treatment with a sublethal 6 GY TBI 2 days later. This pretransplant conditioning effectively eliminates endogenous lung progenitor cells from their niches, thereby reducing stem cell competition between host and donor cells for these niches. Therefore, we first investigated the effect of BLM treatment on endogenous patch-forming cells. To this end, we used a transplantation assay in which recipient mice were conditioned with CY and 6 GY TBI. C57BL / 6-Tdtomato + (B6.129(Cg)-Gt(ROSA)26Sor tm4(ACTB-tdTomato,-EGFP)Luo / J) and C57BL / 6-GFP + C57BL / 6-Tg(CAG-EGFP)1Osb / J mice (green fluorescent protein) were treated with BLM for 1, 2, or 4 weeks, after which lung cells were harvested and injected with different doses (2, 4, or 8 × 10 6 TdTomato and GFP donor mice were transplanted into recipient C57BL / 6 mice conditioned with CY and 6 Gy TBI (Figure 1A). Donor-derived patches were determined in recipient mice 64 days after transplantation. Considering that TdTomato and GFP donor mice are congenic with recipient animals and may be subject to some degree of rejection, a 1:1 mixture of cells from both strains was used to confirm robust engraftment after transplantation of lung cells from both types of donors. Furthermore, due to the limited availability of fluorescent donor mice, this approach was useful for obtaining the large number of fluorescent donor cells required for large-scale transplantation experiments.

[0101] As shown in Figures 2B-2C (respectively, representative immunohistology of a donor-derived patch and a 2 mm 2Quantitative analysis of the number of patches per lung tissue (shown in Fig. 1) demonstrated that after 1-2 weeks of BLM treatment, high levels of patch-forming progenitor cells persisted in donor mice, whereas 4 weeks of BLM treatment significantly reduced patch-forming lung cells. These results strongly suggest that BLM treatment alone is associated with the loss of endogenous patch-forming lung progenitor cells and, when used in recipient animals, can create space for the engraftment of donor-derived patch-forming cells.

[0102] Consistent with the effectiveness of BLM in progressively ablating endogenous lung patch-forming cells, we found that transplantation of lung cells from TdTomato-positive donors into BLM-treated mice (Figure 2A) failed to induce significant levels of donor-derived lung patches after 1–3 weeks of conditioning with BLM, but 4–5 weeks of treatment successfully induced chimerism with robust donor-derived lung patches, which was consistent with a 2 mm increase in the lungs of recipient mice. 2 This was demonstrated by quantitative analysis of the number of donor-derived patches per lung area (Figures 2B-2C). In addition, the different levels of chimerism after transplantation after treatment with BLM for different periods are shown by views of large lung areas in two mice from each group (Figure 2D). Therefore, after lung harvesting, the entire lung tissue was imaged by immunofluorescence microscopy before fixation and freezing. Figure 12 shows eight additional examples of such large views demonstrating the variability of chimerism among 20 mice transplanted 4 weeks after BLM treatment. These results strongly support the concept of stem cell competition and suggest that ablation of endogenous lung-forming progenitor cells obtained upon induction of fibrosis after 4-5 weeks of BLM treatment allows effective engraftment and colonization of donor-derived lung patches without the need for additional conditioning with TBI or other toxic agents.

[0103] Next, we used immunohistology to identify the different cell types comprising donor-derived patches after transplantation into mice treated with BLM for 4 weeks. In these experiments, GFP+ donors were used for transplantation. As can be seen in Figures 3A-3D, immunofluorescence analysis of donor-derived patches using double staining revealed that these patches contained HOPX+ AT1 alveolar cells and LAMP3+ AT2 alveolar cells. This is particularly important for preventing the progression of fibrosis in IPF. These patches also contained ERG+ endothelial cells and PDGRa cells. + Mesenchymal cells were also included. Further validation of staining along the Z axis was performed to rule out potential errors in tracking the boundaries of each donor-derived cell line (Figures 13A-13D). Notably, AT1, AT2, and endothelial cells were integrated into the pulmonary alveolar structures in typical donor-derived patches (Figure 17). As noted above, robust AT1 and AT2 lung chimerism may be particularly relevant for IPF therapy.

[0104] Notably, the significant engraftment of donor-derived patch-forming lung progenitor cells obtained at the time of transplantation 1 week after 4 weeks of BLM treatment (Figures 4A-4B) was associated with reduced fibrosis compared to BLM-treated mice that did not receive lung cell transplantation (Figures 4C-4F). Accordingly, Ashcroft's test (Figure 4D) and CT analysis (Figure 4E) measuring three-color staining revealed significantly lower levels of fibrosis in the transplanted group compared to the non-transplanted group (P<0.002 and P<0.001, respectively).

[0105] Furthermore, reduced levels of fibrosis were associated with statistically improved lung function as measured by forced expiratory volume (P<0.002), resistance (P<0.002), tissue damping (P<0.002), and tissue elastance (P<0.03), which are commonly used to assess function in patients with IPF (Figure 4F).

[0106] Example 3 Proof-of-concept for the efficacy of lung cell transplantation in the TRF1 / SPC mouse model The BLM mouse model has been widely used to test new potential drugs for the treatment of pulmonary fibrosis, but more relevant models have recently been described by Povedano J. et al., Cell Rep, 2015;12(2):286-99 and Naikawadi et al., JCI Insight, 2015;1(14):e86704. While the BLM model can be easily used to simulate clinical fibrosis, it is associated with broad toxicity to other tissues and organs, including the hematopoietic system, and is not sufficient to study lung cell damage alone. In contrast, pulmonary fibrosis spontaneously develops after tamoxifen treatment in the SPC-Cre TRF1 mouse model. fl / fl Although the model provides a more clinically relevant model of pulmonary fibrosis and requires extensive breeding, it can provide a more specific and stable simulation of IPF, which is clearly correlated with telomere shortening and senescence of AT2 alveolar cells. Thus, in this model, deletion of Trf1 in SPC+AT2 cells results in the progression of fibrosis over time, allowing us to evaluate the role of pulmonary stem cell transplantation at later time points after transplantation.

[0107] In this model, TRF1 is specifically knocked down in AT2 cells upon treatment with tamoxifen (TMX), thereby inducing senescence in these cells and subsequently leading to the progression of fibrosis. Thus, this model more closely simulates the fibrosis seen in IPF patients, where disease progression has been found to be associated with alterations in telomere length.

[0108] As shown in Figures 5A-5C, SPC-Cre TRF1 fl / fl Similar to the BLM model, induction of chimerism after transplantation in this model also depended on the progression of fibrosis. Senescence in these cells was shown to lead to progressive pulmonary fibrosis (Figures 5E, 5F, and 5H). Thus, this model more closely reproduces the fibrosis seen in IPF patients, whose disease may be associated with defective telomere maintenance.

[0109] As shown in Figures 5B, 5C, and 5G, similar to the BLM model, the degree of chimerism induction in the SPC-Cre TRF1fl / fl model correlated with the progression of fibrosis. Lung cell transplantation 2 or 4 weeks after the completion of the TMX course (a total of 8–10 weeks from the start of the experiment) did not result in significant chimerism, whereas transplantation 6–8 weeks after the completion of TMX treatment (a total of 12–14 weeks) resulted in significant levels of donor-derived patches without the need for additional conditioning.

[0110] Time course of host progenitor cell ablation was measured using a 2 mm 2 This was demonstrated by quantitative analysis of the number of donor-derived patches per lung area (Figures 5E and 5G). Different levels of chimerism after transplantation at different time points after completion of TMX treatment are also shown by low-magnification views of the lungs for each group (Figure 5C). Figure 14 shows eight additional examples of such views, illustrating the variability of chimerism in 16 mice transplanted 7 weeks after completion of TMX treatment.

[0111] To evaluate engrafted donor-derived patches obtained after transplantation 14 weeks after the initiation of TMX treatment, mice were transplanted with lung cells from C57BL / 6-GFP donors. Chimerism was quantitatively assessed 8 weeks after transplantation as described for the BLM model. Figures 6A-6D show that typical donor-derived patches with epithelial AT1 (HOPX+) and AT2 (LAMP3+) cells, endothelial (ERG+) cells, and PDGRa+ mesenchymal cells were clearly observed by immunohistology. Further verification of staining along the Z axis was used to rule out potential errors in tracking the boundaries of each donor-derived cell (Figures 15A-15D). Figure 6E shows a quantitative analysis demonstrating the distribution of these cell types in over 40 patches from three to five chimeric mice.

[0112] Finally, as outlined in Figure 7A, transplanted SPC-Cre TRF1 fl / flMice demonstrated successful engraftment of donor patch-forming cells with significant prevention of fibrosis progression, as demonstrated by Ashcroft tests (Figures 7B-7C), pulmonary function tests (Figure 7D), hyaluronic acid staining (Figures 16A-16B), and collagen levels (Figure 7F).

[0113] Taken together, based on two different models of fibrosis, we conclude that a certain level of fibrosis induction is necessary to eliminate the endogenous stem cell pool and reduce stem cell competition with donor-derived lung stem cells.

[0114] Thus, in both the BLM and TRF1 / SPC models, a specific minimum level of fibrosis was found to be necessary to enable effective engraftment of donor-derived patches that generate lung-forming progenitor cells without the need for additional conditioning. Furthermore, this minimum level was demonstrated in the BLM model to be associated with significant elimination of host patch-forming cells, consistent with our hypothesis that overcoming stem cell competition is a prerequisite for effective engraftment. Effective induction of chimerism was demonstrated in both models to attenuate fibrosis and provide functional benefits. These results provide proof-of-concept for the clinical use of pulmonary stem cell transplantation in patients with pulmonary fibrosis.

[0115] Example 4 Pretreatment enables engraftment of embryonic lung cells In contrast to the pulmonary fibrosis mouse model described above, we found that preconditioning was necessary for the induction of lung chimerism in the naphthalene (NA) lung injury model (Figure 8). Therefore, mice were treated with NA alone or NA plus 6 Gy total body irradiation (TBI) 48 hours after naphthalene treatment. Mice were transfected with E16 GFP mice. + 1 x 10 donor-derived 6 Lung cells were injected and the engraftment and development of donor-derived cells was monitored using immunohistological staining, morphometric analysis, and two-photon microscopy.

[0116] Figures 9A-9C provide representative fluorescence microscopy images of mouse lungs after transplantation into TBI-treated, NA-treated, or NA+TBI-treated mice. Figure 9A shows lungs conditioned with 6 GY TBI alone and GFP-treated mice. + Figure 9B shows fluorescent microscopy images of the lungs of C57BL / 6 adult mice transplanted with embryonic progenitor lung cells. + Figure 9C shows fluorescent microscopy images of the lungs of C57BL / 6 adult mice transplanted with embryonic progenitor lung cells, treated with NA and conditioned with 6 GY TBI 48 hours later, and GFP + Figure 9D shows fluorescence microscopy images of the lungs of C57BL / 6 adult mice transplanted with embryonic progenitor lung cells. 3 GFP of engrafted cells per lung tissue + Quantitative morphometric analysis of the patches is shown. GFP indicates engraftment of donor-derived cells in the recipient lung. + Patches were significantly enhanced after transplantation in mice preconditioned with naphthalene and TBI compared with those conditioned with TBI or naphthalene alone, suggesting that the combined preconditioning regimen enhanced donor cell engraftment.

[0117] Example 5 Adaptation of the BLM mouse model for evaluation of stem cell therapy Pulmonary fibrosis induced in some BLM mouse models has been shown to be reversible. Therefore, it was important to develop a more permanent model to enable evaluation of the efficacy of pulmonary stem cell transplantation, which requires a period of approximately 6–8 weeks after transplantation to halt the deterioration of lung function. Therefore, this study adopted and optimized the protocol described by Headley et al. (Exp Physiol, 2018;103(12):1692-1703). This protocol uses a low dose of BLM, administered systematically over a 4-week period, to induce more stable pulmonary fibrosis and enable evaluation of the benefits of transplanted cells at a relatively late time point after cell transplantation.

[0118] As shown in Figure 10, intraperitoneal administration of BLM over 4 weeks (a total of eight doses at 0.035 U / b each) resulted in significant pulmonary fibrosis, as detected by CT (Figures 10A-10B) and quantitative hydroxyproline assay for collagen levels 8 weeks after completion of BLM treatment (Figure 10C). Furthermore, significant fibrosis was demonstrated by H&E (Figure 10D), tricolor (Figures 10E and 11A-11B), alpha-SMA (Figures 10F and 11C), fibronectin (Figure 10G), collagen IV staining (Figure 10H), and hyaluronic acid (HA, Figure 16A) staining. Notably, analysis of lung function parameters measured by Flexivent at different time points after completion of BLM treatment revealed the greatest loss of function 6–8 weeks after completion of BLM treatment (Figure 10I). These results demonstrate that BLM-induced lung injury is stable over a long period of time, making it possible to evaluate the therapeutic effects of recommended lung transplantation modalities at 2 months post-transplantation when performed 4 weeks after BLM administration.

[0119] Although the invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.

Claims

1. administering a therapeutically effective amount of lung-forming progenitor cells to a subject having at least about 15% pulmonary fibrosis, thereby treating pulmonary fibrosis in said subject.

10. A method of treating pulmonary fibrosis in a subject, comprising:

2. 10. The method of claim 1, wherein prior to administration, the level of fibrosis in the subject's lungs is measured by comparing the volume of fiber in the lungs to the total lung volume.

3. 3. The method of claim 2, wherein measuring comprises subjecting the subject to a computed tomography (CT) scan and / or assessing the level of fibrosis by lung biopsy sample.

4. 4. The method of claim 3, wherein assessing the level of fibrosis in the lung biopsy sample comprises subjecting the lung biopsy sample to an Ashcroft test.

5. 10. The method of claim 1, wherein the treatment is administered in the absence of a preconditioning treatment prior to the administration of the cells.

6. The method of claim 1, further comprising dissociating the lung tissue to obtain lung-forming progenitor cells.

7. 7. The method of claim 6, wherein dissociating the lung tissue comprises subjecting the lung tissue to enzymatic digestion.

8. 7. The method of claim 6, wherein the lung tissue is fetal lung tissue or adult lung tissue.

9. 7. The method of claim 6, wherein the lung tissue is human lung tissue.

10. The method of claim 1 , wherein the cells are autologous cells.

11. The method of claim 1 , wherein the cells are allogeneic cells.

12. The method of claim 1 , wherein the cells are administered in a cell suspension.

13. 7. The method of claim 6, further comprising enriching the cells for cells expressing endothelial and / or epithelial markers, depleting the cells of cells expressing CD45, or a combination thereof.

14. 14. The method of claim 13, wherein the epithelial marker is selected from CD326, CD324, CD245, aquaporin-5, podoplanin, or receptor for advanced glycation end products.

15. The method of claim 14, wherein the epithelial marker is CD326.

16. The method of claim 13, wherein the endothelial marker is CD31, CD144, or ERG.

17. The method of claim 16, wherein the endothelial marker is CD31.

18. The cells were then subjected to ELISA using a method for detecting CD326 ＋ CD31 ＋ , CD324 ＋ CD31 ＋ , CD326 ＋ CD144 ＋ , and / or CD324 ＋ CD144 ＋ 20. The method of claim 17, further comprising enriching for cells that are

19. 14. The method of claim 13, further comprising depleting T cells from said cells, depleting B cells from said cells, or a combination thereof.

20. 14. The method of claim 13, wherein enriching or depleting the cells comprises contacting the cells with an agent that binds to the epithelial marker, the endothelial marker, or CD45.

21. 21. The method of claim 20, wherein the binding agent is an antibody.

22. 14. The method of claim 13, further comprising growing the isolated lung cells in culture.

23. The method of claim 1, further comprising determining the expression of an epithelial marker, an endothelial marker, or a combination thereof in the cells.

24. 24. The method of claim 23, wherein the epithelial marker is selected from CD326, CD324, CD245, aquaporin-5, podoplanin, or receptor for advanced glycation end products.

25. 25. The method of claim 24, wherein the epithelial marker is CD326.

26. 24. The method of claim 23, wherein the endothelial marker is CD31, CD144, or ERG.

27. 27. The method of claim 26, wherein the endothelial marker is CD31.

28. 24. The method of claim 23, wherein a predetermined percentage of the cells express the epithelial marker and the endothelial marker.

29. 29. The method of claim 28, wherein the predetermined percentage is at least about 0.1%, at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% of the cells.

30. 29. The method of claim 28, wherein the epithelial marker is CD326 and the endothelial marker is CD31.

31. 24. The method of claim 23, wherein the cells are grown for at least about 12 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 72 hours, at least about 120 hours, at least about 180 hours, at least about 240 hours, at least about 300 hours, at least about 600 hours, or at least about 1200 hours prior to said determining.

32. 24. The method of claim 23, wherein the cells are determined to be unsuitable for administration to the subject if the proportion of the cells that do not express the epithelial marker and the endothelial marker exceeds a predetermined threshold.

33. 33. The method of claim 32, wherein the predetermined threshold is at least about 0.1%, at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% of the cells.

34. 10. The method of claim 1, wherein the subject has idiopathic pulmonary fibrosis or chronic obstructive pulmonary disease.

35. The method of claim 1 , wherein the cells are administered in one or more doses.

36. administering a therapeutically effective amount of lung-forming progenitor cells to a subject having at least about 15% pulmonary fibrosis, thereby improving lung function in said subject; 20. A method of improving lung function in a subject in need thereof, comprising:

37. 37. The method of claim 36, wherein prior to administration, the level of fibrosis in the subject's lungs is measured by comparing the volume of fiber in the lungs to the total lung volume.

38. 37. The method of claim 36, wherein improving lung function comprises reducing fibrosis and / or preventing or slowing the progression of fibrosis.

39. 37. The method of claim 36, wherein improving pulmonary function comprises improving Newtonian resistance of the respiratory system, lung tissue compliance, lung tissue elastance, tissue damping and / or forced expiratory volume.

40. 37. The method of claim 36, wherein the subject has pulmonary fibrosis or chronic obstructive pulmonary disease.

41. 37. The method of claim 36, wherein the treatment is administered in the absence of a preconditioning treatment prior to the administration of the cells.

42. 37. The method of claim 36, wherein the cells are administered in a cell suspension.

43. 1. A method for identifying a subject with pulmonary fibrosis as a candidate for treatment with lung-forming progenitor cells, comprising: a) measuring the level of fibrosis in the lungs of said subject; and i) classifying subjects having a level of fibrosis of at least about 15% or greater as likely responders to cell transplantation, thereby identifying said subjects as suitable for cell transplantation; or ii) classifying subjects having a level of fibrosis less than about 0-15% as likely non-responders to lung cell transplantation, thereby identifying said subjects as unsuitable for cell transplantation. Including, thereby identifying subjects with pulmonary fibrosis as candidates for treatment with lung-forming progenitor cells; The method.

44. 44. The method of claim 43, wherein measuring the level of fibrosis is performed by computed tomography (CT) scan and / or by assessing the level of fibrosis in a lung biopsy sample.

45. 44. The method of claim 43, further comprising administering to the subject identified as a likely responder a cell suspension comprising isolated lung-forming progenitor cells.

46. 46. ​​The method of claim 45, wherein administering the cell suspension comprises increasing the number of donor-derived patch-forming cells and / or decreasing the number of host-derived patch-forming cells.

47. 44. The method of claim 43, wherein the donor-derived patch-forming cells express both endothelial and epithelial markers.

48. 48. The method of claim 47, wherein the endothelial marker is CD31, CD144, or ERG.

49. 49. The method of claim 48, wherein the endothelial marker is CD31.

50. 48. The method of claim 47, wherein the epithelial marker is CD326, CD324, CD245, aquaporin-5, podoplanin, or receptor for advanced glycation end products.

51. 51. The method of claim 50, wherein the epithelial marker is CD326.

52. measuring a second level of fibrosis in the lung of the subject at a second time point, and (i) if the second level of fibrosis is less than about 0-15%, maintaining the subject as a likely responder, or (ii) if the second level of fibrosis is at least about 15% or greater, reclassifying the subject as a likely responder.

44. The method of claim 43, further comprising:

53. 44. The method of claim 43, wherein a level of fibrosis of at least about 15% or greater indicates a reduction in the number of endogenous pulmonary stem cells sufficient to ensure engraftment of the lung-forming progenitor cells.

54. 44. The method of claim 43, wherein a level of fibrosis of less than about 1-15% indicates that there is not a sufficient reduction in the number of endogenous pulmonary stem cells to allow engraftment of the lung-forming progenitor cells.

55. 10. The method of claim 1, further comprising administering immunosuppressive therapy to the subject.

56. 56. The method of claim 55, wherein the immunosuppressive therapy comprises tacrolimus, everolimus, sirolimus, rapamycin, cyclosporin A, an antilymphocyte globulin antibody, an antithymocyte globulin (ATG) antibody, an anti-CD3 antibody, a steroid, azathioprine, an anti-IL-2Ra receptor antibody, mycophenolic acid, or an anti-CD20 antibody.

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