Mammal pulmonary spheroids and pulmonary spheroid cells and uses thereof

Abstract

The present invention relates to the discovery and use of novel mammalian pulmonary spheroids and pulmonary spheroid cells.

Description

[0001] This application is a divisional application of the patent application filed on September 18, 2015, with a priority date of September 18, 2014, application number 201580050155.9, entitled "Mammalian Lung Glomerulus and Lung Glomerular Cells and Their Applications".

[0002] Cross-references to related applications

[0003] This application claims priority to U.S. Provisional Application No. 62 / 052,220, filed September 18, 2014, Case No. 127 / 90 PROV. The disclosure of that application is incorporated herein by reference in its entirety. 1. Technical Field

[0005] This invention relates to the discovery of novel mammalian pulmonary glomeruli and pulmonary glomerular cells (LSCs) and their uses. 2. Technical Background

[0007] 2.1 Introduction

[0008] Lung diseases, such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF), are devastating conditions and are among the top five causes of death worldwide, according to the World Health Organization (WHO). Stem cell therapy is a promising approach to lung regenerative medicine [2]. Current preclinical and clinical efficacy focuses on the infusion of stromal cells isolated from human bone marrow, adipose tissue, placental tissue, or umbilical cord blood (called mesenchymal stem cells or bone marrow stromal cells) to treat patients with COPD, bronchopulmonary dysplasia, obliterative bronchiolitis, asthma, or acute lung injury [3-5, 6, 7]. Pluripotent cells, such as embryonic stem cells or induced pluripotent stem cells, have enormous differentiation and proliferation potential, but clinical translation is hampered by the potential teratogenic risks in human trials [8-10]. On the other hand, several types of resident lung stem cells have been identified [11-16]. The isolation and expansion of these resident lung stem cells for clinical use remains a challenge compared to mesenchymal stem cells. Antigen sorting and purification are usually required. Furthermore, it has not yet been determined which surface markers can identify the optimal cell source for lung stem cells used in regenerative therapies.

[0009] The patent published by Antwerp et al. (PCT No. WO2012 / 047951) reported the discovery of c-kit-positive lung stem cells and their use in treating lung diseases or pulmonary conditions.

[0010] Other methods for isolating stem cells or other cell clusters, such as cardiovascular cell clusters, have been reported. For example, U.S. Patent No. 8,815,585 (Biardsley et al.) discloses an automated culture system for embryonic stem cells (ESCs) and the use of ESCs in therapy. U.S. Patent Publication No. 2010 / 0061966 (Maben et al.), Smith et al. 2007, Publication 115, pp. 896-908, and Mukhina et al. 2004, Circulation Research 95, pp. 911-921, disclose methods for preparing myocardial spheroid cell clusters and cell cultures derived from myocardial spheroid cell clusters. These myocardial spheroid cell clusters are self-assembled aggregates of cells possessing some characteristics of cardiomyocytes, such as the ability to beat in vitro. They disclose the use of these myocardial spheroid cell clusters for the treatment of damaged and diseased organs such as the heart. Myocardial spheroid cell clusters are prepared by culturing in a medium containing added growth factors, particularly basic fibroblast growth factor (bFGF), myocardial nutrient-1, and epidermal growth factor (EGF). 3. Summary of the Invention

[0012] According to the World Health Organization, lung disease is a devastating condition and is listed as one of the top five causes of death globally. Stem cell therapy is a promising strategy for lung regeneration. Previous animal and clinical studies have focused on using mesenchymal stem cells (derived from other parts of the body) for lung regeneration. This article primarily discloses a rapid and robust method for generating therapeutically resident lung progenitor cells from adult lung tissue. Exogenous cells derived from adult lung tissue self-aggregate in suspension culture to form three-dimensional lung spheroids. Without antigen sorting / purification, the lung spheroids recreate stem cell niches in vitro and contain a useful new mixture of lung stem cells and supporting cells. In vitro, lung spheroid cells can be extensively expanded and show the potential to differentiate into alveolar structures and acquire a mature lung epithelial phenotype. In severely immunodeficient mice with bleomycin-induced pulmonary fibrosis, intravenous injection of human lung spheroid cells inhibited apoptosis, fibrosis, and infiltration, but promoted angiogenesis. In an syngeneic rat model of pulmonary fibrosis, lung spheroid cells (LSCs) were superior to adipose-derived mesenchymal stem cells (MSCs) in reducing fibrosis proliferation and infiltration. Our data suggest that pulmonary glomeruli are a simple method for producing regenerated lung cells needed to treat lung diseases.

[0013] In a specific non-limiting embodiment, the present invention provides a method for preparing therapeutically useful mammalian lung spheroids, comprising (i) culturing human lung tissue explant cells under adherent culture conditions to form a first lung cell growth culture; (ii) culturing the first lung cell growth culture under low adhesion conditions to form a cluster of lung spheroids; and (iii) collecting the therapeutically useful mammalian lung spheroids.

[0014] The present invention also provides a method for preparing therapeutically useful mammalian pulmonary glomerular cells, comprising (i) culturing human lung tissue explant cells under adherent culture conditions to form a first lung cell growth culture; (ii) culturing the first lung cell product culture under low adhesion conditions to form pulmonary glomeruli; and (iii) culturing pulmonary glomeruli under adherent culture conditions to form therapeutically useful mammalian pulmonary glomerular cells.

[0015] The present invention provides treatment methods. For example, it provides a method for treating lung diseases in mammalian patients, comprising providing the patient with an effective therapeutic amount of mammalian pulmonary glomeruli, wherein the pulmonary glomeruli are obtained by (i) culturing mammalian lung tissue explants under adherent culture conditions to form a first lung cell growth culture; and (ii) culturing the first lung cell growth culture under low adhesion conditions to form pulmonary glomeruli.

[0016] A method for treating a lung disease in a mammalian patient includes providing the patient with an effective therapeutic amount of mammalian pulmonary glomeruli cells, wherein the pulmonary glomeruli cells are obtained by: (i) culturing mammalian lung tissue explants under adherent culture conditions to form a first lung cell growth culture; (ii) culturing the first lung cell growth culture under low adhesion conditions to form pulmonary glomeruli; and (iii) culturing pulmonary glomeruli clusters under adherent culture conditions to form therapeutically useful mammalian pulmonary glomeruli cells.

[0017] A pharmaceutical composition comprising mammalian pulmonary glomeruli prepared from mammalian lung tissue, wherein (a) the pulmonary glomeruli have a diameter of about 25 μM to about 500 μM; (b) (i) are negative or weakly positive for c-Kit; (ii) are negative for at least one hematopoietic marker; and (iii) are negative for at least one myocardial glomeruli cluster marker; and (c) are pharmaceutically acceptable carriers.

[0018] A pharmaceutical composition comprising mammalian pulmonary glomerular cells prepared from mammalian lung tissue, wherein (a) is weakly positive or negative for (i) c-Kit; (ii) is negative for at least one hematopoietic marker; and (iii) is negative for at least one myocardial glomerular cell cluster marker; and (b) is a pharmaceutically acceptable carrier.

[0019] In the above methods or compositions, mammalian pulmonary glomeruli or pulmonary glomerular cells may be human pulmonary glomeruli or human pulmonary glomerular cells. The adherent culture conditions in step (i) may be glycoprotein-coated, protein-coated, or proteoglycan-coated surfaces, such as collagen-coated surfaces, fibronectin-coated surfaces, laminin-coated surfaces, or uncoated plastic surfaces. Low-adhesion conditions include bioreactors or surfaces coated with neutrally charged hydrogels or any surface resistant to cell adhesion.

[0020] In one implementation, the mammalian pulmonary glomeruli or pulmonary glomerular cells may be (i) positive for antibodies against CCSP, CD105, CD90 and Pro-SPC; and (ii) negative for antibodies against hematopoietic markers.

[0021] In one embodiment, flow cytometry showed that the LSCs were positive for the following percentage markers: CD105: 50-100%, CD90: 0-100%, Pro-SPC: 5-100%, and CCSP: 5-100%. In another embodiment, the LSCs were: CD105: 50-75%, CD90: 0-50%, Pro-SPC: 5-50%, and CCSP: 5-50%. In yet another embodiment, the LSCs were: CD105: 75-100%, CD90: 50-100%, Pro-SPC: 50-100%, and CCSP: 50-100%.

[0022] In one embodiment, the culture conditions may include a medium consisting of Iskoff modified Dubel medium (IMDM) and fetal bovine serum (FBS).

[0023] The present invention also provides a pharmaceutical composition comprising pulmonary glomeruli or pulmonary glomerular cells obtained according to the method defined in any of paragraphs [0007, 008, 0013-1016] at a concentration of 90-250 × 10⁻⁶. 3 Cells / ml, preferably 100-120 × 10⁶ 3 The composition is formulated for intravenous injection, comprising cells / ml as the active ingredient, and pharmacologically acceptable adjuvants and / or excipients.

[0024] In the treatment of mammalian patients, there can be human patients or veterinary patients. Treatment can be allogeneic, autologous, or xenogeneic.

[0025] Lung disease can be chronic or acute. Non-limiting examples of chronic lung disease include asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis, emphysema, lung cancer, and pulmonary fibrosis. Non-limiting examples of acute lung disease include bacterial pneumonia, bronchiolitis obliterans tissue pneumonia (BOOP), chemical pneumonia, viral pneumonia, or a combination thereof.

[0026] In the above methods, mammalian pulmonary glomeruli or pulmonary glomerular cells can be injected directly into the lungs, nose, peritoneum, or intravenously via a spray. Mammalian pulmonary glomeruli or pulmonary glomerular cells can be delivered together with biological materials or other carriers that can help cells colonize in the lungs.

[0027] The present invention also provides a method for identifying compounds for the prevention or treatment of lung diseases, the method comprising the steps of: (a) contacting the compound with a sample containing pulmonary glomeruli or pulmonary glomerular cells; (b) measuring the levels of genes, proteins, or metabolites associated with lung diseases; and (c) determining the efficacy of the compound on the levels of the genes, proteins, or metabolites; thereby identifying compounds for the prevention or treatment of lung diseases.

[0028] The present invention also relates to the following embodiments:

[0029] 1. A method for preparing therapeutically useful mammalian lung bulbs, comprising the following steps:

[0030] (i) Culture mammalian lung tissue explant cells under adherent culture conditions to form a first lung cell growth culture;

[0031] (ii) The first lung cell outward growth culture was cultured under low adhesion conditions to form lung glomerule clusters;

[0032] (iii) Collect mammalian lung bulbs that are useful for treatment.

[0033] 2. The method according to embodiment 1, wherein the mammalian pulmonary glomerulus is a human pulmonary glomerulus.

[0034] 3. The method according to embodiment 1, characterized in that the adherent culture conditions in step (i) are a surface coated with glycoprotein, protein, or proteoglycan.

[0035] 4. The method according to embodiment 3, wherein the glycoprotein-coated surface is a collagen-coated surface, a fibronectin-coated surface, or a laminin-coated surface.

[0036] 5. The method according to embodiment 1, characterized in that the adherent culture conditions in step (i) are uncoated plastic surfaces.

[0037] 6. The method according to embodiment 1, wherein the low adhesion conditions include a bioreactor or a surface coated with a neutrally charged hydrogel.

[0038] 7. The method according to embodiment 1, characterized in that the mammalian pulmonary glomeruli:

[0039] (i) Positive for antibodies against CCSP, CD105, CD90 and Pro-SPC;

[0040] (ii) Negative for antibodies against CD31, CD34 and CD45.

[0041] 8. The method according to embodiment 1, characterized in that the culture conditions comprise a culture medium composed of Iskov modified Dubel medium (IMDM) and fetal bovine serum (FBS).

[0042] 9. A method for preparing therapeutically useful mammalian lung glomerular cells, comprising the following steps:

[0043] (i) Human lung tissue explant cells were cultured under adherent culture conditions to form a first lung cell growth culture;

[0044] (ii) The first lung cell growth culture was cultured under low adhesion conditions to form lung glomeruli;

[0045] (iii) The lung glomeruli are cultured under adherent culture conditions to form therapeutically useful mammalian lung glomerular cells.

[0046] 10. The method according to embodiment 9, wherein the mammalian pulmonary glomerular cells are human pulmonary glomerular cells.

[0047] 11. The method according to embodiment 9, wherein the adherent culture conditions in step (i) are a glycoprotein-coated, protein-coated, or proteoglycan-coated surface.

[0048] 12. The method according to embodiment 11, wherein the glycoprotein-coated surface is a collagen-coated surface, a fibronectin-coated surface, or a laminin-coated surface.

[0049] 13. The method according to embodiment 9, wherein the adherent culture condition in step (i) is an uncoated plastic surface.

[0050] 14. The method according to embodiment 9, wherein the low adhesion condition comprises a bioreactor or a surface coated with a neutrally charged hydrogel.

[0051] 15. The method according to embodiment 9, characterized in that the mammalian lung glomerular cells:

[0052] (i) Positive for antibodies against CCSP, CD105, CD90 and Pro-SPC;

[0053] (ii) Negative for antibodies against CD31, CD34 and CD45.

[0054] 16. A method for treating a lung disease in a mammalian patient, comprising providing the patient with an effective therapeutic amount of mammalian pulmonary glomeruli, wherein the pulmonary glomeruli are prepared by the following steps:

[0055] (i) Culture mammalian lung tissue explants under adherent culture conditions to form a first lung cell growth culture;

[0056] (ii) The first lung cell growth culture is cultured under low adhesion conditions to form lung glomeruli.

[0057] 17. The method according to embodiment 16, wherein the mammalian pulmonary glomerulus is a human pulmonary glomerulus.

[0058] 18. The method according to embodiment 17, wherein the mammalian patient is a human patient.

[0059] 19. The method according to embodiment 16, wherein the mammalian patient is a veterinary patient.

[0060] 20. The method according to embodiment 16, wherein the mammalian pulmonary glomeruli are used for allogeneic transplantation therapy.

[0061] 21. The method according to embodiment 16, wherein the mammalian pulmonary glomeruli are used for autologous transplantation therapy.

[0062] 22. The method according to embodiment 16, wherein the mammalian pulmonary glomeruli are used for xenotransplantation therapy.

[0063] 23. The method according to embodiment 16, wherein the lung disease is a chronic lung disease.

[0064] 24. The method according to implementation scheme 23, wherein the chronic lung disease is asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis, emphysema, lung cancer, or pulmonary fibrosis.

[0065] 25. The method according to embodiment 16, wherein the lung disease is an acute lung disease.

[0066] 26. The method according to embodiment 25, wherein the acute lung disease is pneumonia.

[0067] 27. The method according to embodiment 26, wherein the pneumonia is a bacterial pneumonia, bronchiolitis obliterans histiopathic pneumonia (BOOP), chemical pneumonia, viral pneumonia, or a mixture thereof.

[0068] 28. The method according to embodiment 16, wherein the mammalian pulmonary bulbs are delivered by spraying, direct injection into the lungs, nose, abdominal cavity, or intravenous injection.

[0069] 29. A method for treating a lung disease in a mammalian patient, comprising providing the patient with a therapeutically effective amount of mammalian pulmonary glomerular cells, wherein the preparation of the pulmonary glomerular cells comprises the following steps:

[0070] (i) Culture mammalian lung tissue explants under adherent culture conditions to form a first lung cell growth culture;

[0071] (ii) The first lung cell growth culture was cultured under low adhesion conditions to form lung glomeruli;

[0072] (iii) Culture lung glomerule clusters under adherent culture conditions to form therapeutically useful mammalian lung glomerule cells.

[0073] 30. The method according to embodiment 29, wherein the mammalian pulmonary glomerular cells are human pulmonary glomerular cells.

[0074] 31. The method according to embodiment 29, wherein the mammalian patient is a human patient.

[0075] 32. The method according to embodiment 29, wherein the mammalian patient is a veterinary patient.

[0076] 33. The method according to embodiment 29, wherein the mammalian pulmonary glomeruli are used for allogeneic transplantation therapy.

[0077] 34. The method according to embodiment 29, wherein the mammalian pulmonary glomerular cells are used for autologous transplantation therapy.

[0078] 35. The method according to embodiment 29, wherein the mammalian pulmonary glomerular cells are used for xenotransplantation therapy.

[0079] 36. The method according to embodiment 29, wherein the lung disease is a chronic lung disease.

[0080] 37. The method according to embodiment 36, wherein the chronic lung disease is asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis, emphysema, lung cancer, or pulmonary fibrosis.

[0081] 38. The method according to embodiment 29, wherein the lung disease is an acute lung disease.

[0082] 39. The method according to embodiment 38, wherein the acute lung disease is pneumonia.

[0083] 40. The method according to embodiment 38, wherein the pneumonia is bacterial pneumonia, bronchiolitis obliterans histiopathic pneumonia (BOOP), chemical pneumonia, viral pneumonia, or a mixture thereof.

[0084] 41. The method according to embodiment 29, wherein the mammalian pulmonary glomerular cells are delivered by spraying, direct injection into the lungs, nose, peritoneum, or intravenous injection.

[0085] 42. A pharmaceutical composition comprising mammalian pulmonary bulbs prepared from mammalian lung tissue, characterized in that:

[0086] (a) The pulmonary glomeruli have a diameter of 25 μM to 500 μM;

[0087] (b)(i) is negative or weakly positive for c-Kit;

[0088] (ii) Negative for at least one hematopoietic marker;

[0089] (iii) Negative for at least one marker of cardiac globus-like cell clusters;

[0090] (c) Pharmaceutically acceptable carriers.

[0091] 43. A pharmaceutical composition comprising mammalian pulmonary glomeruli cells prepared from mammalian lung tissue.

[0092] (a)(i) Negative or weakly positive for c-Kit;

[0093] (ii) Negative for at least one hematopoietic marker;

[0094] (iii) Negative for at least one cardiac globulus cell cluster marker;

[0095] (b) Pharmaceutically acceptable carriers.

[0096] 44. A method for identifying compounds for the prevention or treatment of lung diseases, the method comprising the following steps:

[0097] (a) Contacting the compound with a sample containing lung explant-derived cells, pulmonary glomeruli, or pulmonary glomerular cells;

[0098] b. Measure the levels of genes, proteins, or metabolites associated with lung disease;

[0099] c. Determine the efficacy of the compound at the levels of these relevant genes, proteins, or metabolites; thereby identifying compounds for the prevention or treatment of lung diseases. 4. Description of the attached drawings

[0100] Figures 1A-1D. Origin and characterization of pulmonary glomeruli and pulmonary glomerular cells.

[0101] Figure 1A shows a schematic diagram of a protocol for culturing pulmonary glomeruli and pulmonary glomerular cells.

[0102] Figure 1B. Cumulative doubling of glomerular cells from three different donors.

[0103] Figure 1C. The edges of the lung tissue explants with growing cells in Figure I become closed and ready for harvesting.

[0104] Figure 1C. Figure II shows cells growing in suspension culture to form lung glomeruli.

[0105] Figure 1C. Figure III shows the electroplating of lung glomeruli onto the surface of a fibronectin coating to generate lung glomerular cells (LSCs).

[0106] Figure 1C. Figure IV. Expansion of pulmonary glomeruli cells in suspension culture.

[0107] Figure 1D. Immunocytochemistry of pulmonary glomeruli. Scale bar = 50 μm.

[0108] Figures 2A-2D. Pulmonary glomerular cells containing lung progenitor cells.

[0109] Figures 2A(1)-2A(8). Representative flow cytometry images of pulmonary glomeruli expressing CD31, CD34, CD45, CD90, CD105, CCSP, Pro-SPC, and aquaporin 5. Black line: isotype control. Gray line: antibody.

[0110] Figure 2B. Immunocytochemical staining of pulmonary glomerular cells (LSCs) with the above-mentioned markers.

[0111] Figure 2C. Pooled data on the expression of the above markers (n = 3 lung donors).

[0112] Figure 2D. Double staining of Pro-SPC and CSSP in lung glomerular cells (LSCs).

[0113] Figures 3A-3E. In vitro differentiation and paracrine assays of pulmonary glomerular cells. Figure 3A. LSCs in matrix gel. TM Growth on the surface and displaying alveolar-like structures (inset). Figure 3B. In the matrix gel. TMLSCs growing on the surface express aquaporin 5 (white). Figure 3C. Human lung epithelial cells cultured in control medium and LSC conditioned medium (CM) and stained for viability / death assay. Figure 3D. Human umbilical vein endothelial cell (HUVEC) tube formation assay on the surface of a matrix gel in control medium or conditioned medium from LSCs. Data are presented as mean ± SD. All experiments were repeated three times unless otherwise noted. Scale bar = 50 μm. * indicates p < 0.05 when compared with the "control medium" group. Figure 3E. Representative antibody array image showing the protein present in CMs from LSCs and NHDF cells.

[0114] Appendix Figures 4A-4F The therapeutic benefit of human LSCs in mice with bleomycin-induced pulmonary fibrosis. (See attached image) Figure 4A Figure 4B shows a schematic diagram of the mouse study design. Macroscopic view of the explanted lungs 14 days after LSC or saline treatment. Hematoxylin & eosin staining was performed on the lungs. Figure 4C ) and Masson's tricolor staining ( Figure 4D Figure 4E. Quantitative determination of Ashkov score for fibrosis thickening based on H&E staining images (n = 6–7 animals / group). Figure 4F. Quantitative determination of tissue infiltration based on H&E staining images (n = 6–7 animals / group). Data are expressed as mean ± SD. Scale bar = 100 μm. * indicates p < 0.05 when compared with the “Control” group; # indicates p < 0.05 when compared with the “Bleomycin + Saline” group.

[0115] Figures 5A-5E. Mechanism of the therapeutic effect of LSCs in pulmonary fibrosis. Figure 5A(1). Representative confocal images showing the number of apoptotic cells (white nuclei, arrows) in regions with and without LSC implantation and quantification. Figure 5A(2). (n = 4 animals per group). Figure 5B Representative confocal images showing vWF-positive vascular systems (white) in the lungs quantified with saline or LSCs. (n = 4 animals per group). Appendix Figure 5C Representative confocal images show that grafted EGFP-positive LSCs co-express the mature lung epithelial cell marker aquaporin 5. (See attached image.) Figure 5D qPCR expression levels of fibrosis-related genes in lungs treated with saline or LSCs. Figure 5E To identify the mechanisms and biomarkers of lung repair in pulmonary fibrosis mediated by LSC. Data are presented as mean ± SD. Scale bar = 20 μm. * indicates p < 0.05 using unpaired t-test.

[0116] Appendix Figures 6A-6DThe therapeutic advantages of ADCs for LSCs. (Appendix) Figure 6A A schematic diagram showing the rat study design. (Attached) Figure 6B Hematoxylin and eosin staining of rat lung sections treated with saline, AD-MSCs, or LSCs for 14 days. Figure 6C Quantitative determination of fibrous thickening using Ashkov scores was performed using H&E staining images (n = 5 animals per group). (See attached image.) Figure 6D Quantitative determination of tissue infiltration was performed from H&E stained images (n = 5 animals per group). Data are expressed as mean ± SD. Scale bar = 100 μm. * indicates p < 0.05.

[0117] Figures 7A(1)-7B(4). Differences between human LSCs (Figures 7A(1)-7A(4)) and BM-MSCs (Figures 7B(1)-7B(4)) in surface marker expression. When expressing CD105 and CD90, MSCs do not express proto-SPC and CCSP.

[0118] Appendix Figures 8A-8C Flow cytometry analysis of cell phenotypes in lung glomeruli.

[0119] Appendix Figure 8A . Flow cytometry diagram.

[0120] Appendix Figure 8B Flow cytometry of dissociated cells to different markers.

[0121] Appendix Figure 8C Bar graphs show the relative expression of CD105, CD90, Pro-SPC, and CCSP. Pulmonary glomeruli were dissociated into single cells by incubation in TryPEL Select™ for 30 minutes, and the expression of CD105, CD90, Pro-SPC, and CCSP was then analyzed.

[0122] Appendix Figure 9 Expression of CD105, CD90, Pro-SPC and CCSP in LSCs induced by EGFP viral particles.

[0123] Appendix Figure 10 Expression of aquaporin 5 and Pro-SPC in LSCs cultured on a matrix gel.

[0124] Figure 11. Co-expression of TUNEL with aquaporin 5, vWF, or CD90 in mice treated with DiO-labeled LSCs.

[0125] Appendix Figure 12 LSCs promote angiogenesis in the lungs of mice with PF.

[0126] Appendix Figure 13 Expression of Pro-SPC or aquaporin 5 in LSCs grafted into mouse lungs.

[0127] Appendix Figure 14 Expression of Pro-SPC, CCSP, CD90 and CD105 in rat LSCs.

[0128] Appendix Figure 15 LSCs cultured in medium containing 25 ng / mL EOF and without FBS or in medium containing 20% ​​FBS and without EOF.

[0129] Appendix Figures 16A-16B LSCs were successfully derived from the lungs of mice with PF.

[0130] Appendix Figure 17 Expression of POP-SPC, CCSP, CD90 and CD105 in LSCs from the lungs of mice with PF.

[0131] Appendix Figure 18 LSCs were successfully derived from human IPF lungs.

[0132] 5. Detailed Description of the Invention

[0133] 5.1 Definition

[0134] The lung diseases used in this article may include acute lung injury (ALI), acute respiratory distress syndrome (ARDS), asthma, autoimmune diseases, bacterial pneumonia, bronchiolitis obliterans with organizing pneumonia (BOOP), chemical pneumonia, chronic bronchitis, chronic obstructive pulmonary disease (COPD), cystic fibrosis, emphysema, interstitial lung diseases including sarcoidosis, idiopathic pulmonary fibrosis (IPF), lung cancer, pneumoconiosis, pneumonia, pulmonary edema, pulmonary hypertension, tuberculosis, or viral pneumonia. For a review of BOOP, see Buck et al., New England Journal of Medicine, Vol. 370, pp. 1820-1828, 2014.

[0135] Lung conditions can be acute, such as ALI, ARDS, or pneumonia. Alternatively, lung conditions can be chronic, such as asthma, COPD, cystic fibrosis, or IPF.

[0136] As used herein, a “patient” can be a human or an animal (most likely a mammal). Animals that may benefit from the techniques disclosed herein can be livestock, such as pets (cats or dogs); working or show animals (camels, horses, llamas); or domestic animals (cattle, goats, sheep, pigs).

[0137] Mammalian pulmonary bulbs can have a diameter ranging from about 25 μm to about 500 μm; about 50 μm to about 400 μm; about 75 μm to about 300 μm; and about 100 μm to about 200 μm. They can contain about 20 to about 10 5 ; or 50 to about 10 4 ; or approximately 100 to approximately 10 3 Approximately 150 to 500 single cells. The cell count in mammalian lung glomeruli can be controlled by the plating density on a low-adhesion surface. Glomerular size can vary depending on the therapeutic indication. Smaller glomeruli will be better suited for delivery (e.g., intravenous). Larger glomeruli can be injected directly or placed at a specific site.

[0138] The lung tissue used to prepare the spheroids can be from a lung biopsy or a whole donor lung. It can be a healthy or diseased lung. The lung tissue can be shredded or cut into small pieces before plating.

[0139] In one implementation, the pulmonary glomeruli or pulmonary glomerular cells (i) are positive for antibodies against CCSP, CD105, CD90 and Pro-SPC, (ii) are negative for antibodies against EpCAM, CD49f, p75 NGF, c-Kit, and (iii) are negative for antibodies against CD31, CD34, CD45, and pan-cytokeratin.

[0140] Culture conditions may include reagents, such as any suitable cell culture medium. It can be IMDM or other medium types (e.g., DMEM). The medium may also include FBS, or other alternatives such as bovine serum, sheep serum, human serum, or serum-alternative chemicals. The medium may also include other supplements, growth factors, and chemicals that promote cell growth and differentiation.

[0141] 5.2 Cell-based therapies

[0142] The pulmonary glomeruli or pulmonary glomerular cells described herein can be used for a variety of therapeutic purposes. Other cell-based therapies provide examples of methods for expanding or delivering these lung cells, such as techniques developed for embryonic stem cells (ESCs), pluripotent stem cells (PSCs), or induced pluripotent stem cells (iPSCs). Culture techniques have been described in Celis et al., 2014, Nature Materials, Vol. 13, pp. 570-579, and Vesner et al., Frontiers in Pharmacology, Vol. 5, No. 15, July 2, 2014. The therapeutic techniques and clinical needs for iPSCs have also been reviewed. See Isubo et al., New Biotechnologies, Vol. 31(5), 2014. For the use of the pulmonary glomeruli described herein, similar clinical approaches to those developed for cells derived from cardiac glomeruli can be used. See Gomos et al., 2014, Cardiology, Vol. 100, pp. 1153-1157; Mario Ross et al., 2014, JAMA, Vol. 63, pp. 110-122; and Chage et al., 2012, Circulation, Vol. 126, pp. 54-64. A recent review of clinical techniques for mesenchymal stem cell transplantation for chronic conditions. See Farini et al., 2014, Stem Cell International Code 306573, published April 30, 2014. For allogeneic transplantation of pulmonary glomeruli or pulmonary glomerular cells, techniques such as HLA matching or other methods can be used to monitor or reduce the risk of graft-versus-host disease (GVHD). These approaches can be similar to those developed for hematopoietic transplantation. See, for example, Carnet et al., 2014, World Journal of Stem Cells 6(2): 69-81.

[0143] Cell-based technologies are also described in the patent literature. Examples include, but are not limited to, U.S. Patent No. 7,682,828 (Jenish and Houcheringer), methods for preparing iPSCs in PCT published applications WO2009 / 006930, WO2009 / 006997, and WO2009 / 007852 (Sakurada); U.S. Publication US2009 / 0246875 (Yannake et al.); US2008 / 0233610 (Thomson et al.); and European Patent EP1040185 (Brewster et al.).

[0144] 5.3 Ex vivo gene therapy

[0145] The pulmonary glomeruli or pulmonary glomerular cells described herein can be used for a variety of purposes, including applications such as generating genetically engineered cells for implantation. Here, pulmonary glomeruli / cells can be generated from the patient's own cells (autologous transplantation), cells from different donors (allogeneic transplantation), or cells from different species, xenografts (e.g., transgenic or other modifications) to reduce or eliminate transplant rejection. For example, suitable porcine pulmonary glomeruli or pulmonary glomerular cells can be used for human transplantation. Pulmonary glomeruli / cells can be genetically modified using established ex vivo techniques. Examples of ex vivo techniques include, but are not limited to, U.S. Patent Nos. 8,741,642 (Mangily et al.); 8,703,121 (Harris et al.); 8,420,380 (Fissman et al.); 8,080,417 (Piried and Hassen); 7,063,960 (Cui and Wang); 7,087,431 (Wu et al.); 6,575,898 (Smith); 6,251,383 (Appadhaye and Madan); 5,674,722 (Marygan et al.); 5,665,350 (Quinson Bailey); and 5,437,994 (Emerson et al.).

[0146] 5.4 Diagnosis

[0147] The pulmonary glomeruli or cells described herein can be used for a variety of purposes, including diagnostic and cell-based assay applications. These pulmonary glomeruli or pulmonary glomerular cells can be used, for example, to culture and test respiratory irritants and microorganisms for diagnostic purposes. For example, pulmonary glomeruli or cells can be used in methods for diagnosing or detecting bacterial, fungal, or viral pathogens (e.g., H1N1 influenza) in samples from a subject, comprising: (a) culturing an extract or aliquot of a sample from a subject on an LSC; (b) detecting the pathogen (H1N1 influenza) in the sample from the subject by appropriate assay of biomarkers specific to the pathogen (H1N1 influenza); (c) comparing the detected level with at least one sample from a training set, wherein the training set includes data on levels from reference samples, and the comparison step includes applying a statistical algorithm to correlate the detection level in the sample from the subject with the detection level from at least one training set; and (d) diagnosing or detecting the pathogen (H1N1 influenza) based on the detection level in the sample from the subject and the results of the statistical algorithm. General technicians can choose appropriate assays for biomarkers associated with the pathogen (H1N1 influenza), such as PCR assays using primers / probes specific to the pathogen (H1N1 influenza); nucleic acid hybridization assays, such as microarrays of nucleic acids specific to the pathogen (H1N1 influenza); or antibody assays using antibodies specific to the pathogen (H1N1 influenza).

[0148] 5.5 Compositions and Kits

[0149] This invention provides compositions and kits for preparing pulmonary glomeruli or pulmonary glomerular cells, prepared by the methods described herein. The compositions may be suitable for therapeutic use, such as injectable formulations prepared by methods known to those skilled in the art.

[0150] Drug screening

[0151] Pulmonary spheroids or pulmonary spheroid cells can also be used for drug screening or to measure the “functional effect” of the modulatory phenotypes or genes described herein that regulate lung disease. This can also be a chemical or phenotypic effect, such as altered expression profiles of genes in pulmonary spheroids or pulmonary spheroid cells; altered expression of genes associated with lung disease; altered transcriptional activity of genes associated with lung disease; altered translational activity of mRNA encoding genes associated with lung disease; or altered activity and downstream effects of proteins encoded by these genes. Functional effects can include transcriptional activation or inhibition, the ability of cells to proliferate, expression in cells during lung disease progression, and other cellular characteristics. “Determining a functional effect” means measuring the increase or decrease of gene transcription by compounds or the translation of proteins indirectly or directly associated with lung disease. These functional effects can be measured by any method known to those skilled in the art, such as changes in spectral characteristics (e.g., fluorescence, absorbance, refractive index); hydrodynamics (e.g., shape), chromatography; or solubility; ligand binding assays. The compound has the ability to bind to antibodies; measure transcriptional activation of inducible markers or markers; measure changes in enzyme activity; increase or decrease cell proliferation, apoptosis, cell cycle arrest; and measure changes in cell surface markers. Validation of the functional effects of the compound on the occurrence or progression of lung disease can also be performed using assays known to those skilled in the art, such as studies using mouse models. Functional effects can be evaluated using many methods known to those skilled in the art, such as microscopy for quantitative or qualitative measurement of morphological changes, measurement of changes in RNA or protein levels of other genes associated with lung disease, measurement of RNA stability, identification of downstream or reporter gene expression (CAT, luciferase, β-gal, GFP, etc.), for example, by chemiluminescence, fluorescence, colorimetric reactions, antibody binding, inducible labeling, etc.

[0152] The terms "inhibitor," "activator," and "regulator" for biomarkers refer to molecules identified as activating, inhibiting, or regulating genes or proteins encoded by them that are associated with lung disease, both in vivo and in vitro. Inhibitors, activators, or regulators also include naturally occurring and synthetic ligands, antagonists, agonists, antibodies, peptides, cyclic peptides, nucleic acids, antisense molecules, ribozymes, shRNA, RNAi molecules, small organic molecules, etc. Such assays of inhibitors and activators include, for example, (1) (a) mRNA expression, or (b) protein expression by genes associated with lung disease; (2) the application of a putative regulator compound; and (3) determining the functional effect on the activities described above.

[0153] Statistical methods

[0154] Data can be ranked based on their ability to distinguish biomarkers in 1-to-all (i.e., disease versus normal) and all paired (i.e., normal versus specific disease) cases. One statistic used for ranking is the area under the receiver operator characteristic (ROC) curve (the curve of sensitivity versus (1-specificity)). While biomarkers are evaluated for reliability across datasets, independent sample sets are not combined for the purpose of ROC ranking. As a result, multiple independent analyses are performed and multiple independent rankings are obtained for the ability of each biomarker to distinguish groups of interest.

[0155] It should be understood that other genetic and / or diagnostic criteria may be used in this invention. For example, patient characteristics, standard blood post-processing, results of imaging tests, and / or histological evaluations may optionally be combined with the biomarkers disclosed herein.

[0156] Such analytical methods can be used to form predictive models, which are then used to classify test data. For example, a convenient and particularly effective classification method employs multivariate statistical analysis modeling, first using data (“modeling data”) from samples of known categories (e.g., from known lung cancer subjects with or without a specific category, subclass, or level) to form a model (a “predictive mathematical model”), and then classifying unknown samples (e.g., “test data”) according to lung cancer status.

[0157] Pattern recognition (PR) methods have been widely used to characterize many different types of problems, such as linguistics, fingerprinting, chemistry, and psychology. In the methods described in this paper, pattern recognition uses parametric and nonparametric multivariate statistics to analyze spectral data and thus classify samples and predict values ​​of some dependent variables based on observed ranges. There are two main approaches. One set of methods is called "unsupervised," and these methods simply reduce data complexity in a reasonable way and also produce display graphs that can be understood by the human eye. The other approach is called "supervised," whereby a mathematical model is generated using a training set of samples with known categories or outcomes, and then evaluated using an independent set of validation data.

[0158] Unsupervised pattern recognition (PR) methods are used to analyze data without referencing any other independent knowledge. Examples of unsupervised pattern recognition methods include Principal Component Analysis (PCA), Hierarchical Cluster Analysis (HCA), and Nonlinear Mapping (NLM).

[0159] Alternatively, for the development of automated classification methods, the use of “supervised” methods for data analysis has proven effective. Here, a “training set” of biomarker expression data is used to build a statistical model that correctly predicts the “class” of each sample. This training set is then tested with independent data (called a test or validation set) to determine the stability of the computer-based model. These models are sometimes called “expert systems,” but may be based on a range of different mathematical procedures. Supervised methods can use datasets with reduced dimensionality (e.g., several principal components), but typically use unreduced data with all dimensions. In all cases, the method allows for a quantitative description of the multivariate boundaries characterizing and separating each class, e.g., lung cancer for each class based on its biomarker expression profile. Confidence intervals can also be obtained for any prediction, e.g., the probability level of the goodness of fit (see, for example, Sharaf, Ilmen, Kowalski, et al. (1986). Chemometrics. New York: Wiley). Cross-validation can also be used to check the stability of the predictive model by omitting selected samples from the analysis.

[0160] Examples of supervised pattern recognition methods include the following recent centroid methods (Dambni 2005 Bioinformatics 21(22): 4148-4154 and Tibshlani et al. 2002 Proceedings of the National Academy of Sciences 99(10): 6576-6572). ); Soft independent modeling of class analysis (SIMCA) (see, for example, Ward (1977) Chemometrics: Theory and Applications 52: 243-282.); Partial Least Squares Analysis (PLS) (see, for example, Ward (1966) Multivariate Analysis 1: 391-420; Jojess (1982) Causality, Structure, Prediction 1: 263-270); Linear Discriminant Analysis (LDA) (see, for example, Nielsen (1965) Learning Machines, New York.); K-Nearest Neighbor Analysis (KNN) (see, for example, Brown and Martin 1996 Journal of Chemical Information and Computation 36(3): 572-584); Artificial Neural Networks (ANN) (see, for example, Wasserman (1993) Advanced Methods in Neural Computation. John Willie & Sains Inc.; O'Hare & Jennings (Eds.) (1996). Foundations of Distributed Artificial Intelligence (Vol. 9). Wiley. Probabilistic Neural Networks (PNN) (see, for example, Bisop & Nasrabadi (2006). Pattern Recognition and Machine Learning (Vol. 1, 740 pages). New York: Springer; Spouchett, (1990). Probabilistic Neural Networks, Neural Networks, 3(1), 109-118; Rule-based Induction (RI) (see, for example, Queland (1986) Machine Learning, 1(1), 81-106); and Bayesian methods (see, for example, Brithorst (1990). Introduction to Parameter Estimation Using Bayesian Probability Theory. Maximum Entropy and Bayesian Methods (pp. 53-79). Springer, Netherlands; Brithorst, GL (1988). Bayesian Spectral Analysis and Parameter Estimation (Vol. 48) New York: Springer); Unsupervised Hierarchical Clustering (see, for example, Herrillo 2001 Bioinformatics 17(2) 126-136). In one embodiment, the classifier is based on a centroid-based approach, as taught in Mullins et al. 2007 Clinical Chemistry 53(7): 1273-9, whose teachings on disease classification are incorporated herein by reference in their entirety.

[0161] For example, preprocessing data by addressing missing data, translation, scaling, weighting, etc., is often useful. Multivariate projection methods such as Principal Component Analysis (PCA) and Partial Least Squares Analysis (PLS) are so-called scaling-sensitive methods. By using existing knowledge and experience about the type of data being studied, the quality of the data can be enhanced by scaling and / or weighting before multivariate modeling. Sufficient scaling and / or weighting can reveal important and interesting variations hidden within the data, and thus make subsequent multivariate modeling more effective. Scaling and weighting can be used to place data in the correct measures based on knowledge and experience of the system being studied, and thus reveal patterns that are already inherent in the data.

[0162] If possible, missing data, such as gaps in column values, should be avoided. However, if necessary, such missing data can be replaced or "filled" with, for example, the column's mean ("mean fill"); random values ​​("random fill"); or values ​​based on principal component analysis ("principal component fill"). Each of these different methods will have a different impact on subsequent PR analysis.

[0163] The "translation" of descriptor axes can be useful. Examples of such translation include normalization and mean centering. Normalization can be used to remove variations between samples. Many normalization methods are possible, and they can often be applied at any of several points in the analysis. Mean centering can be used to simplify interpretation. Typically, for each descriptor, the mean of all descriptors in the samples is subtracted. In this way, the descriptor mean is aligned with the origin, and all descriptors are centered at zero. In unit variance scaling, data can be scaled to be of equal variance. Typically, the value of each descriptor is scaled by 1 / standard deviation, where the standard deviation is the standard deviation of all descriptors in the samples. Pareto scaling is, in a sense, between mean centering and unit variance scaling. In Pareto scaling, the value of each descriptor is scaled by l / square root (standard deviation), where the standard deviation is the standard deviation of all descriptors in the samples. In this way, each descriptor has a variance numerically equal to its initial standard deviation. Pareto scaling can be performed, for example, on raw data or mean-centered data.

[0164] Logarithmic scaling can be used to aid interpretation when data has a positive bias and / or when data spans a large range (e.g., several orders of magnitude). Typically, for each descriptor, the value is replaced by the logarithm of that value. In isorange scaling, each descriptor is divided by the range of descriptors for all samples. In this way, all descriptors have the same range, i.e., 1. However, this method is sensitive to the presence of outliers. In autoscaling, each data vector is mean-centered and unit-variance scaled. This technique is very useful because each descriptor is then equally weighted, and large and small values ​​are given equal importance. This can be important for analytes that exist at very low but still detectable levels.

[0165] Several supervised methods for scaling data are also known. Some of these can provide a measure of the ability of a parameter (e.g., a descriptor) to distinguish between classes and can be used to improve classification by stretching the separation. For example, in "variance-weighted" scaling, the variance weight of a single parameter (e.g., a descriptor) is calculated as the ratio of the sum of the between-class variance to the within-class variance. A large value means that the variable distinguishes between classes. For example, if it is known that samples fall into two classes (e.g., the training set), the mean and variance of each descriptor can be examined. If the descriptors have very different means and small variances, it will be beneficial for separating the classes. "Feature-weighted" scaling is a more general description of variance-weighted scaling, where not only the mean and standard deviation of each descriptor are calculated, but other well-known weighting factors, such as Fisher weights, are used.

[0166] The methods described herein and / or the recording of results can be implemented using any device capable of implementing the methods and / or recording results. Examples of devices that can be used include, but are not limited to, electronic computing devices, including all types of computers. When the methods described herein are implemented and / or recorded in a computer, a computer program that can be used to configure the computer to perform the steps of the methods can be contained in any computer-readable medium capable of containing the computer program. Examples of computer-readable media that can be used include, but are not limited to, floppy disks, CD-ROMs, DVDs, ROMs, RAMs, and other memory and computer storage devices. The computer program that can be used to configure the computer to perform the steps of the methods and / or record results can also be made available on an electronic network, for example, via the Internet, an intranet, or other networks.

[0167] The process of comparing measured values ​​and reference values ​​can be performed in any convenient manner suitable for the type of measured value and reference value of the discriminant gene in question. The “measurement” can be performed using quantitative or qualitative measurement techniques, and the pattern of comparing measured values ​​and reference values ​​can vary depending on the measurement technique employed. For example, when using qualitative colorimetric assays to measure expression levels, the intensity of the colored reaction product can be compared visually, or by comparing data from density measurements or spectral measurements of the colored reaction product (e.g., comparing numerical data or graphical data, such as bar graphs derived from the measuring device). However, the measured values ​​intended to be used in the methods of this invention are most typically quantitative values. In other examples, the measured values ​​are qualitative. As with qualitative measurements, comparisons can be made by examining numerical data or by examining the representation of the data (e.g., examining a graphical representation such as a bar graph or line graph).

[0168] The comparison process can be manual (e.g., a visual inspection performed by a practitioner of the method) or it can be automated. For example, the measuring device (e.g., a luminometer for measuring chemiluminescence signals) may include circuitry and software that enables it to compare the measured values ​​with reference values ​​for the biomarker protein. Alternatively, a separate device (e.g., a digital computer) may be used to compare the measured and reference values. Automated devices for comparison may include stored reference values ​​for the measured biomarker protein, or they may compare the measured values ​​with reference values ​​obtained from a simultaneously measured reference sample (e.g., a sample from a control subject).

[0169] It will be apparent to those skilled in the art that when repeated measurements are performed, the measurement compared to a reference value is the value that takes into account the repeated measurements. Repeated measurements can be considered by using the average or median of the measurements as the "measured value".

[0170] The present invention also includes methods for identifying patients for a particular treatment or for selecting patients for whom a particular treatment would be desired or contraindicated.

[0171] The above methods can be performed by a reference laboratory, a hospital pathology laboratory, or a physician. These methods may also include algorithms and / or statistical analysis.

[0172] Pharmaceutically acceptable compositions

[0173] Any of the aforementioned pulmonary glomeruli or pulmonary glomerular cells may be administered as a composition, i.e., together with one or more additional components, such as physiologically acceptable carriers, excipients, or diluents. For example, the composition may contain pulmonary glomeruli or pulmonary glomerular cells as described herein, plus a buffer, an antioxidant such as ascorbic acid, a low molecular weight polypeptide (e.g., a polypeptide having fewer than 10 amino acids), a protein, amino acids, carbohydrates such as glucose, sucrose, or dextrin, a chelating agent such as EDTA, glutathione, and / or other stabilizers, excipients, and / or preservatives. The composition may be formulated as a liquid or a lyophilized product. Other examples of components that may be used in pharmaceutical preparations are given in Remington’s Complete Book of Pharmacy, 16th edition, Mark Publishing Company, Easton District, (1980), the relevant portions of which are incorporated herein by reference.

[0174] The above-described compositions containing pulmonary glomeruli or pulmonary glomerular cells can be administered by any suitable means, including but not limited to parenteral, topical, oral, nasal, vaginal, rectal, or pulmonary (by inhalation) administration. If injected, the composition can be administered by bolus or continuous infusion into an intra-articular, intravenous, intra-arterial, intramuscular, intraperitoneal, or subcutaneous manner. Topical administration refers to administration at the site of disease, as well as percutaneous delivery and sustained release from implants, skin patches, or suppositories. Inhalation delivery includes, for example, nasal or oral inhalation, inhalation using a nebulizer, inhalation in aerosol form, etc. Administration by suppositories inserted into a body cavity can be achieved, for example, by inserting the composition in solid form into a selected body cavity and dissolving it. Other alternatives include eye drops, oral formulations such as pills, lozenges, syrups, and chewing gum, and topical formulations such as lotions, gels, sprays, and ointments. In most cases, pulmonary glomeruli or pulmonary glomerular cells can be administered topically or by injection or inhalation.

[0175] The aforementioned pulmonary glomeruli or pulmonary glomerular cells can be administered at any dose, frequency, and duration that is effective in treating the condition to be treated. The dosage depends on the molecular nature of the therapeutic molecule and the nature of the condition being treated. Treatment can continue as long as necessary to achieve the desired results. The periodicity of treatment may or may not be constant throughout the duration of treatment. For example, treatment may initially occur at weekly intervals and then every other week thereafter. Treatments with durations of days, weeks, months, or years are included in this invention. Treatment can be stopped and then restarted.

[0176] Maintenance doses can be administered after initial treatment. The dose can be expressed as cells per kilogram of body weight (n / kg) or as cells per square meter of skin surface (n / m²). 2 (This can be used as a fixed dose measurement, independent of height or weight. These are standard dosing units in the art.) A person's skin surface area can be calculated using a standard formula based on her height and weight. For example, therapeutic pulmonary glomeruli or pulmonary globules can be measured in units of approximately 1.0 × 10⁻⁶.4 Cells / kg to approximately 1.0 × 10⁻⁶ 10 Cells / kg or approximately 1.0 × 10⁻⁶ 5 Cells / kg to approximately 1.0 × 10⁻⁶ 8 Cells / kg. Alternatively, approximately 1.0 × 10⁻⁶ cells / kg can be applied. 6 One cell to approximately 1.0 × 10⁻⁶ 11 A dose per cell. Or approximately 2.0 × 10⁻⁶ cells. 6 5.0 × 10 cells 6 1.0 × 10⁶ cells 7 2.0 × 10 cells 7 5.0 × 10 cells 7 1.0 × 10⁶ cells 8 5.0 × 10 cells 8 1.0 × 10⁶ cells 9 5.0 × 10 cells 9 1.0 × 10 cells 10 5.0 × 10⁶ cells or 5.0 × 10⁶ cells 10 Each cell.

[0177] 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 pertains. The articles “a” and “an” are used herein to refer to one or more (i.e., at least one) grammatical object of that article. As an example, “an element” refers to one or more elements.

[0178] Throughout this specification, the word "comprising" or variations thereof shall be construed as implying the inclusion of the stated elements, integers, or steps, or groups of elements, integers, or steps, but does not exclude any other elements, integers, or steps, or groups of elements, integers, or steps. The invention may suitably "comprise," "consist of," or "substantially consist of" the steps, elements, and / or reagents described in the claims.

[0179] It should also be noted that the claims can be drafted to exclude any optional elements. Therefore, this specification is intended to serve as a basis for using exclusive terms such as “alone,” “only,” etc., or for using “negative” limitations in relation to the statement of the claim elements.

[0180] Where a range of values ​​is provided, it should be understood that each intermediate value between the upper and lower limits of that range, up to one-tenth of the lower limit unit, is also specifically disclosed, unless the context clearly indicates otherwise. Each smaller range between any stated value or intermediate value in the range and any other statement or intermediate value in the range is included within the invention. The upper and lower limits of these smaller ranges may independently include or exclude the range, and each range in which any one, two, or both limitations are included is also included within the invention, subject to [the invention's scope / restriction]. When the range includes one or two limitations, ranges excluding one or both of those included limitations are also included in the invention.

[0181] The following embodiments further illustrate the invention and are not intended to limit the scope of the invention. In particular, it should be understood that the invention is not limited to the specific embodiments described, as these can certainly be modified. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting, as the scope of the invention will be limited only by the appended claims.

[0182] 6. Examples

[0183] We sought to develop an improved method for generating clinically applicable amounts of resident lung progenitor cells. Multicellular spheroids represent a three-dimensional cell culture approach commonly used in cancer biology [17,18]. Spheroid approaches have also been used to generate neural stem cells

[19] and cardiac stem cells

[20] . Spheroidal environments have also been reported to mimic in vivo stem cell niches and to reprogram somatic cells into neural progenitor-like cells [21,22]. We hypothesize that lung spheroids can be generated from healthy lung tissue, and that they may contain lung progenitor cells suitable for therapeutic applications. Exogenous cells from adult lung tissue self-aggregate into three-dimensional multicellular lung spheroids. These spheroids resemble stem cell niches. When reseeded onto a fibronectin-coated surface, the lung spheroids dissociate into single cells, which we call lung spheroid cells (LSCs). LSCs and lung spheroids represent a selective mixture of two lung progenitor cells and supporting stromal cells. Intravenous infusion of human LSCs improved bleomycin-induced pulmonary fibrosis in immunodeficient mice. In a rat model of pulmonary fibrosis, head-to-head comparisons showed the therapeutic advantage of LSCs over adipose-derived mesenchymal stem cells with the same genetic background.

[0184] method

[0185] Production of pulmonary bulbs and pulmonary bulb cells

[0186] Rat pulmonary glomeruli and pulmonary glomerular cells

[0187] We derived pulmonary glomeruli and pulmonary glomerular cells from rat lungs, as described in the procedure below. Lungs from Wistar-Kyoto rats were removed and cut into small fragments. Cells grown from the lung explants were collected and forced to form pulmonary glomeruli in suspension culture. The pulmonary glomeruli were then replate onto an adhesive culture to produce pulmonary glomerular cells. The morphology of the rat pulmonary glomeruli and pulmonary glomerular cells is similar to that of their human counterparts.

[0188] Lung tissue from healthy individuals was obtained from the Cystic Fibrosis and Lung Disease Research and Treatment Center at the University of North Carolina at Chapel Hill. Donor comorbidities are shown in Table 1.

[0189] Table 1.

[0190] Distal lung tissue, approximately 6 mm × 6 mm in size, was isolated and washed with phosphate-buffered saline (PBS) (BioTech). The tissue samples were then cut into smaller biopsy-sized fragments, washed three times with PBS, and subsequently enzymatically digested for 5 minutes at 37°C in collagenase IV solution (5 mg / ml) (Sigma-Aldrich). Iskov-modified Dubel medium (IMDM; BioTech) containing 20% ​​fetal bovine serum (FBS; Corning) was then added to the samples to inactivate the collagenase. Subsequently, the tissue samples were further cut into smaller tissue explants (~0.5 × 0.5 mm) before plating. Approximately 50 tissue explants were then placed on fibronectin-coated plates, approximately 1.5 cm apart, and covered overnight with 2 mL of IMDM containing 20% ​​FBS to aid attachment to the plate. The culture was maintained in IMDM with 20% FBS, with the medium changed every other day. Cells began to grow from the tissue explants within approximately one week. Once these outward-growing cells have merged to approximately 70-80%, they are then processed with TryPLE Select. TM(Biotech) Harvest the cells after incubation for 5–10 minutes. Then, seed the cells at a density of 100,000 cells / cm² in Ultra-Low attachment flasks (Corning) and culture in IMDM with 10% FBS to form spheroids. Bright lung spheroids (LSs) begin to form in 3–7 days. LSs are then collected from the suspension culture flasks and re-coated onto fibronectin-coated surfaces to generate adherent lung spheroid cells (LSCs). LSCs are cultured in IMDM containing 20% ​​FBS, 50 μg / mL gentamicin, 2 mmol / L L-glutamine (Biotech), and 0.1 mmol / L 2-mercaptoethanol (Biotech). LSC cohorts are cultured in FBS-free medium containing 25 ng / mL epidermal growth factor (EGF; from Shenandoah Biotechnology, PA) to test the effect of EGF on cell growth. Cells are passaged every 3–5 days. We used channel 2-3 LSCs for all in vitro and in vivo tests. Human alveolar epithelial cells (HPAEpiC; from the ScienCell Research Laboratory) and normal human dermal fibroblasts (NHDF; from ATCC) were cultured into human LSCs in the same medium as control cells. Rat LSCs were generated from 6-week-old syngeneic Wistar-Kyoto rats using a protocol similar to that used for human LSCs. Rat adipose-derived mesenchymal stem cells (AD-MSCs) were derived from the same rat strain as described above. Li et al., 2012 American College of Cardiology Journal, 59942-953. Mouse PF-LSCs were generated from 6-week-old CD1 mice (Charles River Laboratory) 14 days after lymphococcal instillation. Lung biopsy tissue from IPF patients was obtained from the IPF Clinic at the University of North Carolina at Chapel Hill.

[0191] Flow cytometry analysis

[0192] To characterize the antigenic phenotype of LSCs, flow cytometry was performed using a FACSCalibur or LSR II flow cytometer (BD) and analyzed using FlowJo software (TreeStar). Cells were incubated for 60 minutes with antibodies against CD31, CD34, CD45, CD49f, CD90, CD105, c-Kit, EpCAM, p75NGF, CCSP, Pro-SPC, pan cytokeratin, and aquaporin 5. Isotyped antibodies were used as negative controls. Human bone marrow mesenchymal stem cells (BM-MSCs) obtained from Lonza were used as control cells for flow cytometry and cultured in the same LSC medium. To reveal the change in cell phenotype from spherical to adherent cells, analysis was performed using TryPEL Select. TM(Life Sciences, CA) Incubate for 10-15 minutes to dissociate lung glomeruli into single cells, and then perform flow cytometry analysis on the dissociated cells.

[0193] Immunocytochemistry of pulmonary glomeruli and LSCs

[0194] LSCs were plated on fibronectin-coated slides (BD Biosciences) and then fixed with 4% paraformaldehyde (PFA) prior to immunocytochemistry (ICC) for the aforementioned antigens. Lung glomeruli were mounted in OCT (Tissue-Tek) chambers and frozen sections (5 μm) were prepared for immunostaining. Images were captured using an epi fluorescence microscope (Olympus 1X81). LSCs or lung glomeruli sections were stained with antibodies against CD31, CD34, CD45, CD49f, CD90, CD105, c-Kit, EpCAM, p75NGF, CCSP, Pro-SPC, pan cytokeratin, KRT5, p63, and aquaporin 5, and detected using FITC- or Texas Red-conjugated secondary antibodies.

[0195] In vitro alveolar-like structure formation, differentiation, and paracrine assays

[0196] LSCs are laid on the matrix adhesive TM (BD Biosciences) This technique was used to observe the formation of alveolar-like structures in vitro. LSCs were transduced with viral particles containing enhanced green fluorescent protein (EGFP, a vector endonuclease). Additionally, matrix gel... TM LSCs were fixed with 4% PFA and subsequently immunostained with EGFP and aquaporin 5. The nuclei were reverse-stained with DAPI. To reveal the effect of LSC-secreted factors on lung epithelial cell survival, HPAEpiCs were cultured in either control medium (pure IMDM) or LSC-conditioned medium (LSC-CM). After 3 days, live and dead HPAEpiCs were stained with calcein-AM and ethidium homodimer-1 (EthD) (live / dead assay kit, LifeTech). The pro-angiogenic effect of LSC-conditioned medium was investigated by endothelial cell tube formation assay. Human umbilical vein endothelial cells (HUVECs, from ATCC) were seeded at a density of 2 × 10⁴ cells / well in 96-well plates with reduced growth factor matrix gel. TM Add 100 μL of pure IMDM or conditioned medium from human LSCs to each well. After 4 hours, image the wells using a Nikon TE-200 white light microscope. Then measure the average tube length using NIH analysis software. Cytokines and growth factors secreted by LSCs were determined using a protein dot array (Rebio Biotechnology Co., Ltd.). Conditioned medium from normal human skin fibroblasts (NHDF) was used as a control for the protein array.

[0197] Animal program

[0198] All animal work was conducted in accordance with animal health regulations and using the North Carolina State University committee. Six- to eight-week-old female severely combined immunodeficiency (SCID) mice (Charles River Laboratory) were randomly assigned to three treatment groups (n = 6–7 mice per group): 1) Sham-operated control: 50 μl PBS was injected intratracheally into the lungs; 2) Bleomycin + saline: 0.7 U / kg body weight of bleomycin was added to 50 μl PBS (EMD Biosciences) and instilled intratracheally into the lungs, followed by an intravenous injection of 200 μl PBS 24 hours later; 3) Bleomycin + LSC: 0.7 U / kg body weight of bleomycin was added to 50 μl PBS (EMD Biosciences) and instilled intratracheally into the lungs, followed by an intravenous injection of 1 × 10⁶ human LSCs in 200 μl PBS 24 hours later. Animal subpopulations in the bleomycin + LSC group received LSCs transduced with viral particles labeled with either a green fluorescent cell tracker DiO (LifeTech) or EGFP (vector endonuclease), which facilitated histological examination of infused cells in mouse lungs. On day 14, all mice were sacrificed and their lungs were harvested for histological analysis, including hematoxylin and eosin (H&E) staining for alveolar thickening and infiltration, Masson's trichrome staining for fibrosis, terminal deoxynucleotidyl transferase dUTP nick end marker (TUNEL) staining for apoptosis, and other immunohistochemical (IHC) staining for angiogenesis and LSC differentiation. To perform head-to-head comparisons of LSCs and another stem cell type in lung regeneration, 5 × 10⁵ cells were used. 6 Lung fibrosis was induced in 6-week-old female Wistar-Kyoto rats (Charles River Laboratory) by injection of syngeneic rat LSCs or AD-MSCs with bleomycin. Animals were euthanized 14 days later. The same H&E staining was performed to measure the extent of lung damage.

[0199] Histology

[0200] All animals were sacrificed 14 days after treatment. Mouse lungs were harvested and frozen in OCT compounds. Frozen sections (5 μm thick) were prepared. For H&E staining, frozen lung sections were stained in hematoxylin for 2 minutes, eosin for 30 seconds, and then subjected to Masson's trichrome staining according to the manufacturer's instructions (HT15 Trichrome Staining (Mason) Kit; Sigma-Aldrich). For immunofluorescence staining, frozen lung sections were fixed with 4% PFA, blocked / permeabilized with a protein blocking solution containing 1% saponin (DAKO, Carpintria, CA), and then stained with the following antibodies: rabbit anti-von Willebrand factor (Ebin), rabbit anti-aquaporin 5 (Ebin), and chicken anti-GFP (Ebin). FITC or Texas-Red secondary antibodies were also obtained from Ebin. Images were taken using a Zeiss LSM 710 laser scanning confocal microscope system. Apoptotic cells were detected using an in situ cell death assay kit (Roche Diagnostics, Mannheim, Germany) by nick-end labeling of terminal deoxynucleotidyl transferase dUTP (TUNEL) according to the manufacturer’s instructions.

[0201] PCR array

[0202] Using RT 2 Using the Analyzer™ PCR array system (Kiagen), we compared the expression of stem cell-related genes in human LSCs and HPAEpiCs. In short, total RNA was extracted from the lungs of explants and analyzed using RT-PCR. 2 cDNA was prepared from a mixture of total RNA from three independent lungs using a one-chain kit (Qiage). Experimental mixtures were prepared by adding cDNA to an RT2qPCR reaction mixture (Qiage) within a 96-well PCR array. Quantitative real-time PCR was performed using a Roche photocyclic real-time PCR system. A similar fibrosis-related gene PCR array was used to compare the expression of key genes involved in dysregulated tissue remodeling during repair and fibrosis in “bleomycin + saline” and “bleomycin + LSC” lungs.

[0203] Statistical analysis

[0204] Unless otherwise stated, results are expressed as mean ± SD. Comparisons between any two groups were performed using a two-tailed unpaired t-test for significance. Comparisons of more than two groups were performed using one-way ANOVA followed by post-hoc variance correction. A difference was considered statistically significant when p < 0.05.

[0205] result

[0206] Production of pulmonary glomeruli and LSCs

[0207] A three-stage "adhesion-suspension-adhesion" culture method was used. Figure 1AWe derived pulmonary bulbs and LSCs from healthy adult lung tissue. One week after coating onto a fibronectin-coated surface, both stromal-like cells and stromal-like cells began to grow from the lung tissue explants. Those growing cells fused together within 2–3 weeks. Figure 1C Figure I). When seeded on an ultra-low adhesion surface (to prevent cell adhesion), the product cells spontaneously aggregate into three-dimensional lung spheres (Figure I). Figure 1B -ii). When reseeded onto a fibronectin-coated surface, the pulmonary glomeruli dissociate into single cells, which we call pulmonary glomerular cells (LSCs); Figure 1C Figure II). A single biopsy-sized lung tissue sample can yield up to 50-200 million, more typically 10-20 million channels of 0 LSC. When maintained and passaged in an IMDM with 20% FBS ( Figure 1C (Figure IV) LSCs can undergo 5-15 further doublings within 15-50 days ( Figure 1B Such cell yield and growth potential should meet the needs of clinical cell manufacturing.

[0208] Cell phenotypes in pulmonary glomeruli and LSCs

[0209] Immunocytochemistry reveals the histological structure of each lung glomerulus ( Figure 1D Lung stem cells / progenitor cells (e.g., Pro-SPC, p63-positive cells, KRT5-positive cells, and / or CCSP-positive cells) cluster in the center of a sphere surrounded by CD105- or CD90-positive supporting (stromal-like) cells. These architectural features of the lung spheres are similar to stem cell niches previously reported in spheres formed from cardiac stem cells

[23] . The lung spheres do not contain cells expressing hematopoietic markers such as CD45, CD31, and CD34, nor do they contain cells expressing mature lung epithelial markers pan cytokeratin or aquaporins. The lung spheres also contain a small fraction of cells expressing p75NGF, EpCAM, and CD49f, but not c-kit. As derivatives of the lung spheres, LSCs show a similar phenotypic profile. Flow cytometry analysis (Figures 2A and 2B) showed that LSCs were highly positive for CD105, CD90, Pro-SPC, and CCSP, and weekly positive for p75NGF, EpCAM, and CD49f. Double staining confirmed that the LSC subsets were positive for both Pro-SPC and CCSP. Figure 2CLSCs were negative for hematopoietic cell markers CD45, CD31, and CD34, or mature lung epithelial cell markers pan cytokeratin or aquaporin. These compound data suggest that LSCs represent a selective mixture of lung progenitor cells and supporting cells. The phenotype of LSCs differs from that of BM-MSCs because the latter do not express Pro-SPC or CCSP (Figs. 7A(1)–7B(4)). To reveal the change in cell phenotype from spheroids to adherent cells, we dissociated lung spheroids into single cells and then performed flow cytometry analysis. Except for a slightly higher expression of Pro-SPC in the spheroid stage ( Figures 8B-8C The results indicated similar phenotypes. Immunocytochemistry of LSCs confirmed the flow cytometry results. Figure 2B To enable histological analysis, an LSC cohort was transduced with EGFP viral particles. We confirmed that EGFP transduction did not affect the phenotype of the LSCs. Figure 9 Using a strictly correlated gene PCR array, we compared gene expression in human LSCs and (HPAEpiC) (data not shown). Compared to HPAEpiC, a large number of genes were upregulated, such as bone morphogenetic protein 2 (BMP2), stromal cell-derived factor 1 (SDF-1; also known as CXC motif chemokine 12 [CXCL12]), and fibroblast growth factor 2 (FGF2).

[0210] LSC's ability to form alveolar-like structures and promote angiogenesis

[0211] When in the matrix gel TM During culture, LSCs self-assemble into alveolar-like structures. Figure 3A Immunostaining showed that LSCs expressed aquaporin 5 (red / green overlapping with open arrows). Figure 3B ), and obtained the morphology of mature lung epithelial cells. Undifferentiated LSCs are shown in green with solid arrows ( Figure 3B These undifferentiated cells remained positive for Pro-SPC (white arrows). Figure 10 Conditioned medium derived from LSCs promotes the survival / proliferation of human lung epithelial cells (Fig. 3C) and human endothelial cells in the matrix gel. TM Tube formation on LSCs (Fig. 3D; LSC-conditioned medium vs. control medium: 237.8±32.7 μm vs. 108.7±81.1 μm; p < 0.05) indicates the pro-survival and pro-angiogenic effects of LSC-secreted factors. Cytokine array ( Figure 3E The results showed that, compared with control cells (NHDFs), human LSCs secreted higher concentrations of pro-angiogenic factors, such as insulin-like growth factor binding protein 2 (IGFBP2), hepatocyte growth factor (HGF), and brain-derived neurotrophic factor (BDNF).

[0212] Regenerative potential of LSCs in mice with pulmonary fibrosis

[0213] Overview of animal research design, such as Figure 4A Pulmonary fibrosis was induced in severely combined immunodeficient (SCID) mice with intratracheal bleomycin infusion. Twenty-four hours later, animals received intravenous infusion of 1 × 10⁶ individual LSCs or saline as a control. Animals were followed for 14 days and then sacrificed for endpoint analysis. Macroscopic views of the explant lungs revealed significant tissue damage (dense black spots) in the actinomycin-treated lungs. Figure 4B Lungs treated with LSC showed similarities to those in the control group. H&E staining ( Figure 4C The results showed that LSC treatment significantly reduced fiber thickening (via Ashkov score). Figure 4E The infiltration rate of "bleomycin + saline" versus "bleomycin + LSC" was 5.7±1.0 versus 4.3±0.7. Figure 4F The difference between "bleomycin + saline" and "bleomycin + LSC" was 11.6 ± 5.7 versus 4.3 ± 4.8. Masson's trichrome staining confirmed the reduction in fibrosis induced by LSC treatment (blue). Figure 4D ).

[0214] Mechanisms of LSC-mediated lung protection and regeneration

[0215] LSC transplantation reduced tissue apoptosis in bleomycin-treated lung. LSC transplantation (DiO-labeled; Fig. 5A, green) reduced the number of terminally labeled positive apoptotic cells (Fig. 5A, red nuclei) in bleomycin-treated lung (region w / o LSC vs. region w / LSC: 1.9 ± 0.5% vs. 0.7 ± 0.2% of total nuclei). This protection was observed in epithelial, stromal, and endothelial cell types in the lung. Figure 11 Conversely, LSC treatment increased angiogenesis in bleomycin-treated lungs: more vWF-positive vascular systems were detected in LSC-treated lungs. Figure 5B "Bleomycin + saline" vs. "Bleomycin + LSC": 6.0 ± 2.3 vs. 11.8 ± 3.3 / HPF). Furthermore, a greater number of vessels formed around LSCs compared to other areas where LSCs were absent. Figure 12 These results are consistent with the pro-survival and pro-angiogenic effects of LSCs observed in vitro (Figs. 3C(1) and 3C(2)). Although some injected human LSCs acquired a mature lung epithelial cell phenotype: EGFP-positive LSCs co-expressed the lung epithelial cell marker aquaporin 5 (AQ5). Figure 5C White arrow; Figure 13 (Illustration b) Some LSCs retain Pro-SPC ( Figure 13(Illustration b). To reveal the overall effect of LSC treatment on pulmonary fibrosis, we extracted RNA from the lungs of the “bleomycin + LSC” and “bleomycin + saline” groups. Quantitative PCR arrays showed that LSC treatment attenuated the expression of fibrosis genes in bleomycin-treated lungs ( Figure 5D ).

[0216] Rat pulmonary glomeruli and pulmonary glomerular cells

[0217] Using a similar technique, we derived pulmonary glomeruli and pulmonary glomerular cells from rat lungs. Lungs from Wistar-Kyoto rats were removed and cut into small fragments. Cells growing from the lung explants were collected and forced to form pulmonary glomeruli in suspension culture. The pulmonary glomeruli were then replate onto an adhesive culture to produce pulmonary glomerular cells. The morphology of the pulmonary glomeruli and pulmonary glomerular cells is similar to their human counterparts.

[0218] Therapeutic advantages of LSCs compared to AD-MSCs

[0219] Because most current clinical trials use MSCs to treat IPF and COPD, we sought to compare the therapeutic efficacy of LSCs and AD-MSCs. To exclude donor variability, we derived rat LSCs and AD-MSCs from the syngeneic Wistar-Kyoto (WKY) rat strain, as these rats share the same genetic background. Rat LSCs share a similar antigenic phenotype with their human counterparts, exhibiting consistent expression of CD105, CD90, Pro-SPC, and CCSP. Figure 14 ). Twenty-four hours after bleomycin infusion, WKY rats were randomized to receive saline, rat LSCs, or rat AD-MSCs ( Figure 6A Consistent with previous reports, H&E staining showed improvement compared to the saline control ( Figure 6B ; Figure 6C Compared to D (white bar), AD-MSC treatment reduced infiltration 14 days after cell therapy ( Figure 6B ; Figure 6C And D, black bar). A trend was also observed in the reduction of fibrotic thickening (via Ashkov score). However, the highest therapeutic effect was observed in rats receiving LSCs, which expressed the least degree of fibrotic thickening and tissue infiltration (and D, black bar). Figure 6B ; Figure 6C (and D, red bar). These data indicate that LSCs are superior to AD-MSCs in treating PF in rats.

[0220] discuss

[0221] The last decade has witnessed a series of studies on the identification of endogenous lung stem cells

[24] . Many cell types in the lung have been proposed as stem / progenitor cells, including basal cells, spherocytes, and alveolar type II cells. However, mesenchymal cells (derived from bone marrow, adipose tissue, and umbilical cord) remain the main players in ongoing cell-based therapeutic trials for the treatment of lung diseases because these cells are easy to isolate and proliferate. Mesenchymal cells are easier to isolate and expand than resident lung progenitor cells.

[0222] Multicellular spheroids have been used as a method for generating neural and cardiac stem cells. A recent report showed that infusion of pericardial glomeruli in patients with mild to moderate heart attacks reduced scarring and increased biopsy

[25] . Pulmonary spheroids have been used to date as a method for growing and testing lung cancer cells

[26] . In this study, we show pulmonary spheroids as a direct method for generating therapeutic lung progenitor cells. The cell yield and growth potential of pulmonary spheroidal cells (LSCs) make them suitable for both autologous and allogeneic applications (Fig. 1). Antigen sorting is not required because LSCs represent a selective mixture containing lung progenitor cells as well as supporting cells (Fig. 2). The origin of LSCs has not been determined. Expression of CD105, CD90, Pro-SPC, and CCSP in human LSCs suggests that they may contain lung mesenchymal stem cells, alveolar progenitor cells, and airway progenitor cells. LSCs differ from MSCs: human MSCs express CD105 and CD90 but do not express Pro-SPC or CCSP (Fig. 7). A subgroup of LSCs is double-positive for Pro-SPC and CSSP, representing bronchoalveolar stem cells (BASCs). In addition, a small subset of LSCs also express CD49f (Itga6), p75NGF, and EpCAM, previously reported as markers of pluripotent lung stem cells [27,28]. The percentage of positivity for these lung stem cell markers in LSCs is higher than that occurring naturally in adult lungs. We hypothesize that spheroid cultures may artificially enrich these stem cells. This natural mixture of lung progenitor cells may be a result of in vitro cell culture processes. Three-dimensional spheroid cultures can enrich stem cell populations. Alternatively, in vitro cell culture processes can revert mature lung epithelial cells to progenitor cells, a process similar to the in vivo dedifferentiation of lung cells into stem cells

[29] . LSCs can form alveolar-like structures in vitro, indicating their potential to differentiate into mature lung cells. Conditioned media derived from LSCs contain various pro-angiogenic factors and promote tube formation in endothelial cells, suggesting that LSCs can promote lung regeneration through paracrine mechanisms.

[0223] To test the regenerative potential of LSCs in vivo, we established a pulmonary fibrosis model in mice via intratracheal infusion of bleomycin. Using immunodeficient (SCID) mice allowed for testing human LSCs without fear of rejection. No complications were observed in mice receiving LSC infusions. No tumors or abnormal tissues were observed in animals treated with LSCs. Treatment with LSCs inhibited fibrosis, infiltration, and apoptosis, but promoted angiogenesis (see [link to LSC treatment]). Figures 3A-3E LSCs are implanted and acquire a mature lung phenotype in the recipient lung. Figure 4C And D), although such small implantation and differentiation events are insufficient to explain the observed benefits. Evidence suggests that injected stem cells regenerate damaged tissue through indirect paracrine mechanisms

[30] . We hypothesize that LSCs secrete beneficial factors to regulate the environment and recruit endogenous repair mechanisms ( Figure 4F ).

[0224] Among them, LSCs that can form alveolar-like structures and obtain mature lung epithelial phenotypes / morphology in vitro, demonstrating their differentiation potential ( Figure 3A And B). Conditioned culture medium from LSCs promoted lung epithelial cell survival (Fig. 3C) and endothelial cell tube formation (Fig. 3D). Cytokine array data showed that LSCs could promote lung regeneration through the secretion of anti-apoptotic and pro-angiogenic factors and cytokines. Figure 3E ).

[0225] To test the regenerative potential of LSCs in vivo, we created a pulmonary fibrosis model in mice via intratracheal infusion of bleomycin. Using immunodeficient (SCID) mice allowed for testing human LSCs without fear of rejection. No complications were observed in mice receiving LSC infusions. No tumors or abnormal tissues were observed in any animals treated with LSCs. Treatment with LSCs inhibited fibrosis ( Figure 4E ),infiltration( Figure 4F ) and apoptosis (Figure 5A), but promotes angiogenesis ( Figure 5B LSCs are implanted and acquire a mature lung phenotype in the recipient lung. Figure 5C Although such small implantation and differentiation events seem insufficient to explain the overall benefit. Evidence suggests that injected stem cells regenerate damaged tissue through indirect paracrine mechanisms

[28] . We speculate that LSCs secrete beneficial factors that regulate the environment and recruit endogenous repair mechanisms ( Figure 5E ).

[0226] Since mesenchymal stem cells (MSCs) are the most popular cell type in clinical trials for lung diseases, we conducted a head-to-head comparison of rat LSCs and AD-MSCs in the same rat model with PF (Figure 6). Notably, LSCs outperformed AD-MSCs in reducing fibrotic thickening and tissue infiltration in PF lungs. The underlying mechanisms require further elucidation. Given that LSCs are derived from adult lungs rather than adipose tissue, we hypothesize that they are pre-designated to differentiate into lung cells and promote endogenous lung regeneration.

[0227] In summary, we identified pulmonary spheroids from healthy human lungs as a novel source of lung progenitor cells for therapeutic lung regeneration. Pulmonary spheroids represent a simple and highly reproducible method for generating antigen-free, therapeutic lung cells. Typically, FBS-free media containing EOF are used for lung epithelial stem cell culture. We compared LSCs cultured under two conditions: 1) 25 ng / mL EOF and no FBS; 2) 20% FBS, no EOF. The morphology of LSCs was similar under both conditions, although cells cultured in 20% FBS medium grew faster (…). Figure 15 These unresolved issues will guide our future efforts in this research area. As an autologous product, we anticipate that IPF patients will come to the clinic, followed by lung biopsies to generate the tissue needed for LSC culture. These autologous cells will then be reintroduced into the same patient via intravenous injection. To this end, we have demonstrated that LSCs can be derived from mice with PF (Figure 16 and...). Figure 17 ) and people ( Figure 18 )lung.

[0228] Future research needs to elucidate the origins of LSCs and the mechanisms of their therapeutic benefits, and translate these findings into clinically relevant large animal models of lung diseases.

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[0260] It should be understood that although the invention has been described in conjunction with its detailed description, the foregoing description is intended to illustrate and not limit the scope of the invention. Other aspects, advantages, and modifications of the invention are within the scope of the claims set forth below. All publications, patents, and patent applications referenced in this specification are incorporated herein by reference, as if each individual publication or patent application were specifically and individually indicated to be incorporated herein by reference.

Claims

1. A composition comprising a plurality of pulmonary bulbs derived from lung tissue explants, wherein the plurality of pulmonary bulbs comprises: (i) At least one lung progenitor cell; (ii) at least one supporting matrix-like cell; and (iii) At least one pulmonary glomerular cell (LSC) that co-expresses at least one supporting matrix-like cell marker and at least one lung progenitor cell marker; The at least one progenitor cell, the at least one supporting matrix-like cell, and the at least one LSC are derived from the same lung tissue explant.

2. The composition of claim 1, wherein the plurality of pulmonary bulbs are obtained from lung tissue explants cultured under low adhesion conditions.

3. The composition according to claim 1, wherein the at least one lung progenitor cell expresses one or more of the following, or any combination thereof: surface-promoting protein C (SPC), Clara cell secretory protein (CCSP), integrin subunit α6 (ITGA6 or CD49f), p75 nerve growth factor (NGF), epithelial cell adhesion molecule (EpCAM), p63, keratin 5 (KRT5).

4. The composition according to claim 1, wherein the at least one LSC does not express one or more of CD45, CD31, CD34, c-kit, or any combination thereof.

5. The composition according to claim 1, wherein the at least one lung progenitor cell expresses one or more upregulated genes selected from the group consisting of bone morphogenetic protein 2 (BMP2), stromal cell-derived factor 1, fibroblast growth factor 2 (FGF2), or any combination thereof.

6. The composition of claim 5, wherein one or more upregulated genes are upregulated compared to alveolar epithelial cells, dermal fibroblasts, or mesenchymal stem cells.

7. The composition according to claim 1, wherein the at least one supports the expression of one or more of CD105, CD90, or a combination thereof by stromal-like cells.

8. The composition of claim 1, wherein the pulmonary bulbs have a diameter ranging from about 25 μm to about 500 μm.

9. The composition according to claim 1, wherein the at least one LSC secretes one or more angiogenic factors.

10. The composition according to claim 9, wherein the one or more angiogenesis factors are selected from insulin-like growth factor binding protein 2 (IGFBP2), hepatocyte growth factor (HGF), brain-derived neurotrophic factor (BDNF), and any combination thereof.

11. The composition of claim 1, wherein the composition further comprises a pharmaceutically acceptable carrier.

12. The composition according to claim 1, wherein the at least one lung progenitor cell marker comprises surfactant protein C (SPC), Clara cell secretory protein (CCSP), integrin subunit α6 (ITGA6 or CD49f), p75 nerve growth factor (NGF), epithelial cell adhesion molecule (EpCAM), p63, keratin 5 (KRT5) and aquaporin 5 (Aq5), or any combination thereof.

13. The composition of claim 1, wherein the at least one supporting stromal-like cell marker comprises CD105 and / or CD90.

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