Pharmaceutical for lung regeneration and method for producing alveolar organoid

Abstract

Provided is a pharmaceutical for lung regeneration, the pharmaceutical containing vascular endothelial stem cells. Also provided is a method for producing an alveolar organoid, the method comprising a step for three-dimensional co-culture of vascular endothelial stem cells and alveolar epithelial cells. This pharmaceutical for lung regeneration is useful in the treatment of lung disease accompanied by alveolar damage, such as chronic obstructive lung diseases, infectious lung diseases, and pulmonary fibrosis.

Description

Lung regeneration medicine and method for producing alveolar organoids

[0001] The present invention relates to a drug for lung regeneration and a method for producing alveolar organoids.

[0002] When the lungs sustain severe damage, or even mild, sustained damage over a long period of time, their normal repair and regeneration functions fail. As a result, essential parenchymal cells are lost, leading to abnormal repair and regeneration responses, resulting in excessive accumulation of extracellular matrix and an increase in interstitial cells, leading to organ dysfunction. Among lung diseases that cause fibrosis, idiopathic pulmonary fibrosis (IPF) has a poor prognosis, with a median survival time of 3-5 years after diagnosis and a 5-year survival rate of 20-40%. Recently, two antifibrotic drugs, pirfenidone and nintedanib, have been developed and are indicated for the treatment of idiopathic pulmonary fibrosis. However, these drugs only slow the progression of fibrosis and have not improved the prognosis. Therefore, the only curative treatment currently available is transplantation, which requires significant invasive procedures. The development of novel, minimally invasive treatments is urgently needed. In addition, chronic obstructive pulmonary disease, which occurs when alveoli collapse due to long-term exposure to harmful substances such as smoking, reducing the ability to take in oxygen and expel carbon dioxide, can only be managed through symptomatic treatment such as oxygen inhalation and administration of bronchodilators, and no radical treatment that can regenerate the alveoli has yet been developed.

[0003] In recent years, it has become clear that blood vessels not only transport blood but also secrete humoral factors, collectively known as angiocrine factors (AF), into surrounding tissues to promote the maintenance and regeneration of organs (Non-Patent Document 1). In particular, endothelial cells (ECs), which line the vascular lumen, play a central role in this process. In fact, a study using mice has reported that AF secreted from vascular endothelial cells suppresses irreversible and fatal sclerosing pathology (fibrosis) that occurs after severe lung injury and promotes the regeneration of epithelial cells responsible for gas exchange (Non-Patent Document 2).

[0004] In recent years, it has become clear that vascular endothelial cells exhibit diversity within the same organ (Non-Patent Document 3). Prior to the report in Non-Patent Document 3, the present inventors had been conducting research into the possible presence of endothelial stem cells (ESCs) within vascular endothelial cells, which possess high self-renewal capacity while also being capable of differentiating into endothelial cells with other properties. In 2018, they discovered that endothelial cells in the liver possessing a cell surface protein called CD157 function as endothelial stem cells (Non-Patent Document 4). In Non-Patent Document 4, the present inventors demonstrated that transplantation of CD157-positive endothelial cells into a mouse model reproducing hemophilia A, which is caused by a genetic abnormality in liver endothelial cells, improved hemostatic function to normal levels, suggesting the potential for therapeutic applications of endothelial stem cells.

[0005] Regarding the vascular environment in idiopathic pulmonary fibrosis, it is known that capillary density is extremely reduced within the widespread fibrotic lesions (Non-Patent Document 5), but no attempts have been made to regenerate the vascular environment within the fibrotic lesions. Furthermore, although a reduction in pulmonary capillaries is also observed in chronic obstructive pulmonary disease, no attempts have been made to regenerate pulmonary blood vessels.

[0006] Rafii, S et al. Angiocrine functions of organ-specific endothelial cells. Nature. 2016 Jan 21;529(7586):316-25. doi: 10.1038 / nature17040.Cao Z et al. Targeting of the pulmonary capillary vascular niche promotes lung alveolar repair and ameliorates fibrosis. Nat Med. 2016 Feb;22(2):154-62. doi: 10.1038 / nm.4035. Epub 2016 Jan 18.Kalucka J et al. Single-Cell Transcriptome Atlas of Murine Endothelial Cells. Cell. 2020 Feb 20;180(4):764-779.e20. doi: 10.1016 / j.cell.2020.01.015. Epub 2020 Feb 13. Wakabayashi T et al. CD157 Marks Tissue-Resident Endothelial Stem Cells with Homeostatic and Regenerative Properties. Cell Stem Cell. 2018 Mar 1;22(3):384-397.e6. doi: 10.1016 / j.stem.2018.01.010. Epub 2018 Feb 8.Chandru H et al. Angiogenesis in pulmonary fibrosis: too much or not enough? Chest. 2012 Jul;142(1):200-207. doi: 10.1378 / chest.11-1962.

[0007] The objectives of the present invention are to discover unknown uses for vascular endothelial stem cells, to provide a pharmaceutical for lung regeneration, and to provide a method for producing alveolar organoids.

[0008] In order to solve the above-mentioned problems, the present invention encompasses the following inventions. [1] A pharmaceutical for lung regeneration comprising vascular endothelial stem cells. [2] The pharmaceutical according to [1] above, comprising vascular endothelial stem cells as an active ingredient and used so that the vascular endothelial stem cells are delivered to the lung. [3] The pharmaceutical according to [1] above, wherein the vascular endothelial stem cells are CD157-positive vascular endothelial cells. [4] The pharmaceutical according to [1] above, wherein the vascular endothelial stem cells are vascular endothelial cell protein C receptor-positive vascular endothelial cells. [5] The pharmaceutical according to [1] above, wherein the vascular endothelial stem cells are CD157-positive and vascular endothelial cell protein C receptor-positive vascular endothelial cells. [6] The pharmaceutical according to any of [1] to [5] above, wherein the vascular endothelial stem cells are pulmonary vascular endothelial stem cells. [7] The pharmaceutical according to any of [1] to [5] above, wherein the vascular endothelial stem cells are human vascular endothelial stem cells. [8] The pharmaceutical according to any of [1] to [5] above, wherein the pharmaceutical is for treating a pulmonary disease in which alveoli are damaged. [9] The medicament according to [8] above, wherein the pulmonary disease in which alveoli are damaged is chronic obstructive pulmonary disease, infectious pulmonary disease, or pulmonary fibrosis.

[10] A method for producing alveolar organoids, comprising a step of three-dimensionally co-culturing vascular endothelial stem cells and alveolar epithelial cells.

[11] The method according to

[10] above, wherein the vascular endothelial stem cells are CD157-positive vascular endothelial cells.

[12] The method according to

[10] above, wherein the vascular endothelial stem cells are vascular endothelial cell protein C receptor-positive vascular endothelial cells.

[13] The method according to

[10] above, wherein the vascular endothelial stem cells are CD157-positive and vascular endothelial cell protein C receptor-positive vascular endothelial cells.

[14] The method according to any one of

[10] to

[13] above, wherein the three-dimensional co-culturing step further comprises the addition of fibroblasts.

[15] The method according to any one of

[10] to

[13] above, wherein the vascular endothelial cells are pulmonary vascular endothelial cells.

[16] The method according to any one of

[10] to

[13] above, wherein the vascular endothelial cells are human vascular endothelial cells.

[17] The method according to

[14] above, wherein the fibroblasts are pulmonary fibroblasts.

[0009] The present invention provides a pulmonary regeneration drug containing vascular endothelial stem cells, and a method for producing alveolar organoids using vascular endothelial stem cells and alveolar epithelial cells.

[0010] This figure shows the results of flow cytometry analysis of the expression of CD157 and EPCR (endothelial protein C receptor) in vascular endothelial cell fractions (CD31+CD45-Dead Cell-) isolated from mouse lungs from which dead cells had been removed. Vascular endothelial cells from the CD157(+) group, CD157(+)EPCR(-) group, CD157(+)EPCR(+) group, CD157(-) group, CD157(-)EPCR(-) group, and CD157(-)EPCR(+) group were plated at 1.0 x 10 in a 24-well plate. 41 shows the results of immunostaining and observing the colonies formed after 10 days of culture using OP9 stromal cells as feeder cells, seeded at 100 cells / well. This figure also shows the results of counting the number of colonies in each group in FIG. 2. This figure also shows the results of counting the number of cells engrafted in the lungs after intravenous administration of vascular endothelial cells from the CD157(+)EPCR(-) group, CD157(+)EPCR(+) group, CD157(-)EPCR(-) group, and CD157(-)EPCR(+) group to mice that had been given vascular damage by monocrotaline administration. This figure also shows the results of attempting to construct alveolar organoids by co-culturing vascular endothelial cells from the CD157(+)EPCR(-) group, CD157(+)EPCR(+) group, CD157(-)EPCR(-) group, and CD157(-)EPCR(+) group with alveolar epithelial cells and fibroblasts. This is a schematic diagram of the experiment of Example 5. Figure 1 shows the results of an attempt to regenerate damaged alveolar epithelial cells by co-culturing CD157(+)EPCR(+) vascular endothelial cells isolated from untreated mice with alveolar epithelial cells and fibroblasts isolated from mice with bleomycin-induced pulmonary fibrosis. Figure 2 shows the results of an attempt to construct alveolar organoids by co-culturing green-fluorescent CD157-positive EPCR-positive vascular endothelial cells, red-fluorescent type 2 alveolar epithelial cells, and multiple-passaged primary cultured fibroblasts. Figure 3 shows the results of transplanting Matrigel containing green-fluorescent CD157-positive EPCR-positive vascular endothelial cells, red-fluorescent type 2 alveolar epithelial cells, and multiple-passaged primary cultured fibroblasts into the lungs of pulmonary fibrosis model mice, and evaluating the engraftment of alveolar epithelial cells. This figure shows the results of transplanting Matrigel encapsulating green-fluorescent CD157-positive EPCR-positive vascular endothelial cells, red-fluorescent type II pneumocytes, and multiply passaged primary cultured fibroblasts into the lungs of a pulmonary fibrosis model mouse, and evaluating the engraftment of vascular endothelial cells.

[0011] [Drug for lung regeneration] The present invention provides a drug for lung regeneration comprising vascular endothelial stem cells (hereinafter referred to as "drug of the present invention"). The drug of the present invention may contain vascular endothelial stem cells as an active ingredient and be used so that the vascular endothelial stem cells are delivered to the lung. The vascular endothelial stem cells contained in the drug of the present invention refer to cells that exist within vascular endothelial cells and have both the ability to divide in an undifferentiated state (self-renewal ability) and the ability to differentiate into vascular endothelial cells.

[0012] In the pharmaceutical composition of the present invention, the vascular endothelial stem cells may be CD157-positive cells among CD31-positive, CD45-negative vascular endothelial cells. Furthermore, in the pharmaceutical composition of the present invention, the vascular endothelial stem cells may be endothelial protein C receptor (EPCR)-positive cells among CD31-positive, CD45-negative vascular endothelial cells. Furthermore, in the pharmaceutical composition of the present invention, the vascular endothelial stem cells may be CD157-positive and EPCR-positive cells among CD31-positive, CD45-negative vascular endothelial cells. The present inventors have confirmed that CD157-positive, EPCR-positive vascular endothelial cells have higher colony-forming ability than CD157-positive or EPCR-positive vascular endothelial cells (see Example 2).

[0013] The vascular endothelial stem cells used in the pharmaceutical of the present invention are preferably mammalian vascular endothelial stem cells. Mammals are not particularly limited, but examples include humans, monkeys, cows, pigs, sheep, goats, dogs, cats, mice, rats, and rabbits. Human vascular endothelial stem cells are preferred. Human vascular endothelial stem cells can be safely applied to humans.

[0014] Vascular endothelial stem cells can be prepared from any organ or tissue. For example, they can be prepared from the liver, retina, brain, heart, skin, muscle (skeletal muscle), lung, kidney, placenta, fat, etc. Since the pharmaceutical agent of the present invention is a drug for lung regeneration that is delivered to the lung, it is preferable to prepare it from an organ derived from endoderm, and particularly preferable to prepare it from the lung. Endoderm-derived organs other than the lung include the esophagus, stomach, intestine, liver, pancreas, trachea, etc.

[0015] CD31-positive CD45-negative vascular endothelial cells can be prepared, for example, by digesting and dispersing an isolated organ or tissue with a commercially available cell dispersion reagent to prepare a cell suspension, staining the cell suspension with anti-CD31 antibody and anti-CD45 antibody, and then recovering the CD31-positive CD45-negative cells using flow cytometry.CD157-positive vascular endothelial cells can be prepared, for example, by digesting and dispersing an isolated organ or tissue with a commercially available cell dispersion reagent to prepare a cell suspension, staining the cell suspension with anti-CD31 antibody, anti-CD45 antibody, and anti-CD157 antibody, and then recovering the CD31-positive CD45-negative CD157-positive cells using flow cytometry. EPCR-positive vascular endothelial cells can be prepared, for example, by digesting and dispersing an isolated organ or tissue with a commercially available cell dispersion reagent to prepare a cell suspension, staining the cell suspension with anti-CD31 antibody, anti-CD45 antibody, and anti-EPCR antibody, and then recovering CD31-positive, CD45-negative, EPCR-positive cells using flow cytometry.CD157-positive, EPCR-positive vascular endothelial cells can be prepared, for example, by digesting and dispersing an isolated organ or tissue with a commercially available cell dispersion reagent to prepare a cell suspension, staining the cell suspension with anti-CD31 antibody, anti-CD45 antibody, anti-CD157 antibody, and anti-EPCR antibody, and then recovering CD31-positive, CD45-negative, CD157-positive, EPCR-positive cells using flow cytometry.

[0016] The vascular endothelial stem cells used in the pharmaceutical of the present invention may be prepared using a method developed by the present inventors for reprogramming vascular endothelial stem cells from vascular endothelial cells that do not have stem cell properties (see PCT / JP2024 / 009955).

[0017] The vascular endothelial stem cells used in the medicament of the present invention may be vascular endothelial cells containing vascular endothelial stem cells, and the proportion of vascular endothelial stem cells in the vascular endothelial cells may be 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more.

[0018] The vascular endothelial stem cells used in the medicament of the present invention may be vascular endothelial cells containing a mixture of CD157-positive cells and CD157-negative cells. The proportion of CD157-positive cells in the vascular endothelial cells containing a mixture of CD157-positive cells and CD157-negative cells may be 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more.

[0019] The medicament of the present invention may further comprise alveolar epithelial cells. The alveolar epithelial cells may comprise type 2 alveolar epithelial cells. The alveolar epithelial cells can be prepared, for example, by digesting and dispersing isolated lung tissue with a commercially available cell dispersion reagent to prepare a cell suspension, staining this cell suspension with an anti-CD31 antibody, an anti-CD45 antibody, an anti-EpCAM antibody, and an anti-IA / IE antibody, and recovering CD31-negative, CD45-negative, EpCAM-positive, IA / IE-positive cells using flow cytometry technology.

[0020] It has been confirmed that the medicament of the present invention can regenerate damaged alveoli. Therefore, the medicament of the present invention can be suitably used for treating pulmonary diseases in which alveoli are damaged. Examples of pulmonary diseases in which alveoli are damaged include chronic obstructive pulmonary disease (COPD), infectious pulmonary diseases, and pulmonary fibrosis. Examples of infectious pulmonary diseases include bacterial pneumonia, viral pneumonia, pulmonary tuberculosis, pulmonary mycosis, and pulmonary parasitic disease. Examples of pulmonary fibrosis include idiopathic pulmonary fibrosis, radiation pneumonitis, idiopathic organizing pneumonia, and collagen vascular disease.

[0021] The pharmaceutical of the present invention can be administered to a living body in the form of a cell suspension in which vascular endothelial stem cells are suspended in an appropriate solution suitable for administration to the living body. Examples of suitable solutions include physiological saline, phosphate-buffered saline (PBS), and other physiological salt solutions. They may also be suspended in various extracellular matrices. Vascular endothelial stem cells are typically prepared immediately prior to administration, but cryopreserved vascular endothelial stem cells may also be used to prepare them at the time of use. The route of administration of the pharmaceutical of the present invention is not particularly limited as long as it delivers vascular endothelial stem cells to the lungs. Examples include direct administration of a vascular endothelial stem cell suspension to the lungs, administration via the pulmonary artery, intravenous administration close to the lungs, and intrabronchial administration. The dosage cannot be determined unambiguously because it varies depending on the target organ, patient age, weight, etc., but a physician can determine an appropriate dosage based on the above-mentioned circumstances. For example, 1 to 1 x 10 cells per administration. ９ The vascular endothelial stem cells may be administered. The frequency of administration can be appropriately selected from the range of once per day to once per week. The dosage and frequency of administration can be increased or decreased as appropriate depending on the patient.

[0022] The present invention includes the following inventions. - A method for lung regeneration, comprising the step of administering vascular endothelial stem cells so that they are delivered to the lungs of a subject. - A method for treating a lung disease in which the alveoli are damaged, comprising the step of administering vascular endothelial stem cells so that they are delivered to the lungs of a subject. - Vascular endothelial stem cells for use in lung regeneration. - Vascular endothelial stem cells for treating a lung disease in which the alveoli are damaged. - Use of vascular endothelial stem cells in the manufacture of a medicine for lung regeneration. - Use of vascular endothelial stem cells in the manufacture of a medicine for treating a lung disease in which the alveoli are damaged.

[0023] [Method for producing alveolar organoids] The present invention provides a method for producing alveolar organoids (hereinafter referred to as the "method for producing the present invention"). The method for producing the present invention may include a step of three-dimensionally co-culturing vascular endothelial stem cells and alveolar epithelial cells. The vascular endothelial stem cells used in the method for producing the present invention may be the same as the vascular endothelial stem cells used in the above-mentioned pharmaceutical preparation of the present invention.

[0024] The alveolar epithelial cells used in the production method of the present invention can be prepared, for example, by digesting and dispersing isolated lung tissue with a commercially available cell dispersion reagent to prepare a cell suspension, staining this cell suspension with an anti-CD31 antibody, an anti-CD45 antibody, an anti-EpCAM antibody, and an anti-IA / IE antibody, and recovering CD31-negative, CD45-negative, EpCAM-positive, IA / IE-positive cells using flow cytometry. When preparing human alveolar epithelial cells, lung tissue collected by human biopsy can be used.

[0025] In the three-dimensional co-culture step of the production method of the present invention, feeder cells may be added to vascular endothelial stem cells and alveolar epithelial cells for co-culture. Examples of feeder cells include fibroblasts and mesenchymal stem cells. Fibroblasts are preferred, and pulmonary fibroblasts are more preferred. Pulmonary fibroblasts can be prepared, for example, by digesting and dispersing isolated lung tissue with a commercially available cell dispersion reagent to prepare a cell suspension, staining this cell suspension with anti-CD31, anti-CD45, anti-EpCAM, anti-CD146, anti-Lyve1, and anti-CD140a antibodies, and recovering CD31-negative, CD45-negative, EpCAM-negative, CD146-negative, Lyve1-negative, CD140a-positive cells using flow cytometry. When preparing human lung fibroblasts, lung tissue collected by human biopsy can be used.

[0026] The method for three-dimensional co-culture is not particularly limited and can be appropriately selected from known three-dimensional culture methods. For example, a method in which vascular endothelial stem cells and alveolar epithelial cells are embedded in an extracellular matrix (ECM) and then three-dimensionally cultured may be used. Examples of extracellular matrices include Matrigel (trade name), collagen gel, and laminin gel. In the co-culture, the ratio of the number of vascular endothelial stem cells to alveolar epithelial cells is not particularly limited, but it is preferable that the number of vascular endothelial stem cells outnumber the alveolar epithelial cells, and the number of vascular endothelial stem cells may be approximately 3 to 7 times the number of alveolar epithelial cells. Furthermore, when fibroblasts are co-cultured as feeder cells, it is preferable that the number of fibroblasts outnumber the alveolar epithelial cells, and the number of fibroblasts outnumber the alveolar epithelial cells, and the number of fibroblasts may be approximately 8 to 12 times the number of alveolar epithelial cells. The specific number of cells can be appropriately determined depending on the culture system used.

[0027] The medium used for three-dimensional co-culture is not particularly limited, and any known medium that can be used for culturing alveolar cells can be used. For example, a medium prepared by adding an ITS supplement, cholera toxin, EGF, bovine pituitary extract, FGF-7, VEGF, or the like to a basal medium such as DMEM medium or DMEM / Ham's F-12 medium can be suitably used. The culture period for three-dimensional co-culture is not particularly limited, and the morphology of the culture can be observed over time, and the culture can be continued until the desired organoids are produced.

[0028] The alveolar organoids produced by the production method of the present invention have vascular endothelial cells attached thereto, and therefore, when transplanted into a living body, they are advantageous in that they rapidly connect with existing blood vessels, allowing for the rapid supply of oxygen and nutrients to the transplanted alveolar organoids. Furthermore, an epithelial-vascular interface is formed, allowing the organoids to rapidly perform the alveolar function of gas exchange.

[0029] The alveolar organoids produced by the production method of the present invention can be used for transplantation of lung regeneration, can be used to prepare artificial lungs, and can be used to evaluate the effects of drugs on alveoli.

[0030] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0031] Example 1: Analysis of CD157 and EPCR Expression in Murine Lung Vascular Endothelial Cells. 1-1 Experimental Method: Seven- to nine-week-old C57BL / 6 mice (Japan SLC) were used. The excised lungs were minced as finely as possible using ophthalmic scissors. The minced tissue was immersed in a mixed solution of Dispase II (Roche Applied Science), collagenase (Wako), and type II collagenase (Worthington Biochemical) and shaken at 37°C to digest the extracellular matrix. The digested cell solution was passed through a 40 μm pore size filter to obtain a dispersed cell suspension. Red blood cells were lysed using ACK (Ammonium-Chloride-Potassium) solution (0.15 M NH4Cl, 10 mM KHCO3, and 0.1 mM Na2-EDTA), and the remaining cells were used in the following experiments.

[0032] The prepared cells were stained for cell surface antigens with anti-CD31 (BD Biosciences), anti-CD45 (BD Biosciences), anti-CD157 (Biolegend), and anti-EPCR (endothelial protein C receptor) antibodies (Thermo Fisher Scientific), and then stained for dead cells (Thermo Fisher Scientific) before flow cytometry analysis. The presence or absence of CD157 and EPCR expression was confirmed in endothelial cells (CD31+, CD45-negative) after removal of dead cells using a SOAP FACSAria (BD Biosciences) and FlowJo Software (Treestar Software).

[0033] 1-2 Results The results are shown in Figure 1. Among the vascular endothelial cells (CD31 positive, CD45 negative) isolated from mouse lungs, 92.5% were CD157 negative, EPCR negative, 2.2% were CD157 positive, EPCR negative, 2.6% were CD157 negative, EPCR positive, and 2.7% were CD157 positive, EPCR positive.

[0034] Example 2: Colony-forming ability of vascular endothelial cells from the four fractions isolated in Example 1 2-1 Experimental method Using lungs excised from 7- to 9-week-old C57BL / 6 mice, CD157-negative EPCR-negative vascular endothelial cells, CD157-positive EPCR-negative vascular endothelial cells, CD157-negative EPCR-positive vascular endothelial cells, and CD157-positive EPCR-positive vascular endothelial cells were isolated by flow cytometry in the same manner as in Example 1. Separately, CD157-positive vascular endothelial cells and CD157-negative vascular endothelial cells were isolated using only the presence or absence of CD157 expression as an indicator. 1.0 × 10 vascular endothelial cells of each type were placed in a 24-well plate. 4 Seeded at 2.0 x 10 cells / well 4 OP9 stromal cells (RIKEN cell bank) were used as feeder cells and cultured for 10 days in RPMI-1640 medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific), 0.1% 2-mercaptoethanol (Gibco), and 1% penicillin-streptomycin solution (Sigma-Aldrich).

[0035] After the incubation period, the colonies were immunostained with anti-CD31 antibody (BD Biosciences), biotin-conjugated polyclonal anti-Rat IgG (Agilent Technologies), and an ABC kit (Vector Laboratories), and the number of colonies was counted. A colony consisting of multiple vascular endothelial cells was counted as one colony.

[0036] 2-2 Results The results are shown in Figures 2 and 3. Figure 2 shows images of immunostained vascular endothelial cell colonies in each group, and Figure 3 shows the results of counting the number of colonies in each group. Colony formation ability was higher in the CD157-positive group than in the CD157-negative group. Furthermore, within the CD157-positive group, the CD157-positive EPCR-positive group had more colonies than the CD157-positive EPCR-negative group. The CD157-negative EPCR-positive group had the third highest number of colonies. However, when comparing the colony morphology with that of the CD157-positive EPCR-positive group (see the enlarged colony image on the right side of Figure 2), differences in colony morphology were observed: the CD157-positive EPCR-positive group primarily formed sheet-like colonies composed of individual endothelial cells adhered together, whereas the CD157-negative EPCR-positive group primarily formed scattered colonies composed of individual endothelial cells not adhered together.

[0037] Example 3: Engraftment Ability of Transplanted Vascular Endothelial Cells from Four Groups 3-1 Experimental Method C57BL / 6 mice and C57BL / 6-Tg (CAG-EGFP) mice (hereinafter referred to as "Green Mice") were purchased from Japan SLC and used. Monocrotaline (Sigma-Aldrich) 450 μg / g body weight was administered via the ophthalmic vein of 8- to 10-week-old male C57BL / 6 mice to create a pulmonary vascular disorder model. Using lungs excised from Green Mice, CD157-negative EPCR-negative vascular endothelial cells, CD157-positive EPCR-negative vascular endothelial cells, CD157-negative EPCR-positive vascular endothelial cells, and CD157-positive EPCR-positive vascular endothelial cells were isolated by flow cytometry using the same method as in Example 1. Two days after monocrotaline administration, 1 × 10 vascular endothelial cells from each group were cultured. 5 The cells were suspended in 100 μL of PBS(-) and transplanted intravenously into the ophthalmic vein of a mouse model of vascular disorder. Two weeks after transplantation, the lungs were excised from the mice and subjected to flow cytometry analysis in the same manner as in Example 1. The number of EGFP-positive CD31-positive CD45-negative vascular endothelial cells was confirmed and analyzed.

[0038] 3-2 Results The results are shown in Figure 4. The number of vascular endothelial cells engrafted in the lungs per individual was highest for CD157-positive EPCR-positive vascular endothelial cells, demonstrating their high transplantability.

[0039] Example 4: Construction of Alveolar Organoids by Co-Culture of Vascular Endothelial Cells, Alveolar Epithelial Cells, and Fibroblasts (1) 4-1 Experimental Method Using lungs excised from 7- to 9-week-old C57BL / 6 mice, CD157-negative EPCR-negative vascular endothelial cells, CD157-positive EPCR-negative vascular endothelial cells, CD157-negative EPCR-positive vascular endothelial cells, and CD157-positive EPCR-positive vascular endothelial cells were isolated by flow cytometry using the same method as in Example 1. Separately, lungs excised from 7- to 9-week-old C57BL / 6 mice were enzyme-treated, and type 2 alveolar epithelial cells, which are stem cells of alveolar epithelial cells, and fibroblasts, which are feeder cells, were isolated using antibodies. The enzyme treatment was performed in the same manner as in Example 1, except that type I collagenase (Worthington Biochemical) was used instead of type II collagenase, and a cell suspension was prepared. To isolate type 2 alveolar epithelial cells, antibody reactions were performed using anti-CD31 antibody (Biolegend), anti-CD45 antibody (Biolegend), anti-EpCAM antibody (Biolegend), and anti-IA / IE antibody (Biolegend), and CD31(-)CD45(-)EpCAM(+)IA / IE(+) cells were isolated as type 2 alveolar epithelial cells using flow cytometry. To isolate fibroblasts, antibody reactions were performed using anti-CD31 antibody (Biolegend), anti-CD45 antibody (Thermo Fisher Scientific), anti-EpCAM antibody (Biolegend), anti-CD146 antibody (Biolegend), anti-Lyve1 antibody (R&D Systems), and anti-CD140a antibody (Biolegend), and CD31(-)CD45(-)EpCAM(-)CD146(-)Lyve1(-)CD140a(+) cells were isolated as fibroblasts using flow cytometry.

[0040] Vascular endothelial cells 2.5×10 4pcs, type 2 alveolar epithelial cells 5 x 10 3 cells, and 5 x 10 fibroblasts 4 The cells were embedded in 30 μL of 50% Matrigel (Corning) and dropped into a well of a non-coated 6-well dish (Thermo Fisher Scientific) to initiate culture. 3 5 x 10 fibroblasts 4The control was a single cell embedded in 30 μL of 50% Matrigel (Corning). The culture medium used was DMEM-Ham's F-12 medium (Nakarai) supplemented with 10% fetal bovine serum (Gibco), 4 mM L-glutamine (Gibco), 100 U / mL penicillin-100 μg / mL streptomycin solution (Sigma-Aldrich), 10 μg / mL insulin-5 μg / mL transferrin-selenium (Gibco), 0.1 μg / mL cholera toxin (Sigma-Aldrich), 25 ng / mL epidermal growth factor (Corning), 30 μg / mL bovine pituitary extract (Thermo Fisher Scientific), 20 ng / mL FGF-7 (R&D Systems), and 20 ng / mL recombinant human VEGF165 (Pepro Tech). Y27632 (10 μM, Sellec Chemicals) was added for the first 4 days of culture. After 7 days of culture, organoids were fixed with 2% PFA / PBS(-) and immunostained with anti-CD31 antibody (Merck), DAPI (Thermo Fisher Scientific), TOPRO3 (Thermo Fisher Scientific), anti-E-Cadherin antibody (Thermo Fisher Scientific), anti-Rat-AF647 (Thermo Fisher Scientific), and anti-Armenian hamster-AF488 (Jackson Immuno Research).

[0041] 4-2 Results The results are shown in Figure 5. The first row is an immunostained image of vascular endothelial cells (EC), the second row is an immunostained image of alveolar epithelial cells, the third row is a DAPI-stained image of nuclei, and the fourth row is a composite image of the above three rows. An extensive vascular endothelial cell network was formed only when CD157(+)EPCR(+) vascular endothelial cells were co-cultured with alveolar epithelial cells. Some of the network formed seemed to envelop spheres of alveolar epithelial cells. Furthermore, the formation of alveolar organoids was most frequently induced in cultures in which such vascular networks were formed.

[0042] Example 5: Effect of vascular endothelial stem cells on alveolar epithelial cell regeneration using an organoid culture system. 5-1 Experimental Method. A schematic diagram of the experiment in Example 5 is shown in Figure 6. Seven- to nine-week-old C57BL / 6 male mice were intratracheally administered bleomycin (Nippon Kayaku Co., Ltd.) at 1.5 μg / g body weight to induce pulmonary fibrosis. C57BL / 6-Tg(CAG-tdTomato) mice purchased from Jackson Laboratory were also intratracheally administered bleomycin at 1.5 μg / g body weight to induce pulmonary fibrosis. One week after bleomycin administration, lungs were removed from each mouse. Fibroblasts were isolated from the lungs of C57BL / 6 mice, and tdTomato-labeled type 2 alveolar epithelial cells were isolated from the lungs of C57BL / 6 mice and C57BL / 6-Tg(CAG-tdTomato) mice, respectively, using the same method as in Example 4. Separately, lungs were excised from 7- to 9-week-old untreated C57BL / 6 male mice, and CD157-positive, EPCR-positive vascular endothelial cells were isolated in the same manner as in Example 1. As in Example 4, 2.5 × 10 CD157-positive, EPCR-positive vascular endothelial cells were isolated. 4 pcs, type 2 alveolar epithelial cells 5 x 10 3 cells, and 5 x 10 fibroblasts 4 The cells were embedded in 30 μL of 50% Matrigel (Corning) and dropped into a well of a non-coated 6-well dish (Thermo Fisher Scientific) to initiate culture. 3 5 x 10 fibroblasts 4Only the cells were embedded in 30 μL of 50% Matrigel (Corning) as a control. After culturing for 7 days using the same medium as in Example 4, the cells were observed under a phase-contrast microscope and a fluorescent microscope.

[0043] 5-2 Results The results are shown in Figure 7. The top row shows bright-field images, the bottom row shows fluorescent images of type 2 alveolar epithelial cells, the left column shows the co-culture group with CD157-positive EPCR-positive vascular endothelial cells, and the right column shows the control group without co-culture of vascular endothelial cells. In the control group, almost no spheres were formed by the damaged type 2 alveolar epithelial cells, but in the co-culture group with CD157-positive EPCR-positive vascular endothelial cells, sphere formation by the damaged type 2 alveolar epithelial cells was approximately 10-fold higher than in the control group. These results demonstrate that vascular endothelial stem cells have a regenerative effect on damaged type 2 alveolar epithelial cells.

[0044] Example 6: Construction of alveolar organoids by co-culturing vascular endothelial cells, alveolar epithelial cells, and fibroblasts (2) 6-1 Experimental method Cdh5-cre-ERT2 mice (Hisamichi Naito et al., Dev Cell. 2019 Jan 28;48(2):151-166.e7. doi: 10.1016 / j.devcel.2018.12.002. Epub 2019 Jan 10.), which express Cre / ERT2 specifically in vascular endothelial cells, were crossbred with B6.Cg-Gt(ROSA)26Sortm6(CAG-ZsGreen1)Hze / J mice (Jackson Laboratory), whose cells emit green fluorescence upon Cre recombinase expression, to generate mice with ZsGreen1 labeling specifically in vascular endothelial cells. CD157-positive EPCR-positive vascular endothelial cells were isolated by flow cytometry using lungs excised from the mice, as described in Example 1. Furthermore, lungs were excised from C57BL / 6-Tg(CAG-tdTomato) mice, and tdTomato-labeled type 2 alveolar epithelial cells were isolated by the same method as in Example 4. For fibroblasts, lungs excised from 8- to 10-week-old C57BL / 6 mice were minced with scissors and treated with 1 mg / mL collagenase (Wako) at 37°C for 30 minutes. Red blood cells were lysed with ACK solution, and the fibroblasts were passed through a 100 μm mesh filter. Then, the fibroblasts were isolated by flow cytometry using the same antibodies as in Example 4. The isolated fibroblasts were subcultured 3 to 5 times in DMEM (high glucose) medium (Nakarai) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) and 1% penicillin-streptomycin solution (Sigma-Aldrich) before use in the experiments.

[0045] Vascular endothelial cells 5 x 10 4 pcs, type 2 alveolar epithelial cells 5 x 10 3 cells, and 5 x 10 fibroblasts 4The organoids were embedded in 30 μL of 50% Matrigel (Corning) using the same method as in Example 4, dropped into the wells of a non-coated 6-well dish (Thermo Fisher Scientific), and culture was initiated using the same medium as in Example 4. After 6-7 days of culture, the organoids were fixed in 4% PFA / PBS(-) supplemented with glutaraldehyde (Sigma-Aldrich) to a concentration of 0.5%, and nuclear staining was performed with DAPI (Thermo Fisher Scientific).

[0046] 6-2 Results The results are shown in Figure 8. The bottom row is an enlarged view of the top row. Even when primary cultured fibroblasts that had been passaged multiple times were used as feeder cells, we were successful in culturing alveolar organoids that formed a vascular network.

[0047] Example 7 Transplantation of Vascular Endothelial Stem Cells into Pulmonary Fibrosis Model Mice 7-1 Experimental Method Using the same method as in Example 6, CD157-positive EPCR-positive vascular endothelial cells were isolated from the lungs of mice obtained by crossbreeding Cdh5-cre-ERT2 mice with B6.Cg-Gt(ROSA)26Sortm6(CAG-ZsGreen1)Hze / J mice, tdTomato-labeled type 2 alveolar epithelial cells were isolated from C57BL / 6-Tg(CAG-tdTomato) mice, and fibroblasts isolated from the lungs of C57BL / 6 mice were subcultured 3 to 5 times.

[0048] C57BL / 6 mice aged 8-10 weeks were intratracheally administered bleomycin at 2.0 μg / g body weight, and 7 days later, pulmonary fibrosis model mice were used. 4 pcs, type 2 alveolar epithelial cells 5 x 10 3 cells, and 5 x 10 fibroblasts 4The cells were embedded in 30 μL of 50% Matrigel and administered locally to the left lung of a mouse model of pulmonary fibrosis. Specifically, after anesthetizing the mouse with a triple-dose anesthesia and providing respiratory support via a ventilator, the left thoracotomy was performed and the cell-embedded Matrigel was locally injected into the left lung. The mouse was then euthanized after 10 days and the engraftment of the transplanted cells was evaluated. Specifically, the lungs were excised, tissue sections were prepared from the transplanted site, and fluorescent immunohistochemical staining was performed using anti-CD31 antibody (BD Biosciences) and anti-E-cadherin antibody (Thermo Fisher Scientific).

[0049] 7-2 Results Figure 9 shows the results of E-cadherin immunostaining, and Figure 10 shows the results of CD31 immunostaining. In Figures 9 and 10, the lower panels are enlarged views of the upper panels. Figure 9 shows that tdTomato-labeled cells expressed E-cadherin and engrafted as alveolar epithelial cells. Figure 10 shows that ZsGreen1-labeled cells surrounding the tdTomato-labeled cells expressed CD31 and engrafted as vascular endothelial cells. These results demonstrate that transplanting vascular endothelial stem cells into the lungs of pulmonary fibrosis model mice can proliferate type 2 alveolar epithelial cells that remain in the injured lung and have the ability to divide.

[0050] The present invention is not limited to the above-described embodiments and examples, and various modifications are possible within the scope of the claims. The technical scope of the present invention also includes embodiments obtained by appropriately combining the technical means disclosed in different embodiments. Furthermore, all academic literature and patent documents described in this specification are incorporated herein by reference.

Claims

1. Lung regeneration medicine containing vascular endothelial stem cells.

2. The pharmaceutical composition according to claim 1, which contains vascular endothelial stem cells as an active ingredient and is used so that the vascular endothelial stem cells are delivered to the lungs.

3. The pharmaceutical according to claim 1, wherein the vascular endothelial stem cells are CD157-positive vascular endothelial cells.

4. The pharmaceutical according to claim 1, wherein the vascular endothelial stem cells are vascular endothelial cell protein C receptor-positive vascular endothelial cells.

5. The pharmaceutical composition according to claim 1, wherein the vascular endothelial stem cells are CD157-positive and vascular endothelial cell protein C receptor-positive vascular endothelial cells.

6. The pharmaceutical according to any one of claims 1 to 5, wherein the vascular endothelial stem cells are pulmonary vascular endothelial stem cells.

7. The pharmaceutical according to any one of claims 1 to 5, wherein the vascular endothelial stem cells are human vascular endothelial stem cells.

8. The pharmaceutical composition according to any one of claims 1 to 5, for use in treating pulmonary diseases in which the alveoli are damaged.

9. The pharmaceutical composition according to claim 8, wherein the pulmonary disease in which alveoli are damaged is chronic obstructive pulmonary disease, infectious pulmonary disease, or pulmonary fibrosis.

10. A method for producing alveolar organoids, comprising three-dimensional co-culturing of vascular endothelial stem cells and alveolar epithelial cells.

11. The method of claim 10, wherein the vascular endothelial stem cells are CD157-positive vascular endothelial cells.

12. The method of claim 10, wherein the vascular endothelial stem cells are vascular endothelial cell protein C receptor-positive vascular endothelial cells.

13. The production method according to claim 10, wherein the vascular endothelial stem cells are CD157-positive and vascular endothelial cell protein C receptor-positive vascular endothelial cells.

14. The production method according to any one of claims 10 to 13, wherein the three-dimensional co-culturing step further comprises the inclusion of fibroblasts.

15. The method of any one of claims 10 to 13, wherein the vascular endothelial cells are pulmonary vascular endothelial cells.

16. The method of any one of claims 10 to 13, wherein the vascular endothelial cells are human vascular endothelial cells.

17. The method of claim 14, wherein the fibroblasts are pulmonary fibroblasts.

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