Fibroblasts with enhanced erythropoietin production ability, production method, and pharmaceutical composition

Abstract

It was found that by culturing human fibroblasts using a culture medium supplemented with TNF-α and IL-4, the EPO production capacity of the fibroblasts was improved. As a result, a fibroblast with enhanced EPO production capacity was discovered, and a method for using the cells to improve a state of decreased EPO production capacity and a method for treating renal damage or renal anemia were also discovered.

Description

Technical Field

[0001] The present invention relates to a fibroblast with enhanced erythropoietin (hereinafter also referred to as EPO) production ability, a method for producing the cell, and a pharmaceutical composition containing the cell. Background Art

[0002] EPO is a hormone that helps promote the production of red blood cells. Most of EPO is produced in the kidneys, and then acts on erythroblast progenitor cells in the bone marrow to promote the production (differentiation and proliferation) of red blood cells.

[0003] In recent years, the ability of EPO to promote red blood cell production has attracted attention, and it has been found to be effective for kidney diseases such as renal anemia (Non-Patent Document 1). In fact, EPO preparations are widely used as therapeutic agents for renal anemia in the medical field.

[0004] It is also known that EPO has anti-inflammatory, antioxidant, anti-apoptotic, and angiogenesis-promoting effects in various tissues. There are also reports that EPO can protect the kidneys from the decline in renal function caused by renal injury by administering it to subjects with renal injury (acute kidney injury (AKI), chronic kidney disease (CKD), diabetic nephropathy, etc.) (Non-Patent Documents 2 and 3).

[0005] In addition, the inventors have previously found that fibroblasts expressing vascular cell adhesion molecule-1 (VCAM-1, CD106) are useful as cardiac cell culture materials (Patent Document 1), and that cardiac fibroblasts expressing VCAM-1 are useful as injectable compositions for treating cardiac diseases (Patent Document 2). However, the usefulness of fibroblasts expressing VCAM-1 for purposes other than cardiac cell culture materials, or the usefulness of injectable compositions containing fibroblasts expressing VCAM-1 for diseases other than cardiac diseases, is still unclear.

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent document 1: International Publication No. 2016 / 006262.

[0009] Patent document 2: International Publication No. 2018 / 155651.

[0010] Non-patent literature

[0011] Non-patent document 1: Nephron, 1997; 77(2): 176-185.

[0012] Non-patent document 2: Proc Natl Acad Sci., 2004; 101(41): 14907-14912.

[0013] Non-patent literature 3: J Renal Inj Prev., 2013; 2(1): 29-30. Summary of the invention

[0014] Problem that the invention aims to solve

[0015] An object of the present invention is to provide a fibroblast having enhanced EPO production ability and a method of improving a state in which the EPO production ability is decreased using the fibroblast.

[0016] Means used to solve problems

[0017] The present inventors conducted studies to solve the above problems and found that fibroblasts with enhanced EPO production ability can be obtained by culturing human fibroblasts in a medium supplemented with TNF-α and IL-4, thereby completing the present invention.

[0018] That is, the present invention includes the following first aspect (Invention A).

[0019] (A1) A fibroblast with enhanced EPO production ability.

[0020] (A2) The fibroblasts according to (A1), which are CD106-positive fibroblasts.

[0021] (A3) The cell according to (A1) or (A2), wherein the fibroblasts are kidney fibroblasts.

[0022] (A4) The cell according to any one of (A1) to (A3), wherein the amount of EPO produced by the cell is 1.65 times or more higher than that of a control fibroblast.

[0023] (A5) A fibroblast population comprising the cells according to any one of (A1) to (A4).

[0024] (A6) The cell population according to (A5), wherein the ratio of the CD106-positive fibroblasts to all fibroblasts contained in the cell population (based on the number of cells) is 4.7% or more.

[0025] In addition, the present invention includes the following second aspect (Invention B).

[0026] (B1) A method for producing fibroblasts with enhanced EPO production ability, comprising:

[0027] The fibroblasts are contacted with TNF-α and IL-4; and the contacted fibroblasts are cultured under conditions where the EPO production capacity can be enhanced.

[0028] (B2) The method according to (B1), wherein the fibroblasts with enhanced EPO production ability are CD106-positive fibroblasts.

[0029] (B3) The method according to (B2), further comprising selecting or concentrating CD106-positive fibroblasts using an anti-CD106 antibody.

[0030] (B4) The method according to any one of (B1) to (B3), wherein the fibroblasts are kidney fibroblasts.

[0031] (B5) A fibroblast with enhanced EPO production ability, which can be obtained by the production method according to any one of (B1) to (B4).

[0032] (B6) A fibroblast population comprising the cells described in (B5).

[0033] In addition, the present invention includes the following third aspect (Invention C).

[0034] (C1) A pharmaceutical composition comprising the cell according to any one of (A1) to (A4) and (B5) or the cell population according to any one of (A5), (A6) and (B6).

[0035] (C2) The pharmaceutical composition according to (C1), which is used to improve a state in which EPO production capacity is reduced.

[0036] (C3) The pharmaceutical composition according to (C1), which is used for administration to patients suffering from renal damage.

[0037] (C4) The pharmaceutical composition according to (C3), wherein the renal injury is chronic kidney disease (CKD).

[0038] (C5) The pharmaceutical composition according to (C1), which is used for administration to a patient suffering from renal anemia.

[0039] (C6) The pharmaceutical composition according to any one of (C1) to (C5), which is a composition for injection.

[0040] (C7) The pharmaceutical composition according to any one of (C1) to (C5), wherein the cell population is constructed as a planar or three-dimensional cell tissue.

[0041] In addition, the present invention includes the following fourth aspect (Invention D).

[0042] (D1) A method for improving a subject's decreased erythropoietin production ability, comprising administering or transplanting into the subject a cell described in any one of (A1) to (A4) and (B5), a cell population described in any one of (A5), (A6) and (B6), or a composition described in (C1).

[0043] (D2) A method for treating renal damage in a subject, comprising administering or transplanting into the subject the cell described in any one of (A1) to (A4) and (B5), the cell group described in any one of (A5), (A6) and (B6), or the composition described in (C1).

[0044] (D3) A method for treating chronic kidney disease (CKD) in a subject, comprising administering or transplanting into the subject a cell described in any one of (A1) to (A4) and (B5), a cell group described in any one of (A5), (A6) and (B6), or a composition described in (C1).

[0045] (D4) A method for treating renal anemia in a subject, comprising administering or transplanting the cell described in any one of (A1) to (A4) and (B5), the cell group described in any one of (A5), (A6) and (B6), or the composition described in (C1) to the subject.

[0046] (D5) A method of administering EPO to a subject, comprising administering or transplanting fibroblasts with enhanced EPO production capacity to the subject.

[0047] In addition, the present invention includes the following fifth aspect (Invention E).

[0048] (E1) Using the cell described in any one of (A1) to (A4) and (B5), the cell population described in any one of (A5), (A6) and (B6), or the composition described in (C1) as an injection for administration to kidney tissue or its surrounding area.

[0049] (E2) Using the cell described in any one of (A1) to (A4) and (B5), the cell group described in any one of (A5), (A6) and (B6), or the composition described in (C1) as a transplant material for transplantation into kidney tissue or its surrounding area.

[0050] Effects of the Invention

[0051] According to the present invention, it is possible to provide a fibroblast with enhanced EPO production ability, and also to provide a method for improving a state in which the EPO production ability is reduced and a method for treating renal damage or renal anemia using the fibroblast. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1A The figures show the characteristics of kidney fibroblasts. A is a microscopic photograph of cultured kidney fibroblasts (scale bar: 500 μm).

[0053] Figure 1B A is a diagram showing the characteristics of renal fibroblasts. B is a flow cytometry analysis diagram showing the expression of actin alpha 2, smooth muscle (α-SMA), CD106, and EPO. Gating was performed on CD106-positive areas in non-treated (NonTreatment (NT)) cells or cells to which TNF-α and IL-4 (+TNF-a and IL-4 (UP) were added, and the gated cells were defined as CD106-positive NT (CD106-positive in NT: CD106-positive in NT) cells or CD106-positive UP (CD106-positive in UP: CD106-positive in UP) cells. The gray histogram shows the fluorescence intensity of the isotype control antibody as a negative control, and the black histogram shows the fluorescence intensity of the labeled antibody for α-SMA, CD106, or EPO.

[0054] Figure 2 This is a flow cytometry analysis diagram that gates the EPO-positive region in untreated (untreated (NT)) cells or cells with TNF-α and IL-4 (+TNF-α and IL-4 (UP)) to investigate the CD106 positivity in gated cells. The gray histogram shows the fluorescence intensity of the isotype control antibody as a negative control, and the black histogram shows the fluorescence intensity of the labeled antibody for CD106 or EPO.

[0055] Figure 3It is a figure showing the relative comparison of the EPO content in various fibroblast culture supernatants. A sets the EPO production in untreated (untreated (NT)) cells to 1, and expresses the EPO production in TNF-α and IL-4 added (UP) cells as relative values. B shows the EPO content (average optical density Average OD) in various fibroblast culture supernatants. C shows a calibration curve. D shows the EPO content (mIU / mL) in various fibroblast culture supernatants. DETAILED DESCRIPTION

[0056] Hereinafter, the present invention will be described in detail using specific embodiments, but the present invention is not limited to the specific embodiments shown.

[0057] One embodiment of the present invention is a fibroblast with enhanced EPO production ability.

[0058] The fibroblasts of the present embodiment are preferably CD 106 positive. CD 106 is also called VCAM-1, and is a protein known as a cell adhesion molecule expressed in vascular endothelial cells and the like.

[0059] The source of fibroblasts is not limited, and pluripotent stem cells such as embryonic stem cells (ES cells), artificial pluripotent stem cells (iPS cells) and Muse cells or adult stem cells such as mesenchymal stem cells can be differentiated and used. In addition, primary cells collected from animals (including humans) can be used, and cell strains can also be used. Fibroblasts are preferably derived from kidneys.

[0060] Among human-derived fibroblasts, the proportion of CD106-positive fibroblasts is low, and in the case of kidney-derived fibroblasts in a natural state, the proportion is only about 4.7% (based on the number of cells) at most.

[0061] Therefore, a fibroblast population from humans in which the proportion of CD106-positive fibroblasts from humans is increased by the manufacturing method described later may also be included in the present embodiment. For example, as another embodiment of the present invention, it may be a fibroblast population from humans, that is, the proportion of CD106-positive fibroblasts from humans in all fibroblasts from humans (cell number basis) is 4.7% or more. In addition, in the fibroblast population from humans of the present embodiment, the proportion of CD106-positive fibroblasts to all fibroblasts (cell number basis) may be 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100%. In addition, by selecting cells known to be CD106-positive, the process of cell screening can be omitted.

[0062] In addition, the proportion of EPO-positive (hereinafter also referred to as EPO+) cells in human fibroblasts is low in the natural state, and in the case of kidney-derived fibroblasts, it is only about 40.7% (based on the number of cells) at most. In addition, since EPO is a hematopoietic hormone and not a cell membrane-expressed protein, it is difficult to select or concentrate cells using EPO as an indicator.

[0063] Therefore, for example, the proportion of EPO+ fibroblasts in the fibroblast population can be increased by selecting or concentrating CD106-positive fibroblasts from humans according to the method for manufacturing fibroblasts described later. Alternatively, the proportion of EPO+ fibroblasts in the fibroblast population can be increased by other methods. In the fibroblast population from humans of the present embodiment, the proportion of EPO+ fibroblasts to all fibroblasts (based on the number of cells) can be 40.7% or more, 45% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100%.

[0064] The enhancement of EPO production ability refers to, for example, compared with the EPO production in the fibroblast as a control, the EPO production increases. The fibroblast as a control is not particularly limited, and can be a natural kidney fibroblast. The assay method for comparing EPO production is not particularly limited, for example, the EPO positive cell ratio can be measured by flow cytometry as a method known to those skilled in the art, and the concentration of the EPO produced can also be measured by ELISA method (enzyme-linked immunosorbent assay).

[0065] When using flow cytometry, for example, when the proportion of EPO+ fibroblasts in the human fibroblast population of the present embodiment to all fibroblasts (based on cell number) is 40.7% or more, 45% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100%, it can be determined that the EPO production ability is enhanced.

[0066] When using the ELISA method, for example, when the amount of EPO contained in the culture supernatant of the human fibroblast group of this embodiment increases by 1.65 times or more, 2.0 times or more, 3.0 times or more, 5.0 times or more, or 10.0 times or more compared with the fibroblasts used as a control cultured under the same conditions, it can be judged that the EPO production ability is enhanced.

[0067] In addition, for example, 9.2×10 fibroblasts 5 The cells are cultured for 5 days and 3 mL of the culture supernatant is recovered. When the concentration of EPO in the culture supernatant concentrate concentrated 100 times by Amicon-Ultra-2 (centrifugal filter) or the like exceeds 5.08 mIU / mL, it can be judged that the EPO production ability is enhanced. When it is 5.10 mIU / mL or more, 6.00 mIU / mL or more, 7.00 mIU / mL or more, 8.00 mIU / mL or more, 8.39 mIU / mL or more, 10.0 mIU / mL or more, 20.0 mIU / mL or more, 50.0 mIU / mL or more, or 100.0 mIU / mL or more, it can also be judged that the EPO production ability is enhanced.

[0068] In addition, for example, 9.2×10 fibroblasts 5 The cells are cultured for 5 days. When the concentration of EPO in the unconcentrated culture supernatant after recovering 3 mL of the culture supernatant exceeds 5.08E-02mIU / mL, it can be judged that the EPO production ability is enhanced. When it is 5.10E-02mIU / mL or more, 6.00E-02mIU / mL or more, 7.00E-02mIU / mL or more, 8.00E-02mIU / mL or more, 8.39E-02mIU / mL or more, 10.0E-02mIU / mL or more, 20.0E-02mIU / mL or more, 50.0E-02mIU / mL or more, or 100.0E-02mIU / mL or more, it can also be judged that the EPO production ability is enhanced.

[0069] In addition, for example, 9.2×10 fibroblasts 5The cells are cultured for 5 days. When the amount of EPO in the unconcentrated culture supernatant after recovering 3 mL of the culture supernatant exceeds 1.52E-01mIU per 1 dish (3 mL), it can be judged that the EPO production ability is enhanced. When it is 1.60E-01mIU or more, 2.00E-01mIU or more, 2.52E-01mIU or more, 3.00E-01mIU or more, 4.00E-01mIU or more, 5.00E-01mIU or more, 10.0E-01mIU or more, 20.0E-01mIU or more, or 50.0E-01mIU or more, it can also be judged that the EPO production ability is enhanced.

[0070] In addition, for example, 9.2×10 fibroblasts 5 The cells are cultured for 5 days. When the EPO production amount per cell in the unconcentrated culture supernatant after recovering 3 mL of the culture supernatant exceeds 1.66E-07mIU, it can be judged that the EPO production ability is enhanced. When it is 1.70E-07mIU or more, 1.80E-07mIU or more, 1.90E-07mIU or more, 2.00E-07mIU or more, 2.74E-07mIU or more, 3.00E-07mIU or more, 5.00E-07mIU or more, 10.0E-07mIU or more, or 20.0E-07mIU or more, it can also be judged that the EPO production ability is enhanced.

[0071] The present embodiment may also be a fibroblast population containing fibroblasts with enhanced EPO production ability. The fibroblasts are preferably CD106 positive.

[0072] The cell population of this embodiment may contain other fibroblasts or other components in addition to CD106-positive fibroblasts. When containing other fibroblasts, the proportion of CD106-positive fibroblasts relative to the total number of cells in the cell population may be 0.03% or more, 0.1% or more, 1% or more, 2% or more, 4% or more, 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, 98% or more, or 99% or more based on the number of cells.

[0073] The method for producing fibroblasts will be described below. Fibroblasts can be produced by referring to and following the previous report by the present inventors (Patent Document 1).

[0074] (Fibroblast Preparation)

[0075] In the preparation of fibroblasts, the source of fibroblasts is not particularly limited, and can be as described. On the other hand, it can be cells from oneself, such as preparing the following fibroblasts, that is, the kidney fibroblasts separated from the kidney tissue of the patient suffering from kidney disease or the kidney fibroblasts differentiated and separated by the adult (body) stem cells of the patient. In addition, it can also be the fibroblasts differentiated and recovered by the pluripotent stem cells such as the iPS cells from the self cells. In addition, it can be cells other than oneself, such as preparing the following fibroblasts, that is, the kidney fibroblasts separated from the kidney tissue of the donor providing kidney cells or the kidney fibroblasts differentiated and separated by the adult (body) stem cells of the donor. In addition, it can also be the fibroblasts differentiated and recovered by the pluripotent stem cells such as the iPS cells or ES cells from the donor.

[0076] (Isolation of Fibroblasts)

[0077] The prepared fibroblasts can be separated by treating them with enzymes such as dispase and collagenase. In addition to using enzymes such as dispase and collagenase, other treatments such as mechanical treatments may be used if the desired separation can be achieved.

[0078] (Selection or enrichment of CD106 positive fibroblasts)

[0079] In the case where fibroblasts are CD106 positive, fibroblasts can be provided for the selection or concentration of CD106 positive fibroblasts. In the case where CD106 positive fibroblasts can be obtained without selection or concentration, selection or concentration can be omitted. For the selection or concentration of CD106 positive fibroblasts, flow cytometry, magnetic bead method, affinity column method or panning method using anti-CD106 antibody (anti-VCAM-1 antibody) can be exemplified. Specifically, magnetic bead cell sorting (MACS) or fluorescence activated cell sorting (FACS) can be used. Anti-CD106 antibodies can use commercially available antibodies, and antibodies made using known methods can also be used. In addition, monoclonal antibodies or polyclonal antibodies can be used, but from the perspective of specificity, monoclonal antibodies are preferably used. In addition, CD106 negative fibroblasts can also be removed by introducing drug resistance genes, thereby obtaining CD106 positive fibroblasts. In addition, fluorescent protein encoding genes can also be introduced, and fluorescent protein positive cells are separated with FACS, etc. Alternatively, real-time PCR, next generation sequencer, in situ hybridization, and the like can be used to confirm the expression of the CD106 gene in cells and to isolate the CD106 gene expression group.

[0080] (Fibroblast Culture)

[0081] Fibroblasts can be cultured until a desired cell number is reached and / or until a desired function is acquired, etc. The culture conditions are not limited, and the culture can be carried out by a known cell culture method.

[0082] The culture medium used for the culture can be appropriately set according to the type of cells to be cultured, for example, DMEM, α-MEM, RPMI-1640, HFDM-1 (+), etc. can be used. Nutrients such as FCS or FBS, growth factors, cytokines, antibiotics, etc. can be added to the culture medium. In addition, culture can also be carried out using a culture medium containing TNF-α and / or IL-4.

[0083] The number of days of the culture cycle can be appropriately set according to the purpose of, that is, until the cultured cells reach a desired cell number and / or until the cultured cells have a desired function, etc. For example, the cycle may be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 2 weeks, 1 month, 2 months, 3 months, 6 months, etc.

[0084] The culture temperature can be appropriately set according to the type of cells to be cultured, for example, it can be above 10°C, above 15°C, above 20°C, above 25°C, above 30°C, or below 60°C, below 55°C, below 50°C, below 45°C, or below 40°C.

[0085] The culture may be performed multiple times. For example, the purity of the desired fibroblasts may be increased by selection or concentration and culture may be performed each time.

[0086] (Fibroblast recovery)

[0087] The cultured fibroblasts can be recovered by detaching the cells using proteases such as trypsin, by using a temperature-responsive culture dish to detach the cells by temperature changes, or by using an instrument such as a cell scraper to detach the cells.

[0088] Another aspect of the present invention is a method for producing fibroblasts with enhanced EPO production ability, the method comprising contacting fibroblasts with TNF-α and IL-4, and culturing the contacted fibroblasts under conditions where the EPO production ability can be enhanced.

[0089] The fibroblasts are preferably CD106 positive. In this case, the production method of this embodiment may include selecting or concentrating CD106 positive fibroblasts using an anti-CD106 antibody.

[0090] The method for contacting fibroblasts with TNF-α and IL-4 is not particularly limited as long as the induction of fibroblasts with enhanced EPO production ability or the induction of CD106 positive fibroblasts with enhanced EPO production is not significantly damaged. A typical approach is to add TNF-α and IL-4 to a culture medium containing fibroblasts, but is not limited thereto. TNF-α and IL-4 can either contact fibroblasts separately or simultaneously. The method for contacting TNF-α and IL-4 simultaneously can be to mix TNF-α and IL-4 and then add them to the fibroblasts. For example, TNF-α and IL-4 can be dissolved in water, culture medium, serum or dimethyl sulfoxide (DMSO) and then added to the culture medium.

[0091] In this embodiment, since fibroblasts can be cultured in a medium supplemented with TNF-α and IL-4, the fibroblasts can be cultured while maintaining the expression level of CD106 in the fibroblasts. Therefore, fibroblasts having a high CD106 expression level can be produced.

[0092] When TNF-α and IL-4 are added to the culture medium (mL), the amount of TNF-α added is not particularly limited, and is usually 0.1 ng / mL or more, can be 0.5 ng / mL or more, can be 1 ng / mL or more, can be 10 ng / mL or more, can be 50 ng / mL or more. The upper limit is not particularly limited, and is usually 500 ng / mL or less, and can be 100 ng / mL or less.

[0093] In addition, the amount of IL-4 added is not particularly limited, and is usually 0.1 ng / mL or more, and can be 0.5 ng / mL or more, and can be 1 ng / mL or more, and can be 2 ng / mL or more. The upper limit is not particularly limited, and is usually 10 ng / mL or less, and can be 5 ng / mL or less, and can be 1 ng / mL or less.

[0094] The ratio (weight ratio) of TNF-α to IL-4 added is usually 10000:1 to 1:1, and may be 50000:1 to 10:1. Alternatively, it may be 1:1 to 1:10000, or 1:10 to 1:50000.

[0095] By further culturing the fibroblasts in contact with TNF-α and IL-4, fibroblasts with enhanced EPO production ability can be produced.

[0096] For the cultivation of fibroblasts, there is no particular restriction as long as the EPO production ability of fibroblasts can be enhanced, and it can be carried out by known cell culture methods. In addition, it is preferred to cultivate under the condition that CD106 is positive. The method described in the item of (culture of fibroblasts) in the manufacture method of the above-mentioned fibroblasts can be illustrated.

[0097] The recovery of the cultured fibroblasts may be exemplified by the method described in the section (Recovery of Fibroblasts) in the above-mentioned method for producing fibroblasts.

[0098] When the fibroblasts are CD106 positive, the selection or concentration of CD106 positive fibroblasts can be performed by using an anti-CD106 antibody. The selection or concentration can be performed at any time. It can be before contact with TNF-α and IL-4, after contact with TNF-α and IL-4, before culture, or after culture.

[0099] Examples include the method described in the section (Selection and enrichment of CD106-positive fibroblasts) in the above-mentioned method for producing fibroblasts.

[0100] By selecting or enriching with an anti-CD106 antibody, the proportion of CD106-positive fibroblasts in fibroblasts can be increased, thereby obtaining a cell population containing CD106-positive fibroblasts with enhanced EPO production ability that has a high disease therapeutic effect.

[0101] By the manufacturing method of the fibroblast with enhanced EPO production capacity of the present embodiment, the fibroblast with enhanced EPO production capacity can be obtained, and the fibroblast or the fibroblast group containing the cell is another embodiment of the present invention. Because fibroblasts, especially kidney fibroblasts, are stimulated by TNF-α and IL-4, the ratio of CD106 expressing cells (cell number basis) increases, and the ratio of EPO expressing cells (cell number basis) increases, so the fibroblast with enhanced EPO production capacity can be suitable for use as a composition for treating diseases with decreased EPO production.

[0102] Another aspect of the present invention is a pharmaceutical composition comprising the above-mentioned fibroblasts with enhanced EPO production ability or a cell population thereof. The fibroblasts are preferably CD106 positive.

[0103] By administering or transplanting the pharmaceutical composition of the present embodiment into the organism, EPO can be efficiently produced in the organism. Therefore, although there is no particular limitation, it is possible to improve the state of decreased EPO production capacity caused by damage, defect, and dysfunction of renal tissue, for example. In addition, it can be a mode in which administration can be performed to patients with renal injury or renal anemia, or it can be a mode in which renal injury or renal anemia can be treated thereby. As renal injury, there is no particular limitation, and for example, acute renal injury (AKI), chronic kidney disease (CKD), diabetic nephropathy, acute glomerulonephritis (acute nephritis), chronic glomerulonephritis (chronic nephritis), rapidly progressive glomerulonephritis, nephrotic syndrome, acute renal failure, and chronic renal failure can be cited. Preferably, renal injury is chronic kidney disease (CKD). More preferably, renal injury is chronic renal failure. In addition, renal failure refers to a state in which renal function decreases compared to normal, and in the present embodiment, it specifically refers to a state in which renal function decreases to less than 30% compared to normal.

[0104] The method of administering or transplanting the pharmaceutical composition of the present embodiment to a biological body is not particularly limited, for example, it can be administered by injection. The administration site of the pharmaceutical composition is not particularly limited, for example, by directly administering or transplanting the pharmaceutical composition to kidney tissue and / or its surrounding area (for example, under the kidney capsule, etc.), it is possible to improve the above-mentioned disease state or treat renal anemia. In addition, the pharmaceutical composition can also be administered to vein, artery, lymph node, lymphatic vessel, bone marrow, subcutaneous, corpus cavernosum, and intraperitoneal administration. Administration to vein, artery, lymphatic vessel can be by injection into the vascular, can also be injected by catheter, and other known methods can also be used.

[0105] The method of injection is not particularly limited, and known injection methods such as needle injection and needle-free injection can be applied. The method of the catheter used for injection can also be a known method and is not particularly limited.

[0106] In addition, although the pharmaceutical composition of the present embodiment is not particularly limited, it can also be applicable to organisms as artificial organ materials, for example, the artificial organ material can be a cell group constructed as a planar or three-dimensional cell organization. The planar or three-dimensional cell organization can be formed by culturing a fibroblast group alone, or it can be formed by culturing a fibroblast group and other cells such as kidney cells together as a planar or three-dimensional cell organization. As a planar or three-dimensional cell organization, there is no particular limitation, for example, cell sheets, cell fibers, tissues formed using a 3D printer, etc. can be exemplified.

[0107] In this embodiment, the size or shape of the planar or three-dimensional cell tissue is not particularly limited as long as it is suitable for the necrotic kidney tissue and / or its surrounding area. In addition, the so-called planar shape can be either single-layer or multi-layer.

[0108] In addition, the conditions such as the composition of the culture medium, temperature, humidity, etc. in the cell culture are not particularly limited as long as they can cultivate the fibroblasts with enhanced EPO production ability and can construct a planar or three-dimensional cell tissue. For example, the conditions of the culture conditions described in the items of (culturing of fibroblasts) and / or (recovery of fibroblasts) in the manufacturing method of the above-mentioned fibroblasts can be exemplified.

[0109] By applying such a planar or three-dimensional cell tissue to necrotic renal tissue and / or its surrounding area, or by exchanging it with the necrotic renal tissue and / or its surrounding area as an artificial organ, renal damage or renal anemia can be treated.

[0110] The ratio of the fibroblasts with enhanced EPO production ability contained in the pharmaceutical composition can be appropriately set based on the method of administration or transplantation to a patient, as long as the fibroblasts with enhanced EPO production ability are contained as an active ingredient in a therapeutically effective amount.

[0111] When other fibroblasts are contained in the pharmaceutical composition, the proportion of fibroblasts with enhanced EPO production ability relative to the total amount of fibroblasts contained in the pharmaceutical composition can be 0.03% or more, 0.1% or more, 1% or more, 2% or more, 4% or more, 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, 98% or more, or 99% or more based on the number of cells.

[0112] In addition, the number of fibroblasts with enhanced EPO production ability contained in the pharmaceutical composition can be, for example, 1.0×10 2 cells / mL or more, 1.0×10 3 cells / mL or more, 1.0×10 4 cells / mL or more, 1.0×10 5 cells / mL or more, 1.0×10 6 cells / mL or more, 5.0×10 6 cells / mL or more, 1.0×10 7 The number of fibroblasts with enhanced EPO production ability contained in the composition for injection can be further increased or decreased according to the severity of the disease.

[0113] The method for confirming the above-mentioned therapeutic and / or improving effects of the pharmaceutical composition is not particularly limited. For example, an increase in the blood EPO concentration after administration or transplantation of the composition compared to before administration or transplantation of the composition, or an increase in the number of red blood cells after administration or transplantation of the composition compared to before administration or transplantation of the composition can be shown.

[0114] The method for confirming the above-mentioned therapeutic and / or improving effects of the pharmaceutical composition is not particularly limited. For example, it can be illustrated by a decrease in the blood creatinine (Cr) value after administration or transplantation of the composition compared to before administration or transplantation of the composition, or a decrease in the blood urea nitrogen (BUN) value after administration or transplantation of the composition compared to before administration or transplantation of the composition.

[0115] The method for confirming the above-mentioned therapeutic and / or improving effects of the pharmaceutical composition is not particularly limited. For example, it can be shown that the endogenous creatinine clearance (CCr) value after administration or transplantation of the composition is increased compared with that before administration or transplantation of the composition, or that the glomerular filtration rate (GFR) value is increased compared with that before administration or transplantation of the composition.

[0116] The method for confirming the above-mentioned therapeutic and / or improving effects of the pharmaceutical composition is not particularly limited. For example, it can be illustrated by a decrease in urine protein in the urine after administration or transplantation of the composition compared to before administration or transplantation of the composition, or a decrease in urine occult blood compared to before administration or transplantation of the composition.

[0117] The pharmaceutical composition may further contain other physiologically acceptable components as a pharmaceutical composition. Examples of such other components include physiological saline, cell preservation solution, cell culture solution, hydrogel, extracellular matrix, and cryopreservation solution.

[0118] As another embodiment of the present invention, it can also be a method for improving the state of decreased EPO production ability in the following manner, that is, by administering or transplanting the above-mentioned fibroblasts with enhanced EPO production ability, their cell groups, or a pharmaceutical composition containing the fibroblasts or their cell groups to a subject suffering from a state of decreased EPO production ability, thereby improving the state of decreased EPO production ability. The fibroblasts of this embodiment are preferably CD106 positive.

[0119] In addition, as another embodiment of the present invention, it can also be a method for treating renal injury or protecting the kidney in the following manner, that is, by administering or transplanting the above-mentioned fibroblasts with enhanced EPO production ability, their cell groups, or a pharmaceutical composition containing the fibroblasts or their cell groups to a subject suffering from renal injury, thereby treating renal injury or protecting the kidney. The fibroblasts of this embodiment are preferably CD106 positive.

[0120] In addition, as another embodiment of the present invention, it can also be a method for treating renal anemia in the following manner, that is, by administering or transplanting the above-mentioned fibroblasts with enhanced EPO production ability, their cell group, or a pharmaceutical composition containing the fibroblasts or their cell group to a subject suffering from renal anemia. The fibroblasts of this embodiment are preferably CD106 positive.

[0121] In addition, as another embodiment of the present invention, the fibroblasts with enhanced EPO production ability, their cell groups, or compositions containing the fibroblasts or their cell groups can also be used as injections or transplant materials for administration or transplantation to kidney tissue and / or its surrounding areas. The fibroblasts of this embodiment are preferably CD106 positive.

[0122] [Example]

[0123] The present invention will be described in more detail by the following examples, but it should be understood that the scope of the present invention is not limited to the examples.

[0124] <Cultivation of human adult kidney fibroblasts>

[0125] Human adult kidney fibroblasts were purchased from Innoprot. The cells were cultured in serum-free fibroblast medium (HFDM-1(+), Cell Science & Technology) containing recombinant human epidermal growth factor (rh-EGF) supplemented with 1% newborn calf serum (NBCS, Hyclone). After 3 to 5 passages, the fibroblasts were treated with 50 ng / mL TNF-α (Pepro tech) and 2 ng / mL IL-4 (Wako).

[0126] <Flow cytometry analysis>

[0127] Table 1 shows the antibodies and reagents used in flow cytometry analysis. Cells were fixed with 4% paraformaldehyde (Wako) and allowed to stand in PBS (Phosphate buffer saline: buffer solution) (Wako). Permeabilization was performed with 0.1% saponin and reaction was performed with various antibodies for 30 minutes each. Flow cytometry analysis was performed using MACSQuant Analyzer10 (Miltenyi Biotec) according to the manufacturer's instructions.

[0128]

Table 1

[0129]

[0130]

[0131] <Evaluation of erythropoietin production ability>

[0132] Untreated human renal fibroblasts (NT) and human renal fibroblasts (UP) treated with TNF-α (50 ng / mL) and IL-4 (2 ng / mL) for 3 days were cultured at 9.2×10 5 The concentration of cells / dish is inoculated to Culture dishes (Corning, Corning, NY) were cultured with serum-free DMEM medium for 5 days, and 3 mL of the culture supernatant of fibroblasts was recovered respectively. Amicon-Ultra-2 (centrifugal filter) (Merck, Darmstadt, Germany) and Human Erythropoietin ELISA Kit (human erythropoietin ELISA kit) (Proteintech, Rosemont, IL) were used to prepare the supernatant samples according to the manufacturer's recommended protocol. The determination was implemented using SpectraMax M2 (multi-function microplate reader) (San Jose, CA). The EPO production (mIU) before concentration by Amicon-Ultra-2 was calculated based on the minimum concentration volume (15 μL) recorded in the product data sheet. Specifically, the EPO production (mIU) was calculated by dividing the EPO production (mIU / mL) after concentration by the concentration rate calculated from the above minimum volume (100 times, assuming the concentration rate when the relative filling volume is 1.5 mL concentrated to 15 μL), and then multiplying by the total amount of culture medium per dish (3 mL). The EPO production per cell (mIU / cell) was calculated by dividing the EPO production (mIU) by the number of cells inoculated (9.2×10 5 cells) to calculate.

[0133] <Example 1: Enhancement of EPO expression in CD106 (VCAM-1) positive renal fibroblasts by combined use of TNF-α and IL-4>

[0134] Human adult kidney fibroblasts were cultured in a cell culture dish. Human adult kidney fibroblasts are flat and spindle-shaped, which is a typical fibroblast shape ( Figure 1A ).

[0135] After treating renal fibroblasts with 50 ng / mL of TNF-α and 2 ng / mL of IL-4 for 3 days, the cell characteristics were analyzed using FACS. The antibodies used were antibodies against α-SMA, CD106, or EPO. After cell membrane permeabilization with 0.1% Saponin, staining of proteins localized in the cytoplasm was performed.

[0136] In the untreated renal fibroblasts, α-SMA, a fibroblast marker, was expressed in 66.54% of the cells. On the other hand, in the renal fibroblasts, the proportion of CD106-positive cells was low (4.70% based on the number of cells), and the proportion of EPO+ cells was also low (40.68% based on the number of cells) ( Figure 1B ). If CD106-positive cells are gated and analyzed, the proportions of cells expressing α-SMA and EPO (based on the number of cells) are 97.94% and 87.92%, respectively. In other words, it is shown that the expression ratios of α-SMA and EPO in CD106-positive renal fibroblasts are high (based on the number of cells). This result shows that, in the natural state, it is difficult to efficiently select or concentrate EPO+ cells using known methods using anti-CD106 antibodies ( Figure 1B ).

[0137] In addition, by treating renal fibroblasts with TNF-α and IL-4 for 3 days, the proportion of CD106 positive cells increased to 47.50% (based on cell number), and the proportion of α-SMA+ cells increased to 78.71% (based on cell number). Although the proportion of EPO+ cells in the treated renal fibroblasts was 41.15% (based on cell number), the proportion of EPO+ cells in CD106 positive cells was 81.82% (based on cell number). Figure 1B ). That is, it was shown that even after CD106 expression was induced by treatment with TNF-α and IL-4, the expression ratios of α-SMA and EPO (based on the number of cells) in CD106-positive renal fibroblasts became high.

[0138] <Example 2: Increase in CD106-positive rate in EPO-expressing renal fibroblasts by combined use of TNF-α and IL-4>

[0139] Human adult kidney fibroblasts were cultured in a cell culture dish. The kidney fibroblasts were treated with 50ng / mL TNF-α and 2ng / mL IL-4 for 3 days, and their cell characteristics were analyzed using FACS. The antibodies used were antibodies against CD106 or EPO.

[0140] In the untreated renal fibroblasts, the ratio of EPO+ cells was 37.24% based on the number of cells, of which the ratio of CD106-positive cells was 47.78%. On the other hand, in the renal fibroblasts treated with TNF-α and IL-4 for 3 days, the ratio of EPO+ cells was 35.13% based on the number of cells, of which the ratio of CD106-positive cells was 82.86%, which is a very high ratio of CD106-positive cells.

[0141] That is, it is shown that by purifying renal fibroblasts by a known method using an anti-CD106 antibody, it is possible to efficiently obtain highly purified CD106-positive fibroblasts with enhanced EPO production capacity. Based on these results, it is suggested that by selectively making EPO-producing renal fibroblasts positive for CD106 using TNF-α and IL-4, it is possible to efficiently select or concentrate CD106-positive fibroblasts with enhanced EPO production capacity.

[0142] <Example 3: Evaluation of erythropoietin production ability of various fibroblasts>

[0143] To evaluate the erythropoietin-producing capacity of untreated human renal fibroblasts (NT) and human renal fibroblasts (UP) treated with TNF-α (50 ng / mL) and IL-4 (2 ng / mL) for 3 days, the erythropoietin content in the culture supernatant of each fibroblast was evaluated by ELISA ( Figure 3 The results showed that the culture supernatant of UP contained 1.65 times more EPO than the culture supernatant of NT ( Figure 3 In addition, the average optical density (Average.OD) of the culture supernatant of UP was 0.21, containing 8.39mIU / mL of EPO, while the average.OD of the culture supernatant of NT was 0.14, containing 5.08mIU / mL of EPO (after concentration) ( Figure 3 In addition, the culture supernatant of UP before concentration contained EPO at a concentration of 8.39E-02mIU / mL, 2.52E-01mIU, and 2.74E-07mIU / cell, while the culture supernatant of NT before concentration contained EPO at a concentration of 5.08E-02mIU / mL, 1.52E-01mIU, and 1.66E-07mIU / cell.

[0144] Based on the present results, it was found that renal fibroblasts produced by treatment with TNF-α and IL-4 not only had enhanced EPO expression but also improved production capacity, suggesting that by using TNF-α and IL-4 to selectively make EPO-producing renal fibroblasts CD106-positive, it is possible to efficiently select or concentrate CD106-positive fibroblasts with enhanced EPO production capacity.

[0145] Known EPO is a hormone that helps promote the production of red blood cells. Most of the EPO is produced in the kidney, and then acts on the erythroblast progenitor cells in the bone marrow, with the effect of promoting the production of red blood cells (differentiation and proliferation), and in recent years, the ability of promoting red blood cell production possessed by EPO has attracted attention, and it is found that it is effective for renal diseases such as renal anemia (non-patent literature 1). In fact, in the medical field, EPO preparations are widely used as therapeutic agents for renal anemia. In addition, it is known that EPO has anti-inflammatory, anti-oxidative, anti-apoptotic, and angiogenesis-promoting effects in various tissues, and it is also reported that EPO can bring the kidney protection function (non-patent literature 2 and non-patent literature 3) of protecting the kidney from the decline of renal function caused by renal injury by administering to the object suffering from renal injury (acute kidney injury (AKI), chronic kidney disease (CKD), diabetic nephropathy, etc.). That is, if in view of the effect of EPO known by these existing studies, it is revealed that the administration of CD106 positive fibroblasts with enhanced EPO production capacity artificially made according to the present invention is effective for renal injury.

Claims

1. A method for producing fibroblasts with enhanced erythropoietin production ability, wherein the fibroblasts are CD106-positive fibroblasts derived from kidneys, wherein: Based on the number of cells, the ratio of the CD106-positive fibroblasts to all the fibroblasts contained in the fibroblast population is 4.7% or more. The manufacturing method comprises: contacting fibroblasts with TNF-α at a concentration of 50 ng / mL to 100 ng / mL and IL-4 at a concentration of 2 ng / mL to 5 ng / mL; and The contacted fibroblasts are cultured for 3 or 4 days under conditions where the erythropoietin production capacity can be enhanced.

2. The manufacturing method according to claim 1, wherein: The method also includes using anti-CD106 antibodies to select or enrich CD106-positive fibroblasts.

3. A fibroblast with enhanced erythropoietin production ability, wherein: The method can be obtained by the production method according to claim 1 or 2.

4. A pharmaceutical composition, wherein Containing the fibroblasts according to claim 3.

5. Use of the fibroblasts according to claim 3 in the manufacture of a pharmaceutical composition for treating renal injury or renal anemia.

6. The use in manufacturing according to claim 5, wherein: The pharmaceutical composition is a composition for injection.

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