WO2014115912A1 - Pharmaceutical composition for preventing and treating immune diseases, including amniotic fluid stem cells or culture medium thereof - Google Patents

Abstract

The present invention provides a pharmaceutical composition for preventing and treating autoimmune diseases or immune dysfunction-related diseases, including amniotic fluid derived stem cells having an immunoregulatory function or a culture medium thereof.

Description

Pharmaceutical composition for preventing and treating immune diseases, including amniotic stem cells or culture medium thereof

The present invention provides a pharmaceutical composition for preventing and treating autoimmune diseases or immune disorders, including amniotic stem cells having a immunomodulatory function or a culture thereof.

Human Amniotic Fluid Cells (AFCs) have been used in prenatal genetic diagnosis to diagnose fetal malformations caused by fetal genetic changes during pregnancy (Hoehn H et al., Method Cell). Biol., (26) 11-34, 1982). The cells identified in the amniotic fluid are divided into adherent and non-adherent cells that form colonies in a general culture environment. Most of the studies on amniotic cells are carried out through adherent cells. Adherent amniotic cells are classified into epithelial cells (epitheloid E-type), amniotic fluid specific AF-type, and fibroblastic F-type according to morphological characteristics and biochemical criteria.

Amniotic cells have been used for neuronal differentiation and stem cell research since reports of amniotic cells that differentiate into trioderm cells and the expression of Oct-4 gene, a representative undifferentiated marker of stem cells, have been reported. (Hengstschlager, J. Rep. End., (2) 233-238, 2005, Prusa et al., Hum. Reprod. (18) 1489-1493, 2003).

The fact that amniotic cells have the capacity of stem cells is also evidenced by the report that telomerase activity, which proves stem cell proliferation, and long telomere appear in embryonic tissues and germ cells, in addition to Oct-4 gene expression. (Mosquera et al., (36) 494-496, 1999).

Stem cells isolated from amniotic cells maintained long telomere and normal karyotype during 250 population doubling, and even in differentiation experiments, adipocytes, osteoblasts, myocytes, endothelial cells, and neurons (neurocyte) and hepatocyte (differentiation) to hepatocytes were identified through mRNA gene expression analysis (De Coppi et al., Nat. Biotechnol (25) 100-106, 2007).

Amniotic fluid cells, however, are difficult to establish culture conditions, and may be sufficient to secure sufficient quantities of cells, the presence of various cells in the amniotic fluid and the unreported culture factors, the cessation of amniotic cell cycles, the state of cell differentiation capacity, and cellular aging. Despite the diverse functions of amniotic cells, the biomedical industry's use of amniotic cells is limited.

As a conventional technique using amniotic cells, Korean Patent Application No. 10-2009-0024054 discloses a method for mass production of growth factors using amniotic fluid-derived mesenchymal stem cells, and Korean Patent Application No. 10-2011-0014398 discloses mesenchyme. Disclosed is a cell therapy composition for preventing or treating graft-versus-host disease comprising stem cells and immunoregulatory T cells as an active ingredient.

However, these prior arts do not imply any implications or teach about the immunomodulatory activity and biomedical use of amniotic fluid derived stem cells or their cultures.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing and treating autoimmune diseases or immune disorders, including amniotic fluid-derived stem cells having a immunomodulatory function or a culture thereof.

The object of the present invention is to obtain amniotic stem cells after collecting amniotic fluid from the mother; Separately obtaining peripheral blood monocytes from human blood; Amniotic stem cells and peripheral blood mononuclear cells obtained in the above step were co-cultured, and then IFN-γ protein expression analysis, flow cytometry, immunosuppressive gene expression analysis and peripheral blood monocyte proliferation inhibitory effect were achieved.

The present invention has an excellent effect of providing a pharmaceutical composition for the prevention and treatment of autoimmune diseases or immune disorders, including amniotic fluid-derived stem cells or a culture medium having an immunomodulatory function.

1 is a graph showing the results of measuring the cell proliferation rate of peripheral blood monocytes co-cultured with amniotic stem cells according to the present invention. (A) Peripheral blood mononuclear cell proliferation rate according to the ratio of cocultured amniotic fluid stem cells and peripheral blood mononuclear cell counts, (B) Peripheral blood mononuclear cell proliferation rate according to the physical distance of co-cultured amniotic fluid stem cells and peripheral blood monocytes.

Figure 2 is a graph showing the change in IFN-γ protein expression of peripheral blood monocytes co-cultured with amniotic stem cells according to the present invention. (A) Picture showing the result of decreasing IFN- [gamma] protein expression dependent on amniotic stem cell number. (B) It is a graph which quantified the result of decreasing IFN- [gamma] protein expression dependent on amniotic stem cell number.

3 is a graph showing changes in protein expression of human peripheral blood monocytes IFN-γ co-cultured with human fibroblasts according to the present invention. (A) Human fibroblasts are photographs showing that they do not affect IFN-γ protein expression in peripheral blood monocytes. (B) Human fibroblasts do not affect IFN-γ protein expression in peripheral blood monocytes. It is a graph.

Figure 4 is a graph of the results of measuring the expression changes of immunolabeled proteins HLA-ABC, HLA-DR, HLA-G, CD80, CD86 and CD274 of amniotic stem cells following recombinant human IFN-γ treatment through flow cytometry.

Figure 5 is a photograph of the results of analyzing the expression of immunosuppressive genes of amniotic stem cells according to recombinant human IFN-γ treatment (A) and treatment concentration (B) through RT-PCR.

Figure 6 is a graph of the results of analyzing the cell growth rate of peripheral blood monocytes by CD274 and IDO according to the present invention.

The present invention provides a pharmaceutical composition for preventing and treating autoimmune diseases, immune disorders, graft-versus-host disease, including amniotic fluid stem cells having an immunomodulatory function or a culture thereof.

The present invention provides a pharmaceutical composition comprising amniotic stem cells cultured according to the present invention or a culture solution thereof. Amniotic stem cells or the culture medium thereof preferably contain 0.1 to 99% by weight based on the total weight of the pharmaceutical composition.

The pharmaceutical composition according to the present invention may be prepared in various forms including pharmaceutically acceptable carriers, for example, oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and the like. It may be formulated in the form of sterile injectable solutions.

The pharmaceutically acceptable carrier is lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like. Also included are diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, and the like. Oral solid preparations include tablets, pills, powders, granules, capsules and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, sucrose or lactose. ), Gelatin, and the like, and may include a lubricant such as magnesium stearate, talc, and the like. Oral liquid preparations include suspensions, solvents, emulsions, syrups, and the like, and may include water, diluents such as liquid paraffin, wetting agents, sweeteners, fragrances, preservatives, and the like. Parenteral preparations include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories.Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and ethylol. Injectable esters such as late and the like. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycero gelatin and the like can be used.

Hereinafter, the present invention will be described in more detail with reference to the following Examples and Experimental Examples. However, these Examples and Experimental Examples are only for the purpose of helping the understanding of the present invention, and the scope of the present invention is not limited by them in any sense.

Example 1 Isolation and Culture of Amniotic Stem Cells According to the Present Invention

Amniotic fluid was obtained through amniotic fluid puncture from patients who visited Kyungpook National University Obstetrics and Gynecology for prenatal diagnosis. The amniotic fluid derived stem cells (AFSC) were isolated and cultured.

10 mL of amniotic fluid was centrifuged at 1000 rpm for 10 minutes, the supernatant was removed, and only the precipitate was suspended in α-MEM serum free medium. The suspension was centrifuged at 1000 rpm for 10 minutes, the supernatant was removed and the precipitate suspended in culture AmnioMax II complete medium (Gibco, New york, USA) and then primary cultured for 7 days in a cell culture plate. The cells attached to the cell culture plate were cultured for 7 more days and then subjected to the first passage and 15% (v / v) ES-FBS (v / v) in α-MEM medium (Gibco, New York, USA) after three passages. Hyclone, Logan, UT, USA), 1% (v / v) L-glutamine (Hyclone, Logan, UT, USA), 1% (v / v) penicillin / streptomycin (Gibco, New York, USA), 18% (v / v) Chang B, 2% (v / v) Chang C (Irvine Scientific, Waalwijk, Netherlands) added Chang's medium was used as a culture medium. The culture medium was subcultured once every 2 to 3 days with fresh medium and 0.05% Trypsin-EDTA (Gibco, New York, USA) was used for cell collection.

Example 2 Isolation of Peripheral Blood Monocytes According to the Present Invention

Peripheral blood mononuclear cell; PBMC) was used in the present invention by separating a density gradient centrifugation method using Ficoll-paqueTM Plus (GE healthcare, Uppsala, Sweden) in normal blood. Separately used immediately for the experiment and no subculture. The medium used in the experiment was Chang's medium.

Example 3 Immune Response Inhibitory Effect Assay of Peripheral Blood Monocytes According to the Present Invention

Experimental Example 1 Amniotic Stem Cell Proliferation Assay According to the Present Invention

Amniotic stem cells 5x10 isolated and cultured according to Example 1² To 4x10³After dispensing the cells into a 96 well cell culture plate, mitomycin C was treated in 96 wells at a final concentration of 10 μg / mL for 2 hours to suppress proliferation of amniotic stem cells. After the proliferation inhibitory treatment of amniotic stem cells, the culture medium was removed, and the amniotic stem cells were washed three times with PBS. Separately, the final blood mononuclear cells isolated according to Example 2 were diluted in 1 μg / mL of the final concentration of phytohemagglutinin (PHA) (sigma, St. Louis, MO, USA) in Chang's media. Sensitized. Peripheral blood monocytes 1x10 sensitized with PHA per well cultured amniotic stem cells inhibited proliferation⁵ Dispense cells, then 37 ° C CO₂In the incubator gun Co-cultures for 72 hours.

The mixed ratio for co-culture of PHA sensitized peripheral blood mononuclear cells 1x10 ⁵ cells and amniotic stem cells according to the present invention is 5x10 ² cells: 1x10 ⁵ cells (1: 200), 1x10 ³ cells, respectively. : 1x10 ⁵ cells (1: 100), 2x10 ³ cells: 1x10 ⁵ cells (1:50), and 4x10 ³ cells: 1x10 ⁵ cells (1:25) were measured.

After 54 hours of co-culture, 5-Bromo-2-deoxyuridine (BrdU) was treated in the co-culture wells to a final concentration of 10 μM, and then labeled for 18 hours.

The blastocyst growth rate of amniotic stem cells and peripheral blood monocytes co-cultured according to the present invention was measured using Proliferation ELISA, BrdU (colorimetric) (Roche Diagnostics, Mannheim, Germany).

As shown in FIG. 1A, the coculture of amniotic stem cells and peripheral blood monocytes inhibited the proliferation of peripheral blood mononuclear cells, and the proliferation inhibitory effect of the peripheral blood mononuclear cells increased as the number of cocultured amniotic stem cells increased.

Experimental Example 2: Immune cell proliferation assay according to the physical distance of coculture of amniotic fluid stem cells and peripheral blood monocytes according to the present invention

The transwell system was used to investigate the effect of physical isolation between the proliferation-inhibited amniotic stem cells and immune cells on immune cell proliferation of amniotic stem cells.

Amniotic stem cells and peripheral blood monocytes were co-cultured in the same manner as in Experimental Example 1 or cultured with amniotic stem cells in the same manner as in Experimental Example 1, and the proliferation of amniotic stem cells with Mitomycin C. Peripheral blood monocytes sensitized with PHA were inserted into the lower compartment of transwell cultured with amnional proliferation-inhibited amniotic stem cells in the same manner as in 1 to measure the cell proliferation rate of peripheral blood monocytes. The ratio of coculture of amniotic stem cells and peripheral blood mononuclear cells was 4 x 10 ³ cells: the peripheral blood mononuclear cells 1x10 ⁵ cells (1:25) that best inhibited the growth of peripheral blood mononuclear cells according to the results of Experiment 1 above. The experiment was conducted in proportions.

Peripheral blood monocytes incubated with PHA sensitized peripheral blood monocytes as a control (FIG. 1B Activated PBMC). Peripheral blood monocytes co-cultured with contact with amniotic stem cells by the method of Experimental Example 1 (FIG. 1B, hAFSC: PBMC = 1: 25) and 0.4μm pore sized transwell (Corning Life Sciences, Amsterdam, Netherlands) to insert the amniotic stem cells into the insert to inhibit proliferation and then divide PHA sensitized peripheral blood cells Cell proliferation of peripheral blood monocytes co-cultured by physical contact was compared.

As shown in the graph of FIG. 1B, the survival rate of peripheral blood mononuclear cells in the co-cultured cultures by directly contacting amniotic stem cells and peripheral blood monocytes was 10% lower than the survival rate of the physically isolated test cells. It was found to be effective.

However, the survival inhibition rate of peripheral blood monocytes was 70% even in the absence of physical contact, so it was confirmed that the proliferation inhibitory effect of peripheral blood monocytes was not only induced by amniotic fluid but also by amniotic fluid.

Therefore, it is thought that amniotic stem cells inhibit peripheral blood monocyte proliferation through contact with peripheral blood monocytes and soluble amniotic stem cell secretion factors.

Experimental Example 3: Peripheral Blood Monocyte Activity Inhibitory Effect Assay of Amniotic Stem Cells According to the Present Invention: IFN-γ ELISPOT assay

T cell activity in peripheral blood monocytes co-cultured with amniotic fluid stem cells according to the present invention was assayed by IFN-γ ELISPOT assay. In this experiment, the positive control group cultured only peripheral blood mononuclear cells sensitized with PHA, and the negative control group cultured only peripheral blood mononuclear cells not sensitized with PHA.

Experimental Example 1 and 1x10 ⁵ of peripheral blood was sensitized with 5μg / mL PHA in the same manner monocytes with Experimental Example 1 to the pumping stem cell culture in the same manner a positive stem cells: the mixing ratio of the peripheral blood mononuclear cells, respectively 5x10 ⁴ cells : 1x10 ⁵ cells (1: 2), 2.5x10 ⁴ cells: 1x10 ⁵ cells (1: 4), 1.25x10 ⁴ cells: 1x10 ⁵ cells (1: 8), 6.25x10 ³ cells: 1x10 ⁵ cells (1:16 ), 3.125x10 ³ cells: 1x10 ⁵ cells (1:32) and 1.5625x10 ³ cells: 1x10 ⁵ cells (1:64) were incubated in a 37 ° C. incubator for 24 hours in the same manner as in Example 1.

After removing the culture medium of the co-cultured cells, the cells were washed with deionized water and wash buffer (1xPBS containing 0.05% Tween-20). Each well was treated with Biotinylated Anti-Human IFN-γ antibody and then reacted for 2 hours and then washed with wash buffer. Each well was washed with streptavidin-HRP for 1 hour and then washed and put in a substrate to visualize the spot. The number of visualized spots was measured using an ELISPOT meter (Cellular Technology, OH, USA).

As shown in FIG. 2, spots of IFN-γ were reduced depending on the number of amniotic stem cells co-cultured with peripheral blood monocytes. Therefore, amniotic stem cells or amniotic fluid culture secretion is thought to inhibit the activity of T cells.

Comparative Example 1 Assay of Inhibitory Effects of Fibroblasts Peripheral Blood Monocyte Activity on the Invention: IFN-γ ELISPOT Assay

In order to confirm whether the peripheral blood mononuclear cell activity inhibitory effect is a positive stem cell specific reaction, the experiment was performed in the same manner as in Experiment 3 except that the amniotic stem cells were human fibroblasts in Experimental Example 3.

As shown in FIG. 3, human fibroblasts did not affect T cell activity inhibition.

Example 4 Screening of Immune Response Inhibitory Mediators of Amniotic Fluid Stem Cells

Flow cytometry and gene expression analysis were performed to investigate the immune response inhibitory mediators of amniotic fluid stem cells showing the immune response inhibitory effect of peripheral blood monocytes obtained according to Example 2.

Recombinant human IFN-γ (rhIFN-γ) treated in Experimental Example 4 and Experimental Example 5 is a cytokine secreted from peripheral blood monocytes when an immune response is induced and immunity of amniotic stem cells when the rhIFN-γ is secreted. Treatment was performed to confirm the expression level change of the mediator.

Experimental Example 4: Immunomarker Characterization of Amniotic Stem Cells According to the Present Invention: Flow Cytometry

Amniotic stem cells cultured according to Experimental Example 1 were treated with recombinant human IFN-γ (rhIFN-γ) at a concentration of 500ng / mL for 24 hours, followed by washing the cells with PBS and washing the amniotic stem cells with 0.05% Trypsin. -2x10 after return to EDTA⁵ The cells were suspended in 100 μL of PBS containing 2% (v / v) FBS, followed by cold cancer by adding PE-conjugated monoclonal antibodie IgG1, IgG2a, HLA-ABC, HLA-DR, HLA-G, CD80, CD86, and CD274, respectively. The reaction was carried out in a cow for 30 minutes. 5 minutes at 1,500 rpm after completion of the reaction After centrifugation, the supernatant was removed, and the amniotic stem cells reacted with each of the recovered antibodies were washed three times with PBS containing 2% (v / v) FBS and flow cytometry (FACS caliber, BD Biosciences, USA). Analyzed.

As shown in FIG. 4, the characteristics of amniotic stem cells positive for HLA-ABC and negative for HLA-DR and HLA-G and CD80 and CD86 were not changed even after treatment with rhIFN-γ and HLA-ABC. Expression increased further. CD274, also known as a negative regulator of T cell activation, was also expressed on the surface of amniotic stem cells and expressed by recombinant IFN-γ.

Experimental Example 5: Analysis of Immune Response Marker Gene Expression in Amniotic Stem Cells According to the Present Invention

Amniotic stem cells cultured according to Experimental Example 1 were treated with recombinant human IFN-γ (rhIFN-γ) at a concentration of 500 ng / mL for 24 hours, followed by washing the cells with PBS and treating the washed amniotic stem cells with TRIZOL reagent. Amniotic stem cell total RNA was purified by treatment. Using the purified total RNA 1ug as a template, cDNA was synthesized through PrimeScriptTM 1st strand cDNA systhesis kit (TaKaRa Bio Inc., Shiga, Japan), and used as a template for gene expression analysis.

Using the CDNA as a template, RT-PCR was performed using the primers shown in Table 1 below. The PCR product obtained through RT-PCR was subjected to electrophoresis on 1.5% agarose gel and visualized with ethidium bromide (EtBr) to confirm gene expression levels.

Table 1 Primer for Analysis of Immune Response Gene Expression gene Primer sequencing (3`-5`) PCR product size (bp) IDO F GCCTGATCTCATAGAGTCTGG 345 R TTACTGCAGTCTCCATCACG TGF-β F CTATCCACCTGCAAGACTATCGAC 777 R GGAGCTGAAGCAATAGTTGGTGTC IL-10 F GCCTAACATGCTTCGAGATC 366 R CTCATGGCTTTGTAGATGCC GAPDH F GCTTGTCATCAATGGAAATCCC 194 R TCCACACCCATGACGAACATG

Abbreviations: IDO (Indoleamine-pyrrole 2,3-dioxygenase), TGF-β (Transforming growth factor beta), IL-10 (Interleukin 10), GAPDH (Glyceraldehyde 3-phosphate dehydrogenase).

As shown in FIG. 5A, expression of TGF-β and IL-10 was induced by IFN-γ in all of the amniotic fluid cells of different batches. IDO was not expressed in the normal culture state, but the expression was induced by IFN-γ treatment.

As shown in Figure 5B it can be seen that the expression of these immunoregulatory factors is induced in rhIFN-γ concentration-dependent.

Experimental Example 6: Immunosuppressive Effect of Peripheral Blood Monocytes by CD274 or IDO Treatment of Amniotic Response Marker of Amniotic Stem Cells According to the Present Invention

According to the results of Experimental Examples 4 and 5, the inhibitory effect of the CD274 and IDO expressed in the amniotic stem cells was suppressed the immune response of peripheral blood monocytes.

Experimental Example 1 and the peripheral blood mononuclear cells were primed with 4x10 ³ of positive stem cells and PHA 1μg / mL in the same manner by the addition of 1x10 ⁵ cells, and then 1-Methyl tryptophan (1-MT ) (sigma, St. Louis, MO , USA) treated with 4 μg / mL of mouse IgG (R & D systems, Minneapolis, MN, USA) with 0.5 μM and 1 mM, anti-CD274 antibody (eBioscience, San Diego, CA, USA) 4 μg / mL, isotype control and 3 Co-culture for days. After co-culture, the proliferation of each treated cell was measured using a Cell Proliferation ELISA and BrdU (coloretric) in the same manner as in Experimental Example 1.

As shown in FIG. 6, when the IDO inhibitor 1-MT was treated with 0.5 mM, 1 mM, or the anti-CD274 antibody alone or in combination with 1-MT, the concentration of peripheral blood mononuclear cell proliferation of amniotic stem cells was increased. It is confirmed that the recovery is dependent. Anti-CD274 antibody treated group showed about 40% recovery of cell proliferation inhibition compared to isotype control. Combination of 1-MT and anti-CD274 antibody showed the greatest recovery of peripheral blood mononuclear cell proliferation of amniotic stem cells. According to the results, two molecules of IDO and CD274 are involved in the immunomodulatory capacity of amniotic stem cells. do.

As described above, the present invention has an excellent effect in providing a pharmaceutical composition for preventing and treating autoimmune diseases or immune disorders-related diseases including amniotic fluid-derived stem cells having a immunomodulatory function or a culture thereof. to be.

Claims (3)

Immune response suppression method of peripheral blood monocytes characterized in that the co-culture of amniotic stem cells and peripheral blood monocytes Pharmaceutical composition for the prevention and treatment of autoimmune diseases, characterized in that it contains one or more selected from the amniotic stem cells, amniotic stem cell culture medium Pharmaceutical composition for preventing and treating graft-versus-host disease, characterized in that it contains at least one selected from amniotic stem cells and amniotic stem cell culture

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