Method for treating thyroid associated ophthalmopathy

Abstract

Provided is a composition including mesenchymal stem cells as an effective ingredient for prevention, alleviation, or treatment of thyroid-associated ophthalmopathy. A pharmaceutical composition including mesenchymal stem cells as an effective ingredient for treatment of thyroid-associated ophthalmopathy allows patients with thyroid-associated ophthalmopathy to recover from an abnormal increase in hyaluronic acid production in orbital fibroblasts, increased adipocyte differentiation, and increased lipid accumulation, and thus may be useful for the treatment of thyroid-associated ophthalmopathy.

Claims

1. A method of treating or preventing thyroid-associated ophthalmopathy, the method comprising administering mesenchymal stem cells expressing at least one selected gene from the group consisting of forkhead box P3 (FOXP3), human leukocyte antigen G (HLA-G), and toll-like receptor 4 (TLR4), a cell population thereof, or a culture, a lysate, or an extract thereof, as an active ingredient, to a subject in need thereof.

2. The method of claim 1, wherein the stem cells have a characteristic a) or b) below: a) a characteristic of expressing at least one gene selected from the group consisting of chemokine (C-X-C motif) ligand 1 (CXCL-1), monocyte chemotactic protein 1 (MCP-1), and a tissue inhibitor of metalloproteinases (TIMP-1); or b) a surface antigen characteristic of at least one gene selected from the group consisting of CD90, CD146, CD105, and CD73.

3. The method of claim 1, wherein the mesenchymal stem cells are umbilical cord-derived, umbilical cord blood-derived, bone marrow-derived, placenta-derived, or adipose-derived mesenchymal stem cells.

4. The method of claim 1, wherein the mesenchymal stem cells are placenta chorion-derived mesenchymal stem cells.

5. The method of claim 1, wherein the mesenchymal stem cells suppress differentiation of orbital fibroblasts into adipocytes or production of hyaluronic acid.

6. The method of claim 1, wherein the thyroid-associated ophthalmopathy occurs in association with hyperthyroidism.

7. The method of claim 1, wherein the mesenchymal stem cells are for ocular administration.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1A shows levels of neuro-protective cytokines of human placenta-derived mesenchymal stem cells (hPMSCs) measured from a cell culture medium;

(2) FIG. 1B shows markers which identify mesenchymal stem cells (MSCs) identified on the surface of hPMSCs;

(3) FIG. 2 shows the results of immunoassay confirming HAS, HA, and HAdase included in tears of a normal person and a patient with thyroid-associated ophthalmopathy (TAO);

(4) FIG. 3A shows co-culturing of orbital fibroblasts and hPMSCs and the results of measuring changes in HAS2 expression by western blotting;

(5) FIG. 3B shows co-culturing of orbital fibroblasts and hPMSCs and the quantified values of the changes in HAS2 expression;

(6) FIGS. 4A and 4B show the results of observing changes of surface markers of the co-cultured fibroblasts after co-culturing orbital fibroblasts with hPMSCs from a normal person and a TAO patient;

(7) FIG. 5 shows the results of analyzing mRNA expression of PPARγ, ADIPONECTIN, and C/EBPα by using real-time PCR after co-culturing orbital fibroblasts with hPMSCs from a normal person and a TAO patient;

(8) FIG. 6 shows the results of observing changes in lipid accumulation of fibroblasts after co-culturing orbital fibroblasts with hPMSCs from a normal person and a TAO patient; and

(9) FIG. 7 is a graph showing the effect that mesenchymal stem cells according to an embodiment have on immune response regulatory factors of inflammation-induced cells; wherein AD: adipose-derived mesenchymal stem cells, BM: bone marrow-derived mesenchymal stem cells, PD: placenta-derived mesenchymal stem cells, WI-38: fibroblasts; a: FOXP3, b: HLA-G, c: hTRL4; *Control vs. Others: decrease, # Control vs. Others: increase, ** 1 ng vs. 10 ng: decrease, ##1 ng vs. 10 ng: increase.

MODE OF DISCLOSURE

(10) Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Reference Example

Reference Example 1

Isolation of Placenta-Derived Mesenchymal Stem Cells

(11) An informed consent based on sufficient explanation beforehand was received from a healthy mother who normally delivered a baby, and an umbilical cord was separated from placenta tissues collected at the time of normal placenta delivery. The separated tissues (chorioamniotic membranes) were added to a 50-ml tube, DPBS was added to remove excess blood, and in 20 ml of enzyme solution I (1 mg/ml collagenase type I, 2 mg/ml Trypsin, 20 mg/ml DNase I, 1.2 U/ml Dispase, ×1 PS in HBSS) the suspension solids collected by scraping the upper part of the chorioamniotic membranes with sterilized slide glass were gathered on one side. 10 ml of the enzyme solution I was added, homogenously mixed, and a 37° C. enzyme reaction was repeated twice for 15 minutes to isolate stem cells from the tissues. The isolated cell suspension was centrifuged, and the isolated cells were cultured by using DMEM/F12 to which 10% fetal bovine serum, 1% penicillin-streptomycin, 1 ug/ml heparin, and 25 ng/ml fibroblast growth factor-4 (FGF-4) were added. The culture medium was replaced at an interval of 4 to 5 days, and TrypLE available from Invitrogen was passage-cultured for a short period of time (3 minutes) in a 37° C. incubator at the first passage.

Reference Example 2

Orbital Fibroblast Culture and Treatment

(12) Orbital fibroblasts from humans (4 normal people, 4 patients) were obtained and cultured in DMEMFI2 (available from Gibco) (including 10% FBS and 1% penicillin-streptomycin). Two days after distributed and grown in a medium, the fibroblasts were mixed with 5 g/ml insulin, 1 mM dexamethasone, and 0.5 mM IBMX in DMEM (10% FBS), and thus differentiation into adipocytes was started (day 0). After 72 hours (day 3), the medium was replaced with a DMEM medium supplemented with 10% FBS and 5 μg/ml insulin, followed by supplying a DMEM medium supplemented with 10% FBS every other day.

Reference Example 3

Orbital Fibroblast Lipid Accumulation Analysis

(13) Orbital fibroblasts from normal people and patients were placed in differentiation medium inducing adipose production, and then the cells were cultured for 10 days as in co-culturing with human placenta-derived mesenchymal stem cells (hPMSCs) and not co-culturing (for the first 4 days with DMEM supplemented with 10% FBS, 33 uM biotin, 17 uM pantothenic acid, 0.2 nM T3, 10 μg/mL transferrin, 0.2 uM prostaglandin I2, 0.1 mM isobutylmethylxanthine (IBMX), 1 uM dexamethasone, and 5 ug/ml insulin; and the next 5 to 10 days, without IBMX, dexamethaxone, and insulin). After 10 days, change in lipid accumulation of the fibroblasts was observed by Oil-Red-O staining.

Reference Example 4

Tear Sampling

(14) Tears were collected from normal people (n=13) and thyroid-associated ophthalmopathy (TAO) patients (n=13) by using schirmer strips. Then, the schirmer strips were moved to a 0.5 ml-tube having cannula on the bottom, and 30 μL of PBS was added thereto. The content of the tube was moved to a larger tube (1.5 ml), and centrifuged for 5 minutes (13,000 rpm). The tears thus collected were preserved at −20° C.

Reference Example 5

Real-Time PCR

(15) In day 8 of the culturing, hPMSCs (2×10.sup.5) were co-cultured with orbital fibroblasts for 48 hours. The cell lysate was homogenized in TRIzol (available from Invitrogen, Carlsbad, Calif., USA) to extract RNA. 1 μg of the total RNA from the samples was reverse transcript to synthesize cDNA. Conditions for synthesizing cDNA are as follows: RNA fusion (65° C., 1 minute), annealing (25° C., 5 minutes), amplification (42° C., 60 minutes), and enzyme inactivation (85° C., 1 minute).

(16) Normalization was performed by amplifying mRNA of each gene according to the following PCR conditions: initial fusion (95° C., 2 minutes), amplification (95° C., 10 seconds; 55° C., 20 seconds; and 72° C., 20 seconds) 40 cycles. PPARγ, ADIPONECTIN, and C/EBPα, primer sets are as follows:

(17) PPARγ FP: 5′-TTGACCCAGAAAGCGATTCC(SEQ ID NO: 1)-3′, RP: 5′-AAAGTTGGTGGGCCAGAATG(SEQ ID NO: 2)-3′; ADIPONECTIN FP: 5′-GGCCGTGATGATGGCAGAGAT(SEQ ID NO: 3)-3′, RP: 5′-TTTCACCGATGTCTCCCTTAGG(SEQ ID NO: 4)-3′ C/EBPα FP: 5′-TGTATACCCCTGGTGGGAGA(SEQ ID NO: 5)-3′, RP: 5′-TCATAACTCCGGTCCCTCTG(SEQ ID NO: 6)-3′. The mRNA expression of each of the genes was normalized to 18s rRNA. Data are depicted as folds (mean±SEM) of a factor related to adipose differentiation compared to the normal group.

Reference Example 6

Western Blot

(18) Lysates were prepared by using a RIPA buffer. The total protein of the same amount was separated by SDS-PAGE and transferred to a membrane. The membrane was immunoblotted with anti-HAS1 and HAS2 (available from SantaCruz Biotechnology, SA, USA) at 1:1000 dilution, and the same membrane was cultured with GAPDH (available from SantaCruz). After washing, the resultant was cultured with horseradish peroxidase-conjugated anti-goat IgG secondary antibody at 1:10000 dilution at room temperature for 3 hours. The immune response bands were made into images by using an enhanced chemiluminescence solution (available from Animal Genetics, Suwon, Korea) and detected with the ChemiDoc™ XRS+ System Imager (available from Bio-Rad Laboratories, Hercules, Calif., USA). Protein expression levels were normalized to GAPDH. Data are depicted as folds (mean±SEM) of HAS2 compared to the normal group.

Reference Example 7

Enzyme-Linked Immunosorbent Assay (ELISA)

(19) Tears taken from the normal people and TAO patients were prepared, and levels of hyaluronic acid (Ha) and hyaluronidase (Hyal) of the tears were determined by the ELISA. The present analysis was performed according to manual of the manufacturer.

Reference Example 8

FACS Analysis

(20) Human fibroblasts (3×10.sup.5) were dissociated with a cell dissociation buffer (Life Technologies) and washed with PBS (2% (v/v) FBS). The resultant was cultured with an isotype control IgG or an antigen-specific antibody (BD Biosciences, CA, USA) for 20 minutes was used to identify cells. FACS sorting was performed by using a FACS vantage Flow Cytometer (BD Biosciences, CA, USA)

Example 1

Characteristic Analysis of Placenta-Derived Stem Cells

(21) In order to analyze characteristics of the placenta-derived mesenchymal stem cells isolated in the same manner as in Reference Example 1, cytokine secretion characteristics and surface antigen characteristics of the cells were analyzed. In particular, concentrations of neuro-protective cytokines of the placenta-derived mesenchymal stem cells in the culture medium were measured by the ELISA, characteristics of surface antigens (CD34, CD45, CD90, CD31, HLA-DR, CD146, CD106, and CD73) of the placenta-derived mesenchymal stem cells were analyzed by FACS analysis, and the results are shown in FIG. 1.

(22) FIG. 1A shows levels of neuro-protective cytokines of human placenta-derived mesenchymal stem cells (hPMSCs) measured from a cell culture medium. As shown in FIG. 1A, it was confirmed that secretion of cytokines related to inflammatory response and would healing (CXCL-1, MCP-1, and TIMP-1) in the culture solution of culturing hPMSCs was increased as the result of the ELISA.

(23) FIG. 1B shows a surface pattern of hPMSCs. As shown in FIG. 1B, it was confirmed that the mesenchymal cell markers, CD90, CD146, CD105, and CD72, were positive as the result of FACS analysis.

Example 2

Confirmation of Hyaluronic Acid Synthesis Increase in TAO Patients

(24) Tears obtained from normal people and TAO patients (total of 26 samples) were used to perform western blotting, and amounts of hyaluronic acid synthase (HAS) were measured. A total of 10 μg protein was loaded Anti-HAS1 and HAS2 were cultured at 4° C. ELISA analysis was also performed on the tears obtained from the TAO patients.

(25) As a result, it was confirmed that protein expressions of the hyaluronic acid synthases, HAS1 and HAS2, increased in the tears of the TAO patients compared to those of tears obtained from the normal people as shown in FIG. 2A. As shown in FIG. 2B, an increase of the level of Ha was also observed, but it was confirmed that the level of HAdase did not have a significant change as shown in FIG. 2C.

(26) As a result of the ELISA analysis, the level of hyaluronic acid in the tears of the TAO patients increased, but the level of HAdase was not different from that of the normal people. Therefore, it was confirmed that production of hyaluronic acid increased as the HAdase increased in the TAO patients.

(27) *77

Example 3

Confirmation of Hyaluronic Acid Production Decrease Effect of hPMSCs

(28) On day 15 of culturing fibroblasts, the orbital fibroblasts were cultured together with IL-18 (20 ng/mL) as a stimulant during an adipose production inducing process. After co-culturing with hPMSCs, protein expression of HAS2 was determined.

(29) As shown in FIG. 3A, it was confirmed that expression of the hyaluronic acid sythase, HAS2, in the fibroblasts of the TAO patients increased, and that the protein expression of the synthase increased through the co-culture with PMSCs decreased.

(30) Also, as shown in FIG. 3B, the protein expression level of the hyaluronic acid sythase, HAS2, was quantified, and the result showed that the fibroblasts of the TAO patients cultured in an adipose production inducing differentiation medium increased protein expression of HAS2 about 2.3 folds by the IL-1β stimulation, which was reduced by co-culture with PMSCs.

Example 4

Confirmation of Effects on Orbital Fibroblast Surface Antigen Markers of hPMSCs

(31) The orbital fibroblasts obtained from the normal people and TAO patients were co-cultured with hPMSCs, and changes thus occurred were observed. The fibroblasts were analyzed by using FACS.

(32) On day 15 of culturing the fibroblasts, the fibroblasts were treated with IL-1β (20 ng/mL). After 24 hours of culturing, hPMSCs were co-cultured with the resultant. The co-cultured fibroblasts were classified by CD90 or CD105 markers. Also, the fibroblasts were observed through a microscope.

(33) As shown in FIGS. 4A and 4B, it was confirmed that CD90 in the fibroblasts of the TAO patients changed by the co-culturing with hPMSCs as the result of analyzing changes of CD105 and CD90 which are fibroblasts of the normal people and patients.

(34) *87

Example 5

Confirmation of Effect of hPMSCs on Adipogenesis

(35) Orbital fibroblasts obtained from the normal people and TAO patients were cultured with adipose differentiation inducing medium 1 (33 μM biotin, 17 μM pantothenic acid, 0.2 nM T3, 10 μg/mL transferrin, 0.2 μM prostaglandin 12, 0.1 mM isobutylmethylxanthine (IBMX), 1 μM dexamethasone, and 5 μg/ml insulin) for 4 days, and the resultant was cultured with adipose differentiation inducing medium 2 (33 μM biotin, 17 μM pantothenic acid, 0.2 nM T3, 10 μg/mL transferrin, 0.2 μM prostaglandin 12, and 0.1 mM) from the 5th day to 10th day. On day 8 of the culturing, the orbital fibroblasts being cultured in the adipose differentiation medium were co-cultured with hPMSCs (2×10.sup.5) for 48 hours. Thereafter, mRNA expression of PPARγ, ADIPONECTIN, and C/EBPα, which are representative adipogenesis-related factors, were confirmed described in relation to Reference Example 5, and the results are shown in FIG. 5.

(36) As a result, as shown in FIG. 5, it was confirmed that mRNA expression of PPARγ, ADIPONECTIN, and C/EBPα in the fibroblasts of the patients cultured in the adipose differentiation inducing medium increased 21.5 folds, about 80 folds, and 33 folds, each respectively. Whereas, it was confirmed that mRNA expression of PPARγ, mRNA expression of ADIPONECTIN, and mRNA expression of C/EBPα that all increased by the co-culturing with PMSCs decreased to about 8 folds, about 33 folds, and about 12.4 folds, each respectively. Therefore, the results shown above confirmed that the mesenchymal stem cells suppressed expressions of PPARγ, ADIPONECTIN, and C/EBPα of orbital fibroblasts, and this indicates that the mesenchymal stem cells suppress adipogenesis property of the orbital fibroblasts.

Example 6

Confirmation of Lipid Accumulation of Orbital Fibroblasts

(37) Effects of human placenta-derived mesenchymal stem cells (PMSCs) on lipid accumulation of orbital fibroblasts were confirmed. In particular, the orbital fibroblasts obtained from the normal people and TAO patients were co-cultured with PMSCs as well as adipogenesis inducing medium culture. After 10 days, lipid accumulation in the orbital fibroblasts were confirmed as described in Reference Example 3 by using Oil-Red O staining, and the results are shown in FIG. 6.

(38) As a result, it was confirmed that lipid accumulation of the fibroblasts of the TAO patients induced in the differentiation medium reduced by the co-culturing with PMSCs as shown in FIG. 6.

(39) From these results, it was confirmed that the hPMSCs reduce abnormal activity of the fibroblasts of the TAP patients.

Example 7

Confirmation of Immunoregulatory Ability of Mesenchymal Stem Cells

(40) In order to confirm immunoregulatory abilities of orbital fibroblasts of placenta-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, and bone marrow-derived mesenchymal stem cells isolated as described in Reference Example 1, the mesenchymal stem cell in naïve state were treated with inflammation inducing factors, and expression of the factors each expressed in the mesenchymal stem cells were compared with expression according to inflammatory reaction. The adipose-derived mesenchymal stem cells and bone marrow-derived mesenchymal stem cells were provided from the American Type Culture Collection (ATCC).

(41) Particularly, the inflammation inducing factors, LPS and IL-1β, were each treated on the mesenchymal stem cells at 1 ng or 10 ng, respectively. Then, expression levels of immunoregulatory factors, hFOXP3, hHLA-G, and hTRL4, were confirmed by qRT-PCR. In particular, qRT-PCR was performed in steps including collecting cells treated with inflammation inducing factors and performing cell lysis using TRIZOL; synthesizing cDNA by using a reverse transcriptase; amplifying PCR by using a gene specific base sequence and a Tag. DNA polymerase; and performing electrophoresis on the amplified PCR product to on an agarose gel to confirm the presence of the amplified gene. The results of qRT-PCR analysis are shown in FIG. 7.

(42) FIG. 7 is a graph that shows effects of the mesenchymal stem cells according to an embodiment on immune reaction regulatory factors of inflammation induced cells. AD: adipose-derived mesenchymal stem cell, BM: bone marrow-derived mesenchymal stem cell, PD: placenta-derived mesenchymal stem cell, WI-38: fibroblasts; a: FOXP3, b: HLA-G, c: hTRL4; *Control vs. Others: decrease, # Control vs. Others: increase, ** 1 ng vs. 10 ng: decrease, ## 1 ng vs. 10 ng: increase.

(43) As shown in FIG. 7, it may be known that the mesenchymal stem cells according to an embodiment express HLA-G, FOXP3, and TLR4 more than the fibroblasts do. Particularly, the placenta-derived mesenchymal stem cells not only expressed FOXP3 and TLR4 as well as HLA-G known for protecting cells from T-cell attack by involved in immune response regulation than other cells and had high expression level after being treated with inflammation inducing factors. It may be known that mesenchymal stem cells (particularly, placenta-derived mesenchymal stem cells) exhibiting overexpression of an immuneregulatory ability factor, such as HLA-G, may be effectively used as a treating agent of thyroid-associated ophthalmopathy by analyzing change in expression as expression patterns of the immune-associated factors are treated with the inflammation inducing factors in a naive state.