COMPOSITION FOR PREVENTING OR TREATING PULMONARY FIBROSIS CONTAINING EXOSOME ISOLATED FROM ADIPOSE-DERIVED STEM CELL AS ACTIVE INGREDIENT

Abstract

The present disclosure relates to a composition for preventing or treating pulmonary fibrosis, containing an exosome isolated from an adipose-derived stem cell as an active ingredient, a use of the exosome in preparation of a pharmaceutical for preventing or treating pulmonary fibrosis and a method for preventing pulmonary fibrosis, including a step of administering a pharmaceutical composition containing the exosome as an active ingredient to a subject.

Claims

1. A pharmaceutical composition for preventing or treating pulmonary fibrosis, comprising exosomes isolated from adipose-derived stem cells as an active ingredient.

2. The pharmaceutical composition according to claim 1, wherein the exosomes are isolated from adipose-derived stem cells cultured under a normoxia condition.

3. The pharmaceutical composition according to claim 1, wherein the exosomes are isolated from adipose-derived stem cells cultured under a hypoxic condition.

4. The pharmaceutical composition according to claim 3, wherein the hypoxic condition is induced by 0.5-10% oxygen or by a hypoxic cell sensitizer.

5. The pharmaceutical composition according to claim 1, wherein the exosomes comprise hepatocyte growth factor (HGF) and interleukin-10.

6. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is for intratracheal administration or inhalation.

7. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is an injection.

8. The pharmaceutical composition according to claim 7, wherein the injection comprises the exosomes at a concentration of 5×10.sup.8 to 5×10.sup.10 particles/kg.

9-16. (canceled)

17. A method for preventing pulmonary fibrosis, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising exosomes isolated from adipose-derived stem cells as an active ingredient to a subject.

18. The method according to claim 17, wherein the exosomes are isolated from adipose-derived stem cells cultured under a normoxia condition.

19. The method according to claim 17, wherein the exosomes are isolated from adipose-derived stem cells cultured under a hypoxic condition.

20. The method according to claim 19, wherein the hypoxic condition is induced by 0.5-10% oxygen or a hypoxic cell sensitizer.

21. The method according to claim 17, wherein the exosomes comprise hepatocyte growth factor (HGF) and interleukin-10.

22. The method according to claim 17, wherein the pharmaceutical composition is for intratracheal administration or inhalation.

23. The method according to claim 17, wherein the pharmaceutical composition is an injection.

24. The method according to claim 23, wherein the injection comprises the exosomes at a concentration of 5×10.sup.8 to 5×10.sup.10 particles/kg.

Description

DESCRIPTION OF DRAWINGS

[0072] FIG. 1 schematically illustrates an exosome isolated from human adipose-derived stem cells (HASCs) and application thereof according to an exemplary embodiment of the present disclosure.

[0073] FIG. 2 schematically illustrates a method for isolating an exosome from stem cells of various origins according to an exemplary embodiment of the present disclosure.

[0074] FIGS. 3A-3B show a result of analyzing the characteristics of exosomes isolated from a human adipose-derived stem cell (HASC), a human umbilical cord-derived mesenchymal stem cell (UC-MSC) and a human bone marrow-derived mesenchymal stem cell (BM-MSC) according to an exemplary embodiment of the present disclosure: (A) concentration of exosomes isolated from the stem cells of various origins determined by nanoparticle tracking analysis; (B) expression level of HGF (hepatocyte growth factor) and IL-10 (interleukin-10) exosomes determined by ELISA analysis.

[0075] FIGS. 4A-4B show a result of analyzing collagen synthesis inhibition ability of exosomes isolated from a human adipose-derived stem cell (HASC), a human umbilical cord-derived mesenchymal stem cell (UC-MSC) and a human bone marrow-derived mesenchymal stem cell (BM-MSC) using a fibrosis-induced lung fibroblast according to an exemplary embodiment of the present disclosure: (A) schematic of hypoxic condition of lung fibroblast for inducing fibrosis and exosome treatment condition; (B) expression level of COL1A1 (human procollagen type I alpha 1) obtained by treating fibrosis-induced lung fibroblast with exosomes isolated from HASC, UC-MSC and BM-MSC and subjecting culture medium obtained 24 hours later to ELISA analysis. Here, GM represents a positive control group for which a growth medium was used and BM represents a negative control group for which a serum-free basal medium was used.

[0076] FIGS. 5A-5C show a result of analyzing the characteristics of normoxia exosomes and hypoxia exosomes isolated from human adipose-derived stem cells cultured under normoxia and hypoxia conditions, respectively, according to an exemplary embodiment of the present disclosure: (A) schematic of culture condition and condition for isolation of exosomes from human adipose-derived stem cells (DMEM (high glucose) normal culture medium containing 10% FBS and 1% antibiotics is used for isolation of normoxia exosomes and normal culture medium supplemented with 100 μM cobalt chloride(II) hexahydrate (CoCl.sub.2.6H.sub.2O) is used for isolation of hypoxia exosomes. After culturing the human adipose-derived stem cells for 24 hours, the medium is replaced with a serum-free medium and the exosomes are isolated.); (B) transmission electron microscopic images showing the structure and morphology of normoxia exosomes and hypoxia exosomes; (C) concentration of normoxia exosomes and hypoxia exosomes determined by nanoparticle tracking analysis.

[0077] FIGS. 6A-6B show the therapeutic effect for pulmonary fibrosis of an exosome isolated from an adipose-derived stem cell according to an exemplary embodiment of the present disclosure: (A) schematic of establishment of bleomycin-induced pulmonary fibrosis C57BL/6J mouse model and administration schedule of normoxia exosomes and hypoxia exosomes isolated from human adipose-derived stem cells; (B) images of mouse lungs obtained by administering normoxia exosomes and hypoxia exosomes isolated from human adipose-derived stem cells to bleomycin-induced pulmonary fibrosis C57BL/6J mouse for 3 times via intravenous injection with 2-day intervals and imaging the lungs of the mouse 1 day later (PBS represents a negative control group administered with PBS (phosphate buffer saline) (N=3) and HASCs represents a positive control group administered with human adipose-derived stem cells at a concentration of 1×10.sup.6 cells/100 μL (N=3). Normoxia-Exo and Hypoxia-Exo respectively represent test groups administered with normoxia exosomes and hypoxia exosomes at a concentration of 1×10.sup.8 particles/100 μL (N=3). The PBS, human adipose-derived stem cells (HASCs), normoxia exosomes and hypoxia exosomes were administered to the bleomycin-induced pulmonary fibrosis C57BL/6J mouse for 3 times via intravenous injection with 2-day intervals.).

[0078] FIG. 7 shows the therapeutic effect for pulmonary fibrosis of an exosome isolated from an adipose-derived stem cell according to an exemplary embodiment of the present disclosure. After administering normoxia exosomes and hypoxia exosomes isolated from human adipose-derived stem cells to bleomycin-induced pulmonary fibrosis C57BL/6J mouse for 3 times via intravenous injection with 2-day intervals, trichrome staining was conducted on the lungs of the mouse 1 day later (magnification: ×10) (PBS represents a negative control group administered with PBS (phosphate buffer saline) (N=3) and HASCs represents a positive control group administered with human adipose-derived stem cells at a concentration of 1×10.sup.6 cells/100 μL (N=3). Normoxia-Exo and Hypoxia-Exo respectively represent test groups administered with normoxia exosomes and hypoxia exosomes at a concentration of 1×10.sup.8 particles/100 μL (N=3).

[0079] FIG. 8 shows the therapeutic effect for pulmonary fibrosis of an exosome isolated from an adipose-derived stem cell according to an exemplary embodiment of the present disclosure. After administering normoxia exosomes and hypoxia exosomes from human adipose-derived stem cells to bleomycin-induced pulmonary fibrosis C57BL/6J mouse for 3 times via intravenous injection with 2-day intervals, trichrome staining was conducted on the lungs of the mouse 1 day later (magnification: ×40; scale bar: 100 μm).

[0080] FIG. 9 shows the therapeutic effect for pulmonary fibrosis of an exosome isolated from an adipose-derived stem cell according to an exemplary embodiment of the present disclosure. After administering normoxia exosomes and hypoxia exosomes isolated from human adipose-derived stem cells to bleomycin-induced pulmonary fibrosis C57BL/6J mouse for 3 times via intravenous injection with 2-day intervals, trichrome staining was conducted on the lungs of the mouse 1 day later and the stained collagen area, i.e., the fibrotic area (%), was quantified (PBS represents a negative control group administered with PBS (phosphate buffer saline) (N=3) and HASCs represents a positive control group administered with human adipose-derived stem cells at a concentration of 1×10.sup.6 cells/100 μL (N=3). Normoxia-Exo and Hypoxia-Exo respectively represent test groups administered with normoxia exosomes and hypoxia exosomes at a concentration of 1×10.sup.8 particles/100 μL (N=3).).

BEST MODE

[0081] Hereinafter, the constitution and effect of the present disclosure will be described in more detail through examples. The examples are for illustrative purposes only and the scope of the present disclosure is not limited by the examples.

Example 1: Isolation of Exosomes from Stem Cells of Various Origins

[0082] Stem cells exhibit different characteristics depending on the tissue from which they are derived and the culturing condition. Therefore, the exosomes isolated from the stem cell exhibiting different characteristics show difference in structural components (Extracellular vesicles: Exosomes, microvesicles, and friends; G Raposo et al., J. Cell Biol., 2013, Vol. 200, No. 4, pp. 373-383). That is to say, the cell type that is the origin of the exosome is a very important factor determining the characteristics of the exosome.

[0083] Therefore, exosomes were derived from stem cells of various origins in order to isolate exosomes exhibiting effective therapeutic effect for pulmonary fibrosis.

[0084] Specifically, while culturing human adipose-derived stem cells (HASCs), human umbilical cord-derived mesenchymal stem cells (UC-MSCs) and human bone marrow-derived mesenchymal stem cells (BM-MSCs), exosomes were isolated from the respective stem cells.

[0085] The respective stem cells were cultured using a DMEM (Dulbecco's modified Eagle's medium, high glucose; Gibco, Cat #: 11995065) normal culture medium supplemented with 10% FBS (fetal bovine serum) and 1% penicillin/streptomycin.

[0086] 24 hours before isolating exosomes from the respective stem cells, the medium was replaced with a serum-free, antibiotic-free, phenol red-free medium (Gibco, Cat #: 31053028). After culturing for 24 hours, a cell culture supernatant was recovered. The recovered cell culture supernatant was centrifuged at 300 g for 10 minutes to remove cell debris and then a cell strainer with a pore size of 0.4 μm was used to remove the debris larger than the pore size. Then, a filter with a pore size of 0.22 μm was used to remove the cell debris larger than the pore size. After the filtration, the recovered solution was subjected to TFF (tangential flow filtration) at a flow rate of 4 mL/min using a filter with a MWCO (molecular weight cut off) of 500 kD to remove proteins. The recovered solution was sonicated. The TFF process was repeated continuously to obtain the finally isolated and purified exosomes (FIG. 2).

[0087] After recovering the supernatant, the respective stem cells were cultured again after adding a normal culture medium. Subculturing was conducted until passage 7 or 8.

Example 2: Characterization of Exosomes Isolated from Stem Cells of Various Origins

[0088] The characteristics of the exosomes isolated from the stem cells of various origins in Example 1 were analyzed.

[0089] First, the concentration of the exosomes isolated from the human adipose-derived stem cells (HASC-Exo), the exosomes isolated from the human umbilical cord-derived stem cells (UC-MSC-Exo) and the exosomes isolated from the human bone marrow-derived stem cells (BM-MSC-Exo) was investigated by nanoparticle tracking analysis.

[0090] As a result of investigating the concentration of the respective exosomes by nanoparticle tracking analysis, the concentration of the human adipose-derived stem cell exosomes (HASC-Exo) was 2.25×10.sup.9 particles/mL, the concentration of the human umbilical cord-derived stem cell exosomes (UC-MSC-Exo) was 1.38×10.sup.9 particles/mL and the concentration of the human bone marrow-derived stem cell exosomes (BM-MSC-Exo) was 1.81×10.sup.9 particles/mL (FIG. 3A).

[0091] In addition, ELISA (enzyme-linked immunosorbent assay) was conducted in order to compare the characteristics of the exosomes isolated from the human adipose-derived stem cells, the human umbilical cord-derived stem cells and the human bone marrow-derived stem cells. Through this, the expression level of HGF (hepatocyte growth factor) and IL-10 (interleukin-10) known to be helpful in relieving pulmonary fibrosis was determined.

[0092] Specifically, after mixing the respective exosomes isolated from the stem cells of various origins at a concentration of 7×10.sup.7 particles/200 μL with a RIPA (radioimmunoprecipitation assay) buffer at a ratio of 1:1, the expression level of HGF and IL-10 in the exosomes was investigated using a HGF ELISA kit (Abcam, ab100534) and an IL-10 ELISA kit (Abcam, ab100549).

[0093] As a result of investigating the expression level of HGF and IL-10 by ELISA, HGF was expressed at the highest level in the exosomes isolated from the human adipose-derived stem cells (HASC-Exo), showing significant difference from the exosomes isolated from the human umbilical cord-derived stem cells (UC-MSC-Exo). Meanwhile, the expression level was not significantly different from the exosomes isolated from the human bone marrow-derived stem cells (BM-MSC-Exo). As for IL-10, the expression level was similar in all the exosomes isolated from the stem cells of various origins (FIG. 3B).

Example 3: Evaluation of Collagen Synthesis Inhibition Ability of Exosomes Using Fibrosis-Induced Lung Fibroblasts

[0094] Most organs undergo inflammation and healing processes after tissue damage. If the damage is slight, the normal structure and function of the tissue are maintained. But, continued damage leads to fibrosis of the tissue during the healing process. The fibrosis process involves the accumulation of extracellular matrices (ECM) such as collagen, fibronectin, etc. in the tissue, leading to destruction of normal structure and functional disorder (Tuberculosis and Respiratory Diseases, Vol. 54, No. 2, pp. 1-12, February, 2003).

[0095] In order to investigate whether the respective exosomes isolated from the human adipose-derived stem cells, the human umbilical cord-derived stem cells and the human bone marrow-derived stem cells exhibit effective therapeutic effect for pulmonary fibrosis, an in-vitro model was established using lung fibroblasts and the collagen synthesis inhibition ability of the respective exosomes isolated from the stem cells of various origins was evaluated.

[0096] Specifically, primary human lung fibroblasts (Normal, ATCC, PCS-201-013) were subcultured until passage 6 using a fibroblast basal medium (ATCC, PCS-201-030) supplemented with the fibroblast growth kit-low serum (ATCC, PCS-201-041).

[0097] After the subculturing, the medium composition was changed by adding cobalt chloride(II) hexahydrate ((CoCl.sub.2.6H.sub.2O) known as a lung sensitizer inducing a hypoxic condition in cultured cells to the culture medium at a concentration of 100 μM. The medium was treated with the exosomes isolated from the human adipose-derived stem cells (HASC-Exo), the exosomes isolated from the human umbilical cord-derived stem cells (UC-MSC-Exo) or the exosomes isolated from the human bone marrow-derived stem cells (BM-MSC-Exo) at a concentration of 1×10.sup.8 particles/mL or 5×10.sup.8 particles/mL. 24 hours later, the culture medium was harvested and the expression level of COL1A1 (collagen type I, alpha 1) was investigated using an ELISA kit. A growth medium (GM) and a serum-free basal medium (BM) were used as a positive control group and a negative control group, respectively.

[0098] As a result of evaluating the collagen synthesis inhibition ability of the exosomes isolated from the stem cells of various origins by ELISA, the group treated with the exosomes isolated from the human adipose-derived stem cells (HASC-Exo) showed significantly decreased expression of COLIA1 than the groups treated with the exosomes isolated from the human umbilical cord-derived stem cells (UC-MSC-Exo) and the exosomes isolated from the human bone marrow-derived stem cells (BM-MSC-Exo).

[0099] From this result, it was confirmed that the exosomes isolated from the human adipose-derived stem cells (HASC-Exo) exhibit superior collagen synthesis inhibition ability as compared to the exosomes isolated from the human umbilical cord-derived stem cells (UC-MSC-Exo) and the exosomes isolated from the human bone marrow-derived stem cells (BM-MSC-Exo) (FIGS. 4A-4B).

[0100] Because the exosomes isolated from the human adipose-derived stem cells (HASC-Exo) exhibit superior collagen synthesis inhibition ability in the fibrosis-induced lung fibroblasts as compared to the exosomes derived from other stem cells (UC-MSC-Exo and BM-MSC-Exo), it can be inferred that the exosomes isolated from the adipose-derived stem cells (HASC-Exo) may be useful in treatment of pulmonary fibrosis.

Example 4: Isolation of Exosomes from Human Adipose-Derived Stem Cells Under Normoxia and Hypoxia Conditions

[0101] In general, experiments using stem cells are conducted under normal oxygen partial pressure (oxygen 21%). However, it is known that the in-vivo environment where the stem cells are actually exposed in the body has a very low oxygen partial pressure (Exp Hematol., 2002; 30: 67-73).

[0102] In Example 3, it was confirmed that the exosomes isolated from the human adipose-derived stem cells (HASC-Exo) exhibit superior collagen synthesis inhibition ability in fibrosis-induced lung fibroblasts as compared to the exosomes derived from other stem cells (UC-MSC-Exo and BM-MSC-Exo) and the exosomes isolated from the adipose-derived stem cells can be usefully used for treatment of pulmonary fibrosis.

[0103] Based on this result, in order to isolate exosomes effective for treatment of pulmonary fibrosis, human adipose-derived stem cells were proliferated by subculturing until passage 7 and then cultured under a normoxia condition or a hypoxic condition to isolate exosomes (normoxia exosomes or hypoxia exosomes).

[0104] Specifically, the human adipose-derived stem cells were subcultured until passage 7 using a DMEM (Dulbecco's modified Eagle's medium, high glucose; Gibco, Cat #: 11995065) normal culture medium supplemented with 10% FBS (fetal bovine serum) and 1% penicillin/streptomycin. Cobalt chloride(II)hexahydrate (CoCl.sub.2.6H.sub.2O) known as a substance inducing a hypoxic condition in cultured cells was used to induce a hypoxic culture condition. After changing the medium composition by adding the cobalt chloride(II) hexahydrate to the culture medium at a concentration of 100 μM, the cells were cultured for 1 day (FIG. 5A). Meanwhile, for a normoxic culture condition, cobalt chloride(II) hexahydrate was not added to the culture medium.

[0105] 24 hours before isolating exosomes, the medium was replaced with a serum-free, antibiotic-free, phenol red-free medium (Gibco, Cat #: 31053028). After culturing for 24 hours, a cell culture supernatant was recovered. Exosomes were isolated and purified from the recovered cell culture supernatant using a multifilter system as described in Example 1.

[0106] As a result of investigating the concentration of the exosomes isolated from the human adipose-derived stem cells cultured under the normoxia condition or hypoxic condition by nanoparticle tracking analysis, the concentration of the exosomes isolated from the human adipose-derived stem cells cultured under the normoxia condition (normoxia exosomes) was 2.25×10.sup.9 particles/mL and the concentration of the exosomes isolated from the human adipose-derived stem cells cultured under the hypoxic condition (hypoxia exosomes) was 2.45×10.sup.9 particles/mL (FIG. 5C). Also, the isolated exosomes were found to be in the form of round nanoparticles (FIG. 5B).

Example 5: Evaluation of Therapeutic Effect for Pulmonary Fibrosis of Exosomes Isolated from Adipose-Derived Stem Cells

[0107] In order to investigate the therapeutic effect for pulmonary fibrosis of the exosomes obtained in Example 4, i.e., the exosomes obtained from the adipose-derived stem cells cultured under the normoxia condition (Normoxia-Exo) and the exosomes obtained from the adipose-derived stem cells cultured under the hypoxic condition (Hypoxia-Exo), a pulmonary fibrosis animal model was established and the therapeutic effect for pulmonary fibrosis was evaluated by administering the exosomes to the established pulmonary fibrosis animal model.

[0108] Specifically, a bleomycin induced pulmonary fibrosis C57BL/6J mouse model was established by injecting a bleomycin solution, which induces pulmonary fibrosis, directly into the lungs of C57BL/6J mouse (9-week-old, male, Central Lab. Animal) by intratracheal instillation (IT). Normoxia-Exo and Hypoxia-Exo were respectively administered to the established bleomycin-induced pulmonary fibrosis C57BL/6J mouse via intravenous (IV) injection. A group administered with PBS (phosphate-buffered saline) was used as a negative control group and a group administered with human adipose-derived stem cells (HASCs) was used as a positive control group.

[0109] More specifically, the human adipose-derived stem cells (HASCs) were prepared in PBS at a concentration of 1×10.sup.6 cells/100 μL and each of Normoxia-Exo and Hypoxia-Exo was dispersed in PBS to a concentration of 1×10.sup.8 particles/100 μL (corresponding to 5×10.sup.9 particles/kg) for injection.

[0110] The prepared Normoxia-Exo and Hypoxia-Exo were administered to the bleomycin-induced pulmonary fibrosis C57BL/6J mouse for 3 times on days 1, 3 and 5 with 2-day intervals via intravenous injection. The PBS, as the negative control group, and the human adipose-derived stem cells (HASCs) prepared at the concentration of 1×10.sup.6 cells/100 μL, as the positive control group, were also administered for 3 times with 2-day intervals via intravenous injection, as the Normoxia-Exo and Hypoxia-Exo (FIG. 6A).

[0111] On day 6, i.e., on the next day after the third administration of the PBS, HASCs, Normoxia-Exo and Hypoxia-Exo via intravenous injection, the lung tissue was extracted to investigate the therapeutic effect for pulmonary fibrosis (FIG. 6B).

[0112] Trichrome staining was conducted on the lung tissue. The degree of pulmonary fibrosis was investigated by quantifying the blue-stained collagen area (FIGS. 7-9).

[0113] As a result, it was confirmed that the test groups to which the Normoxia-Exo and Hypoxia-Exo respectively isolated from the adipose-derived stem cells cultured under the normoxia condition and the hypoxic condition were administered showed significantly decreased stained collagen area as compared to the negative control group (PBS-administered group) or the positive control group (human adipose-derived stem cells (HASCs)-administered group), to a level similar that of the normal lung tissue.

[0114] In particular, the test group to which the exosomes isolated from the adipose-derived stem cells cultured under the hypoxic condition (Hypoxia-Exo) were administered showed remarkably decreased stained collagen area as compared to the negative control group, the positive control group and the test group administered with Normoxia-Exo, to a level nearly similar that of the normal lung tissue (FIGS. 7-9).

[0115] From these results, it was confirmed that both the exosomes obtained from the adipose-derived stem cells cultured under the normoxia condition (Normoxia-Exo) and the exosomes obtained from the adipose-derived stem cells cultured under the hypoxic condition (Hypoxia-Exo) have an effect of improving or treating pulmonary fibrosis. In particular, it can be seen that the exosomes obtained from the adipose-derived stem cells cultured under the hypoxic condition (Hypoxia-Exo) exhibit remarkably superior therapeutic effect for pulmonary fibrosis.