STEM CELL INDUCTION INTO PRECHONDROCYTES AND DIFFERENTIATION INTO CHONDROCYTES BY CIPROFLOXACIN

Abstract

The present invention relates to the effect of ciprofloxacin of inducing stem cells into chondroprogenitor cells or differentiating stem cells into chondrocytes.

A pharmaceutical composition, chondroprogenitor cells, and chondrocytes according to the present invention may be used for preventing or treating cartilage-related diseases.

Claims

1. A composition for inducing stem cells into chondroprogenitor cells, comprising ciprofloxacin and a growth factor.

2. The composition of claim 1, wherein the stem cells are mesenchymal stem cells.

3. The composition of claim 1, wherein the growth factor is one or more selected from the group consisting of epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF) and skin growth factor (SGF).

4. A composition for inducing differentiation of stem cells into chondrocytes, comprising ciprofloxacin.

5. The composition of claim 4, wherein the stem cells are mesenchymal stem cells.

6.-44. (canceled)

45. A method for treating cartilage-related diseases, the method comprising administering a chondroprogenitor cells to a subject in need thereof, wherein the chondroprogenitor cells are induced from a composition for inducing stem cells into chondroprogenitor cells, comprising ciprofloxacin and a growth factor.

46. The method of claim 45, wherein the stem cells are mesenchymal stem cells.

47. The method of claim 45, wherein the growth factor is one or more selected from the group consisting of epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF) and skin growth factor (SGF).

48. A method for treating cartilage-related diseases, the method comprising administering chondrocytes to a subject in need thereof, wherein the chondrocytes are induced from a composition for inducing differentiation of stem cells into chondrocytes, comprising ciprofloxacin.

Description

DESCRIPTION OF DRAWINGS

[0104] FIG. 1A shows the characteristics of stem cells as the basic characteristics of stem cells.

[0105] FIG. 1B shows positive reactions to CD29, 73, 90, and 105 and negative reactions to CD31, 34, and 45 in the immunophenotype expressed by stem cells, as a basic characteristic of stem cells.

[0106] FIG. 1C confirmed the ability to differentiate into fat, bone, and cartilage to confirm pluripotency in the differentiation ability of mesenchymal stem cells, as a basic characteristic of stem cells.

[0107] FIG. 1D shows the results of showing self-renewal ability through colony forming assay, as a basic characteristics of stem cells.

[0108] FIG. 1E shows that the stem cells of the present invention are stable by confirming that there are no changes that would cause genetic defects even after culturing up to 6 passages.

[0109] FIG. 2 shows the results of confirming the cytotoxicity of ciprofloxacin.

[0110] FIG. 3A shows, using a 2D method, changes in cell characteristics and proteins when ciprofloxacin is used to induce differentiation of mesenchymal stem cells into chondrocytes.

[0111] FIG. 3B shows the results of confirming, using a 3D method, changes in cell characteristics and proteins when ciprofloxacin is used to induce differentiation of stem cells into chondrocytes.

[0112] FIG. 4A shows the characteristics of chondroprogenitor cells induced by ciprofloxacin.

[0113] FIG. 4B shows the karyotyping results of chondroprogenitor cells induced by ciprofloxacin showing there are no genetic mutations in the induced cells.

[0114] FIG. 5A shows the results of confirming the expression rates of stemness markers of chondroprogenitor cells and stem cells according to the present invention.

[0115] FIG. 5B shows the results of confirming the self-renewal abilities of chondroprogenitor cells and stem cells according to the present invention.

[0116] FIG. 5C shows the results of confirming the multipotency (ability to induce fat differentiation, ability to induce osteogenic differentiation, and ability to induce cartilage differentiation) of chondroprogenitor cells and stem cells according to the present invention.

[0117] FIG. 6A shows the results of confirming the mRNA expression level of SOX9 according to the induction of the differentiation of chondroprogenitor cells and stem cells according to the present invention.

[0118] FIG. 6B shows that in the case of chondroprogenitor cells according to the present invention, COMP increases as SOX9 decreases, but in the case of stem cells, COMP increases at the early stage of differentiation induction and then decreases.

[0119] FIG. 7A shows the results of confirming the chondrocyte regeneration effect of chondroprogenitor cells and stem cells according to the present invention.

[0120] FIG. 7B shows the results of analyzing the chondrocyte region regenerated in the chondroprogenitor cell treatment group, stem cell treatment group, normal chondrocytes, and inflammatory chondrocytes according to the present invention.

[0121] FIG. 8 shows the results of confirming the inhibitory effect of the chondroprogenitor cells according to the present invention on a cartilage-degrading enzyme (ADAMTS4).

MODES OF THE INVENTION

[0122] The benefits and features of the present invention, and the methods of achieving the benefits and features will become apparent with reference to experimental examples and preparation examples to be described below in detail. However, the present invention is not limited to the experimental examples and the preparation examples to be disclosed below and may be implemented in various other forms, and the present disclosure is provided for rendering the disclosure of the present invention complete and for fully informing those with ordinary skill in the art to which the present invention pertains of the scope of the present invention.

EXAMPLES

Example 1. Confirmation of Cytotoxicity of Ciprofloxacin

[0123] Stem cells (collected from tissue donated by a hospital for non-profit patient treatment) were cultured by attaching 80% or more of the stem cells to a Dulbecco's modified Eagle's medium (DMEM) or a-MEM containing 1 to 10% FBS.

[0124] Thereafter, the cells were collected using Trypsin-EDTA, counted, seeded at 110.sup.3 cells/well in a 96-well plate, and attached to the plate for 24 hours. Thereafter, ciprofloxacin (Samsung Pharmaceutical, ciprofloxacin injection) was administered according to the concentration, and then cytotoxicity was confirmed for a specific time (up to 166 hours). Next, each well was treated with Cell Counting Kit-8 (hereinafter referred to as CCK, DOJINDO, Cat. No. CK04-13) at 10 l/well, and then cultured at 37 C. in a 5% CO.sub.2 incubator for 1 hour.

[0125] The absorbance was measured using an absorbance meter (Bio-Tek, Epoch2) and quantitatively calculated.

[0126] As a result, it was confirmed that ciprofloxacin in stem cells did not exhibit cytotoxicity even when treated up to 6 M (FIG. 2).

Example 2. Induction of Differentiation of Stem Cells into Chondrocytes Using Ciprofloxacin (2D)

[0127] Stem cells (collected from tissue donated by a hospital for non-profit patient treatment) were cultured by attaching 80% or more of the stem cells to a Dulbecco's modified Eagle's medium (DMEM) or a-MEM containing 1 to 10% FBS.

[0128] Thereafter, the cells were collected using Trypsin-EDTA, counted, seeded at 110.sup.3 cells/well in a 96-well plate, and attached to the plate for 24 hours. Thereafter, ciprofloxacin (Samsung Pharmaceutical, ciprofloxacin injection) was administered according to the concentration, and then cytotoxicity was confirmed for a specific time (up to 166 hours). Next, each well was treated with Cell Counting Kit-8 (hereinafter referred to as CCK, DOJINDO, Cat. No. CK04-13) at 10 l/well, and then cultured at 37 C. in a 5% CO.sub.2 incubator for 1 hour.

[0129] Thereafter, the stem cells induced to differentiate for 16 days were washed with DPBS and fixed with 4% paraformaldehyde for 1 hour.

[0130] The completely fixed cells were stained using an Alcian blue solution (Merck, B8438) for 1 hour to 1 day. Thereafter, the cells were washed once with 1 N HCl, and then further washed three times with sterile distilled water.

[0131] Finally, the washed sample was observed under a microscope.

[0132] As a result, it was confirmed that cells clustered together as stem cells were induced into chondrocytes, and it could be confirmed that Alcian blue staining, which appears depending on SOX9, was present throughout the chondrocytes (FIG. 3A).

Example 3. Induction of Differentiation of Stem Cells into Chondrocytes Using Ciprofloxacin (3D)

[0133] Stem cells (collected from tissue donated by a hospital for non-profit patient treatment) were cultured by attaching 80% or more of the stem cells to a Dulbecco's modified Eagle's medium (DMEM) or a-MEM containing 1 to 10% FBS.

[0134] Thereafter, the cells were collected and counted using Trypsin-EDTA, and 510.sup.5 cells were inoculated into a 15 ml conical tube. A medium containing 0.1 to 6 M ciprofloxacin was centrifuged to settle the cells at 150 g using a centrifuge (Hanil Science Co., Ltd., Combi415). The cells were cultured in a 5% CO.sub.2 incubator at 37 C. while exchanging the medium every 2 to 3 days.

[0135] The stem cells induced to differentiate for 16 days were washed with DPBS and fixed with 4% paraformaldehyde for 1 hour.

[0136] The completely fixed cells were stained using an Alcian blue solution (Merck, B8438) for 1 hour to 24 hours. Thereafter, the cells were washed once with 1 N HCl, and then further washed three times with sterile distilled water.

[0137] Finally, the washed sample was observed.

[0138] As a result, it was confirmed that cells clustered together and became tissue-like as stem cells differentiated into chondrocytes, and it could be confirmed that Alcian blue staining, which appears depending on SOX9, was present throughout the chondrocytes (FIG. 3B).

Example 4. Induction of Stem Cells into Chondrocytes Using Ciprofloxacin

[0139] Stem cells (collected from tissue donated by a hospital for non-profit patient treatment) were cultured by attaching 80% or more of the stem cells to a Dulbecco's modified Eagle's medium (DMEM) or a-MEM containing 1 to 10% FBS. Thereafter, the cells were collected using Trypsin-EDTA and then counted. The cells were suspended in a flask with a medium containing 0.1 to 1 M ciprofloxacin and a growth factor such as 0.1 to 100 ng EGF and 0.1 to 100 ng FGF, and cultured in a 5% CO.sub.2 incubator at 37 C. while exchanging the medium every 2 to 3 days. This process was repeated for 1 to 3 passages.

[0140] As a result, it was confirmed that stem cells were induced into chondroprogenitor cells from the composition including ciprofloxacin, the cell characteristics of the induced chondroprogenitor cells (FIG. 4A) were confirmed, and it was confirmed that no genetic mutations occurred in the cells by performing karyotyping (FIG. 4B).

Example 5. Comparison of Chondroprogenitor Cells and Stem Cells

[0141] Completely cultured stem cells (collected from tissue donated by a hospital for non-profit patient treatment) and chondroprogenitor cells induced in Example 4 were collected and washed with water.

[0142] After the completely washed cells were suspended using a FACS buffer, they were stained for stem cell markers CD73, CD90, CD105, CD31, CD34, and CD45 at 4 C. for 1 hour, and then centrifuged at 1500 RPM for 3 minutes. Thereafter, the cells were washed twice using a FACS buffer, and then expression was confirmed using a FACS (BD, Accuri6) apparatus.

[0143] As a result, it was confirmed that a positive marker for stem cells was shown to be positive even in chondroprogenitor cells, and a negative marker for stem cells was shown to be negative even in chondroprogenitor cells.

[0144] In addition, it was confirmed that when colony forming assay and bone cell differentiation chondrocyte differentiation ability, and adipocyte differentiation ability were performed using commonly used culture media or kits, the cells are shown to have the same or similar value to that of stem cells.

Example 6. Comparison of Chondrogenic Differentiation Abilities of Chondroprogenitor Cells and Stem Cells

[0145] The chondrogenic differentiation abilities of completely cultured stem cells (collected from tissue donated by a hospital for non-profit patient treatment) and the cartilage progenitor cells prepared in Example 4 above were compared.

[0146] As a result, it was confirmed that in the control, SOX9, an early marker, was basically expressed in chondroprogenitor cells about 20-fold more than in stem cells. However, it was confirmed that as differentiation was induced, the value decreased in chondroprogenitor cells, and only the expression of the early marker continuously increased in stem cells (FIG. 6A).

[0147] Thereafter, it was confirmed that COMP, which is a component that constitutes a matrix according to the organization of chondrocytes, increases as SOX9 decreases in chondroprogenitor cells, but in the case of stem cells, COMP increases and then decreases in the early stage of differentiation induction (FIG. 6B).

[0148] For this reason, when chondroprogenitor cells are applied to chondrocyte treatment, it is possible to address cartilage hypertrophy (induced by an exponential increase in SOX9) and the absence of hyaline cartilage (the absence of Col2, which hardens cartilage such as COMP), which are problems that occur when chondrocyte treatment is performed using stem cells.

Example 7. Comparison of Chondrocyte Regeneration Abilities of Chondroprogenitor Cells and Stem Cells

[0149] The chondrocyte regeneration ability effects of completely cultured stem cells (collected from tissue donated by a hospital for non-profit patient treatment) and the cartilage progenitor cells prepared in Example 4 above were compared.

[0150] An inflammatory response was induced in chondrocytes using LPS. Thereafter, physical stress was induced by applying a scratch of 2 mm or more, and then stem cells or chondroprogenitor cells were treated.

[0151] As a result, after an inflammatory response was induced in chondrocytes, some regions were damaged, and when the regions where chondrocytes were regenerated were compared by treating stem cells and chondroprogenitor cells, normal chondrocytes exhibited regeneration of about 61%, but chondrocytes in which inflammation was induced were regenerated by 24%. Furthermore, the group treated with stem cells and chondroprogenitor cells showed a regenerative ability of about 47% or more, and in particular, the group treated with stem cells showed a regenerative ability of about 47% and the group treated with chondroprogenitor cells showed a regenerative ability of about 89% (FIGS. 7A and 7B).

[0152] That is, it can be seen that the chondrocyte regeneration ability of chondroprogenitor cells is better than that of stem cells.

Example 8. Comparison of Cartilage-Degrading Enzyme Inhibitory Effects of Chondroprogenitor Cells

[0153] In order to confirm the paracrine effect of chondroprogenitor cells, an inflammatory response was induced in chondrocytes using a plate including an insert (VWR, 734-2719). After the induction, the inhibitory effect on a cartilage-degrading enzyme was confirmed by performing treatment of chondroprogenitor cells on the insert (indirect treatment) or directly treating the corresponding plate with chondroprogenitor cells.

[0154] Cartilage-degrading enzymes that induce cartilage degradation include enzymes such as ADAMTS4, ADAMTS5, and MMP-13.

[0155] As a result, it was confirmed that the expression levels of ADAMTS4 decreased by about 35% in the case of indirect treatment with chondroprogenitor cells and by about 76% in the case of direct treatment (FIG. 8).