KR20160144780A - A method for differentiation of tonsil-derived mesenchymal stem cell into tenocyte - Google Patents

Abstract

The present invention relates to a method for differentiating tenocytes from tonsil-derived mesenchymal stem cells, and a pharmaceutical composition for preventing or treating tendon diseases using the same. The differentiating method of the present invention can secure a large amount of tenocytes by showing high differentiating ability into the tenocytes, the differentiated cells according to the present invention have high histocompatibility by using abandoned autologous tissues and exhibit excellent usability as a pharmaceutical composition for preventing or treating tendon diseases.

Description

METHOD FOR DIFFERENTIATION OF TONSIL-DERIVED MESENCHYMAL STEM CELL INTO TENOCYTE BACKGROUND OF THE INVENTION 1. Field of the Invention < RTI ID = 0.0 >

TECHNICAL FIELD The present invention relates to a method for differentiating dry cells from mesenchymal-derived mesenchymal stem cells and a cell therapy agent using the same.

Stem cells are the cells that can differentiate into various cells constituting biological tissue and collectively referred to as the undifferentiated cells in the pre-differentiation stage which can be obtained from each tissue of embryo, fetus and adult body. Stem cells are characterized by the ability to differentiate into specific cells by differentiation stimuli (environment), self-renewal by the division of cells by self-renewal, and differentiated into different cells by differentiation stimuli It is characterized by having plasticity.

Stem cells can be divided into pluripotency, multipotency, and unipotency stem cells according to their differentiation potential. Pluripotent stem cells are pluripotent cells with the potential to differentiate into all cells, and some stem cells have the potential of multipotential or mono-differentiability.

The stem cells can be used as a cell therapy agent on the basis of the differentiation ability of the stem cells, and research and development related thereto are actively underway. However, in the case of cell therapy using embryonic stem cells, problems of ethical problems and inconsistency of tissue compatibility are raised, and there is a problem of possibility of tumor development when using the degenerated stem cell as a cell therapy agent.

Thus, studies using mesenchymal stem cells, which are known to have low ability to differentiate, are relatively safe. Mesenchymal stem cells (MSCs) are multi-potential non-hematopoietic progenitor cells in the adult bone marrow. They are cells capable of differentiating into various types of cells such as fat, cartilage, bone, muscle and skin. Clinical studies for regeneration of various tissues using such mesenchymal stem cells are under way, and they are applicable to organ transplantation.

However, among mesenchymal stem cells, some stem cells are difficult to use because of their great limitations in obtaining cells for their use. For example, cells that can be obtained using the most non-invasive method are mesenchymal stem cells through bone marrow harvesting. However, bone marrow harvesting, the most non-invasive method, requires anesthesia and causes pain, which limits its use. In order to isolate patient-tailored stem cells, a cell harvesting method using peripheral blood is required as an alternative. However, the number of mesenchymal stem cells that can be isolated from adults is too small, the separation method is not economical, However, since it is difficult to proliferate by an amount that can be used for cell therapy, an alternative method that can increase practicality is needed.

In addition, adult stem cells obtained from elderly patients have a significantly lower proliferative capacity than the cells obtained from lower ages, and secretion of various factors and migration ability to stem cell lesions are reduced. Therefore, adult stem cells are naturally separated from patients of lower ages There is a need to obtain cells from tissues that can or are discarded.

On the other hand, the tendon is a taut band, usually a fibrous connective tissue that connects the muscle to the bone. The elasticity of the tendon regulates and stabilizes the force during exercise and stores energy and restores it with high efficiency. The tendon has a highly differentiated superfine structure, its vascularization level is low, and collagen conversion is slow, so if it is damaged, the healing rate is very slow and it is very difficult to recover to its original strength. Thus, it is common for the injury to recur at the injury site of the gun.

Gun diseases are chronic disorders or injuries that appear to be caused by an imbalance between the metabolic reaction and the anabolic metabolic reaction resulting from overdose or aging resulting in gradual tendon abrasion and tearing. The consequences of such an imbalance are dryness, weakness, tear, and pain. In contrast, acute dry injury, for example, tearing or separation from the bone, occurs very abruptly and usually requires surgery to restore the tear or reattach the tendon to the bone.

Anyone can develop the disease, but the person is more likely to develop the disease if he or she is a person who tends to repeat the same movements during occupation, exercise, or routine routine activities. For example, the shoulder joint has a large range of motion, but is not very stable, and degeneration occurs rapidly due to wear due to shoulder use. Moreover, the rotating-near functional unit is vulnerable to damage due to weak blood circulation and is a place where complete rupture is common.

Therapeutic therapies have traditionally focused on anti-inflammatory measures, including treatment with non-steroidal anti-inflammatory drugs (NSAIDs), steroid injections, and physical therapy. More recently, shock wave therapy, low-level laser therapy, vascularized therapy, and other experimental therapies have been tested. In general, the long-term benefits of current therapies are still unclear, while they are short-term relief through some remedies (e.g., NSAIDs and cortisone injections). Therefore, there is a need for an improved method of treating dry disease that can provide benefits over a longer period of time as compared to current treatments.

Under these circumstances, it is necessary to research and develop a method for securing a suitable cell for application to human body by confirming the optimal differentiation method from a specific stem cell which can show high ability to differentiate into a dry cell.

Korean Patent Publication No. 10-2014-0103132 Korean Patent Publication No. 10-2014-0031300

The present invention relates to a method of differentiating dry-cell from mesenchymal stem cells, which comprises culturing a mesenchymal-derived mesenchymal stem cell in a medium containing TGF-? 3.

The present invention also relates to a dry cell produced according to the aforementioned method for differentiating a dry cell.

The present invention also relates to a pharmaceutical composition for the prevention or treatment of dry eye disease, comprising a dry cell produced according to the above-mentioned dry cell differentiation method as an active ingredient.

The inventors of the present invention completed the present invention by inventing a method for mass production of dry cells from one-way-derived mesenchymal stem cells in a short period of time while studying a method for mass production of dry cells suitable for human application.

In the present invention, a mesenchymal stem cell originating from one side is located in the back of the throat and the nose, and is a tissue that acts primarily as a lymphocyte immune tissue while defending the body from substances such as bacteria that enter from the outside Means an undifferentiated stem cell having the ability to replicate into two or more new cells while having self-replicating ability derived from one-way.

In the present invention, a tenocyte is a white fibrous connective tissue cell with wing-like protrusions extending between the bundles of fibers, and can be a tethered band, usually a fibrous connective tissue that connects muscles to the bone .

The present invention provides a method for differentiating a dry cell from a mesenchymal stem cell derived from a monocyte, which comprises culturing a mesenchymal stem cell derived from one end in a medium containing TGF-? 3.

In the present invention, TGF-β3 corresponds to a growth factor that plays an important role in the regulation of cell differentiation, and it stimulates mesenchymal stem cells derived from one end as a differentiation factor from mesenchymal stem cells into dry cells, By weight.

In the present invention, cell culture is preferably performed by culturing 2x10 ⁴ to 5x10 ⁴ cells per cm ² in a culture dish for 2 to 13 days, preferably 3 to 10 days. Further, in the present invention, TGF-? 3 is 10 ng / ml to 70 ng / ml. Preferably 20 ng / ml to 50 ng / ml.

One-shot-derived mesenchymal stem cells are cultured for 1 to 3 days, preferably every 3 days, by changing medium containing 10 ng / ml to 70 ng / ml, preferably 20 ng / ml to 50 ng / ml of TGF- Can be re-stimulated.

The culture medium may be any one selected from DMEM (Dulbecco's Modified Eagle Medium), RPMI1640 (Roswell Park Memorial Institute 1640), MEM (Minimum Essential Media) or Ham F10, and preferably DMEM (Dulbecco's Modified Eagle Medium) medium.

The method of the present invention for differentiating dry cells from mesenchymal stem cells employs one-way derived mesenchymal stem cells and shows remarkable differentiation potential compared with the method using adult stem cells derived from other sources. Can be confirmed through a high expression pattern. According to the present invention, the expression of type I collagen, Decorin, Tenomodulin and Scleraxis is greatly increased by the differentiation of the undifferentiated state into the dry cell of stem cells derived from the unilateral stem cells.

The present invention also provides a dry cell produced according to the above differentiation method. The dry cells prepared according to the present invention exhibit high 1 type collagen, decorin, tenomodulin and scleraxis expression levels compared to the one-way stem cells, and have excellent characteristics as dry cells.

The present invention also provides a pharmaceutical composition for preventing or treating dry disease, which comprises dry cells prepared according to the above differentiation method as an active ingredient. That is, it can be used as a drug used for treatment, diagnosis and prevention of diseases and diseases in cells and tissues prepared by isolation, cultivation and special manipulation from an individual.

The dry disease may be any one selected from the group consisting of Achilles tendon disease, patellofacial disease, lateral phase hyperplasia, medial phase hyperplasia, plantar fasciitis, and rotator cuff disease.

The pharmaceutical composition for preventive or therapeutic treatment of dry disease can be formulated into a unit dosage form of a pharmaceutical preparation suitable for administration to a patient in accordance with a conventional method in the pharmaceutical field. The preparation may be administered once or several times ≪ / RTI > As formulations suitable for this purpose, injectable preparations, injecting agents and spraying agents are preferred as parenteral administration preparations. In addition, the pharmaceutical composition for preventing or treating dry disease may include a pharmaceutically acceptable inert carrier. Also, it can be transplanted and administered using a method of administration commonly used in the art, and preferably it can be transplanted or transplanted directly to a diseased part of a patient in need of treatment, but is not limited thereto. In addition, the above administration is possible both in a non-surgical administration using a catheter and in a surgical administration method such as implantation or transplantation after incision of a disease site. The dose may be administered once or several times in the range of 1.0 × 10 ⁵ to 1.0 × 10 ⁹ cells / kg body weight, preferably 1.0 × 10 ⁶ to 1.0 × 10 ⁷ cells / kg body weight. It should be understood, however, that the actual dosage of the active ingredient should be determined in light of various relevant factors such as the disease to be treated, the severity of the disease, the route of administration, the body weight, age and sex of the patient, The scope of the present invention is not limited thereto.

The present invention also provides a method for three-dimensional differentiation of dry cells from a mesenchymal stem cell.

According to an embodiment of the present invention, there is provided a method for three-dimensional differentiation of dry cells from a mesenchymal stem cell, comprising: (a) culturing a stem cell derived from one end in a medium containing hydrogel; And (b) adding TGF-? 3 to the culture medium of step (a). The present invention also provides a method for the three-dimensional differentiation of dry cells from a mesenchymal stem cell.

In the three-dimensional differentiation method according to the present invention, in step (a), it is preferable to cultivate ¹ x 10 ⁶ to 2 x 10 ⁶ cells per cm. In step (b), TGF-? 3 is preferably 10 ng / ml to 70 ng / Preferably, 20 ng / ml to 50 ng / ml is added. Re-stimulation by cell differentiation period and culture medium exchange can be applied by properly modifying the method of differentiation of dry cells.

The three-dimensional differentiation method according to the present invention provides a three-dimensional environment through a hydrogel support to precisely regulate the differentiation direction of the stem cells before transplantation, and shows a high differentiation rate and cell survival rate, Lt; / RTI > Hydrogels include, but are not limited to, natural hydrogels such as alginate, collagen and agarose, and synthetic hydrogels such as polyethyleneglycol (PEG), polylactic-co-glycolic acid (PGLA) . These hydrogels offer advantages such as constructing physiology, pathological models, or enhancing the cellular environment and cell delivery vehicles through three-dimensional cell culture at the tissue level rather than the cell level.

According to another embodiment of the present invention, there is provided a method for the three-dimensional differentiation of dry cells from a mesenchymal stem cell line derived from a single duct, comprising the steps of: (a) culturing a somatic stem cell in a culture medium supplemented with TGF- ; (b) culturing the cells of step (a) in a collagen I-treated culture plate membrane; And (c) subjecting the cells to equi-biaxial pressure through the collagen I-treated culture plate membrane to provide three-dimensional differentiation of dry cells from the mesenchymal stem cells.

In step (a) of the three-dimensional differentiation method of cancer cells according to the present invention, TGF-? 3 may be added at 10 ng / ml to 70 ng / ml, preferably 20 ng / ml to 50 ng / ml. The culture plate membrane treated with collagen I in step (b) may be, for example, Bioflex culture plate membrane with collagen I (Bioflex; BF-3001C), and the TGF- Cells can be cultured.

The step of applying the biaxial-tensile pressure of step (c) is a step of providing mechanical stimulation for activation of the dry cells. Application of biaxial-tensile pressures is described, for example, in Flexcell ^? In the tension system, pressure can be applied through the vacuum to transfer cyclic pressure to the loading post under the membrane via the Bioflex culture plate membrane with collagen I. The application of pressure can be applied by applying a pressure of 0.5 to 2 Hz, preferably 1 Hz for 30 minutes to 180 minutes, preferably about 120 minutes, and the cultivation under biaxial-tensile pressure is carried out for 1 to 7 days, preferably 2 to 4 Lt; / RTI > days. Re-stimulation by cell differentiation period and culture medium exchange can be applied by properly modifying the method of differentiation of dry cells.

The differentiation method according to the present invention shows a high ability to differentiate into a dry cell, and it is possible to secure a large amount of dry cells. The cells differentiated according to the present invention have high tissue compatibility by using an abandoned autologous tissue, And exhibits excellent usability as a composition.

FIG. 1 shows the results of confirming the expression of dry cell-associated genes according to differentiation time and TGF-β3 concentration from one-way derived mesenchymal stem cells.
FIG. 2 is a graph showing the results of immunofluorescence analysis of the expression of type 1 collagen upon differentiation of mesenchymal stem cells into dry cells. FIG.
FIG. 3 is a graph showing the change in expression of tenomodulin from differentiation of mesenchymal stem cells into dry cells by immunofluorescence. FIG.
FIG. 4 is a graph showing changes in expression of Decorin by differentiation of mesenchymal stem cells into dry cells by immunofluorescence.
FIG. 5 is a graph showing the results of immunofluorescence analysis of the expression of Scleraxis according to differentiation of mesenchymal stem cells into dry cells.
FIG. 6 is a graph showing the results of immunohistochemical staining for the expression of type 1 collagen upon differentiation of mesenchymal stem cells into dry cells.
FIG. 7 is a graph showing the results of immunohistochemical staining for the expression of tenomodulin by differentiation of mesenchymal stem cells into dry cells.
FIG. 8 is a graph showing the results of immunohistochemical staining for changes in the expression of Decorin according to differentiation of mesenchymal stem cells into dry cells.
FIG. 9 is a graph showing the results of immunohistochemical staining for the expression of Scleraxis according to the differentiation of mesenchymal stem cells into dry cells.

Examples and production examples are provided to facilitate understanding of the present invention. The following examples and preparation examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples and the production examples.

< Example  1> One way origination Intermediate lobe  Culture of stem cells

Tissue-derived mesenchymal stem cells (TMSCs) were isolated from patients who underwent tonsillectomy at post-nasopharyngeal surgery in Ewha Womans University Hospital (4-20-year-old low-grade tissue, Passing the examination: ECT 11-53-02).

The collected fresh tissue was washed three times with PBS (phosphate buffered saline), cut into small pieces using a sterilized scissor, and incubated with 210 U / mL collagenase type I (Invitrogen) and 10 μg / mL DNAse (Sigma-Aldrich, St. Louis, MO). Louis, MO, USA) for 30 min at 37 ° C. The mixture was filtered through a cell strainer, washed twice in DMEM medium containing 20% normal human serum (PAA Laboratories GmbH, Austria), and washed once in DMEM medium containing 10% human serum. Then, centrifugation was performed in Ficoll-Paque (GE Healthcare, Buckinghamshire, UK) and mononuclear cells were obtained in digestive tonsil tissue. The cells were cultured in a DMEM medium supplemented with 10% fetal bovine serum (FBS), 100 μg / mL streptomycin, and 100 U / mL penicillin at a concentration of 1 × 10 ⁷ per 150 mm diameter. After 24 h, unattached cells were removed and replaced with fresh DMEM supplemented with 50 uM 2-mercaptoethanol, 100 U / mL penicillin, 100 μg / mL streptomycin, 10% FBS. Fresh cells with 3-5 passages were used in the experiment.

< Example  2> One-way derived from two-dimensional culture Intermediate lobe  Differentiation of tenocytes from stem cells

The mesenchymal stem cells prepared in Example 1 were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS), 100 μg / mL streptomycin and 100 U / mL penicillin in a 4 well slide chamber at 2 × 10 ⁴ per well Lt; / RTI &gt; TGF-β3 (Peprotech) -free wells and TGFβ-3 20 ng / ml or 50 ng / ml stimulated wells were cultured and stimulation dates were 1, 3, and 7 days, respectively. Cells were re-stimulated every 3 days.

< Example  3> RT- PCR  Confirmation of cell differentiation through

RT-PCR was performed to confirm differentiation into dry cells. Total RNA was extracted with Trizol (Invitrogen), and the diluted RNA was transferred to a quartz cuvette, and then the absorbance at 260 nm and the absorbance at 280 nm were measured. cDNA synthesis was performed using Maxime RT premix kit (Intron viotechnology) and mRNA expression was confirmed by real time PCR with each Tenocyte primer using FastStart SYBR Green Master (Roche Applied Science).

The primer information used is shown in Table 1 below.

The experimental results are shown in Fig. As shown in FIG. 1, it was confirmed that mRNA expression of type 1 collagen, Decorin, Tenomodulin and Scleraxis, which are related to dry cell-related genes, was increased through TGF-β3 stimulation in one-way derived mesenchymal stem cells, .

< Example  4> Immunity Fluorescence  Confirmation of cell differentiation through

In the same manner as in Example 2, the cells incubated with TGF-? 3 (Peprotech) antibody stimulation were fixed with 20 占 폚 methanol for 5 minutes. The fixed cells were stained with the primary antibody of Decorin (abcam), Collagen type I (abcam), Tenomodulin (santacruz) or Scleraxis (abcam) and then incubated overnight at 4 ° C with fluorescent secondary antibody 647 donkey anti-rabbit IgG (Thermo) and DAPI (Invitrogen). The stained slides were analyzed with a Zeiss microscope (LSM 510 Meta; Carl Zeiss, Oberkochen, Germany).

The results are shown in Figs. 2 to 5. Fig. FIG. 2 shows the results of fluorescence staining of collagen type I (Col-I), FIG. 3 shows Tenomodulin (Tmnd), FIG. 4 shows Decorin and FIG. 5 shows Scleraxis (Scx).

As shown in FIGS. 2 to 5, the expression of Decorin, Collagen type I, Tenomodulin, and Scleraxis, which are genes expressed in dry cells according to TGF-β3 treatment, was increased and the differentiation into dry cells was confirmed.

< Example 5> Immunity  Identification of dry cell differentiation by histochemical staining

In the same manner as in Example 2, the cells incubated with TGFβ3 (Peprotech) antibody stimulation were fixed with 20 ° C methanol for 5 minutes. Methanol was reacted with 3% H ₂ O ₂ at 4 ° C for 30 min for endogenous peroxidase activation. Vectastain ABC kit (Vector Laboratories, Burlingame, Calif.) Was used for immunohistochemical staining. Cells were incubated at 4 ° C overnight with the primary antibodies to Decorin (abcam), Collagen type I (abcam), Tenomodulin (santacruz) or Scleraxis (abcam). The cells incubated overnight were incubated with primary antibody-related biotinylated secondary (Rabbit) antibodies for 1 hour at room temperature after washing with PBS. And incubated with Avidin-Biotinylated HRP for 1 hour at room temperature. After adding DAB substrate, wait until the desired color development was achieved at room temperature, wash with distilled water to stop color development, stain nuclear staining with hematoxylin, and mount the stained slide.

The results are shown in FIG. 6 to FIG. Fig. 6 shows the results of immunohistochemical staining of type 1 collagen (Collagen type I), Fig. 7 shows Tenomodulin, Fig. 8 shows Decorin and Fig. 9 shows Scleraxis.

As shown in FIGS. 6 to 9, it was confirmed that the expression of Decorin, Collagen type I, Tenomodulin and Scleraxis, which are genes expressed in dry cells according to TGF-β3 treatment, Respectively.

From the above results, it was confirmed that the ability to differentiate from mesenchymal stem cells into dry cells was excellent according to the present invention.

<110> Ewha University - Industry Collaboration Foundation <120> A METHOD FOR DIFFERENTIATION OF TONSIL-DERIVED MESENCHYMAL STEM          CELL INTO TENOCYTE <130> P15-077-EWHA <160> 10 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Collagen type 1 forward primer <400> 1 tgacctcaag atgtgccact 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Collagen type 1 reverse primer <400> 2 accagacatg cctcttgtcc 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Collagen type 3 forward primer <400> 3 gctggcatca aaggacatcg 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Collagen type 3 reverse primer <400> 4 tgttacctcg aggccctggt 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TNMD forward primer <400> 5 ttgaagaccc acgaagtaga 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TNMD reverse primer <400> 6 atgacatgga gcacactttc 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Mohawk forward primer <400> 7 tggtttgcta atgcaagacg 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Mohawk reverse primer <400> 8 ccttcgttca tgtgggttct 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Decorin forward primer <400> 9 cgcctcatct gagggagctt 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Decorin reverse primer <400> 10 tactggaccg ggttgctgaa 20

Claims (9)

And culturing the mesenchymal stem cells derived from the one or more tonsils in a medium containing TGF-? 3. The method according to claim 1, wherein the concentration of TGF-? 3 is 10 ng / ml to 70 ng / ml. 3. The method according to claim 2, wherein the TGF-beta3 is re-added every 1 to 3 days for re-stimulation of differentiation. The method according to claim 1, wherein the culturing is performed for 2 days to 13 days. The culture medium according to claim 1, wherein the culture medium comprises at least one selected from the group consisting of DMEM (Dulbecco's Modified Eagle Medium), RPMI1640 (Roswell Park Memorial Institute 1640), MEM (Minimum Essential Media) Differentiation method. 6. A dry cell produced according to the method of any one of claims 1 to 5. A pharmaceutical composition for preventing or treating dry matter disease, comprising the dry cell according to claim 6 as an active ingredient. (a) culturing a one-sided stem cell in a medium containing a hydrogel; And
(b) adding TGF-? 3 to the culture medium of step (a). (a) culturing one-way stem cells in a culture medium supplemented with TGF-? 3;
(b) culturing the cells of step (a) in a collagen I-treated culture plate membrane; And
(c) subjecting the cells to equi-biaxial pressure through the collagen I-treated culture plate membrane.

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