KR102282437B1 - Method for differentiation of fibroblast-like cells from human adipose derived stem cells - Google Patents

Abstract

The present invention relates to a method for differentiating human adipose-derived stem cells into fibroblast-like cells using a plate coated with gelatin and containing a cell differentiation medium, and to a medium composition. The plate containing gelatin of the present invention can exert the effect of inducing direct cross-differentiation from human adipose-derived stem cells to fibroblasts, and efficiently performing mass culture in vitro at low cost by using economical materials. In addition, the fibroblast-like cells differentiated from human adipose-derived stem cells of the present invention can be used in a composition for alleviating skin wrinkles or scars.

Description

Method for differentiation of fibroblast-like cells from human adipose derived stem cells

The present invention relates to a method for differentiation inducing differentiation from human adipose-derived mesenchymal stem cells into fibroblast-like cells in a cell culture plate for inducing differentiation containing gelatin using a medium composition and the medium composition.

Depending on whether the skin surface is destroyed or not, there are no open wounds such as cuts, lacerations, penetrating wounds, abrasions, etc. in which the skin or mucous membrane is damaged and the internal tissues are exposed to air, and there is no rupture of the skin or mucous membrane, but blunt It can be classified as a closed wound that can occur when bruised, twisted, impacted, pulled, or bent.

In the wound healing process, epidermal cells such as fibroblasts, vascular endothelial cells, and keratinocytes initiate proliferation by intracellular factors, and the cells migrate to the wound site, Tissue regeneration occurs through the processes of granulation tissue formation, angiogenesis, and reepithelization.

Factors having a wound healing effect include epithelial growth factor (EGF), acid fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), transforming growth factors (TGF) and growth factors such as insulin-like growth factors (IGF). , adhesion factors such as fibronectin, laminin and vitronectin, and compounds such as retinoids.

In the wound healing process, anabolic and catabolic processes occur in a balanced way for 6 to 8 weeks after the initial wound, and in this stage, the degree of recovery that generally contains 30% to 40% of normal skin tissue indicates The scar is formed in a congested, raised state due to the increase in tensile strength as the collagen fibers gradually cross-link, but gradually returns to a skin-like state over time. In the case of an imbalance between the assimilation and catabolic stages during the wound healing process, the collagen cannot be decomposed and hardened and the scar remains in a raised form, and these tissues are classified as hypertrophic scars or keloids. .

The interest in wound healing and the development of various procedures or drugs is ultimately the greatest goal of beauty to prevent scar formation. Scars can be treated through surgical or non-surgical means, and non-surgical means include silicone gel, occlusive dressings such as Cordran and Scarguard, treatment using compression, and steroid treatment to inhibit collagen formation. Cryotherapy, laser, and surgical excision methods are used as effective means. It is known that the surgical method is not effective because it generally shows a scar recurrence rate of 25% to 100%.

In addition, interferon or verapamil, bleomycin, 5-FU (5-fluorouracil), retinoic acid, imiquimod, tacrolimus Alternatively, there is a method of injecting botulinum toxin, but this method also has disadvantages of low treatment efficiency, high scar recurrence rate, and limited use due to side effects caused by drug administration.

A method using stem cells has been proposed as a method for wound healing. Stem cells are undifferentiated cells that can divide for a long time through self-renewal and can be differentiated into various types of cells under a specific environment. Stem cells are divided into embryonic stem cells and adult stem cells according to the tissue of origin. In terms of research using this, the potential of adult stem cells is limited compared to embryonic stem cells, but there are few ethical issues, so many studies are being conducted on adult stem cells.

Mesenchymal stem cells (MSCs) are multipotent cells, also called mesenchymal stromal cells. Mesenchymal stem cells can be differentiated into various types of cells including osteoblasts, chondroblasts, adipocytes, and the like.

Unlike in the living body, in order to artificially culture animal cells outside the body, nutrients close to the living conditions based on body fluids such as plasma or lymph and environmental conditions such as pH, temperature, and osmotic pressure must be sufficiently satisfied. Therefore, the medium composition was determined based on the body fluid, the determined culture medium was selected, serum was added to the cells at an appropriate ratio, and then used for cell culture. Accordingly, various medium compositions for culturing human stem cells have been proposed, and a method of culturing human stem cells using a medium containing animal serum such as Fetal Bovine Serum (FBS) is common.

However, the differentiation process of stem cells takes a long time due to various factors, or the differentiated cells do not have sufficient effects in many cases.

For example, in the case of Patent Document 1 below, cell differentiation is induced by physical stimulation, but since in vitro culture for several weeks is required, it is not suitable in terms of cost and mass production.

On the other hand, as in these days, artificial temperature control of air conditioning and heating according to changes in the environment or life patterns, skin stress caused by various stresses and environmental pollution occurring in social life, frequent washing due to makeup habits, and natural skin due to increasing age Due to various causes such as aging, the moisture in the stratum corneum decreases, the skin becomes dry, the surface becomes rough, and the skin becomes brittle, loses moisture and looks lifeless. Therefore, there is a great need for skin regeneration and wrinkle improvement. is increasing

Since the method of treating wounds using the stem cells is helpful for skin regeneration, focusing on the fact that it can also help improve scars and wrinkles through skin regeneration from a cosmetic point of view, it is useful for wound healing, scarring and wrinkle improvement. An effective method for economic differentiation of stem cells and a medium composition thereof were invented.

Korean Patent Registration No. 1490235

In order to overcome the limitations of the prior art, it is an object of the present invention to provide a cell differentiation method capable of mass production at low cost by shortening the differentiation induction period, and a medium composition thereof.

The method for differentiation from human adipose-derived mesenchymal stem cells to fibroblast-like cells according to the present invention comprises culturing human adipose-derived mesenchymal stem cells on a gelatin-coated plate, and adding a replaceable medium for cell differentiation, It is characterized in that it differentiates into subtype cells.

In another embodiment, the present invention provides a plate for inducing differentiation of fibroblasts, characterized in that the plate is coated with gelatin and a medium for cell differentiation is added.

Preferably, the cell differentiation medium is transforming growth factor (TGF-β: Transforming growth factor-β), fibroblast growth factor (FGF: Fibroblast growth factor), dexamethasone (Dexamethasone), connective tissue growth factor (CTGF: Connective) tissue growth factor), insulin growth factor (IGF), epidermal growth factor (EGF), fetal bovine serum and penicillin/streptomycin.

In addition, it is preferable that the plate induces differentiation of human adipose-derived stem cells into fibroblasts.

In addition, it is preferable that the plate is a polystyrene plate.

In addition, it is preferable to replace the cell differentiation medium every 2 to 4 days.

In addition, it is preferable to replace the cell differentiation medium 2 to 7 times.

As another embodiment of the present invention, the present invention provides a composition for improving skin wrinkles or scars comprising fibroblast-like cells differentiated using the differentiation method or plate according to the present invention.

The plate containing the gelatin of the present invention can exert the effect of inducing direct cross-differentiation from human adipose-derived mesenchymal stem cells to fibroblasts, and can efficiently perform mass culture in vitro at low cost by using economical materials. effect that makes it possible.

In addition, the differentiated fibroblast-like cells as described above can also be used as a cell therapy composition for the treatment of skin wrinkles or scars.

1 schematically shows the results of the confirmation experiment of human adipose-derived mesenchymal stem cells according to the present invention.
2 and 3 schematically show experimental results comparing marker expression with and without gelatin coating used as a differentiation induction method.
4 and 5 schematically show the experimental results of performing flow cytometry (FACS) for fibroblast (HDF)-specific markers on fibroblast-like cells (dHDF) differentiated from human adipose-derived stem cells (ADSC). did it

Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings.

The present invention provides a plate for inducing differentiation of fibroblasts, characterized in that the plate is coated with gelatin and a medium for cell differentiation is added.

For the plate according to the present invention, an appropriate extracellular matrix molecule should be used for the differentiation and proliferation of stem cells. Examples of the extracellular matrix molecules that can be used include collagen, fibronectin, Matrigel, and gelatin. In the present invention, gelatin having the highest cell differentiation and proliferation rate is preferably used. and a culture plate coated with gelatin was used without including feeder cells. As it does not undergo a co-culture step for proliferation and differentiation, it can be applied quickly to patients by shortening the time required for differentiation, and it can be differentiated by reducing the possibility of disease transmission due to contamination during the culturing of feeder cells. It has the effect of increasing the stability of cells.

In addition, when the plate coated with the 'gelatin' of the present invention is used, the effect of inducing direct cross-differentiation from human adipose-derived mesenchymal stem cells to fibroblast-like cells can be exerted, and the use of economical materials can help reduce costs. It is possible to exert the effect of efficiently enabling mass culture in vitro.

Gelatin for coating the plate according to the present invention may use various concentrations of gelatin, preferably 0.1 to 0.2% by weight. When the concentration is less than the concentration range, the gelatin is hardly coated and thus the coating effect may be almost absent. On the contrary, when the concentration range is exceeded, the thickness of the coated gelatin becomes too thick or hard, so that cell differentiation does not occur well.

Meanwhile, the plate according to the present invention is preferably a polystyrene plate.

In addition, the plate according to the present invention may be for inducing differentiation from human adipose-derived stem cells into fibroblasts.

The human adipose-derived stem cells are preferably human adipose-derived mesenchymal stem cells. In this case, the mesenchymal stem cells, preferably, may be derived from bone marrow, adipose tissue or umbilical cord.

The method for differentiation from human adipose-derived mesenchymal stem cells to fibroblast-like cells according to the present invention comprises culturing human adipose-derived mesenchymal stem cells on a gelatin-coated plate, and adding a replaceable medium for cell differentiation, Differentiation into mimetic cells.

The cell differentiation medium is transforming growth factor (TGF-β: Transforming growth factor-β), fibroblast growth factor (FGF: Fibroblast growth factor), dexamethasone (Dexamethasone), connective tissue growth factor (CTGF: Connective tissue growth factor) , insulin growth factor (IGF: Insulin growth factor), epidermal growth factor (EGF: Epidermal growth factor), preferably including fetal bovine serum and penicillin / streptomycin, any one used for animal cell differentiation in the art Anything can be added.

The cell differentiation medium is preferably formulated with fetal bovine serum (FBS), penicillin and streptomycin added, and more preferably in a commercially available DMEM/HIGH GLUCOSE medium. It is recommended to use a composition containing fetal bovine serum (FBS), penicillin and streptomycin.

The fetal bovine serum contained in the cell differentiation medium of the present invention is preferably 5 to 15%, more preferably 10%.

In addition, the penicillin/streptomycin contained in the cell differentiation medium of the present invention is preferably 0.05 to 2%, more preferably 1%.

In addition, the transforming growth factor (TGF-β: Transforming growth factor-β) contained in the cell differentiation medium of the present invention is preferably 10 to 20 ng/ml, more preferably 15 ng/ml it's good to be

In addition, the fibroblast growth factor (FGF: Fibroblast growth factor) included in the cell differentiation medium of the present invention is preferably 10 to 30 ng/ml, more preferably 20 ng/ml .

In addition, the dexamethasone contained in the cell differentiation medium of the present invention is preferably 0.05 to 0.2 nM, more preferably 0.1 nM.

In addition, the connective tissue growth factor (CTGF) included in the cell differentiation medium of the present invention is preferably 50 to 150 ng/ml, more preferably 100 ng/ml good.

In addition, the insulin growth factor (IGF) contained in the cell differentiation medium of the present invention is preferably 5 to 15 ng/ml, more preferably 10 ng/ml.

In addition, the epidermal growth factor (EGF) contained in the cell differentiation medium of the present invention is preferably 5 to 15 ng/ml, more preferably 10 ng/ml.

The cell differentiation method of the present invention was carried out at a temperature of _{5% CO 2 and 37 °C.} At this time, the optimal temperature for cell differentiation is a condition that mainly depends on the body temperature of the host from which the cells are separated, and 5% CO ₂ is added to monitor the cell metabolism and growth to determine the time of nutrient consumption in the nutrient-limited medium. For the normal action of phenol red, a pH indicator, CO ₂ in the medium generated during cell metabolism is a condition to prevent vaporization into the incubator.

The cell differentiation period according to the present invention is preferably 5 to 14 days, and in the present invention, it was differentiated for 7 days.

The medium for cell differentiation according to the present invention is preferably replaced at intervals of 2 to 4 days, and in the present invention, it is replaced at intervals of 3 days. This is for the purpose of maintaining the freshness of the medium, and in the case of factors treated with the differentiation medium, when maintained at a high temperature for a long time, the activity decreases, so it is recommended to replace the medium.

In previous studies, cells of different origins were induced into IPS cells and then differentiated. However, in the method of inducing differentiation from human adipose-derived stem cells to fibroblasts, the present invention has a feature in that direct conversion (trans-differentiation) is performed from mesenchymal stem cells to fibroblasts. .

The fibroblast-like cells differentiated from human adipose-derived stem cells according to the present invention express fibroblast-specific markers FSP-1 (Fibroblast-specific protein-1), CD91, CD55 and CD10.

According to an experiment to be described later, fibroblast-like cells (dHDF) differentiated from human adipose-derived stem cells according to the present invention are compared with human adipose-derived stem cells (ADSC), FSP-1, a specific marker of fibroblasts (HDF). . The proportion of cells positive for CD91, CD55 and CD10 was found to be more than 90%, which was similar to that of fibroblasts.

As described above, the present invention provides a gelatin-coated plate for direct cross-differentiation from human adipose-derived mesenchymal stem cells into fibroblast-like cells, a cell differentiation medium composition, and a differentiation method using the same in vitro at low cost. Since it can exert the effect of enabling efficient mass culture production, it is possible to provide an alternative material for fibroblasts. In addition, it is possible to provide a composition for improving skin wrinkles or scars by using such an alternative material.

Hereinafter, the content of the present invention will be described in more detail through the following Examples or Experimental Examples.

In the examples below, the induction of differentiation into fibroblasts from human adipose-derived stem cells and the characteristics of the differentiated fibroblasts were confirmed.

(Example)

(1) Securing of human adipose-derived mesenchymal stem cells

Adipose tissue (Goma Biotech, Biopredic) is washed three times with a phosphate buffer solution and cut into 1-3 mm pieces using a scalpel. After that, it was treated with 0.075% collagenase type I and reacted in a shaking-water bath at 37°C for 1 hour, neutralized with DMEM (Dulbecco's Modificaion of Eagle's Medium) medium containing 10% of fetal bovine serum (FBS), and put into a culture vessel. moved and attached. When the cells have proliferated about 80% of the surface of the culture vessel, the cells are removed by treatment with Tryipin-EDTA, adjusted to a concentration of 2 x 105 cells per ml, and the stem cell positive markers CD44 and CD90 and the negative marker CD45 antibodies and 30 reacted for minutes. The stained cells were washed with a staining solution, and the cells were suspended in a new staining solution, and then the cells were analyzed using a flow cytometer (FACScalibur, BD science) and Cellquest software (CELLQUEST software; BD science). .

As a result, as shown in FIG. 1 , it was confirmed that the cells were attached to the culture vessel, the stem cell positive markers CD44 and CD90 were expressed, and the hematopoietic stem cell marker CD45 was hardly expressed, indicating that the cells were adipose-derived stem cells. Confirmed.

(2) Culture of human adipose-derived mesenchymal stem cells

The human adipose-derived mesenchymal stem cells isolated above were transferred to an existing 6 well plate (Costar) and a gelatin-coated polystyrene (PS) plate (6 well clear TC-treated Multiple Well Plates, 3516, Costar, Corning, NY). , USA) (1 X 10 ⁵ cells per well). At this time, the medium used was DMEM/HIGH GLUCOSE medium ((Dulbecco's Modified Eagle's Medium - High Glucose Liquid media (SH30243, Hyclone) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin). , UT, USA), detailed medium composition is shown in Table 1 below), and _{cells were cultured for 1 day at 5% CO 2} and a temperature condition of 37°C.

organic salts mg/L mmol/L Calcium chloride 200 1.8021 Ferric nitrate-9H ₂ 0 0.1 0.0002 Potassium chloride 400 5.3655 Magnesium sulfate 97.67 0.8112 sodium chloride 6400 109.514 Sodium phosphate monobasic H ₂ 0 125 0.9059 amino acids mg/L mmol/L L-Arginine-HCl 84 0.3987 L-Cystine-2HCl 62.57 0.1998 L-Glutamine 584 3.9959 Glycine 30 0.3996 L-Histidine-HCl-H ₂ 0 42 0.2004 L-Isoleucine 104.8 0.7989 L-Leucine 104.8 0.799 L-Lysine-HCl 146.2 0.8004 L-Methionine 30 0.211 L-Phenylalanine 66 0.3995 L-Serine 42 0.3997 L-Threonine 95.2 0.7992 L-Tryptophan 16 0.0783 L-Tyrosine-2Na-2H ₂ 0 103.79 0.3974 L-Valine 93.6 0.799 Vitamins mg/L mmol/L Calcium D-pantothenate 4 0.0084 D-Pantothenic acid Na salt 0 0 Choline chloride 4 0.0286 folic acid 4 0.0091 Myo-inositol 7 0.0389 Niacinamide 4 0.0328 Pyridoxine-HCl 4 0.0195 Riboflavin 0.4 0.0011 Thiamine-HCl 4 0.0119 Other mg/L mmol/L D-Glucose 4500 24.9778 HEPES 0 0 Phenol red-Na 15.9 0.0422 Sodium pyruvate 110 0.9996 Sodium bicarbonate 3700 44.0424

(3) Induction of differentiation from human adipose-derived stem cells to fibroblasts

In order to differentiate cells cultured in gelatin-coated plates into fibroblasts, 15 ng/ml Transforming growth factor-β (TGF-β), 20 ng/ml fibroblast growth factor ( FGF: Fibroblast growth factor), 0.1 nM dexamethasone, 100 ng/ml Connective tissue growth factor (CTGF), 10 ng/ml Insulin growth factor (IGF), 10 ng/ml In DMEM/HIGH GLUCOSE cell differentiation medium supplemented with epidermal growth factor (EGF), 10% fetal bovine serum and 1% penicillin/streptomycin, 5% CO ₂ and a temperature of 37°C for 6 well plate 2 ml and cultured for 7 days. The medium was replaced once every 3 days, and after removing the existing culture medium (suction), it was replaced with 2 ml of DMEM/HIGH GLUCOSE differentiation medium using a pipette.

(Comparative example)

Culture and differentiation were carried out in the same manner as in Example, except that a plate not coated with gelatin was used in the above example.

(Experimental Example 1) Comparison of differentiation rate with or without gelatin coating

In order to compare the differentiation capacity of the differentiated fibroblast-like cells (non-coated) and human adipose-derived stem cells (ADSC) and fibroblasts (HDF) in the comparative example, the fibroblast-specific marker FSP-1 (Fibroblast-specific protein) -1) and CD10 marker expression was confirmed by flow cytometry (FACS).

Fibroblast-like cells (non-coated), human adipose-derived stem cells (ADSC) and fibroblasts (HDF) differentiated in the comparative example were treated with 0.05% trypsin/0.02% EDTA to remove the cells, and then 2 x 10 ^{The concentration was adjusted to 5} cells per ml, and the cell solution was blocked by Fc receptors.

(1) After that, in the case of FSP-1, the cells were fixed using the Fixation/Permeabilization solution kit (BDCytofix/Cytoperm™) kit, (2) in the case of CD10, CD10 After reacting with the antibody of

The stained cells were washed with a staining solution and the cells were suspended in a new staining solution, and then the cells were analyzed with a flow cytometer (FACScalibur, BD science) and Cellquest software (CELLQUEST software; BD science). .

As shown in Figures 2 and 3, it was confirmed that differentiation hardly proceeds when applied to a plate not coated with gelatin coating using the same medium composition.

(Experimental Example 2) Confirmation of characteristics of fibroblast-like cells differentiated from human adipose-derived stem cells through flow cytometry (FACS)

In order to confirm whether it exhibits similar characteristics to human skin fibroblasts, the expression of markers of FSP-1 (Fibroblast-specific protein-1), CD91, CD55 and CD10 specifically expressed in fibroblasts was analyzed by flow cytometry as in Experimental Example 1. FACS) was used for comparison.

As a result of confirming the expression of the fibroblast-specific marker, as shown in FIGS. 4 and 5 , differentiated fibroblast-like cells (dHDF) compared to human adipose-derived stem cells (ADSC) are fibroblast (HDF)-specific markers. In FSP-1. It was confirmed that the proportion of cells positive for CD91, CD55 and CD10 was more than 90%, which was confirmed to exhibit similar or higher expression to fibroblasts.

Although the embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and variations are possible within the scope without departing from the technical spirit of the present invention described in the claims. It will be apparent to those of ordinary skill in the art.

Claims (12)

A method of culturing human adipose-derived mesenchymal stem cells on a gelatin-coated plate and adding a replaceable medium for cell differentiation to differentiate them into fibroblast-like cells, the method comprising:
The cell differentiation medium is transforming growth factor (TGF-β: Transforming growth factor-β), fibroblast growth factor (FGF: Fibroblast growth factor), dexamethasone (Dexamethasone), connective tissue growth factor (CTGF: Connective tissue growth factor) , Insulin growth factor (IGF), epidermal growth factor (EGF), fetal bovine serum and penicillin/streptomycin, characterized in that it contains fibroblasts from human adipose-derived mesenchymal stem cells Method of differentiation into cells. delete The method of claim 1, wherein the cell differentiation medium is replaced every 2 to 4 days from human adipose-derived mesenchymal stem cells to fibroblast-like cells. The method according to claim 1, wherein the medium for cell differentiation is replaced 2 to 7 times from human adipose-derived mesenchymal stem cells to fibroblast-like cells. delete A plate coated with gelatin and added with a cell differentiation medium,
The cell differentiation medium is transforming growth factor (TGF-β: Transforming growth factor-β), fibroblast growth factor (FGF: Fibroblast growth factor), dexamethasone (Dexamethasone), connective tissue growth factor (CTGF: Connective tissue growth factor) , Insulin growth factor (IGF), epidermal growth factor (EGF), fibroblasts from human adipose-derived mesenchymal stem cells comprising fetal bovine serum and penicillin/streptomycin A plate for inducing cell-like differentiation. delete The plate for inducing differentiation of fibroblasts from human adipose-derived mesenchymal stem cells according to claim 6, wherein the plate is for inducing differentiation into fibroblasts from human adipose-derived stem cells. The plate for inducing differentiation of fibroblasts from human adipose-derived mesenchymal stem cells according to claim 6, wherein the plate is a polystyrene plate. The plate for inducing differentiation of fibroblasts from human adipose-derived mesenchymal stem cells according to claim 6, wherein the cell differentiation medium is replaced every 2 to 4 days. The plate for inducing differentiation of fibroblasts from human adipose-derived mesenchymal stem cells according to claim 6, wherein the medium for cell differentiation is replaced 2 to 7 times. delete

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