KR101280911B1 - Process for the differentiation of adult stem cells to chondrocytes or osteocytes using tauroursodeoxycholic acid - Google Patents

Abstract

The present invention provides a method for differentiating adult stem cells into chondrocytes or bone cells, comprising culturing adult stem cells in chondrocytes or osteocytic differentiation medium containing tauurusodeoxycholic acid. The differentiation method according to the present invention can selectively inhibit differentiation into adipocytes without significantly affecting the differentiation of adult stem cells into osteocytes or chondrocytes. Therefore, the differentiation method according to the present invention can differentiate adult stem cells into bone cells or chondrocytes purely, while minimizing the possibility of contamination of adipocytes, and thus can be usefully used for the production of bone cells and chondrocytes for cell therapy. have.

Description

Process for the differentiation of adult stem cells to chondrocytes or osteocytes using tauroursodeoxycholic acid}

The present invention relates to a method of differentiating adult stem cells to chondrocytes or bone cells using taurusodeoxycholic acid. More specifically, the present invention induces the differentiation of adult stem cells in the presence of taurusodeoxycholic acid, thereby purely differentiating adult stem cells into osteocytes or chondrocytes while selectively inhibiting differentiation into adipocytes. It is about a method.

Articular cartilage is a special form of connective tissue consisting of a thousand layers, intermediate layers, deep layers, and lime layers that vary in depth from the joint surface, and is important for preventing extracellular matrix from being damaged by physical force. Play a role. Articular cartilage tissue causes constitutive changes of the matrix in addition to the reduction of cartilage thickness and the number of chondrocytes with aging, and also changes in the function of chondrocytes. Aging is considered to be an important cause of osteoarthritis, one of the most common degenerative diseases in humans (Bobacz K et al, Ann Rheum Dis, 63 (12), pp. 1618-1622, 2004). In addition, articular cartilage is a tissue that is unable to regenerate itself after injury due to the absence of blood vessels, nerves and lymphoid tissues (Brittberg M et al, New England Journal of Medicine, 331, pp. 889-895, 1994). At present, efforts are being made to treat degenerative joint cartilage disease with various medical approaches, but there are still many limitations in normal cartilage tissue regeneration.

The regeneration of cartilage tissue by autologous chondrocyte transplantation has been recently tried and is known to be a clinically effective surgical method. However, it cannot be used for a wide range of cartilage injuries. have. Chondrocytes are already differentiated cells and do not have strong cell division in tissues, but proliferation is promoted during monolayer culture in vitro after separation, thereby obtaining a sufficient number of chondrocytes. However, prolonged monolayer culture in order to obtain sufficient cell numbers, not only causes the dedifferentiation of chondrocytes to be reduced, but also the tissues calcified during transplantation in vivo by hypertrophy of chondrocytes. There is a problem in this formation (Fischer J et al. Arthritis & Rheumatism, 62 (9), pp. 2696-2706, 2010). In addition, in the case of degenerative arthritis, the cells obtained from the chondrocytes are aged, and thus, mass culture of the chondrocytes is very difficult. On the other hand, stem cells have a faster growth rate in monolayer culture than chondrocytes, and stem cells obtained from older people are also actively studied for cartilage regeneration using stem cells because they have excellent mitotic ability and biosynthetic activity.

In addition, osteoblasts, like chondrocytes, are already differentiated cells and have a limitation in that they are difficult to purely separate after the differentiation induction process. It is reported that the use of bone progenitor cells together with bone cells is effective for differentiation of bone cells and regeneration of bone tissues (Chung CP et al. J Controlled Release 12; 51 (2-3) 1998). Therefore, various studies have been conducted to differentiate adult stem cells, including adipose derived stem cells, into osteocytes.

On the other hand, adult stem cells, including adipose-derived stem cells, can overcome the cancer or ethical problems that are generally problematic for embryonic stem cells, fat cells, osteoblasts, chondrocytes, heart cells, muscle cells and It is attracting attention as a cell source for cell therapy because it can differentiate into various cells such as neurons (Park JS et al. Biomaterials, 32 (1), pp. 28-38, 2011, Shetty P et al. Asian J Transfus) Sci, 4 (1), pp. 14-24, 2010, and Kume S et al. J Bone Miner Res, 20 (9), pp. 1647-58, 2005). However, the efficient mass differentiation technology of adult stem cells has not yet been established, it is recognized as a major limitation in actual clinical and industrial use. For example, in order to heal articular cartilage damage, it is essential to develop an efficient differentiation induction technique that can differentiate stem cells into a large amount of chondrocytes in vivo and in a laboratory.

Adult stem cells are known as various adult stem cells including lubricous membranes, umbilical cord blood, stem cells derived from bone marrow or adipose tissue. However, mesenchymal stem cells derived from bone marrow have been mainly used for differentiation into bone cells or chondrocytes. . However, stem cells derived from adipose tissue (commonly referred to as 'fat-derived mesenchymal stem cells' or 'fat-derived stem cells') are advantageous in that they can be easily and largely obtained through liposuction and the like. Many researchers are working to differentiate adipose derived stem cells into osteocytes or chondrocytes. However, in 2006, Kern et al. Reported that adipose-derived stem cells tended to form adipose tissue rather than cartilage or bone tissue (Kern S et al. Stem Cells, 24 (5), pp. 1294-1301, 2005 and Im GI et al. Osteoarthritis Cartilage, 13 (10), pp. 845-853, 2005), compared with bone marrow, it is pointed out that adipose-derived stem cells have a poor ability to differentiate into chondrocytes (Im GI et. Osteoarthritis Cartilage, 13 (10), pp. 845-853, 2005; Rebelatto CK et al.Exp Biol Med, 233 (7), pp. 901-913, 2008; and Bioback K et al. Biomed Mater Eng, 18 (1) pp. 71-76, 2008). Differentiation of adipose-derived stem cells into adipocytes undergoes differentiation into progenitor cells of adipose-derived stem cells, proliferation of progenitor cells, and adipocyte differentiation of progenitor cells, and during the differentiation process, peroxisome-activated receptor γ It is known that transcription factors of fat cells such as) are activated.

The present inventors conducted various studies to develop a differentiation method capable of purely differentiating adipose derived stem cells into osteocytes or chondrocytes while minimizing differentiation into adipose tissue. As a result, surprisingly, tauroursodeoxycholic acid, a type of bile acid, does not affect the differentiation of adult stem cells, such as fat-derived stem cells, into osteoblasts or chondrocytes, but differentiates them into adipocytes. It was found to selectively inhibit.

Accordingly, the present invention provides a method of purely differentiating adult stem cells into bone cells or chondrocytes by inducing differentiation of adult stem cells in the presence of taurusodeoxycholic acid, and selectively inhibiting differentiation into adipocytes. It aims to do it.

According to one aspect of the present invention, there is provided a method for differentiating adult stem cells into chondrocytes, the method comprising culturing adult stem cells, in a medium for chondrocyte differentiation comprising tauurusodeoxycholic acid.

According to another aspect of the present invention, there is provided a method for differentiating adult stem cells into bone cells, comprising culturing adult stem cells, in a medium for osteoblast differentiation comprising taurousodeoxycholic acid.

In the differentiation method of the present invention, the adult stem cells may be preferably adipose-derived stem cells, the concentration of taurousodeoxycholic acid may be in the range of 5 to 500 μM.

In one embodiment of the present invention, the chondrocyte differentiation medium is DMEM medium containing taurusodeoxycholic acid, TGF-β1, dexamethasone, ascorbic acid, insulin, transferrin, and selenium salt; More preferably, Taururusodeoxycholic acid 50-150 μg / mL, TGF-β1 5-20 ng / mL, dexamethasone 50-200 nM, ascorbic acid 10-100 μg / mL, insulin 0.001-1 mg / mL , DMEM medium containing 0.055 to 500 mg / mL transferrin, and 500 to 670 μg / mL selenium salt.

In another embodiment of the present invention, the osteoblast differentiation medium is a DMEM medium comprising tauurosodeoxycholic acid, dexamethasone, ascorbic acid, L-alanyl-L-glutamine, and glycerol 2-phosphate; More preferably, Taururusodeoxycholic acid 50-150 μg / mL, dexamethasone 50-200 nM, ascorbic acid 10-100 μg / mL, L-alanyl-L-glutamine 50-200 nM, and glycerol 2- DMEM medium containing phosphate 5-20 mM.

The differentiation method according to the present invention induces the differentiation of adult stem cells in the presence of taurousodeoxycholic acid, thereby differentiating into adult cells without significantly affecting the differentiation of adult stem cells into osteocytes or chondrocytes. Can be selectively suppressed. Therefore, the differentiation method according to the present invention can differentiate adult stem cells into bone cells or chondrocytes purely, while minimizing the possibility of contamination of adipocytes, and thus can be usefully used for the production of bone cells and chondrocytes for cell therapy. have.

Figure 1 shows the results of evaluating the cell proliferation rate and survival rate of human adipose-derived stem cells according to Taururusodeoxycholic acid (TUDCA) treatment. The upper part of FIG. 1 is the result of cell proliferation for 3 days in the TUDCA untreated (0) group and the TUDCA treated (50, 100, 150 μM) group (n = 3, p > 0.01). The dead assay shows live cells (green) and dead cells (red) (Scale bar = 100 μm).
Figure 2 shows the results of measuring the gene expression pattern of bone cells, chondrocytes, adipocytes-related marker genes of human adipose derived stem cells following TUDCA treatment by RT-PCR. Runx2, Col I and ALP represent osteoblast differentiation marker genes, ColII and Agg represent chondrocyte differentiation marker genes, and PPARγ represents adipocyte differentiation marker genes. GAPDH is a control gene.
3 shows the results of evaluating the adipocyte differentiation inhibitory activity of human adipose derived stem cells following TUDCA treatment. FIG. 3A is a schematic diagram showing a method of dividing and culturing a proliferation phase and an adipocyte differentiation phase. Figure 3 B is the result of measuring the lipid drop (Lipid drop) through the oil red O staining on the cells of each group (Scale bar = 100 ㎛). 3C shows the results of quantitative analysis of lipid loading generated using Oil red O extraction quantification method (n = 3, *, p <0.01). 3D shows the RT-PCR results of the adipocyte differentiation marker genes GPDH and PPARγ.
Figure 4 shows the results of evaluating osteoblast differentiation of human adipose derived stem cells following TUDCA treatment. 4A shows the results of measuring the activity of alkaline phosphatase (Alkaline Phosphatase, ALP) on days 3 and 7 in each experimental group (n = 3, *, p <0.01). 4B is a result of measuring the expression of marker genes OCN, Col I and ALP by performing RT-PCR on osteoblast differentiation of each group (n = 4). 4C is a result of evaluating the degree of calcium salt deposition of cells obtained from each group by von Kossa staining (Scale bar = 100 μm). 4D is a result of quantifying the degree of calcium deposition of cells obtained in each group (n = 4, p > 0.01).
Figure 5 shows the results of evaluating chondrocyte differentiation of human adipose derived stem cells following TUDCA treatment. 5A is a result of measuring the glycosaminoglycan (GAG) matrix of cells obtained from each group by Alcian blue staining (Scale bar = 100 μm). 5B shows the results of quantifying the GAG Matrix of each group through Alcian blue extraction quantification method (n = 3, p > 0.01), and FIG. 5C shows the degree of chondrocyte differentiation of each group by RT-PCR. Expression of the marker genes Col I, Col II and Agg was measured.

The present invention provides a method for differentiating adult stem cells into chondrocytes, comprising culturing adult stem cells in chondrocyte differentiation medium containing tauurusodeoxycholic acid. The present invention also provides a method for differentiating adult stem cells into osteoblasts, comprising culturing adult stem cells in osteoblast differentiation medium containing tauurusodeoxycholic acid.

Tauroursodeoxycholic acid is a bile acid produced by the liver with cholesterol as a precursor. Taururusodeoxycholic acid has been reported to regenerate islet cells of diabetic rats and is known to protect cardiomyocytes by inhibiting the death of cardiomyocytes induced by myocardial infarction. However, it has not been reported that taurusodeoxycholic acid may be associated with the differentiation of adult stem cells.

According to the present invention, it has been found that taurorusodeoxycholic acid can selectively inhibit differentiation into adipocytes without significantly affecting the differentiation of adult stem cells into osteocytes or chondrocytes. Therefore, the differentiation method according to the present invention can differentiate adult stem cells into bone cells or chondrocytes purely, while minimizing the possibility of contamination of adipocytes, and thus can be usefully used for the production of bone cells and chondrocytes for cell therapy. have.

In the differentiation method of the present invention, the adult stem cells may be stem cells having various origins such as synovial membrane, umbilical cord blood, bone marrow, placenta, alveolar bone, fat (fat tissue), preferably fat-derived stem cells. . In particular, adipose derived stem cells have been reported to have a strong tendency to form adipose tissue (Kern S et al. Stem Cells, 24 (5), pp. 1294-1301, 2005 and Im GI et al. Osteoarthritis Cartilage, 13 (10), pp. 845-853, 2005), the differentiation method according to the present invention can be usefully applied to differentiation of adipose derived stem cells into chondrocytes or bone cells.

In the differentiation method of the present invention, the concentration of taurousodeoxycholic acid may be in the range of 5 to 500 μM, more preferably in the range of 50 to 150 μM.

The chondrocyte differentiation medium is not particularly limited as long as it contains tauurusodeoxycholic acid, and conventionally known chondrocyte differentiation medium may be used (for example, Rebelatto CK et al.Exp Biol Med). , 233 (7), pp. 901-913, 2008, etc.). In one embodiment, the medium for chondrocyte differentiation used in the differentiation method of the present invention is DMEM medium containing tauurosodeoxycholic acid, TGF-β1, dexamethasone, ascorbic acid, insulin, transferrin, and selenium salt; More preferably, Taururusodeoxycholic acid 50-150 μg / mL, TGF-β1 5-20 ng / mL, dexamethasone 50-200 nM, ascorbic acid 10-100 μg / mL, insulin 0.001-1 mg / mL , DMEM medium containing 0.055 to 500 mg / mL transferrin, and 500 to 670 μg / mL selenium salt. More specifically, the chondrocyte differentiation medium used in the differentiation method of the present invention is 50 μM of tauurosodeoxycholic acid, 10 ng / mL of TGF-β1, 100 nM of dexamethasone, 50 ng / mL of ascorbic acid, 1 g / insulin. DMEM medium containing L, transferrin 0.5 g / L, and 0.67 mg / L selenium salt. The insulin, transferrin, and selenium salts may use commercially available ITS (Gibco, USA). In addition, the chondrocyte differentiation medium may further include fetal calf serum (FBS) and antibiotics, if necessary, and may include, for example, about 10% FBS and about 1% penicillin-streptomycin. .

The osteoblast differentiation medium is not particularly limited as long as it contains tauurusodeoxycholic acid, and conventionally known osteoblast differentiation medium may be used (for example, Cheng SL et al (1994), Endocrinology 134: 277-286, etc.). In one embodiment, the medium for osteoblast differentiation used in the differentiation method of the present invention is DMEM comprising tauurosodeoxycholic acid, dexamethasone, ascorbic acid, L-alanyl-L-glutamine, and glycerol 2-phosphate badge; More preferably, Taururusodeoxycholic acid 50-150 μg / mL, dexamethasone 50-200 nM, ascorbic acid 10-100 μg / mL, L-alanyl-L-glutamine 50-200 nM, and glycerol 2- DMEM medium containing phosphate 5-20 mM. More specifically, the osteoblast differentiation medium used in the differentiation method of the present invention is 50 μM of tauurosodeoxycholic acid, 10 ng / mL of TGF-β1, 100 nM of dexamethasone, 50 ng / mL of ascorbic acid, L-Ala DMEM medium containing Neil-L-glutamine 200 mM, and 10 mM glycerol 2-phosphate. The L-alanyl-L- glutamine may be used commercially available Glutamax (Invitrogen, USA). In addition, the osteoblast differentiation medium may further include fetal calf serum (FBS) and antibiotics, if necessary, and may include, for example, about 10% FBS and about 1% penicillin-streptomycin. .

In the differentiation method of the present invention, the culture may be performed on a conventional culture plate in the form of pellet culture, for example, may be cultured at 37 ℃, 5% CO ₂ conditions. The incubation period is not particularly limited, but may be performed, for example, for about three weeks. After completion of the culture, chondrocytes and osteoblasts are obtained in pellet form and can be separated by removing the medium.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrating the present invention, and the scope of the present invention is not limited to these examples.

Example 1. Isolation and Culture of Human Adipose-Derived Stem Cells

Under the permission of the Ethics Committee of the College of Medical Sciences, the College of Medicine, with the consent of the patient, the fat tissue was removed through liposuction. To remove contaminated blood, the resulting adipose tissue was washed three times using (phosphate buffered saline, PBS) (Sigma, St. Louis, MO). Adipose tissue was then digested with PBS containing 0.2 w / v% bovine albumin and 2 mg / mL type II collagenase (Sigma) for 45 minutes at 37 ° C. Filtration was performed with a 70 μm filter, and the filtrate was centrifuged to remove floating adipocytes. Isolated adipose-derived stem cells (ASCs) were collected from stem cell culture [1% Penicillin-Streptomycin] and Dulbecco's modified Eagle medium (DMEM) with 10% Fetal Bovine Serum (FBS) (Invitrogen, USA). , Gibco BRL, Gaithersburg, MD)] was incubated in 37 ℃, 5% CO ₂ incubator. Cells passaged 3 or 4 were used in the next experiment.

Example 2 Evaluation of Cell Proliferation and Survival Rate of Human Adipose-Derived Stem Cells Treated with Taururusodeoxycholic Acid

Human adipose derived stem cells obtained in Example 1 were seeded at a density of 1 × 10 ⁴ cells / cm ² , and stem cell culture [1% penicillin-Streptomycin and 10% fetal bovine cells were cultured for 3 days. Dulbecco's modified Eagle medium (DMEM, Gibco BRL, Gaithersburg, MD) with serum (FBS) added (Invitrogen, USA). At this time, the cell proliferation rate was evaluated by treating tauroursodeoxycholic acid (TUDCA) (Calbiochem, San Diego, Calif.) At 0, 50, 100, 150 μM concentration in the stem cell culture. Cells of each group of concentrations incubated for 0, 1, 2 and 3 days were washed once with PBS, suspended in 1 mL of stem cell culture, and then Cell Counting Kit-8 (CCK-8, Dojindo, Tokyo, Japan) 100 μL of the solution was added and reacted at 37 ° C. for 2 hours. Take 100 μL from each group of culture and place it in a 96-well plate, and the absorbance of the sample was measured at 450 nm using an ELISA plate reader (Power Wave X340; Bio-Tek Instruments, Inc., Winooski, VT). It was. The cell proliferation rate was converted to the ratio of absorbance to cell proliferation time (Absorbance / day), and the result is shown in FIG. 1 (upper). As can be seen from the results in FIG. 1 (top), the treatment concentrations of TUDCA showed values of 0.2045 ± 0.0149, 0.2268 ± 0.0221, 0.22443 ± 0.01, and 0.2257 ± 0.0195 at 0, 50, 100, and 150 μM, respectively. However, it was not statistically significant (n = 3, p > 0.01). In addition, the cell viability was confirmed by Live / Dead assay for each group cultured for 3 days. Each group was washed three times with PBS, stained with 4 mM calcein AM (Invitrogen) and 4 μM ethidium homodimer (Ethidium Homodimer, EthD-1, Invitrogen) for 5 minutes. Live cells (green) and dead cells (red) were observed with a fluorescence microscope (Confocal Laser Scanning Microscope, CLSM), and the results are shown in FIG. 1 (bottom). As shown in FIG. 1 (bottom), only live cells stained green were observed in both the TUDCA treated group (50, 100, 150 μM) and the untreated group (0 μM), and the dead cells stained red. Was not observed. Therefore, from the results using the Cell Counting Kit-8 and the results of the Live / Dead assay, it was confirmed that the cell proliferation rate and the survival rate of the adipose-derived stem cells were not significantly affected in the TUDCA concentration used, that is, in the range of 50 to 150 μM. It became.

Example 3. Osteoblast, Chondrocyte, Adipocyte-related Marker Gene Expression Pattern of Human Adipose-Derived Stem Cells by TUDCA Treatment

The human adipose derived stem cells obtained in Example 1 were seeded at a density of 1 × 10 ⁴ cells / cm ² , and stem cell culture solution [1% penicillin-Streptomycin and 10% fetal bovine serum (FBS) (Dvibecco's modified Eagle medium (DMEM, Gibco BRL, Gaithersburg, MD)) added to (Invitrogen, USA)] was incubated for 5 days divided into TUDCA-treated and untreated groups. The cells obtained after the culture were subjected to Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) to measure the expression of osteoblast, chondrocyte, and adipocyte differentiation marker genes. After washing the cells of each group three times with PBS, the cells were recovered by trysin-EDTA, and RNA was extracted through the Trizol (Life Technologies, Inc. Grand Island, NY) method. . After RT-PCR was performed on 5 ㎍ of extracted RNA using the RT-PCR kit (Bioneer, Seoul, Korea), the change of gene expression was measured by electrophoresis using 1.2% agarose gel. . Primer sets and respective differentiation markers used in RT-PCR are shown in Table 1 below.

Differentiated cells Marker order Origin ^* Bone cells Runx2 sense 5'-TTG CAG CCA TAA GAG GGT AG-3 ' NM_001015051.2 Antisense 5'-GTC ACT TTC TTG GAG CAG GA-3 ' Col i sense 5'-CTG ACC TTC CTG CGC CTG ATG TCC-3 ' NM_000088 Antisense 5'-GAC TGG GGC ACC AAC GTC CAA-3 ' ALP sense 5'-ACG TGG CTA AGA ATG TCA TC-3 ' NM_000478 Antisense 5'-CTG GTA GGC GAT GTC CTT A-3 ' Cartilage cell ColⅡ sense 5'-TTC AGC TAT GGA GAT GAC AAT C-3 ' NM_001844 Antisense 5'-AGA GTC CTA GAG TGA CTG AG-3 ' Aggrecan
(Agg) sense 5'-GAA TCT AGC AGT GAG ACG TC-3 ' NM_013227 Antisense 5'-CTG CAG CAG TTG ATT CTG AT-3 ' Fat cell PPARγ sense 5'-AGA CAA CAG ACA AAT CAC CAT-3 ' NM_138711 Antisense 5'-CTT CAC AGC AAA CTC AAA CTT-3 ' GAPDH
(Housekeeping gene) sense 5'-CGG ATT TGG TCG TAT TGG GC-3 ' NM_002046 Antisense 5'-CAG GGA TGA TGT TCT GGA GA-3 '

* Source: NCBI accession number

As a result of performing the RT-PCR as shown in FIG. From the results of FIG. 2, the difference in expression of Runx2, Col I, ALP and osteocholia differentiation markers Col II and Agg, which are osteoblast differentiation markers, was not found in the TUDCA-treated and untreated groups. On the other hand, PPARγ, an adipocyte differentiation marker, showed low expression in the TUDCA-treated group, but not in the TUDCA-treated group. Therefore, TUDCA treatment does not affect osteoblast differentiation and chondrocyte differentiation, but is expected to have activity that inhibits differentiation differentiation into adipocytes.

Example 4 Evaluation of Adipocyte Differentiation Inhibitory Activity of Human Adipose-Derived Stem Cells by TUDCA Treatment

Human adipose derived stem cells obtained in Example 1 were cultured by dividing into a proliferation phase and a differentiation cell differentiation phase. In the proliferation phase, human adipose-derived stem cells were cultured in stem cell culture medium for 5 days in the same manner as in Example 3, and in the adipocyte differentiation phase, adipose cell differentiation medium (StemProAdipogenesis Differentiation Kit, Incubated for 10 days in Gibco BRL). Culture of human adipose derived stem cells was divided into five groups as shown in FIG. The comparative group (Not induced group) was cultured in stem cell culture for 15 days; Group 1 was cultured in stem cell culture for 5 days and then cultured in adipocyte differentiation medium for 10 days; The second group was cultured for 5 days in stem cell culture medium to which 50 μM of TUDCA was added, and then cultured for 10 days in a medium for adipocyte differentiation without treatment of TUDCA; Group 3 (Group 3) was incubated for 5 days in stem cell culture medium without TUDCA treatment and then cultured for 10 days in adipocyte differentiation medium added with 50 μM TUDCA; The fourth group (Group 4) was incubated by adding 50 μM TUDCA in both the growth stage and the differentiation stage.

The effect of TUDCA on the differentiation of adipocytes was measured by the oil red O staining method. Oil red O staining was performed according to the method of Ramirez-Zacarias et al. [Histochemistry 97, 493-497, (1992)]. Cells of each group were washed twice with PBS and 0.5 ml of Oil Red O solution (0.36% Oil Red O in 60% isopropanol) was added and treated for 15 minutes at room temperature. The stain was then removed and washed three times with 60% isopropanol. The result is the same as B of FIG. In FIG. 2B, purple indicates adherent cells stained with Hematoxylin, and red portion shows Lipid drop (Scale bar = 100 μm). From the results of B of FIG. 3, a considerable amount of lipid loading occurred in the first and second groups, particularly in the first group not treated with TUDCA during both proliferation and differentiation. drop was observed very much. On the other hand, relatively little geological loading was observed in the third and fourth groups including TUDCA in the differentiation stage compared to the first and second groups.

Quantitative analysis of the resulting lipid loading was performed using Oil red O extraction quantification. After microscopic observation after oil red O staining, each group was washed twice with PBS, lipid loading was extracted with 60% isopropanol, and absorbance at 500 nm was measured and quantitatively analyzed. From the results of FIG. 3C, the absorbances of 0.149 ± 0.007 and 0.093 ± 0.017 for the first group and the second group, respectively, were 0.068 ± 0.006 for the third group and the fourth group treated with TUCDA in the differentiation step, respectively. , A relatively low absorbance of 0.068 ± 0.001. Statistically significant results were found among all groups except the statistical value between group 3 and group 4 (*, p <0.01).

In addition, the degree of adipocyte differentiation of each group was measured using the RT-PCR method in the same manner as in Example 3 to measure the expression of adipocyte differentiation-related markers (FIG. 3D). Primer sets used in RT-PCR are shown in Table 1 above, GPDH primer sets are sense primers: 5'-TGA TGG CCT GGG CTT TGG CG-3 'and antisense primers: 5'-GCT GGG CCC CAT AGC CTC CT- The sequence of 3 '(Source: NM_005276) was used. RT-PCR analysis results are the same as in FIG. From the results of FIG. 3D, the expression of PPARγ, which is an initial marker for adipocyte differentiation, and GPDH, which is a late marker, in the fourth group, were compared with those of the first, second, and third groups including the not induced group. Low expression pattern was shown. Therefore, from the results of Oil red O staining, Oil red O extraction, and RT-PCR results, it was confirmed that TUDCA treatment in the culture of adipose-derived stem cells, in particular, can effectively inhibit differentiation into adipocytes. Can be.

Example 5 Evaluation of Bone Cell Differentiation of Human Adipose-Derived Stem Cells Following TUDCA Treatment

Human adipose derived stem cells obtained in Example 1 were seeded at a density of 1 × 10 ⁴ cells / cm ² , and osteoblast differentiation with and without TUDCA treatment was evaluated in stem cell culture and osteoblast differentiation culture. Stem cell cultures were prepared with Dulbecco's modified Eagle medium (DMEM, Gibco BRL, Gaithersburg, MD) supplemented with 1% Penicillin-Streptomycin and 10% Fetal Bovine Serum (FBS) (Invitrogen, USA). ] Was used as a basic medium (Basal Medium, BM). Osteocyte differentiation cultures were 10% FBS, 100 nM dexamethasone, 1% Glutamax (Invitrogen, USA), 50 μg / mL ascorbic acid, 10 mM Glycerol 2-Phosphate, 1% penicillin DMEM medium with streptomycin added was used as a medium for osteoblast differentiation. Incubation was performed every 21 days for 21 days. The experimental group included the stem cell culture group (Basal medium, BM), the TUDCA 50 μM treated group (BM + TUDCA 50 μM), the osteoblast differentiation culture group (Osteogenic medium, OM), and the osteoblast differentiation culture. The group was treated with TUDCA 50 μM (OM + TUDCA 50 μM).

4A is a result of measuring the activity of alkaline phosphatase (Alkaline Phosphatase, ALP) on the 3rd and 7th day in each experimental group (n = 3). Each experimental group (all groups collected on days 0, 3, and 7) was added to 50 μl lysis buffer (Cell Culture Lysis Reagent 5X, Promega) and cell lysate was added for 10 minutes. The protein was recovered by ultracentrifugation (Micro 17R, Hanil Science Industrial, Seoul, Korea) at 13000 rpm. 50 μL of each group of protein and 150 μL of p-Nitrophenyl Phosphate (pNPP) were treated, and then absorbed at 405 nm at 15 ° C. for 15 minutes. The measured values were evaluated by the values in the calibration lines obtained using 4-nitrophenol 10 mM solution (Sigma). 0.8785 ± 0.01 and 0.7457 ± 0.01 were measured in the BM and BM + TUDCA 50 μM groups, respectively, and 3.3705 ± 0.1 and 3.4235 ± 0.2 in the OM and OM + TUDCA 50 μM groups, respectively. The OM and OM + TUDCA 50 μM groups showed statistically higher ALP activity than the BM and BM + TUDCA 50 μM groups, but did not show a significant effect on ALP activity by TUDCA treatment in each culture (* , p <0.01).

4B is a result of measuring the marker gene expression by performing RT-PCR in the same manner as in Example 3 to determine the degree of osteoblast differentiation of each group (n = 4). The primer sets of Col I, ALP, and GAPDH used in RT-PCR are the same as in Table 1 above, and the Osteocalcin (OCN) primer set is sense primer: 5'-ATG AGA GCC CTC ACA CTC CT- The sequences of 3 'and antisense primers: 5'-GCC GTA GAA GCG CCG ATA GG-3' (Source: NM_199173) were used. Compared with the BM and BM + TUDCA 50 μM groups, the marker genes related to osteoblast differentiation, OCN, ColI and ALP, were higher in the OM and OM + TUDCA 50 μM groups. There was no significant effect on the expression level of osteoblast differentiation genes following TUDCA treatment.

4C is a result of evaluating the degree of calcium salt deposition of cells obtained from each group by von Kossa staining (n = 4). Cells obtained from each group were fixed with 10% formalin and washed three times with PBS. Then 5% silver nitrate solution was added and exposed to strong light for 1 hour. The overstained portion was removed with 5% sodium thiosulfate solution and contrast stained using Nuclear fast red. In FIG. 4C, black indicates calcium deposition and red indicates cell nuclei (Scale bar = 100 μm). Calcium deposition was not found in the BM and BM + TUDCA 50 μM groups, but calcium deposition was observed in the OM and OM + TUDCA 50 μM groups, respectively.

4D is a result of quantifying the degree of calcium deposition of cells obtained from each group. Cells obtained from each group were gently washed with PBS and then measured according to the manufacturer's instructions using a commercially available calcium quantification kit (Bioassays, USA) (n = 4). The BM and BM + TUDCA 50 μM groups showed 0.0052 ± 0.0007 and 0.0061 ± 0.0006, respectively, and the OM and OM + TUDCA 50 μM groups showed 0.1722 ± 0.0176 and 0.1949 ± 0.0367, respectively. The calcium deposition was higher in OM, a bone cell differentiation medium than in culture medium BM, but there was no significant difference in calcium deposition according to TUDCA treatment in each culture ( p > 0.01).

From the above ALP activity, RT-PCR, von Kossa staining, and calcium quantitative results, it was confirmed that the treatment of TUDCA had no effect on osteoblast differentiation in culturing human adipose derived stem cells and osteoblast differentiation. Therefore, purely differentiated bone cells can be obtained while excluding the possibility of contamination due to differentiation into adipocytes.

Example 6 Evaluation of Chondrocyte Differentiation of Human Adipose-Derived Stem Cells by TUDCA Treatment

Human adipose derived stem cells obtained in Example 1 were seeded at a density of 1 × 10 ⁴ cells / cm ² , and chondrocyte differentiation with and without TUDCA treatment was evaluated in stem cell culture and chondrocyte differentiation culture. Stem cell cultures were prepared with Dulbecco's modified Eagle medium (DMEM, Gibco BRL, Gaithersburg, MD) supplemented with 1% Penicillin-Streptomycin and 10% Fetal Bovine Serum (FBS) (Invitrogen, USA). ] Was used as a basic medium (Basal Medium, BM). Chondrocyte differentiation cultures were 10% FBS, 100 nM dexamethasone, 50 μg / mL ascorbic acid, 1% penicillin-streptomycin, 1% ITS (insulin 1 g / L, sodium selenite 0.67 mg / L, transferrin 0.5 g / L), DMEM medium to which 10 ng / mL TGF-β1 was added was used as a medium for osteoblast differentiation. Incubation was performed every 21 days for 21 days. The experimental group included the stem cell culture group (Basal medium, BM), the TUDCA 50 μM treated group (BM + TUDCA 50 μM), the chondrocyte differentiation culture group (Chondrogenic medium, CM), and the chondrocyte differentiation medium. TUDCA 50 μM treated groups (CM + TUDCA 50 μM) were performed.

5A is a result of measuring the glycosaminoglycan (GAG) matrix of cells obtained from each group by Alcian blue staining (n = 3). Cells obtained from each group were washed twice with PBS, placed in 4% Alcian blue solution and treated for 30 minutes at room temperature. The stain was removed and contrast stained using Nuclear fast red. In FIG. 5A, red is the nuclear site of the cell, and blue is the GAG matrix observed in chondrocytes (Scale bar = 100 μm). The BM and BM + TUDCA 50 μM groups could not identify the GAG matrix, but the CM and CM + TUDCA 50 μM groups were able to identify many GAG matrices.

5B is a result of quantifying the GAG Matrix of each group by Alcian blue extraction quantification method. Cells obtained from each group were reacted with 6 M guanidine-HCl for 2 hours at room temperature to extract the blue stained GAG Matrix portion, and the absorbance was measured at 600 nm wavelength (n = 3). The BM and BM + TUDCA 50 μM groups were 0.064 ± 0.004 and 0.057 ± 0.002, respectively. The CM and CM + TUDCA 50 μM values were 0.328 ± 0.008 and 0.388 ± 0.018, respectively. Although the expression level of GAG matrix was higher in CM, a chondrocyte differentiation medium than in culture medium BM, it was evaluated that there was no significant difference in the expression level of GAG matrix according to TUDCA treatment in each culture medium ( p > 0.01).

5C shows the results of measuring marker gene expression by performing RT-PCR in the same manner as in Example 3 for the degree of chondrocyte differentiation of each group (n = 3). Primer sets of Col I, Col II, Agg, and GAPDH used in RT-PCR are the same as in Table 1 above. Col II and Agg were identified as chondrocyte-associated marker genes, and Col I was used as the negative factor. Compared with the BM group and the BM + TUDCA 50 μM group, the chondrocyte differentiation marker genes Col II and Agg were more expressed in the CM group and the CM + TUDCA 50 μM group, and the negative factor Col I Was expressed low. However, there was no significant effect on the expression level of chondrocyte differentiation-related genes by TUDCA treatment in each culture.

From the above Alcian blue staining, Alcian blue extraction quantification, and RT-PCR results, it was confirmed that TUDCA treatment had no effect on chondrocyte differentiation in culturing and adjuvant chondrocyte differentiation of human adipose derived stem cells. Therefore, purely differentiated chondrocytes can be obtained while excluding the possibility of contamination due to differentiation into adipocytes.

Claims (8)

A method for differentiating adipose derived stem cells into chondrocytes, comprising culturing adipose derived stem cells in a medium for chondrocyte differentiation comprising tauroursodeoxycholic acid. A method for differentiating fat-derived stem cells into bone cells, comprising culturing fat-derived stem cells in bone cell differentiation medium containing tauurusodeoxycholic acid. delete The differentiation method according to claim 1 or 2, wherein the concentration of taurousodeoxycholic acid is in the range of 5 to 500 µM. The method of claim 1, wherein the chondrocyte differentiation medium is DMEM medium containing tauurosodeoxycholic acid, TGF-β1, dexamethasone, ascorbic acid, insulin, transferrin, and selenium salt. According to claim 1, wherein the chondrocyte differentiation medium is taururusodeoxycholic acid 50-150 μg / mL, TGF-β1 5-20 ng / mL, dexamethasone 50-200 nM, ascorbic acid 10-100 μg / A differentiation method comprising: DMEM medium comprising mL, insulin 0.001-1 mg / mL, transferrin 0.055-500 mg / mL, and selenium salt 500-670 μg / mL. The medium for osteoblast differentiation is a DMEM medium comprising tauurosodeoxycholic acid, dexamethasone, ascorbic acid, L-alanyl-L-glutamine, and glycerol 2-phosphate. Differentiation method. The medium for osteoblast differentiation according to claim 2, wherein the medium for osteoblast differentiation is 50 to 150 µg / mL of taurorusodeoxycholic acid, 50 to 200 nM of dexamethasone, 10 to 100 µg / mL of ascorbic acid, and L-alanyl-L-glutamine. A differentiation method of DMEM medium comprising 50 to 200 nM and glycerol 2-phosphate 5 to 20 mM.

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