KR100627695B1 - Animal serum-free medium composition for culturing human stem cells and method for differentiating from the cultured human stem cells to hematocytes - Google Patents

Abstract

The present invention relates to a cell culture medium composition comprising human serum without animal serum, more specifically 50-70% (v / v) DMEM, 30-50% (v / v) Nutrient Mixture, 4 -6 μg / ml insulin, 4-6 μg / ml transferrin, 4-6 ng / ml sodium selenite, 10 ^-7 -10 ^-9 M dexamethasone, 80- Human stem cells comprising 120 U penicillin / 800-1,200 U streptomycin, 5-15 ng / ml EGF, 5-15 ng / ml PDGF-BB, 5-15% human serum A culture medium composition, a method for culturing human stem cells using the same, and a method for inducing differentiation into hepatocytes.

According to the present invention, by using human serum from the early stage of cell separation, it was possible to culture and preserve the mesenchymal stem cells without animal serum, thereby reducing the safety problems of fetal bovine serum used in mesenchymal stem cell culture. Solved. In addition, it reduced the proliferative capacity and differentiation capacity of stem cells appearing when using human serum. In addition, the use of hepatocytes differentiated from human mesenchymal stem cells can effectively develop a cell therapy for the treatment of liver disease.

Description

Animal serum-free medium composition for culturing human stem cells and method for differentiating from the cultured human stem cells to hematocytes}

Figure 1 is a graph showing the growth rate of fat matrix cells (ATSC) in a medium containing fetal bovine serum and human serum.

Figure 2 is a graph showing the growth rate of bone marrow mesenchymal stem cells (BMSC) in a medium containing fetal bovine serum and human serum.

Figure 3 is a photograph showing the fat differentiation and bone differentiation in adipose matrix cells and bone marrow mesenchymal stem cells cultured in fetal bovine serum and human serum.

Figure 4 is a photograph analyzing the karyotype after 10 times crab culture of mesenchymal stem cells isolated from adipose tissue.

Figure 5 is a photograph showing the morphological changes when the mesenchymal stem cells isolated from adipose tissue when induced hepatocyte differentiation with various cytokines.

Figure 6 shows the expression changes of genes (abumin and a-fetoprotein (AFP)) in undifferentiated fat substrate cells (hATSC) and hepatocytes induced differentiation (HO).

Figure 7 is the result of measuring the low density lipoprotein (LDL) uptake in cells induced to differentiate into hepatocytes.

8 is a graph showing the increase in urea production measured in the medium of cells induced to differentiate into hepatocytes.

The present invention relates to a cell culture medium composition comprising human serum without animal serum, and more particularly to a specific concentration of DMEM, nutrition mixture (Nutrient Mixture), insulin-transferrin-selenium, EGF, PDGF It relates to a human stem cell culture medium composition comprising a -BB, human serum and the like, a method for culturing human stem cells using the same and a method for inducing hepatocyte differentiation.

Unlike in vivo, in order to supply culture medium to artificially cultivate animal cells in vitro, it is necessary to satisfactorily satisfy the nutrients close to the biological conditions based on body fluids such as plasma or lymph and environmental conditions such as pH, temperature and osmotic pressure. . Therefore, the culture medium composition was determined based on the body fluid, and the determined culture medium was selected and serum was added to the cells at an appropriate ratio, and then used for cell culture. In this way, early in the development of the medium, Morgan (Morgan) et al. 1950 produced M199 medium based on the body fluid composition and cultured the primary chick (Morgan, et al., 1950). Subsequently, the main medium developed was Eagle basal medium (Eagle, 1955) used for culturing human and rat embryo cells, and culture medium for culturing mice fetal cells (Dulbecco's modified eagles medium, Dulbecco, et al., 1959), Ham's F-12 (Ham, 1965) and Morore et al. RPMI-1640 (Moore et al., 1967) used to culture hamster tumor cells.

In cell culture, 5 to 20% of animal serum is added to the basal medium according to the nutritional requirements of the cells. The serum contains insulin, which promotes cell growth and function, various hormones of the polypeptide system, growth factors that regulate cell division and function, and adhesion and diffusion factors such as fibronectin, transferrin as a binding protein, lipids and minerals. Many trace elements are included. Serum is also a buffer that regulates the osmotic pressure and pH of the culture, and has a viscous action that protects cells from proteolytic inhibition and physical shock (Griffiths, 1987). Therefore, a constant concentration of serum is added to the basal medium to form a culture medium. Animal cells can be cultured using serums containing these various functions.

Stem cells have the property of continuously producing the same cells as themselves for a certain period of time in an undifferentiated state, and differentiate into specific cells under suitable conditions. Stem cells can be divided into embryonic stem cells and adult stem cells, depending on their origin. Human embryonic stem cells have many ethical problems because they are made from embryos that can occur in human life, but they are known to have superior cell proliferation and differentiation capacity as adult stem cells. Adult stem cells can be obtained from bone marrow, blood, brain, skin, etc., so there are few ethical problems, but they have limited multipotency compared to embryonic stem cells. The most researched adult stem cells are hematopoietic stem cells, and recent studies on mesenchymal stem cells are also invigorating.

The first bone marrow stromal cells found among mesenchymal stem cells were first identified by a study by Fiedenstein et al. (1968) (Friedenstein et al, 1968). And various connective tissues such as adipose tissue (Owen, 1988). Experiments have been attempted to differentiate bone marrow stromal cells into cardiomyocytes (Makino et al, 1999), hepatocytes (Oh et al, 2001) and neurons (Woodbury et al, 2000; Deng et al, 2001) ex vivo. Mesenchymal stem cells, including bone marrow stromal cells, can be used for a variety of purposes, including cell therapy of various organs, gene transfer, and regulation of immune responses (Tocci & Forte, 2003).

However, when a medium containing animal serum (fetal bovine serum) is used to culture such human stem cells, a considerable amount of fetal bovine serum remains in the cells even if the cells cultured with fetal bovine serum are washed with a solution without fetal serum. As a powerful heterologous antigen, human transplantation of cells cultured with fetal bovine serum is prohibited in the United States due to the possibility of human infection of bovine diseases such as mad cow disease.

Techniques for the isolation of mesenchymal stem cells from human bone marrow and for the separation of mesenchymal stem cells from adipose tissue are described by Pittenger et al. (Science 284: 143-147, 1997) and van et al. (J Clin Invest. 58: 699-). 704, 1976). In these cells, α-MEM or DMEM medium and 10-20% fetal bovine serum were used for cell culture. However, the use of animal serum (fetal bovine serum) in these media has caused many safety problems in human transplantation.

To overcome this, there have been attempts to use human serum instead of fetal bovine serum in conventional cell culture media (Kuznetsov SA, Mankani MH, Robey PG.Effect of serum on human bone marrow stromal cells: ex vivo expansion and in vivo bone formation) Transplantation. 2000 Dec 27; 70 (12): 1780-7) Due to the loss of characteristics of stem cells, such as decreased proliferative capacity of stem cells, decreased differentiation capacity and accelerated differentiation into bone cells, Human serum was difficult to use as it is.

Accordingly, the present inventors have made intensive studies to overcome the problems of the prior art, using a medium composition containing a specific concentration of DMEM, Nutrient Mixture, insulin-transferrin-selenium, EGF, PDGF-BB, human serum, etc. In this case, human stem cells can be cultured at a high growth rate, and the cell type can be maintained even after proliferation for more than one month, and it can be induced to differentiate into derivatives of bone, fat, cartilage and liver tissue. Was completed.

Therefore, the main object of the present invention is to provide a new human stem cell culture medium composition and hepatocyte-induced medium composition containing human serum without animal serum that can solve the conventional safety problems.

Another object of the present invention is to provide a method for culturing human stem cells using the culture medium composition and a method for inducing differentiation into hepatocytes.

In order to achieve the object of the present invention, the present invention provides a cell culture medium composition comprising human serum without animal serum, 50-70% (v / v) DMEM, 30-50% (v / v) nutrient mixture ( Nutrient Mixture, 4-6 μg / ml insulin, 4-6 μg / ml transferrin, 4-6 ng / ml sodium selenite, 10 ^-7 -10 ^-9 M dexamethasone ( dexamethasone), including 80-120 U penicillin / 800-1,200 U streptomycin, 5-15 ng / ml EGF, 5-15 ng / ml PDGF-BB, 5-15% human serum It provides a human stem cell culture medium composition, characterized in that.

In the medium composition of the present invention, DMEM (Dulbecco's modified Eagle's medium) is a basic medium used for cell culture, and in the examples of the present invention, low-glucose DMEM (L-glutamine) (Life Technologies) was used. .

In the medium composition of the present invention, the nutrition mixture (Nutrient Mixture) is a mixture of various amino acids, vitamins, inorganic salts and the like commonly used in cell culture, in the embodiment of the present invention specifically F-12 Nutrient Mixture (Life Technologies, Inc.). ) Was used.

In the medium composition of the present invention, in order to provide insulin, transferrin and sodium selenite, insulin-transferrin-selenium (Sigma) was specifically used in the Examples.

In the medium composition of the present invention, dexamethasone (dexamethasone) means 9-Fluoro-16 -Methylprednisolone (C ₂₂ H ₂₉ FO ₅ ), EGF means epidermal growth hormone (Epidermal Growth Factor), PDGF-BB The homodimer of the B polypeptide chain of platelet-derived growth factor (PDGF).

In the medium composition of the present invention, human serum may be obtained from a blood source or the like, but is preferably obtained by centrifugation from autologous blood collected from a patient who is the origin of cells to be cultured.

In the medium composition of the present invention, when human serum is 5% or less, there is a limitation of proliferation rate and a significant increase in proliferative capacity does not occur at 15% or more, and there is a disadvantage of suppressing the differentiation capacity of cells.

In order to achieve another object of the present invention, the present invention provides a method for culturing human stem cells using the cell culture medium composition of the present invention.

In the method of culturing human stem cells of the present invention, the step of inoculating human stem cells in a culture vessel comprising the cell culture medium composition and the culture vessel in a CO ₂ incubator 5 to 15% of the carbon dioxide Culturing at 35-39 ° C. in skill.

In the culture method of the present invention, the culture vessel is, for example, 100 mm tissue culture dish (IWAKI, Japan), 60 mm tissue culture dish (IWAKI, Japan), 6 well tissue culture dish (Nuncoln ^™ , Denmark), 12 well tissue culture dish (TPP). _, Europe), etc., and CO ₂ incubators include Forma 3130 CO ₂ incubator (Formascientific Inc, USA), SANYO CO ₂ incubator (Japan), NUAIR US Autoflow CO ₂ incubator (Hansol SM Korea). Can be used.

In the culturing method of the present invention, the human stem cells may be both human-derived fetal stem cells and adult stem cells, but is preferably isolated from mesenchymal stem cells, more preferably bone marrow and adipose tissue among adult stem cells It is characterized by one mesenchymal stem cell.

In the culturing method of the present invention, preferably, the human stem cells maintain the ability to differentiate into derivatives of bone, fat and cartilage, and proliferate while maintaining the karyotype of human stem cells for at least one month. do.

To achieve the other object of the present invention, the present invention provides 15-25% (v / v) human serum, 40-44% (v / v) DMEM, 26-30% (v / v) nutrition mixture ), 8-12% (v / v) provides a freeze-dried medium composition of human stem cells containing DMSO (Dimethyl Sulfoxide).

In order to achieve the other object of the present invention, the present invention is in place of 5-15 ng / ml EGF, 5-15 ng / ml PDGF-BB and 5-15% human serum in the human stem cell culture medium composition of the present invention Initial differentiation from human mesenchymal stem cells to hepatocytes, comprising 5-15 ng / ml hepatocyte growth factors (HGF), 5-15 ng / ml oncostatin M (OSM) Provided is a medium composition for induction.

In the medium composition of the present invention, if any one of HGF and OSM is missing or out of the concentration range, early differentiation from human mesenchymal stem cells to hepatocytes is not well induced.

In order to achieve the other object of the present invention, the present invention induces the initial differentiation using the medium composition for inducing differentiation with the ancestor of the present invention, and then the medium from the day 8-15 days after inducing differentiation It provides a method of inducing differentiation from human mesenchymal stem cells to hepatocytes, which induces final differentiation by adding 0.05-0.2% DMSO to the composition.

In the differentiation-inducing method of the present invention, when DMSO is omitted or out of the concentration range, final differentiation from human mesenchymal stem cells to hepatocytes is not well induced.

The present invention specifically provides a method and a medium composition for separating mesenchymal stem cells and culturing in a medium to which human serum is added from the beginning, and subculture at least five times in a state of maintaining differentiation capacity and enabling cryopreservation. There has been no successful culturing of mesenchymal stem cells with human serum, but in the present invention, it was cultured so as not to be exposed to fetal bovine serum from the initial stage for human use, and five passages necessary for securing cells for human transplantation. Differentiation capacity was maintained for a while.

In addition, the present invention provides a medium composition capable of inducing differentiation from human mesenchymal stem cells cultured with human serum to hepatocytes and a method of inducing differentiation, thereby effectively developing a liver disease cell therapy using the above method.

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Cell Culture and Storage

Mesenchymal stem cells were isolated from human bone marrow and fat and cultured with conventional fetal bovine serum containing medium and human serum replacement medium. Ficoll was added to bone marrow to separate stromal stem cells from human bone marrow, and centrifuged at 3500 rpm for 30 minutes. The supernatant, a mononuclear cell layer, was washed with HBSS to remove Ficoll. After centrifugation, mesenchymal stem cells were cultured using the following three culture solutions. Adipose tissues are obtained from patients undergoing subcutaneous fat removal surgery in plastic surgery to separate stromal stem cells from adipose tissue. The isolated adipose tissue was washed with HBSS, and the adipose tissue was thinly sliced and then treated in a serum-free α-MEM medium containing 0.075% collagenase at 37 ^° C. for 30 minutes. After centrifugation, the stromal stem cells were cultured using the following three culture solutions in pellets. ① Culture solution consisting of α-MEM (L-glutamine), 10% fetal bovine serum, ② Culture solution consisting of α-MEM (L-glutamine), 10% human serum, ③ Fibronectin (10 ng / ml) 50-70% low-glucose DMEM (L-glutamine) (Life Technologies), 30-50% Nutrient Mixture (Life Technologies), 1x insulin-transferrin-selenium (Sigma), 10 ^-8 M dexamethasone (Sigma) , 100 U penicillin / 1,000 U streptomycin (Sigma), 10 ng / ml EGF (Daewoong Pharmaceuticals), 10 ng / ml PDGF-BB (R & D system), and 10% human serum. The separated cells were plated in a dish to remove unattached cells after 24 hours. Cells were grown to 70-80% and treated with trypsin / EDTA to separate the cells into single cells and spread them to a density of 2000 / cm 2 for crab culture. Cell proliferation rate was measured by counting the total cell number during crab culture on a 3-day basis. In order to separate human serum, blood was collected in a container treated with anticoagulant (heparin) to prevent blood coagulation, and centrifuged at 500 rpm for 5 minutes and serum was separated. Serum was filtered and sterilized in a 0.2 μm filter and used for cell culture.

To freeze the mesenchymal stem cells, first remove the cells and suspend them in 900 µl of cell culture medium containing 20% human serum (DMEM-low glucose / F-12 medium). Put 100µl of DMSO into the freezing vial, add suspended cells, store at -20 ℃ for 1 hour 30 minutes, and transfer to -70 ℃.

In order to measure the proliferation capacity, the cells were passaged at three-day intervals, and the cells were collected at each passage, and the time taken to double the number of cells was measured by counting the number of cells in a certain volume using a haematometer under a microscope. . As a result, as shown in Figure 1, there was no significant difference in the initial proliferative capacity of mesenchymal stem cells isolated from bone marrow and adipose tissue in the medium added with fetal bovine serum, but if the fetal bovine serum was replaced with 10% human serum Growth rate was significantly decreased in MEM medium, but no significant difference was observed in DMEM-LG / F-12 medium. In addition, even though these cells were cultured up to 10 times, no significant difference in growth capacity was observed, and no significant difference in growth capacity was observed after thawing after freeze-drying using a freeze medium containing human serum. The composition of low-glucose DMEM (L-glutamine) (Life Technologies) used in the experiment is shown in Table 1 below, and the composition of F-12 Nutrient Mixture (Life Technologies) is shown in Table 2 below.

Example 2 Expression of Cell Surface Antigens

Flow cytometry was performed to determine whether the surface antigen expression changes in human mesenchymal stem cells cultured in fetal bovine serum (composition 1 of Example 1) and human serum (composition 3 of example 1). For flow cytometry, incubate the cells for 72 hours, remove the cells with 0.25% trypsin / EDTA, and fix for 30 minutes with 4% formaldehyde. Immobilized cells were washed with flow cytometry buffer (FCB; 1 x PBS, 2% FBS, 0.05% sodium azide), divided into 10 ⁶ cells, followed by CD29, CD44, CD90, CD105, CD34, CD14b, and HLA-DR antibodies. Each containing flow cytometry buffer was treated. Cells treated with antibodies were measured using a FACScan argon laser cytometer (Becton Dickson, San Jose, Calif.).

As shown in Table 3, the expression of surface antigen in mesenchymal stem cells cultured with human serum did not show a significant difference from mesenchymal stem cells cultured in fetal bovine serum.

Comparison of Surface Protein Expression in Adipose Matrix and Bone Marrow Mesenchymal Stem Cells in Fetal Bovine Serum and Human Serum ATSC BMSC + FBS  + HS + FBS + HS CD44 ++ ++ ++ ++ CD29 ++ ++ ++ ++ CD105 + + + + CD34 - - - - CD14b - - - - HLA-DR - - - - CD90 ++ ++ ++ ++

  ++, strong positive; +, weak positive; -, negative

Example 3 Induction of Adipose and Bone Differentiation

Differentiation into adipose and osteocytes was induced to confirm whether the differentiation ability of mesenchymal stem cells cultured with human serum (composition 3 of Example 1) is maintained. Differentiation of mesenchymal stem cells into adipocytes (10% fetal bovine serum, 1 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, 10 μM insulin, and 200 ?? M indomethacin in α-MEM) and osteoblast differentiation Differentiation was performed using medium (10% fetal bovine serum, 0.1 μM dexamethasone, 10 mM β-glycerophosphate, and 50 μM ascorbic acid in α-MEM). Differentiated fat cells were quantified by staining lipids accumulated on the cell surface with Oil Red O, treated with isopropyl alcohol, and measuring absorbance at 510 nm. After staining with Oil Red O, remove the differentiation medium, fix with 4% formaldehyde at room temperature for 15 minutes and wash twice with HBSS. After staining for 1 hour at room temperature with 2% Oil Red O, washed with HBSS and observed the degree of differentiation under a microscope. Differentiated bone cells were stained with alizarin red S and quantified by measuring and quantifying the percentage of differentiated cells in total cells using Meta Morph program. After staining with Alizarin red S, the differentiation medium was removed and fixed with 70% ethanol at room temperature for 15 minutes and washed twice with distilled water. After staining with Alizarin red S for 10 minutes at room temperature, the degree of differentiation was observed under a microscope.

In order to determine whether these cells maintain differentiation capacity, differentiation of mesenchymal stem cells proliferated in each culture condition (human serum and FBS) into bone cells (OM) and adipocytes (AM) induced significant differentiation. Not observed (FIG. 3). In addition, after one or five crab cultures of bone marrow mesenchymal mesenchymal stem cells and adipocytes in fetal bovine serum (FBS) or human serum (HS), the differentiation of fat and bone cells into two weeks was induced by different methods. Differentiation capacity of the four samples was quantified by the above method, and the average value was measured. There was no significant difference between the differentiation capacity and the proliferative capacity after crab culture up to 5 times (Table 4).

Quantitative Analysis of Adipose and Bone Differentiation in Adipose Matrix and Bone Marrow Mesenchymal Stem Cells Cultured in Fetal Bovine Serum and Human Serum BMSC ATSC + FBS + HS + FBS + HS passage 1 passage 5 passage 1 passage 5 passage 1 passage 5 passage 1 passage 5 Adipogenic differentiation (OD / 12 well, n = 4) 1.287 1.342 1.321 1.234 1.58 1.63 1.45 1.54 Osteogenic differentiation (% area after 2 wks of differentiation, n = 4) 62.2 57.3 62.7 59.8 52.6 49.3 56.3 50.8

Example 4 Karyotyping

After cell proliferation 10 times after cell proliferation, the karyotype of mesenchymal stem cells isolated from adipose tissue was analyzed. Cover glass in a 30 mm dish and incubate for 24 hours by plating the appropriate cells. In order to stop cell division in mitosis, the cells were incubated for 4 hours with 0.05 ml of colcemid (Gibco, 10 μg / ml), and 2 ml of a stock solution (KCl, 75 mM) was added thereto and treated at 37 ° C. for 20 minutes. Fix with Carnoy Fixing Solution (methanol: acetic acid = 3: 1) at 4 ℃ for 30 minutes and take out cover glass and fix it on slide glass. After drying for 4 hours in a slide dryer was treated for 25 seconds in a 0.025% trypsin solution and treated for 2 minutes in a Giemsa solution was subjected to Giemsa-banding and analyzed by a 1000 times optical microscope. Cells showed normal XX karyotype (FIG. 4).

Example 5 Induction of Differentiation into Hepatocytes

Differentiation into hepatocytes was induced to confirm whether the differentiation capacity of mesenchymal stem cells cultured with human serum (composition 3 of Example 1) is maintained. First, using 2x10 ⁴ ~ 3x10 ⁴ / culture medium composition of the composition 3 of the cells of cm ² Example 1 fibronectin (10ng / ㎖) is a plating on the coating culture plate, then the serum is added after 12-20 hours The prepared medium was removed and washed with HBSS. Then 50-70% low-glucose DMEM (L-glutamine) (Life Technologies), 30-50% F-12 Nutrient Mixture (Life Technologies), 1x insulin-transferrin-selenium (Sigma), 10 ^-8 M dexamethasone ( Sigma), 100 U penicillin / 1,000 U streptomycin (Sigma), 10 ng / ml hepatocyte growth factors (HGF) (R & D system), 10 ng / ml oncostatin M (OSM) (R & D system) Differentiation was induced, and final differentiation was induced by adding 0.1% DMSO (Sigma) to the previous culture solution from the 11th day after induction of differentiation.

In order to induce differentiation of hepatocytes, cytokines, which are known to be involved in hepatocyte differentiation, have been treated with various conditions in cells to establish appropriate conditions for differentiation. Differentiation was not induced by HGF alone or OSM alone (top of FIG. 5). When HGF and OSM were treated together, some initial differentiation was induced (middle of FIG. 5), and by addition of 0.1% DMSO It was confirmed that the complete change in form (bottom of Figure 5). Hepatocytes differentiated under the above conditions were morphologically similar to actual hepatocytes (FIG. 5).

Example 6 Functional Test of Differentiated Hepatocytes

Various experiments were performed to determine whether differentiated hepatocytes actually maintain hepatocyte function. Total RNA was isolated from hepatocytes differentiated in Example 5 using Tri Reagent (Sigma), and then total RNA was converted to cDNA using reverse transcriptase and oligo (dT) primer. PCR was carried out to express the amount of albumin and α by using primers for Taq polymerase and α-fetoprotein (AFP) (Sense: CTCGTTGCTTACACAAAGAAAG, Anti-sense: ATGGAAAATGAACTTGTCATCA) and Albumin (Sense: TTTGCTCAGTATCTTCAGCAGT, Anti-sense: AGTAAGGATGTCTTCTGGCAAT) The change was measured. As a result, it was confirmed that expression of AFP was increased and albumin was expressed as compared to control cells (hATSCs) that were not differentiated from differentiated cells (HO) (FIG. 6).

Next, we determined whether differentiated hepatocytes take up low density lipoprotein (LDL) using the Dil-Ac-LDL staining kit (Biomedical Technologies: Stoughton, Massachusetts, USA). Dil-Ac-LDL was diluted to 10 ug / ml in the medium used for differentiation, and then it was added to cells and incubated at 37 ° C for 4 hours. The medium containing Dil-Ac-LDL was removed, washed several times with clean medium, and observed using a fluorescence microscope. As a result, differentiated hepatocytes were uptake (green stain) of LDL (FIG. 7). The left side of the figure is a phase contrast microscope of the cells on the right side.

Finally, each time the culture solution was changed from the differentiated cells, the culture solution was collected and then examined to see if the differentiated cells produced urea. First, the collected 1 ml culture solution and 1 ml chromogenic reagent (1 part of 3% 2,3-butanedione monoxime in ethanol + 29 parts of acid mixture (H ₂ SO ₄ : H ₃ PO ₄ : H ₂ 0 = 1: 3: 7)) was mixed and boiled at 100 ° C for 30 minutes. Then, absorbance was measured at 492 nm using a UV Spectrophotometer. As a result, as the differentiation progressed, it was confirmed that the amount of urea produced increased. (FIG. 8) These results show that the cells induced by this differentiation method function as hepatocytes.

As described above, according to the present invention, when using a medium composition containing human serum without animal serum, human stem cells can be cultured at a high growth rate, and the cell karyotype can be maintained even after proliferating for 1 month or more. Differentiation can be induced with derivatives of bone, fat, cartilage and hepatocytes. Therefore, by using human serum from the early stage of cell separation, it was possible to culture and preserve mesenchymal stem cells without animal serum, thereby solving the safety problem by fetal bovine serum used in mesenchymal stem cell culture. In addition, the use of hepatocytes differentiated from human mesenchymal stem cells can effectively develop a cell therapy for the treatment of liver disease.

Claims (8)

For cell culture medium compositions comprising human serum without animal serum, 50-70% (v / v) DMEM, 30-50% (v / v) Nutrient Mixture, 4-6 μg / ml insulin ( insulin), 4-6 μg / ml transferrin, 4-6 ng / ml sodium selenite, 10 ^-7 -10 ^-9 M dexamethasone, 80-120 U penicillin / Human mesenchymal stem cell culture medium composition comprising 800-1,200 U streptomycin, 5-15 ng / ml EGF, 5-15 ng / ml PDGF-BB, 5-15% human serum . delete delete delete delete 15-25% (v / v) human serum, 40-44% (v / v) DMEM, 26-30% (v / v) Nutrient Mixture, 8-12% (v / v) DMSO Freeze-dried medium composition of human stem cells comprising. delete 5-15 ng / ml hepatocyte growth factors (HGF) in place of 5-15 ng / ml EGF, 5-15 ng / ml PDGF-BB and 5-15% human serum in the media composition of claim 1 Induction of initial differentiation using a media composition comprising -15 ng / ml oncostatin M (OSM), followed by 0.05-0.2% in the media composition from 8-15 days after starting to induce differentiation A method of inducing differentiation from human mesenchymal stem cells to hepatocytes, characterized by inducing final differentiation by adding DMSO.

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