KR101810014B1 - Method for generating induced pluripotent stem cells from human-derived somatic cells using histone deacetylase inhibitor and bone morphogenetic protein pathway blocker - Google Patents

Abstract

The present invention relates to a method for producing a recombinant human somatic cell, comprising the steps of: (a) culturing a human-derived somatic cell into which a gene encoding a dediffering inducer is introduced in the presence of at least one selected from the group consisting of a protein kinase C inhibitor, a histone deacetylase inhibitor, Culturing in a medium containing a differentiation-enhancing agent; And (b) separating embryonic stem cell-like colonies from the culture obtained from step (a). The present invention also provides a method for producing iPS cells. The production method of the present invention is capable of producing reprogrammed pluripotent stem cells at high efficiency and also capable of producing reprogrammed pluripotent stem cells under xenopathogen-free and feeder cell-free conditions Can be manufactured.

Description

Histone deacetylase inhibitor and bone morphogenetic protein pathway inhibiting agent United States Patent Application 20060162991 Kind Code: A1 A method for producing stem cells from human-derived somatic cells using a histone deacetylase inhibitor and an osteogenic protein pathway inhibitor blocker}

More particularly, the present invention relates to a method for producing a pluripotent stem cell comprising a protein kinase C inhibitor, a histone deacetylase inhibitor, and / or an osteogenic protein pathway inhibitor bone morphogenetic protein (BMP) pathway blocker) as a reprogramming-enhancing agent to produce or prepare reprogrammed pluripotent stem cells at high efficiency. In addition, the manufacturing method according to the present invention includes a method for producing reprogramming pluripotent stem cells under conditions of xenopathogen-free and feeder cell-free.

Induced pluripotent stem cells (iPS cells) refer to pluripotency cells obtained by differentiation from differentiated cells (for example, somatic cells) and are capable of differentiating into various organ cells. Since iPS cells can be obtained by reprogramming the cells differentiated by the differentiation inducing factors, it is possible to generate patient immunocompetent universal cell lines without somatic cell transfer. Therefore, iPS cells can be derived from the patient's cells, so immunization rejection can be avoided when applied to clinical applications. In addition, iPS cells do not use oocytes or embryos, so there is no bioethical controversy or religious criticism.

Takahashi, K., and Yamanaka, S., et al., In August 2006, first reported the formation of iPS cells by degeneration using mouse cells (Takahashi, K., and Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. cell 126, 663-676) one since, Takahashi, K., etc., and Yu, J. et target human cells reported the formation of iPS cells by the de-differentiation Induction of pluripotent stem cells from adult cells (Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., and Yamanaka, S. human fibroblasts by defined factors Cell 131 , 861-872 and Yu, J., Vodyanik, MA, Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, JL, Tian, S., Nie, J. , Jonsdottir, GA, Ruotti, V., Stewart, R. , et al. (2007) Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells. Takahashi, K. et al. Have found that induction factors of Sox2, Oct3 / 4, and Klf4 are essential for obtaining iPS cells, and additionally, c-Myc plays a role in promoting the formation of iPS cells. In addition, Yu, J. et al. Found that the induction factor of Sox2, Oct3 / 4, and Nanog is essential for obtaining iPS cells, and the formation of iPS cells is increased in the presence of Lin28.

On the other hand, according to a conventional method of producing iPS cells by reprogramming, for example, according to Takahashi, K. et al (2007) Cell 131 , 861-872, 5 x 10 ⁴ human dermal fibroblasts There is a problem of low efficiency in which only about 10 iPS cells are obtained. In addition, since it requires the use of xenopathogen such as fetal bovine serum and the use of animal-derived feeder cells in the process of inducing the differentiation, There are limits to safety in clinical application.

The present inventors have conducted various studies to develop a method for producing iPS cells with high efficiency. As a result, it has been found that a specific substance, that is, a proteine kinase C inhibitor, a histone deacetylase inhibitor, and / or an osteogenic protein pathway blocker has an activity of promoting the differentiation of somatic cells and can produce iPS cells with high efficiency Respectively. In addition, when the protein kinase C inhibitor, the histone deacetylase inhibitor, and / or the osteogenic protein pathway blocker are used, it is possible to obtain a reverse transcription reaction under xenopathogen-free and feeder cell- We have found that pluripotent stem cells can be produced.

Accordingly, it is an object of the present invention to provide a process for producing iPS cells using a protein kinase C inhibitor, a histone deacetylase inhibitor, and / or an osteogenic protein pathway inhibitor as a reprogramming-enhancing agent.

According to one aspect of the present invention, there is provided a method for producing a recombinant human somatic cell comprising: (a) culturing human-derived somatic cells into which a gene encoding a reprogramming inducing factor has been introduced from a group consisting of a protein kinase C inhibitor, a histone deacetylase inhibitor, In a medium containing at least one selected reprogramming-enhancing agent; And (b) separating embryonic stem cell-like colonies from the culture obtained from step (a).

In a preferred embodiment of the present invention, the de-differentiation-enhancing agent is a mixture of a histone deacetylase inhibitor and an osteogenic protein pathway blocker, and the concentration of the de-differentiation-enhancing agent is in the range of 0.001 to 1000 μM Lt; / RTI >

In one embodiment of the present invention, said culturing of step (a) comprises the steps of: (i) culturing a medium obtained by adding said de-differentiation-enhancing agent to the medium used for introduction of the gene encoding the dedifferentiation inducing factor (" and performing a first incubation for 3-6 days in, (ii) in the presence of a substrate protein (extracellular matrix protein) other cells, and performing a secondary culture for 1.5~3 days in the first medium, and ( iii) performing a tertiary culture for 15 to 30 days in a culture medium obtained by adding the de-differentiation-enhancing agent to a human embryonic stem cell culture medium ("second medium").

The medium used for introducing the gene encoding the dediffering inducer and / or the culture medium for human embryonic stem cell culture may be a non-xenogeneic infectious agent medium, and the culture of (i) to (iii) Can be preferably performed under feeder cell-free conditions.

INDUSTRIAL APPLICABILITY According to the present invention, a method for producing stem cells capable of producing differentiated pluripotent stem cells can be carried out by using a protein kinase C inhibitor, a histone deacetylase inhibitor, and / or an osteogenic protein pathway blocker as a reprogramming- Can be manufactured. In addition, when the de-differentiation enhancer is used, the entire process for producing the dedifferentiated pluripotent stem cells can be performed under xenopathogen-free and feeder cell-free conditions. Thus, the method of the present invention can produce highly reprogrammed pluripotent pluripotent stem cells suitable for clinical use, that is, excellent safety.

Fig. 1 shows the results obtained when a medium treated with a combination of a protein kinase C inhibitor (Go6983, Go), a histone deacetylase inhibitor (tricostatin A, TSA) and an osteogenic protein pathway blocker (dorsomorphin, DM) , And the production efficiency (number of iPS cell colonies obtained per 10,000 cells) of the resulting human de-differentiating pluripotent stem cells.
FIG. 2 shows a medium treated with a combination of a protein kinase C inhibitor (Go6983, Go) and a histone deacetylase inhibitor (trichostatin A, TSA); Or Goetene kinase inhibitor (Go6983, Go) was used as a culture medium, the resultant immunodefined human pluripotent stem cells were immunostained using Tra1-60 antibody, which is an undifferentiated marker.
Figure 3 shows a medium treated with a combination of a protein kinase C inhibitor (Go6983, Go) and a histone deacetylase inhibitor (trichostatin A, TSA); Oct4, SSEA4, Tra1-60, and Sox2 when the medium treated with the protein kinase C inhibitor or the protein kinase C inhibitor (Go6983, Go) was used.

In the present specification, the term " induced pluripotent stem cells (iPS cells) ", also referred to as "reprogrammed pluripotent stem cells ", reprograms (i.e., quot; pluripotency "). The STEMs can be variously differentiated into long-term cells such as the brain and heart.

The present invention relates to a method for producing a recombinant human somatic cell, comprising the steps of: (a) culturing a human-derived somatic cell into which a gene encoding a dediffering inducer is introduced in the presence of at least one selected from the group consisting of a protein kinase C inhibitor, a histone deacetylase inhibitor, Culturing in a medium containing a differentiation-enhancing agent; And (b) separating embryonic stem cell-like colonies from the culture obtained from step (a).

The de-differentiation inducing factor includes all factors having a function to induce reprogramming, and preferably includes a combination of de-differentiation inducing factors known to be involved in reprogramming. For example, the dedifferentiation factor may be selected from the group consisting of Sox2, Oct3 / 4, Nanog, Klf4, Lin28, and Myc, which are known to induce reprogramming. The amino acid sequences and base sequences of Sox2, Oct3 / 4, Nanog, Klf4, Lin28, and c-Myc are known in GenBank and the like.

Derived somatic cells into which the gene encoding the dedifferentiation inducing factor is introduced can be obtained by a known method such as the method of Takahashi, K. et al. (Takahashi, K., et al., (2007) Cell 131 , 861- 872) and / or Yu, J. et al. (Yu, J., et al., (2007) Science, New York, NY). That is, somatic cells isolated from a human body are inoculated into a medium (for example, DMEM medium) containing fetal bovine serum and antibiotic substance (penicillin / streptomycin) Can be transferred to the human-derived somatic cells. The transfer of the gene encoding the dedifferentiation inducer can be carried out by culturing for one day, but is not limited thereto. On the other hand, the gene encoding the dedifferentiation inducer is transferred using a xenopathogen-free medium without xenopathogen such as fetal bovine serum, It is preferable in terms of clinical applicability of pluripotent stem cells. Therefore, it is preferable to use a xenopathogen-free medium such as MesenGro ^™ hMSC Medium (StemRD, USA) for the introduction of the gene encoding the de-differentiation inducer. Can be used.

In the method of the present invention, the protein kinase C inhibitor includes all substances having a protein kinase C inhibitory activity. The protein kinase C inhibitor is selected from the group consisting of 3- [1- [3- (dimethylamino) propyl] -5-methoxy-1H-indol-3-yl] -4- (1H- (1H-indol-3-yl) -1H-pyrrole-2-carboxylic acid 2,5-dione, < / RTI > 3- (lH-indol-3-yl) -4- [2- (4-methylpiperazin- 1 -yl) quinazolin- 3-yl) -4- [2- (4-methylpiperazin-1-yl) quinazolin-4-yl] pyrrole-2,5-dione, Sotrastaurin, AEB 071); 3-yl} -3- (l-methyl-lH-indol-3-yl) maleimide methanesulfonate (3- {1- - [3- (amidinothio) propyl] -1H-indol-3-yl} -3- (1-methyl-1H-indol-3-yl) maleimide methane sulfonate, Ro-31-8220); 13-hydroxyoctadecadienoic acid; 3- (dimethylamino) propyl] indol-3-yl] -3- (indol- (indol-3-yl) maleimide, GF 109203X, Go6850); (E, K) pyrrole (3,4-dimethoxyphenyl) -1,3,4-tetrahydro- H) (1,4,13) oxadiazacyclohexadecene-1,3 (2H) -dione (13 - ((dimethylamino) methyl) -10,11,14,15-tetrahydro- 1,3-dioxo-1,3-dioxo-1,3-diene, LY-333531, Ruboxistaurin, PKC beta inhinitor) ; Diamino-N - ([1- (1-oxotridecyl) -2-piperidinyl] methyl) 2-piperidinyl] methyl) hexanamide, NPC 15437); And 4'-demethylamino-4'-hydroxystaurosporine (RK-286C), but the present invention is not limited thereto.

The histone deacetylase inhibitor may be N-hydroxy-3- (3-phenylsulfamoylphenyl) acrylamide, Belinostat, PXD101, PX105684 ); 7- (4- (3-ethynylphenylamino) -7-methoxyquinazolin-6-yloxy) -N-hydroxyheptanamide -yloxy) -N-hydroxyheptanamide, < / RTI >CUDC-101); [R- (E, E)] - 7- [4- (dimethylamino) phenyl] -N-hydroxy-4,6-dimethyl-7-oxo-2,4-heptadienamide , E)] - 7- [4- (dimethylamino) phenyl] -N-hydroxy-4,6-dimethyl-7-oxo-2,4-heptadienamide, Trichostatin A, TSA); N-hydroxy-N'-phenyloctanediamide (Vorinostat, SAHA, Zolinza, MK-0683); Valproic acid or its salt; Sodium butyrate; Benzo [d] imidazol-5-yl) -N-hydroxyacrylamide (3- (2-butyl-1- (2- (diethylamino) ethyl) -1H-benzo [d] imidazol-5-yl) -N-hydroxyacrylamide, SB939); N-hydroxy-N'-3-pyridinyloctanediamide (Pyroxamide, NSC 696085); 3- (dimethylaminomethyl) -N- [2- [4- (hydroxycarbamoyl) phenoxy] ethyl] -1-benzofuran-2-carboxamide [4- (hydroxycarbamoyl) phenoxy] ethyl] -1-benzofuran-2-carboxamide, PCI-24781, CRA-02478); N - [[4 - [[(2-aminophenyl) amino] carbonyl] phenyl] methyl] carbamic acid 3-pyridinyl methyl ester phenyl] methyl] carbamic acid 3-pyridinylmethyl ester, Entinostat, MS-275, SNDX-275, MS-27-275); (2-aminophenyl) -4 - [[(4-methylpiperazin-1-yl) pyridin-3-ylpyrimidin-2-yl) amino] methyl] benzamide, Mocetinostat, MGCDO103); 1-methyl-1H-pyrrol-2-yl] -N-hydroxy-2-propenamide (3- [5- (3- (3-Fluorophenyl) -3-oxopropen-1-yl) -1-methyl-1H-pyrrol-2-yl] -N-hydroxy-2-propenamide, MC1568; (N-hydroxy-3- [4 (4-fluorophenyl) ethyl] (E) -propenamide, Panobinostat, LBH-589, NVP-LBH589, LBH589); Phenyl] -2 (E) -propenhydrooxamic acid (3 < RTI ID = 0.0 > (E) -propenohydroxamic acid, Dacinostat, LAQ824, NVP-LAQ824) was prepared by the method described in Example 1, ; Methyl) piperazin-1 -yl) pyrimidine-5-carboxamide (prepared from N -hydroxy-2- (4 - (((1-methyl-1H-indol-3-yl) methyl) amino) methyl) piperidin-1-yl) pyrimidine-5-carboxamide, JNJ-26481585); [(6 - [(diethylamino) methyl] naphthalen-2-yl} methyl [4- (hydroxycarbamoyl) phenyl] carbamate [4- (hydroxycarbamoyl) phenyl] carbamate, Givinostat, Gavinostat, ITF2357, ITF 2357); And 4- (4-chloro-2-methylphenoxy) -N-hydroxybutanamide, Droxinostat). But is not limited to.

Also, the bone morphogenetic protein (BMP) pathway blocker may be noggin; Chordin; Follistatin; A) pyrimidine (6- (4- (2-tert-butoxycarbonylamino) phenyl) piperidin-1-ylethoxy) phenyl) -3-pyridin-4-ylpyrazolo (1,5-a) pyrimidine and dorsomorphin.

In the method of the present invention for producing dedifferentiated pluripotent stem cells, the de-differentiation-enhancing agent may be selected from the group consisting of a protein kinase C inhibitor, a histone deacetylase inhibitor, and an osteogenic protein pathway inhibitor, Especially preferably, a combination of a histone deacetylase inhibitor and an osteogenic protein pathway inhibitor can be used to produce a degenerated pluripotent stem cell with high efficiency.

The amount of the de-differentiation-enhancing agent, that is, the concentration in the medium may preferably be in the range of 0.001 to 1000 μM, more preferably 0.01 to 10 μM. Of course, the concentration range may be used if necessary.

The culture in the presence of the de-differentiation-enhancing agent, i.e. the culture of step (a)

(i) de-differentiation inducing the de-differentiation on the medium used for the introduction of a gene encoding a factor-enhancing agents added to the resulting medium (the "first medium") in a step of performing a first incubation for 3-6 days, (ii (Ii) carrying out a secondary culture in the first medium for 1.5 to 3 days in the presence of an extracellular matrix protein, and (iii) culturing the transformant in a medium for human embryonic stem cell culture, Followed by carrying out a third culture for 15 to 30 days in a medium obtained by adding the medium ("second medium").

In said step (i), said primary culture can be carried out for 3 to 6 days, for example for about 4 days. The medium used for the introduction of the gene encoding the dedifferentiation inducing factor is preferably a xenopathogen-free medium, for example, MesenGro ^™ hMSC Medium (StemRD, USA) have.

The step (ii) is carried out in the presence of extracellular protein using the same medium as that used in step (i), and serves to pre-adapt the cells to the subsequent tertiary culture. Examples of the extracellular matrix proteins include coating proteins commonly used in cell culture such as vitronectin, Matrigel (BD Biosciences, USA), CellStart (Invitrogen, USA), gelatin gelatin, etc. may be used without limitation. The secondary culture may be performed for 1.5-3 days, for example about 2 days.

In the step (iii), the tertiary culture may be performed for 15 to 30 days, for example, for about 18 to 20 days. The culture medium for culturing human embryonic stem cells may be a culture medium commonly used for culturing human embryonic stem cells, but it is preferable to use a xenopathogen-free medium. For example, F-12 medium containing a genotype-free serum replacement, glutamax, non-essential amino acid, beta-mercaptoethanol, antibiotic, and basic fibroblast growth factor (bfgf). The basal medium may be a conventional cell culture medium as well as DMEM / F-12 medium such as DMEM (Dulbecco's Modified Eagle's Medium, GIBCO, USA); MEM (Minimal Essential Medium, GIBCO, USA); BME (Basal Medium Eagle, GIBCO, USA); RPMI 1640 (GIBCO, USA); DMEM / F-10 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-10; GIBCO, USA); α-MEM (α-Minimal Essential Medium; GIBCO, USA); G-MEM (Glasgow ' s Minimal Essential Medium, GIBCO, USA); IMDM (Isocove ' s Modified Dulbecco ' s Medium, GIBCO, USA); KnockOut DMEM (GIBCO, USA), and the like.

The cultivation of the above (i) to (iii) can be carried out particularly preferably without using animal-derived support cells, that is, under feeder cell-free conditions.

The production method of the present invention includes a step of separating embryonic stem cell-like colonies from the culture obtained from step (a) (i.e., step (b)).

Since the colonies of the pluripotent pluripotent stem cells have a morphologically distinct form in which the nuclei are relatively large and the cytoplasm is small and the round cells are clustered in the culture, They can be separated by physical methods using Pasteur pipettes.

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.

The two 6-well plates MesenGro ^TM hMSC Medium (StemRD, USA) was added to (-A medium), human somatic cells the fat stromal cells (Adipose-derived stromal cell; ADSC ) (Lonza, USA) to a 1.5x10 ⁵ cells per well (Takahashi, K. et al. (2007) Cell 131 , 861-872), and a gene encoding four kinds of dediffering inducers (Oct4 , Sox2, Klf4, c-Myc) (1 day incubation). As shown in the following Table 1, the depletion-enhancing agent was added to the medium-A to prepare the medium-A1 to the medium-A7, and the medium was changed to medium-A1 to medium-A7 every day for 4 days, Respectively.

Each well was treated with trypsin / EDTA and centrifuged, and the cells obtained from each well were inoculated into a 6 cm culture dish coated with vitronectin (5 / / ml, BD Biosciences, USA) to obtain about 5 × 10 ⁴ cells Then, the cells were incubated for 2 days with the same daily medium (i.e. medium-A1 to medium-A7).

(GIBCO, USA), 1x glutamax (GIBCO, USA), 1% (v / v) nonessential amino acid (GIBCO, USA) Supplemented with 0.1 mM beta-mercaptoethanol (GIBCO), 1% (v / v) penicillin / streptomycin (GIBCO, USA) and 8 ng / ml bfgf (Chiba and Diostech, Korea) Cultured for 20 days while the culture medium of each well was changed daily with medium B1 to medium B7 (see Table 1) prepared by adding a differentiation-enhancing agent to DMEM / F-12 (GIBCO, USA) Respectively.

Treated group badge badge De-differentiation-enhancing agent Group 1 Badge-A1 Badge-B1 No treatment (control group) Group 2 Badge-A2 Badge-B2 Go 5 μM Group 3 Badge-A3 Badge-B3 TSA 10 nM Group 4 Badge-A4 Badge-B4 DM 1 μM Group 5 Badge-A5 Badge-B5 Go 5 μM + TSA 10 nM Group 6 Badge-A6 Badge-B6 Go 5 μM + DM 1 μM Group 7 Badge-A7 Badge-B7 TSA 10 nM + DM 1 μM

- Go: 3- [1- [3- (Dimethylamino) propyl] -5-methoxy-lH-indol-3-yl] -4- (lH- 5-dione) (Go6983)

- TSA: Trichostatin A (Trichostatin A)

- DM: dorsomorphin.

After the induction of differentiation induction factors, iPS cell colonies were observed to form in each well on the 20th day after the incubation, and hESC-like colonies were counted on the 25th day of culture. The results of measuring the number of iPS cell colonies obtained per 10,000 cells are shown in Fig. As can be seen from the results of FIG. 1, the number of iPS cell colonies in the group treated with the protein kinase C inhibitor, the histone deacetylase inhibitor, or the BMP pathway inhibitor increased by at least 2 times as compared with the control group. In particular, the number of iPS cell colonies in the group treated with both the histone deacetylase inhibitor (trichostatin A) and the BMP pathway inhibitor (dorsomorphin) was remarkably increased to 7 times or more as compared with the control group.

Test Example 1. Identification of iPS cells

In Example 1, iPS cell colonies in the culture medium of the fifth group (Go6983 + TSA) and the second group (Go6983) were stained using a Tra1-60 antibody (Millipore, USA) The expression of alkaline phosphatase, Oct4, SSEA4, Tra1-60, and Sox2, which are undifferentiated markers of iPS cell colonies, was measured.

That is, on the 25th day of culture, the colonies were washed with phosphate-buffered saline and fixed with 4% formaldehyde solution for 10 minutes. The cells were washed three times with phosphate-buffered saline for 10 minutes, and then blocked with 10% normal donkey serum at room temperature for 1 hour. The primary antibody, mouse Tra1-60 antibody (1: 500, Millipore, USA) was treated at room temperature for 1 hour, washed three times with phosphate-buffered saline for 10 minutes, and then incubated with a secondary antibody, biotinylated target anti-mouse IgG 1: 100, Vector Laboratory, USA) and incubated at room temperature for 30 minutes. After washing three times for 10 minutes with phosphate buffered saline, streptavidin-horseradish peroxydase conjugate (Vector Laboratory, USA) was treated at room temperature for 30 minutes, washed again with phosphate-buffered saline for 10 minutes three times, and then stained with DAB Peroxidase Substrate Kit Laboratory, USA).

In the same manner, the colonies were washed with phosphate-buffered saline, fixed with 4% formaldehyde solution for 10 minutes, and then subjected to alkaline phosphatase staining as follows. iPS cell colonies were washed three times for 10 minutes each and then washed with 100X NBT solution (50 mg / ml nitro blue tetrazolium (Sigma, USA) in a mixed solvent of 70% dimethylformamide and 30% water) and 100x XPHOS solution (100 mM Tris pH 8.5, 100 mM NaCl, 50 mM MgCl ₂ ) was added to 10 mg / ml of 5-bromo-4-chloro-3-indolyl phosphate, disodium salt And iPS cell colonies were incubated for 30 minutes or more, followed by washing with H ₂ O and observed.

In order to measure the expression of Oct4, SSEA4, Tra1-60, and Sox2, immunostaining was performed as follows. First, iPS cell colonies were washed with phosphate-buffered saline and fixed with 4% formaldehyde solution for 10 minutes. Mouse anti-Oct4 antibody (Santa Cruz, USA), mouse anti-SSEA4 antibody (Millipore, USA), mouse anti-Tra1-60 antibody (Millipore, USA) and mouse anti-Sox2 antibody (1: 200, Invitrogen, USA) for 1 hour at room temperature. The reaction mixture was washed three times with phosphate-buffered saline for 10 minutes at room temperature. The cells were washed three times with phosphate-buffered saline for 10 minutes, and stained with 30 nM 4 ', 6-diamidino-2-phenylindole (DAPI) (Invitrogen, USA). After washing three times with phosphate-buffered saline for 10 minutes, the cells were observed under a fluorescence microscope

The results obtained are shown in FIG. 2. The results are shown in FIG. 2. The results are shown in FIG. 2. The undifferentiated markers of the produced iPS cell colonies, alkaline phosphatase, Oct4, SSEA4, Tra1-60, and The results of measurement of Sox2 expression are shown in FIG. From the results of FIGS. 2 and 3, iPS cells produced according to the present invention showed positive responses to Tra1-60 antibody, and the undifferentiated markers of iPS cells, alkaline phosphatase, Oct4, SSEA4, Tra1-60 , And Sox2 were expressed well.

Claims (1)

(a) culturing a human-derived somatic cell into which a gene encoding a dedifferential inducer has been introduced, as a de-differentiation-enhancing agent, in a medium comprising a mixture of a histone deacetylase inhibitor and an osteogenic protein pathway blocker; And
(b) isolating embryonic stem cell-like colonies from the culture obtained from step (a), said method comprising the steps of:
Wherein the histone deacetylase inhibitor is [R- (E, E)] -7- [4- (dimethylamino) phenyl] -N-hydroxy-4,6-dimethyl- Amide and the osteogenic protein pathway inhibitor is selected from the group consisting of 6- (4- (2-piperidin-1-ylethoxy) phenyl) Lt;
Wherein the concentration of the de-differentiation-enhancing agent is in the range of 0.01 to 10 [mu] M,
Wherein said human-derived somatic cell is a lipid stromal cell,
Wherein the gene encoding the dedifferentiation inducing factor is Oct4, Sox2, Klf4 and c-Myc,
Wherein the culture of step (a) is (i) cultured in a medium ("first medium") obtained by adding the de-differentiation-enhancing agent to a medium used for introducing a gene encoding a reprogramming inducing factor for 3 to 6 days, (Ii) performing a secondary culture in the first medium for 1.5 to 3 days in the presence of an extracellular matrix protein, and (iii) culturing a human embryonic stem cell culture Performing a third culture for 15 to 30 days in a medium ("second medium") obtained by adding the de-differentiation-enhancing agent to the medium,
The culture medium for culturing human embryonic stem cells according to (iii) above, wherein the medium for culturing human embryonic stem cells is selected from the group consisting of knockout genotype free serum substitute, glutamax, nonessential amino acid, beta-mercaptoethanol, antibiotic, and basic fibroblast growth factor (bfgf) It is a xenopathogen-free medium,
Wherein the concentration of the non-essential amino acid is 1% (v / v), the concentration of the beta-mercaptoethanol is 0.1 mM, the bfgf (v / v) (iPS cells) having a concentration of basic fibroblast growth factor of 8 ng / ml.

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