KR101490017B1 - Medium Composition for Establishing Novel Epiblast Stem Cell and Method for Establishing Novel Epiblast Stem Cell Using Thereof - Google Patents

Abstract

The present invention relates to a medium composition for the establishment of a somatic differentiation-specific somatic embryonic stem cell comprising chimera-forming ability and FGF4 (Fibroblast growth factor 4) as an active ingredient in a basic medium, a chimeric formative somatic differentiation-specific somatic embryonic stem cell And a chimera-forming ability-specific somatic differentiation-specific luminal stem cell established by the method.

Description

[0001] The present invention relates to a medium composition for establishing a novel cervical stem cell, and a method for establishing a cervical stem cell using the same. ≪ Desc / Clms Page number 1 >

The present invention relates to a novel composition for culturing stem cells of chondrocyte having chimeric ability and specificity for somatic differentiation, and a method for preparing cervical stem cells using the same.

Stem cells have the ability to infinitely divide unlike normal somatic cells and have the ability to differentiate into specific cells under suitable environmental conditions. The differentiation capacity is defined as pluripotency, multipotency, or unipotency depending on the characteristics of stem cells.

Among them, pluripotent stem cells are able to differentiate into all kinds of cells constituting an individual and infinitely proliferate in vitro. To date, known pluripotent stem cells include embryonic stem cells, embryonic germ cells, embryonic carcinoma cells, and epiblast stem cells from the developing mesenchymal stem cells , EpiSC).

Embryonic stem cells, embryonic germ cells, embryonic tumor cells can easily form teratomas and chimeras. However, it has been reported that embryonic stem cells (EpiSCs) rarely form chimeras, and that they do not differentiate into germ cells, resulting in poorer differentiation than embryonic stem cells (Brons, IG, Smithers, LE, Trotter, MW, Rugg Nature 448, < / RTI > < RTI ID = 0.0 > Davis, TJ, Evans, EP, Mack, DL, Gardner, RL, and McKay, RD (2007) Nature 448, 196-199). Therefore, embryonic cells such as embryonic stem cells are said to have naive pluripotency and EpiSC have primed pluripotency.

However, it is possible to change EpiSC to fully pluripotent state by in vitro culture condition and genetic manipulation. Especially, in vitro culture conditions affecting EpiSC differentiation ability are continuously being studied.

The present invention provides a medium composition for establishing somatic differentiation-specific somatic embryonic stem cell (EpiSC) having chimeric ability different from that of a cervical epithelial stem cell having little existing chimeric ability, and a method for establishing a cervical stem cell using the same.

In order to solve the above problems, the present invention provides a medium composition for establishing a somatic differentiation-specific somatic embryonic stem cell comprising chimera-forming ability comprising FGF4 (Fibroblast growth factor 4) as an active ingredient in a basic medium.

The present invention also relates to a method for the production of chimeric formative somatic differentiation-specific luminal stem cell, comprising culturing epidermal stem cells in a medium containing FGF4 (Fibroblast growth factor 4) as an active ingredient in a basic medium And a chimera-forming ability-specific somatic differentiation-specific lumenal stem cell established by the method.

According to the present invention, it is possible to establish a somatic differentiation-specific lumenal stem cell having chimeric ability to regulate culture conditions. Thus, it can be useful for studying chimerism, somatic and germ cell differentiation.

1 is a photograph of an experimental result of Example 1 in which EpiSC (luminal epithelial stem cells) were prepared from embryonic developmental 5.5-6.5 (E 5.5-6.5) day sac capsules. A is a photograph of E6.5 embryos obtained from GOF18 transgenic mice. GFP-positive cyst epithelial cells were used for the production of EpiSCs. B is a photograph of the GFP-positive part extended from the sacral cortex. C is a GFP-negative state for all cells up to T cell culture, and only a small amount of cells and colonies showed GFP-positive. D is a photograph showing that GFP-negative cells are established.
Figure 2 is a photograph of the result of Example 2 examining the primed pluripotency of F4-EpiSC. A is a photograph showing that GFP-positive F4-EpiSC was stained positive for Oct4 and weakly stained for anti-NANOG antibody. B is a photograph of an experimental result showing that F4-EpiSC is a universal state showing weak alkaline phosphatase activity. C is a graph of RT-PCR analysis using ESC-specific and EpiSC-specific markers for F4-EpiSC. D Stella And the methylation pattern of the promoter region of Dppa5 . E is a graph of the results for Oct4 promoting activity in ESC and F4-EpiSC. As a control group, the activity of an empty vector was used.
Figure 3 shows the experimental results of Example 3 examining the distinguishing characteristics of F4-EpiSC from EpiSC. A is a heat map showing the entire gene expression profile of ESC (male, female), E3, T9, EpiSC and F4-EpiSC. B is a heatmap for universal and EpiSC-associated genes. C is a heat map for germ cells and meiosis markers.
4 is a photograph of the result of the experiment of Example 4 examining the chimeric ability of F4-EpiSC. A is a photograph of the hematoxylin and eosin staining of the teratoma derived from F4-EpiSC. B is a photograph of an experiment showing RFP and GFP expression of F4-EpiSC infected with lentivirus. C is a photograph showing E6.5 chimeric embryonic fibroblast cells expressing both RFP and GFP. D shows that E13.5 chimeric embryos differentiated into skin tissue (left) and testis (right) are RFP-positive. However, Oct4-GFP was not detected in gonadal tissues at all. In addition to RFP (red fluorescence), GFP (green fluorescence) must be seen to indicate that it is differentiated into germ cells. Since no green fluorescence is seen, it can be seen that it does not differentiate into germ cells.

The present invention provides a medium composition for establishing a somatic differentiation-specific somatic embryonic stem cell having chimeric ability to form, which comprises FGF4 (Fibroblast growth factor 4) as an active ingredient in a basic medium.

The inventors of the present invention have been studying the differentiation ability according to the culturing conditions of the cervical stem cell, and it has been found that when cultured in the FGF4 condition in which the chimeric ability and the differentiation of the germinal stem cells are not differentiated into germ cells, . However, it has been confirmed that the chimeric formation rate is maintained at a high level even though it does not change to a fully pluripotent state, and it is possible to use the established novel stem cell line for chimera formation, somatic and germ cell differentiation studies The present invention has been completed.

Conventionally, the medium supplemented with Activin and FGF2 was used for culturing stem cells of the endoplasmic reticulum but in the present invention, a medium containing FGF4 (Fibroblast growth factor 4) was used. In one embodiment of the present invention, the FGF4 may be contained in the base medium at a concentration of 10 to 100 ng / ml, 15 to 50 ng / ml or 20 to 30 ng / ml, but is not limited thereto.

The basal medium may be a conventional cell culture medium, for example, 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); DMEM / F-12 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12; 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.

More specifically, the basal medium may be, but not limited to, DMEM medium containing glutamine, non-essential amino acid, and folate serum that do not contain any growth factors besides FGF4.

The present invention also relates to a method for the production of chimeric formative somatic differentiation-specific luminal stem cell, comprising culturing epidermal stem cells in a medium containing FGF4 (Fibroblast growth factor 4) as an active ingredient in a basal medium ≪ / RTI >

The endoplasmic reticulum stem cells used in the cultivation of the present invention can be obtained from GOF18 (genomic Oct4 fragment, 18 kb) mouse containing the GFP transgene under the control of the entire regulatory region of the Oct4 gene, By selecting and culturing only benign cells, somatic differentiation specific chimeric stem cell with chimeric ability can be established. The culture is carried out in a medium containing FGF4 (Fibroblast growth factor 4) as an active ingredient in a basic medium, wherein FGF4 is administered at a concentration of 10 to 100 ng / ml, 15 to 50 ng / ml or 20 to 30 ng / ml in the basic medium But is not limited thereto.

The basal medium may be a conventional cell culture medium, for example, 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); DMEM / F-12 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12; 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.

More specifically, the basal medium may be, but not limited to, DMEM medium containing glutamine, non-essential amino acid, and folate serum that do not contain any growth factors besides FGF4.

Through the cultivation of the parasitic outer stem cells under the above conditions, somatic differentiation-specific somatic embryonic stem cells with chimera-forming ability are established, and the present invention provides a somatic differentiation-specific somatic embryonic stem cell having the ability to form chimeras established by the above method .

As can be seen from the following examples, the F4-EpiSC of the present invention cultured and established in the presence of FGF4 was confirmed to be a novel cell type that maintained the quasi-universality constantly and distinguished from the conventional EpiSC. In particular, referring to Example 4, it was confirmed that when injected into a mouse blastocyst, it showed chimeric ability to form, and showed no specificity to germ cells and showed specificity for somatic cell differentiation.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Experimental Example >

The following experimental examples are intended to provide experimental examples that are commonly applied to the respective embodiments according to the present invention.

1. Mouse and cell culture

All mouse species were either raised and stored in the Max Planck Institute (MPI, Germany) or purchased from Harlan or Jackson laboratories (CA, USA). Animal experiments were conducted in accordance with MPI's Animal Protection Guidelines and German Animal Protection Act. Establishment of GOF18 EpiSC was performed as described in Greber et al., 2010. More specifically, E6.5 embryos (129 / Sv female X C57 / BL6 and DBA / 2 background GOF18 + / + male) were obtained and transferred to mouse embryonic fibroblast (MEF) medium. For incision, the decidua was removed with a needle and an extraembryonic ectoderm was removed from the epiblast using a manual glass pipette. After washing with PBS, extracellular bovine blood was cultured in an FGF-based medium on inactivated MEF. The medium used was DMEM (GIBCO BRL Grand Island, NY, USA) medium containing 20% FBS, 2 mM glutamine, nonessential amino acid and 25 ng / ml FGF4.

The EpiSC (cortical stem cell) medium was DMEM / F12 (GIBCO BRL) containing 20% knockout serum replacement (GIBCO BRL), 2 mM glutamine, nonessential amino acids, Activin A and 5 ng / ml bFGF. After initial incubation for 3 to 5 days on MEF, colonies of cystic stem cell colonies were isolated and gelatin was transferred to a culture dish pre-coated for 30 minutes. For conditional media, irradiated MEFs were dispensed at a density of 5 x 10 ⁴ cells / cm ² and cultured in MEF medium for 96 h. 0.45 [mu] l of the conditioned medium was filtered, and FGF4 (25 ng / ml) or bFGF (5 ng / ml) was added. Cell populations were subdivided into gelatin-coated culture dishes, and the medium was replaced every 48 hours.

2. Blastocyst  Injection( Blastocyst Injection )

Blastocyst was obtained from B6C3F1 mice. F4-EpiSC was recovered by treatment with 0.25% trypsin EDTA and was placed in PBS drop with mineral oil. The blastocysts were placed in a PBS drop containing adjoining 10% fetal bovine serum (v / v). GFP and RFP positive cells (n = 10-15) were loaded into the injection pipette and injected into the B6C3F1 blastocyst through a Piezo Micromanipulator (Prime-tech Ltd, Tsuchiura, Ibaragi, Japan). Each 8-15 implanted blastocyst was implanted into the uterus of a pseudopregnant ICR mouse.

3. DNA  Methylation analysis

DNA was isolated from ESC and EpiSC. Sequentially, the DNA samples were bisulfite treated using the EpiTect Bisulfite Kit (QIAGEN) according to the manufacturer's instructions. Stella And Dppa5 PCR amplification of the promoter region was performed using the following primer set and annealing temperature.

Stella 1 ^st forward 5'-TTTTTTTATTTTGTGATTAGGGTTG-3 ';

Stella 1 ^st Reverse 5'-CTTCACCTAAACTACACCTTTAAAC-3 '(161 bp, 45 ° C);

Stella 2 ^nd forward 5'-TTTGTTTTAGTTTTTTTGGAATTGG-3 ';

Stella 2 ^nd reverse 5'-CTTCACCTAAACTACACCTTTAAAC-3 '(116 bp, 55 ° C);

Dppa5 1 ^st forward 5'-GGTTTGTTTTAGTTTTTTTAGGGGTATA-3 ';

Dppa5 1 ^st reverse 5'-CCACAACTCCAAATTCAAAAAAT-3 '(140 bp, 45 ° C);

Dppa5 2 ^nd Forward 5'-TTTAGTTTTTTTAGGGGTATAGTTTG-3 ';

Dppa5 2 ^nd reverse 5'-CTTCACCTAAACTACACCTTTAAAC-3 '(132 bp, 55 ° C).

The PCR product was then subcloned into the pGEM-T Easy vector (Promega, Madison, Wis.) And sequenced. The BiQ Analyzer software (Max Planck Society, Germany) was used for visualization and quantification of bisulfite sequence data.

4. Flow cell  analysis

For FACS classification, the hybrid cells were separated with 0.25% trypsin EDTA, neutralized with DMEM medium containing 10% FCS, washed with PBS and then filtered to remove large cell aggregates with 40 mm nylon mesh. Cells were resuspended in a suitable culture medium and 5 × 10 ⁶ cells / ml specimens were transferred to polystyrene tubes and analyzed using a FACSAria ™ cell sorter (BD Biosciences). High-intensity GFP-positive cells were isolated by fluorescence-activated cell sorting (FACS) using FACSDiVa ™ software (Becton Dickinson, San Jose, Calif.).

5. Immunocytochemistry ( Immunocytochemistry )

For immunocytochemistry, F4-EpiSC was fixed in 4% paraformaldehyde for 20 minutes at room temperature. After washing with PBS, cells were treated with PBS containing 10% normal goat serum and 0.03% Triton X-100 for 46 minutes at room temperature. The cells were then incubated overnight at 4 ° C with primary antibodies (anti-Nanog (Cosmo Bio, REC-RCAB0002PF, 1: 500) and anti-Oct4 (Abcam, ab19857, 1: 500) Were visualized after incubation for 3 hours according to the manufacturer's instructions with a suitable fluorescent-labeled secondary antibody (Alexa Fluor 488 or 568; Molecular Probes, Eugene, OR, USA). Specimens were analyzed with an Olympus Fluorescence microscope and images were acquired using a Zeiss Axiocam camera.

6. Luciferase  analysis( Luciferase Assay )

In order to quantify the relative Oct4 promoting activity of F4-EpiSC, the Oct4 upstream sequence ~ 2 kb (Oct4-2kb), including the distal enhancer (DE) and the proximal enhancer (PE), and the ΔDE, (Promega, USA). The Oct4 upstream sequence (~ 2 kb) containing the distal enhancer (DE) and the proximal enhancer (PE) was derived from the pOct4-GFP plasmid, which was cut and conjugated to the Kpn I / Bgl II cleavage site of the pGL3 basic vector.

The pGL3-Oct4 / ΔDE or pGL3-Oct4 / ΔPE reporter constructs were prepared in two steps. First, DE 5 'or PE 5' fragment pOct4-GFP were PCR- amplified using specific primers from the plasmid, it is digested with Kpn I and Mlu I restriction enzymes and cloned into the pGL3 basic vector pGL3 respectively DE-5 ' Or pGL3-PE 5 'plasmid. The DE 5 'or PE 5' fragments were then PCR-amplified using specific primer pairs from Oct4-GFP plasmids with Mlu I and Bgl II restriction sites, respectively. The amplified fragment was cleaved and pGL3-DE5 &apos; or pGL3-PE5 &apos; Was ligated to the Mlu I / Bgl II site of the plasmid .

Luciferase assays were performed on MEF, ESC and F4-EpiSC using the Dual-Luciferase Reporter Assay System (Promega, USA). For analysis of Oct4 reporter promoting activity, pGL3-Oct4-2 kb vector, pGL3-Oct4-DΔDE, or pGL3-Oct4 / ΔPE vector (firefly luciferase activity) and pRL-TK vector (Renilla luciferase activity was transfected into MEF, ESC and F4-EpiSC. After 48 hours of transfection, the growth medium was removed and the cells were washed with 1X PBS. Cells were then lysed with 1X passive lysis buffer (PLB) and incubated for 10 minutes at room temperature with shaking. The cell lysate was transferred into a 1.5 ml fresh tube and centrifuged at 10,000 rpm for 5 minutes at 4 ° C. 10 [mu] l of the supernatant was transferred to a 96-well plate, and luciferase expression was analyzed by luminometry. Each experiment was performed in triplicate, and the measurements were recorded in relative light units (RLU).

7. Microarray  analysis( Microarray Analysis )

RNA samples to be analyzed via microarray were prepared using Qiagen RNeasy column with on-column DNA digestion. 300 ng total RNA per sample was used as the input of the linear amplification protocol (Ambion, Austin, TX, http://www.ambion.com ), which included T7-linked double strand cDNA and 12 hours in vitro Warrior, Biotin-labeled nucleotide injection. The purified and labeled RNAs were ligated according to the manufacturer's instructions using MouseRef-8 v2 gene expression BeadChips (Illumina, Inc., San Diego, http://www.illumina.com ) for 18 hours. After washing, the chip was stained with streptavidin-Cy3 (GE Healthcare, Chalfont St. Giles, UK, http://www.gehealthcare.com ), and the iScan reader (Illumina, Inc., San Diego, Lt; / RTI &gt;

The intensity of each bead was mapped to the genetic information using BeadStudio 3.2 (Illumina). Background correction was performed with the background correction model of the Affymetrix Robust Multi-array Analysis (RMA), the mutation control was performed using log ₂ scaling, and the gene expression normalization was calculated as the quantile method implemented in the R-Bioconductor lumi package .

< Example  1> Embryogenesis  5.5-6.5 (E 5.5-6.5) days From the sacral envelope EpiSC  ( Embryonic stem cell )

E Embryos at 5.5-6.5 were GOF18 ( Oct4 - GFP transgenic system) transgenic mice. GFP-positive ectodermal blood was obtained by isolating the embryonic / cervical envelope from the embryonic outer ectoderm (see Figure 1A). Isolated GFP-positive ectodermal blood was cultured in FGF4-containing medium (F4 medium). The GFP-positive portion expanded from the sacral cortex was cut out and dispensed into a gelatin-coated culture dish containing F4 medium (see Fig. 1B). GFP-positive cortical stem cells were established by continuous selection and culture. By 3 days of culture, all cells were GFP-negative and only a small number of cells and colonies were GFP-positive. However, it was confirmed that pure clusters of GFP-expressing cervical stem cells were formed by repeating FACS purification three times for each group (see Fig. 1C). These F4-lined embryonic stem cells (F4-EpiSC) were found to maintain homogeneous GFP-positive colonies after prolonged (more than one year) proliferation. When F4-EpiSCs were transferred to a medium for general cultured stem cell culture containing Activin and bFGF, GFP-negative cells were regenerated and lost uniformity (see FIG. 1D). These results suggest that F4 medium is essential for maintaining the uniformity of Oct4-GFP-positive clustering EpiSCs.

< Example  2> F4- EpiSC  Quasi-universal ( primed pluripotency ) Review

GFP-positive F4-EpiSC stained positively for Oct4 and lightly stained for anti-NANOG antibody (see FIG. 2A). Compared to ESC, F4-EpiSC was found to be very weakly stained by alkaline phosphatase (see FIG. 2B). Real-time RT-PCR analysis was performed using mouse ESC-specific marker Nanog And Rex1 expression levels are significantly lower (about 900 and 100-fold, respectively) in EpiSC when compared to ESC (see FIG. 2C). These results are consistent with previous reports that Oct4 expression remains the same in primed pluripotent stem cells, but the level of expression of Nanog is significantly reduced in EpiSCs. In particular, the F4-EpiSC of the present invention was found to express higher levels of Cer1 , Eomes , Fgf5 , Sox17 and T caspase epiblast markers (see FIG. 2C).

The DNA methylation status of the germ cells and the Stella And Dppa5 . As a result, Stella And Dppa5 were not all methylated in ESC, but EpiSC was hypermethylated (see FIG. 2D). As expected, Stella And the promoter region of Dppa5 were not methylated in mouse ESC and were hypermethylated in F4-EpiSC. Luciferase assay also confirmed that F4-EpiSC uses Oct4 luminal envelope-specific PE (see FIG. 2E). Taken together, these results indicate that F4-EpiSC is in a quasi-pluripotential state and that this quasi-pluripotent state is maintained in the absence of external Activin / Nodal pathway stimulation in the presence of FGF4.

< Example  3> F4- EpiSC With EpiSC  Review distinctive features

We examined whether F4-EpiSC showed similar characteristics to other EpiSC cell lines such as T9 and E3. As a result of the examination, it was confirmed that F4-EpiSC differs from EpiSC (see FIG. 3A), and EpiSC cell markers were expressed more extensively than T9 and E3 cell lines (see FIG. 3B). In addition, F4-EpiSC cells did not express germ cells and meiosis markers except for lfitm3 (see Fig. 3C). Therefore, F4-EpiSC is very similar in character to Epiblast, and in It is expected to act as an Epiblast in vivo , with the exception of germ cell precursors. Taken together, these results indicate that F4-EpiSC is a novel cell type differentiated from EpiSC.

< Example  4> F4- EpiSC chimera Formability  Review

F4-EpiSC was similar to that of ciliated epidermal cells that differentiated into mesenchymal cells in the process of vesicle formation, and F4-EpiSC was found to have better differentiation potential than other EpiSC cell lines lacking chimeric ability. The inventors of the present invention have found that in In vivo , we examined whether F4-EpiSCs could differentiate into various lineages of cells. First, F4-EpiSC was implanted to examine the ability of the dorsal subdermal subcutaneously in the immunosuppressed (SCID) mouse to examine the ability of the teratoma to form, and the results are shown in FIG. Referring to FIG. 4A, three months after transplantation, it was found that a teratoma including various tissues such as internal organs, muscle and nerve tissue was formed.

In addition, the cells were injected into a mouse blastocyst to examine the chimeric ability of F4-EpiSC. Previously, EpiSCs have been reported to be quasi-universal but not chimeric (Tesar et al., 2007; Bao et al., 2009), and chimeric ability is a typical characteristic of full universality (Nichols and Smith, 2009). On the other hand, the F4-EpiSC of the present invention maintained Oct4-GFP during the continuous subculture period and showed more similar characteristics to the epidermis, and it was expected to show a better ability to perform compared to EpiSC. To confirm the presence of F4-EpiSCs in chimeric embryos, F4-EpiSCs were infected with lentivirus expressing red fluorescence protein (RFP). Oct4 - GFP And CMV - infected with RFP F4-EpiSC expressed GFP in the presence of active Oct4 and showed sporadic expression of RFP (see FIG. 4B).

Initially, the inventors of the present invention confirmed chimerism in the early developmental E6.5 embryo. Chimera E6.5 embryos showed a high F4-EpiSC distribution (positive for both GFP and RFP) in the cortical shell, and low distribution in the ectopic tissue (only RFP positive, see FIG. 4C). Chimeric formation was also observed in E13.5 embryos (see Figure 4D). In particular, F4-EpiSCs were distributed in somatic embryos containing testis tissue, but not in RPP-positive cells, but not in germline cells, since Oct4-GFP was not activated. The efficiency of somatic cell chimera formation, which can be judged as RFP - positive cells in the embryo, was about 21.4%. Therefore, it was found that F4-EpiSC showed better differentiation ability compared to the previously reported EpiSC without chimeric ability.

These results indicate that F4-EpiSC is differentiated into trichomes other than germ cells, and ES cells lacking Nanog are able to differentiate into trichomes in regeneration and chimera, , It is believed that this characteristic of F4-EpiSC is due to the low level of Nanog expression of F4-EpiSC.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that such detail is solved by the person skilled in the art without departing from the scope of the invention. will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (7)

delete delete delete delete Culturing the epiblast stem cells in a culture medium containing FGF4 (Fibroblast growth factor 4) as an active ingredient in a basic medium which is a DMEM medium containing glutamine, nonessential amino acids and fetal calf serum, And the ability to differentiate into somatic cells without the ability to differentiate into germ cells. 6. The method of claim 5,
Wherein the FGF4 is contained at a concentration of 10 to 100 ng / ml in the basic medium. The method of claim 1, wherein the FGF4 is present in a concentration of 10 to 100 ng / ml in the basic medium. 6. The stem cell according to claim 5 or 6, wherein the stem cell has chimeric ability through the blastocyst injection method and is differentiated into somatic cells without the ability to differentiate into germ cells.

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