WO2005068615A1

WO2005068615A1 - Method for inhibiting differentiation of pluripotent stem cell

Info

Publication number: WO2005068615A1
Application number: PCT/KR2004/003119
Authority: WO
Inventors: Hyun-Sook Park; Mi-Ra Kim; Do-Yeon Kim
Original assignee: Korea Atomic Energy Research Institute
Priority date: 2004-01-20
Filing date: 2004-11-30
Publication date: 2005-07-28
Also published as: KR20050076461A; KR100632851B1

Abstract

Provided is a method of culturing a pluripotent stem cell while inhibiting differentiation of the pluripotent stem cell by suppression of Wnt/beta-catenin signaling.

Description

METHOD FOR INHIBITING DIFFERENTIATION OF PLURIPOTENT STEM CELL

TECHNICAL FIELD The present invention relates to a method of inhibiting the differentiation of cells in which differentiation is induced by Wnt beta-catenin signaling. More particularly, the present invention relates to a method, of inhibiting cell differentiation through suppression of the Wnt/beta-catenin signaling.

BACKGROUND ART Mouse embryonic stem cells were first cultured in vitro in 1981. In 1988, gene knockout mice were first created by combination of the in vitro culture technology of mouse embryonic stem cells with a gene knockout technology. Thereafter, the gene knockout technology has played a leading role in development of biotechnology/medical science as an important technology indispensable for study of gene functions and establishment of human disease models [Smith AG, Annu Rev Cell Dev Biol 17:435-62, 2001]. In 1998 which is 17 years later from development of the in vitro culture technology of mouse embryonic stem cells, human embryonic stem cell lines were first established by Dr. Thomson at the University of Wisconsin [Thomson JA, Science 282(5391 ): 1145-7, 1998]. With respect to human embryonic stem cells, in vitro culture is difficult and manipulation is inconvenient, relative to mouse embryonic stem cells, which causes many technical difficulties in application as cellular therapeutic agents by gene manipulation or in vitro manipulation of large-scale cultured cells. To use currently available pluripotent stem cells as cellular therapeutic agents, establishment of new technology able to culture in large-scale and to induce differentiation into desired cells is much needed. Large-scale culture of pluripotent stem cells requires complete inhibition of differentiation so that loss of pluripotency by differentiation does not occur. Oct-3/4 and Nanog have been known as transcription factors playing the most important role in maintenance of the pluripotency of embryonic stem cells. With respect to the Nanog, the gene was not cloned until May, 2003 [Chambers I et al, Cell 113(5):643-55, 2003, Mitsui K et al, Cell 113(5): 631-42, 2003]. The pluripotency maintenance mechanism of the Oct-3/4 and the Nanog is in an early stage of the study and thus further study is still needed. LIF (Leukemia Inhibitory Factor) and BMP (Bone Morphogenic Factor) have been known as factors that can be externally supplied in pluripotent stem cell culture condition for maintenance of pluripotency. However, these factors are applicable to mice. With respect to human embryonic stem cells, there are no effects (LIF) or no reports (BMP) [Ying QL et al, Cell 115: 281-92, 2003]. In January, 2004, there was a report that artificial induction of Wnt signaling by a drug called BIO(6-bromoindirubins) maintains the expression of Oct-3/4 [Sato N et al, Nature Medicine 10(1): 55-63]. However, there is no mention to a BIO treatment duration in this experimental procedure. Judging from the experimental conditions, the report result is resulted from BIO treatment of 2-5 days. However, with respect to an embryoid body formation method which is a method for inducing natural differentiation of pluripotent stem cells, it has been reported that the expression of Oct-3/4 is maintained even at five days after the formation of embryoid bodies. In this respect, assertion of pluripotency maintenance based on no loss of Oct-3/4 after the BIO treatment for 2-5 days is unreasonable. To conclude that the Wnt signaling by BIO treatment can inhibit cell differentiation, the maintenance of cell pluripotency by BIO treatment over several generations must be demonstrated. In this report, lithium, another drug which induces Wnt signaling, provided a contrary result to BIO. In this respect, there is a need to demonstrate whether the effect of BIO is based on Wnt signaling or another action. In addition, this report asserts the maintenance of cell pluripotency by the BIO treatment from the fact that embryonic cells after the BIO treatment for 2-5 days are differentiated into all cells, including ectoblast, mesoblast, and endoblast in mice embryonic stem cells only, not in human embryonic stem cells. However, it is unreasonable to conclude that the BIO treatment maintains cell pluripotency based on the experimental result after the BIO treatment for a short time and with mouse embryonic stem cells only. Wnt, which is a starting material inducing Wnt signaling, is a secreted glycoprotein and 19 types of the Wnt protein are known. The Wnt proteins begin signaling by binding with Frizzled receptors of cell surfaces. The Frizzled receptors are composed of about 700 amino acids. CRDs (cysteine-rich domains) of the Frizzled receptors binding with the Wnt proteins are located outside cells and 7 transmembrane (TM) domains are piercingly located in cell membranes. Frizzled proteins that are secreted extracellularly were found. These Frizzled proteins contain CRDs binding with the Wnt proteins but not the TM domains and are called sFRPs (secreted Frizzled-related proteins). The sFRPs are responsible for suppressing the Wnt signaling by binding with the Wnt proteins. In addition to the Frizzled receptors and the sFRPs, as other proteins with CRDs, there exist collagen 18(XVIII) [Rhen M and Pihlajaniemi T, J Biol Chem 270(9): 4705-11 , 1995], endostatin [Hanai J et al, J Cell Biol 156(3): 529-39, 2002], carboxypeptidase Z [Song L and Fricker LD, J Biol Chem 272(16): 10543-50, 1997], receptor tyrosine kinase [Xu YK, Nusse R, Curr Biol 8(12): R405-6, 1998, Masiakowski, P and Yancopoulos GD, 8(12): R407, 1998], and transmembrane enzyme Corin [Yan W et al, J Biol Chem 274(21 ): 14926-35, 1999]. These proteins bind with the Wnt proteins. At this time, it is known that the Wnt/beta-catenin signaling is suppressed. In addition, it is reported that WIF-1(Hsieh JC et al, Nature 398(6726): 431-6, 1999), Cerberus(Picolo et al, Nature 397:707-10), Dickkopf-1 (Tian et al, N Engl J Med: 349(26): 2483-94, 2003), etc. are extracellular inhibitors of the Wnt signaling. They bind with the Wnt proteins outside cells and inhibit binding of the Wnt proteins with the Frizzled receptors of cell membranes, thereby suppressing the Wnt signaling. When the Wnt proteins bind with the Frizzled receptors, intracellular enzymes called GSK-3 (glycogen synthase kinase-3) are inactivated (phosphorylated). As a result, the inactivated GSK-3 enzymes cannot decompose beta-catenin any more, and thus, accumulation of beta-catenin in cytoplasms occurs. The accumulated beta-catenin is translocated into cell nuclei and induces transcription of various genes together with a Lef/TCF transcription factor. This pathway is called Wnt/beta-catenin signaling. In this respect, the most reliable marker of the Wnt/beta-catenin signaling is the presence of beta-catenin in cell nuclei. The Wnt/beta-catenin signaling can also be induced by drugs that directly inactivate the GSK-3 enzymes, such as lithium, retinoic acid, or BIO, in addition to the Wnt proteins. There are conflicting reports about effects of the Wnt/beta-catenin signaling on embryonic stem cells: some reports state that the Wnt/beta-catenin signaling induces neural differentiation of mouse embryonic stem cells [Lyu J et al, J Biol Chem 278(15): 13487-95, 2003], whereas other reports state that the Wnt/beta-catenin signaling must be suppressed to induce neural differentiation [Ying QL et al, Nat Biotechnol 21(2): 183-6, 2003]. In addition, it is known that the Wnt/beta-catenin signaling plays a very important role in generation/differentiation/stem cell-related phenomenon and facilitates or inhibits cell differentiation according to the types of tissues and the degree of cell differentiation. Also, there are reports that the Wnt/beta-catenin signaling provides maintenance of the undifferentiated state of hematopoietic tissue stem cells [Eaves CJ, Nat Immunol 4(6): 511-2, 2003], division of gastrointestinal epithelial stem cells [Kuhnert F et al, PNASU 101 (1 ): 266-71 , 2004], differentiation induction of neural stem cells [Muroyama Y etal, Biochem Biophys Res Commun, 313(4): 915-21 , 2004], differentiation of muscular tissue [Polesskaya A et al, Cell 113(7): 841-52, 2003] and regeneration of muscular tissue [Seale P et al, Cell Cycle 2(5): 418-9, 2003], differentiation of hair follicles [Alonso L and Fuchs E, Genes Dev 17(10): 1189-200, 2003], differentiation induction of lens epithelial cells [Ryu et al, summary of Korean Journal of Biochemistry, Winter Conference, 2003], etc. Further, there is a report that the Wnt/beta-catenin signaling induces ventral formation rather than dorsal formation during formation of the body axis in embryogenesis [Kofron M et al, Dev Biol 237(1 ): 183-210, 2001]. In spite of many researches as described above, there are no reports about a method of culturing stem cells with no loss of cell pluripotency by inhibition of cell differentiation. Therefore, while studying the Wnt/beta-catenin signaling system and differentiation of pluripotent stem cells, the present inventors surprisingly elucidated the relationship between the Wnt/beta-catenin signaling system and the differentiation of pluripotent stem cells and deduced a cell culture method with no loss of pluripotency.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a phase contrast microscopic image of a pluripotent stem cell line (SnuhES3) differentiation-induced by retinoic acid treatment, lithium treatment, or feeder cell removal, and an immunostaining image of beta-catenin. FIG. 2 is immunostaining images for beta-catenin, SSEA-4, and BrdU of a pluripotent stem cell line (MizhESI) at 4 days after retinoic acid treatment. FIG. 3 is a reverse-transcription PCR (RT-PCR) result for mRNA expression of Oct-3/4 and Nanog which are pluripotency markers after differentiation induction of a pluripotent stem cell line (SnuhES3) by retinoic acid treatment, lithium treatment, or feeder cell removal. FIG. 4 is a RT-PCR result for mRNA expression pattern of Wnts and Frizzled-4 at 4 days after differentiation induction of a pluripotent stem cell line by treatment of 10^"6 M retinoic acid or 10 mM lithium. FIG. 5 is a RT-PCR result for mRNA expression pattern of sFRP-1 , sFRP-2, and WIF-1 which are extracellular Wnt antagonists at 4 days after differentiation induction of a pluripotent stem cell line by treatment of 10^"6 M retinoic acid or 10 mM lithium.

DETAILED DESCRIPTION OF THE INVENTION Technical Goal of the Invention The present invention provides a method of inhibiting the differentiation of pluripotent stem cells. The present invention also provides a method of culturing new embryonic stem cells useful as a cellular therapeutic agent which maintains pluripotency and dose not affect desired cell differentiation. The present invention also provides a method of culturing cells differentiable by Wnt/beta-catenin signaling, in addition to the pluripotent stem cells, while inhibiting cell differentiation.

Disclosure of the Invention According to an aspect of the present invention, there is provided a method of inhibiting differentiation of a pluripotent stem cell by suppression of Wnt beta-catenin signaling. According to another aspect of the present invention, there is provided a method of culturing a pluripotent stem cell while inhibiting cell differentiation by suppression of Wnt/beta-catenin signaling. The pluripotent stem cell is mainly derived from an embryo and may be derived from an adult. The suppression of the Wnt/beta-catenin signaling may be performed by any method known in the pertinent art. The suppression of the Wnt/beta-catenin signaling may be performed by addition of a Wnt/beta-catenin signaling suppressor to a cell culture medium. The suppression of the Wnt/beta-catenin signaling may also be performed by mixing a cell culture medium with a cell culture medium of a Wnt/beta-catenin signaling suppressor gene-transfected cell. The suppression of the Wnt/beta-catenin signaling may also be performed by mixing a cell culture medium with a cell culture medium of a Wnt/beta-catenin signaling suppressor-producing cell. The suppression of the Wnt/beta-catenin signaling may also be performed using a Wnt/beta-catenin signaling suppressor gene-transfected cell as a feeder cell for cell culture or using a Wnt/beta-catenin signaling suppressor-producing cell as a feeder cell for cell culture. The suppression of the Wnt/beta-catenin signaling may also be performed by transfection of a Wnt/beta-catenin signaling suppressor gene into a cell to be cultured. In addition, the suppression of the Wnt/beta-catenin signaling may be performed by coating a Wnt/beta-catenin signaling suppressor on a cell culture plate, a three-dimensional cell culture bead, a culture support, or a combination thereof. Examples of the Wnt/beta-catenin signaling suppressor include but are not limited to sFRP, collagen XVIII, endostatin, carboxypeptidase Z, receptor tyrosine kinase, transmembrane enzyme Corin, WIF-1, Cerebus, Dickkopf-1 , and a combination thereof. The Wnt/beta-catenin signaling suppression method or the cell culture method using it may also be applied to a cell differentiable by the Wnt/beta-catenin signaling, in addition to the pluripotent stem cell. Hereinafter, the present invention will be described in more detail. The method of inhibiting the differentiation of a pluripotent stem cell according to the present invention is performed by suppression of the Wnt/beta-catenin signaling. Therefore, stem cell culture is possible while maintaining pluripotency by inhibition of cell differentiation. The suppression of the Wnt/beta-catenin signaling is an essential process of the method of inhibiting cell differentiation according to the present invention. The present inventors found that based on numerous experimental results, cell differentiation can be inhibited by the suppression of the Wnt/beta-catenin signaling. The present inventors investigated cell differentiation when the Wnt/beta-catenin signaling is activated or inactivated. Activation of the Wnt/beta-catenin signaling at a differentiated state of stem cells was demonstrated by expression of beta-catenin in cell nuclei. When the Wnt/beta-catenin signaling is inactivated, human embryonic stem cells have beta-catenin in cellular membranes. On the other hand, when the Wnt/beta-catenin signaling is activated, beta-catenin is translocated into cell nuclei. Meanwhile, differentiation of stem cells was determined by expression of SSEA-4, BrdU, Oct-3/4, or Nanog. When stem cells are differentiated, SSEA-4, Oct-3/4, and Nanog disappear, and a BrdU level is remarkably reduced. When stem cells were treated with lithium (LiCI) during cell culture to facilitate the Wnt/beta-catenin signaling, the presence of beta-catenin in cell nuclei supporting the Wnt/beta-catenin signaling in the stem cells was observed. At the same time, SSEA-4, Oct-3/4, and Nanog disappeared and a BrdU level was remarkably reduced. At this time, mRNA expression of sFRP-1 , sFRP-2, and WIF-1 which are Wnt antagonists was completely prevented. Further, in the case of an untreated control, no evidences supporting cell differentiation were observed. These results demonstrate that the Wnt/beta-catenin signaling facilitates the differentiation of stem cells. Therefore, it can be seen that stem cells can be cultured while inhibiting the differentiation of the stem cells by suppression of the Wnt/beta-catenin signaling. As a result of addition of retinoic acid (all-trans-retinoic acid), which is the commonest differentiation induction factor of stem cells, or removal of feeder cells, the present inventors identified differentiation induction by disappearance of SSEA-4, Oct-3/4, and Nanog, and remarkable reduction of a BrdU level. At this time, presence of beta-catenin in cell nuclei supporting the Wnt/beta-catenin signaling was also observed and mRNA expression of sFRP-1 , sFRP-2, and WIF-1 which are Wnt antagonists was completely prevented. In addition, in the case of an untreated control, no evidences supporting cell differentiation were observed. These results demonstrate that cell differentiation by the Wnt/beta-catenin signaling is a common phenomenon of stem cell differentiation. Therefore, it can be seen that stem cells can be cultured while inhibiting the differentiation of the stem cells by suppression of the Wnt/beta-catenin signaling.

Effect of the Invention According to the present invention, a method of inhibiting differentiation of pluripotent stem cells is provided. The present invention also provides a method of culturing new embryonic stem cells useful as a cellular therapeutic agent which maintains pluripotency and dose not affect desired cell differentiation. In addition, the present invention provides a method of culturing cells differentiable by Wnt/beta-catenin signaling, in addition to the pluripotent stem cells, while inhibiting cell differentiation.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described more specifically by Experimental Examples. However, the following Experimental Examples are provided only for illustrations and thus the present invention is not limited to or by them. Experimental Example 1 : cell culture and differentiation induction Experiments were performed for 19 passages of distributed human embryonic stem cells (SnuhES3-63 generations, Dept. of Obstetrics and Gynecology, Seoul National University Hospital; MizhES1-163 generations, MizMedi Hospital). When expression of SSEA-4, alkaline phosphatase, and Oct-3/4, which are undifferentiation markers, was investigated for every 5 passages, it was confirmed that the stem cells maintained an undifferentiated state. First, STO cell lines (ATCC, CRL-1503), which are mouse embryonic fibroblast cells, whose growth had been inhibited by radiation, were inoculated as feeder cells onto culture dishes. After one day of the inoculation, the cell masses of the human embryonic stem cells were inoculated onto the feeder cells. Cell differentiation induction began at 3 days after the inoculation at which the human embryonic stem cells were localized and formed colonies. Cell differentiation was induced by retinoic acid (all-trans-retinoic acid) treatment, lithium treatment, or feeder cell removal. Retinoic acid was dissolved in a DMSO solution and mixed with culture media to make two types cultures in which the final concentration of retinoic acid was 10^"6 M and 10^"7M, respectively. The retinoic acid treatment was continued daily for 4 days. Lithium was dissolved in water and mixed with culture media so that the final concentration of lithium was 10 mM. The lithium treatment was performed once and then daily exchange of a fresh medium for 4 days was performed. In the case of differentiation induction by feeder cell removal, the feeder cells were almost completely removed with micropipettes from 3 days after the inoculation of the human embryonic cells. With respect to the above differentiation-induced groups, medium exchange was performed daily during experiments. As a control, medium exchange was performed daily for 7 days without treatment. Experimental Example 2: immunostaining The human embryonic stem cells of the control and the differentiation-induced groups (retinoic acid treatment group, lithium treatment group, and feeder cell removal group) obtained in Experimental Example 1 were fixed with 4% formalin solution at 7 days after the inoculation at which the experiments were terminated, at room temperature for 30 minutes. To evaluate Wnt/beta-catenin signaling activity, the fixed cells were stained with a beta-catenin antibody (see FIGS. 1 and 2 and Experimental Result 1 ). Furthermore, to evaluate maintenance of the undifferentiated state, the fixed cells were stained with a SSEA-4 antibody which is an undifferentiated cell marker (see FIG. 2 and Experimental Result 1 ). To evaluate cell division capability, the fixed cells were stained with a BrdU antibody (see FIG. 2 and Experimental Result 1 ). The beta-catenin antibody, the SSEA-4 antibody, and the BrdU antibody were mouse monoclonal antibodies for beta-catenin (Chemicon), SSEA-4 (Davor Solter), and BrdU(Roche), respectively. Primary antibodies were incubated at a low temperature overnight and then the staining was performed with ABC kit (Vector Laboratories) for standard staining. Experimental Example 3: reverse-transcription polvmerase chain reaction (RT-PCR) for evaluation of tRNA expression of Oct-3/4. Nanog. Wnts. Frizzled, sFRP. and WIF-1 The human embryonic stem cells were extracted from the experimental groups of Experimental Example 1 , washed with a phosphate buffered solution, and then centrifuged with a centrifuge at 3,000 rpm for 5 minutes. Supernatants were discarded and RNAs were isolated from cell masses using RNeasy kit (Qiagen). About 500 ng of RNAs were subjected to RT-PCR with random hexamer primers using superscript II first-strand synthesis system for RT-PCR (Invitrogen, Rockville, MD). 100-200 ng of resulting cDNAs were mixed with a sense primer, an antisense primer, Perfect Premix (Takara), and water, and then PCR was performed for 30-40 cycles. Electrophoretic analysis for 5-10 μl of each PCR product was performed on 1.2-2% agarose gel (see FIGS. 3, 4, and 5, and Experimental Results 1 and 3). Experimental Results> 1. Cell differentiation rate According to RT-PCR results for mRNA expression of Oct-3/4 and Nanog which are pluripotency markers after differentiation induction of the pluripotent stem cell line (SnuhES3) by retinoic acid treatment, lithium treatment, or feeder cell removal, disappearance of Oct-3/4 and Nanog was observed. The amount of cDNAs was adjusted to the amount of GAPDH with a constant expression level regardless of conditions. In comparison with the control, in all the differentiation-induced groups, disappearance of Oct-3/4 and Nanog was observed (FIG. 3). According to a phase contrast microscopic image analysis for the differentiation-induced pluripotent stem cell line (SnuhES3), the control exhibited an increased nuclear/cytoplasmic ratio and dense cell nuclei, which are specific morphology of undifferentiated cells. On the other hand, the retinoic acid treatment group, the lithium treatment group, and the feeder cell removal group exhibited typical characteristics of differentiated cells, such as sparse nuclei and large cytoplasms (FIG.

1 ). As a result of observation of the pluripotent stem cell line (MizhESI) treated with

10^"7 M and 10^"6 M of retinoic acid at 4 days after the treatment, SSEA-4 which is an undifferentiation marker was expressed in all colonies of the undifferentiated pluripotent stem cell line used as a control, whereas partial expression was observed in a 10^"7 M retinoic acid treatment group and no expression was observed in a 10^"6 M retinoic acid treatment group (FIG. 2). BrdU expression was observed in most cells of the undifferentiated pluripotent stem cell line colonies used as the control. With respect to the retinoic acid treatment groups, in the 10^"7 M retinoic acid treatment group, as differentiation partially proceeded in colonies, BrdU expression also partially occurred. Therefore, a high level area and a low level area for BrdU-positive cells coexisted. On the other hand, in the 10^"6 M retinoic acid treatment group, differentiation uniformly proceeded over all colonies.

Therefore, the BrdU-positive cells were maintained at a low level. This illustrates that as cell differentiation proceeds, cell division capability is lowered by retinoic acid treatment. 2. Activation of Wnt/beta-catenin signaling In undifferentiated pluripotent stem cell lines, beta-catenin (indicated by brown color) was observed like an intercellular boundary line in cell membranes. On the other hand, after differentiation induction of the pluripotent stem cell lines by retinoic acid treatment, lithium treatment, or feeder cell removal, it was observed that beta-catenin was translocated into cell nuclei (FIGS. 1 and 2). With respect to the behavior of beta-catenin according to the concentration of retinoic acid, in 10^~7 M retinoic acid treatment, differentiation partially proceeded in colonies and translocation of beta-catenin into cell nuclei occurred in the same pattern as the differentiation. That is, beta-catenin was partially observed in colonies. On the other hand, in 10^"6 M retinoic acid treatment, differentiation uniformly proceeded in all colonies and translocation of beta-catenin into cell nuclei occurred in the same pattern as the differentiation. That is, beta-catenin was uniformly observed in all colonies. These results illustrate that activation of Wnt/beta-catenin signaling occurs simultaneously with cell differentiation. 3. Expression pattern of Wnts, Wnt antagonists, and Frizzled receptors according to differentiation As differentiation of pluripotent stem cell lines proceeded, Wnts exhibited different expression patterns (FIG. 4). After differentiation induction, Wnt-1 and -2 exhibited an increased expression level, whereas Wnt-3, -3a, -5a, and -5b exhibited a decreased expression level. sFRP-1 , sFRP-2, and WIF-1 , which are extracellular Wnt antagonists, exhibited a very high expression level at an undifferentiated state, whereas no expression was observed as differentiation was induced (FIG. 5). With respect to mRNAs of the Frizzled-4 which is a Wnt receptor, no expression was observed at an undifferentiated state but expression was induced after differentiation induction (FIG. 4 (A)). Therefore, it is judged that in pluripotent stem cells, Wnt/beta-catenin signaling is activated by inducing the expression of Wnt receptors or by inhibiting the expression of Wnt antagonists, thereby inducing cell differentiation.

Claims

1. A method of inhibiting differentiation of a cell by suppression of Wnt/beta-catenin signaling.

2. A method of culturing a cell while inhibiting differentiation of the cell by suppression of Wnt/beta-catenin signaling.

3. The method of claim 1 or 2, wherein the cell is a pluripotent stem cell.

4. The method of claim 3, wherein the pluripotent stem cell is derived from an embryo.

5. The method of claim 3, wherein the pluripotent stem cell is derived from an adult.

6. The method of claim 2, wherein the suppression of the Wnt/beta-catenin signaling is performed by addition of a Wnt/beta-catenin signaling suppressor to a cell culture medium.

7. The method of claim 2, wherein the suppression of the Wnt/beta-catenin signaling is performed by mixing a cell culture medium with a cell culture medium of a Wnt/beta-catenin signaling suppressor gene-transfected cell.

8. The method of claim 2, wherein the suppression of the Wnt/beta-catenin signaling is performed by mixing a cell culture medium with a cell culture medium of a Wnt/beta-catenin signaling suppressor-producing cell.

9. The method of claim 2, wherein the suppression of the Wnt/beta-catenin signaling is performed using a Wnt/beta-catenin signaling suppressor gene-transfected cell as a feeder cell for cell culture.

10. The method of claim 2, wherein the suppression of the Wnt/beta-catenin signaling is performed using a Wnt/beta-catenin signaling suppressor-producing cell as a feeder cell for cell culture.

11. The method of claim 2, wherein the suppression of the Wnt/beta-catenin signaling is performed by transfection of a Wnt/beta-catenin signaling suppressor gene into a cell to be cultured.

12. The method of claim 2, wherein the suppression of the Wnt/beta-catenin signaling is performed by coating a Wnt/beta-catenin signaling suppressor on a cell culture plate, a three-dimensional culture bead, a culture support, or a combination thereof.

13. The method of any one of claims 6 through 12, wherein the Wnt/beta-catenin signaling suppressor is sFRP, collagen XVIII, endostatin, carboxypeptidase Z, receptor tyrosine kinase, transmembrane enzyme Corin, WIF-1 , Cerebus, Dickkopf-1 , or a combination thereof.