WO2006115080A1 - Growth promoting agent for pluripotent stem cell - Google Patents

Abstract

A growth promoting agent for a pluripotent stem cell, comprising visfatin or a visfatin gene as the active ingredient; a culture medium or culture kit for a pluripotent stem cell, comprising visfatin as a component; and a method for the culture of a pluripotent stem cell using the culture medium or culture kit.

Description

 Specification

 Pluripotent stem cell proliferation promoter

 Technical field

 The present invention relates to a proliferation promoter for pluripotent stem cells.

 Background art

 [0002] Embryonic stem cells (ES cells) are pluripotent stem cells derived from the inner cell mass present in the blastocyst before implantation, and were first established in 1981 in mice. (See Non-Patent Document 1). ES cells can proliferate indefinitely in culture, presumably retaining their ability and normal chromosome type. By injecting it into the blastocyst, it can participate in embryonic development and create a chimeric animal. Since the first knockout mouse was created in 1987 (see Non-Patent Document 2), a large number of knockout mice have been created so far, and the functions of their gene products have been analyzed in detail.

 The power that mouse ES cells have been used as a tool for developmental engineering so far The establishment of human ES cells was reported in 1998 (see Non-Patent Document 3), which is expected to be applied in regenerative medicine. Began to be. If human ES cell strength can be differentiated into blood cells, nerve cells, cardiomyocytes, etc. in the future, cell transplantation therapy will enable treatment of diseases that currently have no cure. This expectation is due to the ability of ES cells to differentiate into all somatic cells (potentiation of differentiation) and the ability to proliferate indefinitely while maintaining an undifferentiated state (self-renewal ability).

[0003] In this way, ES cells are becoming increasingly important for research and clinical applications in regenerative medicine, but at present, the presence of serum or feeder cells is essential for their culture. is there. In other words, mouse ES cells can be maintained without using feeder cells with a serum-free medium supplemented with LIF when the number of cells is large. However, at low density, serum or feeder cells are essential. In addition, serum and feeder cells are essential for culturing human ES cells. Furthermore, unlike mouse ES cells, LIF is ineffective in human ES cells. When human ES cells are clinically applied, it is essential to culture them without using animal serum or feeder cells from the viewpoint of safety. Therefore, ES in serum-free medium There is a desire to identify factors that maintain cell growth.

 [0004] Visfatin is also known as PBEF (Pre-B cell colony-enhancing factor), and in previous studies, it has been reported that it is a differentiation and proliferation stimulating factor of B cell progenitor cells. (See Patent Document 1 and Non-Patent Document 4). Recently, it has been clarified that visfatin has an insulin mimic effect of binding to the insulin receptor and lowering the blood gnolecose concentration (see Non-Patent Document 5). However, the relevance of ES cells to embryo development has not been clarified.

 [0005] Patent Document 1: Japanese Patent Publication No. 8-505373

 Non-Patent Document 1: Evans, M. J. and Kauftnan, M. H. Nature, 292 (5819): pl54-156 (198 1)

 Non-Patent Document 2: Hooper, M. et al, Nature, 326 (6110): p292-295 (1987)

 Non-Patent Document 3: Thomson, JA et al., Science, 282 (5391): pi 145-1147 (1998) Non-Patent Document 4: Samal, B. et al., Mol. Cell. Biol, Feb; 14 (2 ): pl431-1437 (1994) Non-Patent Document 5: Fukuhara A. et al., Science, vol.307, p426-430 (2005)

 Disclosure of the invention

 Problems to be solved by the invention

[0006] An object of the present invention is to provide a proliferation promoter for pluripotent stem cells containing visfatin or a visfatin gene as an active ingredient. Another object of the present invention is to provide a culture solution or culture kit for pluripotent stem cells containing visfatin, or a method for culturing pluripotent stem cells using these.

 Means for solving the problem

[0007] The present inventors analyzed the expression of the visfatin gene in ES cells. The bisfatin gene was highly expressed in undifferentiated ES cells, whereas its expression was greatly increased by differentiation. Decreasing was clearly a force. At the protein level as well, it became clear that the abundance of visfatin similarly decreased with ES cell sorting.

[0008] Next, the present inventors produced a knockout mouse in order to analyze the function of visfatin in vivo. Heterogeneous mutant mice developed normally and had fertility, but sugar Abnormalities in metabolic capacity were observed. On the other hand, homozygous mice were not born from crosses between heterozygous mice. As a result of fetal analysis, homozygous mutant mice were found to be lethal immediately after implantation. When blastocysts homo-deficient in visfatin are cultured, an inner cell mass is normally formed, and this inner cell mass force ES cell colony is formed, whereas visfatin-deficient blastocyst is cultured in vitro. However, the inner cell mass and the subsequent ES cells could not grow. From this result, it became clear that visfatin is essential for the proliferation of pluripotent stem cells.

 [0009] In addition, the present inventors also used a visfatin gene hetero-knockout ES cell (bisfatin hetero KO-ES cell) using a serum-free medium containing no insulin !, a serum-free medium containing insulin, and a serum medium. Was cultured. As a result, there was no difference in the growth rate of wild-type ES cells and bis-fatin hetero KO-ES cells in serum medium. Serum-free medium containing insulin and serum-free medium containing insulin! In the first place, the growth rate of visfatin hetero KO-ES cells decreased compared to wild-type ES cells, and the degree of the decrease was remarkable in serum-free medium without insulin. . From the above results, it became clear that visfatin is a factor that maintains the proliferation of ES cells.

 [0010] Further, the present inventors have introduced a siRNA of visfatin into ES cells to knock down the amount of protein and examined the growth rate. As a result, the growth rate was significantly reduced compared to wild-type ES cells. From the above, it was confirmed that visfatin was a factor that maintained the proliferation of ES cells as described above.

 The visfatin of the present invention can be used as a proliferation promoter for pluripotent stem cells such as ES cells. Visfatin is particularly useful because it is a factor expressed (produced) by pluripotent stem cells themselves.

 The present invention has been completed based on such findings.

[0011] That is, the present invention provides:

 (1) A proliferation promoter for pluripotent stem cells containing visfatin or a visfatin gene as an active ingredient,

 (2) The proliferation promoter according to (1) above, wherein the pluripotent stem cell is an embryonic stem cell,

(3) a culture solution or culture kit for pluripotent stem cells containing visfatin as a component, (4) The culture solution or culture kit according to (3) above, wherein the pluripotent stem cells are embryonic stem cells,

(5) A method for culturing pluripotent stem cells, characterized by using the culture solution or culture kit according to (3) or (4) above,

 (6) Use of visfatin or visfatin gene as a proliferation promoter for pluripotent stem cells, and

 (7) The use according to (6) above, wherein the pluripotent stem cell is an embryonic stem cell.

 The invention's effect

 The visfatin and visfatin gene of the present invention are useful as a proliferation promoter for pluripotent stem cells such as ES cells. In particular, by adding the growth promoter of the present invention to a serum-free medium (serum-free medium), pluripotent stem cells can be cultured even in the absence of serum. It is used effectively. BEST MODE FOR CARRYING OUT THE INVENTION

[0013] Hereinafter, in the present specification, the abbreviations of amino acids, (poly) peptides, (poly) nucleotides, etc. are defined by IUPAC—IUB [IUPAC-IUB Communication on Biological No menclature, Eur. Biochem., 138: 9 (1984)], “Guidelines for the preparation of texts including base sequences or amino acid sequences” (edited by the Japan Patent Office), and conventional symbols in this field.

 In the present invention, “visfatin (visfatin protein)” is not particularly limited as long as pluripotent stem cell proliferation promoting activity is maintained, but specifically, the following (a) to (1) :

(a) the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4,

 (b) an amino acid sequence in which one or more amino acids are deleted, added or substituted in the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4,

 (c) in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, an amino acid sequence in which the amino terminal force is also deleted of 26 amino acids,

 (d) in the amino acid sequence of (c), an amino acid sequence in which methionine is added to the amino terminus;

(e) an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4, (f) an amino acid sequence encoded by DNA having the base sequence represented by nucleotides 18 to 1493 in the base sequence represented by SEQ ID NO: 1;

 (g) an amino acid sequence encoded by DNA having a base sequence represented by nucleotides 96 to 1571 in the nucleotide sequence represented by SEQ ID NO: 3;

 (h) an amino acid sequence encoded by a DNA having a base sequence having 80% or more sequence identity with the DNA of (f) or (g),

 (i) an amino acid sequence encoded by a DNA complementary to the DNA described in (f) or (g) above, and a DNA that is hybridized under stringent conditions; and

 (j) a partial amino acid sequence of any one of the amino acid sequences of (a) to (i),

 Examples of the protein having any amino acid sequence and having the activity of promoting proliferation of pluripotent stem cells are exemplified.

 [0015] Here, the amino acid sequence represented by SEQ ID NO: 2 is the amino acid sequence of human visfatin, which is described in the literature (Mol. Cell. Biol, Feb; 14 (2): pl431-1437 (1994)) and GenBanc. Acc. No. U02020 is a known sequence. The amino acid sequence represented by SEQ ID NO: 4 is the amino acid sequence of mouse bisfatin, and is a known sequence in GenBank Acc. No. AF234625.

 The “amino acid deletion, addition or substitution” in (b) and “80% or more sequence identity” in (e) and (h) include, for example, SEQ ID NO: 2 or 4 It includes the processing that a protein having the amino acid sequence shown in the cell undergoes in the cell, the species that the protein is derived from, species differences, individual differences, differences between tissues, and the like, and artificial amino acid variations.

[0017] As a technique for artificially performing the “amino acid deletion, addition, or substitution” in the above (b) (hereinafter sometimes referred to as amino acid modification as a whole), for example, SEQ ID NO: 2 or 4 For example, a conventional site-directed mutagenesis can be performed on the DNA encoding the amino acid sequence shown in FIG. Here, as a site-specific mutation introduction method, for example, a method using amber mutation (gapped 'duplex' Method, Nucleic Acids Res., 12,9441-9456 (1984)), and a PCR method using a mutagenesis primer.

 The number of amino acids modified as described above is at least one residue, specifically one or several, or more. The number of such modifications may be within a range where the pluripotent stem cell proliferation promoting activity of the protein can be found.

 Of the deletions, additions or substitutions, the modification relating to amino acid substitution is particularly preferable. The substitution is more preferably substitution with an amino acid having similar properties such as hydrophobicity, charge, pK, and structural features. Examples of such substitution include (1) glycine, alanine; (2) parin, isoleucine, leucine; (3) aspartic acid, glutamic acid, asnoragin, glutamine, (4) serine, threonine; (5) Substitution within the group of lysine, arginine; (6) ferulanine, tyrosine.

In the above (e) and (h), “sequence identity” refers to sequence identity or homology between two DNAs or two proteins. The “sequence identity” is determined by comparing two sequences that are optimally aligned over the region of the sequence to be compared. Here, the DNA or protein to be compared may have an addition or a deletion (for example, a gap) in the optimal alignment of the two sequences. Such sequence identity can be calculated, for example, by creating an alignment using the ClustalW algorithm (Nucleic Acid Res., 22 (22): 4673_4680 (1994)) using Vector NTI. Note that sequence identity is measured using sequence analysis software, specifically analysis tools provided by Vector NTI, GENETY X-MAC, and public databases, such as the website address http It is generally available at: //www.ddbj.nig.ac.jp.

 The sequence identity may be 80% or more, preferably 90% or more, more preferably 95% or more.

Regarding the “hybridization under stringent conditions” in (i) above, the hybridization performed here is, for example, by Sambrook J., Frisch EF, Maniatis T., Molecular Claw- 2nd edition (Molecular Cloning 2nd edition), Cold Spring Harbor Laboratories (Cold Spring Harbor Laboratory press), etc. It can be performed according to the usual method. In addition, “under stringent conditions” means, for example,

A hybrid containing 6 X SSC (1.5 M NaCl, 0.1M trisodium citrate solution as 10 X SSC), 50% formamide in a solution at 45 ° C, 2 X SSC (Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6). The salt concentration in the washing step can be selected from, for example, 2 X SSC, 50 ° C conditions (low stringency conditions) to 0.2 X SSC, 50 ° C conditions (high stringency conditions). The temperature in the washing step can be selected, for example, from room temperature (low stringency conditions) to 65 ° C. (high stringency conditions). It is also possible to change both the salt concentration and the temperature.

 [0020] The "partial amino acid sequence" in (j) above is any amino acid sequence of (a) to (i) above, preferably 15 to L00 residues in the amino acid sequence of (a) above, preferably Represents a partial amino acid sequence of 15 to 50 residues, and is not particularly limited as long as the peptide fragment having the amino acid sequence ability has the activity of promoting the proliferation of pluripotent stem cells.

 The visfatin of the present invention shown above may be derived not only from humans and mice but also from any species, and specifically, mammals such as humans, mice, rats, monkeys, marmosets, horsetails, horses, etc. Animal-derived visfatin is exemplified.

 In the present invention, “visfatin gene” refers to a gene encoding visfatin. Specifically, a gene encoding a protein having the amino acid sequence ability of any of the above (a) to (j) and having the activity of promoting proliferation of pluripotent stem cells is shown.

 More specifically, the following (A) to (K):

 (A) a base sequence encoding the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4,

(B) a base sequence encoding an amino acid sequence in which one or more amino acids have been deleted, added or substituted in the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4,

(C) in the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4, a base sequence encoding an amino acid sequence in which the amino terminal force is also deleted of 26 amino acids,

 (D) in the amino acid sequence of (C), a base sequence encoding an amino acid sequence in which methionine is added to the amino terminus;

(E) 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 A base sequence encoding an amino acid sequence having

 (F) a nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3,

 (G) a base sequence represented by nucleotides from the 18th nucleotide to the 1493th nucleotide in the base sequence represented by SEQ ID NO: 1,

 (H) a nucleotide sequence represented by nucleotides 96 to 1571 in the nucleotide sequence represented by SEQ ID NO: 3;

 (I) a base sequence having 80% or more sequence identity with the DNA having the base sequence described in (G) or (H),

 C D complementary to DNA having the base sequence described in (G) or (Η) above, a DNA base sequence that hybridizes under stringent conditions, and (Κ) (Α) to ω The partial base sequence of any of the base sequences,

 Examples thereof include a gene encoding such a protein, wherein the protein encoded by the nucleotide sequence is a protein having a proliferation promoting activity of pluripotent stem cells.

[0022] Hereinafter, the visfatin gene and the method for preparing visfatin of the present invention will be described.

 The visfatin gene can be obtained by conventional genetic engineering methods (for example, Sambrook J., Frisch EF, Maniatis T., Molecular Cloning 2nd edition), published by Konored Spring Nono 1 Laboratory (Cold Acquired according to the method described in Spring Harbor Laboratory pres). Specifically, in the case of the human 'visfatin gene represented by SEQ ID NO: 1, for example, a cDNA library derived from human intraperitoneal adipose tissue was used as a cage, and an appropriate part of the base sequence described in SEQ ID NO: 1 was used as a primer. Cloning can be performed by performing PCR. In the case of the mouse bisfatin gene shown in SEQ ID NO: 3, for example, by performing a PCR using a mouse liver-derived cDNA library as a cage and using an appropriate part of the nucleotide sequence shown in SEQ ID NO: 3 as a primer. Can be cloned.

[0023] A visfatin protein is produced and obtained according to a normal genetic engineering method by using the visfatin gene. For example, it can be obtained by preparing an expression vector that can express the visfatin gene in a host cell, introducing it into the host cell, transforming it, and then culturing the transformed host cell (transformant). Visfatin protein can be obtained from the culture medium. The visfatin gene expression vector includes, for example, genetic information that can be replicated in a host cell, can be propagated autonomously, can be easily isolated and purified from the host cell, and functions in the host cell. Examples include those in which a gene encoding the visfatin of the present invention is inserted into an expression vector having a detectable promoter and a detectable marker. The expression vector can be appropriately selected according to the host to be used and the purpose, and examples thereof include plasmids, phage vectors, and virus vectors.

 For example, when the host is Escherichia coli, examples of the vector include plasmid vectors such as pUC118, pUC119, pBR322, pCR3, and pETlla, and phage vectors such as λ and λgtll. When the host is yeast, examples of the vector include pYES2, pYEUra3 and the like. When the host is an insect cell, pAcSGHisNT-A and the like can be mentioned. When the host is an animal cell, plasmid vectors such as pCEP4, pKCR, pCDM8, pGL2, pcDNA3.1, pRc / RSV, pRc / CMV, pcDL-SRa296, retrovirus vector, adenovirus vector, adeno Virus vectors such as related virus vectors. These are vectors available to those skilled in the art.

 [0025] The vector may appropriately have factors such as a promoter capable of inducing expression, a gene encoding a signal sequence, a marker gene for selection, and a terminator. In addition, a sequence expressed as a fusion protein with thioredoxin, His tag, or GST (Dartathione S-transferase) may be added to facilitate isolation and purification. In this case, a GST fusion protein vector (such as pGEX4T) that has an appropriate promoter (such as lac, tac, trc, tr p, CMV, or SV40 early promoter) that functions in the host cell, or a tag sequence such as Myc or His Vectors (such as pcDNA3.1 / Myc-His) and vectors that express fusion proteins with thioredoxin and His tags (pET32a) can be used.

[0026] By transforming a host with the visfatin gene expression vector prepared above, a transformant (transformed cell) containing the expression vector can be prepared. Examples of the host used here include Escherichia coli, yeast, insect cells, animal cells and the like. Examples of E. coli include E. coli K-12 strains HB101 strain, C600 strain, JM109 strain, DH5α strain, AD494 (DE3) strain, and the like. Examples of yeast include Saccharomyces cerevisiae. Examples of animal cells include L929 cells, BALB / c3T3 cells, C127 cells, CHO cells, COS cells, Vero cells, Hela cells, and 293-EBNA cells. Insect cells include sl9.

 [0027] As a method for introducing an expression vector into a host cell, a conventional method suitable for the host cell may be used. Specific examples include a calcium phosphate method, a DEAE-dextran method, an electo-poration method, a method using a lipid for gene transfer (Lipofectamine, Lipofectin; Gibco-BRL), and a method using a virus vector. After the introduction, a transformant in which the expression vector is introduced into the host cell can be selected by culturing in a normal medium containing a selection marker.

 [0028] The transformant can be cultured by a usual method used for culturing microorganisms, yeast, insect cells or mammalian cells. For example, in the case of Escherichia coli, culturing is performed in a medium appropriately containing a suitable carbon source, nitrogen source, and micronutrients such as vitamins. The culture method may be either solid culture or liquid culture, and liquid culture such as aeration and agitation culture is preferable.

 A visfatin protein can be produced by continuing to culture the transformant under suitable conditions. The obtained protein can be further isolated and purified by a general biochemical purification means. Examples of the purification means include salting out, ion exchange chromatography, adsorption chromatography, affinity chromatography, gel filtration chromatography, and the like. When visfatin protein is expressed as a fusion protein with the aforementioned thioredoxin, His tag, GST or the like, it can be isolated and purified by a purification method utilizing the properties of these fusion protein and tag.

 Specific examples of expression and purification of human visfatin are shown below as representative examples.

 [0029] 1) Expression and purification in mammalian cells

Expression vector expressing human visfatin protein (SEQ ID NO: 2) prepared as described above 1. Transform mammalian cells such as COS-1. By culturing this transformant in OPTI-MEM medium (GIBCO) containing antibiotics, human visfatin is secreted into the medium. Collect cell culture supernatants on the 2nd to 8th days after transformation. The culture supernatant is filtered through an ultrafiltration membrane, and purified on human bisfatin proteins by using DEAEsepharose, ANX- Sepharose, Octy Oct Sepharose, Mono-Q columns (all manufactured by Amersham Pharmacia).

 [0030] 2) Expression and purification in E. coli

 This part is encoded to produce a protein having an amino acid sequence with methionine added to the amino terminus of the amino acid sequence shown in amino acid numbers 27 to 491 of human visfatin protein (SEQ ID NO: 2). The gene to be cloned is cloned into an expression vector for Escherichia coli such as pETl la (Novagen). Next, E. coli (DE3 strain: Novagen, etc.) is transformed with the expression plasmid. The obtained transformant was cultured at 37 ° C until the OD 600 reached 0.6, and isopropyl mono-β-D-thiogalatatopyranoside (hereinafter referred to as IPTG) at a final concentration of ImM was added. And further overnight culture. The cells were then collected by centrifugation, and the cells were collected from lOOmM Tris-hydrochloric acid (ρΗ7.6), 5 mM ethylenediammine tetraacetate ninatrime (hereinafter referred to as EDTA '2Na), 5 mM dithiothreitol (hereinafter referred to as DTT). ) And ImM phenol methylsulfur fluoride (hereinafter referred to as PMSF) and suspended in a buffer (hereinafter referred to as buffer A) and sonicated (3 times for 5 minutes under ice-cooling). ), And centrifuge the lysate at 12,000 Xg for 15 minutes at 4 ° C to collect the sediment (hereinafter referred to as the inclusion body fraction).

[0031] To the inclusion body fraction, buffer A containing 2M urea is added, suspended, and subjected to ultrasonic treatment (XI times for 5 minutes under ice-cooling). The solution after sonication is centrifuged at 12,000 Xg for 15 minutes at 4 ° C, and the resulting precipitate is suspended by adding buffer A containing 4M urea, sonication, and centrifugation. The operation of separating is performed in the same manner as described above. Furthermore, buffer A containing 6M urea is added to the resulting precipitate, followed by suspension, sonication, and centrifugation as described above. The obtained precipitate was suspended in a buffer containing 20 mM Tris-HCl (pH 8.5), 2 mM DTT and 8 M urea, and the suspension was centrifuged at 12,000 Xg for 15 minutes at 4 ° C. Collect the supernatant. The obtained supernatant was added to HiLoad Superdex 200pg (A flow rate; 1. OmlZ min., Detection wavelength: 280 nm). Peak fractions eluting between 45 and 55 minutes are collected and concentrated with Centricon (Grace Japan, molecular weight cut off 30 000), then Mono Q HR10 / 10 ion exchange column (Falmacia) For chromatography (flow rate 1. OmlZ min, 0-1 M NaCl gradient, detection wavelength; 280 nm). Fractions eluted with about 100 to about 200 mM NaCl are collected and concentrated with Centricon (Grace Japan, molecular weight cut off 30,000) to lmg protein Zml.

 [0032] To the obtained protein fraction, 1OO3 amount of lOOmM Tris-HCl (pH 8.5) in the volume of the fraction is added with gentle stirring. Further, gentle stirring was continued overnight at room temperature. Next, the supernatant was recovered by centrifugation at 18,000 xg for 20 minutes at 4 ° C, and 7 mL of 2 M urea, 20 mM Tris-HCl (pH 8.5), 4 mM was added to the supernatant. Add a buffer containing reduced glutathione and 0.4 mM acid-type dartathione and stir gently. Place the resulting solution in a dialysis tube with a molecular weight cut off of 25,000, and add 1000 times the volume of 2M urea, 20 mM Tris-HCl (pH 8.5), 4 mM reduced glutathione and 0.4 mM oxidized dartathione. Dialyze against the containing buffer at 4 ° C for 8-16 hours. Next, against a buffer containing 20 mM Tris-HCl (pH 8.5), 4 mM reduced glutathione, and 0.4 mM oxidized glutathione, 1000 times the volume in the dialysis tube, 8-16 hours at 4 ° C. Dialyze. In addition, dialyze against a buffer containing 20 mM Tris monohydrochloride (PH8.5), ImM reduced glutathione, and 0. ImM oxidized glutathione, 1000 times the volume in the dialysis tube, at 4 ° C for 8 to 16 hours. . In this way, human 'bisfatin can be obtained from E. coli.

 Refer to WO02 / 010772 and the like for the details of clotting of the visfatin gene, expression vector preparation, preparation of transformants and expression / purification of visfatin protein described above.

[0033] In the present invention, "pluripotent stem cells" refer to cells that maintain undifferentiated / pluripotent typified by ES cells. The ES cell may be an ES-like cell generated by nuclear reprogramming from a somatic cell. In addition to ES cells, Embryonic Germ Cell (EG cell) derived from primordial germ cells, mutipotent germline stem cell (mGS cell) isolated from testis, bone marrow Examples include multipotent adult progenitor cells (MAPC) that are released. These pluripotent stem cells may be derived from any source such as human, monkey, rat, mouse, marmoset and the like.

 In the present invention, the “proliferation promoting activity of pluripotent stem cells” possessed by visfatin or a visfatin gene is compared to the proliferation of pluripotent stem cells in the absence of visfatin or without introduction of a visfatin gene. Thus, it means such activity that the growth is promoted when visfatin is added to the medium or when the bisfatin gene is transfected into cells.

 In the present invention, the proliferation promoting activity of pluripotent stem cells possessed by visfatin or visfatin gene can be measured, for example, as follows.

 First, in the case of visfatin (or its candidate substance), pluripotent stem cells are cultured in a serum-free medium supplemented with visfatin (or its candidate substance) at a concentration of about lng / ml to 10 μg / ml. It can be examined by measuring the cell growth promoting activity. At that time, it is preferable to cultivate the same pluripotent stem cells in a serum-free medium without bisfatin as a control (negative control cells) and to compare with these negative control cells. When cell growth is promoted when visfatin (or its candidate substance) is added compared to negative control cells, it is judged that the bisfatin (or candidate substance) used has cell growth promoting activity.

 [0036] More preferably, the same pluripotent stem cells are cultured using a serum medium (positive control cells) and compared with the positive control cells. In cells cultured in serum-free medium supplemented with visfatin, if the growth of about 10% or more of the cells was maintained as compared to positive control cells and the cells maintained pluripotency, the visfatin used (or (Candidate substance) is judged to have cell growth promoting activity. The evaluation of maintaining pluripotency can be easily evaluated by analyzing the expression of marker genes such as Oct3 / 4 and ECAT genes by Northern blot, RT-PCR, Western plot, and immunostaining. It can also be determined by examining whether a chimeric mouse is born by microinjecting cells into a blast cyst.

[0037] The serum-free medium used for the measurement is any medium that does not contain serum. Any serum-free medium can be used. It may also include growth factors and site power in. The serum-free medium is a serum-free medium in which the proliferation of pluripotent stem cells (pluripotent stem cells used for measurement) is not normally maintained in the absence of visfatin.

 [0038] ES cells are preferred as pluripotent stem cells used for measurement. Specifically, RF8 cells (Meiner'V. Et al., Proc. Natl. Acad. Sci. USA, 93: 14041-14046 (1996)), JI cells (Li, E. et al., Cell, 69 : 915-926 (1992)), CGR8 cells (NicholsJ. Et al, Development, 110: 1341-1348 (1990), MG1.19 cells (Gassmann, M. et al., Proc. Natl. Acad. Sci., USA, 92: 1292-1296 (l 995)), commercially available mouse ES cells 129SV (No.R-CMTI-l-15, R-CMTI-1A), mouse ES cells C57 / BL6 (No. R-CMTI-2A), mouse ES cells DBA-l (No.R-CMTI-3A) (Dainippon Pharmaceutical Co., Ltd.), etc. Human ES cells include KhES-l, KhES- 2 or KhES-3 (the Center for Stem Cell Medicine, Kyoto University Research Institute), and monkey ES cells include force-quizal ES cells (Asahi Techno Glass).

 [0039] Next, in the case of a visfatin gene (or a candidate gene thereof), a transformant prepared by transfecting a pluripotent stem cell with a visfatin gene or candidate gene incorporated into an expression vector is used. Serum-free medium! It can be performed by culturing and measuring whether cell growth is promoted.

 At this time, it is preferable to culture cells without visfatin gene introduced in serum-free medium as a control (negative control cells) and compare with these negative control cells. When cell growth is promoted by transfecting the visfatin gene or its candidate substance compared to negative control cells, the used visfatin gene (or its candidate substance) has cell growth promoting activity. to decide.

[0040] More preferably, pluripotent stem cells are cultured using a serum medium (positive control cells) and compared with the positive control cells. Visfatin gene-introduced cells maintain approximately 10% or more of the growth compared to positive control cells, and the cells maintain pluripotency. ) Is judged to have cell growth promoting activity. The evaluation of maintaining pluripotency can be easily performed by analyzing the expression of marker genes such as Oct3 / 4 and ECAT gene group by Northern blot, RT-PCR, Western blot, and immunostaining. In addition, the cells become blast cysts It can be determined by examining whether a small mouse is injected and a chimeric mouse is born.

 [0041] Gene transfer to pluripotent stem cells can be performed by a generally known technique. Specifically, for example, a microinjection method, a calcium phosphate method, an electoporation method, a DEAE-dextran method, a method using a lipid for gene transfer (Lipofectamine, Lipofectin; G¾co-BRL), or a method using a virus vector (virus And the like).

 The ES cells and serum-free medium used for the measurement are the same as in the case of visfatin.

 [0042] In addition to the above-described measurement methods, cell proliferation promoting activity can also be measured by a technique using bisfatin gene hetero knockout ES cells as described in Examples 6 and 7.

 [0043] The proliferation promoter for pluripotent stem cells containing visfatin of the present invention as an active ingredient is used as it is or a known pharmaceutically acceptable carrier (excipient, diluent, extender, binder, lubricant) Agents, flow aids, disintegrants, surfactants, etc.), conventional additives, stabilizers, and the like can be mixed to prepare a reagent composition or a pharmaceutical composition. The composition can be produced by blending and dissolving bisfatin, which is an effective component, in an acceptable normal carrier, excipient, binder, stabilizer, diluent, buffer and the like.

 [0044] The amount of visfatin of the present invention added to the medium (culture solution) may be a concentration of about 1 ng / ml to 10 μg / ml. The concentration is preferably about 0.1 μg / ml to 10 g / ml.

 [0045] Specific examples of pluripotent stem cells to which visfatin of the present invention is applied include ES cells. The ES cell may be an ES cell generated by reprogramming a somatic cell nucleus. In addition to ES cells, Embryonic Germ Cell (EG cell) derived from primordial germ cells, mutipotent germline stem cell (mGS cell) isolated from testis, Multipotent adult progenitor cell (MAPC) isolated from bone marrow, etc. Can be mentioned. The origin of these pluripotent stem cells may be derived from humans, monkeys, rats, mice, marmosets, and the like.

[0046] The serum-free medium to which visfatin is added may be any serum-free medium as long as it does not contain serum and is a practical medium for research and clinical applications. It is also acceptable to include factors such as growth factors and site power-in. [0047] The visfatin of the present invention may be added to arabic bullying medium (culture medium), prepared as a culture medium for pluripotent stem cells, and then added to the cells for cell culture. Alternatively, the visfatin of the present invention may be added to the medium after contacting the serum-free medium with the cells. The visfatin of the present invention may be included as one component of a pluripotent stem cell culture kit which may be packaged alone. In the case of a kit, as components in the kit other than visfatin, other factors that can be simultaneously contained in the serum-free medium, such as BMP4 and LIF, can be listed.

 [0048] The proliferation promoter for pluripotent stem cells containing the visfatin gene of the present invention as an active ingredient is used in a form incorporated into an expression vector that is expressed in pluripotent stem cells that are mammalian cells. The visfatin gene (visfatin gene expression vector) can be formulated as it is or using a commonly used stabilizer, buffer, solvent and the like. The visfatin gene may be chemically modified! Examples of the chemically modified compounds include, for example, phosphorothioate, phosphorodithioate, alkylphosphotriester, alkylphosphonate, and alkylphosphoamidate, which enhance the ability to migrate into cells or stability in cells. Derivatives ("Antisense RNA and DNA" published by WILEY-LISS, 1992, pp.l-50, J. Med. Chem. 36: 1923-1937, 1993) are included. These can be synthesized according to conventional methods.

 [0049] Transfection into cells can be performed using either a non-viral introduction method or a viral introduction method.

 Examples of the non-viral introduction method include a microinjection method, a calcium phosphate method, an electo-poration method, a DEAE-dextran method, and a method using a lipid for gene introduction (Lipofect amine, Lipofectin; Gibco-BRL). Examples of the method using a recombinant virus include a method using a virus vector such as a retrovirus, an adenovirus, and an adeno-associated virus.

 [0050] The amount of the visfatin gene of the present invention introduced into the cell may be an amount known to those skilled in the art used for normal transfection. The pluripotent stem cells to be applied are the same as in the case of the bisfatin supplemented card.

[0051] Hereinafter, the ability to specifically explain the present invention by way of examples. It is not limited at all.

 Example 1

 [0052] Current analysis of Visfatin transmission in ES cells and mouse tissues

90 ng / ml retinoic acid was added to undifferentiated mouse ES cell RF8 (derived from 129ZSvJae mouse), serum medium (DMEM (Nacalai), 10 ⁷ Unit LIF (Chemicon)), 1.0% L-glutamine ( Invitorogen), 0.5% penisline streptomycin (Invitorogen), 0.2% 2-mercaptoethanol (Invitorogen), 1.0% MEM non-essential amino acid solution (Invitorogen), 14.8% FCS (Biowest)) ES cells were differentiated by culturing for 6 days. The differentiated ES cells were treated with TRIzoKGIBCO BRL), extracted with chloroform and ethanol precipitated, and suspended in L DEPC-treated water to obtain total RNA. In addition, each tissue extracted from B6 strain mice was also treated with TRIzoKGIBCO BRL), and total RNA was similarly obtained according to the protocol attached to the reagent. The extracted total RNA (lysate) was quantified by measuring the absorbance with a UV spectrometer (260 nm, 280 nm).

 [0053] The reverse transcription reaction was carried out using REVERTRA ACE a (Toyobo Co., Ltd.), and the obtained cDNA was used as a saddle to reflect the expression level of the mouse Visfatin gene (SEQ ID NO: 3). By performing RT-PCR under conditions where amplification was observed, the presence or absence and expression level of the mouse Visfatin gene in each ES cell and tissue were confirmed.

 The PCR method used here was a primer GE-U8 (SEQ ID NO: 5; TTCCTACTTTGAATG CCGTGAA) and primer AntiL-15Sall (SEQ ID NO: 6; GCTGTCGACTGGAACAG AATAGCCTGGAA). The reaction was performed. The PCR is followed by DNA denaturation (94 ° C, 1 min), 30 cycles of denaturation (94 ° C, 10 sec), annealing (55 ° C, 30 sec), one extension (68 ° C, 1 min), Further, the final elongation (68 ° C, 5 minutes) was performed. As a positive control, the same reaction was performed for the NAT1 gene (Yamanak a, S. et al., Embo J., 19, 5533-5541 (2000)).

[0054] The results are shown in FIG. The mouse Visfatin gene was highly expressed in undifferentiated ES cells, whereas its expression was greatly reduced in the separated ES cells. In mouse tissues, high expression was observed in brown fat, mesenteric fat, peritesticular fat, kidney, and muscle. Example 2

 [0055] Visfatin hetero KO ES cell growth rate

 According to the method described in Examples 1 to 4 of JP-A-2004-154135, a homologous thread lacking a part of the genomic gene encoding the amino acid sequence of the mouse Visfatin gene (SEQ ID NO: 3) is replaced with an ES cell (Visfatin Gene heteroknockout ES cells) Obtained # 8-7 and # 8-8

[0056] A total of 3 clones of # 8—7, # 8—8, and one clone of wild-type ES cell RF8 were placed in 24 well plates (Gibco), 2.5 x ⁴ each, V ヽ N2 medium without insulin (F12—GMEM (Invitrogen), N2 mixture (100 μg / ml Apo—transferin (Sigma)), 50 μg / ml BSA (G¾co), 6 ng / ml Progesterone (Sigma), 16 μg / ml Putrescine (Sigma), 30 nM SodiumSelenite (Sigma)), lOng / ml humanBMP4 (R & D), 10 ³ Unit LIF (Chemicon), 1.0% L-glutamine (Invitorogen), 0.5% penicillin streptomycin (Invitorogen), 0.2% 2-mercaptoethanol (Invitorogen)), N2 medium containing insulin (Insulin (Sigma) 25 μg / ml added medium) or serum medium (DMEM (Nacalai), 10 ⁷ Units LIF (Chemicon), 1.0% L-glutamine (Invitorogen) , 0.5% penisline streptomycin (Invitorogen), 0.2% 2-mercaptoethanol (Invitorogen), 1.0% MEM non-essential amino acid solution (Invitorogen), 14.8% FCS (Biowest)) Incubate for 6 days and measure growth rate every 2 days.

 As a result, as shown in FIG. 2, there was no difference in the growth rate between wild type ES cells and # 8-7, # 8-8 ES cells in serum medium. N2 medium containing insulin In the N2 medium without insulin and insulin, the growth rate of # 8-7 and # 8-8 ES cells decreased compared to wild-type ES cells. It was remarkable in the medium. These results indicate that Visfatin is involved in promoting the proliferation of ES cells.

 Example 3

[0058] Embryonic analysis of Visfatin KO mice

Visfatin heterozygous mutant (KO) mice are mated and blastocysts (blastocysts) 3.5 days after fertilization From the female uterus, transferred to a gelatin-coated 4-well dish (manufactured by NUNC), ES medium (DMEM (Nacalai), 10 ⁷ Unit LIF (Chemicon), 1.0% L-glutamine (Invito rogen) ), 0.5% penisrin streptomycin (Invitorogen), 0.2% 2-mercaptoethanol (Invitorogen), 1.0% MEM non-essential amino acid solution (Invitorogen), 14.8% FCS (Biowest)) Culture was performed. The culture was performed for 6 days. The results are shown in Figure 3.

[0059] In the case of wild-type mice, the inner cell mass adhered to the bottom of the dish about 2nd day after the start of culture. From about 3 days after the start of the culture, proliferation of blast cyst-derived cells was observed under the microscope, and on the 2nd day, proliferation of the inner cell mass was observed in the central part. Nutritional ectoderm growth was observed to surround the inner cell mass. On the other hand, when blast cysts homo-deficient in Visfatin were cultured under the same conditions, neither the inner cell mass nor the trophectoderm grew. This result revealed that visfatin is essential for the growth of embryos (pluripotent stem cells).

 Example 4

 [0060] Changes in Visfatin protein level during ES cell differentiation

90 ng / ml retinoic acid was added to undifferentiated mouse ES cell RF8 (derived from 129ZSvJae mouse), serum medium (DMEM (Nacalai), 10 ⁷ Unit LIF (Chemicon)), 1.0% L-glutamine ( Invitorogen), 0.5% penisline streptomycin (Invitorogen), 0.2% 2-mercaptoethanol (Invitorogen), 1.0% MEM non-essential amino acid solution (Invitorogen), 14.8% FCS (Biowest)) ES cells were differentiated by culturing for 6 days. ES cells lacking the Visfatin gene heterozygously (JP-A 2004-154135) were also differentiated under the same conditions.

[0061] Undifferentiated ES cells, differentiated ES cells, undifferentiated visfatin hetero KO ES cells, and differentiated visfatin hetero KO ES cells were collected and subjected to SDS-PAGE. The protein on the SDS-PAGE gel was transferred to PVDF membrane (Millipore) by the semi-dry method. The membrane thus prepared was washed with TTBS buffer (20 mM Tris—HCl (pH 7.4), 150 mM NaCl, 0.05% Tween 20, 0.05% Na3N), and then in TTBS buffer containing 3% gelatin. And kept at 37 ° C for 1 hour. Thereafter, the membrane was treated with the rabbit anti-visfatin polyclonal antibody described in JP-A-2000-356637. 1 / 1,000 dilution with TTBS buffer containing ₀ urushi serum albumin The solution was incubated at 37 ° C for 1 hour. The membrane was then washed 3 times with TTBS buffer at room temperature for 5 minutes x 3 times in a solution of horseradish peroxidase-labeled anti-rabbit Ig G antibody diluted 1,000-fold with TTBS containing 1% ushi serum albumin. And kept at 37 ° C for 1 hour. Thereafter, the membrane was washed 3 times with TTBS buffer at room temperature for 5 minutes, and a signal derived from visfatin protein was detected using LAS-3000 (Fuji Film). The results are shown in Fig. 4.

 [0062] The visfatin protein band specifically recognized by the anti-visfatin antibody was quantified using analysis software of LAS-3000 (Fuji Film). As a result, a large amount of visfatin protein was found in undifferentiated ES cells. In contrast to the expression, the amount of protein was significantly reduced after separation. Moreover, in visfatin hetero KO ES cells, the amount of visfatin protein in an undifferentiated state was significantly reduced as compared to wild type ES cells, and further decreased by sorting. Based on the above, it was confirmed that the abundance of visfatin significantly decreased even at the protein level as ES cells differentiated.

 Example 5

 [0063] Visfatin knockdown ES cell proliferation

 l) Introduction of visfatin-RNAi retrovirus into ES cells

 The siRNA sequence for the Visfatin gene (GTTGTTGCTGCCTTGTCTT; SEQ ID NO: 9) was synthesized. This is a retroviral vector with a U6 promoter (PMOW3.3-PGK-

EGFP) (Gene Ther. 2002 Apr; 9 (8): 477-87).

[0064] 8 × 10 ⁶ Plat-E cells were seeded in a 10 cm dish and infected with the retrovirus prepared above according to the manufacturer's protocol using FuGENE6 reagent (Roche). Supernatants were collected 24 hours after infection.

Serum medium (DMEM (Nacalai), 10 ⁷ Units LIF (Chemicon), 1.0% L-glutamine (Invitrogen), 0.5% penisline streptomycin (Invitorogen), 0.2% 2-mercaptoethanol (Invitrogen) Invitorogen), 1.0% MEM non-essential amino acid solution (Invitorogen), 14.8

Mouse ES cells MG1.19 cultured in FCS (Biowest)) were infected with the retrovirus in the supernatant collected above, and the ES cells were further cultured for 20 hours.

[0065] 2) Confirmation of visfatin protein level in Visfatin-RNAi-introduced ES cells After 20 hours, the cells were collected by trypsin treatment, and the collected cells were collected in ES medium (Meiner, V 丄. Et al., Proc. Natl. Acad. Sci. USA, 93 (24): pl4041-14046 (1996)). After suspending, 1,000 cells were spread on a gelatin-coated dish in a 10 cm dish and cultured for 7 days, and after 7 days, a single colony that had grown on the dish was picked up.

 [0066] Each colony was cultured in ES medium for 3 days. After culturing, the cells were collected and subjected to SDS-PAGE. The protein on the SDS-PAGE gel was transferred to a PVDF membrane (Millipore) by the semi-dry method. The membrane thus prepared was washed with TTBS buffer (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.05% Tween20, 0.05% Na3N), and then in TTBS buffer containing 3% gelatin. Incubated at 37 ° C for 1 hour. Thereafter, the membrane was incubated at 37 ° C. for 1 hour in a solution obtained by diluting the rabbit anti-visfatin polyclonal antibody described in JP-A-2000-356637 1,000 times with TTBS buffer containing 1% ushi serum albumin. Next, the membrane was washed 3 times with TTBS buffer at room temperature for 5 minutes × 3 times, and the anti-rabbit IgG antibody labeled with horseradish peroxidase was diluted 1,000 times with TTBS containing 1% ushi serum albumin. Incubated for 1 hour at ° C. Thereafter, the membrane was washed three times with TTBS buffer at room temperature for 5 minutes, and a signal derived from visfa tin protein was detected using LAS-3000 (Fuji Film). The results are shown in FIG.

 As a result of quantifying the visfatin protein band specifically recognized by the anti-visfatin antibody using the analysis software of LAS-3000 (Fuji Film), vi sfatin of the three types of knockdown cells No. 1, 2 and 3 The amount of protein was reduced to 10% of control wild type ES cells (WT).

 [0067] 3) Visfatin knockdown Measurement of ES cell proliferation rate

Three types of knock-down ES cells and control wild-type ES cells were inserted into the serum-containing medium (DMEM (Nacalai)), 10 ⁷ Unit LIF (Chemicon), 1.0% L-glutamine (Invitorogen), 0.5% penisline streptomycin (Invitorogen), 0.2% 2-mercaptoethanol (Invitorogen), 1.0% MEM non-essential amino acids 4 days culture using the solution (Invitorogen), 14.8% FCS (Biowest)), and serum-free medium for ES cell culture N2B27 (Ying et al. Cell 115: 281-292, 2003) (SCS) did. The result is shown in Fig. 6. As a result, the growth rate of the three types of Visfatin knockdown ES cells in the serum-containing medium was significantly lower than that of the control wild-type ES cells. In the serum-free medium, the decrease in the growth rate was more remarkable, about half that of the control cells. From the above results, it was shown that the proliferation of ES cells was suppressed by reducing the protein amount of Visfatin, that is, Visfatin was involved in promoting the proliferation of ES cells.

 Example 6

 [0068] Rejuvenation of RS cells by Visfatin

 1) Preparation of pCAG- IRES-puro

 pCX-EGFP was treated with Ndel / EcoRI, and the CAG promoter part excised was inserted into the Ndel / EcoRI site of pIRE S puro (clontech).

 [0069] 2) Preparation of plasmid containing mouse VisfatinORF

 PCR was performed using a cDNA library derived from mouse liver (clontech) to amplify the sequence encoding the entire mouse Visfatin cDNA. The obtained fragment was incorporated into PCR2.1 (Invitrogen) using the TA cloning method. As PCR conditions, RNA (1 μg) of RF8 ES cells was made into a saddle and an RT product amplified by Oligo dT primer was made into a saddle. STMO-mou-S-ORF-1 (SEQ ID NO: 7; ATGAATGCTGCGGCAGAAGCCG AGTT) and STMO-mou-AS-770 (SEQ ID NO: 8; TGGTCTTTCCCCCAAGCCGTTA TGGT) were used as primers. KOD Plus (Takara) was used for the PCR extension reaction. PCR is followed by DNA denaturation (94 ° C, 10 seconds) followed by 35 cycles of denaturation (94 ° C, 2 seconds) —annealing (55 ° C, 2 seconds) and one extension (68 ° C, 1 minute 30 seconds) Further, the final elongation (68 ° C, 5 minutes) was performed.

 [0070] 3) Preparation of pCAG-visfatin- IRES-puro

 Visfatin cDNA ORF was excised from PCR2.1 using EcoRI and incorporated into pCAG-IRES-puro treated with EcoRI.

[0071] 4) Introduction of pCAG-visfatin- IRES-puro into ES cells

According to Example 2, spread a total of 3 clones of 1 clone of # 8-7, # 8-8, and wild-type ES cell RF8 in a 10 cm dish (Gibco). Next, the expression plasmid pCAG-Visfatin-IRES-puro prepared in 3) above was electroporated at 20 g per dish. Introduce into cells by one ration.

 Specifically, after treating cells with trypsin, neutralize with ES medium and pipette well until single cells are obtained. Centrifuge it at 800 rpm for 5 minutes at room temperature, discard the supernatant, and add 800 μl of PBS. Mix 20 μg of DNA (pCAG-Visfatin-IRES-puro) well into this cell suspension, immediately transfer the sample to the cuvette, and use the Bio Rad instrument Gene Pulser II model 1052105 to apply Volts ;; Electroporation is performed under the conditions of 0.25 kV, CAP; 0.5, uF xlOOO.

[0072] From the second day after electoral poration, cells that have been introduced with the Visfatin expression plasmid are selected by culturing the cells in a serum medium containing puromycin at a final concentration of 2 μg / mL for 10 days. For ES cells, Visfatin hetero KO-ES cells, and wild-type ES cells in which the Visfatin heterozygous KO-ES cells were returned to Visfatin hetero KO-ES cells as described above, 2.5xl0 ⁴ Individually spread on 24 well plates (G¾co). Then, the cells are cultured in N2 medium without insulin, N2 medium with insulin, and serum medium, respectively. Incubate cells for 6 days and measure growth rate every 2 days.

 [0073] As a result, there is no difference in the growth rate of any cell in the serum medium, but N2 medium containing insulin and N2 medium not containing insulin! / ヽ Visfatin hetero K Returning Visfatin-expressing plasmid to 0-ES cells! The growth rate of ES cells is significantly higher than the growth rate of KO-ES cells, and approaches the growth rate of wild-type ES cells. Example 7

 [0074] Recovery of ES cell proliferation by addition of Visfatin

In accordance with Example 2, a total of 3 clones of 1 clone of # 8-7, # 8-8, and wild type ES cell RF8 are spread on each 24 well plate (manufactured by G¾co) with 2.5 x ^{4 4} clones. Mouse Visfatin protein (SEQ ID NO: 4) prepared according to the method described in Examples 1 to 3 of WO2002 / 10772 was added to N2 medium containing no insulin and N2 medium containing insulin at final concentrations of 0.1, 1, and The cells are cultured in the same manner as described in Example 2 except that 10 g / mL is added. Incubate the cells for 6 days and measure the growth rate every 2 days.

N2 medium with insulin, no insulin! / ヽ N2 medium! /, Mouse Visf on misaligned medium Even when atin is added, the cell growth rate is increased compared with the case where mouse Visfatin is not added, and approaches the growth rate of wild-type ES cells.

 Industrial applicability

[0075] The present invention provides a pluripotent stem cell proliferation promoter containing visfatin or a visfatin gene as an active ingredient. By adding the growth promoter of the present invention to a serum-free medium (blood-free culture solution), pluripotent stem cells can be cultured even in the absence of serum. Therefore, the growth promoter of the present invention is effectively used in ES cell research and clinical application in regenerative medicine.

 Brief Description of Drawings

[0076] FIG. 1 shows the results of praying for the expression of visfatin gene in ES cells and mouse tissues by RT-PCR. rvisfatinj indicates the analysis result of the visfatin gene, “NATI J indicates the analysis result of NAT1, which is a positive control. Each of the RT-PCR amplification cycles was repeated 30 times. From the left side of the lane, the negative transcriptase was excluded. Control (RT-), Undifferentiated ES cell, Undifferentiated ES cell, Differentiated ES cell, Differentiated ES, Ovary, Testis, Lung, Heart, Liver, Kidney (kidney), brain (brain), spleen (spleen), thymus (thymus), small intestine (intestine ^ skin (skin), muscle (mu _SC le), mesenteric fat, brown fat, MG1.19 cells, liver (liver) and peri-testicular fat expression results are shown respectively.

 FIG. 2 is a graph showing the results of examining the proliferation rate of visfatin gene hetero knockout ES cells (# 8-7 and # 8-8). For comparison, the same experiment was performed on wild-type ES cell RF8. In the figure, A) is a serum medium, B) is an N2 medium containing no insulin, and C) is a change in the number of cells when the cells are cultured in an N2 medium containing insulin.

 FIG. 3 is a photomicrograph showing the culture results of blast cysts on day 3.5 after fertilization obtained by mating visfatin hetero KO mice. The results on the 1st, 3rd and 5th days of culture were shown. For comparison, the same experiment was performed on wild-type mice. Top: Results from bisphatin homo-deficient blast cysts. Bottom: Wild-type mouse results.

[Fig. 4] Western stamp analysis showing that the amount of visfatin protein decreases with ES cell sorting. In the figure, “Visfatin” indicates the band of visfatin protein. , “Β actinj indicates the control β-actin band.“ WT ”indicates the results for wild-type ES cells, and“ Vis +/− ”indicates the results for the visfatin gene hetero-knockout ES cells.

 [Fig. 5] Western blot analysis showing that visfatin siRNA was introduced into wild-type ES cells and the amount of visfatin protein was knocked down. In the figure, “Visfatin” indicates the band of bisfatin protein, “13 actinj indicates the band of 13 actin as a control.“ WT ”indicates the result of wild-type ES cells, and Knock down (l, 2,3) indicates The results of three types of knockdown ES cells are shown.

 FIG. 6 is a graph showing that the growth rate of Visfatin knockdown cells was suppressed. In the figure, the vertical axis indicates the number of cells, and the horizontal axis indicates the number of culture days. The left figure shows the experimental results in the presence of serum, and the right figure shows the experimental results in N2B27 serum-free medium (Ying et al. Cell 115: 281-292, 2003). WT (white bar) shows the results for wild-type ES cells, and KD (color bar) shows the results for knockdown cells.

Sequence listing free text

 The base sequence set forth in SEQ ID NO: 5 is an oligonucleotide primer designed for gene amplification.

 The nucleotide sequence set forth in SEQ ID NO: 6 is an oligonucleotide primer designed for gene amplification.

 The nucleotide sequence set forth in SEQ ID NO: 7 is an oligonucleotide primer designed for gene amplification.

 The nucleotide sequence set forth in SEQ ID NO: 8 is an oligonucleotide primer designed for gene amplification.

 The nucleotide sequence set forth in SEQ ID NO: 9 is an oligonucleotide designed for siRNA.

Claims

The scope of the claims  [1] A proliferation promoter for pluripotent stem cells containing visfatin or a visfatin gene as an active ingredient.  [2] The growth promoter according to claim 1, wherein the pluripotent stem cell is an embryonic stem cell. [3] A culture solution or culture kit for pluripotent stem cells containing visfatin as a component. [4] The culture solution or culture kit according to claim 3, wherein the pluripotent stem cells are embryonic stem cells. [5] A method for culturing pluripotent stem cells, comprising using the culture solution or culture kit according to claim 3 or 4.