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WO2006115080A1 - Agent favorisant la croissance pour cellule souche pluripotente - Google Patents

Agent favorisant la croissance pour cellule souche pluripotente Download PDF

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Publication number
WO2006115080A1
WO2006115080A1 PCT/JP2006/307898 JP2006307898W WO2006115080A1 WO 2006115080 A1 WO2006115080 A1 WO 2006115080A1 JP 2006307898 W JP2006307898 W JP 2006307898W WO 2006115080 A1 WO2006115080 A1 WO 2006115080A1
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Prior art keywords
cells
visfatin
cell
pluripotent stem
gene
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Japanese (ja)
Inventor
Shinya Yamanaka
Mirei Murakami
Masako Nishizawa
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Nara Institute of Science and Technology NUC
Sumitomo Chemical Co Ltd
Sumitomo Pharma Co Ltd
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Nara Institute of Science and Technology NUC
Sumitomo Dainippon Pharma Co Ltd
Sumitomo Chemical Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]

Definitions

  • the present invention relates to a proliferation promoter for pluripotent stem cells.
  • Embryonic stem 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.
  • Non-Patent Document 3 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).
  • 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.
  • 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.
  • serum or feeder cells are essential.
  • serum and feeder cells are essential for culturing human ES cells.
  • LIF is ineffective in human ES cells.
  • 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.
  • 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.
  • PBEF Pre-B cell colony-enhancing factor
  • 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)
  • 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.
  • 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.
  • homozygous mice were not born from crosses between heterozygous mice.
  • homozygous mutant mice were found to be lethal immediately after implantation.
  • 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.
  • 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.
  • 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.
  • a visfatin gene hetero-knockout ES cell bisfatin hetero KO-ES cell
  • serum-free medium containing no insulin a serum-free medium containing insulin
  • serum-free medium containing insulin serum-free medium containing insulin
  • 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.
  • the present invention provides:
  • a method for culturing pluripotent stem cells characterized by using the culture solution or culture kit according to (3) or (4) above,
  • pluripotent stem cell is an embryonic stem cell.
  • the visfatin and visfatin gene of the present invention are useful as a proliferation promoter for pluripotent stem cells such as ES cells.
  • pluripotent stem cells such as ES cells.
  • a serum-free medium serum-free medium
  • 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) :
  • Examples of the protein having any amino acid sequence and having the activity of promoting proliferation of pluripotent stem cells are exemplified.
  • 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.
  • 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.
  • amino acid modification 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
  • a conventional site-directed mutagenesis can be performed on the DNA encoding the amino acid sequence shown in FIG.
  • 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.
  • 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.
  • sequence identity refers to sequence identity or homology between two DNAs or two proteins.
  • sequence identity is determined by comparing two sequences that are optimally aligned over the region of the sequence to be compared.
  • 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.
  • 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.
  • sequence identity may be 80% or more, preferably 90% or more, more preferably 95% or more.
  • 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.
  • 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.
  • 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.
  • 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.
  • (H) a nucleotide sequence represented by nucleotides 96 to 1571 in the nucleotide sequence represented by SEQ ID NO: 3;
  • 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.
  • 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.
  • 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.
  • 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.
  • examples of the vector include plasmid vectors such as pUC118, pUC119, pBR322, pCR3, and pETlla, and phage vectors such as ⁇ and ⁇ gtll.
  • examples of the vector include pYES2, pYEUra3 and the like.
  • the host is an insect cell, pAcSGHisNT-A and the like can be mentioned.
  • 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.
  • 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.
  • a sequence expressed as a fusion protein with thioredoxin, His tag, or GST may be added to facilitate isolation and purification.
  • a GST fusion protein vector such as pGEX4T
  • an appropriate promoter such as lac, tac, trc, tr p, CMV, or SV40 early promoter
  • 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)
  • a transformant By transforming a host with the visfatin gene expression vector prepared above, a transformant (transformed cell) containing the expression vector can be prepared.
  • the host used here include Escherichia coli, yeast, insect cells, animal cells and the like.
  • E. coli include E. coli K-12 strains HB101 strain, C600 strain, JM109 strain, DH5 ⁇ strain, AD494 (DE3) strain, and the like.
  • yeast include Saccharomyces cerevisiae.
  • 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.
  • 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.
  • 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.
  • the transformant can be cultured by a usual method used for culturing microorganisms, yeast, insect cells or mammalian cells.
  • 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.
  • the purification means include salting out, ion exchange chromatography, adsorption chromatography, affinity chromatography, gel filtration chromatography, and the like.
  • 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.
  • Expression vector expressing human visfatin protein (SEQ ID NO: 2) prepared as described above 1.
  • Transform mammalian cells such as COS-1.
  • OPTI-MEM medium containing antibiotics
  • human visfatin is secreted into the medium.
  • 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).
  • 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).
  • 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- ⁇ -D-thiogalatatopyranoside (hereinafter referred to as IPTG) at a final concentration of ImM was added. And further overnight culture.
  • IPTG isopropyl mono- ⁇ -D-thiogalatatopyranoside
  • the cells were then collected by centrifugation, and the cells were collected from lOOmM Tris-hydrochloric acid ( ⁇ 7.6), 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).
  • buffer A a buffer
  • sonicated 3 times for 5 minutes under ice-cooling
  • 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.
  • 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.
  • Embryonic Germ Cell derived from primordial germ cells
  • mGS cell mutipotent germline stem cell isolated from testis
  • bone marrow Examples include multipotent adult progenitor cells (MAPC) that are released.
  • MPC multipotent adult progenitor cells
  • 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.
  • 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.
  • the proliferation promoting activity of pluripotent stem cells possessed by visfatin or visfatin gene can be measured, for example, as follows.
  • 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 ⁇ g / 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.
  • the same pluripotent stem cells are cultured using a serum medium (positive control cells) and compared with the positive control cells.
  • a serum medium positive control cells
  • 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.
  • 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.
  • 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.
  • mouse 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).
  • 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.
  • 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.
  • 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; G3 ⁇ 4co-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.
  • 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.
  • 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.
  • 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 ⁇ g / ml.
  • the concentration is preferably about 0.1 ⁇ g / ml to 10 g / ml.
  • 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.
  • Embryonic Germ Cell derived from primordial germ cells
  • mGS cell mutipotent germline stem cell isolated from testis
  • MPC Multipotent adult progenitor cell isolated from bone marrow, etc.
  • the origin of these pluripotent stem cells may be derived from humans, monkeys, rats, mice, marmosets, and the like.
  • 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.
  • 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.
  • 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.
  • Transfection into cells can be performed using either a non-viral introduction method or a viral introduction method.
  • non-viral introduction method examples 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).
  • methods using a recombinant virus include a method using a virus vector such as a retrovirus, an adenovirus, and an adeno-associated virus.
  • 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.
  • ES cell RF8 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.
  • 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).
  • 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).
  • REVERTRA ACE a Toyobo Co., Ltd.
  • 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)).
  • mice 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.
  • 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 ⁇ N2 medium without insulin (F12—GMEM (Invitrogen), N2 mixture (100 ⁇ g / ml Apo—transferin (Sigma)), 50 ⁇ g / ml BSA (G3 ⁇ 4co), 6 ng / ml Progesterone (Sigma), 16 ⁇ g / 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 ⁇ g
  • 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.
  • ES cell RF8 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.
  • 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.
  • 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.
  • 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.
  • 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-
  • 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
  • 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.
  • TTBS buffer 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.05% Tween20, 0.05% Na3N
  • 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.
  • 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.
  • 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).
  • 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.
  • RNA (1 ⁇ g) 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
  • STMO-mou-AS-770 SEQ ID NO: 8; TGGTCTTTCCCCCAAGCCGTTA TGGT
  • 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.
  • Visfatin cDNA ORF was excised from PCR2.1 using EcoRI and incorporated into pCAG-IRES-puro treated with EcoRI.
  • 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).
  • the expression plasmid pCAG-Visfatin-IRES-puro prepared in 3) above was electroporated at 20 g per dish. Introduce into cells by one ration.
  • 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 ⁇ g / mL for 10 days.
  • a serum medium containing puromycin at a final concentration of 2 ⁇ g / mL for 10 days.
  • 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 (G3 ⁇ 4co). 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.
  • 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 G3 ⁇ 4co) 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.
  • the present invention provides a pluripotent stem cell proliferation promoter containing visfatin or a visfatin gene as an active ingredient.
  • 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.
  • 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.
  • 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.
  • A) is a serum medium
  • B) is an N2 medium containing no insulin
  • 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.
  • “Visfatin” indicates the band of visfatin protein.
  • “ ⁇ actinj indicates the control ⁇ -actin band.
  • “ WT ” indicates the results for wild-type ES cells
  • “ 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.
  • “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
  • 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.
  • 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
  • KD color bar
  • 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.

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Abstract

La présente invention concerne un agent favorisant la croissance destiné à une cellule souche pluripotente, comprenant de la visfatine ou un gène de visfatine comme principe actif ; un milieu de culture ou un kit de culture pour une cellule souche pluripotente, comprenant de la visfatine comme composant ; et un procédé de culture d'une cellule souche pluripotente utilisant le milieu de culture ou le kit de culture.
PCT/JP2006/307898 2005-04-19 2006-04-14 Agent favorisant la croissance pour cellule souche pluripotente Ceased WO2006115080A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPWO2008133141A1 (ja) * 2007-04-24 2010-07-22 東洋紡績株式会社 オスモチン組換えタンパク質およびその製造方法、並びにその利用
JP2011505814A (ja) * 2007-12-14 2011-03-03 ビオエヌテヒ・アクチエンゲゼルシャフト 体細胞を再プログラムするためのｒｎａの使用
WO2015112581A1 (fr) * 2014-01-21 2015-07-30 The Medical College Of Wisconsin, Inc. Procédés d'inhibition sélective de cellules souches pluripotentes

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
FURUHARA A. ET AL.: "Visfatin: A protein secreated by visceral fat that mimics the effects of insulin", SCIENCE, vol. 307, 2005, pages 426 - 430, XP003001023 *
SAMAL B. ET AL.: "Cloning and characterization of the cDNA encoding a novel mutant pre-B-cell colony-enhancing factor", MOL. CELL. BIOL., vol. 14, no. 2, 1994, pages 1431 - 1437, XP002947539 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPWO2008133141A1 (ja) * 2007-04-24 2010-07-22 東洋紡績株式会社 オスモチン組換えタンパク質およびその製造方法、並びにその利用
JP2011505814A (ja) * 2007-12-14 2011-03-03 ビオエヌテヒ・アクチエンゲゼルシャフト 体細胞を再プログラムするためのｒｎａの使用
WO2015112581A1 (fr) * 2014-01-21 2015-07-30 The Medical College Of Wisconsin, Inc. Procédés d'inhibition sélective de cellules souches pluripotentes
US10316287B2 (en) 2014-01-21 2019-06-11 The Medical College Of Wisconsin, Inc. Methods for selective inhibition of pluripotent stem cells
US10808222B2 (en) 2014-01-21 2020-10-20 The Medical College Of Wisconsin, Inc. Methods for selective inhibition of pluripotent stem cells
US11959096B2 (en) 2014-01-21 2024-04-16 The Medical College Of Wisconsin, Inc. Methods for selective inhibition of pluripotent stem cells

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