WO2012057052A1 - Utilisation de la peptidyl-prolyle isomérase pin1 pour contrôler la préservation et le renouvellement stables de cellules souches - Google Patents

Utilisation de la peptidyl-prolyle isomérase pin1 pour contrôler la préservation et le renouvellement stables de cellules souches Download PDF

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WO2012057052A1
WO2012057052A1 PCT/JP2011/074384 JP2011074384W WO2012057052A1 WO 2012057052 A1 WO2012057052 A1 WO 2012057052A1 JP 2011074384 W JP2011074384 W JP 2011074384W WO 2012057052 A1 WO2012057052 A1 WO 2012057052A1
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pin1
cells
stem cells
pluripotent stem
peptidylprolyl isomerase
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明秀 梁
真由子 西
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公立大学法人横浜市立大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/533—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving isomerase
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    • 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
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
    • C12N2501/405—Cell cycle regulated proteins, e.g. cyclins, cyclin-dependant kinases
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/99—Isomerases (5.)

Definitions

  • the present invention relates to the use of peptidylprolyl isomerase Pin1 to control stable maintenance and replication of stem cells.
  • ES cells embryonic stem cells
  • ES cells are isolated from embryonic cells that are the origin of life. Therefore, ethical problems arise in its clinical use.
  • Yamanaka et al. Succeeded in reprogramming fibroblasts directly into induced pluripotent stem (iPS) cells by transducing four transcription factors (Oct3 / 4, Sox2, Klf-4, c-Myc).
  • iPS induced pluripotent stem
  • the current method is problematic in that the stem cells (iPS cells) prepared have low stability and the dedifferentiation efficiency is extremely low.
  • gene transfer methods such as retroviral vectors are used, genes are randomly introduced into the genome of the cell, and as a result, gene mutations such as activation of endogenous oncogenes occur due to gene insertion. The possibility of becoming malignant has been pointed out.
  • it is considered to be extremely important to develop a method for effectively controlling the maintenance and proliferation of stem cells and a method for efficiently removing cancerous cells.
  • iPS cells Stem cells prepared by a conventional method are difficult to replicate while maintaining undifferentiation, and have a problem that their efficiency of initialization and dedifferentiation is extremely low.
  • An object of the present invention is to provide a technique capable of establishing, maintaining and replicating iPS cells with high efficiency and stability.
  • the present inventors dramatically increased iPS cells by co-expressing peptidylprolyl isomerase Pin1 in addition to the four transcription factors (Oct3 / 4, Sox2, Klf-4, c-Myc) used in the conventional method. Succeeded in raising the efficiency of the establishment of In iPS cells, when Pin1 expression was suppressed by Pin1-specific inhibitors, iPS colony formation ability was suppressed, and when iPS colonies were treated with Pin1 inhibitors, abnormal differentiation was observed. Furthermore, we found that Oct4 is a Pin1 substrate, and Pin1 binds to the Serine 12 proline site of Oct4 to increase the stability of Oct4 protein, thereby enhancing the function of Oct4 as a transcription factor. The present invention has been completed based on these findings. The gist of the present invention is as follows.
  • a method for producing an induced pluripotent stem cell from a somatic cell comprising a step of enhancing the expression of peptidylprolyl isomerase Pin1 in the somatic cell.
  • (3) A somatic cell reprogramming promoter comprising a substance that enhances the expression of peptidylprolyl isomerase Pin1 and / or peptidylprolyl isomerase Pin1 in somatic cells.
  • a drug that suppresses self-renewal of pluripotent stem cells comprising a substance that inhibits peptidylprolyl isomerase Pin1.
  • a drug that destroys the pluripotency of pluripotent stem cells including a substance that inhibits peptidylprolyl isomerase Pin1.
  • An agent for killing pluripotent stem cells and / or cancer stem cells comprising a substance that inhibits peptidylprolyl isomerase Pin1.
  • a stabilizer for Oct4 protein which contains a peptidylprolyl isomerase Pin1 and / or Pin1 gene.
  • a transcriptional activity enhancer for Oct4 protein comprising peptidylprolyl isomerase Pin1 and / or Pin1 gene.
  • An induced pluripotent stem cell produced by the method according to (1).
  • a method for removing undifferentiated cells from differentiation-treated pluripotent stem cells comprising the step of suppressing peptidylprolyl isomerase Pin1.
  • a method for promoting somatic cell reprogramming comprising a step of enhancing the expression of peptidylprolyl isomerase Pin1 in somatic cells.
  • a method for activating pluripotent stem cell self-replication which comprises the step of enhancing the expression of peptidylprolyl isomerase Pin1 in somatic cells.
  • a method for maintaining the pluripotency of pluripotent stem cells comprising a step of enhancing the expression of peptidylprolyl isomerase Pin1 in somatic cells.
  • (21) A method for killing pluripotent stem cells and / or cancer stem cells using a substance that inhibits peptidylprolyl isomerase Pin1.
  • (22) A method for stabilizing Oct4 protein using peptidylprolyl isomerase Pin1 and / or Pin1 gene.
  • (23) A method for enhancing the transcriptional activity of Oct4 protein using peptidylprolyl isomerase Pin1 and / or Pin1 gene.
  • the present invention it has become possible to stably and easily culture a large amount of stem cells while maintaining an undifferentiated state.
  • the present invention provides a reprogramming promoter for somatic cell dedifferentiation (iPS cell conversion), which has been extremely low in efficiency, so that stem cells can be prepared more efficiently than in the past.
  • iPS cell conversion somatic cell dedifferentiation
  • Pin1 is highly expressed in human iPS cells.
  • A Immunoblot analysis of Oct4, SOX2 and Pin1 in MRC5 and MRC5-derived iPS cells. Actin was used as a control.
  • B Immunofluorescence staining analysis of Pin1 and SOX2 in human iPS cells. Immunofluorescence staining for SOX2 (red) or Pin1 (green) is shown by phase contrast microscopy. Nuclei were stained with 4 ′, 6-diamidino-2-phenylindole® (DAPI) ® (blue). Note that Pin1 is highly expressed in SOX2-positive cells.
  • DAPI 6-diamidino-2-phenylindole®
  • C, D and Pin1 promotes the induction of iPS cells by “Reprogramming 4 factors” (4F; A photograph showing a representative colony stained with alkaline phosphatase (C). The number of colonies positive for alkaline phosphatase staining was measured by three independent experiments (D). Note that the co-induction of Pin1 and 4F increases the frequency of iPS colony formation.
  • Teratoma tissue derived from human iPS induced by E, 4F and Pin1. Cells were injected subcutaneously into NOD-SCID mice. Representative photographs of tumors stained with hematoxylin and eosin are shown. Loss of self-renewal ability of human iPS cells due to Pin1 inhibition.
  • A-C and human iPS cells were dissociated with actinase and seeded at isolated concentrations on feeder cells supplemented with various concentrations of Juglone. Colony formation was confirmed with a phase contrast microscope (A). The number of colonies was counted 3 days after Juglone treatment (B). The number of cells per colony was counted by DAPI staining (C). Data are shown as mean values ⁇ SEM. D, Human iPS cells were seeded at various concentrations in the presence of Juglone at various concentrations, and alkaline phosphatase staining was performed.
  • E, F, human iPS cells are dissociated with actinase and isolated in the presence of 50 mg / ml of Pin1-inhibited phosphorylated peptide PINTIDE (RRRRRRRRRWFYpSPR) (SEQ ID NO: 5) or non-phosphorylated peptide (RRRRRRRRRWFYAPR) (SEQ ID NO: 6) Seeded in a feeder-free dish at the indicated concentration (E). (F) from which the number of alkaline phosphatase positive colonies was counted. Data are shown as mean values ⁇ SEM. Pin1 inhibition in mouse ES cells suppresses colony formation. A.
  • BDF2DFand R1 Two mouse ES cell lines (BDF2DFand R1) were seeded on a gelatin-coated dish and treated with DMSO or Juglone (10 ⁇ M). Colonies were stained with alkaline phosphatase (red). B-D and mouse ES cells (R1) were infected with an adenoviral vector encoding GFP or GFP-dominant negative (dn) -Pin1 (3000 viral particles / cell). The cells were stained with alkaline phosphatase (red) and DAPI (blue) and observed by immunofluorescence staining. The total number of colonies and the number of cells per colony were counted. Data are shown as mean values ⁇ SEM.
  • Inhibition of Pin1 causes abnormal differentiation of human iPS cells.
  • A Human iPS cells were cultured for 5 days until colonies were formed, and treated with DMSO® or Juglone® (10 ⁇ M) for 3 days. Thereafter, cells were stained with alkaline phosphatase (red). Representative images of phase contrast microscopy and immunofluorescence staining analysis are shown.
  • B Human iPS cells were cultured for 5 days until colonies were formed, and infected with an adenoviral vector encoding GFP or GFP-dominant negative (dn) -Pin1 (3000 viral particles / cell). The cells were stained with alkaline phosphatase (red) and DAPI (blue) and observed by immunofluorescence staining.
  • Pin1-binding protein in human iPS cells A, B, human iPS sputum cell lysates were immunoprecipitated using non-immune control mouse IgG (IgG) or mouse anti-Pin1 monoclonal antibody. Proteins bound to protein A / G agarose beads were subjected to SDS-PAGE and silver staining was performed (A). “M” indicates a protein marker. The excised gel band was digested with trypsin, analyzed by linear ion trap (LTQ) Orbitrap hybrid mass spectrometer, and protein was identified by peptide mass fingerprinting (PMF), Mascot, and Aldente search algorithms (B). Pin1 interacts with phosphorylated Oct4 and promotes its transcriptional activity.
  • IgG non-immune control mouse IgG
  • M indicates a protein marker.
  • the excised gel band was digested with trypsin, analyzed by linear ion trap (LTQ) Orbitrap hybrid mass spectrometer, and protein
  • CIP Dephosphorylation
  • GST pull-down analysis using GST ⁇ or GST-Pin1
  • immunoblot analysis with anti-Oct4 antibody was performed (upper panel). Coomassie staining of GST or GST-Pin1 used in this assay is shown in the lower panel.
  • B Human iPS cells were fixed with 4% paraformaldehyde, and double-stained with anti-Oct4 monoclonal antibody (green) and anti-Pin1 polyclonal antibody (red). Then, it analyzed with the confocal microscope.
  • C HeLa cells were transfected with the indicated vector and HA-LacZ, and collected at the indicated time after cycloheximide (CHX) treatment. Immunoblot analysis was performed using anti-Oct4, anti-Pin1 and anti-HA antibodies (left panel). Quantitative data is shown in the right panel.
  • D The Oct-SOX reporter gene and pRL-CMV were transiently expressed together with Oct4, SOX2 or Pin1 in HeLa cells. Cells were harvested 24 hours after transfection and a gene reporter assay was performed.
  • E HeLa cells were transiently co-expressed with Oct4-SOX2 and Oct4-SOX reporter gene and wild-type Pin1 or W34A or K63A mutant.
  • FIG. 1 Schematic diagram of the Oct4-deficient mutant produced in this study (left panel). HeLa cells were transfected with the indicated Oct4-deficient mutant for 24 hours. Sputum cell lysates were subjected to GST pull-down analysis using GST or GST-Pin1, and immunoblot analysis was performed using Oct4 antibody (right panel). B, Amino acid sequence alignment of human, rabbit, mouse and rat Oct4 proteins. The conserved Ser12-Pro motif is shown enclosed. C, HeLa cells were transfected with Oct4 site-specific mutant Oct4-S12A, and GST pull-down analysis was performed.
  • Pin1 is highly expressed in cancer stem cells in human breast cancer tissue. Paraffin sections of human breast cancer patient tissues were immunostained using anti-Pin1 antibody. Pin1 is brown, the cancer stem cell marker CD44 is blue, and the cell outline is light green. High expression of brown stained Pin1 is seen in CD44 positive cancer stem cells. A, Pin1 inhibition induces cell death of cancer stem cells.
  • the mammary epithelial cell line MCF-10A and the cancer stem cell line derived from this cell (CSC10A) were each treated with 10 ⁇ M of Juglone, and apoptosis was detected 24 hours later by the Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method (brown ).
  • TUNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling
  • Juglone was administered to breast cancer stem cells (CSC10A), MCF-10A-Ras cells, and MCF-7 cells at 5 ⁇ M or 10 ⁇ M, and cell survival was confirmed 72 hours later using Cell Counting Kit-8 (Dojindo Laboratories, # CK07) did.
  • CSC breast cancer stem cells
  • MCF-10A-Ras cells MCF-7 cells
  • cell survival was confirmed 72 hours later using Cell Counting Kit-8 (Dojindo Laboratories, # CK07) did.
  • CSC cell death due to Juglone is observed compared to the other two cells.
  • the present invention provides a method for producing induced pluripotent stem (iPS) cells from somatic cells, which comprises a step of enhancing the expression of peptidylprolyl isomerase Pin1 in somatic cells.
  • iPS induced pluripotent stem
  • Examples of methods for enhancing the expression of peptidylprolyl isomerase Pin1 in somatic cells include the following methods.
  • peptidylprolyl isomerase Pin1 gene into somatic cells. Introduce peptidylprolyl isomerase Pin1 protein into somatic cells. Introduce peptidylprolyl isomerase Pin1 mRNA into somatic cells. -Treating somatic cells with protein kinase C inhibitor or protein kinase A inhibitor activates the function of peptidylprolyl isomerase Pin1.
  • protein kinase C inhibitor include Calphostin C, Polymixin B, Rottlerin, Y-27632, PD 173074, GF 109203X, and the like.
  • protein kinase A inhibitors include Staurosporine, SP600125, Apigenin, LY 294002, KT 5823, and KT5720.
  • ⁇ Pin1 expression is induced by administration of cell growth factor.
  • cell growth factors include epidermal growth factor, fibroblast growth factor, vascular endothelial growth factor and the like.
  • ⁇ Pin1 expression is induced by expressing the transcription factor E2F.
  • somatic cell is not particularly limited, and any animal species (eg, humans and mice, rats, rabbits, pigs, cats, sheep, horses, goats, birds, etc.) of any age (eg, fetus, newborn, adult, etc.)
  • Non-human animals from any tissue (eg, nerve, hematopoiesis, dental pulp, skin, liver, stomach, intestine, spleen, pancreas, brain, lung, kidney, etc.) (eg, fibroblasts, epithelial cells, Nerve cells, blood cells, muscle cells, mesothelial cells, etc.).
  • Somatic cells may be differentiated cells or undifferentiated cells such as stem cells and progenitor cells.
  • any of a primary culture cell, a subculture cell, and a cell line may be sufficient.
  • Pin1 may be transduced into Pin 1 simultaneously with or before the transduction of factors necessary for reprogramming of the somatic cells.
  • Pin1 protein may be introduced into somatic cells at the same time as or before or after the transduction of factors necessary for reprogramming of somatic cells.
  • Pin1 binds to a phosphorylated Ser / Thr-Pro ⁇ ⁇ ⁇ ⁇ motif and regulates the function of phosphorylated proteins by isomerizing the protein structure into cis and trans via the peptide bond Type regulator.
  • This novel “post-phosphorylation” regulatory mechanism is known to play an important role in the functional expression of phosphorylated proteins by altering target protein activity, protein-protein binding, subcellular localization, and stability. It has been.
  • Pin1 binds to a phosphorylated protein and its structure changes, so that ON / OFF of other post-translational modification switches such as ubiquitination and SUMOylation is regulated.
  • Pin1 has been shown to play an extremely important role in the pathogenesis of intractable diseases such as cancer, immune diseases and neurodegenerative diseases. Phosphoproteins targeted by Pin1 vary widely and differ depending on the cell and tissue differences. Moreover, even in the same cell / tissue, the repertoire varies greatly between the normal state and the disease state due to the different phosphorylation state of the substrate. We have now found that Pin1 regulates stem cell self-renewal and pluripotency maintenance by using Oct4 as a substrate in pluripotent stem cells.
  • the DNA is incorporated into a vector capable of expressing DNA encoding factors necessary for somatic cell reprogramming and introduced into somatic cells.
  • a vector in which the DNA encoding Pin1 is expressed can be introduced into the somatic cells. All of the DNA encoding factors necessary for somatic cell reprogramming may be incorporated into a single vector and introduced into somatic cells, or DNA encoding each factor required for somatic cell reprogramming may be introduced. It may be incorporated into another vector and introduced into somatic cells.
  • DNA encoding Pin1 may be introduced into a somatic cell by incorporating it into a vector incorporating a DNA encoding a factor necessary for somatic cell reprogramming, or a factor necessary for somatic cell reprogramming. May be incorporated into a vector different from the vector into which the DNA encoding is incorporated and introduced into somatic cells.
  • vectors capable of expressing DNA encoding factors required for reprogramming of somatic cells and vectors encoding Pin1 include viral vectors, plasmids, artificial chromosomes, etc., but viral vectors (for example, , Retrovirus vectors, lentivirus vectors, adenovirus vectors, adeno-associated virus vectors, Sendai virus vectors, and the like), and retrovirus vectors are more preferable.
  • retrovirus vector pMXs retrovirus vector, pBabe retrovirus vector, pRetro retrovirus vector and the like can be used.
  • the vector may contain elements such as promoters, enhancers, transcription terminators, initiation codons, splicing signals, polyadenylation sites, stop codons, and other gene expression regulatory sequences, cloning sites, drug resistance genes, reporter genes and the like.
  • Pin1 The DNA sequence and amino acid sequence information of Pin1 can be obtained from the database GenBank accession numbers Human Pin1 NM006221, Mouse Pin1 NM023371, Rat Pin1 NM00110670.
  • the DNA sequence and amino acid sequence of human Pin1 are shown in SEQ ID NOs: 1 and 2.
  • Pin1 may be either a natural type or a mutant as long as the object of the present invention is achieved.
  • somatic cells In order to transduce somatic cells with factors necessary for reprogramming somatic cells and Pin1, known techniques such as microinjection, liposome, lipofection, electroporation, calcium phosphate method, and viral infection can be used. After transducing factors or Pin1 necessary for somatic cell reprogramming into somatic cells, the cells may be collected, replated on feeder cells, and cultured. As the feeder cells, mouse fibroblasts, SNL76 / 7, human mesenchymal cells and the like can be used.
  • ecotropic receptors that infect only rodents are targeted to target cells (for example, it may be introduced into a human somatic cell), and an ecotropic retrovirus may be used to introduce a factor or Pin1 necessary for somatic cell reprogramming.
  • Infecting target cells by transfecting retroviral vectors into packaging cells such as PLAT-E cells (designed to express ecotropic virus-derived envelope glycoproteins (env)) Can be increased.
  • ES cell-specific marker genes (Oct3 / 4, Nanog, Lin28, PH34, Dnmt3b, Noda1, SSEA3, SSEA4, Tra-1-60, Tra-1-81 , Alkaline phosphatase, etc.) expression, semi-permanent cell growth, differentiation pluripotency (trioderm formation), etc. (Cell ⁇ ⁇ 131: 861-872 (2007)).
  • the method used for ES cells may be applied.
  • the present invention also provides an induced pluripotent stem cell produced by the above method.
  • the present invention provides a somatic cell reprogramming promoter comprising a substance that enhances the expression of peptidylprolyl isomerase Pin1 in somatic cells and / or peptidylprolyl isomerase Pin1.
  • the present invention also provides a method for promoting somatic cell reprogramming, which comprises the step of enhancing the expression of peptidylprolyl isomerase Pin1 in somatic cells.
  • the substance that enhances the expression of peptidylprolyl isomerase Pin1 in somatic cells may be the Pin1 gene.
  • the Pin1 gene should be incorporated into the vector.
  • the vector may be a vector capable of expressing DNA encoding Pin1, and such a vector has been described above.
  • Pin1 also plays an important role in the self-renewal and maintenance of pluripotency of pluripotent stem cells (eg, iPS cells, ES cells (Embryonic® Stem® Cell), embryonic stem cells, hematopoietic stem cells, neural stem cells and cancer stem cells). all right.
  • pluripotent stem cells eg, iPS cells, ES cells (Embryonic® Stem® Cell), embryonic stem cells, hematopoietic stem cells, neural stem cells and cancer stem cells.
  • the present invention also provides a substance that enhances the expression of peptidylprolyl isomerase Pin1 in somatic cells and / or a drug that activates self-renewal of pluripotent stem cells, including peptidylprolyl isomerase Pin1.
  • the present invention also provides a method for activating pluripotent stem cell self-replication, which comprises the step of enhancing the expression of peptidylprolyl isomerase Pin1 in somatic cells.
  • the present invention provides a substance that enhances the expression of peptidylprolyl isomerase Pin1 in somatic cells and / or an agent that maintains the pluripotency of pluripotent stem cells, including peptidylprolyl isomerase Pin1.
  • the present invention also provides a method for maintaining the pluripotency of pluripotent stem cells, comprising the step of enhancing the expression of peptidylprolyl isomerase Pin1 in somatic cells.
  • pluripotent stem cells that are unnecessary after differentiation induction can be killed.
  • a substance that inhibits peptidylprolyl isomerase Pin1 to suppress self-renewal of pluripotent stem cells or destroy pluripotent stem cells specifically kill pluripotent stem cells Can be made.
  • Pin1 inhibitors can also kill cancer stem cells.
  • the present invention provides a drug that suppresses self-renewal of pluripotent stem cells, including a substance that inhibits peptidylprolyl isomerase Pin1.
  • the present invention also provides a method for suppressing self-renewal of pluripotent stem cells using a substance that inhibits peptidylprolyl isomerase Pin1.
  • the present invention also provides an agent that destroys the pluripotency of pluripotent stem cells, including a substance that inhibits peptidylprolyl isomerase Pin1.
  • the present invention also provides a method for destroying the pluripotency of pluripotent stem cells using a substance that inhibits peptidylprolyl isomerase Pin1.
  • the present invention provides an agent for killing pluripotent stem cells and / or cancer stem cells, comprising a substance that inhibits peptidylprolyl isomerase Pin1.
  • the present invention also provides a method of killing pluripotent stem cells and / or cancer stem cells using a substance that inhibits peptidylprolyl isomerase Pin1.
  • Substances that inhibit Pin1 include Juglone (5-Hydroxy-1,4-naphthlenedione; 5-Hydroxy-p-naphthoquinone), PPIase-Parvulin Inhibitor (Diethyl-1,3,6,8-tetrahydro-1,3, 6,8-tetraoxobenzo [lmn] [3,8] phenanthroline-2,7-diacetate; PiB), Pin1 inhibitory peptide (eg, PINTIDE), vector incorporating dominant negative Pin1 that suppresses endogenous Pin1 , Anti-Pin1 antibody, siRNA, miRNA, antisense RNA, antisense oligonucleotide, ribozyme and the like.
  • a substance that inhibits Pin1 may be added, introduced, or transduced into pluripotent stem cells and / or cancer stem cells by a known method.
  • the present invention also provides a method for removing undifferentiated cells from differentiated pluripotent stem cells, the method comprising a step of inhibiting peptidylprolyl isomerase Pin1.
  • the differentiation treatment of pluripotent stem cells can be performed by a known method.
  • a substance that inhibits Pin1 may be brought into contact with differentiated pluripotent stem cells.
  • a substance that inhibits Pin1 is added to, introduced into, or transduced into pluripotent stem cells. To do. By suppressing Pin1, pluripotent stem cells will be killed and removed.
  • Pin1 binds to Oct4 and contributes to stabilization of Oct4 protein or enhances transcriptional activity of Oct4 protein.
  • the present invention provides a stabilizer for Oct4 protein containing peptidylprolyl isomerase Pin1 and / or Pin1 gene.
  • the present invention also provides a method for stabilizing Oct4 protein using peptidylprolyl isomerase Pin1 and / or Pin1 gene.
  • the present invention also provides an agent for enhancing the transcriptional activity of Oct4 protein, which contains a peptidylprolyl isomerase Pin1 and / or Pin1 gene.
  • the present invention also provides a method for enhancing the transcriptional activity of Oct4 protein using peptidylprolyl isomerase Pin1 and / or Pin1 gene.
  • the Pin1 and / or Pin1 gene may be contacted with Oct4 protein, a cell expressing Oct4 protein, or the like for the purpose of stabilizing Oct4 protein or enhancing the transcription activity of Oct4 protein.
  • Cells expressing Oct4 protein can be any cell (eg, fibroblast, epithelium) from any tissue (eg, nerve, hematopoiesis, dental pulp, skin, liver, stomach, intestine, spleen, pancreas, brain, lung, kidney, etc.) Cell, nerve cell, blood cell, muscle cell, endocrine cell, skin keratinocyte).
  • the cells that express Oct4 protein may be differentiated cells or undifferentiated cells such as stem cells and progenitor cells. Moreover, any of a primary culture cell, a subculture cell, and a cell line may be sufficient.
  • the present invention provides a method for screening a substance effective for at least one of the following using the inhibitory activity against peptidylprolyl isomerase Pin1 as an index.
  • pluripotent stem cells 1) Inhibits self-renewal of pluripotent stem cells. 2) destroy the pluripotency of pluripotent stem cells. 3) Kill pluripotent stem cells and / or cancer stem cells.
  • the “inhibitory activity against peptidylprolyl isomerase Pin1” as an index in the screening method of the present invention is any of the effect of suppressing the expression of Pin1 gene, the effect of suppressing the expression of Pin1 protein, and the effect of inhibiting the enzyme activity of Pin1. There may be.
  • the screening method of the present invention can be performed using cultured cells.
  • cells are cultured in the presence or absence of a test substance, and the expression level of Pin1 gene or Pin1 protein is measured. If the expression level of Pin1 gene or Pin1 protein in cells cultured in the presence of the test substance is reduced compared to cells cultured in the absence of the test substance, the test substance is the above 1) It can be determined that at least one of (3) to (3) is effective.
  • Expression level of Pin1 gene is determined by nucleic acid hybridization method, RT-PCR method, real-time PCR method, subtraction method, differential display method, differential hybridization method, cross hybridization method, northern blot hybridization method, RNA protection method Etc. can be measured.
  • the expression level of Pin1 protein can be measured by Western blotting, dot blotting, slot blotting, ELISA, RIA, mass spectrometry, or two-dimensional electrophoresis.
  • the enzyme activity of Pin1 may be measured by culturing cells in the presence or absence of a test substance. If the enzyme activity of Pin1 in cells cultured in the presence of the test substance is reduced compared to the cells cultured in the absence of the test substance, the test substance is as described in 1) to 3) above. It can be determined that at least one is effective.
  • the enzymatic activity of Pin1 is Zhang Y, Fussel S, Reimer U, Schutkowski M, Fischer G. Substrate-based design of reversible Pin1 inhibitors. Biochemistry. 2002 Oct 1; 41 (39): 11868-77. PubMed PMID: 122 (# 5 Zhang Y. Biochemistry 2002).
  • the cells used in the screening method of the present invention may be any cells that express Pin1, and examples include HeLa, 293T, COS-7, HOS, U2OS, MCF-7, PC3, LNCaP, MCF-10A, and the like. .
  • the screening method of the present invention can also be performed using Pin1 protein.
  • the enzyme activity of Pin1 protein is measured in the presence or absence of the test substance. If the enzyme activity of Pin1 in the presence of the test substance is reduced compared to the enzyme activity in the absence of the test substance, the test substance is at least one of the above 1) to 3) Can be determined to be effective.
  • the method for measuring the enzyme activity of Pin1 was described above.
  • the test substance may be any substance, for example, protein, peptide, polysaccharide, oligosaccharide, monosaccharide, lipid, low molecular weight compound, nucleic acid (DNA, RNA, oligonucleotide, mononucleotide, etc.), etc. it can.
  • These substances may be natural products, chemically or biochemically synthesized products, or may be products produced by genetic engineering.
  • Example 1 (wrap up) There are two important features of stem cells: self-renewal and pluripotency.
  • peptidylprolyl isomerase Pin1 is important for pluripotent stem cell self-renewal and pluripotency maintenance.
  • Proteomic analysis also revealed that Pin1 is involved in the transcription factor Oct4 and other substrates. It was found that the expression of Pin1 increases with the induction of iPS cells, and that the induction efficiency of iPS cells increases when Pin1 is expressed together with Yamanaka 4 factors.
  • iPS cells when Pin1 expression was suppressed by Pin1-specific inhibitors, iPS colony formation ability was suppressed, and when iPS colonies were treated with Pin1 inhibitors, abnormal differentiation was observed.
  • Oct4 is a Pin1 substrate, and Pin1 binds to the Serine 12 proline site of Oct4 to increase the stability of Oct4 protein, thereby enhancing the function of Oct4 as a transcription factor.
  • Pin1 plays an important role in the maintenance and induction of pluripotent stem cells, and uses Pin1 as a molecular switch as a tool to control proliferation and cell death of pluripotent stem cells Is also expected.
  • Stem cells are characterized by their ability to self-renew, and self-replication occurs with cell division.
  • Stem cells have pluripotency capable of differentiating into various cells. Proliferation of pluripotent stem cells is caused by activation of several transcription factors such as c-Myc, Klf4, Oct4, and SOX2, which have the function of reprogramming cells, but only in the presence of bFGF, which is a growth factor It has been known. It has been shown that the intracellular signal of bFGF is essential for maintaining pluripotency, and if this factor is removed, cell growth is inhibited, and abnormal cell differentiation or cell death occurs. It has been.
  • bFGF has the effect of causing cell division, which activates intracellular signals originating from tyrosine kinases on the cell membrane in target cells. These tyrosine kinases activate various phosphorylation pathways in cells, and it is known that the phosphorylation is mainly phosphorylation of serine threonine. It is already known that this intracellular phosphorylation signal is important for the self-renewal of pluripotent stem cells and the maintenance of pluripotency, but how the phosphorylated protein is controlled to transmit the signal I don't know about that. Protein phosphorylation is known to be a very important basic method in intracellular signal transduction systems, and is known to be important in cell proliferation, differentiation, morphogenesis and the like.
  • Pin1 is important for pluripotent stem cell self-renewal and pluripotency. It was investigated using human iPS cells and mES cells that Pin1 expression was induced with iPS cell induction, and that inhibition of Pin1 inhibited pluripotency and self-renewal of pluripotent stem cells. Proteomic analysis also revealed that Pin1 binds to the Serine 12 proline motif of Oct4, which is important as a reprogramming and transcription factor, and enhances its stability and transcriptional activity. This data not only shows that Pin1 is important for self-renewal and proliferation of pluripotent stem cells, but also enhances proliferation and maintenance of pluripotent stem cells by controlling Pin1 activity and function It is proposed that it can be done.
  • iPS cells were obtained from RIKEN BioResource Center (clone number 201B7).
  • iPS cells are hESC culture medium (KNOCKOUT Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 20% KNOCKOUT SR (Invitrogen), 1% GlutaMAX (Invitrogen), 100 ⁇ M Non-essential amino acids (Invitrogen), 50 ⁇ M ⁇ -mercaptoethanol and 10 ng / ml basic FGF) ⁇ Takahashi, 2007 # 58 ⁇ .
  • Mouse ES cells were cultured in mESC culture medium (KNOCOUT Dulbecco's modified Eagle's medium supplemented with 15% KNOCKOUT SR, 1% GlutaMAX (Invitrogen), 100 ⁇ M Non-essential amino acids, 50 ⁇ -mercaptoethanol and 1000 U / ml rhLIF) # 6 Yamada M. Hum Mol Genet 2010).
  • Colony formation was scored by counting the number of alkaline phosphatase (AP) staining positive colonies as previously described (# 7 Liu Y. Stem cells 2008). The number of cells per colony was determined by counting DAPI stained cells (# 7 Liu Y. Stem cells 2008).
  • Retroviral cells using retroviral vector pMXx-Pin1 or empty vector pMXx together with VSV-G gene using Effectene transfection reagent (Qiagen; http://www.qiagen.com/products/transfection/transfectionreagents/effectenetransfectionreagent.aspx)
  • Effectene transfection reagent (Qiagen; http://www.qiagen.com/products/transfection/transfectionreagents/effectenetransfectionreagent.aspx)
  • the cell supernatant containing the virus was collected and filtered through a 0.45 ⁇ m filter, and 10 ⁇ g / ml hexadimethrine bromide (polybrene) was added to obtain a virus solution.
  • the target cells MRC5 cells
  • the target cells were seeded at 6 ⁇ 10 5 cells in a 100 mm dish and infected with a virus solution containing virus / polybrene for 16 hours. After 24 hours, the virus solution was replaced with DMEM cell medium, and the culture was further continued. Six days later, MRC5 cells were plated on mouse fibroblasts (MEF; feeder cells), and after 24 hours, the DMEM medium was replaced with hESC medium. When cells were cultured at 37 ° C. and 5% CO 2 for 30 days, multiple iPS cell colonies appeared. These were stained with alkaline phosphatase ( http://www.funakoshi.co.jp/node/15683 ; Funakoshi), and the number of positive colonies stained in red was counted.
  • alkaline phosphatase http://www.funakoshi.co.jp/node/15683 ; Funakoshi
  • Oct4 cDNA was subcloned into pcDNA3-HA expression vector (Invitrogen).
  • Oct4 expression constructs are shown below: pcDNA-HA-Oct4 wild type: aa 1-360, pcDNA-HA-Oct4 ⁇ C: aa 1-297, pcDNA-HA-Oct4 ⁇ N1: aa 138-360, pcDNA-HA- Oct4 ⁇ N2: aa 113-360, pcDNA-HA-Oct4 ⁇ N3: aa 34-360.
  • PcDNA-HA-Oct4-S12A was performed using the KOD-Plus Mutagenesis Kit (TOYOBO, Osaka, Japan) according to the operating procedure.
  • the primers used are shown below; Forward: 5′-CGCCCCCTCCAGGTGGT-3 ′ (SEQ ID NO: 3); Reverse: 5′-CGAAGGCAAAATCTGAAGCC-3 ′ (SEQ ID NO: 4).
  • Gene reporter analysis The pGL3-fgf4 reporter plasmid containing the OCT-SOX binding cassette and the firefly luciferase gene were transfected with 50 ng of pRL-CMV (# 8 Masui S. Natl Cell boil 2007). After 24 hours, the cells were dissolved in passive lysis buffer (Promega) and incubated at room temperature for 15 minutes. Luciferase activity was measured by Dual-Luciferase reporter assay system (Promega) according to the operating procedure.
  • GST pull-down analysis and immunoprecipitation analysis HeLa cells were treated with GST pull-down buffer (50 mM HEPES [pH 7.4], 150 mM NaCl, 10% glycerol, 1% Triton-X100, 1.5 mM MgCl 2 , 1 mM EGTA, 100 mM NaF, 1 mM Na 3 VO 4 , 1 mM DTT, 0.5 ug / ml leupeptin, 1.0 ug / ml pepstatin and 0.2 mM PMSF) and incubated with 30 ul glutathione agarose beads containing GST-Pin1 or GST for 2 hours at 4 ° C.
  • GST pull-down buffer 50 mM HEPES [pH 7.4], 150 mM NaCl, 10% glycerol, 1% Triton-X100, 1.5 mM MgCl 2 , 1 mM EGTA, 100 mM NaF, 1 mM Na 3
  • the recovered protein was washed three times with lysis buffer and subjected to SDS-PAGE.
  • NP-40 lysis buffer (10 mM Tris HCl [pH 7.5], 100 mM NaCl, 0.5% NP-40, 1 mM Na 3 VO 4 , 100 mM NaF, 0.5 ug / ml leupeptin, 1.0 ug / ml pepstatin and 0.2 mM PMSF).
  • Cell lysates were incubated with Protein A / G Sepharose / non-immune IgG complex for 1 hour. The supernatant was immunoprecipitated with 5ug HA antibody and Protein A / G Sepharose. After washing with Lysis buffer three times, it was applied to SDS-PAGE gel and proteomic analysis was performed.
  • MS Proteomics analysis Mass spectrometry
  • Teratoma formation Cells were dissociated using actinase, collected in tubes, and centrifuged. The precipitated cells were suspended in human ESC culture medium. NOD-SCID mice (CREA, Tokyo, Japan) 2 ⁇ 10 6 cells and an equal amount of Matrigel (BD Biosciences) were mixed and injected subcutaneously. The tumor was removed after 9 weeks. The frozen tumor tissue was embedded with an optimum cutting temperature compound (OCT), and frozen sections were stained with hematoxylin and eosin.
  • OCT optimum cutting temperature compound
  • Double immunohistochemical staining double immunohistochemical staining
  • Paraffin tissue sections were deparaffinized using xylene and ethanol, and then autoclaved in 121 mM citrate buffer (pH 6.0) (121C for 15 min). Then, it was immersed in 0.3% hydrogen peroxide (hydrogen peroxidase) for 30 minutes. Blocking was performed for 30 minutes at room temperature using 10% normal goat serum (DAKO). Next, an anti-Pin1 antibody (anti-PIN1 polyclonal antibody (Santa Cruz Biotechnology, diluted to 100-fold)) was reacted overnight at 4 ° C.
  • the Pin1 protein labeled with the Pin1 antibody was treated with the Histofine Kit-PO (Nichirei, Tokyo, Japan) and AEC plus reaction (AECplus; DAKO, Campinteria, CA), and then developed brown color, followed by overnight reaction of mouse anti-CD44 monoclonal antibody (Cell Signaling, diluted to 100-fold) at 4 ° C. After that, it was colored blue using Histofine kit-AP (Nichirei) and BCIP / NBT system (Dako). Next, the cells were stained with methyl green in green and observed with an optical microscope.
  • TUNEL method 1 ⁇ 10 5 cells were seeded in a 12-well cell culture plate and treated with Juglon (5 ⁇ M) or DMSO (negative control). After 24 hours, apoptotic cells were detected using Promega's DeadEnd TM Colorimetric TUNEL System. Representative apoptotic cell pictures are presented.
  • FIG. 9B Cells were treated with various concentrations of Juglone or DMSO (control) in the same manner as A, and the proportion of apoptotic cells in all cells was measured 24 hours later using a light microscope.
  • Pin1 is induced with cell reprogramming and promotes iPS cell induction.
  • MRC5 which is a human fibroblast
  • MRC5 expression was significantly increased in the induced iPS cells (FIG. 1A).
  • Analysis by fluorescent immunostaining also revealed that Pin1 expression was predominantly increased in SOX2-positive cells that are iPS cell markers.
  • the expression of Pin1 was also reduced in the differentiated part of SOX2 negative (FIG. 1B).
  • iPS cells were injected subcutaneously into immunodeficient mice (NOD / SCID mice), and tumors formed after 9 weeks were excised and stained with HE. A histological examination revealed that it was a teratoma. Intestinal-like epithelial cells (endoderm), spindle-shaped muscle cells (mesoderm), cartilage tissue (mesoderm), nerve tissue (ectoderm), skin epithelium (ectoderm), etc. were observed (Fig. 1E). . From these results, it was found that the expression efficiency and reprogramming of iPS cells are enhanced by expressing Pin1 together with Yamanaka 4 factors.
  • Pin1 is required for iPS cell self-renewal and colony formation. From the previous results, it was found that Pin1 positively controls the formation of iPS cells. Therefore, the role of Pin1 functionally was examined using iPS cells and ES cells. First, we looked at whether Pin1 is involved in self-replication. The iPS colonies were made into individual cells, and the self-replicating ability was examined by checking whether each formed a colony. Human iPS cells were made into individual cells using the enzyme actinase, seeded on feeder cells, and Pin1 inhibitor Juglone was added at each concentration.
  • Pin1 Functions of Pin1 in maintaining pluripotency
  • Human iPS cells were cultured for 5 days, and Juglone was added while colonies were formed to inhibit Pin1.
  • differentiated cells that were negative for alkaline phosphatase appeared in a mosaic pattern (FIG. 4A).
  • differentiated cells that were negative for alkaline phosphatase increased (FIG. 4B).
  • Pin1 was first subjected to GST pull-down. Human iPS cell lysate and recombinant GST or GST-Pin1 protein were mixed, collected with glutathione beads, and immunoblot analysis using Oct4 antibody revealed that Oct4 does not bind to GST but binds to GST-Pin1 ( Figure 6A). Moreover, since the binding was not seen when iPS cell lysate was previously treated with CIP, a dephosphorylating enzyme, it was suggested that Pin1 binds to the phosphorylation site of Oct4. Next, iPS cells were fluorescently immunostained and the intracellular localization of Pin1 and Oct4 was examined.
  • Pin1 is known to be involved in the stability of substrate proteins. Therefore, we investigated whether Pin1 is involved in the stability of Oct4. HeLa cells were transfected with Oct4 and Pin1 or empty vector, and then treated with cycloheximide to temporarily stop protein synthesis, and the half-life of Oct4 protein synthesized so far was observed over time. . Compared to control cells, cells in which Pin1 was forcibly expressed significantly inhibited the degradation of Oct4 protein and were stabilized (FIG. 6C). Next, in order to examine the transcriptional activity of Oct4 in cells, luciferase assay was performed.
  • a luciferase construct in the gene promoter region of a target gene called FGF4 having a cassette that binds Oct4 and SOX2 together with Oct4, SOX2, and Pin1 was used in HeLa cells. Pin1 alone did not increase FGF4 transcriptional activity, but it was found that co-expression of Pin1 and FGF4 increased Oct4 transcriptional activity depending on the amount of Pin1 (Fig. 6D).
  • a similar luciferase assay was performed using a WW domain mutant that is a Pin1 binding domain or a PPIase domain mutant that is an enzyme activation domain. As a result, both mutants were found to be less effective in increasing the transcriptional activity of Oct4 compared to the wild type (FIG. 6E). This indicates that both the binding domain and the enzyme activation domain are important for the function of Pin1 on Oct4.
  • Pin1 was investigated to which site of Oct4 protein Pin1 that binds to Serine 12 proline of Oct4 binds.
  • Pin1 is known to bind only to serine-proline or threonine-proline sites. Therefore, a mutant in which the serine-proline or threonine-pronin site existing in 6 positions in Oct4 was deleted was prepared, and GST pull-down was performed. As a result, it was found that even the mutant in which the C-terminal of Oct4 protein was deleted binds to Pin1, whereas it does not bind when the N-terminal is deleted (FIG. 7A). In the mutant in which the N-terminal three Pin1 binding sites were deleted, Pin1 did not bind, suggesting that it binds to any of amino acids 1 to 34.
  • Pin1 can bind only to the serine 12 proline site, and serine 12 proline is a sequence that is conserved not only in humans, but also in rabbits, mice, and rats. Pin1 was thought to bind to this site (Fig. 7B). When an S12A mutant in which serine was substituted with alanine was prepared and GST pull-down was performed, the prepared Oct4-S12A mutant did not bind to Pin1 (FIG. 7C). Next, it was examined whether Pin1 functions against the Oct4-S12A mutant. When Pin1 and Oct4 were co-expressed, the amount of protein increased in wild-type Oct4, but no change was observed in the Oct4-S12A mutant, indicating that there was no reactivity to Pin1 (Fig. 7D). .
  • Pin1 showed high expression in cancer stem cells in human breast cancer tissue (FIG. 8).
  • CSC breast cancer stem cells
  • MCF-10A-Ras cells MCF-7 cells
  • Fig. 10 Cell Counting Kit-8 (Dojindo Laboratories, # CK07)
  • Pin1 is essential for pluripotent stem cell maintenance and stemness.
  • Pin1 is induced with the induction of human iPS cells.
  • Co-expression of reprogramming factor 4 and Pin1 significantly promotes iPS cell induction efficiency.
  • Pin1 inhibition using Juglone, AdV-dnPin1, and PINTIDE as specific Pin1 inhibitors markedly inhibits colony formation of human iPS cells and mouse ES cells.
  • Pin1 inhibition induces abnormal differentiation in human iPS cells after colony formation.
  • Proteomic analysis revealed that Oct4 is a putative substrate for Pin1 in human iPS cells.
  • Pin1 interacts with the Serine 12 proline motif of Oct4 and promotes stability and transcriptional activity.
  • Our findings reveal a novel role for Pin1 as a regulator of self-renewal and survival through the function of Oct4 in pluripotent stem cells.
  • Pin1 is a multifunctional protein that catalyzes various phosphorylated proteins, including a variety of cellular processes such as cell cycle and apoptosis.
  • the diverse mechanistic actions of Pin1 on many phosphorylated proteins along various cellular functions can be shown as modulators in multiple signal pathways depending on different cell types and environments.
  • Pin1 was positioned as an important regulator in the transcription factor network that controls pluripotent stem cell self-renewal and stem cell nature through the regulation of Oct4.
  • iPS cells induced by the expression of Oct4, SOX2, Klf4 and c-Myc induce high Pin1 expression levels, and these cells are dependent on Pin1 function.
  • Pin1 is one of the important executive factors that induces iPS cells from somatic cells in cooperation with reprogramming transcription factors.
  • Pin1 knockout mice grow normally but have some developmental abnormalities including weight loss, retinal degeneration and mammary developmental disorders. Pin1 knockout mice also show testicular atrophy and progressive loss of spermatogenic cells with marked proliferation of primordial germ cells (PGC). These phenotypes are attributed to germline stem cell maintenance and proliferation disorders due to loss of Pin1 function.
  • PPC primordial germ cells
  • Pin1 acts as a suppressor of substrate proteolysis or as a promoter.
  • Our current data showed that Pin1 prolongs the half-life of Oct4 protein and thereby promotes its transcriptional activity.
  • Oct4 is known to be controlled by post-translational modifications such as SUMOylation.
  • Our findings also show that Oct4 is regulated by phosphorylation followed by prolyl isomerization. Identification of binding kinases that act on the binding of Pin1 and Oct4 will advance understanding of the regulatory pathways that regulate pluripotency during and after induction.
  • pluripotent stem cells such as iPS cells can be used for future regenerative medicine.
  • iPS cells may be used for clinical treatment because of the possibility of forming tumors.
  • Pin1 inhibition can effectively inhibit the proliferation of iPS cells in the undifferentiated stage. Pin1 can thus be a molecular switch that can reversibly control the proliferation and survival of iPS cells, thereby reducing the risk of cell transformation and tumor formation.
  • the present invention can be applied to applications for regenerative medicine, stem cell engineering, gene therapy, and search for new regulatory factors using Pin1.
  • SEQ ID NO: 1 shows the cDNA sequence of human Pin1 (492 bp; underlined: stop codon).
  • atggcgga cgaggagaag ctgccgccg gctgggagaagcgcatgagc cgcagctcag gccgagtgta ctacttcaac cacatcacta acgccagccagtgggagcgg cccagcggca acagcagcag tggtggcaaa aacgggcagg gggagggagccctgc cagggtccgc tgcacc tgctggtgaa gcacagccag tcacggcggc cctctg gcggcaggag aagatcaccc ggaccaaggaggaggccctg gagct
  • SEQ ID NO: 3 shows the sequence of the primer.
  • SEQ ID NO: 4 shows the sequence of the primer. 5'-CGAAGGCAAAATCTGAAGCC-3 ' ⁇ SEQ ID NO: 5> The amino acid sequence of Pin1-inhibited phosphorylated peptide PINTIDE is shown. RRRRRRRRRWFYpSPR ⁇ SEQ ID NO: 6> The amino acid sequence of a non-phosphorylated control peptide is shown. RRRRRRRRRWFYAPR

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Abstract

La présente invention concerne une technique permettant une génération, une préservation et un renouvellement stables et très efficaces de cellules souches pluripotentes induites (iPS). La présente invention concerne un procédé de production de cellules souches pluripotentes à partir de cellules somatiques, ledit procédé comprenant une étape d'amplification de l'expression de la peptidyl-prolyle isomérase Pin1 dans les cellules somatiques. La présente invention concerne également des cellules souches pluripotentes induites obtenues par le procédé décrit ci-avant. La présente invention concerne en outre un agent d'amplification de la reprogrammation des cellules somatiques, un agent thérapeutique d'activation de l'auto-renouvellement de cellules souches pluripotentes induites et un agent thérapeutique de préservation du caractère pluripotent de cellules souches pluripotentes induites, chacun contenant une substance amplifiant l'expression de la peptidyl-prolyle isomérase Pin1 dans des cellules somatiques et/ou la peptidyl-prolyle isomérase Pin1. La présente invention concerne également un agent thérapeutique d'inhibition de l'auto-renouvellement de cellules souches pluripotentes induites, un agent thérapeutique d'inhibition du caractère pluripotent de cellules souches pluripotentes induites et un agent thérapeutique provoquant la mort de cellules souches pluripotentes induites et/ou de cellules souches cancéreuses, chacun contenant une substance inhibant la peptidyl-prolyle isomérase Pin1. La présente invention concerne un stabilisateur de la protéine Oct-4 et un agent d'amplification de l'activité de transcription de la protéine Oct-4, chacun contenant la peptidyl-prolyle isomérase Pin1 et/ou un gène Pin1.
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012165287A1 (fr) * 2011-05-27 2012-12-06 公立大学法人横浜市立大学 Procédé de production pour une cellule souche cancéreuse artificielle et procédé de différenciation induite associé
US10731134B2 (en) 2011-05-27 2020-08-04 Public University Corporation Yokohama City University Production method for artificial cancer stem cell and induced differentiation method therefor
WO2018074457A1 (fr) * 2016-10-17 2018-04-26 学校法人慶應義塾 Agent d'élimination de cellules souches indifférenciées, et procédé associé
JPWO2018074457A1 (ja) * 2016-10-17 2019-07-18 学校法人慶應義塾 未分化幹細胞除去剤、及び未分化幹細胞除去方法
WO2020130077A1 (fr) * 2018-12-21 2020-06-25 国立大学法人北海道大学 Composition pour éliminer des cellules souches pluripotentes et procédé d'élimination de cellules souches pluripotentes
JP7409670B2 (ja) 2018-12-21 2024-01-09 国立大学法人北海道大学 多能性幹細胞を除去するための組成物、及び多能性幹細胞の除去方法

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