Method for preparing induced pluripotent stem cells using synthetic peptide

Abstract

Provided is a method of preparing induced pluripotent stem cells using a synthetic peptide, and more particularly, to a method of preparing induced pluripotent stem cells using a peptide capable of inhibiting the activity of NF-B and promoting mesenchymal-epithelial transition (MET). Since undifferentiated multipotent stem cells may be efficiently prepared under xenopathogen-free or feeder cell-free conditions without requiring co-culture with animal serum or xenogeneic cells, the method for preparing the induced pluripotent stem cells using the synthetic peptide according to the present disclosure is very useful for developing stem cell therapeutic agents that are clinically applicable.

Claims

1. A method for preparing induced pluripotent stem (iPS) cells, the method comprising: (a) introducing one or more nucleic acid sequences encoding reprogramming factors Oct3/4, Sox2 and Klf4 into isolated mammalian somatic cells and then culturing; and (b) introducing a peptide having any one of amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3 into the isolated mammalian somatic cells to which the reprogramming factor gene has been introduced and then culturing, such that iPS cells are obtained.

2. The method of claim 1, wherein the peptide has a concentration of 0.01 to 100 M.

3. The method of claim 1, wherein the somatic cells are human somatic cells.

4. The method of claim 1, wherein the iPS cells are prepared under xenopathogen-free or feeder cell-free conditions.

5. The method of claim 1, wherein the reprogramming factor further comprises c-Myc or Lin28.

6. The method of claim 1, wherein step b) comprises introducing the peptide in the presence of a biomaterial.

7. The method of claim 6, wherein the biomaterial is a synthetic polymer or a natural polymer.

8. The method of claim 7, wherein the synthetic polymer is any one selected form the group consisting of poloxamer, polyethylene glycol and polypropylene glycol.

9. The method of claim 7, wherein the natural polymer is any one selected from vitronectin, collagen, gelatin, alginic acid, chondroitin sulfate, fibronectin and an extracellular matrix protein.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

(2) FIG. 1 is a colony staining result verified through alkaline phosphatase staining (AP), after treating functional peptides P1, P2, and P3 and control peptides C1 and C2 for 10 days at 24-hr intervals in human dermal fibroblasts introduced with a reprogramming gene;

(3) FIG. 2 is a result of verifying the expression of Oct4 as a marker of embryonic stem cells (ES cells) using immunofluorescence (IF) after treating the peptide by the same method as illustrated in FIG. 1; and

(4) FIG. 3 is a result of verifying the degree of mesenchymal-epithelial transition (MET), which is a dedifferentiation process of differentiated somatic cells, by a marker THY1 of human dermal fibroblasts and an epithelial cell adhesion molecule (EPCAM) as a marker of epithelial cells using flow cytometry (FACS) after treating the peptide by the same method as illustrated in FIG. 1.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

(5) Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as those commonly understood by those skilled in the art. In general, the nomenclature used in this specification is well-known and commonly used in the art.

(6) In the present disclosure, a functional peptide for inhibiting the activity of NFB protein is found and it is verified that the peptide inhibits epithelial mesenchymal transition (EMT) and furthermore induces mesenchymal-epithelial transition (MET) to promote dedifferentiation.

(7) Accordingly, the present disclosure relates to a composition for promoting dedifferentiation of induced pluripotent stem cells from differentiated cells comprising a peptide represented by an amino acid sequence of Formula 1 below as an active ingredient.

(8) TABLE-US-00005 [Formula1] (SEQIDNO:6) GKCSTRGRKX.sup.1X.sup.2RRKK

(9) Where X.sup.1 and X.sup.2 are cysteine (C) or methionine (M).

(10) In another aspect, the present disclosure relates to a new use of a peptide represented by an amino acid sequence of Formula 1 below for promoting dedifferentiation of induced pluripotent stem cells from differentiated cells.

(11) TABLE-US-00006 [Formula1] (SEQIDNO:6) GKCSTRGRKX.sup.1X.sup.2RRKK

(12) Where X.sup.1 and X.sup.2 are cysteine (C) or methionine (M).

(13) In another aspect, the present disclosure relates to a method for preparing induced pluripotent stem cells from differentiated cells, comprising (a) introducing a reprogramming gene into differentiated cells; and (b) treating a peptide represented by an amino acid sequence of Formula 1 below in the cells introduced with the reprogramming gene and culturing the cells.

(14) TABLE-US-00007 [Formula1] (SEQIDNO:6) GKCSTRGRKX.sup.1X.sup.2RRKK

(15) Where X1 and X2 are cysteine (C) or methionine (M).

(16) In the present disclosure, the peptide may be any one selected from the group consisting of SEQ ID NOs: 1 to 3, but is not limited thereto.

(17) TABLE-US-00008 PeptideP1: (SEQIDNO:1) GKCSTRGRKCCRRKK PeptideP2: (SEQIDNO:2) GKCSTRGRKCMRRKK PeptideP3: (SEQIDNO:3) GKCSTRGRKMCRRKK

(18) In the present disclosure, the peptide has a concentration of preferably 0.01 to 100 M and more preferably 10 M, but is not limited thereto. Furthermore, the peptide is preferably treated for 10 days at 24-hr intervals, but is not limited thereto.

(19) In the present disclosure, the differentiated cells are preferably somatic cells or precursor cells, and are preferably derived from the human, but are not limited thereto.

(20) The somatic cells of the present disclosure refers to human dermal fibroblasts having a mesenchymal characteristic.

(21) In the present disclosure, it is preferred to use an Eagle's minimum essential medium (EMEM, ATCC 30-2003) for somatic cell culture and use Essential 8 Medium (Gibco, A15169-01) after peptide treatment, but it is not limited thereto.

(22) In the present disclosure, a reprogramming gene may be any one selected from the group consisting of Oct3/4, Sox2, c-Myc, Klf4 and Lin28, but is not limited thereto. A reprogramming gene of Oct3/4, Sox2, Klf4, c-Myc or Lin28 means a gene capable of reprogramming differentiated cells, and in particular, Oct4, Sox2, Klf4 and c-Myc are called Yamanaka genes.

(23) In the present disclosure, according to a known method, the reprogramming genes are divided into three groups and cloned into three different episomal vectors and expressed together with p53 shRNA (pCXLE-hOCT3/4-shp53, pCXLE-hSK, and pCXLE-hUL) to enhance the establishment efficiency of dedifferentiation of stem cells (Okita, K. et al., Nat Methods. 8(5):409-12, 2011).

(24) In the present disclosure, a method of transferring the reprogramming gene to the differentiated cells may use a method of administering a reprogramming gene to a culture medium of differentiated cells, a method of directly injecting a reprogramming gene, or a method of infecting differentiated cells by viruses obtained from packaging cells transfected with a viral vector inserted with a reprogramming gene, but is not limited thereto. As the method of directly injecting the reprogramming gene into the differentiated cells, microinjection, electroporation, insulator, particle bombardment, and the like may be used, but it is not limited thereto.

(25) In the present disclosure, the reprogramming gene is injected by an electroporation method using a nucleofector (Amaxa, US/Nucleofector, Electroporation Gene Transfer, Lonza) instrument from Lonza, and it is preferred that the somatic cells introduced with the reprogramming gene are stabilized by replacing a medium daily for about 3 to 5 days in a culture dish and then treated with the peptide.

(26) The present disclosure may be used as a cell therapeutic agent by preparing the induced pluripotent stem cells under xenopathogen-free or feeder cell-free conditions. In the present disclosure, the problems of the xenopathogen are solved by coating a vitronectin recombinant human protein (VTN-N) on a surface of the culture dish to exclude the use of the feeder cell as a xenogeneic cell. The somatic cells introduced with the reprogramming gene are cultured in a culture dish coated with a vitronectin protein and treated with the functional peptide to promote the efficiency of dedifferentiation into induced pluripotent stem cells.

(27) In the present disclosure, the peptide may be directly treated in a cell culture medium or treated by mixing with a biomaterial for culture, and the biomaterial means a synthetic polymer or a natural polymer. The synthetic polymer in the present disclosure may preferably be poloxamer, polyethylene glycol or polypropylene glycol, and the natural polymer may be vitronectin, collagen, gelatin, alginic acid, chondroitin sulfate, fibronectin or an extracellular matrix protein, but it is not limited thereto. In the present disclosure, vitronectin is used as the natural polymer, and the biomaterial may be used to be coated on a culture container, but it is not limited thereto.

(28) Furthermore, the present disclosure may exclude a xenopathogen generated by supplying various genes from MEF feeder cells to the induced pluripotent stem cell by using a biomaterial for culture capable of replacing mouse embryonic fibroblasts (MEF), which are animal-derived feeder cells used in a conventional culture of induced pluripotent stem cells (iPSC). Therefore, the preparation of iPSC using the peptides of the present disclosure may improve a dedifferentiation-inducing efficiency problem with the xenopathogen problem to be considered to use undifferentiated multipotent stem cells as a cell therapeutic agent.

(29) In the present disclosure, the functional peptide may be characterized by inhibiting nuclear translocation of NF-B protein in somatic cells into which a reprogramming gene is introduced and inhibiting the activity of the NF-B by inhibiting an NF-B signaling mechanism.

(30) In the present disclosure, it is verified that when the peptide is treated with the somatic cells introduced with the reprogramming gene, the nuclear translocation of the NF-B protein is inhibited by the peptide to inhibit epithelial mesenchymal transition (EMT) and promote mesenchymal-epithelial transition (MET), and thus the dedifferentiation efficiency of the induced pluripotent stem cells (iPSC) from the human somatic cells is increased.

(31) In the present disclosure, the maintenance of the undifferentiated state of the pluripotent stem cells is verified by increased expression of one or more genes selected from the group consisting of alkaline phosphatase (ALP), OCT4, SOX2, human telomerase reverse transcriptase (hERT) and SSEA-4. That is, the somatic cells introduced with the reprogramming gene are treated with the functional peptide, and then an initial process in which a colony is generated in the dedifferentiation process is observed through alkaline phosphatase staining (AP staining), and furthermore, expression of Oct4 is verified by immunofluorescence (IF) using an Oct4 antibody. Finally, the MET degree in the dedifferentiation process of the somatic cells is verified by flow cytometry (FACS) using antibodies of THY1 as a marker of human dermal fibroblasts and an epithelial cell adhesion molecule (EPCAM) as a marker of the epithelial cell.

(32) The present disclosure may improve dedifferentiation-inducing efficiency and the induced pluripotent stem cells prepared by the method are multipotent stem cells in an undifferentiated state which normally represent characteristics as multipotent stem cells. Therefore, the present disclosure is very useful to efficiently prepare multipotent stem cells which are clinically applicable and develop a mass culture system capable of ensuring multipotent stem cell resources which are clinically applicable.

(33) Hereinafter, the present disclosure will be described in more detail with Examples. These Examples are just to exemplify the present disclosure, and it is apparent to those skilled in the art that it is interpreted that the scope of the present disclosure is not limited to these Examples.

Example 1: Synthesis of Dedifferentiation Induction-Promoting Peptide

(34) A peptide P1 (GKCSTRGRKCCRRKK: SEQ ID NO: 1) was synthesized from a C terminal using a synthesis device by an F-moc solid chemical synthesis method. That is, the peptide was synthesized by using Rink resin (0.075 mmol/g, 100 to 200 meshes, 1% DVB crosslinking) bounded with 9-fluorenylmethoxycarbonyl (Fmoc-) as a blocking group, and 50 mg of Rink Amide MBHA resin was added in a synthesizer, the resin was swollen with DMF, and then a 20% piperidine/DMF solution was used to remove the Fmoc-group. In order of the sequence from the C terminal, 0.5 M amino acid solution (solvent: DMF), 1.0 M DIPEA (solvent: DMF&NMP), 0.5 M HBTU (solvent: DMF) were added by 5, 10 and 15 equivalents, respectively, and reacted for 1 to 2 hrs under a nitrogen stream. Whenever the deprotection and the coupling steps were completed, a washing process was performed twice with DMF and methanol. After the last amino acid was coupled, the Fmoc-group was removed by deprotection.

(35) Verification of the synthesis used a ninhydrin test method, and the tested and synthesized resin was dried and shaken with a reagent K cleavage cocktail at a ratio of 20 ml per 1 g of resin for 3 hrs and then the cocktail in which the resin and the peptide were dissolved was separated by filtering. Cold ether was added to the filtered solution and the peptide was crystallized by a solid phase and centrifuged and separated. In this case, the peptide reagent K cleavage cocktail was washed with ether several times and centrifuged to completely remove reagent K cleavage cocktail. The crude obtained above was dissolved in distilled water and separated and purified by using liquid chromatography and the purified peptide was lyophilized.

(36) TABLE-US-00009 PeptideP1: (SEQIDNO:1) GKCSTRGRKCCRRKK

(37) Furthermore, a peptide P2 (SEQ ID NO: 2) in which the fifth cysteine of C terminal of the peptide P1 (SEQ ID NO: 1) was substituted with methionine and a peptide P3 (SEQ ID NO: 3) in which the sixth cysteine of the C terminal of the peptide P1 (SEQ ID NO: 1) was substituted with methionine were synthesized by a F-moc solid-phase chemical synthesis method using a synthesizer.

(38) TABLE-US-00010 PeptideP2: (SEQIDNO:2) GKCSTRGRKCMRRKK PeptideP3: (SEQIDNO:3) GKCSTRGRKMCRRKK

Comparative Example 1: Control Peptide C1 of Functional Peptides P1, P2, and P3

(39) The peptide C1 was synthesized by a F-moc solid-phase chemical synthesis method using the same synthesizer as in Example 1.

(40) TABLE-US-00011 PeptideC1: (SEQIDNO:4) HRRCNKNNKKR

Comparative Example 2: Control Peptide C2 of Functional Peptides P1, P2, and P3

(41) The peptide C2 was synthesized by a F-moc solid-phase chemical synthesis method using the same synthesizer as in Example 1.

(42) TABLE-US-00012 PeptideC2: (SEQIDNO:5) GLRSKSKKFRRPDIQYPDA

Example 2: Verification of Induction Efficiency of Peptides for Dedifferentiation of Human Somatic Cells

2-1: Verification of Colony Number of Induced Pluripotent Stem Cells Using ALP Staining

(43) In order to verify the dedifferentiation induction efficiency of each peptide, the DNAs of reprogramming genes Oct3/4, Sox2, c-Myc, Klf4, and Lin28 were introduced to human dermal fibroblasts (hDF) of 1.510.sup.5/well on a 6-well coated with 2 g of vitronectin recombinant human protein (VTN-N) by an electroporation method. According to a known method, the reprogramming genes were divided into three groups and cloned into three different episomal vectors and expressed together with p53 shRNA(pCXLE-hOCT3/4-shp53, pCXLE-hSK, and pCXLE-hUL) to enhance the establishment efficiency of dedifferentiation of stem cells (Okita, K. et al., Nat. Methods. 8(5):409-12, 2011). The cells introduced with the reprogramming genes were cultured in an Eagle's minimal essential medium (EMEM, ATCC) and the medium was replaced at 24-hr intervals until cell confluency became 50%. When the confluency became 50%, 10 M of a dedifferentiation induction-promoting peptide and a control peptide were treated, respectively, and the medium was replaced with Essential 8 Medium and the peptides were treated at the same time as the medium replacement every 24 hrs. The peptide was treated for 10 days at 24-hr intervals, and in order to verify dedifferentiation efficiency per peptide, alkaline phosphatase (AP), known as a marker of embryonic stem cells, was used. For AP staining, an alkaline phosphatase detection kit(Milipore) was used. The medium was removed, 4% paraformaldehyde was added to the cells and they were immobilized for 2 minutes, a Naphthol/Fast Red Violet staining agent was added in a well and reacted at room temperature in a dark space for 20 minutes or more, and then the staining agent was washed with Dulbecco's phosphate buffered saline (DPBS) and the number of stained colonies was verified with a microscope.

(44) As a result, as compared with cells without treating the peptide and control groups treated with peptides C1 or C2, in cells treated with peptides P1, P2, or P3, it was verified that more colonies were stained, as illustrated in FIG. 1. Even in a graph counting and quantifying the number of colonies, similarly, it was verified that the peptides P1, P2, and P3 more efficiently induced dedifferentiation (see FIG. 1).

(45) 2-2: Verification of Formation of Induced Pluripotent Stem Cells Using Embryonic Stem Cells Marker Staining

(46) Using the same method as in Example 2-1, reprogramming genes were introduced into the cells by an electroporation method in human dermal fibroblasts and divided in a 6-well coated with vitronectin. Thereafter, expression levels of Oct4 as a marker of embryonic stem cells were compared with each other by using an immunofluorescence (IF) method.

(47) The cells treated with 10 M of each peptide for 10 days were fixed with 4% paraformaldehyde for 10 minutes at room temperature, and then 0.5% Triton-X 100 was added and they were cultured for 15 minutes at room temperature to pass through a cell wall and to penetrate into nuclei in the cells, and blocked with a buffer PBS in which 3% bovine serum albumin (BSA) was dissolved for 30 minutes. An Oct4 primary antibody was diluted with a buffer PBS in which 1% BSA was dissolved at a ratio of 1:100 and reacted for 16 hrs at 4 C. and a secondary antibody bound with fluorescein isothiocyanate (FITC) was diluted at a ratio of 1:200 and reacted for 1 hr at room temperature. Finally, a dye (Hoechst 33342, blue) for staining nuclei was treated for 10 minutes at room temperature and washed with a buffer PBS, and colonies with the clearest fluorescent expression in the well were photographed with a confocal scanning microscope (IX 70, Olympus Co., Tokyo, Japan).

(48) As a result, as compared with cells without treating the peptide and control groups treated with peptides C1 or C2, in cells treated with peptides P1, P2, or P3, it was verified that more fluorescence was expressed, as illustrated in FIG. 2. It can be seen that an expression amount of the Oct4 as a marker of embryonic stem cells is large and the peptides P1, P2, and P3 promote dedifferentiation. It was verified that the AP staining was clearer in the group treated with the peptides P1, P2, or P3 and the functional peptides P1, P2, and P3 promoted dedifferentiation to colonies as a type of iPSC (see FIG. 2).

(49) 2-3: Verification of Difference in Dedifferentiation Induction of Peptides Using Flow Cytometry (FACS)

(50) Using the same method as in Example 2-1, reprogramming genes were introduced into the cells by an electroporation method in human dermal fibroblasts and divided in a 6-well coated with Vitronectin. After 10 days of peptide treatment in which the cells were stabilized, the cells were collected in a 15 ml Falcon tube by using a scrapper and then centrifuged for 3 minutes at 1500 rpm. The cells were washed with a buffer (PBS) and then 1.510.sup.5 cells were transferred to each tube by using cold PBS. THY1 (cat: sc-59398) and EPCAM (cat: ab20160) antibodies at a concentration of 10 g/ml were diluted with a 3% BSA/PBS solution in the tube and reacted for 30 minutes at 4 C., and then centrifuged three times for 5 minutes at 1500 rpm. After washing with cold PBS, the cells were transferred to a round tube and analyzed by flow cytometry (FACS). Before measuring samples, a control reference value was fixed using THY1 for human dermal fibroblasts and EPCAM for a human mammary epithelial cell (HMEC).

(51) As a result, as compared with control groups treated with peptides C1 or C2, in a group treated with peptides P1, P2, or P3, it can be verified that a mesenchymal shape of dermal fibroblasts is significantly changed to an epithelial shape (see FIG. 3).

(52) Although the specific part of the present disclosure has been described in detail, it is obvious to those skilled in the art that such a specific description is just a preferred embodiment and the scope of the present disclosure is not limited. Thus, the substantial scope of the present disclosure will be defined by the appended claims and equivalents thereof.

INDUSTRIAL APPLICABILITY

(53) Since undifferentiated multipotent stem cells may be efficiently prepared under xenopathogen-free or feeder cell-free conditions without requiring co-culture with animal serum or xenogeneic cells, the method for preparing the induced pluripotent stem cells using the synthetic peptide according to the present disclosure is very useful for developing stem cell therapeutic agents that are clinically applicable.