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WO2009131262A1 - Method of manufacturing induced pluripotent stem cell originated from human somatic cell - Google Patents

Method of manufacturing induced pluripotent stem cell originated from human somatic cell Download PDF

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Publication number
WO2009131262A1
WO2009131262A1 PCT/KR2008/002361 KR2008002361W WO2009131262A1 WO 2009131262 A1 WO2009131262 A1 WO 2009131262A1 KR 2008002361 W KR2008002361 W KR 2008002361W WO 2009131262 A1 WO2009131262 A1 WO 2009131262A1
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gene
cells
virus
genes
somatic
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PCT/KR2008/002361
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French (fr)
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Se Pill Park
Eun Young Kim
Kilsoo Jeon
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Mirae Biotech Co., Ltd.
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Publication of WO2009131262A1 publication Critical patent/WO2009131262A1/en

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    • 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
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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
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • 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
    • C12N2501/60—Transcription factors
    • C12N2501/602—Sox-2
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C12N2501/603—Oct-3/4
    • 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
    • C12N2501/60—Transcription factors
    • C12N2501/605—Nanog
    • 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
    • C12N2501/60—Transcription factors
    • C12N2501/608—Lin28
    • 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
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/027—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a retrovirus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES

Definitions

  • the present invention relates to a method of manufacturing induced pluripotent stem cells originated from somatic cells, and more particularly, to a method of manufacturing of induced pluripotent stem cells originated from somatic cells which may dramatically effectively manufacture the induced pluripotent stem cells originated form somatic cells.
  • Embryonic Stem (ES) cells originated from inner cell masses of blastocyst of mammalia may branch into about 210 different organs of the human and have characteristics of being endlessly proliferated while maintaining pluripotency. Accordingly, human ES cells may be expected to be used for disease studies, efficiency/stability testing of drugs, disease treatment (childhood diabetes, spinal damage), and the like.
  • An aspect of the present invention provides a method of manufacturing of induced pluripotent stem cells originated from human somatic cells which may dramatically and effectively manufacture the induced pluripotent stem cells originated from somatic cells.
  • a method for manufacturing stem cells including: preparing Oct-4 gene, Sox2 gene, Nanog gene, and a fourth gene from human embryonic stem cells, and allowing each of the genes to be infected in host cells using a lentiviral vector system to generate viruses in which each of the genes are induced; concentrating or mixing each of the viruses to prepare a virus concentrated mixture, and mixing the virus concentrated mixture and a first culture solution to prepare a virus solution; floating human somatic cells having been cultivated in advance in a first culture dish, and mixing and reacting the floated somatic cells and the virus solution to prepare a somatic cell-virus mixture; adding and retaining the somatic cell-virus mixture as is in a second culture dish including a second culture solution to induce the genes in the somatic cells; and cultivating the somatic cells in which the genes are induced in a third culture dish including a third culture solution.
  • the fourth gene may be at least one of Lin28 gene and XIAP gene (anti-apoptotic gene).
  • the allowing of each of the genes to be infected in host cells may include: preparing the Oct-4 gene, the Sox2 gene, the Nanog gene, and the fourth gene from the human embryonic stem cells to clone the genes in a lentiviral vector, respectively; and allowing the cloned lentiviral vectors to be infected in the host cells to generate viruses in which the genes are induced by the cloned lentiviral vectors, respectively.
  • the concentrating of each of the viruses may be achieved by centrifugation, and the mixing of each of the viruses may be performed in such a manner that an amount of each of the viruses is the same.
  • virus concentrated mixture and the first culture solution may be mixed with a ratio of about 1 :1 to 5.
  • the floating of human somatic cells may include: separating the somatic cells from the first culture dish using a cell separation solution; and centrifuging the separated somatic cells.
  • a volume ratio of the somatic cell-virus mixture and the second culture solution may be about 1 : 10 to 20.
  • composition of the first and second culture solutions may be the same. Also, the reacting may be performed for about 5 to 15 minutes.
  • FIG. 1 is a photograph showing a DNA band detected by electrophoresis after performing a Polymerase Chain Reaction (PCR);
  • FIG. 2 is a schematic diagram illustrating a mechanism of pGEM-T Easy Vector
  • FIG. 3 is an electrophoretic photograph showing genes cloned in T- vector
  • FIG. 4 is a schematic diagram illustrating a mechanism of pENTR4 vector
  • FIG. 5 is a schematic diagram illustrating homologous recombination according to an exemplary embodiment of the present invention.
  • FIG. 6 is a mimetic diagram illustrating an envelope plasmid, a packaging plasmid, and a target vector each for producing viruses;
  • FIG. 7 is a microphotograph illustrating a state where a lentiviral vector is infected in a 239T cell
  • FIG. 8 is a microphotograph (A) and a fluorescence microphotograph (B) each showing stem cells 24 hours after inducing genes;
  • FIG. 9 is a fluorescence microphotograph showing stem cells 48 hours after inducing genes according to Comparative Example (A) and Example (B);
  • FIG. 10 is microphotographs (A) and (C) and fluorescence microphotographs (B) and (D) each showing two types of stem cells originated from somatic cells 20 to 25 days after inducing genes;
  • FIG. 11 is an electrophoretic photograph showing endogenous and exogenous gene expression within two types of stem cells originated from somatic cells;
  • FIG. 12 is a microphotograph showing stem cells in which an Alkaline phosphatase (AP) is activated
  • FIG. 13 is microphotographs showing a state of expression of SSEA-I, SSEA-4, TRA-I -60, and TRA- 1-81;
  • FIG. 14 is a microphotograph (A) and a fluorescence microphotograph (B) each showing the differentiation of stem cells where differentiation has been induced for four days;
  • FIG. 15 is fluorescence microphotographs showing the differentiation of stem cells where differentiation has been induced for fourteen days.
  • the present invention relates to a method for establishing induced pluripotent stem cells originated from human somatic cells by inducing genes specifically over- expressed in stem cells to somatic cells, unlike the human somatic cells, to thereby cause de-differentiation of the somatic cells.
  • the present invention may directly induce a reprogramming process in the somatic cells having been differentiated to thereby successfully manufacture stem cells having pluripotency.
  • Oct3/4, Sox2, Nanog Four transcription factors related to the above are Oct3/4, Sox2, Nanog, and a fourth gene.
  • the Oct3/4 and Sox2 are main transcription factors determining the pluripotency, which may function to up-regulate genes concerning sternness and suppress genes concerning the differentiation.
  • the Nanog gene may increase cloning efficiency of human embryonic stem cells, and thus a survival rate of a cell having been incipiently de-differentiated may be increased.
  • XIAP gene may be used as the fourth gene, and they are also simultaneously used.
  • Lin28 gene may function to maintain cloning effect.
  • XIAP gene may function to prevent a survival rate of a cell from being reduced, which is known as an anti-apoptotic gene.
  • a lentiviral vector in order to validate a method for effectively inducing the above-mentioned four genes into somatic cells, a lentiviral vector may be used, and presence/absence of adhesiveness of cell may be regulated at the time of inducing the genes to thereby maximize manufacturing efficiency of stem cells.
  • Lin 28 gene is used as the fourth gene.
  • the four transcription factors that is, Oct-4 gene, Sox2 gene, Nanog gene, and Lin 28 gene are required to be generated from human embryonic stem cells.
  • total RNA may be extracted from the human embryonic stem cells and cDNA may be composed from the extracted total RNA.
  • the composed cDNA may be cloned by predetermined primers and amplified by RT-PCT.
  • the prepared transcription factors may be cloned in a T-vector, and then the cloned transcription factors may be sub-cloned in an entry cloning vector such as a pENTR4 vector (manufactured by Invitrogen) so that the cloned transcription factors are again homologus recombinated with the lentiviral vector.
  • an entry cloning vector such as a pENTR4 vector (manufactured by Invitrogen) so that the cloned transcription factors are again homologus recombinated with the lentiviral vector.
  • Each of the transcription factors cloned in the T-vector may be ligated with the entry cloning vector to thereby be sub-cloned in the entry cloning vector.
  • the entry cloning vector in which the transcription factors are sub-cloned may be induced into the lentiviral vector through the homologus recombination with the lentiviral vector.
  • the lentiviral vectors of four types including the respective transcription factors may be infected by respective viruses to generate transgenic viruses in which respective genes are induced.
  • viruses of the present invention as described above may be generated by a lentiviral vector system.
  • Each of the four type-viruses in which the four type-genes are effectively induced may be concentrated to prepare a virus concentrated mixture.
  • the above- described concentration process may be performed by centrifuging the respective viruses. Through the concentration process, gene transfer efficiency into human somatic cells which will be described below may be significantly increased.
  • a content of each virus for preparing the virus concentrated mixture may be preferably maintained to be identical to each other so that the four genes are effectively expressed.
  • the prepared virus concentrated mixture may be mixed with a first culture solution to thereby prepare a virus solution.
  • a mixture ratio between the virus concentrated mixture and the first culture solution is about 1 :1 to 5.
  • the mixture ratio of the first culture solution to the virus concentrated mixture exceeds '5', the gene transfer efficiency may be significantly reduced. Conversely, when the mixture ratio thereof is less than T, problems may occur in stability of somatic cells, that is, objects of the gene transfer.
  • the mixture ratio between the virus concentrated mixture and the first culture solution is preferably about 1 :1.
  • the human somatic cells in which the four type-gene combinations will be induced may be cultivated in advance in a first culture dish before performing the gene transfer, and attached on the first culture dish.
  • a cell separation solution such as a triple solution and the like is required to be used in the first culture dish where the human somatic cells are cultivated to thereby separate the somatic cells from the first culture dish and float the separated somatic cells.
  • the floated somatic cells may be separated to prepare only a solid content of the somatic cells.
  • the purpose of floating the somatic cells is to increase a reaction surface area between the virus solution and the cells.
  • a time required when the floated cells are completely attached on the culture dish may be about two to three hours.
  • superior efficiency may be acquired along with an increase in a probability that viruses are penetrated into spherical cells in three-dimensions, in comparison with a method of gene transfer of attached somatic cells.
  • the floated somatic cells and virus solution as described above may be reacted with each other for about 5 to 15 minutes after being added to a reaction dish such as a conical tube and the like and mixed together.
  • a somatic cell-virus mixture may be prepared through the reaction.
  • the somatic cell-virus mixture is moved to a second culture dish including a second culture solution and retained as is for about 24 hours, and thereby infection of the somatic cells may be achieved.
  • genes included in the viruses may be induced into the somatic cells.
  • a ratio of the second culture solution to the somatic cell-virus mixture may be preferably about 10 to 20:1. When the ratio thereof exceeds '20', the gene transfer efficiency of the somatic cells may be deteriorated.
  • a composition of the first culture solution and second culture solution may be preferably the same, and thereby gene expression may be facilitated by maintaining metabolism and function of the somatic cells.
  • the somatic cells in which gene transfer is carried out in about 24 hours may be separated from the second culture dish using the cell separation solution, and moved to a third culture dish including a third culture solution to be cultivated for several weeks, and thereby obtaining stem cells.
  • a basic composition of the third culture solution may be obtained by mixing the second culture solution and a culture solution of the human embryonic stem cells with a ratio of 1 : 1 therebetween, and additionally include an undifferentiated inducer and the like.
  • a trizol reagent manufactured by Sigma
  • 200 ⁇ l of chloroform was inserted, mixed together in an inverted state, retained for about 15 minutes at a room temperature, and then centrifuged under a condition of 1,300 rpm, for about 15 minutes, and at about 4 ° C , thereby collecting only a supernatant except for precipitation, that is, solid of DNA and protein.
  • RNA inhibitor diethylpyrocarbonate (DEPC) water
  • cDNA In order to compose cDNA, 3 ⁇ l(l ⁇ g/ ⁇ l) of the complete RNA and 2 ⁇ l(10pmols/ ⁇ l) of oligo dT(dT) were mixed together, reacted for about 5 minutes at about 70 ° C , and retained for about 5 minutes at 4 ° C .
  • a primer used for gene cloning was h ⁇ ct4(forward primer: 5'-GAATTC-CCATGGCGGGACACCT-S' (22mer), reverse primer: 5'-GCGGCCGC-AGTTTGAATGCATGGGAG-3 l (26mer)), hSox2(forward primer: 5'-GAATTC-GCATGTACAACATGATGG-S' (24mer)), reverse primer: 5'- GCGGCCGC-TC AC ATGTGTGAGAGG-3' (24mer), hNanog(forward primer: 5'- GAATTC-ACATGAGTGTGGATCCAGCT-3' (26mer), reverse primer: 5'- GCGGCCGC-TC AC ACGTCTTCAGGTTG-S' (26mer)), hLin28(forward primer: 5'- GAATTC-CCATGGGCTCCGTGT-3' (21mer), reverse primer: 5'-GCGGCCGC- GCTCAATTCTGTGCCTCC-3' (26mer)).
  • a DNA band was subject
  • FIG. 1 is a photograph showing a DNA band detected by electrophoresis after performing the PCR. Referring to FIG. 1, it can be seen that bands of four types of genes were accurately detected.
  • FIG. 2 is a schematic diagram illustrating a mechanism of pGEM-T easy vector.
  • pENTR4 vector h ⁇ ct4, hSox2, hNanog and hLin28 each cloned in T-vector were cut using EcoRI enzyme (see FIG. 3), and then carried out ligation with pENTR4 vector (see FIG. 4).
  • FIG. 3 is an electrophoretic photograph showing genes cloned in T-vector
  • FIG. 4 is a schematic diagram illustrating a mechanism of pENTR4 vector.
  • each 4 ⁇ l of pENTR4/hOct4, hSox2, hNanog and hLin28 DNA, 2 ⁇ l of lentiviral vector, 2 ⁇ l of water, and 2 ⁇ l of LR clonase (manufactured by Invitrogen) enzyme were mixed to perform an overnight reaction at 20 ° C . Then, 1 ⁇ l of Proteinase K solution was inserted to be reacted for 10 minutes at 137 ° C .
  • FIG. 5 is a schematic diagram illustrating homologous recombination according to an exemplary embodiment of the present invention. Referring to FIG. 5, a genetic region of an entry cloning vector and a ccdB region of the lentiviral vector were replaced with each other, and thereby the homologous recombination was carried out.
  • FIG. 6 is a mimetic diagram illustrating an envelope plasmid, a packaging plasmid, and a target vector each for producing viruses
  • FIG. 7 is a microphotograph illustrating a state where a lentiviral vector is infected in a 239T cell. 5. gene injection in human somatic cells using lenti viral infection
  • 0.5 x 10 6 numbered human somatic cells prepared in a culture dish of 100 mm the previous day were detached from the culture dish to thereby be floated.
  • the floating of the human somatic cells was carried out such that the somatic cells were detached using the triple solution, and solid contents of the somatic cells were separated from the detached somatic cells using centrifugation.
  • 50 ⁇ l of each virus concentrated solution corresponding to the respective genes was mixed with a first culture solution at a ratio of 1 :1 (200 ⁇ l of a virus mixture; 200 ⁇ l of the culture solution), reacted with the floated somatic cells for 5 to 10 minutes in a conical tube of 15 ml, and then placed in a culture dish of 100 mm where 5,600 ⁇ l of a second culture solution was contained.
  • Gene transfer was performed by cultivating the virus concentrated solution for 24 hours. At the time of injecting the gene, a total of 6 ml of the culture solution was used, and 0.6 ⁇ g/ml of polybrene (manufactured by Sigma) was processed.
  • the somatic cell- culture solution (first culture solution) and the culture solution (second culture solution) used at the time of injecting the gene were obtained by adding each of 0.1 mM of ⁇ - mercaptoethanol (manufactured by Sigma), a non-essential amino acid of 1%, 50 U/ml of penicillin, 50 ⁇ g /ml of streptomycinm, and FBS of 10% (manufactured by Hyclone) to a DMEM culture solution (No. 11995, manufactured by Invitrogen) where 4.5 g/L of high-glucose, 0.11 g/L of Na-pyruvate, and 2mM of L-glutamine were contained.
  • DMEM culture solution No. 119
  • Each of the cells obtained by methods of performing the gene transfer was detached from the culture dish using the triple solution, and placed on five culture dishes of each being 60 mm where MEF (Mouse Embryonic Fibroblast) feeder cells prepared the previous day were contained, to thereby be cultivated.
  • the used culture solution was a somatic cell-culture solution.
  • the somatic cell-culture solution was obtained by adding 4 ⁇ g/ml of basic Fibroblast Growth Factor, that is, an undifferentiated inducer required for maintenance of stem cell, to a standard culture solution.
  • the standard culture solution was obtained by adding 0.1 mM of ⁇ - mercaptoethanol (manufactured by Sigma), 1% of non-essential amino acid, 50 U/ml of penicillin, 50 ⁇ g/ml of streptomycin, and 20% of serum replacement (SR, manufactured by Gibco) to DMEM/F12 culture solution (No. 11320, manufactured by Invitrogen) in which 3.15 g/L of D-glucose, O.llg/L of Na- pyruvate, and 2 niM of L-glutamine are contained.
  • the virus infection was observed through fluorescence 24 to 48 hours after the cultivation.
  • the gene transfer was performed on somatic cells attached on the culture dish.
  • Each 50 ⁇ l (total 200 ⁇ l) of the virus concentrated solution was directly sprayed on 5,800 ⁇ l of the culture solution.
  • the virus concentrated solution was obtained such that each of the Oct4, Sox2, Nanog, and Lin28 was contained in 0.5 x 10 6 numbered human somatic cells prepared in the culture dish of 100 mm the previous day.
  • SSEA stage- specific embryonic antigen, manufactured by Santacruz
  • SSEA-4 each recognizing undifferentiated embryonic stem cells
  • TRA tumorigenic antigen, manufactured by Santacruz- 1 -60 and TRA- 1-81.
  • a colony presumed to be embryonic stem cells was fixed for 20 minutes with paraform-aldehyde (PFA, manufactured by Sigma) of 4%, washed three times using the PBS, and performed non-specific blocking for one hour using a normal goat serum of 10%. Then, the colony was reacted overnight with a first antibody for 6 minutes at 4 ° C with a concentration ratio of 1 :20.
  • PFA paraform-aldehyde
  • the colony was washed three times using the PBS, and a second antibody (product name: rhodamine (TRITC)-conjugated goat anti- human IgM, 1:200, manufactured by Jackson Lab) on which TRITC is attached was processed.
  • a second antibody product name: rhodamine (TRITC)-conjugated goat anti- human IgM, 1:200, manufactured by Jackson Lab
  • TRITC rhodamine
  • DAPI 4'-6-diamidino-2-phenylindole
  • cDNA composed from two types of iPS cells was extended with 30 cycles each for 3 minutes at 94 ° C , 30 seconds at 94 ° C, 30 seconds at 51 to 53 ° C, and one minute at 72 ° C, and also extended for 5 minutes at 72 ° C using polymersase (AccuPrime DNA Taq polymerase, manufactured by Invitrogen).
  • the primer used for ascertaining endogenous gene expression was the same as the primer used at the time of cloning of the gene, and the primer used for ascertaining exogenous gene expression was the forward primer and a reverse primer (5 '- ATGCTCGTC AAGA AGAC AGG-3) originated from gene carrier.
  • a DNA band was subjected to electrolysis after performing the PCR, dyed with ethidum bromide, and observed under ultraviolet (UV) light. The observed result can be shown in FIG. 11.
  • a plurality of colonies was made into an Embryoid Body (EB) having triploblastic characteristics for four days, attached on a culture dish on which gelatin is coated, and then performed dye by inducing spontaneous differentiation for two weeks within a culture solution containing a serum.
  • the differentiated cell was fixed for 15 minutes using PFA of 4%, washed using the PBS, penetrated for 10 minutes using the triton X- 100 solution of 0.2%, and then performed blocking for one hour using the normal goat serum of 10%.
  • EB Embryoid Body
  • an anti-nestin polyclonal antibody (nestin, 1 :500, manufactured by Chemicon) of a nerve cell factor was used for examining ectoderm potency
  • an anti- ⁇ - smooth muscle actin monoclonal antibody (SMA, 1;25, manufactured by Santacruz) was used for examining mesoderm potency
  • an anti- ⁇ -fetoprotein polyclonal antibody (AFP, l;200, manufactured by Sigma) was used for examining endoderm potency.
  • SMA smooth muscle actin monoclonal antibody
  • AFP anti- ⁇ -fetoprotein polyclonal antibody
  • a second antibody (TRITC conjugated goat anti-rabbit IgG, 1 :200, manufactured by Jackson Lab) on which TRITC is attached was processed.
  • DAPI (1 : 1,000) was processed, reacted for one hour at a room temperature, sufficiently washed using the PBS, and then observed using the fluorescence microscope.
  • FIG. 8 is a microphotograph (A) and a fluorescence microphotograph (B) each showing stem cells 24 hours after inducing genes. Referring to FIG. 8, it can be seen that a Venus marker-gene was expressed.
  • FIG. 9 is a fluorescence microphotograph showing stem cells 48 hours after inducing genes according to Comparative Example (A) and Example (B). Referring to
  • FIG. 9 it could be found that gene transfer efficiency of the case of somatic cells where the gene transfer was performed by Example was superior to that of the case of somatic cells where the gene transfer was performed by Comparative Example.
  • iPS induced pluripotent stem
  • FIG. 10 is microphotographs (A) and (C) and fluorescence microphotographs (B) and (D) each showing two types of stem cells (hips 1-1 and hips 1-2) originated from somatic cells 20 to 25 days after inducing genes.
  • stem cells of a colony was established.
  • the number of colonies formed by Example (B) was 5.1 times greater than that by Comparative Example (A). 3. ascertainment of expression of four genes from iPS cells
  • FIG. 11 is an electrophoretic photograph showing endogenous and exogenous gene expression within two types of stem cells originated from somatic cells. Referring to FIG. 11, it could be found that four types of endogenous and exogenous genes, that is, four transcription factors where two types of stem cells (hips 1-1 and hips).
  • FIG. 12 is a microphotograph showing stem cells in which an Alkaline phosphatase (AP) is activated, (substantial microscope-Fast Red Violet/Naphthol AS-BI dye verification).
  • AP Alkaline phosphatase
  • FIG. 13 is microphotographs showing a state of expression of SSEA-I, SSEA-4, TRA-1-60, and TRA-1-81.
  • A, B, C, and D were photographs obtained by examining SSEA-3 expression.
  • E, F, G, and H were photographs obtained by examining SSEA-4 expression.
  • I, J, K, and L were photographs obtained by examining TRA-1-60 expression.
  • M, N, O, and P were photographs obtained by examining TRA-1-81 expression.
  • A, E, I, and M are fluorescence microphotographs showing expression of the Venus marker-gene.
  • B, F, J, and N are fluorescence microphotographs obtained by DAPI dye
  • C, G, K, and O are photographs showing expression of SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 through TRITC dye.
  • D, H, L, and P are photographs obtained by combining DAPI and TRITC results. Referring to FIG. 13, surface factor of the stem cells originated from human somatic cells were well expressed.
  • FIG. 14 is a microphotograph (A) and a fluorescence microphotograph (B) each showing the differentiation of stem cells where differentiation has been induced for four days. Referring to FIG. 14, it can be ascertained that the embryoid body was generated and the Venus marker-gene was expressed.
  • FIG. 15 is fluorescence microphotographs showing the differentiation of stem cells where differentiation has been induced for fourteen days. Red parts of each of photographs are regions where the TRITC dye is performed, and blue parts thereof are regions where the DAPI dye is performed.
  • human induced pluripotent stem (iPS) cells may be effectively manufactured without using an egg cell, and thus can be expected to contribute to maximize the process efficiency when the mass production is attained in the future.

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Abstract

Disclosed is a method for manufacturing stem cells including preparing Oct-4 gene, Sox2 gene, Nanog gene, and fourth gene from human embryonic stem cells, and allowing each of the genes to be infected in host cells using a lentiviral vector system to generate viruses in which each of the genes are induced; concentrating or mixing each of the viruses to prepare a virus concentrated mixture, and mixing the virus concentrated mixture and a first culture solution to prepare a virus solution; floating human somatic cells having been cultivated in advance in a first culture dish, and mixing and reacting the floated somatic cells and the virus solution to prepare a somatic cell- virus mixture; adding and retaining the somatic cell-virus mixture as is in a second culture dish including a second culture solution to induce the genes in the somatic cells; and cultivating the somatic cells.

Description

METHOD OF MANUFACTURING INDUCED PLURIPOTENT STEM CELL ORIGINATED FROM HUMAN SOMATIC CELL
Technical Field The present invention relates to a method of manufacturing induced pluripotent stem cells originated from somatic cells, and more particularly, to a method of manufacturing of induced pluripotent stem cells originated from somatic cells which may dramatically effectively manufacture the induced pluripotent stem cells originated form somatic cells.
Background Art
Embryonic Stem (ES) cells originated from inner cell masses of blastocyst of mammalia may branch into about 210 different organs of the human and have characteristics of being endlessly proliferated while maintaining pluripotency. Accordingly, human ES cells may be expected to be used for disease studies, efficiency/stability testing of drugs, disease treatment (childhood diabetes, spinal damage), and the like.
However, the use of human embryos for the purpose of manufacturing the ES cells raises ethical debates, and disadvantageously has limitations due to a significantly smaller probability of manufacturing stem cells for specific patients and specific diseases.
Disclosure of Invention Technical Goals An aspect of the present invention provides a method of manufacturing of induced pluripotent stem cells originated from human somatic cells which may dramatically and effectively manufacture the induced pluripotent stem cells originated from somatic cells.
Technical solutions
According to an aspect of the present invention, there is provided a method for manufacturing stem cells, the method including: preparing Oct-4 gene, Sox2 gene, Nanog gene, and a fourth gene from human embryonic stem cells, and allowing each of the genes to be infected in host cells using a lentiviral vector system to generate viruses in which each of the genes are induced; concentrating or mixing each of the viruses to prepare a virus concentrated mixture, and mixing the virus concentrated mixture and a first culture solution to prepare a virus solution; floating human somatic cells having been cultivated in advance in a first culture dish, and mixing and reacting the floated somatic cells and the virus solution to prepare a somatic cell-virus mixture; adding and retaining the somatic cell-virus mixture as is in a second culture dish including a second culture solution to induce the genes in the somatic cells; and cultivating the somatic cells in which the genes are induced in a third culture dish including a third culture solution.
In this instance, the fourth gene may be at least one of Lin28 gene and XIAP gene (anti-apoptotic gene).
Also, the allowing of each of the genes to be infected in host cells may include: preparing the Oct-4 gene, the Sox2 gene, the Nanog gene, and the fourth gene from the human embryonic stem cells to clone the genes in a lentiviral vector, respectively; and allowing the cloned lentiviral vectors to be infected in the host cells to generate viruses in which the genes are induced by the cloned lentiviral vectors, respectively.
Also, the concentrating of each of the viruses may be achieved by centrifugation, and the mixing of each of the viruses may be performed in such a manner that an amount of each of the viruses is the same.
Also, the virus concentrated mixture and the first culture solution may be mixed with a ratio of about 1 :1 to 5.
Also, the floating of human somatic cells may include: separating the somatic cells from the first culture dish using a cell separation solution; and centrifuging the separated somatic cells.
Also, a volume ratio of the somatic cell-virus mixture and the second culture solution may be about 1 : 10 to 20.
Also, composition of the first and second culture solutions may be the same. Also, the reacting may be performed for about 5 to 15 minutes.
Brief Description of Drawings FIG. 1 is a photograph showing a DNA band detected by electrophoresis after performing a Polymerase Chain Reaction (PCR);
FIG. 2 is a schematic diagram illustrating a mechanism of pGEM-T Easy Vector; FIG. 3 is an electrophoretic photograph showing genes cloned in T- vector;
FIG. 4 is a schematic diagram illustrating a mechanism of pENTR4 vector;
FIG. 5 is a schematic diagram illustrating homologous recombination according to an exemplary embodiment of the present invention;
FIG. 6 is a mimetic diagram illustrating an envelope plasmid, a packaging plasmid, and a target vector each for producing viruses;
FIG. 7 is a microphotograph illustrating a state where a lentiviral vector is infected in a 239T cell;
FIG. 8 is a microphotograph (A) and a fluorescence microphotograph (B) each showing stem cells 24 hours after inducing genes; FIG. 9 is a fluorescence microphotograph showing stem cells 48 hours after inducing genes according to Comparative Example (A) and Example (B);
FIG. 10 is microphotographs (A) and (C) and fluorescence microphotographs (B) and (D) each showing two types of stem cells originated from somatic cells 20 to 25 days after inducing genes; FIG. 11 is an electrophoretic photograph showing endogenous and exogenous gene expression within two types of stem cells originated from somatic cells;
FIG. 12 is a microphotograph showing stem cells in which an Alkaline phosphatase (AP) is activated;
FIG. 13 is microphotographs showing a state of expression of SSEA-I, SSEA-4, TRA-I -60, and TRA- 1-81;
FIG. 14 is a microphotograph (A) and a fluorescence microphotograph (B) each showing the differentiation of stem cells where differentiation has been induced for four days; and
FIG. 15 is fluorescence microphotographs showing the differentiation of stem cells where differentiation has been induced for fourteen days.
Best Mode for Carrying Out the Invention Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to the like elements throughout. The embodiments are described below in order to explain the present invention by referring to the figures. The present invention relates to a method for establishing induced pluripotent stem cells originated from human somatic cells by inducing genes specifically over- expressed in stem cells to somatic cells, unlike the human somatic cells, to thereby cause de-differentiation of the somatic cells.
Specifically, the present invention may directly induce a reprogramming process in the somatic cells having been differentiated to thereby successfully manufacture stem cells having pluripotency.
Four transcription factors related to the above are Oct3/4, Sox2, Nanog, and a fourth gene. The Oct3/4 and Sox2 are main transcription factors determining the pluripotency, which may function to up-regulate genes concerning sternness and suppress genes concerning the differentiation. Also, the Nanog gene may increase cloning efficiency of human embryonic stem cells, and thus a survival rate of a cell having been incipiently de-differentiated may be increased. Either Lin28 gene or
XIAP gene may be used as the fourth gene, and they are also simultaneously used.
Lin28 gene may function to maintain cloning effect. XIAP gene may function to prevent a survival rate of a cell from being reduced, which is known as an anti-apoptotic gene.
According to the present invention, in order to validate a method for effectively inducing the above-mentioned four genes into somatic cells, a lentiviral vector may be used, and presence/absence of adhesiveness of cell may be regulated at the time of inducing the genes to thereby maximize manufacturing efficiency of stem cells.
According to the present invention, an example in which Lin 28 gene is used as the fourth gene will be herein described in detail.
According to the present invention, in order to manufacture stem cells, the four transcription factors, that is, Oct-4 gene, Sox2 gene, Nanog gene, and Lin 28 gene are required to be generated from human embryonic stem cells.
For this purpose, total RNA may be extracted from the human embryonic stem cells and cDNA may be composed from the extracted total RNA. The composed cDNA may be cloned by predetermined primers and amplified by RT-PCT.
The prepared transcription factors may be cloned in a T-vector, and then the cloned transcription factors may be sub-cloned in an entry cloning vector such as a pENTR4 vector (manufactured by Invitrogen) so that the cloned transcription factors are again homologus recombinated with the lentiviral vector.
Each of the transcription factors cloned in the T-vector may be ligated with the entry cloning vector to thereby be sub-cloned in the entry cloning vector.
The entry cloning vector in which the transcription factors are sub-cloned may be induced into the lentiviral vector through the homologus recombination with the lentiviral vector.
The lentiviral vectors of four types including the respective transcription factors may be infected by respective viruses to generate transgenic viruses in which respective genes are induced.
The viruses of the present invention as described above may be generated by a lentiviral vector system.
Each of the four type-viruses in which the four type-genes are effectively induced may be concentrated to prepare a virus concentrated mixture. The above- described concentration process may be performed by centrifuging the respective viruses. Through the concentration process, gene transfer efficiency into human somatic cells which will be described below may be significantly increased.
A content of each virus for preparing the virus concentrated mixture may be preferably maintained to be identical to each other so that the four genes are effectively expressed.
The prepared virus concentrated mixture may be mixed with a first culture solution to thereby prepare a virus solution. A mixture ratio between the virus concentrated mixture and the first culture solution is about 1 :1 to 5.
When the mixture ratio of the first culture solution to the virus concentrated mixture exceeds '5', the gene transfer efficiency may be significantly reduced. Conversely, when the mixture ratio thereof is less than T, problems may occur in stability of somatic cells, that is, objects of the gene transfer.
The mixture ratio between the virus concentrated mixture and the first culture solution is preferably about 1 :1. The human somatic cells in which the four type-gene combinations will be induced may be cultivated in advance in a first culture dish before performing the gene transfer, and attached on the first culture dish.
For the gene transfer, a cell separation solution such as a triple solution and the like is required to be used in the first culture dish where the human somatic cells are cultivated to thereby separate the somatic cells from the first culture dish and float the separated somatic cells. Through centrifugation, the floated somatic cells may be separated to prepare only a solid content of the somatic cells.
The purpose of floating the somatic cells is to increase a reaction surface area between the virus solution and the cells. A time required when the floated cells are completely attached on the culture dish may be about two to three hours. According to the present invention, superior efficiency may be acquired along with an increase in a probability that viruses are penetrated into spherical cells in three-dimensions, in comparison with a method of gene transfer of attached somatic cells. The floated somatic cells and virus solution as described above may be reacted with each other for about 5 to 15 minutes after being added to a reaction dish such as a conical tube and the like and mixed together.
A somatic cell-virus mixture may be prepared through the reaction.
The somatic cell-virus mixture is moved to a second culture dish including a second culture solution and retained as is for about 24 hours, and thereby infection of the somatic cells may be achieved. Specifically, genes included in the viruses may be induced into the somatic cells.
A ratio of the second culture solution to the somatic cell-virus mixture may be preferably about 10 to 20:1. When the ratio thereof exceeds '20', the gene transfer efficiency of the somatic cells may be deteriorated.
Also, a composition of the first culture solution and second culture solution may be preferably the same, and thereby gene expression may be facilitated by maintaining metabolism and function of the somatic cells.
The somatic cells in which gene transfer is carried out in about 24 hours may be separated from the second culture dish using the cell separation solution, and moved to a third culture dish including a third culture solution to be cultivated for several weeks, and thereby obtaining stem cells. A basic composition of the third culture solution may be obtained by mixing the second culture solution and a culture solution of the human embryonic stem cells with a ratio of 1 : 1 therebetween, and additionally include an undifferentiated inducer and the like. Hereinafter, the present invention will be described in detail by examples. It is to be understood, however, that these examples are for illustrative purpose only, and are not construed to limit the scope of the present invention. [Example]
1. preparation of T-vector in which Oct4, Sox2, Nanog gene and Lin 28 genes are induced
(1) total RNA extraction from human embryonic stem cells
1 ml of a trizol reagent (manufactured by Sigma) was inserted in recovered embryonic stem cells and retained for about five minutes at room temperature to destroy the cells, thereby eluting contents of the cells. Next, 200 μl of chloroform was inserted, mixed together in an inverted state, retained for about 15 minutes at a room temperature, and then centrifuged under a condition of 1,300 rpm, for about 15 minutes, and at about 4 °C , thereby collecting only a supernatant except for precipitation, that is, solid of DNA and protein. Next, 500 μl of isopropanol was inserted in the obtained mixture, retained at a room temperature for about 10 minutes, centrifuged under a condition of 1,300 rpm, for about 10 minutes, and at about 4 "C, thereby removing the remaining chloroform. Next, the obtained mixture was washed using 1 ml of EtOH of 75%, centrifuged under a condition of 8,000 rpm, for about 5 minutes, and at about 4°C, a supernatant was removed, and a pellet was dried. Next, the pellet was melted in 20 μl of an RNA inhibitor (diethylpyrocarbonate (DEPC) water), thereby preparing a complete RNA.
(2) cDNA composition
In order to compose cDNA, 3 μl(lμg/μl) of the complete RNA and 2 μl(10pmols/μl) of oligo dT(dT) were mixed together, reacted for about 5 minutes at about 70 °C , and retained for about 5 minutes at 4 °C . 15 μl of a reverse transcription mixture (water 5.6 μl, ImProm-II 5X buffer 4 μl, 25 mM MgCl2 2.4 μl, 10 mM dNTP 1 μl, RNase inhibitor 1 μl(40 unit), Improm-II reverse transcriptase 1 μl, manufactured by Promega) was inserted in the obtained mixture, annealed for about 5 minutes at 25 °C , extended for 60 minutes at 37°C, and inactivated the Improm- II reverse transcriptase for about 15 minutes at 70 °C .
(3) RT-PCR The obtained mixture was extended with 30 cycles each for 15 minutes at 95 °C ,
1 minute at 95 °C, 1 minute at 51 to 53 °C , 1 minute at 72 °C, and also extended for 5 minutes at 72 °C using the composed cDNA (product name: AccuPrime DNA Taq polymerase, manufactured by Invitrogen). A primer used for gene cloning was hθct4(forward primer: 5'-GAATTC-CCATGGCGGGACACCT-S' (22mer), reverse primer: 5'-GCGGCCGC-AGTTTGAATGCATGGGAG-3l (26mer)), hSox2(forward primer: 5'-GAATTC-GCATGTACAACATGATGG-S' (24mer)), reverse primer: 5'- GCGGCCGC-TC AC ATGTGTGAGAGG-3' (24mer), hNanog(forward primer: 5'- GAATTC-ACATGAGTGTGGATCCAGCT-3' (26mer), reverse primer: 5'- GCGGCCGC-TC AC ACGTCTTCAGGTTG-S' (26mer)), hLin28(forward primer: 5'- GAATTC-CCATGGGCTCCGTGT-3' (21mer), reverse primer: 5'-GCGGCCGC- GCTCAATTCTGTGCCTCC-3' (26mer)). A DNA band was subjected to electrolysis after performing a Polymerase Chain Reaction (PCR), dyed with ethidum bromide, and observed under ultraviolet (UV) light. The observed result can be shown in FIG. 1.
FIG. 1 is a photograph showing a DNA band detected by electrophoresis after performing the PCR. Referring to FIG. 1, it can be seen that bands of four types of genes were accurately detected.
(4) T- vector cloning
In order to carry out cloning in T-vector, only PCR band was eluted using a gel extraction kit (product name: QIAquick gel extraction kit, manufactured by Qiagen), and 1 μl of pGEM-T easy vector, 3 μl of target DNA, 1 μl of ligase buffer, 4 μl of water, and 1 μl of ligase (manufactured by Promega) were mixed together to perform overnight reaction at 16°C . It could be seen that each of the four genes was cloned using a DNA sequencing device (sequencing, Applied biosystems company's 3730XL Capillary DNA sequencer machine). FIG. 2 is a schematic diagram illustrating a mechanism of pGEM-T easy vector. 2. sub-cloning in pENTR4 vector hθct4, hSox2, hNanog and hLin28 each cloned in T-vector were cut using EcoRI enzyme (see FIG. 3), and then carried out ligation with pENTR4 vector (see FIG. 4). FIG. 3 is an electrophoretic photograph showing genes cloned in T-vector, and FIG. 4 is a schematic diagram illustrating a mechanism of pENTR4 vector.
3. homologus recombination with lentiviral vector
In order to perform recombination of pENTR4/hOct4, hSox2, hNanog and hLin28 vector and lentiviral vector (see, FIG. 5), each 4 μl of pENTR4/hOct4, hSox2, hNanog and hLin28 DNA, 2 μl of lentiviral vector, 2 μl of water, and 2 μl of LR clonase (manufactured by Invitrogen) enzyme were mixed to perform an overnight reaction at 20 °C . Then, 1 μl of Proteinase K solution was inserted to be reacted for 10 minutes at 137 °C . Next, the mixture was injected in competent cells, smeared in LB/Apm agar plate, and was cultured overnight at 37 "C . After the overnight culture, a DNA sample was extracted, and observed using the DNA sequencing device (sequencing, Applied biosystems company's 373 OXL Capillary DNA sequencer machine), which homologus recombination was carried out. FIG. 5 is a schematic diagram illustrating homologous recombination according to an exemplary embodiment of the present invention. Referring to FIG. 5, a genetic region of an entry cloning vector and a ccdB region of the lentiviral vector were replaced with each other, and thereby the homologous recombination was carried out.
4. virus production
A transient transfection was performed with a 293T cell using calcium phosphate transfection, thereby producing the virus. The calcium phosphate transfection was replaced with a medium (DMEM; manufactured by Sigma) added with a Foetal Bovine Serum (FBS) of 10% 12 to 16 hours after the transfection was performed, and virus particles were produced (See, FIG. 7). Then, 50,000 g of the virus particles were centrifuged for 4 hours at 4°C, thereby concentrating the virus. FIG. 6 is a mimetic diagram illustrating an envelope plasmid, a packaging plasmid, and a target vector each for producing viruses, and FIG. 7 is a microphotograph illustrating a state where a lentiviral vector is infected in a 239T cell. 5. gene injection in human somatic cells using lenti viral infection
0.5 x 106 numbered human somatic cells prepared in a culture dish of 100 mm the previous day were detached from the culture dish to thereby be floated. The floating of the human somatic cells was carried out such that the somatic cells were detached using the triple solution, and solid contents of the somatic cells were separated from the detached somatic cells using centrifugation. 50 μl of each virus concentrated solution corresponding to the respective genes was mixed with a first culture solution at a ratio of 1 :1 (200 μl of a virus mixture; 200 μl of the culture solution), reacted with the floated somatic cells for 5 to 10 minutes in a conical tube of 15 ml, and then placed in a culture dish of 100 mm where 5,600 μl of a second culture solution was contained. Gene transfer was performed by cultivating the virus concentrated solution for 24 hours. At the time of injecting the gene, a total of 6 ml of the culture solution was used, and 0.6 μg/ml of polybrene (manufactured by Sigma) was processed. The somatic cell- culture solution (first culture solution) and the culture solution (second culture solution) used at the time of injecting the gene were obtained by adding each of 0.1 mM of β- mercaptoethanol (manufactured by Sigma), a non-essential amino acid of 1%, 50 U/ml of penicillin, 50 μg /ml of streptomycinm, and FBS of 10% (manufactured by Hyclone) to a DMEM culture solution (No. 11995, manufactured by Invitrogen) where 4.5 g/L of high-glucose, 0.11 g/L of Na-pyruvate, and 2mM of L-glutamine were contained.
Each of the cells obtained by methods of performing the gene transfer was detached from the culture dish using the triple solution, and placed on five culture dishes of each being 60 mm where MEF (Mouse Embryonic Fibroblast) feeder cells prepared the previous day were contained, to thereby be cultivated. In this instance, the used culture solution was a somatic cell-culture solution. The somatic cell-culture solution was obtained by adding 4 μg/ml of basic Fibroblast Growth Factor, that is, an undifferentiated inducer required for maintenance of stem cell, to a standard culture solution. Here, the standard culture solution was obtained by adding 0.1 mM of β- mercaptoethanol (manufactured by Sigma), 1% of non-essential amino acid, 50 U/ml of penicillin, 50 μg/ml of streptomycin, and 20% of serum replacement (SR, manufactured by Gibco) to DMEM/F12 culture solution (No. 11320, manufactured by Invitrogen) in which 3.15 g/L of D-glucose, O.llg/L of Na- pyruvate, and 2 niM of L-glutamine are contained. The virus infection was observed through fluorescence 24 to 48 hours after the cultivation.
[Comparative Example]
According to the present Comparative Example, the remaining processes except for the gene transfer process were performed in the same way as the above-described Example in comparison with the Example.
According to the present Comparative Example, the gene transfer was performed on somatic cells attached on the culture dish. Each 50μl (total 200 μl) of the virus concentrated solution was directly sprayed on 5,800 μl of the culture solution. In this instance, the virus concentrated solution was obtained such that each of the Oct4, Sox2, Nanog, and Lin28 was contained in 0.5 x 106 numbered human somatic cells prepared in the culture dish of 100 mm the previous day.
Analysis on characteristics of somatic cells
1. Alkaline Phosphatase (AP) activity measurement
In order to examine characteristics of an undifferentiated somatic cell colony shaped in a form, activity of the AP widely used as a marker of undifferentiated cells was measured. The colony was fixed for one minute at formaldehyde (manufactured by Sigma) of 4%, washed using Tris-HCl, reacted for 15 minutes with a dye kit (product name: Fast Red Violet/Naphthol AS-BI, manufactured by Chemicon) to thereby be washed, and then a degree of the reaction was observed using a microscope.
2. verification of presence/absence of gene expression
(1) verification of presence/absence of gene expression using SSEA-3, SSEA-4, TRA- 1-60, and TRA- 1-81
The presence/absence of the gene expression was verified using SSEA (stage- specific embryonic antigen, manufactured by Santacruz)-3 and SSEA-4 each recognizing undifferentiated embryonic stem cells, and TRA (tumorigenic antigen, manufactured by Santacruz)- 1 -60 and TRA- 1-81. A colony presumed to be embryonic stem cells was fixed for 20 minutes with paraform-aldehyde (PFA, manufactured by Sigma) of 4%, washed three times using the PBS, and performed non-specific blocking for one hour using a normal goat serum of 10%. Then, the colony was reacted overnight with a first antibody for 6 minutes at 4°C with a concentration ratio of 1 :20. Next, in order to a degree of the reaction, the colony was washed three times using the PBS, and a second antibody (product name: rhodamine (TRITC)-conjugated goat anti- human IgM, 1:200, manufactured by Jackson Lab) on which TRITC is attached was processed. For nuclear staining, 4'-6-diamidino-2-phenylindole (DAPI, 1 :1,000, manufactured by Sigma) was processed, reacted for one hour at room temperature, sufficiently washed using the PBS, and then observed using a fluorescence microscope.
(2) ascertainment of expression of four genes induced from induced pluripotent stem (iPS) cells
Expression of exogenous four genes and endogenous four genes was ascertained from iPS cells generated by gene transfer. cDNA composed from two types of iPS cells was extended with 30 cycles each for 3 minutes at 94 °C , 30 seconds at 94 °C, 30 seconds at 51 to 53 °C, and one minute at 72 °C, and also extended for 5 minutes at 72 °C using polymersase (AccuPrime DNA Taq polymerase, manufactured by Invitrogen). The primer used for ascertaining endogenous gene expression was the same as the primer used at the time of cloning of the gene, and the primer used for ascertaining exogenous gene expression was the forward primer and a reverse primer (5 '- ATGCTCGTC AAGA AGAC AGG-3) originated from gene carrier. A DNA band was subjected to electrolysis after performing the PCR, dyed with ethidum bromide, and observed under ultraviolet (UV) light. The observed result can be shown in FIG. 11.
3. examination of in vitro differentiation of stem cells
In order to examine in vitro differentiation potency of embryonic stem cells, a plurality of colonies was made into an Embryoid Body (EB) having triploblastic characteristics for four days, attached on a culture dish on which gelatin is coated, and then performed dye by inducing spontaneous differentiation for two weeks within a culture solution containing a serum. The differentiated cell was fixed for 15 minutes using PFA of 4%, washed using the PBS, penetrated for 10 minutes using the triton X- 100 solution of 0.2%, and then performed blocking for one hour using the normal goat serum of 10%. An anti-nestin polyclonal antibody (nestin, 1 :500, manufactured by Chemicon) of a nerve cell factor was used for examining ectoderm potency, an anti-α- smooth muscle actin monoclonal antibody (SMA, 1;25, manufactured by Santacruz) was used for examining mesoderm potency, and an anti-α-fetoprotein polyclonal antibody (AFP, l;200, manufactured by Sigma) was used for examining endoderm potency. Each of the above-mentioned antibodies performed overnight reaction at 4 °C . In order to a degree of the reaction with respect to each of the first antibodies, a second antibody (TRITC conjugated goat anti-rabbit IgG, 1 :200, manufactured by Jackson Lab) on which TRITC is attached was processed. For nuclear staining, DAPI (1 : 1,000) was processed, reacted for one hour at a room temperature, sufficiently washed using the PBS, and then observed using the fluorescence microscope.
Results analysis
1. examination of presence/absence of occurrence of gene transfer in human somatic cells
FIG. 8 is a microphotograph (A) and a fluorescence microphotograph (B) each showing stem cells 24 hours after inducing genes. Referring to FIG. 8, it can be seen that a Venus marker-gene was expressed.
FIG. 9 is a fluorescence microphotograph showing stem cells 48 hours after inducing genes according to Comparative Example (A) and Example (B). Referring to
FIG. 9, it could be found that gene transfer efficiency of the case of somatic cells where the gene transfer was performed by Example was superior to that of the case of somatic cells where the gene transfer was performed by Comparative Example.
2. production of induced pluripotent stem (iPS) cells
FIG. 10 is microphotographs (A) and (C) and fluorescence microphotographs (B) and (D) each showing two types of stem cells (hips 1-1 and hips 1-2) originated from somatic cells 20 to 25 days after inducing genes. Referring to FIG. 10, it can be seen that stem cells of a colony was established. When comparing a number of colonies formed by the gene induced by Comparative Example (A) and Example (B), respectively, the number of colonies formed by Example (B) was 5.1 times greater than that by Comparative Example (A). 3. ascertainment of expression of four genes from iPS cells
FIG. 11 is an electrophoretic photograph showing endogenous and exogenous gene expression within two types of stem cells originated from somatic cells. Referring to FIG. 11, it could be found that four types of endogenous and exogenous genes, that is, four transcription factors where two types of stem cells (hips 1-1 and hips
1-2) originated from human somatic cells were induced were expressed.
4. examination of characteristics of iPS cells (1) Alkaline Phosphatase (AP) activity measurement
FIG. 12 is a microphotograph showing stem cells in which an Alkaline phosphatase (AP) is activated, (substantial microscope-Fast Red Violet/Naphthol AS-BI dye verification).
(2) expression of SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81
FIG. 13 is microphotographs showing a state of expression of SSEA-I, SSEA-4, TRA-1-60, and TRA-1-81. A, B, C, and D were photographs obtained by examining SSEA-3 expression. E, F, G, and H were photographs obtained by examining SSEA-4 expression. I, J, K, and L were photographs obtained by examining TRA-1-60 expression. M, N, O, and P were photographs obtained by examining TRA-1-81 expression. Referring to FIG. 13, A, E, I, and M are fluorescence microphotographs showing expression of the Venus marker-gene. Also, B, F, J, and N are fluorescence microphotographs obtained by DAPI dye, and C, G, K, and O are photographs showing expression of SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 through TRITC dye. D, H, L, and P are photographs obtained by combining DAPI and TRITC results. Referring to FIG. 13, surface factor of the stem cells originated from human somatic cells were well expressed.
(3) verification of characteristics of triploblastic differentiation of iPS cells -induction of embryoid body generation
FIG. 14 is a microphotograph (A) and a fluorescence microphotograph (B) each showing the differentiation of stem cells where differentiation has been induced for four days. Referring to FIG. 14, it can be ascertained that the embryoid body was generated and the Venus marker-gene was expressed.
- induction of triploblastic differentiation
FIG. 15 is fluorescence microphotographs showing the differentiation of stem cells where differentiation has been induced for fourteen days. Red parts of each of photographs are regions where the TRITC dye is performed, and blue parts thereof are regions where the DAPI dye is performed.
Referring to FIG. 15, it can be ascertained that differentiation of each of ectoderm (nerve cells, A), mesoderm (muscle cells, B), and endoderm (liver cells, C) was performed.
According to the present invention, human induced pluripotent stem (iPS) cells may be effectively manufactured without using an egg cell, and thus can be expected to contribute to maximize the process efficiency when the mass production is attained in the future. Although a few embodiments of the present invention have been shown and described, the present invention is not limited to the described embodiments. Instead, it would be appreciated by those skilled in the art that changes may be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.

Claims

1. A method for manufacturing stem cells, the method comprising: preparing Oct-4 gene, Sox2 gene, Nanog gene, and a fourth gene from human embryonic stem cells, and allowing each of the genes to be infected in host cells using a lentiviral vector system to generate viruses in which each of the genes are induced; concentrating or mixing each of the viruses to prepare a virus concentrated mixture, and mixing the virus concentrated mixture and a first culture solution to prepare a virus solution; floating human somatic cells having been cultivated in advance in a first culture dish, and mixing and reacting the floated somatic cells and the virus solution to prepare a somatic cell-virus mixture; adding and retaining the somatic cell-virus mixture as is in a second culture dish including a second culture solution to induce the genes in the somatic cells; and cultivating the somatic cells in which the genes are induced in a third culture dish including a third culture solution.
2. The method of claim 1, wherein the fourth gene is Lin28 gene.
3. The method of claim 1 , wherein the fourth gene is XIAP gene.
4. The method of claim 1, wherein the allowing of each of the genes to be infected in host cells includes: preparing the Oct-4 gene, the Sox2 gene, the Nanog gene, and the fourth gene from the human embryonic stem cells to clone the genes in a lentiviral vector, respectively; and allowing the cloned lentiviral vectors to be infected in the host cells to generate viruses in which the genes are induced by the cloned lentiviral vectors, respectively.
5. The method of claim 1, wherein the concentrating of each of the viruses is achieved by centrifugation.
6. The method of claim 1, wherein the mixing of each of the viruses is performed in such a manner that an amount of each of the viruses is the same.
7. The method of claim 1, wherein the virus concentrated mixture and the first culture solution are mixed with a ratio of about 1 : 1 to 5.
8. The method of claim 1, wherein the floating of human somatic cells includes: separating the somatic cells from the first culture dish using a cell separation solution; and centrifuging the separated somatic cells.
9. The method of claim 1, wherein a volume ratio of the somatic cell-virus mixture and the second culture solution is about 1 : 10 to 20.
10. The method of claim 1, wherein composition of the first and second culture solutions is the same.
11. The method of claim 1, wherein the reacting is performed for about 5 to 15 minutes.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9228204B2 (en) 2011-02-14 2016-01-05 University Of Utah Research Foundation Constructs for making induced pluripotent stem cells

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007069666A1 (en) * 2005-12-13 2007-06-21 Kyoto University Nuclear reprogramming factor

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007069666A1 (en) * 2005-12-13 2007-06-21 Kyoto University Nuclear reprogramming factor

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
PARK, I.H. ET AL.: "Reprogramming of human somatic cells to pluripotency with defined factors", NATURE, vol. 451, 10 January 2008 (2008-01-10), pages 141 - 148 *
TAKAHASHI, K. ET AL.: "Induction of Pluripotent Stem cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors", CELL, vol. 126, August 2006 (2006-08-01), pages 663 - 676 *
YU, J. ET AL.: "Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells", SCIENCE, vol. 318, 21 December 2007 (2007-12-21), pages 1917 - 1919 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9228204B2 (en) 2011-02-14 2016-01-05 University Of Utah Research Foundation Constructs for making induced pluripotent stem cells

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