WO2009006930A1 - Human pluripotent stem cells induced from undifferentiated stem cells derived from a human postnatal tissue - Google Patents

Human pluripotent stem cells induced from undifferentiated stem cells derived from a human postnatal tissue Download PDF

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WO2009006930A1
WO2009006930A1 PCT/EP2007/010019 EP2007010019W WO2009006930A1 WO 2009006930 A1 WO2009006930 A1 WO 2009006930A1 EP 2007010019 W EP2007010019 W EP 2007010019W WO 2009006930 A1 WO2009006930 A1 WO 2009006930A1
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Prior art keywords
human
stem cell
cells
pluripotent stem
tissue
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PCT/EP2007/010019
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English (en)
French (fr)
Inventor
Kazuhiro Sakurada
Tetsuya Ishikawa
Hideki Masaki
Shunichi Takahashi
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iZumi Bio Inc
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iZumi Bio Inc
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Priority to EP07856194.1A priority Critical patent/EP2164952B1/en
Priority to US12/663,840 priority patent/US20110039332A1/en
Priority to US12/157,967 priority patent/US8211697B2/en
Priority to MX2009013706A priority patent/MX2009013706A/es
Priority to NZ582018A priority patent/NZ582018A/xx
Priority to EP08807182.4A priority patent/EP2171045B1/en
Priority to JP2010511743A priority patent/JP5547064B2/ja
Priority to KR1020107001046A priority patent/KR101657318B1/ko
Priority to CN200880100396.XA priority patent/CN102317448B/zh
Priority to MYPI20095379A priority patent/MY159971A/en
Priority to PCT/IB2008/002540 priority patent/WO2009007852A2/en
Priority to AU2008273817A priority patent/AU2008273817B2/en
Priority to AP2010005102A priority patent/AP2010005102A0/en
Priority to CA2690629A priority patent/CA2690629A1/en
Publication of WO2009006930A1 publication Critical patent/WO2009006930A1/en
Priority to US12/465,633 priority patent/US20090304646A1/en
Priority to US12/484,152 priority patent/US9213999B2/en
Priority to US12/484,163 priority patent/US8257941B2/en
Priority to US12/564,836 priority patent/US20100120069A1/en
Priority to US12/580,216 priority patent/US20100105100A1/en
Priority to IL202678A priority patent/IL202678A/en
Anticipated expiration legal-status Critical
Priority to US12/685,569 priority patent/US20100267135A1/en
Priority to US12/709,379 priority patent/US20100240090A1/en
Priority to US14/195,698 priority patent/US9714433B2/en
Ceased legal-status Critical Current

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    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
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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
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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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
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • 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

Definitions

  • the present invention relates to human pluripotent stem cells induced from stem cells present in a human postnatal tissue and its inducing method.
  • diseases associated with tissue degeneration and tissue injury are rapidly increasing. Said diseases include cerebral infarction, myocardial infarction and renal failure that develop in an age- dependent manner due to the metabolic syndrome, Alzheimer's disease, Parkinson's disease and osteoporosis that are induced by age-related internal changes of the tissue, and the like.
  • Type I diabetes, multiple sclerosis and rheumatoid arthritis induced by autoimmune diseases as well as burns and spinal injuries induced by wounds are also diseases characterized by tissue degeneration and tissue injury. As methods of treating such diseases resulting from tissue degeneration and injury, various regenerative medical techniques are being developed now.
  • Regenerative medicine is roughly divided into two methods: the induced regeneration method in which endogenous stem cells in patients are activated with a drug etc., and the cell replacement therapy in which stem cells or somatic cells induced from stem cells or tissues are transplanted.
  • the induced regeneration method does not work well due to reduced function of stem cells from the patient per se, and thus the development of the cell replacement therapy is imperative.
  • a large amount of stem cells or somatic cells induced from stem cells generally need to be prepared as materials for transplantation.
  • stem cells that can differentiate into various tissues and that can self- replicate for a long time are indispensable for the development of a cell replacement therapy.
  • stem cells from cells derived from a human postnatal tissue, said stem cells having properties close to that of ES cells and comprising the genome of the patient per se thereby circumventing immunological rejection of transplanted cells.
  • human pluripotent stem cells can be induced by introducing three genes of Oct3/4, Sox2 and KIf4 or three genes of Oct3/4, Sox2 and KIf4 plus the c-Myc gene or a histone deacetylase (HDAC) inhibitor into undifferentiated stem cells present in a human postnatal tissue in which each gene of Tert, Nanog, Oct3/4 and Sox2 has not undergone epigenetic inactivation.
  • HDAC histone deacetylase
  • Human postnatal tissues are preferably tissues immediately after birth (various tissues of neonates) , umbilical cord tissues (the umbilical cord, cord blood) , the amnion, the placenta etc., and more preferably various neonatal tissues and umbilical cord tissues.
  • Post-natal tissues include tissues of various timings during the period from the birth of an individual to its death.
  • the undifferentiated stem cells refer to stem cells in which at least four genes of Nanog, Oct3/4, Sox2 and Tert have not undergone epigenetic modification by heterochromatin formation due to DNA methylation or histone modification, among the primordial cells in the tissue of somatic stem cells established in vitro, such as mesenchymal stem cells (Science, 1999, Apr 2; 284 (5411) : 143-7) and MAPCs (multipotent adult progenitor cells) (Stem Cell Rev. 2005; 1(1): 53-9), and MIAMI (marrow- isolated adult multilineage inducible) cells (J. Cell Sci . 2004 Jun 15; 117 (Pt 14) : 2971- 81) .
  • ES cell-like pluripotent stem cells refer to cells having an in vitro long-term self-renewal ability and the pluripotency of differentiating into three germ layers, and said pluripotent stem cells may form teratoma when transplanted into a test animal such as mouse.
  • the present invention is thought to provide a useful technique for the cell replacement therapy for the treatment of diseases resulting from tissue degeneration or injury.
  • cells that can be transformed into ES cell -like pluripotent stem cells by introducing four genes of Oct3/4, Sox2 , KIf4 and c-Myc are undifferentiated stem cells in which each gene of Tert , Nanog, Oct3/4 and Sox2 has not undergone epigenetic inactivation;
  • ES cell-like pluripotent stem cells can be induced from undifferentiated stem cells in which the Tert, Nanog, Oct3/4 and Sox2 genes have not undergone epigenetic inactivation present in postnatal tissues, and thus, it is expected that, in the case of humans as well, by adding a histone deacetylase inhibitor in stead of the c-Myc gene to undifferentiated stem cells in which the Tert, Nanog, Oct3/4 and Sox2 genes have not undergone epigenetic inactivation, they could be transformed into ES cell-like pluripotent stem cells.
  • the present invention provides the following (1) to (35) :
  • a human pluripotent stem cell having an in vitro long- term self-renewal ability and the pluripotency of differentiating into ectoderm, mesoderm and endoderm, that was induced from an undifferentiated stem cell present in a human postnatal tissue in which each gene of Tert, Nanog, Oct3/4 and Sox2 has not undergone epigenetic inactivation.
  • the human pluripotent stem cell according to the above (1) induced from an undifferentiated stem cell present in a human postnatal tissue, wherein said undifferentiated stem cell present in the human postnatal tissue was subjected to a primary culture or a second subculture, or a subculture in a low serum concentration.
  • Oct3/4, Sox2 and KIf4 in an undifferentiated stem cell present in a human postnatal tissue wherein said undifferentiated stem cell present in the human postnatal tissue was subjected to a primary culture or a second subculture or to a subculture in a low serum concentration.
  • Oct3/4, Sox2, KIf4 and c-Myc in an undifferentiated stem cell present in a human postnatal tissue, wherein said undifferentiated stem cell present in the human postnatal tissue was subjected to a primary culture or a second subculture or to a subculture in a low serum concentration.
  • the human pluripotent stem cell according to the above (1) induced by combining the forced expression of each of three genes of Oct3/4, Sox2 and KIf4 and a histone deacetylase inhibitor treatment in an undifferentiated stem cell present in a human postnatal tissue, wherein said undifferentiated stem cell present in the human postnatal tissue was subjected to a primary culture or a second subculture or to a subculture in a low serum concentration.
  • the human pluripotent stem cell according to the above (1) induced by combining the forced expression of each of three genes of Oct3/4, Sox2 and KIf4 and a MS-275 treatment in an undifferentiated stem cell present in a human postnatal tissue, wherein said undifferentiated stem cell present in the human postnatal tissue was subjected to a primary culture or a second subculture or to a subculture in a low serum concentration.
  • the human pluripotent stem cell according to any one of the above (1) to (12) wherein said human pluripotent stem cell comes to have teratoma-forming potential when it is transplanted into a test animal .
  • the human pluripotent stem cell according to any one of the above (1) to (13) wherein said human postnatal tissue is a tissue immediately after birth and is a tissue derived from a neonatal tissue or an umbilical cord tissue.
  • the human pluripotent stem cell according to any one of the above (1) to (13) wherein said human postnatal tissue is a tissue immediately after birth and is a tissue derived from a neonatal skin or a blood vessel derived from the umbilical cord.
  • said human postnatal tissue is a tissue immediately after birth and is a tissue derived from a neonatal tissue or an umbilical cord tissue.
  • said human postnatal tissue is a tissue immediately after birth and is a tissue derived from a neonatal skin or a blood vessel of the umbilical cord.
  • a method of inducing a human pluripotent stem cell wherein an undifferentiated stem cell present in a human postnatal tissue, in which each gene of Tert, Nanog, Oct3/4 and Sox2 has not undergone epigenetic inactivation, is subjected to a primary culture or a second subculture or to a third or fourth subculture in a low serum concentration at 0 to 5%, and then each of three genes of Oct3/4, Sox2 and Klf4 is subjected to forced expression.
  • the undifferentiated stem cells of the present invention present in a human postnatal tissue refer to stem cells that have not undergone epigenetic modification by heterochromatin formation due to DNA methylation or histone modification of at least four genes of Nanog, Oct3/4, Sox2 and Tert among the primordial cells in the tissue of various somatic stem cells established in vitro, such as mesenchymal stem cells, MAPCs and MIAMI cells.
  • stem cells that have not undergone epigenetic modification by heterochromatin formation due to DNA methylation or histone modification of at least four genes of Nanog, Oct3/4, Sox2 and Tert among the primordial cells in the tissue of various somatic stem cells established in vitro, such as mesenchymal stem cells, MAPCs and MIAMI cells.
  • Mesenchymal stem cells refer to those cells having the potential of differentiating into mesenchymal cells (bone, cartilage, fat) among the cells (interstitial cells) obtained as nonhematopoietic cells that are adherent to a plastic culture tray when tissues of bone marrow, fat, muscle, skin etc. are cultured in a culture medium containing a high- concentration serum (5% or more) .
  • mesenchymal stem cells are the cells obtained by the above culturing, and thus their properties are different from those of the undifferentiated cells (stem cells in which at least four genes of Nanog, Oct3/4, Sox2 and Tert have not undergone epigenetic modification by heterochromatin formation due to DNA methylation or histone modification, among the primordial cells in the tissue of somatic stem cells established in vitro, such as mesenchymal stem cells, MAPCs and MIAMI cells) immediately after isolation from human postnatal tissues.
  • the undifferentiated cells stem cells in which at least four genes of Nanog, Oct3/4, Sox2 and Tert have not undergone epigenetic modification by heterochromatin formation due to DNA methylation or histone modification, among the primordial cells in the tissue of somatic stem cells established in vitro, such as mesenchymal stem cells, MAPCs and MIAMI cells
  • each tissue at various timings during the period from the birth of an individual to its death (bone marrow fluid, muscle, adipose tissue, peripheral blood, skin, skeletal muscle etc.) and tissues concomitant to birth such as cord tissues (umbilical cord, cord blood) , the amnion, the placenta and the like, preferably there can be mentioned tissues (bone marrow fluid, muscle, adipose tissue, peripheral blood, skin, skeletal muscle etc.) immediately after birth such as various neonatal tissues, and more preferably there can be mentioned various neonatal tissues such as neonatal skin and cord tissues (umbilical cord, cord blood) such as tissues derived from cord-derived blood vessels.
  • Undifferentiated stem cells present in the human postnatal tissues of the present invention can be cultured for a certain period from a primary culture in a culture medium containing or not containing a low concentration serum (preferably 2% or less) and to which cell growth factors (PDGF, EGF, FGF-2 etc.) have been added or not added, and have properties different from those of mesenchymal stem cells that are characterized by a long time culturing in the serum (concentrations exceeding 5%) .
  • a low concentration serum preferably 2% or less
  • cell growth factors PDGF, EGF, FGF-2 etc.
  • FGF-2 As the above cell growth factors, there can be mentioned FGF-2, PDGF, EGF, IGF, insulin, TGFb-I, activin A, noggin, BDNF, NGF, NT-I, NT-2, NT-3 and the like, and the addition of FGF-2 alone or the addition of both PDGF and EGF is preferred.
  • FGF-2 stands for basic fibroblast growth factor
  • PDGF platelet -derived growth factor
  • EGF epidermal growth factor
  • IGF insulin-like growth factor
  • TGF ⁇ -1 stands for transforming growth factor ⁇ -1
  • BDNF brain-derived neurotrophic factor
  • NGF nerve growth factor
  • NT-I neurotrophin-1
  • NT-2 stands for neurotrophin-2
  • NT-3 stands for neurotrophin-3.
  • the above primary culture represents immediately after isolation from a human, the primary culture cells subcultured once represent the second subculture, the primary culture cells subcultured twice represent the third subculture, and the primary culture cells subcultured three times represent the fourth subculture.
  • Culturing for a certain period from the above primary culture generally means from the primary culture to the fourth subculture, preferably from the primary culture to the second subculture.
  • Human pluripotent stem cells induced from undifferentiated stem cells present in a human postnatal tissue in which the Tert, Nanog, Oct3/4 and Sox2 genes have not undergone epigenetic inactivation represent stem cells that have a long- term self-renewal ability under the condition for culturing human ES cells and an in vitro pluripotency of differentiating into ectoderm, mesoderm and endoderm under the condition for inducing in vitro differentiation of human ES cells, and the above human pluripotent stem cells may further have a potential of differentiating into primordial germ cells under the condition for inducing in vitro differentiation of human ES cells.
  • human pluripotent stem cells of the present invention induced from undifferentiated stem cells present in a human postnatal tissue in which the Tert , Nanog, Oct3/4 and Sox2 genes have not undergone epigenetic inactivation may be stem cells that have an ability of forming teratoma when transplanted into a test animal such as mouse.
  • the low concentration serum encompassed by the present invention is generally serum at a concentration of 5% or less, preferably serum at a concentration of 2% or less, and the low density as used herein is a concentration of about 10% or less.
  • the following method may be mentioned.
  • the cells are fixed in a 10% formaldehyde solution at room temperature for 2 to 5 minutes, washed with a phosphate buffer etc., a solution of nitroblue tetrazolium chloride/5-bromo-4-chloro-3 ' -indolyl phosphate p- toluidine salt (hereinafter referred to as the NBT/BCIP solution) , a chromogenic substrate of alkaline phosphatase, is added, and reacted at room temperature for 20-30 minutes.
  • NBT/BCIP solution a solution of nitroblue tetrazolium chloride/5-bromo-4-chloro-3 ' -indolyl phosphate p- toluidine salt
  • the human pluripotent stem cells were expressed cell surface antigens SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, CD9, CD24, and CD90, and ES cell marker genes Nanog, Oct3/4, TDGFl, Dnmt3b, GABRB3 , GDF3 , Zfp42, ALP, CD9, and Thy-1.
  • the promoter regions of Nanog and Oct3/4 in the human pluripotent stem cells were demethylated compared to the parental fibroblasts.
  • the human pluripotent stem cells carries at least a single copy of Oct3/4, Sox2 , Klf4, and c-Myc transgene .
  • the induced human pluripotent stem cells and the parental cells had almost the same SNP genotype each other, and HLA type of the induced human pluripotent stem cell was completely identical to that of the parental cell (undifferentiated stem cell present in a human postnatal tissue) .
  • a histone deacetylase inhibitor and MS-275 and a treatment method using them are as describe later:
  • the method of forced expression as used herein comprises a method for external expression in which a gene is expressed by introducing it with a vector etc. and a method for internal expression in which internal expression is promoted by the stimulation of a drug etc.
  • forced expression as used herein also encompasses a method in which the genes of Oct3/4, Sox2 , Klf4 and c-Myc are extracellularly expressed, and then the proteins produced of Oct3/4, Sox2 , KIf4 and c-Myc are introduced directly into the cell using a method for introducing protein.
  • the method for introducing protein there can be mentioned in case of a method that employs a commercially available carrier reagent (Chariot, BioPorter, GenomONE) , the PTD (protein transduction domain) fusion protein method, the electroporation method, the microinjection method and the like.
  • the external expression method in which each gene of Oct3/4, Sox2 , KIf4 and c-Myc is introduced into a vector etc. for forced expression is as follows:
  • the donor In order to harvest a bone marrow fluid from human bone marrow, the donor is given a general anesthetic, then placed on a prone position, and from the posterior border of the ilium, a needle called the bone marrow collection needle is stuck directly into the skin to lead the needle through the iliac surface to the bone marrow, and the liquid of the bone marrow is aspirated with a syringe.
  • the mononuclear cell fraction separated by density centrifugation is collected.
  • the collected cell fraction as crude purified cells containing the undifferentiated stem cells, is cultured according to the method described in 6. , and used for the induction of human pluripotent stem cells of the present invention.
  • a skin tissue containing the epidermis and the dermis is harvested. This skin tissue is immersed in 0.6% trypsin (manufactured by Invitrogen) /DMEM (Dulbecco's Modified Eagle's Medium) /F- 12 (manufactured by Invitrogen) /1% antibiotics, antimycotics (manufactured by Invitrogen) with the inner side of the skin facing downward, and treated at 37°C for 30 minutes.
  • the skin tissue is finely cut into about 1 mm 2 sections using scissors, which are then centrifuged at 1200 rpm and room temperature for 10 minutes. The supernatant is removed, and to the tissue precipitate is added 25 ml of 0.1% trypsin/DMEM/F-12/1% antibiotics, antimycotics, and stirred using a stirrer at 37°C and 200-300 rpm for 40 minutes.
  • fetal bovine serum (manufactured by JRH) is added, and filtered sequentially with gauze (Type I manufactured by PIP) , a 100 ⁇ m nylon filter (manufactured by FALCON) and a 40 ⁇ m nylon filter (manufactured by FALCON) .
  • gauze Type I manufactured by PIP
  • DMEM/F-12/1% antibiotics, antimycotics is added to wash the precipitate, and then centrifuged at 1200 rpm and room temperature for 10 minutes.
  • the cell faction thus obtained may be cultured according to the method described in 6. below as crude purified cells containing undifferentiated stem cells, and used for the induction of human pluripotent stem cells of the present invention.
  • a connective tissue containing muscle such as a lateral head of biceps brachii muscle and a sartorius muscle of the leg is cut and the muscle is excised, it is sutured.
  • the whole muscle obtained is minced with scissors or a scalpel, and then suspended in DMEM (high glucose) containing 0.06% collagenase type IA (manufactured by SIGMA) and 10% FBS, and incubated at 37°C for 2 hours.
  • the cell fraction obtained may be cultured according to the method described in 6. below as crude purified cells containing undifferentiated stem cells, and used for the induction of human pluripotent stem cells of the present invention.
  • adipose tissue for use in the present invention may be isolated by various methods known to a person skilled in the art. For example, such a method is described in U.S. Pat. No. 6,153,432, which is incorporated herein in its entirety.
  • a preferred source of adipose tissue is omental adipose tissue.
  • adipose cells are typically- isolated by fat aspiration.
  • adipose tissue is treated with 0.01% to 0.5%, preferably 0.04% to 0.2%, and most preferably about 0.1% collagenase, 0.01% to 0.5%, preferably 0.04%, and most preferably about 0.2% trypsin and/or 0.5 ng/ml to 10 ng/ml dispase, or an effective amount of hyaluronidase or DNase (DNA digesting enzyme) , and about 0.01 to about 2.0 mM, preferably about 0.1 to about 1.0 mM, most preferably 0.53 mM concentration of ethylenediaminetetraacetic acid (EDTA) at 25 to 50°C, preferably 33 to 40°C, and most preferably 37°C for 10 minutes to 3 hours, preferably 30 minutes to 1 hour, and most preferably 45 minutes.
  • EDTA ethylenediaminetetraacetic acid
  • Cells are passed through nylon or a cheese cloth mesh filter of 20 microns to 800 microns, more preferably 40 microns to 400 microns, and most preferably 70 microns. Then the cells in the culture medium are subjected to differential centrifugation directly or using Ficoll or Percoll or another particle gradient. The cells are centrifuged at 100 to 3000xg, more preferably 200 to 1500xg, most preferably 500xg for 1 minute to 1 hours, more preferably 2 to 15 minutes and most preferably 5 minutes, at 4 to 50°C, preferably 20 to 40°C and more preferably about 25°C.
  • the adipose tissue-derived cell fraction thus obtained may be cultured according to the method described in 6. below as crude purified cells containing undifferentiated stem cells, and used for the induction of human pluripotent stem cells of the present invention.
  • a method of separating a cell fraction that contains undifferentiated stem cells from a human postnatal peripheral blood or human cord blood As a method of obtaining the undifferentiated stem cells of the present invention present in human postnatal tissue from human postnatal peripheral blood or human cord blood, the following method may be mentioned.
  • a RPMI 1640 medium manufactured by Invitrogen
  • an essential medium for culturing peripheral blood stem cells containing 10% fetal bovine serum (manufactured by JRH Biosciences) , 100 ⁇ g/ml streptomycin and 100 units/ml penicillin (manufactured by Invitrogen) , and after washing twice, the cells are recovered.
  • the recovered cells are suspended again in the essential medium for culturing peripheral blood stem cells, which is then plated in a 100 mm plastic culture dish at 1 x 10 7 cells/dish, and incubated in a 37°C incubator under a condition of 8% CO 2 . After 10 hours, suspended cells are removed and the attached cells are only harvested by pipetting.
  • peripheral blood-derived or cord blood-derived adherent cell fraction thus obtained may be cultured according to the method described in 6. below as crude purified cells containing undifferentiated stem cells, and used for the induction of human pluripotent stem cells of the present invention.
  • Examples of culture media useful in culturing the undifferentiated stem cells of the present invention present in a human postnatal tissue include the ES medium [40% Dulbecco ' s Modified Eagle's Medium (DMEM), 40% F12 medium, 2 mM L- glutamine, 1% non-essential amino acids, 0.1 mM ⁇ - mercaptoethanol (the above are manufactured by SIGMA) , 20% Knockout Serum Replacement (manufactured by Invitrogen) , 10 ⁇ g/ml gentamycin (manufactured by Invitrogen) ] (hereinafter referred to as the ES medium), the MAPC medium [60% Dulbecco ' s Modified Eagle's Medium-low glucose (manufactured by Invitrogen) , 40% MCDB 201 (manufactured by Invitrogen) , IxITS medium supplement (manufactured by SIGMA) , Ix linolenic acid albumin (manu
  • growth factors growth factors, cytokines, hormones
  • FGF-2 growth factors, cytokines, hormones
  • PDGF vascular endothelial growth factor
  • EGF EGF
  • IGF insulin
  • TGFb-I activin A
  • Noggin BDNF
  • NGF NGF
  • NT-I NT-2
  • NT-3 NT-3 and the like.
  • the cell fraction obtained by the above methods 1. to 5. is cultured in a medium containing the above additives for about 1 to 12 days at a low density of about 10 3 cells/cm 2 to 10 4 cells/cm 2 .
  • retrovirus vectors including lentivirus vectors
  • adenovirus vectors preferably adenovirus vectors are used to introduce a mouse-derived cationic amino acid transporter (mCAT) gene, and then a retrovirus vector is used to introduce the Oct3/4, Sox2 , KIf4 and c-Myc genes.
  • mCAT mouse-derived cationic amino acid transporter
  • virus vector plasmids there can be mentioned pMXs, pMXs-IB, pMXs-puro, pMXs-neo (pMXs-IB is a vector carrying the blasticidin-resistant gene in stead of the puromycin-resistant gene of pMXs-puro) [Experimental Hematology, 2003, 31 (11) : 1007-14], MFG [Proc. Natl. Acad. Sci . U.S.A.
  • any cells may be used that can supply a lacking protein of a recombinant virus vector plasmid deficient in at least one gene encoding a protein required for virus packaging.
  • HEK-293 cells derived from human kidney, packaging cells based on a mouse fibroblast NIH3T3, and the like.
  • retrovirus-derived proteins such as gag, pol , and env in the case of retrovirus vectors
  • HIV-derived proteins such as gag, pol, env, vpr, vpu, vif, tat, rev, and nef in the case of lentivirus vectors
  • adenovirus-derived proteins such as ElA and ElB in the case of adenovirus vectors.
  • recombinant virus vectors By introducing any of the above recombinant virus vector plasmid into the above packaging cells, recombinant virus vectors can be produced.
  • various gene introduction methods are known including, but not limited to, the calcium phosphate method [Kokai (Japanese Unexamined Patent Publication) No. 2-227075] , the lipofection method [Proc. Natl. Acad. Sci . U.S.A. 84: 7413 (1987)], the electroporation method and the like, and any suitable method may be used from the known gene introduction methods.
  • histone acetylase inhibitors there can be mentioned those described in the following A to E, and among them MS-275 is preferred.
  • Trichostatin A and its analogs for example: trichostatin A (TSA); and trichostatin C (Koghe et al . 1998, Biochem. Pharmacol. 56: 1359-1364).
  • Hybrid polar compounds based on hydroxamic acid, for example: salicyl hydroxamic acid (SBHA) (Andrews et al . , International J. Parasitology 30: 761-8 (2000)); suberoylanilide hydroxamic acid (SAHA) (Richon et al . , Proc . Natl. Acad. Sci. U.S.A. 95: 3003-7 (1998)); azelaic bishydroxamic acid (ABHA) (Andrews et al . , supra); azelaic-1- hydroxamate-9-anilide (AAHA) (Qiu et al . , MoI. Biol.
  • SBHA salicyl hydroxamic acid
  • SAHA suberoylanilide hydroxamic acid
  • SAHA suberoylanilide hydroxamic acid
  • SAHA suberoylanilide hydroxamic acid
  • ABHA azelaic bishydroxa
  • SFA Short chain fatty acid
  • Na butyrate Cousens et al . , J. Biol. Chem. 254: 1716-23 (1979)
  • isovalerate McBain et al . , Biochem. Pharm. 53: 1357- 68 (1997)
  • valproic acid valerate
  • valerate McBain et al . , supra
  • 4- phenyl butyric acid (4 -PBA) (Lea and Tulsyan, Anticancer RESearch 15: 879-3 (1995)
  • PB phenyl butyric acid
  • Benzamide derivatives for example: MS-275 [N- (2- aminophenyl) -4- [N- (pyridine-3 -yl- methoxycarbonyl) aminomethyl] benzamide] (Saito et al . , Proc. Natl. Acad. Sci. U.S.A. 96: 4592-7 (1999)); and a 3 ' -amino derivative of MS-275 (Saito et al . , supra); and CI-994.
  • a histone deacetylase inhibitor treatment may be carried out, for example, as follows:
  • the concentration of the histone deacetylase inhibitor used depends on a particular inhibitor, but is preferably 0.001 nM to about 10 mM, and more preferably about 0.01 nM to about 1000 nM.
  • the effective amount or the dosage of a histone deacetylase inhibitor is defined as the amount of the histone deacetylase inhibitor that does not significantly decrease the survival rate of cells, specifically undifferentiated stem cells. Cells are exposed for 1 to 5 days or 1 to 3 days. The exposure period may be less than one day. In a specific embodiment, cells are cultured for about 1 to 5 days, and then exposed to an effective amount of a histone deacetylase inhibitor. However, the histone deacetylase inhibitor may be added at the start of culturing. Within such a time frame, a gene-carrying vehicle such as a vector containing a nucleic acid encoding three genes (Oct3/4, Sox2 and KIf4) is introduced into cultured cells by a known method.
  • Examples of culture media useful for culturing human pluripotent stem cells induced from undifferentiated stem cells present in a human postnatal tissue of the present invention include, but not limited to, the ES medium, and a culture medium suitable for culturing human ES cells such as MEF- conditioned ES medium (hereinafter referred to as the MEF- conditioned ES medium) which is a supernatant obtained by adding 10 ng/ml FGF-2 to the ES medium and then mouse embryonic fibroblasts (hereinafter referred to as MEF) were added thereto and cultured for 24 hours to obtain the supernatant .
  • MEF- conditioned ES medium MEF- conditioned ES medium
  • MEF mouse embryonic fibroblasts
  • growth factors As “growth factors, cytokines, hormones” to be added to the above culture media, there can be mentioned ingredients involved in the growth and maintenance of human ES cells including FGF-2, TGFb-I, activin A, Nanoggin, BDNF, NGF, NT-I, NT-2, NT-3 and the like.
  • Y-27632 Calbiochem; water soluble
  • Fasudil HA1077: Calbiochem
  • Rho associated kinase an inhibitor of Rho associated kinase (Rho associated coiled coil -containing protein kinase) is also useful for culturing the human pluripotent stem cells of the present invention.
  • the cells are subcultured every 5 to 7 days in a culture medium containing the above additives on a MEF-covered plastic culture dish or a matrigel -coated plastic culture dish to 1:3 to 1:6 or plated at 10 3 cells/cm 2 to 3 x 10 4 cells/cm 2 .
  • undifferentiated stem cells present in a human postnatal tissue are detected at high rates in young individuals, preferred undifferentiated stem cells for the cell bank are cord blood, the umbilical cord, the placenta, skin obtained from neonates and the like. Even in adults, undifferentiated stem cells for the cell bank may be harvested from the bone marrow, adipose tissue, peripheral blood, skin and the like depending on the physical status of the donor.
  • the undifferentiated stem cells of the present invention obtained from each donor may be stored frozen as they are, or may be transformed into human pluripotent stem cells according to the above-mentioned method of the present invention prior to storing frozen.
  • the undifferentiated stem cells of the present invention or the human pluripotent stem cells of the present invention thus stored may be used for the treatment of the donor per se or of immunohistologically compatible recipients as well.
  • the human pluripotent stem cells of the present invention In treatment, depending on the amount of cell replacement required for the treatment of the subject disease, the human pluripotent stem cells of the present invention must be subcultured according to the method of the above 8.
  • the required number of the human pluripotent stem cells of the present invention obtained by subculturing can be used for the treatment of various diseases by a method described below.
  • Diseases of the central nervous system using the human pluripotent stem cells of the present invention include Parkinson's disease, Alzheimer's disease, multiple sclerosis, cerebral infarction, spinal injury and the like.
  • Parkinson's disease a therapeutic method is possible in which human pluripotent stem cells are differentiated into dopamine-acting neurons and then transplanted into the striate body of the patient with Parkinson's disease. Differentiation into dopamine-acting neurons can be effected by coculturing the PA6 cell which is a mouse stromal cell line and the human pluripotent stem cells of the present invention under a serum-free condition.
  • the human pluripotent stem cells of the present invention are induced to differentiate into neural stem cells followed by transplantation into the injured site is effective.
  • the human pluripotent stem cells of the present invention are cultured in suspension to form an embryoid body, and the embryoid body obtained is cultured in a serum- free medium containing FGF-2 for use in the culture of neural stem cells.
  • the human pluripotent stem cells of the present invention are cocultured with the PA6 cell which is a mouse stromal cell line, and then cultured in a serum- free medium containing FGF-2 for use in the culture of neural stem cells .
  • the human pluripotent stem cells of the present invention are transferred to a serum- free medium containing FGF-2 to directly induce differentiation.
  • treatment can be effected by further inducing the differentiation of neural stem cells induced from the human pluripotent stem cells of the present invention into oligodendrocytes or progenitors of oligodendrocytes, which are then transplanted to the injured site.
  • oligodendrocytes or progenitors of oligodendrocytes from neural stem cells induced from the human pluripotent stem cells of the present invention
  • a method of culturing said neural stem cells in the presence of a fusion protein between a soluble interleukin-6 receptor and interleukin-6 there can be mentioned a method of culturing said neural stem cells in the presence of a fusion protein between a soluble interleukin-6 receptor and interleukin-6.
  • the human pluripotent stem cells of the present invention can be used for the treatment of hepatic diseases such as hepatitis, cirrhosis and liver failure.
  • the human pluripotent stem cells of the present invention are preferably differentiated to hepatic cells or hepatic stem cells, and then are transplanted.
  • Hepatic cells or hepatic stem cells may be obtained by culturing the human pluripotent stem cells of the present invention in the presence of activin A for 5 days, and then culturing in the presence of the hepatocyte growth factor (HGF) for about a week to obtain hepatic cell or hepatic stem cells.
  • HGF hepatocyte growth factor
  • the human pluripotent stem cells of the present invention can be used for the treatment of pancreatic diseases such as type I diabetes mellitus.
  • pancreatic diseases such as type I diabetes mellitus.
  • the human pluripotent stem cells of the present invention are preferably differentiated to pancreatic beta cells, and then are transplanted to the pancreas.
  • the human pluripotent stem cells of the present invention can be differentiated to pancreatic beta cells in following six steps of culturing: (1) culturing in the presence of a serum-free medium, activin A and Wnt protein for 1 to 2 days; (2) culturing in the presence of 0.2% FBS and activin A for 1 to 2 days; (3) culturing in the presence of 2% FBS, FGF- 10 and KAAD- cyclopamine (keto-N-aminoethylaminocaproyl dihydrocinnamoylcyclopamine) for 2 to 4 days; (4) culturing in the presence of 1% B27 (manufactured by Invitrogen) , FGF-10, KAAD-eyelopamine and retinoic acid for 2 to 4 days; (5) culturing in the presence of 1% B27, gamma secretase inhibitor and extendin-4 for 2 to 3 days; (6) culturing in the presence of 1% B27, extendin-4
  • the human pluripotent stem cells of the present invention can be used for the treatment of heart failure associated with ischemic heart diseases.
  • the human pluripotent stem cells of the present invention are preferably differentiated into cardiac muscle cells prior to transplanting to the injured site.
  • cardiac muscle cells can be obtained from the human pluripotent stem cells of the present invention in about 2 weeks after forming the embryoid body.
  • the present invention provides for the first time human pluripotent stem cells induced from undifferentiated stem cells present in a human postnatal tissue and having an in vitro long-term self-renewal ability and the pluripotency of differentiating into ectoderm, mesoderm and endoderm, and further said human pluripotent stem cells may have a potential of differentiating into primordial germ cells.
  • Cells in a tissue that was lost in diseases etc. can be supplied by inducing human pluripotent cells from the undifferentiated stem cells harvested from a patient by using the induction method of the present invention, followed by inducing to differenciate into a necessary cell depending on diseases and then transplanting the cells to the patient.
  • the undifferentiated stem cells of the present invention present in a human postnatal tissue can be used to search drugs that promote the induction from said undifferentiated stem cells to human pluripotent stem cells by using markers such as Tert, Nanog, Sox2 , Oct3/4 and alkaline phosphatase that direct the induction to human pluripotent stem cells. Said drugs can be used in stead of gene introduction and can enhance the induction efficiency of human pluripotent stem cells.
  • Fig. 1 Four genes of Oct3/4, Sox2 , KIf4 and c-Myc were introduced into cells established under a low serum condition from mononuclear cells derived from a human adult bone marrow, and RNA was extracted from the colonies obtained, and the amount expressed of the human Nanog and human Tert genes was demonstrated by quantitative PCR. Fibroblasts and mesenchymal stem cells in which the four genes were not introduced were used as the control in the experiment . The amount expressed of the gene was expressed by a relative value in which the amount expressed was normalized by the amount expressed of the human HPRT gene, and by setting as one the amount expressed of the gene in alkaline phosphatase-positive colonies induced from a neonatal skin fibroblast established by example 6. It was confirmed that the expression of Nanog and Tert was significantly high in colonies in which four genes were introduced and which were positive for alkaline phosphatase.
  • Fig. 2 Four genes of Oct3/4, Sox2 , Klf4 and c-Myc were introduced into the primary culture fibroblasts derived from a neonatal skin, RNA was extracted from the colonies obtained, and the amount expressed of the human Nanog and human Tert genes was demonstrated by quantitative PCR. Its parental fibroblasts and mesenchymal stem cells in which four genes were not introduced were used as the control in the experiment. The amount expressed of genes was normalized by the amount expressed of the human HPRT gene, and further was expressed by a relative value by setting as one the amount expressed of the gene in alkaline phosphatase-positive colonies induced from a neonatal skin fibroblast established by example 6. It was confirmed that the expression of Nanog and Tert was significantly high in colonies in which four genes were introduced and which were positive for alkaline phosphatase.
  • Fig. 3 After three gene introduction and treatment with MS-275 (0.1 or 1.0 ⁇ M) , a histone deacetylase (HDAC) inhibitor by using cells derived from a mouse bone marrow established under a low serum condition, RNA was extracted from the colonies obtained, and the amount expressed of Nanog was demonstrated by quantitative PCR. From the cells in which three genes were introduced and which were treated with a histone deacetylase inhibitor, alkaline phosphatase-positive cell group (colonies) was formed, and it was confirmed that the expression of Nanog in these colonies was significantly higher than the alkaline phosphatase-negative colonies.
  • HDAC histone deacetylase
  • Wl, W2 , W3 , W4 , W5 and W6 represent the number of each well of the 6-well plate used in Example 12.
  • FIG. 4 Figure shows the characterization of human iPS clone 1-8.
  • a-e Morphology of its parental fibroblast (lot. 5F0438)
  • a) human iPS clone 1-8 cells cultured on MEF feeder cells
  • b) human iPS clone 1-8 cells in mTeSRl medium
  • c) human iPS clone 1-8 cells in mTeSRl medium
  • clone 2-4 cells clone 2-4 cells
  • clone 3-2 cells e
  • mTeSRl medium f-g
  • Growth curve of clone 1-8 Growth curve of clone 1-8.
  • Arrows indicate the dates of examinations. Square indicates the period for counting cell numbers to estimate cell proliferation rate
  • h Multicolor karyogram image indicates normal karyotype of iPS clone 1-8 derived cell at day 101.
  • FIG. 5 Figure shows characterization of transcription factor, cell surface antigens and alkaline phosphatase activity in human iPS clone 1-8 cell.
  • a-h Immunohistochemical staining of human iPS cells (clone 1-8) with Nanog (a) , SSEA-3 (b) , SSEA-4 (c) , TRA-1-60 (d) , TRA-1-81 (e) , CD9 (f) , CD24 (g) , Thy- 1 (also called CD90) (h) .
  • Green fluorescent staining indicates that human iPS clone 1-8 expresses all of these surface antigens, i, Alkaline phosphatase staining indicates that iPS clone 1-8 is alkaline phosphatase positive.
  • FIG. 6 Figure shows gene expression analysis of human iPS clone 1-8 cells, a, RT-PCR analysis of hES marker gene expression in clone 1-8 and its parental fibroblast (NeoFB) . Genes were detected at 30 cycles except for CYP26A1 (35 cycles) . b, Silencing of four transgenes in clone 1-8. Crude fibroblasts obtained on 17 days after gene transduction were used as control . "exo" primer sets selectively detected exogenous expression and “total” primer sets included endogenous expression.
  • FIG. 7 Figure shows global gene expression analysis of human iPS clonel-8 cells. Scatter plots show comparison of global gene expression between human iPS clone-1-8 cells cultured in mTeSR and H14 hES cells with MEFs (GSM151741 from public database GEO) (a) , or between clone 1-8 and its parental fibroblasts (b) . Symbols of ES cell specific genes were pointed with lines in both scatter plots. Expression intensity was shown in colorimetric order from red (high) to green (low) .
  • FIG. 8 Figure shows global gene expression analysis by gene trees.
  • Cells were clustered in the gene tree based on a set of genes by the International Stem Cell Initiative (except PTFlA because of no array in the chip) .
  • Samples were designated l-8mTeSR for clone-1-8 cultured in mTeSR, 1-8CM for clone 1-8 cultured in MEF-conditioned medium, 5F0438 for the parental fibroblasts, hESl, hES2 , hES3 (GSM194307, GSM194308, GSM194309) for Sheff 4 line cultured on MEF, hES4, hES5 (GSM194313, GSM194314) for Sheff 4 line cultured on matrigel, hES6, hES7
  • GSM151739, GSM151741) for H14 line cultured on MEF
  • Fibroblastsl for GSM96262
  • Fibroblasts2 for GSM96263, and Fibroblasts3 for GSM96264, respectively.
  • Expression intensity was shown in colorimetric order from red (high) to green (low) .
  • FIG. 9 Figure shows global gene expression analysis by gene trees.
  • Cells were clustered in the gene tree based on a set of genes correlated with Nanog gene expression in human ES cells (seven GEO data) between the ratio of 0.99 and 1 when compared with fibroblasts (three GEO data) .
  • Samples were designated l-8mTeSR for clone-1-8 cultured in mTeSR, 1-8CM for clone 1-8 cultured in MEF-conditioned medium, 5F0438 for the parental fibroblasts, hESl, hES2 , hES3 (GSM194307, GSM194308, GSM194309) for Sheff 4 line cultured on MEF, hES4 , hES5
  • GSM194313, GSM194314 for Sheff 4 line cultured on matrigel
  • hES6, hES7 GSM151739, GSM151741
  • Fibroblastsl for GSM96262, Fibroblasts2 for GSM96263, and Fibroblasts3 for GSM96264, respectively.
  • Expression intensity was shown in colorimetric order from red (high) to green (low) .
  • Fig. 10 The parts of the Oct3/4 promoter including the distal enhancer (Oct3/4-Zl) and the proximal promoter region
  • FIG. 11 Figure shows teratoma that was derived from human iPS-l-8mTeSR cells cultured for 94 days.
  • Human iPS-l-8mTeSR cells were injected into SCID mouse testes and analyzed 56 days after injection, a, HE and alcian blue staining of formaldehyde fixed teratoma tissues.
  • the teratomas contained tissues representative of the three germ layers, ne : neural epitherium, ca: cartilage, et : endodermal tract, b-d, tissues originated from transplant were distinguished from host tissues by HuNu staining.
  • Nestin expressing neural epitherium (b) Collagen II expressing chondrocyte (c) , alpha-fetoprotein expressing endodermal tract (d) .
  • Fig. 12 Figure shows teratoma formation.
  • Teratoma 1 (T- 1) was derived from human iPS-l-8mTeSR cells cultured for 94 days. The human iPS-l-8mTeSR cells were injected into SCID mouse testes and analyzed 56 days after injection.
  • Teratoma 2 (T-2) was derived from human iPS-l-8mTeSR cells cultured for 102 days. The human iPS-l-8mTeSR cells were injected into SCID mouse testes and analyzed 48 days after injection.
  • smooth muscle cells positive for ⁇ -SMA
  • secretary epithelium positive for MUC-I
  • Fig. 13 Figure shows teratoma formation.
  • Teratoma 3 (T3) was derived from human iPS-l-8mTeSR cells cultured for 114 days. Human iPS-l-8mTeSR cells were injected into SCID mouse testis and analyzed 42 days after injection. Three germ layers similar to Fig.11 and 12 were observed.
  • T-Fl and F2 figure shows teratoma that were derived from freeze-thawed iPS-l-8mTeSR cells cultured for 134 days (passage 19) .
  • Human iPS-l-8mTeSR cells were injected into SCID mouse testes and analyzed 46 days (T-Fl) and 48 days (T-F2) after injection. Tissues consisting of three germ layers were observed. Melanocytes were also observed in T-F2 experiment. Pluripotency were maintained even via freezing and thawing.
  • Fig. 14 Figure shows the existence of four transgenes in human iPS clone 1-8. Oct3/4, Sox2 , and KIf4 transgenes were detected by Southern blot analysis. Human iPS clone-1-8 was estimated to have approximately ten copies of both Oct3/4 transgenes and Sox2 transgenes, and a single copy of KIf4 transgene. Genomic PCR proved c-Myc transduction. Primer set was designed to include whole second intron. Black arrows indicate the position of transgene. White arrow indicates the position of endogenous c-Myc.
  • FIG. 15 Figure shows hES maker gene expression profile in ALP positive colonies induced by four genes (Oct4, Sox2 , KIf4 and c-Myc) . Colonies were stained for alkaline phosphatase at 17 days post 4 genes transduction. All ALP (+) colonies were dissected and determined their hES marker gene expressions, a, the number of colonies expressing Nanog, TDGFl, Dnmt3b, Zfp42, FoxD3, TERT, CYP26A1, and GDF3. b, morphologies of octa- positive colonies, c-d, the number of hES cell marker genes categorized by individual experiments.
  • Fig. 16-Fig. 22 Figure shows morphologies of four gene (Oct4, Sox2 , Klf4 and c-Myc) induced colonies categorized by gene expression profile of ES cell related 8 genes (Nanog, TDGFl, Dnmt3b, Zfp42, FoxD3 , TERT, CYP26A1, and GDF3) as well as alkaline phosphatase activity. Circles indicate the picked- up colony.
  • Undifferentiated stem cells present in a human postnatal tissues are undifferentiated stem cells which are present in human postnatal skin, bone marrow, adipose tissue, skeletal muscle tissue, and peripheral blood, and tissues concomitant to birth such as placenta, umbilical cord and cord blood and in which the Tert, Nanog, Oct3/4 and Sox2 genes have not undergone epigenetic inactivation, and, by using a combination of induced expression of the three genes of Oct3/4, Sox2 and KIf4 and the induced expression of c-Myc or the addition of a histone deacetylase inhibitor, can induce human pluripotent stem cells having a long-term self-renewal ability and the pluripotency of differentiating into ectoderm, mesoderm and endoderm.
  • the above human pluripotent stem cells may further have a potential of differentiating into primordial germ cells.
  • Undifferentiated stem cells present in a human postnatal tissue can be cultured using a plastic culture dish.
  • a 2% serum is used, PDGF and EGF or FGF-2 may be added to the culture medium, to which IGF or insulin may further be added.
  • a culture medium containing serum is used for a long term culture, properties of undifferentiated stem cells present in a human postnatal tissue may change, and thus it is important to limit the serum concentration to 2% or less and the number of passages to about twice.
  • the MAPC medium or the FBM medium for example, is used as the culture medium.
  • an incubator at 37°C and 5% CO 2 is used similarly to common culture cells. It is also possible to use low concentration oxygen, for example a 3% oxygen concentration.
  • Culture plates are preferably coated with fibronectin etc.
  • the human pluripotent stem cells of the present invention induced from undifferentiated stem cells present in a human postnatal tissue may be cultured using a plastic culture dish.
  • cells after the four genes of Oct3/4, Sox2 , KIf4 and c-Myc were introduced therein are cultured in a MEF-conditioned human ES cell medium to which 10 ng/ml bFGF and 10 ng/ml activin A had been added, and the medium is changed every 1 to 2 days.
  • the pluripotent stem cells induced are detached with dispase, collagenase, trypsin or the like, and subcultured.
  • the induced human pluripotent stem cells are plated on a MEF-covered plastic culture dish, and cultured in a human ES cell medium supplemented with 10 ng/ml bFGF.
  • the supporting cells are not used, the induced human pluripotent stem cells are plated on a matrigel -coated plastic culture dish, and cultured in a MEF-conditioned human ES cell medium supplemented with 10 ng/ml bFGF and 10 ng/ml activin A. In either of the culture methods, the medium is changed every 1 to 2 days .
  • an adenovirus vector is constructed carrying cDNA having the sequence of coding region of the mouse-derived cationic amino acid transporter (mCAT) gene (see Example 2, Table 1), which is then introduced into the packaging cell based on the HEK293 cell to prepare a virus solution of the adenovirus vector.
  • mCAT mouse-derived cationic amino acid transporter
  • the virus solution is added at a multiplicity of infection (m.o.i.: the ratio of the number of virus particles to the number of cells) of 1 to 20 to undifferentiated stem cells present in a human postnatal tissue in which each gene of Tert, Nanog, Oct3/4 and Sox2 has not undergone epigenetic inactivation, and thus undifferentiated stem cells expressing mCAT are prepared.
  • m.o.i. the ratio of the number of virus particles to the number of cells
  • a retrovirus vector carrying cDNA encoding human Oct3/4, a retrovirus vector carrying cDNA encoding human Sox2 , a retrovirus vector carrying cDNA encoding human Klf4, and a retrovirus vector carrying cDNA encoding human c-Myc are constructed (Table 1) , and then each of them is introduced into the packaging cell capable of producing an ecotropic recombinant virus constructed based on the HEK293 cell to prepare a virus solution of retrovirus vectors.
  • the human pluripotent stem cells of the present invention after being suspended in the Cryopreservation Medium For Primate ES Cells (manufactured by ReproCELL) , preferably are rapidly frozen in liquid nitrogen, and stored in a liquid nitrogen storage vessel.
  • the pluripotent stem cells of the present invention that were stored frozen are rapidly thawed by suspending in a medium that had been warmed to 37°C, removing the medium from the suspension by centrifugation, and then suspending again in a fresh medium to start culturing.
  • siRNA represents a double stranded RNA that comprises about 19 base pairs which is part of the sequence of a gene, and that has an effect of inhibiting the translation of the gene to the protein due to RNA interference.
  • siRNA library it is possible to identify a gene that inhibits the induction from a undifferentiated stem cell present in a human postnatal tissue in which the Tert, Nanog, Oct3/4 and Sox2 genes have not undergone epigenetic inactivation to a human pluripotent stem cell.
  • an inhibitor of the gene using this method it is possible to induce human pluripotent stem cells from undifferentiated stem cells present in a human postnatal tissue .
  • siRNA library those in which four siRNA's are synthesized for each gene of a total of about 25,000 human genes, mixed in equal amounts, and dispensed in a 384 -well culture plate are used, and subjected to screening
  • siRNA manufactured by Qiagen. Details of it are as follows. Four siRNA's synthesized for each gene are mixed in equal amounts, and 2.5 pmol each is dispensed in each well of a 384-well culture plate. In order to cover all of about 25,000 genes, seventy three 384-well culture plates are needed. To predetermined wells of each plate, 2.5 pmol each of the positive and negative control siRNAs is dispensed in order to determine the introduction efficiency of siRNA into the cell and to correct for efficiency of each plate. The final concentration of siRNA is 50 nM.
  • siRNA was prepared, a primary screening is conducted.
  • methods of detecting the activation of genes that could be an index for differentiation into the pluripotent stem cells of the present invention such as Tert, Nanog, Oct3/4 and Sox2 in the cell to be targeted there are the promoter reporter assay of the gene of interest [as the reporter gene, EGFP (enhanced green fluorescence protein), luciferase etc.], the immunocytochemical staining method to said gene product, and the like.
  • the lipofection method may be used for transfection of siRNA to the cell.
  • the lipofection method may be used to each well of a total of 73 plates in which siRNA has been dispensed.
  • 0.1 ⁇ l of LipofectAMINE RNAiMax To each well of a total of 73 plates in which siRNA has been dispensed, 0.1 ⁇ l of LipofectAMINE RNAiMax
  • Opti-MEM manufactured by Invitrogen
  • target cells prepared at 20 to 25 cells/ ⁇ l in up to 40 ⁇ l of the medium are dispended to every well on the 73 plates to introduce siRNA into the cell.
  • the number of cells and the amount of the medium are determined as appropriate depending on the cell used for screening.
  • a reporter system In conducting a reporter assay, cells in which a reporter system has been permanently integrated with a retrovirus vector (including lentivirus) or cells 1 to 7 days after infection with an adenovirus vector carrying the reporter system of interest are used for cells such as adult stem cells for which gene introduction by the lipofection method or the calcium phosphate method is difficult.
  • the reporter system of the present invention When the reporter system of the present invention is applied to cultured lined cells such as HEK293 cells and HeIa cells, the reporter system should be introduced one day in advance or simultaneously with siRNA by a gene introduction method suitable for respective cells.
  • the entire 73 plates to which transfection reagents and cells have been dispensed are cultured in a culturing equipment maintained at 37°C and 5% CO 2 for 2 to 7 days.
  • the culturing time may vary as appropriate depending on the type of the cell, the gene to be detected, and the like.
  • alkaline phosphatase staining As a method of selecting siRNA that promotes the induction from undifferentiated stem cells present in a human postnatal tissue to human pluripotent stem cells, alkaline phosphatase staining can be used. As the alkaline phosphatase staining method, the following method can be mentioned.
  • cells are fixed in a 10% formaldehyde solution at room temperature for 2 to 5 minutes, washed with a phosphate buffer etc., and a chromogenic substrate of alkaline phosphatase, nitroblue tetrazolium chloride/5-bromo-4-chloro-3 ' -indolyl phosphatase para-toluidine salt solution (hereinafter referred to as the NBT/BCIP solution) is added and reacted at room temperature for 20 to 30 minutes .
  • NBT/BCIP solution a chromogenic substrate of alkaline phosphatase, nitroblue tetrazolium chloride/5-bromo-4-chloro-3 ' -indolyl phosphatase para-toluidine salt solution
  • the method used is conducted similarly to the above screening used for siRNA.
  • the compound in stead of siRNA is spotted in each well, the cell is dispensed and cultured, and similarly determined. The transfection procedure is not necessary.
  • the retrovirus vector plasmids for the four genes of Oct3/4-pMx, Sox2-pMx, Klf4-pMx and c-Myc-pMx constructed as in Table 1 were introduced into the packaging cell, the Plat-E cell [Experimental Hematology, 2003, 31 (11): 1007-14], using Fugene HD (manufactured by Roche) .
  • the medium was replaced with a medium suitable for the cell to which gene is to be introduced.
  • the supernatant was recovered and passed through a filter of 45 ⁇ m in diameter (manufactured by Millipore) .
  • the retrovirus vector solutions of the four genes (Oct3/4, Sox2 , KIf4 and c-Myc) were prepared.
  • the retrovirus vector plasmids for the three genes of Oct3/4-pMx, Sox2-pMx, Klf4-pMx and c-Myc-pMx were introduced into the packaging cell, the Plat-E cell, using Fugene HD (manufactured by Roche) .
  • the medium was replaced with a medium suitable for the cell to which gene is to be introduced.
  • the supernatant was recovered and passed through a filter of 45 ⁇ m in diameter (manufactured by Millipore) .
  • the retrovirus vector solution of the three genes (Oct3/4, Sox2 and KIf4) were prepared.
  • Example 2 Preparation of adenovirus vector
  • an oncogene c-Myc
  • c-Myc an oncogene
  • mCATl mouse-derived cationic amino acid transporter 1
  • an adenovirus vector carrying cDNA having the sequence of coding region of the mouse-derived cationic amino acid transporter (mCATl) gene was constructed. Specifically, Adeno-X Expression System 1 kit (manufactured by TakaraBio Clontech) was used. In Adeno-X Expression System 1 kit, based on the experimental method attached to the kit by TakaraBio, the mCATl gene was subcloned into the multi-cloning site of a vector called pShuttle.
  • an expression cassette was excised by the PI -See I site and the I-Ceu I site, cleavage sites on both ends of the expression cassette of pShuttle, and a DNA fragment containing the desired gene was inserted in between the PI -See I site and the I-Ceu I site in the Adeno-X Viral DNA in the above kit, which was then treated with a restriction enzyme Swa I to remove adenovirus DNA for which integration was unsuccessful.
  • the plasmid was transformed into an E. coli DH5 strain, whether the desired gene was correctly introduced into adenovirus DNA or not was confirmed by restriction enzyme treatment, PCR etc.
  • the plasmid was prepared in large quantities, and cleaved with the Pac I restriction enzyme.
  • the gene was introduced into the HEK293 cells (MicroBix) plated in six wells using Lipofectamin 2000 (manufactured by Invitrogen) , and two weeks later when the cell exhibited a cytopathic effect (CPE) , the cells were collected as they are in the medium.
  • CPE cytopathic effect
  • the virus suspension thus prepared was added to one 100 mm plastic culture dish equivalent of HEK293 cells (5 x 10 6 cells) to infect the cells, the virus was propagated. Furthermore, after virus was prepared in large quantities using four 150 mm plate equivalent of HEK293 cells, virus was purified using the Adenovirus Purification kit (manufactured by Clontech) , and stored frozen at -80°C.
  • the titer (plaque forming units, PFU) of the mCATl adenovirus vector was determined using the Adeno-X Rapid Titer kit.
  • HEK293 low cells were plated at a concentration of 5 x 10 4 cells/500 ⁇ l per well.
  • Fifty ⁇ l of serially diluted (from 10 "2 to 10 "7 ) virus vector was mixed with 500 ⁇ l of the medium, and then used to infect the cells. After culturing at 5% CO 2 and 37°C for 48 hours, the medium was aspirated off, the cells were dried for 5 minutes, and then using 500 ⁇ l of cold 100% methanol the cells were fixed by allowing to stand at -20°C for 10 minutes.
  • DAB diaminobenzidine
  • phosphate buffer 250 ⁇ l of the DAB (diaminobenzidine) solution (10 -fold DAB concentrate was diluted with a stable peroxidase buffer) was added to wells, and was allowed to stand at room temperature for 10 minutes. After aspirating off DAB, 500 ⁇ l of phosphate buffer was added. Using a 2Ox objective lens, the number of brown positive cells in six viewing fields was counted.
  • target gene of each colony including an alkaline phosphatase-positive colonies was determined using quantitative PCR in the following manner. Colonies developed by the induction of pluripotent stem cells were harvested, and RNA was extracted using the Recoverall total nucleic acid isolation kit for FFPE (manufactured by Ambion) . After synthesizing cDNA from the extracted RNA, the target gene was amplified using the Taqman Preamp mastermix (manufactured by Applied Biosystems) .
  • the Taqman gene exprESsion assay (manufactured by Applied Biosystems) was used. The following shows the name of the target gene and the product code of each primer.
  • Human Hprt Hs99999909_ml, human Nanog : Hs02387400_gl, human Tert : Hs00162669_ml, Mouse Hprt: Mm01545399_ml, mouse Nanog: MaO2O1955O_sl .
  • cDNA extracted from mesenchymal stem cells established by the following manner was used.
  • the cell mass thus obtained was resuspended in 10ml of MSCGM medium, and plated on a 100 mm plate at a concentration of 10 5 cells/cm 2 and cultured at 37°C. Seven days later, the medium was changed. At this time, the suspended cells in the old medium were collected by centrifuging at 300 g and 4 °C for five minutes, and were returned to the cells together with the fresh medium. On day 13 when the adherent cells became confluent, the supernatant was removed, non-adherent cells were washed off with a phosphate buffer, and adherent cells were collected by detaching with a 0.05% trypsin-EDTA solution and plated at a concentration of 3000 cells/cm 2 . RNA was collected from the cells of the third subculture, and cDNA was synthesized. Example 5. Induction of human pluripotent stem cells from undifferentiated stem cells present in a postnatal human adult bone marrow tissue
  • human adult bone marrow-derived cells (trade name: Human Bone Marrow-Derived Mononuclear Cell) containing undifferentiated stem cells present in a postnatal human adult bone marrow tissue, the cells were established under the low serum (2%) and the high serum (10%) culture conditions, and were used in the experiment for inducing pluripotent stem cells.
  • hBMMNCs frozen human bone marrow-derived mononuclear cells
  • Lot 060809B female, 20 years old, white/ and hBMMNCs
  • the cell mass thus obtained was resuspended, and plated at a concentration of 10 5 cells/cm 2 on a 100 mm plate coated with 10 ng/ml fibronectin.
  • Growth factors [10 ng/ml PDGF-BB
  • the cells were established using a MSCGM medium
  • the Human Bone Marrow-Derived Mononuclear Cells were plated at a concentration of 10 5 cells/cm 2 in a 100 mm plate to which 10 ml of the MSCGM medium had been added, and cultured at 37°C. Seven days later, the suspended cells and the medium were collected except the adherent cells, and centrifuged at 300 g and 4°C for five minutes, and after the supernatant was removed, the cells were resuspended in a fresh medium. The cell suspension was returned to the original 10 cm dish, and culturing was continued.
  • Adherent cells were collected by detaching with a 0.05% trypsin-EDTA solution, and using a cell banker (manufactured by Juj i Field), the primary culture was stored frozen.
  • the wells were washed with the Hank's balanced salt solution, and then colonies were surrounded by a cloning ring
  • the medium was removed, washed with the Hank's balanced salt solution, the Detachment Medium For Primate ES Cells
  • Nanog and Tert were expressed by the colony of alkaline phosphatase activity-positive pluripotent stem cells.
  • the amount expressed of Nanog was as much as 30 -fold higher.
  • the expression of Tert was noted only in said pluripotent stem cells, and not in the mesenchymal stem cells. In the cells that did not form colonies despite the introduction of the four genes, Nanog or Tert was not expressed (Fig. 1) .
  • the pluripotent stem cells were obtained from the low serum culture group but not at all from the high serum culture group (Lot 060809B and Lot 060470B) (Table 2) . Also, culturing under the low serum condition was suitable for the maintenance of the undifferentiated cells.
  • Example 6 Induction of human pluripotent stem cells from undifferentiated stem cells present in human neonatal skin
  • Neonatal Normal Human Skin Fibroblasts primary culture
  • Neonatal Normal Human Skin Fibroblasts primary culture, manufactured by Lonza, Lot 5F0438
  • MCDB202 modified medium a medium containing 2% fetal bovine serum, 5 ⁇ g/ml insulin, 50 ⁇ g/ml gentamycin, 50 ng/ml amphotericin-B (FBM medium, manufactured by Lonza) to obtain 12 ml of a cell suspension.
  • Example 2 Fourteen hours later, the medium was removed, and the mCATl adenovirus vector prepared in Example 2 at an amount equivalent to a m.o.i. of 5 in 500 ⁇ l of the Hank's balanced salt solution per well was added, and was infected at room temperature for 30 minutes. To each well, 2 ml of the FBM medium was added respectively, and cultured at 37°C.
  • the medium of each well was replaced with 2 ml of the retrovirus vector solution (polybrene at a final concentration of 4 ⁇ g/ml was added) of the four genes (Oct3/4, Sox2 , Klf4 and c-Myc) prepared in Example 1, and cultured at 37°C for 4 hours.
  • the virus supernatant was removed and replaced with the MEF-conditioned ES medium. Then medium change with the MEF- conditioned ES medium was continued every two days, and fourteen days after the introduction of the four genes, one well of the 6-well plate was subjected to alkaline phosphatase staining. As a result, six pluripotent stem cell -like alkaline phosphatase-positive colonies were obtained. Alkaline phosphatase-positive colonies were composed of markedly smaller cells than the neonatal normal human skin fibroblasts.
  • Nanog and Tert were expressed by the colonies of alkaline phosphatase activity-positive pluripotent stem cells.
  • the neonatal normal human skin fibroblasts before the introduction of the four genes did not express Nanog, whereas in the case of the cells after the introduction of the four genes, 9-fold as much in the cells that are not forming colonies and 18 -fold as much expression of Nanog in the alkaline phosphatase activity- positive colonies were observed (Fig. 2) .
  • the expression of Tert was only noted in the alkaline phosphatase activity-positive colonies.
  • the pluripotent stem cells are defined by the characteristics of alkaline phosphatase activity-positive and Nanog-positive and Tert -positive .
  • the neonatal normal human skin fibroblasts were confirmed to be the cells that have a relatively high efficiency of inducing the pluripotent stem cells and that can express Nanog by the introduction of the four genes.
  • Colonies of the pluripotent stem cells were isolated in the following manner. On day 17 after gene introduction, six colonies with a characteristic shape were selected from the remaining wells. After washing the wells with the Hank's balanced salt solution, colonies were surrounded by a cloning ring (manufactured by Iwaki) to the bottom of which silicone grease had been applied.
  • One hundred ⁇ l of the Detachment Medium For Primate ES Cells (manufactured by ReproCELL) was added in the ring and cultured at 37°C for 20 minutes.
  • the cell suspension in the ring containing the detached colonies was added to 2 ml of the MEF-conditioned ES medium, and plated in one well of a MEF-coated 24-well plate. After culturing at 37°C for 14 hours, the medium was changed, and subsequently medium change was continued every two days, and 8 days later a second subculture was carried out. The medium was removed, the cells were washed with the Hank's balanced salt solution, the Detachment Medium For Primate ES Cells was added and cultured at 37°C for 10 minutes, and 2 ml of the medium was added to stop the reaction.
  • the cell suspension was transferred to a centrifuge tube, and centrifuged at 4°C and 200 g for 5 minutes, and the supernatant was removed.
  • the cells were resuspended in the MEF-conditioned ES medium, and plated on four wells of a MEF- coated 24-well plate. Seven days after the second subculture, in a subculturing method described below, the cells were plated on a 60 mm plastic culture dish of which bottom had been coated with matrigel at a concentration of 20 ⁇ g/cm 2 .
  • the induced pluripotent stem cells were subcultured every 5 to 7 days for maintenance and growth. From the plastic culture dish on which subculturing is to be conducted, the medium was removed, the cells were washed with the Hank's balanced salt solution, dispase or the Detachment Medium For Primate ES Cells was added, and cultured at 37°C for 5 to 10 minutes. When more than half of the colonies were detached, the ES medium was added to stop the reaction, and the cell suspension was transferred to a centrifuge tube. When colonies precipitated on the bottom of the tube, the supernatant was removed, and the ES medium was added again for suspension. After examining the size of the colonies, any extremely large ones were divided into appropriate sizes by slowly pipetting. Appropriately sized colonies were plated on a matrigel -coated plastic culture dish with a base area of about 3 to 6 times that before subculture. The colony-derived pluripotent stem cells are being grown and maintained now.
  • the Neonatal Normal Human Skin Fibroblasts in the lot (Lot 5F0474) other than the above lot 5F0438 exhibited a favorable induction of pluripotent stem cells. From comparison to Example 5, cells derived from young individuals or cells of which culturing time is short were thought to be suitable for the induction of the pluripotent stem cells.
  • Example 7 Induction of human pluripotent stem cells from undifferentiated stem cells present in a human adult skin
  • human adult tissue-derived cells (trade name: Adult Normal Human Skin Fibroblasts, primary culture) containing undifferentiated stem cells present in a human adult skin, the induction of pluripotent stem cells of the present invention was carried out.
  • Example 2 Fourteen hours later, the medium was removed, and the mCATl adenovirus vector prepared in Example 2 at an amount equivalent to a m.o.i. of 5 in 500 ⁇ l of the Hank's balanced salt solution per well was added, and was infected at room temperature for 30 minutes. To each well, 2 ml of the FBM medium was added, and cultured at 37°C.
  • the medium of each well was replaced with 2 ml of the retrovirus vector solution (polybrene at a final concentration of 4 ⁇ g/ml was added) of the four genes (Oct3/4, Sox2 , KIf4 and c-Myc) prepared in Example 1, and cultured at 37°C for 4 hours.
  • the virus supernatant was removed and replaced with the MEF- conditioned ES medium.
  • medium change with the MEF- conditioned ES medium was continued every two days, and thirteen days after the introduction of the four genes, alkaline phosphatase staining was carried out.
  • Example 8 Examination using neonatal normal human skin fibroblasts of the third subculture
  • Neonatal Normal Human Skin Fibroblasts primary culture, manufactured by Lonza, Lot 5F0439
  • a second subculture two 100 mm plastic culture dishes.
  • the cells were detached using a 0.025% trypsin-EDTA solution (manufactured by Lonza) , centrifuged at 4°C and 200 g for 5 minutes, and the supernatant was removed.
  • the second subcultured cells collected were stored frozen using the cell banker.
  • the frozen second subculture cells were thawed in a 37°C incubator, suspended in 12 ml of the FBM medium, centrifuged at 4°C and 200 g for 5 minutes, and the supernatant was removed.
  • the cells were suspended, and plated at a concentration of 10 4 cell/cm 2 on a 100 mm plastic culture dish of which bottom had been coated with matrigel at a concentration of 20 ⁇ g/cm 2 (a third subculture) .
  • the medium was removed, and the mCATl adenovirus vector prepared in Example 2 at an amount equivalent to a m.o.i. of 5 in 2 ml of the Hank's balanced salt solution was added, and was infected at room temperature for 30 minutes.
  • 10 ml of the FBM medium was added, and cultured at 37°C.
  • the medium Forty eight hours after the introduction of the mCAT-1 adenovirus vector, the medium was removed, and replaced with 10 ml of the retrovirus vector solution (polybrene at a final concentration of 4 ⁇ g/ml was added) of the four genes (Oct3/4, Sox2 , Klf4 and c-Myc) prepared in Example 1, and cultured at 37°C for 4 hours.
  • the virus supernatant was removed and replaced with the MEF-conditioned ES medium.
  • medium change with the MEF-conditioned ES medium was continued every two days, and fourteen days after the introduction of the four genes, alkaline phosphatase staining was carried out. As a result, five pluripotent stem cell-like alkaline phosphatase- positive colonies were obtained. By calculating based on the area of the bottom, this indicates that 0.83 colony per well of the 6 -well plate was obtained (Table 2) .
  • Example 9 Induction of human pluripotent stem cells from undifferentiated stem cells present in the umbilical cord (1)
  • the induction of the human pluripotent stem cells of the present invention from undifferentiated stem cells present in the umbilical cord was attempted.
  • EBM-2 Endothelial Cell Medium kit-2 manufactured by Lonza (2% serum) (hereinafter referred to as EBM-2) to obtain 12 ml of the cell suspension.
  • EBM-2 Endothelial Cell Medium kit-2 manufactured by Lonza (2% serum)
  • EBM-2 2% serum
  • each of the EBM-2 medium 2.5 ml each of the EBM-2 medium was added to each well, and cultured at 37°C. Forty eight hours after the introduction of the mCAT-1 adenovirus vector, the medium of each well was replaced with 2 ml each of the retrovirus vector solutions (polybrene at a final concentration of 5 ⁇ g/tnl was added) of the four genes (Oct3/4, Sox2 , KIf4 and c-Myc) prepared in Example 1, and cultured at 37°C for 4 hours. The virus supernatant was removed and replaced with the MEF-conditioned ES medium. Then medium change with the MEF-conditioned ES medium was continued every two days. Twelve days after the introduction of the four genes, colonies were confirmed.
  • the induction of the human pluripotent stem cells of the present invention from undifferentiated stem cells present in the umbilical cord was attempted.
  • the medium was removed, and the mCATl adenovirus vector at an amount equivalent to a m.o.i. of 1.25 to 5 in 500 ⁇ l of the Hank's balanced salt solution per well was added, and infected at room temperature for 30 minutes. 2.5 ml each of the SmGM-2 medium was added to each well, and cultured at 37°C.
  • the medium of each well was replaced with 2 ml each of the retrovirus vector solutions (polybrene at a final concentration of 5 ⁇ g/ml was added) of the four genes (Oct3/4, Sox2 , KIf4 and c-Myc) prepared in Example 1, and cultured at 37°C for 4 hours.
  • the virus supernatant was removed and replaced with the MEF-conditioned ES medium.
  • medium change with the MEF-conditioned ES medium was continued every two days. Thirteen days after the introduction of the four genes, colonies were confirmed. However, the induced colonies were not stained with alkaline phosphatase activity.
  • Example 11 Induction of mouse pluripotent stem cells from undifferentiated stem cells present in a mouse postnatal tissue
  • Femurs and tibias were extracted from 4 to 6 week-old mice (c57BL/6N lineage, 4 -week-old, female) taking utmost care not to bring in any other tissue.
  • the collected bone was soaked in 70% ethanol for a short period of time, the cells that attached to the outside of the bone were killed to prevent the contamination of cells other than the bone marrow.
  • the bone was immediately transferred to IMDM (Iscove's Modified Dulbecco ' s Medium) (manufactured by SIGMA) to prevent the effect of the cells inside of the bone marrow.
  • IMDM Iscove's Modified Dulbecco ' s Medium
  • All of the treated bone was transferred to a mortar having IMDM, and was smashed with a pestle. After washing several times with IMDM, the bone was cut into pieces with scissors. After further washing with IMDM several times, bone fragments were transferred to centrifuge tubes.
  • the cells derived from mouse deep bone marrow were suspended in the MAPC medium, and plated at a concentration of 10 5 cells/cm 2 .
  • a dish previously coated with a phosphate buffer containing 10 ng/ml fibronectin (Becton Dickinson) was used.
  • growth factors 10 ng/ml PDGF-BB (manufactured by Peprotech) , 10 ng/ml EGF (manufactured by Peprotech) , 1000 units/ml LIF (manufactured by Chemicon) ] were added at the time of use. Three days after plating, growth factors were only added without changing the medium.
  • non-adherent cells were washed off with the phosphate buffer, and adherent cells were collected by detaching with a 0.05% trypsin-EDTA solution (manufactured by Invitrogen) , and using a cell banker (manufactured by Juj i Field), the cells were stored frozen as the primary culture.
  • the primary culture cells that had been stored frozen were thawed in a 37°C water bath, and suspended in 10 ml of the MAPC medium that is a medium containing 2% FBS. In order to remove DMSO in the frozen solution, it was centrifuged at 4°C and 300 g for 7 minutes, and the supernatant was removed. The cell mass obtained was resuspended, and plated at a concentration of 2.5 x 10 3 cells/cm 2 on a 12 -well plastic plate having the bottom which had been gelatin-coated with 0.1% gelatin/phosphate buffer, and 2 ml each of the MAPC medium was added (the second subculture) .
  • the medium was removed, and 2 ml each of the four gene retrovirus vector solution prepared as in Example 1 was added thereto and cultured at 37°C for 4 to 14 hours. Then the virus solution was removed, and replaced with the mouse ES medium [the ES medium to which a final concentration of 0.3% FBS (manufactured by Invitrogen) , 1000 units/ml LIF (manufactured by Chemicon) , and 0.1 mM 2- mercaptoethanol were added] . Then medium change with the mouse ES medium was continued every three days, and 5 to 7 days after the introduction of the four genes, said pluripotent stem cells formed colonies comprising mouse ES cell-like small cells. The colonies of the induced pluripotent stem cells were stained blue violet by alkaline phosphatase activity.
  • mice The mouse pluripotent stem cells of the seventh subculture were subcutaneously transplanted to the back of three syngeneic C57BL/6N mice at 3 x 10 5 cells/mouse, and 38 days later the teratoma that formed was extracted. Teratoma was formed in all three mice. From the extracted teratoma, slices were prepared, and differentiation potential into three germ layers was analyzed by immunological staining and histological staining
  • MAP2 -positive cells the nervous system
  • GFAP-positive cells the nervous system
  • mesodermic system and cartilage tissues as the mesodermic system, and intestinal tract tissues as the endodermic system were observed.
  • the mouse pluripotent stem cells were subcultured every 3 to 4 days.
  • the medium was removed from the plastic culture dish in which subculture is carried out, washed with phosphate buffer, a 0.05% trypsin-EDTA solution was added, and cultured at 37°C for 5 minutes.
  • the ES medium was added to stop the reaction, and the cell suspension was transferred to a centrifuge tube. By centrifuging at 200 g for 5 minutes, the supernatant was removed, and after suspending the precipitate in the mouse ES medium, the cells were plated in a gelatin- coated plate at a concentration of 10 4 cells/cm 2 .
  • the pluripotent stem cells induced from the cells derived from the mouse bone marrow cultured in low serum in the same subculture method could be cultured for a long time.
  • pluripotent stem cells were induced from the postnatal mouse bone marrow-derived cells established under the low serum condition.
  • Example 12 Induction of mouse pluripotent stem cells by the introduction of three genes and histone deacetylase inhibitor treatment
  • pluripotent stem cells Using cells derived from mouse bone marrow that is a mouse postnatal tissue, the induction of pluripotent stem cells was carried out with the introduction of three genes and histone deacetylase inhibitor treatment.
  • the primary culture cells derived from mouse bone marrow containing undifferentiated stem cells that had been stored frozen after preparing in a manner similar to Example 11 were plated at a concentration of 5 x 10 3 cells/cm 2 on a 24 -well plastic plate (manufactured by Becton Dickinson) having the bottom which had been gelatin-coated with a 0.1% gelatin/phosphate buffer, and 2ml each of the IVIAPC medium was added .
  • Example 2 Eight hours later, the medium was removed, 2ml each of the three gene (human Oct3/4, Sox2 and KIf4) retrovirus vector solution prepared as in Example 1 were added, and after further adding MS-275, a histone deacetylase inhibitor, at a final concentration of 1 or 0.1 ⁇ M, they were cultured at 37°C for 14 hours. Then after removing the virus solution, 2ml each of the MAPC medium containing MS-275, a histone deacetylase inhibitor, at a final concentration of 1 or 0.1 ⁇ M was added.
  • mice ES medium Three days later, the medium was replaced with the mouse ES medium [a final concentration of 0.3% FBS (manufactured by Invitrogen) , 1000 units/ml LIF (manufactured by Chemicon) and 0.1 mM 2- mercaptoethanol were added to the ES medium at the time of use] .
  • mice ES medium change with the mouse ES medium was continued every 2 to 3 days. Twelve days after the introduction of three genes (human Oct3/4, Sox2 and KIf4) retrovirus vector, the cells were subcultured from each well of the 24 -well plastic plate to each well of a 6-well plastic plate. A portion of it was also cultured in a 24-well plastic plate. Fifteen days after said three gene introduction and MS-275 treatment, the pluripotent stem cells formed colonies composed of mouse ES cell-like small cells. The colonies of said pluripotent stem cells were stained blue violet by alkaline phosphatase activity.
  • the amount expressed of the Nanog gene was confirmed by quantitative PCR, and the expression of mouse Nanog of colonies of pluripotent stem cells having alkaline phosphatase activity was confirmed (Fig. 3) .
  • the pluripotent stem cells were subcultured from each well of the 6-well plate to a gelatin-coated 100 mm plate. Subculture was continued similarly.
  • mice Twenty nine days after said three gene introduction and MS-275 treatment, the mouse pluripotent stem cells were subcutaneously transplanted to the back of syngeneic C57BL/6N mice at 2x 10 7 cells/mouse, and 34 days later the teratoma that formed was extracted. From the extracted teratoma, slices were prepared, and differentiation potential into three germ layers was analyzed by immunological and histological staining (HE stain, alcian blue stain) . As a result, GFAP-positive cells
  • keratin producing cells skin cells
  • smooth muscle actin-positive cells smooth muscle actin-positive cells
  • Example 13 Induction of mouse pluripotent stem cells by the introduction of three genes
  • mouse pluripotent stem cells was carried out with the introduction of three genes.
  • the primary culture cells derived from mouse bone marrow containing undifferentiated stem cells that had been stored frozen after preparing in Example 11 were plated at a concentration of 1 x 10 4 cells/cm 2 on a 24-well plastic plate
  • the pluripotent stem cells formed colonies composed of mouse ES cell -like small cells.
  • the medium was removed and then a 10% formalin neutral buffer solution was added to wells, and fixed at room temperature for 5 minutes. After washing with a phosphate buffer etc., the 1 step NBT/BCIP solution (manufactured by Pierce) comprising a chromogenic substrate of alkaline phosphatase was added and reacted at room temperature for 20 to 30 minutes. The colonies of said pluripotent stem cells were stained blue violet by alkaline phosphatase activity.
  • the amount expressed of the Nanog gene was confirmed by quantitative PCR, and the expression of mouse Nanog of colonies of pluripotent stem cells having alkaline phosphatase activity was confirmed.
  • pluripotent stem cells were carried out with the introduction of three genes.
  • the primary culture cells derived from mouse bone marrow containing undifferentiated stem cells that had been stored frozen after preparing in Example 11 were plated at a concentration of 1 x 10 4 cells/cm 2 on a 6-well plastic plate (manufactured by Becton Dickinson) the bottom of which had been gelatin-coated with a 0.1% gelatin/phosphate buffer solution, and the MAPC medium was added in 2 ml portions. Two days later, the medium was removed, the three gene (human Oct3/4, Sox2 and KIf4) retrovirus vector solution prepared as in Example 1 were added in 2 ml portions, and after culturing at 37°C for 1 day, the virus solution was removed, and the MAPC medium was added in 2 ml portions.
  • mice ES medium Three days later, the medium was replaced with the mouse ES medium [a final concentration of 0.3% FBS (manufactured by Invitrogen) , 1000 units/ml LIF (manufactured by Chemicon) and 0.1 mM 2- mercaptoethanol were added to the ES medium at the time of use] .
  • Medium change with the mouse ES medium was continued every 2 to 3 days.
  • the cells were subcultured from each well of the 6 -well plastic plate to each well of a 10 cm plastic dish.
  • the pluripotent stem cells formed colonies composed of mouse ES cell-like small cells.
  • the medium was removed and then a 10% formalin neutral buffer solution was added to wells, and fixed at room temperature for 5 minutes. After washing with a phosphate buffer etc., the 1 step NBT/BCIP (manufactured by Pierce) , a chromogenic substrate of alkaline phosphatase, was added and reacted at room temperature for 20 to 30 minutes. The colonies of said pluripotent stem cells were stained blue violet by alkaline phosphatase activity.
  • the amount expressed of the Nanog gene was confirmed by quantitative PCR, and the expression of mouse Nanog of colonies of pluripotent stem cells having alkaline phosphatase activity was confirmed.
  • the mouse pluripotent stem cells were subcutaneously transplanted on the back of syngeneic C57BL/6N mice at 2x 10 7 cells/mouse, and 13 and 17 days later the teratoma that formed was extracted. Slices were prepared from the extracted teratoma, and differentiation potential into three germ layers was analyzed by immunological and histological staining (HE stain, alcian blue stain) .
  • GFAP-positive cells the nervous system
  • smooth muscle actin-positive cells smooth muscle cells
  • bone tissues and cartilage tissues as the mesodermic system
  • intestinal tract tissues endodermal epithelium positive for MUC-I
  • mouse pluripotent stem cells which were single-sorted based on GFP and SSEA-I positive with FACSAria, were subcutaneously transplanted on the back of syngeneic C57BL/6N mice at 2x 10 7 cells/mouse, and 13 and 14 days later the teratoma that formed was extracted.
  • pluripotent stem cell were obtained by the forced expression of each of three genes of Oct3/4, Sox2 , and Klf4 in undifferentiated stem cell present in a postnatal tissue.
  • the pluripotent stem cells showed an in vitro long-term self-renewal ability, and were expressed ES cell marker, Nanog expression and alkaline phosphatase activity, and the ability of differentiation of tissues derivative from all three germ layers (ectoderm, mesoderm and endoderm) .
  • Example 14 Long term expansion and characterization of human induced pluripotent stem cells
  • iPS-1-1 Human induced pluripotent stem
  • cloning cylinder 0.25% trypsin-EDTA
  • human iPS-1-1 Nine sub-clones which were termed human iPS-1-1, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7,1-8 and 1-9 were obtained.
  • human iPS-1-8 clone was successfully expanded on MEF feeder cells in human ES medium supplemented with 0.1 mM 2- mercaptoethanol and 10 ng/ml bFGF or in mTeSRl defined medium
  • human induced pluripotent stem cells were washed with Hanks 's balanced solution, incubated in 0.25% trypsin-EDTA (Gibco) at 37°C for 3 minutes, and then added the culture medium to terminate the trypsin activity.
  • Human induced pluripotent stem cells were centrifuged at 300 x g at room temperature or 4°C for 5 minutes and the supernatant was removed. Precipitated human induced pluripotent stem cells were re-suspended into culture medium. The pluripotent stem cells were usually split into new culture dishes using 1:4 to 1:6 splits. Human iPS-1-8 clone was frozen using Cell freezing solution for ES cells
  • Human iPS-1-8 clone was morphologically indistinguishable from typical human ES cell colonies that consist of small, round, and compact cells with defined edges when cultured on mitomycin-C treated mouse embryonic fibroblasts (MEFs) (Fig. 4) .
  • Human iPS-1-8 clone actively proliferated in mTeSRl medium.
  • Human iPS-1-8 clone derived cells cultured in mTeSRl medium was termed human iPS-l-8mTeSR cells.
  • Human iPS-1-8 clone was able to be passaged more than 30 times, and cultured for more than half year after four factor infections (Fig . 4f,g) .
  • Human iPS-1- 8mTeSR cells were able to be stored in liquid nitrogen and re- cultured in mTeSR medium in the presence of 5 to 20 ⁇ M of Y- 27632.
  • Population doubling time of human iPS-l-8mTeSR cells was approximately 48.5 hours when analyzed between passages 19 to 26 which correspond to days 123 to 148 after four factor infection.
  • l-8mTeSR Karyotype analysis of long-term cultured human iPS-1-8 clone was performed using giemsa stain and multicolor-FISH analysis.
  • Human iPS cells were pretreated with 0.02 ⁇ g/ml colecemid for 2 hours, followed by incubation with 0.075 M KCl for 20 minutes, and then fixed with Carnoy's fixative.
  • multicolor-FISH analysis cells were hybridized with the multicolor FISH probe (Cambio) and analyzed under DMRA2 fluorescent microscope (Leica) .
  • Human iPS-l-8mTeSR cells mainly maintained a normal karyotype (46XY) after long-term culture in mTeSR (68%) without any chromosomal translocation or deletion (Fig. 4h, Table 3) .
  • alkaline phosphatase staining cells were fixed with 10% formalin neutral buffer solution (Wako) at room temperature for 5 minutes, washed with PBS, and incubated with alkaline phosphatase substrate 1 step NBT/BCIP (Pierce) at room temperature for 20-30 minutes. Cells having alkaline phosphatase activity were stained in blue violet.
  • NBT/BCIP Pieris-Bassham
  • the cells were incubated overnight at 4°C with primary antibodies against SSEA-3 (MC- 631; Chemicon) , SSEA-4 (MC813-70; Chemicon) TRA-1-60 (abeam), TRA-1-81 (abeam), CD9 (M-L13; R&D systems), CD24 (ALB9; abeam), CD90 (5E10; BD bioscience) , or Nanog (R&D systems) .
  • SSEA-3 MC- 631; Chemicon
  • SSEA-4 MC813-70
  • TRA-1-60 TRA-1-60
  • TRA-1-81 TRA-1-81
  • CD9 M-L13; R&D systems
  • CD24 ALB9; abeam
  • CD90 5E10; BD bioscience
  • Nanog staining cells were permeabilized with 0.1 % Triton X-100/PBS before blocking.
  • the cells were washed with PBS for three times, and then incubated with AlexaFluor 488 -conjugated secondary antibodies (Molecular Probes) and Hoechst 33258 at room temperature for 1 hour. After further washing, fluorescence was detected with an Axiovert 200M microscope (Carl Zeiss) .
  • Human iPS-l-8mTeSR cells were positive for alkaline phosphatase (hereinafter referred to as "ALP") activity and the glycolipid antigens SSEA-3 and SSEA-4, the keratin sulfate antigens TRA-1-60 and TRA-1-81, and the protein antigens CD9, CD24, Thy-1 (CD90) staining (Fig. 5) .
  • ALP alkaline phosphatase
  • Gene expressions were detected by PCR using Extaq (Takara) . Sequences of the primers were described in Table 4.
  • human iPS-1- 8 clone down-regulated forced expression of four genes Fig. 6b
  • human induced pluripotent stem cell clone-1-8 cultured in mTeSR on matrigel (l-8mTeSR) and its parental fibroblasts (5F0438) were analyzed based on a set of 21,080 genes with present flag call (P ⁇ 0.04) or marginal flag call (0.04D P ⁇ 0.06) for both clone 1-8 and H14 hES line which is data from GEO (GSM151741) , were used as a representative of human ES cells for comparison.
  • DNA microarray data for clone-1-8 cultured in mTeSR (l-8mTeSR) , clone 1-8 cultured in MEF- conditioned medium (1-8CM) and its parental fibroblasts (5F0438) were compared with DNA microarray data for Sheff 4 line cultured on MEF (hESl :GSM194307 , hES2 : GSM194308, hES3 : GSM194309) , Sheff 4 line cultured on matrigel (hES4 : GSM194313, hES5: GSM194314) , H14 line cultured on MEF (hES6 : GSM151739, hES7: GSM151741) , and three fibroblasts (GSM96262 for Fibroblastsl, GSM96263 for Fibroblasts2 and GSM96264 for Fibroblasts3) .
  • the global gene expression profile of the human iPS cell line (clone 1-8) and its parental fibroblasts were analyzed.
  • Cluster analysis using the gene set defined by the International Stem Cell Initiative revealed that the human iPS cell line 1-8 clustered with human ES cell lines but separated from the parental fibroblasts (Fig. 8) .
  • the pearson correlation coefficient was 0.675 between human ES cell lines sheff4 and H14
  • the coefficient was 0.835 between human iPS cell line 1-8 and human ES cell line H14 (Fig. 8) .
  • This analysis indicate that human iPS cell line 1-8 had a similar gene expression pattern to the human ES cell lines H14.
  • the promoter regions of Nanog and Oct3/4 were analyzed for methylation of individual CpG sites.
  • Ten nanograms of bisulfite-treated genomic DNA was PCR-amplified with primers containing a T7 -promoter and transcripts treated with RNase A.
  • fragments originating from a methylated CpG sequence contained a G instead of an A-base, they had a 16 Da higher molecular weight than those resulting from the corresponding non-methylated CpG.
  • This mass difference was detected using a MALDI-TOF mass spectrometer (Autoflex, Bruker Daltonics) .
  • the spectra produced by the mass spectrometer were analyzed using the EpiTYPER (Sequenom) .
  • the percentage methylation of individual CpG sites was calculated using the area under the peak of the signal from the unmethylated and methylated fragments.
  • the percentage methylation of individual CpG sites were calculated using the area under the peak of the signal from the unmethylated and methylated fragments.
  • Table 9 lists up locations and sizes in genome corresponding to amplicon using for methylation analyses.
  • Table 10 lists up the primer sets using for methylation analyses.
  • the Oct3/4 proximal promoter including conserved region 1 (CRl) , the Oct3/4 promoter distal enhancer including CR4 and the Nanog proximal promoter including Oct3/4 and Sox2 binding sites were examined (Figure 10a) .
  • Figure 10b cytosine-phosphate- guanosine (CpG) dinucleotides in these regions are demethylated in clone 1-8 derived cells compared to the parental fibroblasts.
  • Human iPS-l-8mTeSR cell-suspension (0.5 to 2 x 10 6 cells/mouse) was injected into the medulla of left testis of 7 to 8 week old SCID mice (CB17, Oriental Yeast) using a Hamilton syringe. After 6 to 8 weeks, the teratomas were excised under perfusion with PBS followed with 10% buffered formalin, and subjected to the histological analysis. Human iPS-l-8mTeSR cells gave rise to teratomas 4 to 8 weeks after transplantation into testes of SCID mice.
  • HE hematoxylin-eosin
  • Type II collagen For Type II collagen, before the treatment with primary antibody a section was incubated with Hyaluronidase (25 mg/mL) for 30 minutes. Localization of antigens was visualized by using appropriate secondary antibodies (Alexa fluor 594 and 688, Molecular Probes, 1:600) . Nuclei were stained with DAPI. Immunostained teratoma sections were analyzed under a fluorescence microscope (Axio Imager Zl, Zeiss).
  • Teratomas of human iPS-l-8mTeSR cells contained tissues representative of three germ layers, neuroectoderm, mesoderm, and endoderm.
  • Fig. 11 shows teratoma that was derived from human iPS-l-8mTeSR cells cultured for 94 days (Tl) .
  • Human iPS- 1- ⁇ mTeSR cells were injected into SCID mouse testes and analyzed 56 days after injection.
  • HE and alcian blue staining of teratoma tissues revealed that teratomas contained neural epitherium (positive for nestin) cartilage (positive for collagen II), endodermal tract (alpha-fetoprotein) .
  • Human iPS-1- 8mTeSR cell derived tissues were distinguished from host tissues by HuNu staining. In Tl teratoma, smooth muscle cells
  • Human iPS-l-8mTeSR cells which were cultured for 102 days and 114 days, were injected into SCID mouse testes and analyzed 48 days and 42 days(T3) after injection, respectively (T2 , Fig. 12, T3 , Fig. 13). Tissues representative of three germ layers, neuroectoderm, mesoderm and endoderm, were observed. To confirm whether human iPS can be cryopreserved, human iPS-l-8mTeSR cells were frozen down, stored in liquid nitrogen and recultured.
  • T-Fl 46 days
  • T-F2 48 days
  • Genomic DNA was prepared from the human iPS clone 1-8 and its parental fibroblasts. Five ⁇ g of each genomic DNA was digested with Kpnl (POU5F1, Sox2 , and KIf4) . Fragments were separated on a 0.8% agarose gel, blotted onto HybondXL membrane
  • SNP genotyping was performed with the use of the GeneChip Human Mapping 500K Array Set (Affymetrix) according to the manufacture's protocol.
  • Human iPS-l-8mTeSR cells cultured in mTeSR on matrigel, its parental fibroblasts (5F0438) , and fibroblast (5F0416) derived from a different donor were analyzed for this assay.
  • the array set includes a Styl and a Nspl chip. Two aliquots of 250 ng of DNA each were digested with Nspl and Styl, respectively. Each enzyme preparation was hybridized to the corresponding SNP array (262,000 and 238,000 on the Nspl and Styl array respectively) .
  • HLA DNA typing was performed by utilizing hybridization of PCR-amplified DNA with sequence specific oligonucleotide probes (SSOP) (Luminex) . Assays were performed to determine the HLA-A, HLA-B, HLA-Cw, HLA-DR, HLA-DQ, HLA-DP and Bw loci according to manufacturer's instructions.
  • SSOP sequence specific oligonucleotide probes
  • Human iPS cells are promising materials in cell transplantation therapies, they would overcome immune rejection, because human iPS cells can be directly generated from patients' cells and must be the identical HLA type.
  • HLA typing of human iPS-1-8 clone (l-8mTeSR) , parental cells (5F0438) , and unrelated fibroblasts (5F0416) .
  • HLA type of iPS-1-8 clone was completely identical to that of 5F0438 but not 5F0416 (Table 6) .
  • human pluripotent stem cell were obtained by the forced expression of each of four genes of Oct3/4, Sox2 , KIf4, and c-Myc in undifferentiated stem cell present in a human postnatal tissue.
  • the human pluripotent stem cells showed an in vitro long-term self-renewal ability and the pluripotency of differentiation into ectoderm, mesoderm and endoderm.
  • the human pluripotent stem cells were expressed cell surface antigens SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, CD9, CD24, and CD90, and ES cell marker genes Nanog, Oct3/4, TDGFl, Dnmt3b, GABRB3 , GDF3 , Zfp42, ALP, CD9, and Thy-1.
  • the promoter regions of Nanog and Oct3/4 in the human pluripotent stem cells were demethylated compared to the parental fibroblasts.
  • the human pluripotent stem cells carries at least a single copy of Oct3/4, Sox2 , KIf4, and c-Myc transgene.
  • the induced human pluripotent stem cells and the parental cells had almost the same SNP genotype each other, and HLA type of the induced human pluripotent stem cell was completely identical to that of the parental cell (undifferentiated stem cell present in a human postnatal tissue) .
  • Example 15 Gene expression profile of primary culture of 4 genes introduced neonatal fibroblast
  • Two lots of neonatal fibroblasts (5F0416 and 5F0474) were seeded at 10 3 cells/cm 2 or 10 4 cells/cm 2 into 35 mm diameter wells of 6 well plates and cultured in FBM supplemented with FGM-2 SingleQuots (manufactured by Lonza) before the four genes transduction.
  • Cells were infected with mCATl -adenovirus vectors at 2 x 10 s ifu/well and then infected with the retroviral vectors carrying four genes as described in Example 6. Eight wells were prepared for this study (2 different lot and 2 different densities in duplicate) .
  • Real-time quantitative PCR was performed with ABI PRISM 7900HT (manufactured by Applied Biosystems) using PCR primer sets (manufactured by Applied Biosystems, Nanog, Hs02387400_gl, Dnmt3b, Hs00171876_ml , FoxD3 , Hs00255287_sl , Zfp42, HsO1938187_sl, TDGFl, Hs02339499_gl , TERT, Hs00162669_ml , GDF3 , Hs00220998_ml, CYP26A1, Hs00175627_ml , GAPDH, Hs99999905_ml) to determine gene expression of human ES cell markers in colonies.
  • mice ES cells and mouse iPS cells form multilayered / aggregated colonies.
  • mouse ES cell like aggregated colonies which were induced by ectopic expression of four gene in human fibroblasts (e.g. colony #l-2-F and #l-2-B in Fig. 22) .
  • these colonies are all ALP (-) .
  • Nanog gene expression was observed in 161 out of 163 ALP positive colonies and 16 out of 18 ALP negative colonies.
  • TERT and CYP26A1 genes were observed only in 26 and 24 colonies out of 163 ALP positive colonies respectively (Fig. 15a) .
  • Genes such as Nanog, TDGF, and Dnmt3b which are well know to be close association with the pluripotent state in human ES cells, and to be strongly downregulated upon their differentiation had higher tendency to be induced by the four gene transduction.
  • ALP positive colonies can be categorized into 40 groups based on the gene expression pattern of the eight human marker genes (Table 7) .
  • colonies are categorized by the total number of eight marker genes expression, the distribution of colony number followed a normal distribution suggesting the presence of a stochastic process in the colony induction (Fig. 15c, d).
  • the efficiency of human ES cell marker gene expression in human fibroblasts was affected by the donor difference .
  • induced pluripotent stem cells can be isolated from small monolayer colonies comprising small cells with high nucleus to cytoplasm ratio not from fibroblastic colonies, defused colonies or multilayered colonies.
  • Table 8 summarizes all of experiments and results on the ALP positive colony number using human neonatal fibroblasts .
  • Example 16 Generation of human iPS-2-4 clone from human neonatal skin fibroblasts
  • Adenovirus vector plasmids for mCATl were transfected into 293lo cells.
  • the mCATl -adenoviruses were isolated from these cells by three freeze-thaw cycles, purified using Adenovirus purification kit (Clontech) and stored at -80 0 C.
  • the titer of the vector stocks was determined by Adeno-X rapid titer kit
  • the replication deficient MMLV derived retrovirus vector pMx was used for the ectopic expression of human Oct3/4, Sox-2, c- Myc and KIf4.
  • Recombinant retroviruses were generated by transfecting vectors to the Plat-E packaging system (Morita et al . , 2000) followed by incubation in FBM (Lonza) supplemented with FGM-2 SingleQuots (Lonza) . Between 24 and 48 hours after the transfection, supernatant from the Plat-E culture was collected several times at intervals of at least 4 hours and passed through a 0.45 ⁇ m filter.
  • MEF-conditioned medium human ES medium (DMEM/F12 (Gibco) supplemented with 20% Knockout Serum Replacement (KSR, Invitrogen) , 2 mM L-glutamine (Sigma) , Ix nonessential amino acids (Sigma) , 10 ⁇ g/ml gentamycin) , 10 ng/ml bFGF was conditioned on mitomycin-C treated MEF
  • Reprocell for 20-24 hours, harvested, filtered through a 0.45 ⁇ m filter and supplemented with 0.1 mM 2- ⁇ nercaptoethanol (Sigma) and 10 ng/ml bFGF before use.
  • Neonatal Normal Human Skin Fibroblasts primary culture
  • Human neonatal dermal fibroblasts (Lonza; lot 5F0416) were cultured in FBM supplemented with FGM-2 SingleQuots. Three days before the 4 gene introduction, fibroblasts were seeded at 10 3 cells/cm 2 into 6 well plates. Eighteen hours later, the cells were mixed with the mCATl adenovirus vector solution in 500 ⁇ l Hanks' balanced salt solution, and incubated at room temperature for 30 min. The cells were then added to 2 ml of medium and cultured for 48 hrs .
  • the cells were incubated in 2 ml of the retrovirus/polybrene solution (mixture of equal volumes of the retrovirus vector suspension for each of the four genes (Oct3/4, Sox2 , KIf4 and c-Myc) prepared in Example 1, supplemented with 5 ⁇ g/ml of polybrene) at 37°C for 4 hrs to overnight .
  • the virus supernatant was replaced with the MEF-conditioned ES medium. Then medium was changed every days.
  • a colony with a characteristic shape was picked with forceps from a well.
  • the picked colony was transferred into a matigel -coated well in a 24 -well plate and maintained in mTeSR defined medium supplemented with 10 ⁇ M Y-27632. Fourteen hours later the medium was changed. Medium change was continued every days.
  • a second culture was carried out.
  • human iPS-2-4 clone was sub-cloned and designated as human iPS-2-4 sub-clone.
  • iPS-2-4mTeSR cells were morphologically indistinguishable from typical human ES cells and human iPS-1-8 mTeSR cells consisting of small, round, and high nucleus to cytoplasm ratio cells with defined edges.
  • ALP alkaline phosphatase
  • Real-time quantitative PCR was performed with ABI PRISM 7900HT (manufactured by Applied Biosystems) using PCR primer sets (manufactured by Applied Biosystems, Nanog, Hs02387400_gl, Dnmt3b, Hs00171876_ml , FoxD3 , Hs00255287_sl , Zfp42, HsO1938187_sl, TDGFl, Hs02339499_gl , TERT, Hs00162669_ml, GDF3 , Hs00220998_ml , CYP26A1, Hs00175627_ml , GAPDH, Hs99999905_ml) to determine gene expression of human ES cell markers in colonies.
  • Human pluripotent stem cell were obtained by the forced expression of each of four genes of Oct3/4, Sox2, KIf4, and c-Myc in undifferentiated stem cell present in a human postnatal tissue.
  • the human pluripotent stem cells showed an in vitro long-term self-renewal ability, and were expressed ES cell marker genes Nanog, Oct3/4, TDGFl, Dnmt3b, GABRB3 , GDF3 , Zfp42, ALP, CD9, and Thy-1.
  • Example 17 Generation of human iPS-3-2 clone from human neonatal skin fibroblasts
  • human neonatal dermal fibroblasts (Lonza; lot 5F0438) were cultured in FBM supplemented with FGM- 2 SingleQuots. Three days before the 4 gene introduction, fibroblasts were seeded at 10 3 cells/cm 2 into 6 well plates. Eighteen hours later, the cells were mixed with the mCATl adenovirus vector solution in 500 ⁇ l Hanks' balanced salt solution, and incubated at room temperature for 30 min. The cells were then added to 2 ml of medium and cultured for 48 hrs .
  • the cells were incubated in 2 ml of the retrovirus/polybrene solution (mixture of equal volumes of the retrovirus vector suspension for each of the four genes (Oct3/4, Sox2 , KIf4 and c-Myc) prepared in Example 1, supplemented with 5 ⁇ g/ml of polybrene) at 37°C for 4 hrs to overnight.
  • the virus supernatant was replaced with the MEF- conditioned ES medium.
  • medium was changed every days.
  • a colony with a characteristic shape was directly picked with forceps from one of dishes. The picked colony was transferred into a matigel- coated well in a 24 -well plate and maintained in mTeSR defined medium supplemented with 10 ⁇ M Y-27632.
  • Human iPS-3-2 clone actively proliferated in mTeSRl medium.
  • mTeSRl medium we termed these cells derived from human iPS-3-2 clone which culture in mTeSRl medium as human iPS-3-2mTeSR cells.
  • RNA from colonies were extracted using a RecoverAll Total Nucleic Acid Isolation kit (manufactured by Ambion) .
  • genes of interest were amplified using Taqman preamp (manufactured by Applied Biosystems) .
  • Realtime quantitative PCR was performed with ABI PRISM 7900HT (manufactured by Applied Biosystems) using PCR primer sets (manufactured by Applied Biosystems, Nanog, Hs02387400_gl , Dnmt3b, Hs00171876_ml , FoxD3 , Hs00255287_sl , Zfp42, HsO1938187_sl, TDGFl, Hs02339499_gl , TERT, Hs00162669_ml , GDF3 , Hs00220998_ml, CYP26A1, Hs00175627_ml , GAPDH, Hs99999905_ml) to determine gene expression of human ES cell markers in colonies.
  • ES cell marker gene expressions (Table 12) .
  • human pluripotent stem cell were obtained by the forced expression of each of four genes of Oct3/4, Sox2 , KIf4, and c-Myc in undifferentiated stem cell present in a human postnatal tissue.
  • the human pluripotent stem cells showed an in vitro long-term self-renewal ability, and were expressed ES cell marker genes Nanog, Oct3/4, TDGFl, Dnmt3b, GABRB3 , GDF3 , Zfp42, ALP, CD9, and Thy-1.
  • Table 1 shows the name of gene, the NCBI number, the virus vector in which said gene was inserted, insert size, the restriction site at the 5 '-end, the restriction site at the 3 1 end, the length of the translated region, the length of the 3 ' untranslated region, clone ID, and the supplier of the four genes or the three genes and the receptor of mouse ecotropic retrovirus vector (mCAT: mouse-derived cationic amino acid transporter) used in Examples.
  • mCAT mouse-derived cationic amino acid transporter
  • Table 2 summarizes the number of alkaline phosphatase- positive colonies of Examples 4 to 7.
  • the day of four gene introduction is a day when a retrovirus vector was infected.
  • Lot No. is that of Lonza products.
  • Age of donors is based on the donor information of Lonza products.
  • the number of colonies is the number of colonies composed of alkaline phosphatase-positive small cells per 10 cm 2 .
  • Table 3 summarizes the distribution of the karyotype of clone 1-8 at day 101. After the Giemsa stain, chromosome numbers were counted. 67 of 100 cells showed normal karyotype.
  • Table 4 shows primer sequences used in Fig. 6 and Fig. 14.
  • Table 5 summarizes SNP genotyping of human iPS clone 1-8 and fibroblasts (5F0438 and 5F04156) which were analyzed using the GeneChip Human Mapping 500K Array Set.
  • SNPs of clone 1-8 were consistent to that of parental cells in 464,069 (99.17%) of 467,946 of called SNPs and different from that of parental cells in 3,877 (0.83%) of them.
  • SNPs of clone 1- 8mTeSR were consistent to that of unrelated donor cells (5F0416) only in 284,950 (60.50%) of 470,960 of called SNPs and different from that of the unrelated cells in 186,010 (39.50%) of them.
  • HLA-A, HLA-B, HLA-Cw and HLA-DR types of human iPSl-8 (1-8 mTeSR) and fibroblasts (5F0438 and 5F0416) were classified using hybridization of PCR-amplified DNA with sequence specific oligonucleotide probes (SSOP) (Luminex) .
  • SSOP sequence specific oligonucleotide probes
  • Table 7 summarized hES cell marker gene expression patterns in colonies. Colonies were stained for alkaline phosphatase at 17 days post 4 genes transduction. All ALP (+) colonies and 18 ALP (-) colonies were dissected and determined their hES marker gene expression by RT-PCR. Each colony was categorized and counted the number. "+” represents gene expression, and "-" represents no detection by a 40 cycle RT-PCR using amplified cDNA samples.
  • Table 8 summarizes the number of alkaline phosphatase-positive colonies of the experiments using neonatal fibroblasts.
  • the date of four gene introduction is a day when a retrovirus vector was infected.
  • the donor indicates lot number of Lonza products.
  • the number of colonies is the number of colonies composed of alkaline phosphatase-positive small cells per 10 cm 2 . ND: not determined.
  • Table 9 lists up locations and sizes in genome corresponding to amplicons using for methylation analyses of the promoter regions of Nanog and Oct3/4. Columns A, B and C indicate amplicon name, locations and sizes in genome corresponding to amplicons, respectively.
  • Promoter regions in methylation analysis amplicon name location in genome corresponding to amplicon size of amplicon
  • Table 10 lists up the primer sets using for methylation analyses of the promoter regions of Nanog and Oct3/4.
  • Columns A and B indicate names of primers and sequences of primers (capital for gene-specific sequences, lower case for tag sequences) , respectively.
  • Nanog-zl-L aggaagagagGGAATTTAAGGTGTATGTATTTTTTATTTT cagtaatacgactcactatagggagaaggctATAACCCACCCCTATAATC
  • Nanog-zl-R CCAATA Nanog-z2-L aggaagagagGTTAGGTTGGTTTTAAATTTTTGAT cagtaatacgactcactatagggagaaggctTTTATAATAAAAACTCTAT
  • Nanog-z2-R CACCTTAAACC Oct3/4-zl-L aggaagagagTAGTAGGGATTTTTTGGATTGGTTT cagtaatacgactcactatagggagaaggctAAAACTTTTCCCCCACTCT
  • Oct3/4-zl-R TATATTAC Oct3/4-z2-L aggaagagagGGTAATAAAGTGAGATTTTGTTTTAAAAA cagtaatacgactcactatagggagaaggctCCACCCACTAACCTTAACC
  • Table 11 summarizes relative mRNA expression in ALP positive colonies of Examples 15. Numbers of colonies are corresponding to Fig.15-22. Colony #5-2-32, #5-2-49, #5-2-51, #7-2-37 expressed all analyzed human ES cell markers. In contrast, fibroblastic colonies #3-1-212, #3-1-215, #5-1-4 expressed only Nanog though it highly expressed transgenes.
  • Table 12 summarizes relative mRNA expression in clone-2-4 and 3-2.
  • Total RNA was extracted from clones 2-4 and 3-2.
  • Expression of ES cell marker genes were determined by qRT-PCR as described in Example 16 and 17. Both clone-2-4 and -3-2 showed ES cell marker gene expression. All expression values were normalized against human iPS clone-1-8 (day94) .
  • Cells in a tissue that was lost in diseases etc. can be supplied by inducing human pluripotent cells from the undifferentiated stem cells harvested from a patient by using the induction method of the present invention, followed by inducing to differenciate into a necessary cell depending on diseases and then transplanting the cells to the patient.
  • the undifferentiated stem cells of the present invention present in a human postnatal tissue can be used to search drugs that promote the induction from said undifferentiated stem cells to human pluripotent stem cells by using markers such as Tert , Nanog, Sox2 , Oct3/4 and alkaline phosphatase that direct the induction to human pluripotent stem cells. Said drugs can be used in stead of gene introduction and can enhance the induction efficiency of human pluripotent stem cells.

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PCT/EP2007/010019 2007-06-15 2007-11-20 Human pluripotent stem cells induced from undifferentiated stem cells derived from a human postnatal tissue Ceased WO2009006930A1 (en)

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EP07856194.1A EP2164952B1 (en) 2007-06-15 2007-11-20 Human pluripotent stem cells induced from undifferentiated stem cells derived from a human postnatal tissue
US12/663,840 US20110039332A1 (en) 2007-06-15 2007-11-20 Human pluripotent stem cells induced from undifferentiated stem cells derived from a human postnatal tissue
AU2008273817A AU2008273817B2 (en) 2007-06-15 2008-06-13 Multipotent/pluripotent cells and methods
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