WO2016148253A1 - ナイーブ型多能性幹細胞の製造方法 - Google Patents
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Definitions
- the present invention relates to a method for producing and / or maintaining a na ⁇ ve pluripotent stem cell (hereinafter sometimes abbreviated as the method of the present invention).
- the present invention also relates to a naive pluripotent stem cell having an excellent differentiation pluripotency produced and / or maintained by the method of the present invention.
- iPS cells induced pluripotent stem cells
- iPS cell is a type of pluripotent stem cell.
- iPS cells include embryonic stem cells (hereinafter sometimes abbreviated as ES cells), epiblast stem cells (Epiblast Stem cells). Is also included. These pluripotent stem cells have recently been found to have different basic properties depending on the animal species from which they are derived and the types of pluripotent stem cells. Specifically, mouse ES cells and mouse iPS cells are cells that are close to the initial stage of development, are easy to culture and genetically manipulate, have high proliferation ability, and can efficiently induce differentiation. is called.
- Non-patent Document 3 a method in which two factors Nanog and Klf2 are introduced, and LIF (Leukemia Inhibitory Factor), PD0325901 (MEK inhibitor), CHIR99021 (GSK3 inhibitor), Go6983 (PKC inhibitor) is added to the medium (Non-patent Document 3) Reference) reports that the expression of KLF4 and TFCP2L1, which are known as na ⁇ ve gene markers, is increased, and thus human iPS cells in a state close to na ⁇ ve can be obtained.
- LIF Leukemia Inhibitory Factor
- PD0325901 MEK inhibitor
- CHIR99021 GSK3 inhibitor
- Go6983 PLC inhibitor
- An object of the present invention is to produce and / or maintain a pluripotent stem cell capable of highly expressing a gene important for maintaining na ⁇ ve undifferentiated ability.
- the present inventors introduced six genes (Oct3 / 4, Klf4, c-Myc, Sox2, Nanog and Klf2) among so-called reprogramming factors.
- reprogramming factors include LIF, MEK inhibitor, GSK3 inhibitor, cAMP production promoter, TGF- ⁇ inhibitor and PKC inhibitor.
- the present inventors have found a method for producing pluripotent stem cells capable of maintaining the undifferentiated ability of the present invention and completed the present invention.
- the present invention provides [1] the following two genes: Nanog and Klf2 to be temporarily expressed in primed pluripotent stem cells, and LIF, MEK inhibitor, GSK3 inhibitor, cAMP production promoter, TGF
- a method for producing naive pluripotent stem cells comprising culturing in a medium containing a ⁇ inhibitor and a PKC inhibitor, [2] The production method according to the above [1], wherein the prime-type pluripotent stem cells are prime-type induced pluripotent stem cells or prime-type embryonic stem cells, [3] The method for producing a naive human pluripotent stem cell according to [1] or [2] above, wherein the prime type pluripotent stem cell is a prime type human induced pluripotent stem cell or a prime type human embryonic stem cell, [4] The method for producing a naive human induced pluripotent stem cell according to any one of the above [1] to [3], wherein the prime type pluripotent
- the production method according to [16] The production method according to any one of [13] to [15], wherein the medium is an N2B27 medium, [17] Naive pluripotent stem cells comprising culturing naive pluripotent stem cells in a medium containing LIF, MEK inhibitor, GSK3 inhibitor, cAMP production promoter, TGF- ⁇ inhibitor and PKC inhibitor Stem cell maintenance methods, [18] The maintenance method according to [17] above, wherein the naive pluripotent stem cell is a naive induced pluripotent stem cell or a naive embryonic stem cell, [19] The method for maintaining a naive human pluripotent stem cell according to [17] or [18] above, wherein the naive pluripotent stem cell is a naive human induced pluripotent stem cell or a naive human embryonic stem cell, [20] The method for maintaining a naive human induced pluripotent stem cell according to any one
- the maintenance method according to any one of the above, [22] The maintenance method according to any of [17] to [21], wherein the medium is an N2B27 medium, [23] A naive pluripotent stem cell produced according to the method of any one of [1] to [16], [24]
- the present invention relates to a naive pluripotent stem cell and the like maintained according to the method described in any of [17] to [22].
- pluripotent stem cells cultured under specific conditions of the present invention it is possible to stably produce pluripotent stem cells having naive characteristics and having differentiation pluripotency.
- FIG. 1 shows a gene expression vector used for gene transfer.
- FIG. 2A shows a picture of each cell colony.
- FIG. 2B shows the gene expression level in each cell.
- DOX indicates doxycycline.
- P2 represents a page2 cell
- P3 represents a page3 cell.
- FIG. 3 shows the expression level of ESRRB gene in 201B7 cells (passage 1) cultured in each medium.
- KSR 2iLFA and KSR 2iLFA + Go6983 indicate the media indicated in Table 1, and DOX indicates doxycycline.
- FIG. 4A shows photographs of colonies of 201B7 cells (left: passage2, right: passage3) cultured in each medium.
- FIG. 4B shows the expression level of the ESRRB gene in 201B7 cells (left: passage2, right: passage3) cultured in each medium.
- 2iL + Go6983 and 2iLFA + Go6983 indicate the media shown in Table 1, and DOX indicates doxycycline.
- FIG. 5 shows the expression level of ESRRB gene in each combination of reprogramming factors.
- FIG. 6A shows the results of cell staining (green: MAP2, blue: nuclear staining with Hoechst) of each iPS cell induced to undergo neuronal differentiation for 10 days by the SDIA method.
- FIG. 6B shows the proportion of colonies in which MAP2-positive cells on day 10 of differentiation induction exist in each iPS cell.
- FIG. 7A shows the results of cell staining (green: ⁇ III-Tubulin, red: GFAP) of each iPS cell induced to differentiate into neurons by a method mediated by Neurosphere.
- FIG. 7B shows the rate of differentiation of each iPS cell into astrocytes and neurons.
- FIG. 8 shows each stage (P1 to P3) of na ⁇ ve. P1 represents a cell of page1, P2 represents a cell of page2, and P3 represents a cell of page3.
- FIG. 9 shows the gene expression vector used for gene transfer in Example 5.
- the method for maintaining a na ⁇ ve pluripotent stem cell is one in which the na ⁇ ve pluripotent stem cell is cultured and subcultured in a naive medium supplemented with various compounds.
- the somatic cell is not particularly limited, but any somatic cell can be used.
- tissue stem cells such as (1) sputum neural stem cells, hematopoietic stem cells, mesenchymal stem cells, dental pulp stem cells, (2) sputum tissue progenitor cells, (3) sputum fibroblasts (skin cells, etc.)
- Epithelial cells hepatocytes, lymphocytes (T cells, B cells), endothelial cells, muscle cells, hair cells, gastric mucosa cells, intestinal cells, spleen cells (pancreatic exocrine cells, etc.), brain cells, lungs
- differentiated cells such as cells, kidney cells, and skin cells.
- it is a human-derived somatic cell.
- the pluripotent stem cell is not particularly limited.
- an embryonic stem cell ES cell
- an embryonic stem cell derived from a clone embryo obtained by nuclear transfer (ntES) cell a sperm stem cell (GS cell)
- Examples include epiblast cells, embryonic germ cells (embryonic germ cells: EG cells), pluripotent germ stem cells (multipotent germline stem cells: mGS cells), and induced pluripotent stem cells (iPS cells).
- ES cells, ntES cells, and iPS cells are preferable, ES cells and iPS cells are more preferable, and iPS cells are particularly preferable.
- the living body from which these somatic cells or pluripotent stem cells are derived is not particularly limited, and examples thereof include humans and non-human animals (for example, monkeys, sheep, cows, horses, dogs, cats, rabbits, rats, Mouse). Preferably, it is a human.
- the prime-type pluripotent stem cells are not particularly limited, but mean pluripotent stem cells, excluding mouse ES cells and mouse iPS cells recognized as naive types.
- ES cells and iPS cells derived from humans, monkeys, pigs, sheep, dogs or cows, and epiblast stem cells derived from humans and non-human animal cells.
- Human ES cells and human iPS cells are preferable, and human iPS cells are particularly preferable.
- cell lines of human ES cells used as prime-type pluripotent stem cells are not particularly limited.
- the cell line of human iPS cells used as prime-type pluripotent stem cells is not particularly limited.
- WD39 derived from fibroblasts
- aTKA4 derived from T cells
- WD39 or 201B7 is preferable.
- the gene to be introduced into somatic cells or prime-type pluripotent stem cells is not particularly limited as long as it is a factor known as a so-called reprogramming factor (reprogramming factor).
- reprogramming factor for example, Oct3 / 4, Klf4, c-Myc, Sox2, Nanog, Klf2, L-Myc, N-Myc, Klf5, Lin28, Tert, Fbx15, Eras, ECAT15-1, ECAT15-2, Tcl1, ⁇ -catenin, ECAT1, Esg1, Dnmt3L, ECAT8, Gdf3, Sox15, Fthl17, Sall4, Rex1, UTF1, Stella, Stat3, Grb2, Prdm14, Nr5a1, Nr5a2, E-cadherin.
- two or more genes can be selected from these gene groups and introduced in any combination.
- combinations of Oct3 / 4, Klf4, c-Myc, Sox2, Nanog, and Klf2 are preferable.
- the cell into which the gene is introduced is a prime-type pluripotent stem cell, since any of the above reprogramming factors is expressed, reprogramming factors other than those may be introduced.
- a combination of Nanog and Klf2 is preferable, and a combination of Oct3 / 4, Klf4, c-Myc, Sox2, Nanog and Klf2 is also preferable. preferable.
- the species of the gene to be introduced is preferably the same as the species of the cell to which the gene is introduced.
- the gene introduced into a human-derived cell is a human gene.
- a gene introduced into human-derived somatic cells human ES cells, or human iPS cells
- a combination of human Nanog (NANOG) and human Klf2 (KLF2) is preferable, and human Oct3 / 4 (OCT3 / 4)
- a combination of human Klf4 (KLF4), human c-Myc (c-MYC), human Sox2 (SOX2), human Nanog (NANOG) and human Klf2 (KLF2) is preferred.
- the gene expression vector is not particularly limited, and examples thereof include viral vectors, plasmid vectors, artificial chromosome vectors, and transposon vectors.
- virus vectors include retrovirus vectors, adenovirus vectors, Sendai virus vectors, lentivirus vectors, and adeno-associated virus vectors.
- somatic cells or primed pluripotent stem cells after gene introduction into somatic cells or primed pluripotent stem cells, they may be cultured with feeder cells.
- feeder cells A mouse embryonic fibroblast (MEF cell) and a mouse embryonic fibroblast cell line (STO cell) are mentioned.
- the prime medium is not particularly limited.
- DMEM Dynamic Eagle medium
- F12 a mixed medium of DMEM and F12
- Knockout TM D-MEM Replacement serum (KSR; Knockout TM Serum Replacement (Invitrogen)
- FBS fetal bovine serum
- NEAA non-essential amino acids
- 2-mercaptoethanol antibiotics (eg, streptomycin) , Penicillin, puromycin, mitomycin), bFGF (basic fibroblast growth factor), and other additive components, which are arbitrarily combined and added to any of the basal media.
- cloning refers to a method of selecting a cell in which expression of the gene has been confirmed from somatic cells or prime-type pluripotent stem cells into which the reprogramming factor has been introduced.
- a selection method for example, a fluorescent protein gene previously incorporated into a gene expression vector, for example, a green fluorescent protein (GFP) gene, a yellow fluorescent protein (YFP) gene (for example, Venus), a cyan fluorescent protein (A method of confirming fluorescence of a CFP) gene (for example, Cerulean), a red fluorescent protein (RFP) gene (for example, TOMATO) under a fluorescence microscope can be mentioned.
- GFP green fluorescent protein
- YFP yellow fluorescent protein
- RFP red fluorescent protein
- TOMATO red fluorescent protein
- various compounds in the step (iii) and the maintenance method include LIF (Leukemia Inhibitory Factor), MEK inhibitor, GSK3 inhibitor, cAMP production promoter, TGF- ⁇ inhibitor, or PKC inhibitor. . It is preferred to add all these compounds to the naive medium.
- LIF Leukemia Inhibitory Factor
- MEK inhibitor MEK inhibitor
- GSK3 inhibitor GSK3 inhibitor
- cAMP production promoter TGF- ⁇ inhibitor
- TGF- ⁇ inhibitor TGF- ⁇ inhibitor
- PKC inhibitor PKC inhibitor
- the MEK inhibitor is not particularly limited.
- PD0325901 N-[(2R) -2,3-dihydroxypropoxy] -3,4-difluoro-2-[(2-fluoro-4- Iodophenyl) amino] -benzamide; CAS registry number: 391210-10-9), U0126 (1,4-diamino-2,3-dicyano-1,4-bis [2-aminophenylthio] butadiene; CAS registry number : 109511-58-2), PD98059 (2- (2-amino-3-methoxyphenyl) -4H-1-benzopyran-4-one; CAS Registry Number: 167869-21-8), PD184352 (2- (2 -Chloro-4-iodophenylamino) -N-cyclopropylmethoxy-3,4-difluorobenzamide; CA Registration number:. 212631-79-3 and the like Among them,
- the GSK3 inhibitor is not particularly limited.
- CHIR99021 (6-[[2-[[4- (2,4-dichlorophenyl) -5- (5-methyl-1H-imidazole-2- Yl) -2-pyrimidinyl] amino] ethyl] amino] -3-pyridinecarbonitrile; CAS Registry Number: 252917-06-9), BIO (6-bromoindirubin-3′-oxime; CAS Registry Number: 667463- 62-9), Kenpaulrone (9-bromo-7,12-dihydroindolo [3,2-d] [1] benzazepine-6 (5H) -one; CAS Registry Number: 142273-20-9), IM- 16 (3- (4-Fluorophenylethylamino) -1-methyl-4- (2-methyl-1H-indol-3-yl) -1H Pyrrole-2,5-dione; CAS Registry Number: 1129669
- the cAMP production promoter is not particularly limited, and examples thereof include forskolin (CAS registration number: 66428-89-5).
- the TGF- ⁇ inhibitor is not particularly limited.
- A83-01 (3- (6-methyl-2-pyridinyl) -N-phenyl-4- (4-quinolinyl) -1H-pyrazole CAS No. 909910-43-6), SB431542 (4- [4- (1,3-benzodioxol-5-yl) -5- (2-pyridinyl) -1H-imidazole) -2-yl] -benzamide; CAS Registry Number: 301836-41-9). Of these, A83-01 is preferable.
- the PKC inhibitor is not particularly limited.
- Go6983 (3- [1- [3- (dimethylamino) propyl] -5-methoxy-1H-indol-3-yl] -4- ( 1H-Indol-3-yl) -1H-pyrrole-2,5-dione; CAS Registry Number: 133053-19-7), GF109203X (3- (1- (3-dimethylamino) propyl) -1H-indole- 3-yl) -4- (1H-indol-3-yl) -1H-pyrrole-2,5-dione; CAS Registry Number: 133052-90-1). Of these, Go6983 is preferable.
- the naive medium is not particularly limited.
- N2B27 medium N2 medium in which N2 supplement is added to DMEM / F12 medium and B27 medium in which B27 supplement is added to Neurobasal medium is 1 Medium mixed with 1
- NEAA non-essential amino acids
- L-glutamine L-glutamine
- 2-mercaptoethanol 2-mercaptoethanol
- antibiotics eg streptomycin, penicillin, puromycin, mitomycin
- BSA bovine serum albumin
- the concentration of various compounds added to the naive medium in the medium is not particularly limited.
- LIF is 1 ng / mL to 100 ng / mL
- MEK inhibitor is 50 nM to 100 ⁇ M
- GSK3 inhibitor It is preferable to add 50 nM to 100 ⁇ M in the case of cAMP, 50 nM to 100 ⁇ M in the case of cAMP production promoter, 10 nM to 100 ⁇ M in the case of TGF- ⁇ inhibitor, and 50 nM to 100 ⁇ M in the case of PKC inhibitor.
- the culture conditions using a naive medium are obvious to those skilled in the art, but it is preferable to culture under conditions of low oxygen (oxygen concentration: 5%).
- “temporary” in the step (iii) is about 5 days or more, or about 10 days or more, preferably about 10 to 20 days, particularly preferably about 10 to 14 days.
- “expressing the gene introduced in step (i)” in step (iii) is an act of inducing the expression of the transgene, for example, by adding doxycycline.
- the expression level of the naive marker of the cell is determined by quantitative PCR.
- the method of confirming is mentioned.
- examples of the naive marker include DPPA3, ESRRB, TFCP2L1, KLF4, KLF5, and TBX3.
- prime-type pluripotent stem cells mainly form monolayer flat colonies, whereas naive-type pluripotent stem cells mainly form stratified (dome-shaped) colonies, It can also be confirmed by its form.
- the pluripotency marker of the cell is obtained by quantitative PCR.
- examples of the pluripotency marker include OCT3 / 4, Nanog, Sox2, and SSEA4.
- an embryoid body (EB) method and a teratoma (teratoma) formation method can be mentioned.
- the expression of the transgene is not induced and cultured in a naive medium supplemented with various compounds.
- the produced naive pluripotent stem cells maintain an undifferentiated state, that is, highly express ESRRB, which is one of naive markers, and have very high differentiation pluripotency. Therefore, (1) differentiation induction into various desired cells, (2) screening of drug candidate compounds using differentiation-induced cells, (3) preparation of tissue for regenerative medicine from differentiation-induced cells, (4) preparation It can be used for transplantation of the obtained tissue to a patient, and (5) organ regeneration by naive iPS cell transplantation to a blastocyst.
- the cells capable of inducing differentiation of naive pluripotent stem cells are not particularly limited.
- the method for inducing differentiation of naive pluripotent stem cells is not particularly limited.
- the SDIA (Stromal cell-Derived Inducing Activity) method (Proceedings of the National Academy of Sciences of the United States of America, Vol.99, No.3, 1580-1585, 2002).
- cells induced to differentiate using naive pluripotent stem cells can be used for screening drug candidate compounds for treatment of various diseases. For example, by adding a drug candidate compound alone or in combination with other drugs to the differentiated cell, changes in the morphology or function of the cell, increase / decrease in various factors, gene expression profiling, etc. are detected. Therefore, evaluation can be performed.
- the cell is preferably a cell having a phenotype similar to the disease to be treated, and more preferably induced to differentiate from a naive pluripotent stem cell produced from a somatic cell derived from a patient suffering from the disease. It is a cell.
- a tissue can be prepared from cells induced to differentiate using naive pluripotent stem cells and used in the field of regenerative medicine.
- damaged nerve tissue can be normalized by replacing the damaged nerve tissue with normal tissue derived from the present invention. Thereby, the disease derived from the damage of a nerve cell can be treated.
- Such diseases include, for example, Parkinson's disease, Alzheimer's disease, retinitis pigmentosa, amyotrophic lateral sclerosis, optic neuritis, optic neuritis, acute disseminated (disseminated) encephalomyelitis, allergic cerebrospinal cord Inflammation, spinal cord injury, transverse myelitis, spinocerebellar degeneration, chronic inflammatory demyelinating polyneuritis (CIDP), Guillain-Barre syndrome, multiple sclerosis, epilepsy, Parkinsonism, Down syndrome, Examples include schizophrenia, autonomic dysfunction, Huntington's disease, age-related macular degeneration, and inner ear hearing loss.
- Parkinson's disease Alzheimer's disease, retinitis pigmentosa, amyotrophic lateral sclerosis, optic neuritis, optic neuritis, acute disseminated (disseminated) encephalomyelitis, allergic cerebrospinal cord Inflammation, spinal cord injury, transverse myelitis, spino
- a method for transplanting the prepared tissue into a patient is obvious to those skilled in the art.
- a method for transplanting nerve cells Nature Neuroscience, Vol. 2, No. 12, pp. 1137-1140, 1999.
- Example 1 Naiveization of iPS cells 1.1 Culture of prime-type human iPS cells WD39 cells (Imaizumi et al. Molecular Brain 2012, 5:35) as human iPS cells and 201B7 cells widely used as control lines (RIKEN) BioResource Center) was used.
- Human iPS cells were cultured using feeder medium with mitomycin C or radiation-treated mouse embryonic fibroblast cell line (STO cell) or mouse embryonic fibroblast (MEF cell) using the prime-type condition medium shown in Table 1. . The passage was performed once every 5-7 days. At the time of subculture, the colonies were peeled off using a dissociation solution (0.25% Trypsin, 1 mg / ml Collagenase IV, 20% KSR, 1 mM CaCl 2 / PBS), and seeded after finely breaking the colonies by pipetting. IPS cells seeded in 10 cm dishes were used for gene transfer tests.
- STO cell radiation-treated mouse embryonic fibroblast cell line
- MEF cell mouse embryonic fibroblast
- the iPS cells were centrifuged at 200 g for 5 minutes to remove the supernatant, and 1 mL of TrypLE TM Select (Life Technologies) was added and reacted at 37 ° C. for 5 minutes. After adding 2 mL of Trypsin Inhibitor (Life Technologies), it was dissociated into single cells by pipetting 10-20 times with a P1000 pipetman. The volume was increased by adding 7 mL of medium and passed through a 70 ⁇ m cell strainer. IPS cells that became single cells were used for gene transfer.
- Gene Juice registered trademark
- Transfection Reagent Novagen
- rtTA reverse tetracycline-regulated transactivator
- rtTA RiggyBac Transposasetl expression vector
- KLF2 and NANOG doxycycline dependent Yamanaka 4 factors
- a vector that expresses fluorescent protein TOMATO OKSM, KLF2, NANOG and TOMATO
- Doxycycline was added 5 to 10 days after gene introduction, and observed with a fluorescence microscope 8 to 12 hours later. A colony in which fluorescence of TOMATO was observed was judged to be a cell line in which the doxycycline-dependent expression induction system was functioning, and was cloned by colony pickup. Thereafter, doxycycline was not added, and the cells were cultured in the same manner as in “1.1 Culture of prime type human iPS cells”.
- iPS cell naiveization Dissociate the iPS cells transfected and cloned in "1.2 Gene-transfected cell line" into single cells using dissociation solution and TrypLE TM Select, and feed MEF cells to the feeder.
- the medium is a 2iLFA + Go6983 medium shown in Table 1 (hereinafter sometimes abbreviated as the medium of the present invention), and doxycycline (1 ⁇ g / mL) is added to induce the expression of the transgene.
- the cells were cultured with oxygen (Passage 1; P1).
- the cell death was suppressed by adding Y27632 (ROCK inhibitor) so that it might become 10 micromoles on the first day of the start of na ⁇ ve. Passaging was performed 5 to 7 days after the start of naiveization. After removing the medium, 350 ⁇ L of 0.25% Trypsin-EDTA was added to each well and allowed to react at 37 ° C. for 1 minute. The reaction was stopped with Trypsin Inhibitor and 5 mL of medium was added. By pipetting several times with a pipette aid, colonies of iPS cells were peeled from the feeder cell sheet, and the colonies were collected in a 15 mL tube.
- Y27632 ROCK inhibitor
- 201B7 cells were used as iPS cells.
- the cells used for the analysis were 201B7 cells cultured in a medium under prime-type conditions (see “1.1 Cultivation of Prime-type Human iPS Cells”), Passage2 prepared in “1.3 Naiveization of iPS Cells” and It is 201B7 cell of Passage3.
- RNA was collected from each cell using RNeasy mini kit (QIAGEN), reverse transcribed using RiverTra Ace (registered trademark), and then qPCR was performed using SYBR (registered trademark) Premix Ex TaqII (clontech). The primer set is shown in Table 2.
- NANOG and OCT3 / 4 which are pluripotency markers, was also maintained by nailing.
- genes such as KLF4, DPPA3, ESRRB, TFCP2L1, KLF5, and TBX3, which are highly expressed in mouse ES cells that are naive pluripotent stem cells have increased expression due to naiveization, and after the addition of doxycycline is stopped.
- Passage3 also maintained high expression compared to the prime type.
- the expression of Lefty which is a prime-type gene marker, was reduced by na ⁇ ve formation. From the above results, it became clear that iPS cells migrate to a naive type at least after passage 2 by using the method of the present invention. In addition, it was found that inducing the expression of the transgene is not necessary for the maintenance after the transition to the naive type, and only the medium of the present invention is sufficient.
- Example 3 Effect on Na ⁇ ve by Go 6983 Addition Timing In this experiment, the effect on na ⁇ ve by Go 6983 addition timing was examined.
- the passage 1 201B7 cell culture medium (2iLFA + Go6983 medium in Table 1) By changing the passage 1 201B7 cell culture medium (2iLFA + Go6983 medium in Table 1) to the KSR 2iLFA medium in Table 1 or the KSR 2iLFA + Go6983 medium in Table 1 in “1.3 Nabling of iPS cells” and adding doxycycline. After induction of transgene expression, the cells were cultured in hypoxia. Passage 1 cultured under each condition was collected 5 to 7 days after passage and subjected to analysis by qPCR. The result is shown in FIG. As is clear from FIG.
- Example 4 Effect of forskolin and A83-01 addition on navalization
- the ESRRB gene is an important gene that controls self-renewal in mouse ES cells (see Cell Stem Cell, 11,491-504 (2012)).
- the expression of ESRRB is maintained in a high state regardless of the presence or absence of transgene expression induction.
- the reset cells described in Non-Patent Document 3 the expression of ESRRB is hardly recognized (see Non-Patent Document 3).
- One of the differences between the present invention and the conditions described in Non-Patent Document 3 is that forskolin and A83-01 are added to the medium conditions described in Non-Patent Document 3 in the present invention. Therefore, the effect of this difference on na ⁇ ve formation (ESRRB expression and colony morphology) was examined.
- the medium of the present invention (the medium of 2iLFA + Go6983 in Table 1) and the medium of 2iL + Go6983 described in Table 1 were used as the medium for studying the naiveization. This was performed in the same manner as in “Naiveization of iPS cells”. Na ⁇ ve formation was evaluated using ESRRB gene expression and colony morphology in 201B7 cells of Passage 2 and Passage 3 cultured in each medium.
- Non-Patent Document 3 Go6983 is added at the timing of removing doxycycline. However, in this test, since the focus is on the effect of forskolin and A83-01, Go6983 is added at the time of doxycycline addition. From. As is clear from Example 2, the expression of ESRRB is higher when Go6983 is added from the time of doxycycline addition. Therefore, the expression of ESRRB in reset cells in Non-Patent Document 3 is even lower than the results of this experiment, and its expression is hardly observed as described in Non-Patent Document 3, and the transcription factor network in reset cells is weak. (See Non-Patent Document 3).
- naive iPS cells produced and maintained by the method of the present invention showed the same transcription factor expression as naive mouse ES cells, and formed the same transcription factor network as naive mouse ES cells. It is thought that. Therefore, it can be seen that the na ⁇ ve iPS cells produced and maintained by the method of the present invention are more na ⁇ ve than the reset cells described in Non-Patent Document 3.
- Example 5 Comparison of Reprogramming Efficiency by Combination of Reprogramming Factors
- the reprogramming factors (KLF2 and NANOG) described in Non-Patent Document 3 are used. It is the point which added 4 factors in Yamanaka. Therefore, the effect of this difference on na ⁇ ve (expression of ESRRB) was examined.
- the WD39 cells cultured under the above-mentioned conditions “1.1 Cultivation of prime-type human iPS cells” were transfected using the same procedure as “1.2 Preparation of transgenic cell line”.
- rtTA reverse tetracycline-regulated transactivator
- rtTA reverse tetracycline-regulated transactivator
- rtTA-Advanced sequence a reverse tetracycline-regulated transactivator (rtTA: using clontech's rtTA-Advanced sequence)
- PiggyBac Transposon expression vector Proc Natl Acad Sci US A. 2011 Jan 25; 108 (4): 1531 -6
- three types of vectors expressing a reprogramming factor and a fluorescent protein in a doxycycline-dependent manner were introduced (FIG. 9), and cultured in the same manner as in “1.1 Culture of prime type human iPS cells” above. .
- KLF2, NANOG and Venus, (2) Yamanaka 4 factor and Cerulean, and (3) Yamanaka 4 factor, KLF2, NANOG and TOMATO were used as combinations of reprogramming factors and fluorescent proteins.
- Doxycycline was added 4 days after the gene transfer, and drug selection was performed from the day after the doxycycline addition.
- neomycin 100 ⁇ g / mL
- puromycin 1 ⁇ g / mL
- the naive pluripotent stem cells produced and maintained by the method of the present invention are in a state in which naiveization has progressed to several stages more than the reset cells described in Non-Patent Document 3, It functions well as a naive pluripotent stem cell.
- Example 6 Induction of neural differentiation from na ⁇ ve iPS cells
- the SDIA method is a method for inducing differentiation into nerves by culturing iPS / ES cells using mouse stromal cell lines (PA6 cells, RIKEN BioResource Center) as feeder cells (Kawasaki et al. Neuron. 2000 Oct; 28 ( 1): See 31-40).
- PA6 cells mouse stromal cell lines
- RIKEN BioResource Center mouse stromal cell lines
- FBS 10% FBS
- Y27632 a ROCK inhibitor, was added so that the concentration became 10 ⁇ M from 1 hour before the start of the experiment.
- Y27632 was not added after seeding the cells on PA6 cells. The number of cells was seeded on a 12-well plate at 2 ⁇ 10 3 to 2 ⁇ 10 4 / well.
- MAP2 antibody M4403, sigma. Each colony was observed with a fluorescence microscope to determine whether MAP2-positive nerves were induced. The result is shown in FIG.
- Na ⁇ ve iPS cells had a significantly higher proportion of colonies with MAP2-positive cells than prime-type iPS cells. This result was observed in both 201B7 cells and WD39 cells used. From this result, it was found that iPS cells na ⁇ ve by the method of the present invention have excellent differentiation ability.
- Example 7 Induction of astrocyte differentiation from na ⁇ ve iPS cells Prime differentiation and naive iPS cells were induced to undergo neuronal differentiation by a method mediated by Neurosphere (see Stem Cells. 2008 Dec; 26 (12): 3086-98).
- a colony is peeled off in a lump state using a dissociation solution, and an embryoid body is formed by suspension culture in a medium of the prime type condition shown in Table 1 except for bFGF. I let you.
- the colony was peeled off in a lump by repeating pipetting strongly, and an embryoid body was formed by culturing in N2B27 medium (basic medium of naive medium). Except for the method of peeling the colony during embryoid body formation and the culture medium, the same operation was performed for all primed and na ⁇ ve iPS cells.
- LDN193189 was added to 100 nM in order to promote differentiation into the nervous system during embryoid body formation. Seven days after the formation of the embryoid body, the embryoid body was recovered, 1 mL of TrypLE TM Select was added, and the mixture was reacted at 37 ° C. for 10 minutes.
- the dissociated cells were adjusted to 2 ⁇ 10 5 cells / ml, and secondary Neurospheres were formed by suspension culture in MHM medium supplemented with 2% B27 and 20 ng / ml bFGF. Seven days after the formation of secondary Neurosphere, secondary Neurosphere was seeded on a cover glass coated with poly-L-ornithine and fibroctin to promote differentiation.
- MHM medium supplemented with 2% B27, 2% FBS and 20 ng / ml hLIF was used.
- ⁇ III-tubulin which is a neuronal marker
- GFAP which is an astrocyte marker (antibody T8660, sigma and antibody 2. 2B10, Thermo Fisher Scientific).
- the result is shown in FIG.
- the proportion of cells that differentiate into astrocytes was significantly higher in naive iPS cells than in prime-type iPS cells. This result was observed in both 201B7 cells (B7 in the figure) and WD39 cells (WD in the figure) used. From this result, it was found that iPS cells na ⁇ ve by the method of the present invention have excellent differentiation ability.
- pluripotent stem cells having naive characteristics and maintaining pluripotency can be stably produced, so that practical applications such as clinical use and drug evaluation are possible.
- Sequence number 1 Primer sequence number 2: Primer sequence number 3: Primer sequence number 4: Primer sequence number 5: Primer sequence number 6: Primer sequence number 7: Primer sequence number 8: Primer
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Abstract
Description
すなわち、本発明は、[1] 下記の2種の遺伝子:NanogおよびKlf2をプライム型多能性幹細胞に一時的に発現させ、かつLIF、MEK阻害剤、GSK3阻害剤、cAMP産生促進剤、TGF-β阻害剤およびPKC阻害剤を含む培地で培養することを含む、ナイーブ型多能性幹細胞の製造方法、
[2] プライム型多能性幹細胞がプライム型誘導多能性幹細胞またはプライム型胚性幹細胞である、前記[1]に記載の製造方法、
[3] プライム型多能性幹細胞がプライム型ヒト誘導多能性幹細胞またはプライム型ヒト胚性幹細胞である、前記[1]または前記[2]記載のナイーブ型ヒト多能性幹細胞の製造方法、
[4] プライム型多能性幹細胞がプライム型ヒト誘導多能性幹細胞である、前記[1]~[3]のいずれかに記載のナイーブ型ヒト誘導多能性幹細胞の製造方法、
[5] さらに下記の4種の遺伝子:Oct3/4、Klf4、c-MycおよびSox2を一時的に発現させる工程を含む、前記[1]~[4]のいずれかに記載の製造方法、
[6] MEK阻害剤がPD0325901、GSK3阻害剤がCHIR99021、cAMP産生促進剤がフォルスコリン、TGF-β阻害剤がA83-01およびPKC阻害剤がGo6983である、前記[1]~[5]のいずれかに記載の製造方法、
[7] 培地がN2B27培地である、前記[1]~[6]のいずれかに記載の製造方法、
[8] 下記の6種の遺伝子:Nanog、Klf2、Oct3/4、Klf4、c-MycおよびSox2を一時的にプライム型多能性幹細胞に発現させる工程を含む、ナイーブ型多能性幹細胞の製造方法、
[9] プライム型多能性幹細胞がプライム型誘導多能性幹細胞またはプライム型胚性幹細胞である、前記[8]に記載の製造方法、
[10] プライム型多能性幹細胞がプライム型ヒト誘導多能性幹細胞またはプライム型ヒト胚性幹細胞である、前記[8]または前記[9]記載のナイーブ型ヒト多能性幹細胞の製造方法、
[11] プライム型多能性幹細胞がプライム型ヒト誘導多能性幹細胞である、前記[8]~[10]のいずれかに記載のナイーブ型ヒト誘導多能性幹細胞の製造方法、
[12] 下記の6種の遺伝子:Nanog、Klf2、Oct3/4、Klf4、c-MycおよびSox2を一時的に体細胞に発現させる工程を含む、ナイーブ型誘導多能性幹細胞の製造方法、
[13] 下記の6種の遺伝子:Oct3/4、Klf4、c-Myc、Sox2、NanogおよびKlf2を一時的に体細胞に発現させ、かつLIF、MEK阻害剤、GSK3阻害剤、cAMP産生促進剤、TGF-β阻害剤およびPKC阻害剤を含む培地で培養することを含む、前記[12]記載の製造方法、
[14] 体細胞がヒト由来の体細胞である、前記[12]または前記[13]に記載のナイーブ型ヒト誘導多能性幹細胞の製造方法、
[15] MEK阻害剤がPD0325901、GSK3阻害剤がCHIR99021、cAMP産生促進剤がフォルスコリン、TGF-β阻害剤がA83-01およびPKC阻害剤がGo6983である、前記[13]または前記[14]に記載の製造方法、
[16] 培地がN2B27培地である、前記[13]~[15]のいずれかに記載の製造方法、
[17] ナイーブ型多能性幹細胞を、LIF、MEK阻害剤、GSK3阻害剤、cAMP産生促進剤、TGF-β阻害剤およびPKC阻害剤を含む培地で培養することを含む、ナイーブ型多能性幹細胞の維持方法、
[18] ナイーブ型多能性幹細胞がナイーブ型誘導多能性幹細胞またはナイーブ型胚性幹細胞である、前記[17]記載の維持方法、
[19] ナイーブ型多能性幹細胞がナイーブ型ヒト誘導多能性幹細胞またはナイーブ型ヒト胚性幹細胞である、前記[17]または前記[18]記載のナイーブ型ヒト多能性幹細胞の維持方法、
[20] ナイーブ型多能性幹細胞がナイーブ型ヒト誘導多能性幹細胞である、前記[17]~[19]のいずれかに記載のナイーブ型ヒト誘導多能性幹細胞の維持方法、
[21] MEK阻害剤がPD0325901、GSK3阻害剤がCHIR99021、cAMP産生促進剤がフォルスコリン、TGF-β阻害剤がA83-01およびPKC阻害剤がGo6983である、前記[17]~[20]のいずれかに記載の維持方法、
[22] 培地がN2B27培地である、前記[17]~[21]のいずれかに記載の維持方法、
[23] 前記[1]~[16]のいずれかに記載の方法にしたがって製造された、ナイーブ型多能性幹細胞、
[24] 前記[17]~[22]のいずれかに記載の方法にしたがって維持された、ナイーブ型多能性幹細胞等に関する。
1.1 プライム型ヒトiPS細胞の培養
ヒトiPS細胞としてWD39細胞(Imaizumi et al. Molecular Brain 2012, 5:35)およびコントロールラインとして広く使用されている201B7細胞(理研バイオリソースセンター)を用いた。
上記「1.1 プライム型ヒトiPS細胞の培養」条件で培養されたiPS細胞(10cmディッシュ)の培地を除去し7mLのPBSで一回洗浄後、解離液を1mL添加した。1分~2分後に解離液を除去し、7mLのPBSを加えた。軽くディッシュを揺すってフィーダー細胞のみを剥がした後にPBSを除去した。さらにPBSで一度洗浄し、表1のプライム型条件の培地3mLをディッシュに添加しスクレイパーによってiPS細胞のコロニーを剥がした後に15mLチューブに回収した。iPS細胞を200gで5分遠心して上清を除き、TrypLETM Select(Life Technologies)を1mL添加して37℃にて5分反応させた。Trypsin Inhibitor(Life Technologies)2mLを加えた後に、P1000のピペットマンで10回~20回ピペッティングすることで単一細胞に解離した。7mLの培地を足して容量を増やし70μmのセルストレイナーを通した。単一細胞となったiPS細胞を遺伝子導入に使用した。
「1.2 遺伝子導入細胞株の作製」で遺伝子導入およびクローン化されたiPS細胞を解離液およびTrypLETM Selectを用いて単一細胞に解離し、MEF細胞をフィーダーとして播いた6well plateに1×105/wellにて播種した。その際、培地は表1の2iLFA+Go6983の培地(以下、本発明の培地と略記することがある。)を使用し、さらにドキシサイクリン(1μg/mL)の添加によって導入遺伝子の発現を誘導したうえで低酸素にて培養した(Passage1;P1)。なお、ナイーブ化開始の初日は10μMとなるようY27632(ROCK阻害剤)を加え、細胞死を抑えた。ナイーブ化開始の5日~7日後に継代を行った。培地を除いたのち、各ウェルに0.25%Trypsin-EDTAを350μL加え1分間37℃で反応させた。Trypsin Inhibitorによって反応を止め、培地を5mL加えた。ピペットエイドで数回ピペッティングすることでフィーダー細胞のシートからiPS細胞のコロニーを剥がし、コロニーを15mLチューブに回収した。遠心後、上清を除いて200μLの培地を添加しP200のピペットマンで40回程度ピペッティングすることで単一細胞に解離した。MEF細胞をフィーダー細胞として培養している新しい6well plateに細胞全量を播種した(Passage2)。継代後もP1と同様に、本発明の培地にドキシサイクリンを添加した条件で培養した。継代から5日~7日後に顕微鏡下でiPS細胞のコロニー形態を観察し2回目の継代を実施した。2回目の継代以降ドキシサイクリンは添加しなかった。2回目の継代から5日~7日後に顕微鏡下でiPS細胞のコロニー形態を観察し、3回目の継代を実施した。4回目以降も同様に継代した。なお、2回目および3回目の継代時に、それぞれPassage2およびPassage3の細胞の一部をqPCRによる解析用に回収した(図8参照)。
本実験では、iPS細胞として201B7細胞を使用した。
解析に使用する細胞は、プライム型条件の培地で培養された201B7細胞(「1.1 プライム型ヒトiPS細胞の培養」参照)、「1.3 iPS細胞のナイーブ化」で作製されたPassage2およびPassage3の201B7細胞である。各細胞からRNeasy mini kit(QIAGEN)を用いてRNAを回収し、ReverTra Ace(登録商標)を用いて逆転写した後にSYBR(登録商標) Premix Ex TaqII(clontech)を用いてqPCRを行った。プライマーセットは表2に示した。
本実験では、Go6983添加タイミングによるナイーブ化への効果を検討した。「1.3 iPS細胞のナイーブ化」におけるpassage1の201B7細胞培養培地(表1の2iLFA+Go6983の培地)を、表1のKSR 2iLFAの培地または表1のKSR 2iLFA+Go6983の培地に変更し、ドキシサイクリンの添加によって導入遺伝子の発現を誘導したうえで低酸素にて培養した。それぞれの条件で培養したpassage1を継代から5日~7日後に回収し、qPCRによる解析に供した。その結果を図3に示す。図3から明らかであるように、Go6983を添加した培地で培養したiPS細胞のほうが、ESRRBの発現は高かった。本結果から、Go6983をドキシサイクリンによる導入遺伝子の発現誘導と同時に添加することにより、iPS細胞のナイーブ化は促進されることが分かった。
ESRRB遺伝子はマウスES細胞において自己複製を制御する重要な遺伝子である(Cell Stem Cell, 11,491-504 (2012)参照)。本発明では、実施例1の結果から明らかであるように、導入遺伝子の発現誘導の有無にかかわらず、ESRRBの発現は高い状態で維持されている。それに対し、非特許文献3に記載のリセット細胞では、ESRRBの発現はほとんど認められていない(非特許文献3参照)。本発明と非特許文献3に記載の条件における相違点のひとつが、本発明では非特許文献3に記載の培地条件にフォルスコリンとA83-01を加えた点である。そこで、本相違点が、ナイーブ化(ESRRBの発現およびコロニー形態)に及ぼす影響について検討した。
本結果から、導入遺伝子の発現誘導を止めた後もiPS細胞のナイーブ化状態を維持するためにはフォルスコリンとA83-01の添加が必要であることが分かった。
本発明と非特許文献3に記載の条件における相違点のひとつが、本発明では非特許文献3に記載のリプログラミング因子(KLF2及びNANOG)に山中4因子を加えた点である。そこで、本相違点が、ナイーブ化(ESRRBの発現)に及ぼす影響について検討した。
上記「1.1 プライム型ヒトiPS細胞の培養」条件で培養されたWD39細胞に対し「1.2 遺伝子導入細胞株の作製」と同様の手技を用いて遺伝子導入した。遺伝子導入ではリバーステトラサイクリン制御性トランス活性化因子(rtTA)発現ベクター(rtTA:clontechのrtTA-Advanced配列使用)、PiggyBac Transposon発現ベクター(Proc Natl Acad Sci U S A. 2011 Jan 25;108(4):1531-6参照)、ドキシサイクリン依存的にリプログラミング因子及び蛍光タンパクを発現するベクターの3種類のベクターを導入し(図9)、上記「1.1プライム型ヒトiPS細胞の培養」と同様に培養した。なお、リプログラミング因子及び蛍光タンパクの組み合わせは(1)KLF2、NANOG及びVenus、(2)山中4因子及びCerulean、(3)山中4因子、KLF2、NANOG及びTOMATOの三種類を用いた。遺伝子導入の翌日~4日後にドキシサイクリンを添加し、ドキシサイクリン添加の翌日から薬剤によるセレクションを行った。セレクションとして、ドキシサイクリン依存的に発現する耐性遺伝子の種類に従い、ネオマイシン(100μg/mL)又はピューロマイシン(1μg/mL)を添加した。ドキシサイクリン添加5日後に細胞を回収し、qPCRによる遺伝子発現解析を実施したところ、(3)山中4因子、KLF2、NANOG及びTOMATOの遺伝子を導入した細胞では、(1)KLF2、NANOG及びVenusや(2)山中4因子及びCeruleanを導入した細胞に比べてESRRBの発現が相乗的に高かった(図5)。したがって、山中4因子、KLF2、NANOGの6遺伝子の導入によって製造されたナイーブ型iPS細胞は、非特許文献3に記載のリセット細胞よりもナイーブ化が進んだ状態であることがわかる。
(1)PKC阻害剤(例えば、Go6983)を導入遺伝子の発現誘導時に添加することにより、ナイーブ化は促進する。
(2)KLF2、NANOGの2遺伝子のみの導入より、山中4因子を加えた6遺伝子の導入によってナイーブ化はより促進する。
(3)cAMP産生促進剤(例えば、フォルスコリン)とTGF-β阻害剤(例えば、A83-01)の添加により、細胞はナイーブ化状態を維持する。
以上より、本発明方法によって製造され維持されたナイーブ型多能性幹細胞(例えば、ヒトiPS細胞)は、非特許文献3に記載のリセット細胞よりもナイーブ化が数段に進んだ状態であり、ナイーブ型多能性幹細胞として十分に機能する。
プライム型およびナイーブ化したiPS細胞をSDIA法によって神経分化誘導した。SDIA法はマウスストローマ細胞株(PA6細胞、理研バイオリソースセンター)をフィーダー細胞としてiPS/ES細胞を培養することで神経への分化を誘導する方法である(Kawasaki et al. Neuron. 2000 Oct;28(1):31-40参照)。PA6細胞の通常の培養は、αMEM+10%FBSを使用した。SDIA法を行う際に、培地を表1のSDIA mediumに変更した。プライム型からSDIA法を実施する際には上記「1.2 遺伝子導入細胞株の作製」と同様の方法でコロニーを単一細胞にしたが、細胞死を抑える目的で実験開始1時間前から10μMとなるようROCK阻害剤であるY27632を添加した。PA6細胞上に細胞を播種した後にはY27632を添加しなかった。細胞数は12well plateに2×103~2×104/wellで播種した。ナイーブ型からSDIA法を実施する際には上記「1.3 iPS細胞のナイーブ化」で作製したPassage3またはPassage4の細胞をTrypsin-EDTAを用いた方法でコロニーを単一細胞にした。なお、細胞死を抑える目的で実験開始1時間前から10μMとなるようROCK阻害剤であるY27632を添加した。PA6細胞上に細胞を播種した後にはY27632を添加しなかった。細胞数は12well plateに2×103~2×104/wellで播種した。SDIA法の10日目に4%PFAによって細胞を固定し、MAP2に対して免疫組織染色を実施した(抗体M4403、sigma)。1つ1つのコロニーを蛍光顕微鏡で観察し、MAP2陽性の神経が誘導されているか判別した。その結果を図6に示す。
プライム型およびナイーブ化したiPS細胞をNeurosphereを介する方法によって神経分化誘導した(Stem Cells. 2008 Dec;26(12):3086-98参照)。プライム型からアストロサイト分化を実施する際には解離液を用いてコロニーを塊の状態で剥がし、表1のプライム型条件の培地からbFGFを除いた培地で浮遊培養することで胚様体を形成させた。ナイーブ型からアストロサイト分化を実施する際には強くピペッティングを繰り返すことでコロニーを塊の状態で剥がし、N2B27培地(ナイーブ培地の基礎培地)で培養することで胚様体を形成した。なお、胚様体形成時のコロニーの剥がし方と培地以外はすべてプライム型とナイーブ化したiPS細胞で全く同じ操作をした。また、胚様体形成期間には神経系への分化を促進するため、100nMとなるようLDN193189を添加した。胚様体形成の7日後に胚様体を回収し、TrypLETM Selectを1mL添加して37℃にて10分反応させた。Trypsin Inhibitor2mLを加えた後に、P1000のピペットマンで20回~30回ピペッティングすることで単一細胞に解離した。解離した細胞は2×105cells/mlとなるよう調整し、2%B27及び20ng/ml bFGFを添加したMHM培地で浮遊培養することで一次Neurosphereを形成した。一次Neurosphere形成の7日後にNeurosphereを回収し、TrypLETM Selectを1mL添加して37℃にて10分反応させた。Trypsin Inhibitor2mLを加えた後に、P1000のピペットマンで20回~30回ピペッティングすることで単一細胞に解離した。解離した細胞は2×105cells/mlとなるよう調整し、2%B27及び20ng/ml bFGFを添加したMHM培地で浮遊培養することで二次Neurosphereを形成した。二次Neurosphere形成の7日後にpoly-L-ornithine及びfibronectinコートしたカバーガラス上に二次Neurosphereを播種し分化を促した。分化用の培地には2%B27,2%FBS及び20ng/ml hLIFを添加したMHM培地を用いた。カバーガラス上に播種した10日後に4%PFAによって細胞を固定し、神経マーカーであるβIIIーTubulin及びアストロサイトマーカーであるGFAPに対して免疫組織染色を実施した(抗体T8660、sigma及び抗体2.2B10、Thermo Fisher Scientific)。
その結果を図7に示す。ナイーブ化したiPS細胞はプライム型のiPS細胞に比べてアストロサイトへ分化する細胞の割合が顕著に高かった。この結果は、使用した201B7細胞(図中、B7)およびWD39細胞(図中、WD)の両方で認められた。本結果から、本発明の方法によってナイーブ化されたiPS細胞は、優れた分化能を有していることが分かった。
配列番号2:プライマー
配列番号3:プライマー
配列番号4:プライマー
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Claims (24)
- 下記の2種の遺伝子:NanogおよびKlf2をプライム型多能性幹細胞に一時的に発現させ、かつLIF、MEK阻害剤、GSK3阻害剤、cAMP産生促進剤、TGF-β阻害剤およびPKC阻害剤を含む培地で培養することを含む、ナイーブ型多能性幹細胞の製造方法。
- プライム型多能性幹細胞がプライム型誘導多能性幹細胞またはプライム型胚性幹細胞である、請求項1記載の製造方法。
- プライム型多能性幹細胞がプライム型ヒト誘導多能性幹細胞またはプライム型ヒト胚性幹細胞である、請求項1または請求項2記載のナイーブ型ヒト多能性幹細胞の製造方法。
- プライム型多能性幹細胞がプライム型ヒト誘導多能性幹細胞である、請求項1~3のいずれか1項に記載のナイーブ型ヒト誘導多能性幹細胞の製造方法。
- さらに下記の4種の遺伝子:Oct3/4、Klf4、c-MycおよびSox2を一時的に発現させる工程を含む、請求項1~4のいずれか1項に記載の製造方法。
- MEK阻害剤がPD0325901、GSK3阻害剤がCHIR99021、cAMP産生促進剤がフォルスコリン、TGF-β阻害剤がA83-01およびPKC阻害剤がGo6983である、請求項1~5のいずれか1項に記載の製造方法。
- 培地がN2B27培地である、請求項1~6のいずれか1項に記載の製造方法。
- 下記の6種の遺伝子:Nanog、Klf2、Oct3/4、Klf4、c-MycおよびSox2を一時的にプライム型多能性幹細胞に発現させる工程を含む、ナイーブ型多能性幹細胞の製造方法。
- プライム型多能性幹細胞がプライム型誘導多能性幹細胞またはプライム型胚性幹細胞である、請求項8記載の製造方法。
- プライム型多能性幹細胞がプライム型ヒト誘導多能性幹細胞またはプライム型ヒト胚性幹細胞である、請求項8または請求項9記載のナイーブ型ヒト多能性幹細胞の製造方法。
- プライム型多能性幹細胞がプライム型ヒト誘導多能性幹細胞である、請求項8~10のいずれか1項に記載のナイーブ型ヒト誘導多能性幹細胞の製造方法。
- 下記の6種の遺伝子:Nanog、Klf2、Oct3/4、Klf4、c-MycおよびSox2を一時的に体細胞に発現させる工程を含む、ナイーブ型誘導多能性幹細胞の製造方法。
- 下記の6種の遺伝子:Oct3/4、Klf4、c-Myc、Sox2、NanogおよびKlf2を一時的に体細胞に発現させ、かつLIF、MEK阻害剤、GSK3阻害剤、cAMP産生促進剤、TGF-β阻害剤およびPKC阻害剤を含む培地で培養することを含む、請求項12記載の製造方法。
- 体細胞がヒト由来の体細胞である、請求項12または請求項13に記載のナイーブ型ヒト誘導多能性幹細胞の製造方法。
- MEK阻害剤がPD0325901、GSK3阻害剤がCHIR99021、cAMP産生促進剤がフォルスコリン、TGF-β阻害剤がA83-01およびPKC阻害剤がGo6983である、請求項13または請求項14に記載の製造方法。
- 培地がN2B27培地である、請求項13~15のいずれか1項に記載の製造方法。
- ナイーブ型多能性幹細胞を、LIF、MEK阻害剤、GSK3阻害剤、cAMP産生促進剤、TGF-β阻害剤およびPKC阻害剤を含む培地で培養することを含む、ナイーブ型多能性幹細胞の維持方法。
- ナイーブ型多能性幹細胞がナイーブ型誘導多能性幹細胞またはナイーブ型胚性幹細胞である、請求項17記載の維持方法。
- ナイーブ型多能性幹細胞がナイーブ型ヒト誘導多能性幹細胞またはナイーブ型ヒト胚性幹細胞である、請求項17または請求項18記載のナイーブ型ヒト多能性幹細胞の維持方法。
- ナイーブ型多能性幹細胞がナイーブ型ヒト誘導多能性幹細胞である、請求項17~19のいずれか1項に記載のナイーブ型ヒト誘導多能性幹細胞の維持方法。
- MEK阻害剤がPD0325901、GSK3阻害剤がCHIR99021、cAMP産生促進剤がフォルスコリン、TGF-β阻害剤がA83-01およびPKC阻害剤がGo6983である、請求項17~20のいずれか1項に記載の維持方法。
- 培地がN2B27培地である、請求項17~21のいずれか1項に記載の維持方法。
- 請求項1~16いずれか一項に記載の方法にしたがって製造された、ナイーブ型多能性幹細胞。
- 請求項17~22いずれか一項に記載の方法にしたがって維持された、ナイーブ型多能性幹細胞。
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JPWO2016148253A1 (ja) | 2018-01-11 |
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US20200325453A1 (en) | 2020-10-15 |
US20180080009A1 (en) | 2018-03-22 |
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US10738280B2 (en) | 2020-08-11 |
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