WO2012144582A1 - 角膜上皮分化指向性iPS細胞 - Google Patents
角膜上皮分化指向性iPS細胞 Download PDFInfo
- Publication number
- WO2012144582A1 WO2012144582A1 PCT/JP2012/060667 JP2012060667W WO2012144582A1 WO 2012144582 A1 WO2012144582 A1 WO 2012144582A1 JP 2012060667 W JP2012060667 W JP 2012060667W WO 2012144582 A1 WO2012144582 A1 WO 2012144582A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cells
- corneal epithelial
- cell
- stem cells
- pluripotent stem
- Prior art date
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N5/0618—Cells of the nervous system
- C12N5/0621—Eye cells, e.g. cornea, iris pigmented cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N5/0618—Cells of the nervous system
- C12N5/0623—Stem cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/60—Transcription factors
- C12N2501/602—Sox-2
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/60—Transcription factors
- C12N2501/603—Oct-3/4
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/60—Transcription factors
- C12N2501/604—Klf-4
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/60—Transcription factors
- C12N2501/606—Transcription factors c-Myc
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
- C12N2502/1394—Bone marrow stromal cells; whole marrow
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/09—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from epidermal cells, from skin cells, from oral mucosa cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
Definitions
- the present invention mainly relates to a method for inducing differentiation of pluripotent stem cells into corneal epithelial stem cells and / or corneal epithelial cells, a method for producing corneal epithelial stem cells and / or corneal epithelial cells, and corneal epithelial cells derived from the corneal epithelial stem cells
- the present invention relates to a sheet and induced pluripotent stem cells (iPS cells) used for induction of differentiation into corneal epithelial stem cells and / or corneal epithelial cells.
- corneal epithelial stem cell exhaustion As a group of intractable eye diseases, diseases such as Stevens-Johnson syndrome, ophthalmic pemphigus, and alkaline corrosion are known as corneal epithelial stem cell exhaustion. In corneal epithelial stem cell exhaustion, corneal epithelial stem cells present in the corneal limbus of the eye fall into irreversible dysfunction or disappear due to chronic inflammatory reaction due to trauma or autoimmunity, etc. Symptoms are present.
- corneal transplantation has been performed as a treatment for restoring the visual acuity of patients with severe corneal diseases such as corneal epithelial stem cell exhaustion.
- severe corneal diseases such as corneal epithelial stem cell exhaustion.
- corneal transplantation has been performed as a treatment for restoring the visual acuity of patients with severe corneal diseases such as corneal epithelial stem cell exhaustion.
- corneal epithelial stem cell exhaustion For a binocular patient who develops a disease in both eyes, there is no normal cornea derived from the patient himself, and therefore, only the keratoplasty of another family can be performed.
- there is a problem that there is a shortage of donors that can supply the cornea necessary for other corneal transplants, and there is a problem of rejection that can occur against the donor-derived cornea.
- Development of a new treatment method is desired.
- Non-Patent Document 1 A corneal sheet regenerated from its own corneal epithelial stem cells has the advantage that no rejection occurs because it is derived from its own cells.
- Non-patent Document 2 an autologous oral mucosal epithelial cell sheet transplantation method using stem cells of the patient's own oral mucosa has been developed (Non-patent Document 2).
- the autologous oral mucosal epithelial cell sheet transplantation method has already been clinically applied, and has achieved good clinical results.
- the transparent healing rate during the one-year observation period is 93% (14/15 eyes), and the visual acuity improvement rate is 80% (12/15 eyes). That is, it has been reported that the visual acuity of many eye disease patients can be achieved by the autologous oral mucosal epithelial cell sheet transplantation method.
- the method for inducing differentiation of specific cells is known only for some specific cells.
- the SDIA method is known as a method for inducing differentiation of embryonic stem cells into neural stem cells, neural cells, neural crest cells, nerve officers, etc. (Patent Document 2 and Non-Patent Document 3).
- the SDIA method is characterized by performing embryonic stem cells in the presence of stromal cells, and is considered to be a method for suppressing differentiation of embryonic stem cells into epidermal cells and the like and inducing differentiation into neurons. ing.
- differentiation is induced by the SDIA method to nerve cells and the like, which is different from the method for inducing differentiation of corneal epithelial cells and corneal epithelial stem cells, which are considered to be developmentally close to epidermal cells.
- iPS cells artificially induced pluripotent stem cells
- ES cells embryonic stem cells
- Regenerative medicine using iPS cells as a cell source is expected as a technique that can simultaneously solve the problem of donor shortage and rejection.
- iPS cells there is no ethical problem related to the destruction of early embryos because the process of destroying early embryos, which is essential in the case of ES cells, is not necessary.
- corneal epithelium differentiation from human ES cells or human iPS cells there are no reports of corneal epithelium differentiation from human ES cells or human iPS cells.
- the present invention provides a corneal epithelial stem cell for obtaining a corneal epithelial cell sheet that is as easy to obtain as a conventional autologous oral mucosal epithelial cell sheet and has higher safety in terms of the possibility of blood vessel invasion and the like, and Another object is to provide a method for inducing and producing corneal epithelial cells. Another object of the present invention is to provide a corneal epithelial cell sheet derived from the corneal epithelial stem cells and / or corneal epithelial cells, a cell material for inducing differentiation into corneal epithelial stem cells and / or corneal epithelial cells, and the like. To do.
- the present inventor has conducted extensive studies to solve the above problems, and surprisingly, by inducing pluripotent stem cells in the presence of stromal cells, induced pluripotent stem cells can be converted into corneal epithelial cells and corneas. It was found that differentiation can be induced into epithelial stem cells. Moreover, differentiation induction into corneal epithelial stem cells and / or corneal epithelial cells can be achieved more efficiently by inducing differentiation using induced pluripotent stem cells prepared from cells derived from ocular surface epithelium. I also found. The present invention has been completed by further studies based on such findings.
- this invention includes the invention of the aspect hung up below.
- Item 1 A method for inducing differentiation of pluripotent stem cells into corneal epithelial stem cells and / or corneal epithelial cells, comprising culturing pluripotent stem cells in the presence of stromal cells or amnion-derived factors.
- Item 2. The differentiation induction method according to Item 1, wherein the culture is performed in a serum-free medium.
- Item 3 The differentiation induction method according to Item 2, wherein the serum-free medium is substantially free of BMP4 for at least 1 to 2 weeks after the start of culture from the start of culture.
- the differentiation inducing method according to any one of Items 1 to 3, wherein the pluripotent stem cell is an embryonic stem cell (ES cell) or an induced pluripotent stem cell (iPS cell).
- Item 5 The induced pluripotent stem cell according to Item 4, wherein the induced pluripotent stem cell is an induced pluripotent stem cell obtained by introducing a reprogramming factor inducible into a pluripotent stem cell into a cell derived from an ocular surface epithelium.
- Item 6 a method for producing corneal epithelial stem cells and / or corneal epithelial cells, comprising the following steps: (1) culturing pluripotent stem cells in the presence of stromal cells or amnion-derived factor, and (2) selecting corneal epithelial stem cells and / or corneal epithelial cells from the cultured cells.
- Item 7 The method according to Item 6, wherein the step of selecting the corneal epithelial stem cell is a step of selecting a cell having a self-proliferating ability and expressing a corneal epithelial stem cell-specific marker.
- Item 9. An induced pluripotent stem cell obtained by introducing a reprogramming factor inducible into a pluripotent stem cell into a cell derived from an ocular surface epithelium.
- Item 10. The induced pluripotent stem cell according to Item 9, which is used for induction of differentiation into corneal epithelial stem cells and / or corneal epithelial cells.
- Item 11 A cell material for inducing differentiation into corneal epithelial stem cells and / or corneal epithelial cells, comprising the induced pluripotent stem cells according to Item 11, Item 9 or 10.
- a method for inducing differentiation into corneal epithelial stem cells and / or corneal epithelial cells and producing them using the patient's own cells is provided.
- Corneal epithelial stem cells obtained according to the present invention and corneal epithelial cell sheets derived therefrom are as easy to obtain as oral mucosal epithelial cell sheets.
- corneal epithelial cells, corneal epithelial stem cells derived from the patient's own cells, and corneal epithelial cell sheets derived therefrom are rejected because they are the patient's own cells when transplanted into the patient itself (autologous transplantation). There is no reaction.
- a corneal epithelial cell sheet that is safer than the prior art that does not have the problem of blood vessel invasion as in the case of the oral mucosal epithelial cell sheet.
- an induced pluripotent stem cell suitably used for induction of differentiation into a corneal epithelial stem cell and / or a corneal epithelial cell is provided.
- the corneal epithelial stem cells and / or corneal epithelial cells artificially produced according to the present invention can be a source of corneal epithelial stem cells and / or corneal epithelial cells that have been conventionally obtained only by collecting from patients. Therefore, it can be used as a useful tool for contributing to further technical improvement in regenerative medicine in the ocular cornea field.
- B a phase contrast microscopic image of human iPS cell line 253G1 (lower right) is shown.
- ES cell marker ES Marker stained image of B41 cell line.
- (A) shows the results of karyotype analysis of B34, B41, C51 and D43 cell lines.
- B Teratoma formed by B41 cell line (upper), and ectoderm (middle left: pigment epithelium and epidermis, middle right: nerve), mesoderm (lower left), and inner formed in teratomas
- the germ layer (bottom right) is shown.
- the results of measuring the gene expression levels of pax6 and K12 (keratin 12) in B41 strain, 201B7 strain and 253G1 strain under differentiation-inducing conditions by RT-PCR are shown.
- the bottom row shows the results of gene expression analysis of pax6 and K12 when BMP4 was added in the early stage of induction of B41 strain (2 weeks).
- the horizontal axis shows the elapsed period after differentiation induction in weeks, and the vertical axis shows the relative ratio to the expression level of the standard gene GAPDH. Shows the expression of pax6 (upper right and lower right) and K12 (upper left and lower left) in the B41 strain under differentiation-inducing conditions.
- phase contrast micrograph (upper left), K12 (upper right), K14 (lower left), K14 + K12 (lower right)
- phase contrast micrograph (upper left), p63 (upper right), K14 (interrupted left), K12 (interrupted right), p63 + K12 + K14 (bottom left).
- (A) shows a phase contrast image of corneal epithelial stem cells (or progenitor cells) induced to differentiate from the B41 strain, and immunostained images of pax6 and K14 (keratin 14): phase contrast micrograph (upper left) ), Pax6 (upper right), K14 (lower left), K14 and pax6 (lower right).
- (B) shows the phase contrast image of the same cell and the immunostained image of p63 + K14 (keratin 14): phase contrast micrograph (upper left), pax6 (upper right), K14 (lower left), K14 + pax6 (right) under).
- the present invention is mainly intended to provide a method for inducing differentiation of pluripotent stem cells into corneal epithelial stem cells and / or corneal epithelial cells, a production method, a corneal epithelial cell sheet, an induced pluripotent stem cell, and a cell material.
- a differentiation induction method, the production method, the corneal epithelial cell sheet, the induced pluripotent stem cell, and the cell material will be described in this order.
- the method for inducing differentiation of pluripotent stem cells into corneal epithelial stem cells and / or corneal epithelial cells includes a step of culturing pluripotent stem cells in the presence of stromal cells or in the presence of an amnion-derived factor. Differentiation induction method.
- differentiation-inducing method of the present invention will be described in detail, this description is common to the corneal epithelial stem cells and / or corneal epithelial cell production methods described later.
- pluripotent stem cells are induced to differentiate into corneal epithelial stem cells and / or corneal epithelial cells.
- the differentiation induction method of the present invention includes a differentiation induction method for corneal epithelial stem cells and corneal epithelial cells, a differentiation induction method for corneal epithelial stem cells, and a differentiation induction method for corneal epithelial cells.
- Preferred is a method for inducing differentiation into corneal epithelial stem cells, but is not limited thereto.
- the cornea is a film that forms an outer layer of a portion in contact with the outside at the front surface of the eyeball wall that exists in many mammalian eyeballs including humans, and has a transparent dish shape.
- the cornea is anatomically known to be predominantly composed of corneal epithelium, corneal stroma, and corneal endothelium from the outside.
- Corneal epithelium is ectodermally derived in development and is thought to differentiate from the epidermis. Normal corneal epithelium is known to have regenerative ability, and regeneration of the corneal epithelium is achieved by repeated self-proliferation and differentiation of corneal epithelial stem cells. That is, a corneal epithelial stem cell is a cell in which a daughter cell has a self-proliferation ability that has the same characteristics as itself and a differentiation ability that finally differentiates into a corneal epithelial cell. Corneal epithelial stem cells are thought to differentiate into corneal epithelial cells via corneal epithelial progenitor cells and transient amplifying cells (TA cells).
- TA cells transient amplifying cells
- Corneal epithelial stem cells are present in the corneal limbus located at the boundary between the corneal epithelium and the conjunctiva, particularly in the basal portion of the limbal epithelium in a normal cornea.
- corneal epithelial stem cell exhaustion such as Stevens-Johnson syndrome, ophthalmic pemphigoid, and alkaline corrosion
- the corneal epithelial stem cells are irreversibly dysfunctional or disappear.
- pluripotent stem cell may be used as long as the pluripotent stem cell induced to differentiate into corneal epithelial stem cells and / or corneal epithelial cells in the present invention has differentiation pluripotency and proliferative ability. be able to.
- the pluripotency includes the ability to differentiate into ectodermal cells, preferably epidermal cells, particularly preferably corneal epithelial cells.
- the pluripotent stem cell is a mammal in which a corneal epithelial stem cell exists (eg, human, mouse, monkey, cow, dog, cat, rabbit) depending on the intended use of the corneal epithelial stem cell and / or corneal epithelial cell that is induced to differentiate. , Pigs, goats, etc.) can be appropriately selected.
- the pluripotent stem cells are preferably human-derived.
- Human-derived pluripotent stem cells may be derived from humans of all generations (infants, infants, boys, adolescents, adults, middle-aged, elderly). Moreover, the sex of a human is not ask
- corneal epithelial stem cells and / or corneal epithelial cells that are induced to differentiate are used for the treatment of human eye diseases, they are preferably derived from cells collected from patients with eye diseases.
- collected from the patient it is desirable to perform what is called informed consent.
- pluripotent stem cells include embryonic stem cells and induced pluripotent stem cells, but are not limited thereto.
- the embryonic stem cells are stem cells derived from the inner cell mass of the early embryo and having differentiation pluripotency and self-proliferation ability that can be differentiated into virtually any cell.
- a cell derived from any embryonic stem cell strain currently established or newly established in the future can be used.
- an embryonic stem cell is obtained, for example, by removing an early embryo (for example, an early embryo between a fertilized egg and a blastocyst) from a mother fetus, and using an inner cell mass of the early embryo as a feeder cell (for example, mouse embryonic fibroblast).
- a feeder cell for example, mouse embryonic fibroblast
- the method for producing embryonic stem cells used in the present invention is not limited thereto.
- the induced pluripotent stem cell is derived from a somatic cell derived from a somatic cell, which has been provided with a differentiation pluripotency and a self-proliferating ability by introducing a reprogramming factor inducible into the pluripotent stem cell into the somatic cell. It is a cell (see, for example, Patent Document 1).
- induced pluripotent stem cells can be produced by introducing a reprogramming factor that can be induced into pluripotent stem cells into somatic cells.
- an already established induced pluripotent stem cell line can be used as the induced pluripotent stem cell used in the present invention.
- the reprogramming factor capable of inducing somatic cells into pluripotent stem cells is not particularly limited as long as it can reprogram somatic cells. Can do.
- the OCT family includes, but is not limited to, at least one selected from the group consisting of OCT3 / 4, OCT1A, and OCT6.
- the SOX family includes, but is not limited to, at least one selected from the group consisting of SOX1, SOX2, SOX3, SOX7, SOX15, SOX17 and SOX18, preferably SOX1 and / or SOX2.
- KLF4 include, but are not limited to, one or more selected from the group consisting of KLF1, KLF2, KLF4, and KLF5, and preferably at least one selected from the group consisting of KLF2, KLF4, and KLF5.
- Examples of the MYC family include, but are not limited to, at least one selected from the group consisting of C-MYC, N-MYC, and L-MYC.
- the reprogramming factor capable of inducing the above somatic cells into pluripotent stem cells is preferably widely known.
- the combination of OCT3 / 4, SOX2, KLF4 and C-MYC (Oct3 / 4, Sox2, Klf4 and c-Myc) is also known as “Yamanaka factor”.
- the reprogramming factor capable of inducing somatic cells into pluripotent stem cells is either a gene (nucleic acid molecule) or a gene product (protein) as long as somatic cells can be induced into pluripotent stem cells.
- a gene nucleic acid molecule
- both the base sequence (gene) and amino acid sequence (protein) of the specific inducer described above are known to those skilled in the art.
- the base sequence and amino acid sequence are disclosed in, for example, a database published by the National Center for Biotechnology Information (NCBI).
- the reprogramming factor capable of inducing the somatic cell into a pluripotent stem cell may be derived from the same or different species of the somatic cell into which the reprogramming factor is introduced. From the viewpoint of enhancing the establishment efficiency of induced pluripotent stem cells, it is preferably derived from the same kind of organism.
- a person skilled in the art can appropriately select a method for introducing a reprogramming factor capable of inducing the somatic cells into pluripotent stem cells into the somatic cells to be initialized.
- the agent program factor is a gene
- the reprogram factor can be introduced into a somatic cell by a method commonly used in transfection of animal cells.
- examples of the method for introducing the reprogramming factor into somatic cells include a method using a vector; a calcium phosphate method; a lipofection method; an electroporation method; a microinjection method and the like.
- the method using a vector is preferable from the viewpoint of introduction efficiency.
- a viral vector, non-viral vector, artificial virus, or the like can be used as the vector.
- Viruses such as lentivirus, adenovirus, and retrovirus A vector is preferably used from the viewpoint of safety.
- each gene may each be integrated in a different vector, and two or more types of genes may be integrated in one vector.
- the somatic cell from which the induced pluripotent stem cell is derived any somatic cell can be used as long as the induced pluripotent stem cell is induced.
- the somatic cell from which the induced pluripotent stem cell is derived is a normal somatic cell among normal and neoplastic.
- the cells used for obtaining the induced pluripotent stem cells are not particularly limited, and any cells (for example, fibroblasts) for which iPS cell production has been confirmed so far and any cells to be reported in the future. including. From the viewpoint that differentiation efficiency into corneal epithelial cells is particularly high, induced pluripotency obtained by introducing a reprogramming factor that can be induced into pluripotent stem cells into cells derived from ocular surface epithelium It is preferable to use stem cells.
- Cells derived from the ocular surface epithelium can be obtained from, for example, a tissue piece of about 2 mm 2 collected from the corneal limbal epithelium, corneal epithelium, or conjunctival epithelium, but is not limited thereto.
- the cell derived from the ocular surface epithelium may be a cell obtained from a tissue piece collected as described above, or a cell obtained by appropriately culturing the cell.
- the somatic cell into which the reprogramming factor inducible to the pluripotent stem cell is introduced is induced into the induced pluripotent stem cell by culturing.
- the medium to be used, culture conditions, and culture period can be appropriately selected by those skilled in the art from a wide range.
- induced pluripotent stem cells When producing induced pluripotent stem cells, the production of induced pluripotent stem cells can be confirmed by methods known to those skilled in the art. For example, it is possible to confirm that induced pluripotent stem cells are produced by expressing embryonic stem cell markers such as SSEA3, SSEA4, E-Cadherin, Nanog, Oct3 / 4, and Sox2 for convenience. Yes, but not limited to this.
- pluripotent stem cells are cultured in the presence of stromal cells or in the presence of an amnion-derived factor.
- culturing in the presence of stromal cells can be performed by using stromal cells as feeder cells.
- the culture in the presence of stromal cells can be performed by means of culturing pluripotent stem cells in a state where the stromal cells have reached a confluent state in a culture vessel.
- the “confluent state” refers to a state known to those skilled in the art, in which the cells have grown to the number of cells that almost cover the surface of the culture vessel.
- stromal cells are used as feeder cells, stromal cells can be subjected to cell division inactivation treatment by means such as mitomycin C (MMC) treatment or X-ray irradiation as necessary.
- MMC mitomycin C
- stromal cells can be appropriately selected by those skilled in the art within the range in which the object of the present invention can be achieved.
- stromal cells generally refer to supporting cells surrounding a tissue (eg, epithelium and endothelium) or organ.
- stromal cells used by a person skilled in the art as a method known as the SDIA method can be preferably used, but the stromal cell is not limited thereto.
- the stromal cells include mouse calvaria-derived MC3T3-G2 / PA6 cells, M-CSF-deficient mouse calvaria-derived OP9 cells, and mouse fetus-derived NIH / 3T3 cells, but are not limited thereto. is not.
- the stromal cells can be used alone or in combination of two or more.
- the stromal cell is a mouse calvaria-derived MC3T3-G2 / PA6 cell.
- all the said cells are well-known to those skilled in the art, and those skilled in the art can obtain easily.
- the SDIA method is a method for inducing differentiation of embryonic stem cells into ectoderm cells (neural stem cells, nerve cells, neural crest cells or nerve officers), and is a method known from Patent Document 2 and Non-Patent Document 3. It is.
- the SDIA method is characterized by performing embryonic stem cells in the presence of stromal cells, and is considered to be a method for suppressing differentiation of embryonic stem cells into epidermal cells and the like and inducing differentiation into neurons. ing.
- Cultivation in the presence of the amnion-derived factor can be performed according to the method disclosed in Patent Document 4.
- Cultivation in the presence of the amnion-derived factor can be achieved by culturing in the presence of amniotic membrane or a processed product derived from amniotic membrane, but is not limited thereto.
- the above-mentioned amniotic membrane or a processed product derived from the amniotic membrane may be derived from the same organism or a different organism from the organism from which the pluripotent stem cells to be co-cultured are derived.
- the amniotic membrane or a processed product derived from the amniotic membrane can be appropriately obtained by those skilled in the art based on a known method.
- the above culture is performed in the presence of stromal cells or in the presence of an amnion-derived factor.
- “Culture in the presence” is not particularly limited as long as the stromal cell or amnion-derived factor and the pluripotent stem cell are cultured in a state in which the substance can be transferred. That is, when culturing in the presence of stromal cells, the stromal cells and pluripotent stem cells may be in contact with each other or in a state where they cannot be contacted.
- the state of pluripotent stem cells is not particularly limited, and even in an aggregated state (clump), non-aggregated Any of the states may be used.
- the pluripotent stem cell is an induced pluripotent stem cell or an embryonic stem cell
- the induced pluripotent stem cell is preferably cultured in an aggregated state, but is not limited thereto.
- the “aggregation state” means that the cultured pluripotent stem cells are not completely dispersed by enzyme treatment or the like, but are cultured in a state where there is adhesion between cells (clump state) It means to continue culturing in a state where adhesion between cells exists.
- Means for bringing cells into an aggregated state are known to those skilled in the art, and those skilled in the art can appropriately select from the known means.
- the clump state of pluripotent stem cells includes about 50 to 10,000 pluripotent stem cells, but is not limited thereto.
- the “aggregation state” includes “embryoidrybodies”.
- the abundance ratio between pluripotent stem cells and stromal cells or amnion-derived factors is not particularly limited, and can be appropriately set by those skilled in the art.
- the abundance ratio of pluripotent stem cells to stromal cells is preferably about 1 to 150 clumps with respect to a 3.8 cm 2 layer of stromal cells that has reached confluence. More preferably, it can be about 15 to 40 clumps.
- 3.8 cm ⁇ 2 > is an area per well of a 12-well culture container (12 well plate) normally used as a culture container.
- the culture is performed over a period in which differentiation induction into corneal epithelial stem cells and / or corneal epithelial cells is achieved.
- the culture period can vary depending on the type of pluripotent stem cells used and can be appropriately set by those skilled in the art and is not particularly limited. For example, it can be cultured until expression of a marker gene specific for corneal epithelial stem cells and / or corneal epithelial cells described below is observed. From the standpoint that sufficient differentiation induction is achieved, for example, the culture is preferably performed for about 6 weeks or longer, preferably about 8 weeks or longer, particularly preferably about 12 weeks or longer.
- the culture period can be about 6 to 20 weeks, preferably about 8 to 16 weeks, more preferably about 8 to 14 weeks.
- a serum-free medium In the culture process of the present invention, it is preferable to use a serum-free medium.
- the serum-free medium those skilled in the art can appropriately select from known media and those newly developed in the future from media that do not substantially contain serum that can be cultured by mammalian cells.
- Glasgow Minimum Essential Medium, G-MEM, Dulbecco's Modified Eagle Medium, D-MEM, etc. are exemplified as the above serum-free medium, but are not limited thereto. is not.
- the serum-free medium contains about 5 to 20%, preferably about 10% of Knockout® Serum® Replacement (KSR, trade name, Invitogen); about 0.5 to 5%, preferably about 2% of L- Glutamine: about 0.5-5%, preferably about 1% pyruvic acid (sodium pyruvate, Gibco), about 0.5-5%, preferably about 1% non-essential amino acid solution (nonessential amino acids, Gibco About 0 to 1%, preferably about 0.1% by weight, such as 2-mercaptoethanol.
- KSR Knockout® Serum® Replacement
- the serum-free medium can be exchanged by a method known to those skilled in the art.
- the serum-free medium used before and after the exchange can be the same composition or a different composition.
- a person skilled in the art can appropriately set the period between exchanges and the frequency of exchange.
- the medium is changed once every about 1 to 3 days, more preferably once every about 2 days.
- the feeder cells are not usually exchanged, but this is not a limitation.
- the serum-free medium is substantially free of BMP4 at least from about 1 to 2 weeks after the start of culture (for example, about 2 weeks).
- BMP4 (Bone Morphorogenic Protein 4) is a known protein. Prior to the present invention, BMP4 was thought to have an action of suppressing neuronal differentiation of ectodermal cells and inducing differentiation into epithelial cells (such as epidermis and corneal epithelium). It is particularly preferable that the serum-free medium is substantially free of BMP4 for at least about 1 to 2 weeks from the start of the culture process.
- substantially free of BMP4 is sufficient if the content of BMP4 in the medium is less than an amount that does not affect the effect of the differentiation induction method of the present invention, for example, less than 0.5 nM. More preferably, it is less than 0.1 nM, more preferably less than 0.05 nM, and from another point of view, it is preferably not intentionally contained.
- a commercially available medium reagent sold as a medium not containing BMP4 and a combination thereof can be used as a serum-free medium substantially free of BMP4.
- the serum-free medium may be substantially free of retinoic acid, but is not limited thereto.
- the culture can be performed by a technique known to those skilled in the art, for example, using the serum-free medium.
- suitable culture techniques include a technique of culturing under conditions of about 37 ° C. and a carbon dioxide concentration of about 5%, but is not limited thereto. Cultivation under the above conditions can be performed using, for example, a known CO 2 incubator.
- the corneal epithelial stem cell subjected to the differentiation induction method according to the present invention may include a corneal epithelial progenitor cell.
- corneal epithelial stem cells and / or corneal epithelial cells are induced.
- a part of cells in the culture is collected periodically (for example, every 2 weeks), and a marker gene (for example, an eye) is obtained by a quantitative RT-PCR method.
- K12 Keratin 12 gene expressed specifically in differentiated corneal epithelium.
- confirmation of the expression of the marker gene can be used as an indicator that corneal epithelial stem cells and / or corneal epithelial cells have been induced to differentiate.
- the production method of corneal epithelial stem cells and / or corneal epithelial cells of the present invention includes the following steps: (1) a step of culturing pluripotent stem cells, and (2) a step of selecting corneal epithelial stem cells and / or corneal epithelial cells from the cultured cells.
- the step of culturing pluripotent stem cells in step (1) is a step of culturing pluripotent stem cells described in the above section “1. Differentiation induction method”. That is, the step of culturing pluripotent hepatocytes in the presence of stromal cells or in the presence of amnion-derived factor. Step (1) can be continued until, for example, a marker specific to corneal epithelial stem cells and / or corneal epithelial cells is expressed in cultured pluripotent stem cells.
- corneal epithelial stem cells and / or corneal epithelial cells are selected from the cells (population) cultured in step (1).
- Means for selecting corneal epithelial stem cells and / or corneal epithelial cells can be appropriately set by those skilled in the art.
- corneal epithelial stem cells when selecting a corneal epithelial stem cell, as one aspect of the selection means, selecting from the cells (population) cultured in the step (1) using as an index the expression of a self-proliferating ability and / or a corneal epithelial stem cell-specific marker.
- corneal epithelial stem cells can be selected by performing a step of selecting cells having self-proliferating ability and a step of selecting cells expressing a corneal epithelial stem cell-specific marker. It is not limited to.
- the order of performing the step of selecting cells having self-proliferating ability and the step of selecting cells expressing a corneal epithelial stem cell-specific marker is not limited. Also corneal epithelial stem cells can be selected.
- the evaluation of the ability of the cells to proliferate can be performed by methods known to those skilled in the art.
- the cells can be seeded in a culture system, subcultured, and confirmed by cell proliferation, but the present invention is not limited thereto.
- the proliferation of the cells can be confirmed by forming colonies by subculture.
- the subculture for confirming the formation of colonies can be performed in a co-culture system with feeder cells (for example, NIH / 3T3 cells) or in a medium for epithelial stem cells.
- feeder cells for example, NIH / 3T3 cells
- the present invention is not limited to this.
- the medium for performing the subculture is a medium that can culture corneal epithelial stem cells (for example, keratinocyte-conditioned medium (KCM medium), keratinocyte serum-free medium (KSFM medium, Invitrogen, etc.) known to those skilled in the art) ), CnT-20 medium (CELLnTEC), CnT-50 medium (CELLnTEC), etc.).
- KCM medium keratinocyte-conditioned medium
- KSFM medium keratinocyte serum-free medium
- CnT-20 medium CELLnTEC
- CnT-50 medium CELLnTEC
- the reseeding density is preferably about 2000 to 16000 cells / well.
- the proliferation ability is preferably self-replication ability.
- the evaluation of the ability of the cell to proliferate is not limited to this.
- the characteristics of the cell after growth do not change by subculture. Can be used as an indicator.
- the characteristics of the cells after proliferation can be suitably confirmed by a corneal epithelial stem cell marker described later, but are not limited thereto.
- corneal epithelial stem cell-specific marker can be performed by methods known to those skilled in the art.
- corneal epithelial stem cell markers include, but are not limited to, K14 (keratin 14) protein and / or p63 protein and / or N-cadherin protein, which are specific markers for stratified epithelial stem cells (and progenitor cells). Is not to be done.
- ocular tissue specific markers eg, pax6 protein
- corneal epithelial specific differentiation markers eg, K12 (keratin 12) protein
- cell surface markers for example, Integrin alpha 6, N-cadherin, etc.
- a specific detection method for expressing a corneal epithelial stem cell marker is known, for example, detection by immunocytochemistry is preferable. More specifically, examples include detection by microscopic observation using an antibody staining method, detection of cell surface markers by a cell sorter (flow cytometer), and the like.
- a reporter gene comprising a pluripotent stem cell that induces differentiation linked to a reporter gene of the corneal epithelial stem cell marker (for example, a promoter region of a gene encoding a corneal epithelial stem cell marker and a fluorescent protein such as green fluorescent protein (GFP)). Gene
- GFP green fluorescent protein
- a step of selecting a cell expressing a corneal epithelial cell marker can be mentioned.
- the corneal epithelial cell marker include, but are not limited to, an eye tissue-specific marker (for example, pax6 protein), a corneal epithelium-specific differentiation marker (for example, K12 (keratin 12) protein), and the like.
- a means for detecting the expression of a corneal epithelial cell marker for example, a reporter gene of the corneal epithelial cell marker described above (for example, a promoter region of a gene encoding a corneal epithelial cell marker and green fluorescence)
- examples include means for introducing a reporter gene linked with a fluorescent protein such as protein (GFP) and detecting the expression of the reporter gene after induction of differentiation.
- GFP fluorescent protein
- corneal epithelial stem cells When selecting corneal epithelial stem cells, the following embodiments (a) and (b) are exemplified as specific embodiments of the above step (2), but are not limited thereto: (A) After induction of differentiation in step (1), a cell that expresses a corneal epithelial stem cell marker is confirmed, and then the cell is recovered by enzyme treatment or the like, and a corneal epithelial stem cell can be selectively cultured (for example, The corneal epithelial stem cells are purified by re-seeding in a co-culture system using NIH / 3T3 cells as feeder cells, etc., and selecting cells that proliferate using colony formation as an index.
- step (1) After induction of differentiation in step (1), cells expressing a corneal epithelial specific marker are confirmed, and thereafter a cell expressing a cell surface marker specifically expressed in corneal epithelial stem cells using a cell sorter or the like To purify corneal epithelial stem cells.
- corneal epithelial stem cells and / or corneal epithelial cells are produced.
- the corneal epithelial stem cells obtained by the production method of the present invention may include corneal epithelial progenitor cells.
- the corneal epithelial cell sheet of the present invention is a corneal epithelial cell sheet derived from a corneal epithelial stem cell and / or a corneal epithelial cell obtained by the method described in "2. Production method" above.
- the corneal epithelial cell sheet may be a stratified sheet of corneal epithelial cell sheets.
- the means for producing the corneal epithelial cell sheet can be appropriately selected by those skilled in the art from known techniques and techniques developed in the future.
- the following method disclosed in Patent Document 3 or 4 or Non-Patent Document 1 or 2 can be used.
- means for producing a corneal epithelial cell sheet include, but are not limited to, a step of culturing the corneal epithelial stem cells on a temperature-responsive culture dish or carrier in the presence of feeder cells, and Means including a step of recovering the cell sheet obtained by the above step may be mentioned.
- NIH / 3T3 cells treated with Mitomycin ⁇ C can be used, but are not limited thereto.
- the temperature-responsive culture dish used in the above culture step is not particularly limited as long as it is a culture dish that can peel the cell sheet by changing (preferably lowering) the temperature.
- a culture dish coated with a temperature-responsive polymer such as poly (N-isopropylacrylamide) is exemplified.
- a commercially available temperature-responsive culture dish may be used.
- the carrier examples include, but are not limited to, an extracellular matrix such as collagen, laminin, and fibronectin (preferably collagen).
- the conditions for performing the above culture step can be appropriately set by those skilled in the art.
- the culture can be performed under conditions of about 37 ° C. and a carbon dioxide concentration of about 5%, but is not limited thereto.
- the culture period can be about 2 weeks.
- the above-described recovery can be performed by lowering the temperature and peeling the formed cell sheet from the temperature-responsive culture dish.
- the formed sheet can be collected together with the carrier.
- the corneal epithelial cell sheet can be suitably used for the treatment of intractable eye diseases such as Stevens-Johnson syndrome, ophthalmic pemphigus, and alkaline corrosion.
- Induced pluripotent stem cells derived from corneal epithelial stem cells and induced pluripotent stem cells that efficiently differentiate into corneal epithelial cells by introducing reprogramming factors inducible into pluripotent stem cells Stem cells are beneficial because they can be induced to differentiate into corneal epithelial cells and / or corneal epithelial cells faster and more efficiently than induced pluripotent stem cells obtained by introducing reprogramming factors into fibroblasts and the like. is there.
- the cells derived from the eye surface epithelium As the cells derived from the eye surface epithelium, the cells derived from the eye surface epithelium described in the above-mentioned “1. Differentiation induction method” column are used. Specifically, for example, cells derived from an ocular surface epithelium that can be obtained from a tissue piece of about 2 mm 2 collected from the corneal limbal epithelium, corneal epithelium, or conjunctival epithelium are exemplified, but the present invention is not limited thereto.
- the cell derived from the ocular surface epithelium may be a cell obtained from a tissue piece collected as described above, or a cell obtained by appropriately culturing the cell.
- the reprogramming factor inducible in the pluripotent stem cell described in the above-mentioned “1. Differentiation induction method” column can be preferably used.
- the means for introducing into the cell described in the above-mentioned “1. Differentiation induction method” column is preferably used. Can be used.
- the induced pluripotent stem cells of the present invention have high directivity that is induced to differentiate into corneal epithelial stem cells, as shown in the examples described later.
- high directivity induced to differentiate into corneal epithelial stem cells for example, the expression directivity of keratin epithelium-specific marker keratin 12 (K12) can be used.
- K12 keratin epithelium-specific marker keratin 12
- the high directivity of differentiation induction into corneal epithelial stem cells is the expression directivity of K12, K12 is highly expressed at an early stage after the start of differentiation induction. Therefore, the induced pluripotent stem cells of the present invention can be suitably used for inducing differentiation into corneal epithelial stem cells.
- the induced pluripotent stem cells of the present invention have high directivity that is induced to differentiate into corneal epithelial stem cells. Therefore, the present invention also provides a cell material for inducing differentiation into corneal epithelial stem cells, including the induced pluripotent stem cells described in the above-mentioned section “4. Induced pluripotent stem cells”.
- the cell material can be suitably used for induction of differentiation into corneal epithelial stem cells and production thereof. Furthermore, the obtained corneal epithelial cells can be further used for production of a corneal epithelial cell sheet, used as a drug discovery tool, and the like, but are not limited thereto.
- the grown ocular surface-derived epithelial cells were infected with a lentiviral vector loaded with human Oct3 / 4, Sox2, Klf4 and c-Myc (Yamanaka 4 factor), respectively.
- a lentiviral vector loaded with human Oct3 / 4, Sox2, Klf4 and c-Myc (Yamanaka 4 factor), respectively.
- the cells were replated on an iPS cell culture system (on a MEF feeder in iPS medium) and cultured for about 3 to 6 weeks. After confirming the disappearance of exogenous 4 factors in the mountains, colonies that were positive with the SSE4 antibody were picked up and iPS cell lines were established (B31, B34, B41, C51 and D43; 5 strains in total).
- Phase contrast micrographs of the established iPS cell line and the existing iPS cell line 253G1 are shown in FIG.
- any human iPS cell line established this time formed a cobblestone colony characteristic of pluripotent stem cells on MEF feeder cells.
- ES cell marker E-cadherin H-108 rabbit polyclonal antibody, SantaCruz, diluted 50 times
- Nanog R & D, goat polyclonal antibody, 100 times
- Oct3 / 4 R & D, Goat Polyclonal Antibody, 100-fold dilution
- Sox2 245610, mouse monoclonal antibody, R & D, 100-fold dilution
- Alexa568 antibody Invitrogen, diluted 200 times
- nuclei were stained with Hoechest 33342. An immunostained image is shown in FIG.
- the produced iPS cells were confirmed to express any of four ES cell markers, E-cadherin, Nanog, Oct3 / 4 and Sox2. That is, it is considered that induction into iPS cells was performed normally.
- the karyotype of the four iPS cell lines produced (B34, B41, C51 and D43) was verified according to a conventional method. The result of karyotype analysis is shown in FIG. All of the verified four human iPS cell lines had a karyotype of 46, XX [20] in 50 out of 50 cells, and it was confirmed to have a normal human karyotype.
- a teratoma formation test was performed on the obtained iPS cells by a conventional method. The results are shown in FIG. In teratomas, ectoderm (retinal pigment epithelium, epidermis and nerves), mesoderm and endoderm were identified.
- each human iPS cell was collected as a clump (cell mass) by adding a dissociation solution.
- Corneal induction medium G-MEM 10% KSR (Invitrogen) 2mM L-Glutamine (Invitrogen) 1% Pyruvate (Invitrogen, 100x) 1% nonessential amino acids (Invitrogen, 100x) 0.1% 2-mercaptoethanol (Invitrogen, 1000x).
- the expression of the eye tissue-specific marker pax6 was induced, and further, the expression of the mature corneal epithelium-specific marker K12 was increased 6 to 8 weeks after the induction.
- expression of the eye tissue-specific marker pax6 was observed in the same manner as in the B41 strain, and K12 expression was delayed about 4 weeks from the B41 strain at the 8th week. It was accepted from.
- BMP4 was added to the B41 strain at the initial stage of induction (2 weeks), expression of pax6 and expression of K12 were not confirmed.
- the expression level by the real-time PCR method was determined as a relative quantity with respect to the expression level of the standard gene GAPDH.
- commercially available TaqMan Gene Expression Assays (Applied Biosystem) was used as a probe.
- the probes used are as follows: ⁇ TaqMan probe> Pax6: Hs00240871_m1 K12: Hs00165015_m1 GAPDH: Hs99999905_m1.
- the fibroblast-derived 253G1 strain was also examined for expression and co-localization of p63, K12, and K14, as with the B41 strain, after longer-term differentiation induction (about 10 weeks or more). It was confirmed that the cells co-localized and some cells individually existed (FIG. 4c). On the other hand, cells expressing any of K12, K14, and pax6 were not confirmed in the B41 and 253G1 strains that were induced to differentiate by adding BMP4 at the initial stage of culture (FIGS. 4d and 4e). This indicates that addition of 0.5 nM BMP4 suppressed the induction to the corneal epithelium.
- iPS cells (B41 strain) 8 to 12 weeks after differentiation induction were collected by trypsin treatment and seeded in a co-culture system (epithelial stem cell culture system) of feeder cells with NIH / 3T3 cells.
- the reseeding density was about 8000 cells / well (12 well plate).
- KCM Medium Composition 69% Dulbecco's Modified Eagle's Medium (DMEM) (Sigma-Aldrich) 23% Nutrient Mixture F-12 Ham (Sigma-Aldrich) 5% Fetus Bovine Serum (FBS) (Japan Bio Serum) 1% Penicillin-Streptomycin (Invitrogen, 100x) 0.4 ⁇ g / ml Hydrocortisone Succinate (Wako) 2 nM 3,3 ', 5-Triiodo-L-thyronine sodium salt (MP BIOMEDICALS) 100 nM Cholera Toxin (Calbiochem) 2mM L-Glutamine (Invitrogen) 0.5% Insuline Transferrin Selenium (GIBCO, 200x) 10 ng / ml Recombinant Human EGF (R & D Systems).
- DMEM Dulbecco's Modified Eagle's Medium
- F-12 Ham Sigma-Aldrich
- Fetus Bovine Serum F
- Fig. 5 shows the phase difference image and stained image.
- the obtained colonies derived from the B41 strain showed a cobblestone shape peculiar to epithelial cells and expressed K14 and pax6, which are corneal epithelial progenitor cell markers (FIG. 5 (a)).
- K14 and pax6 which are corneal epithelial progenitor cell markers
- FIG. 5 (b) shows expression of p63 and K14, which are corneal epithelial progenitor cell markers.
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Neurosurgery (AREA)
- Neurology (AREA)
- Developmental Biology & Embryology (AREA)
- Ophthalmology & Optometry (AREA)
- Transplantation (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Materials For Medical Uses (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
Description
項1、多能性幹細胞をストローマ細胞または羊膜由来因子の存在下において培養する工程を含む、多能性幹細胞の角膜上皮幹細胞および/または角膜上皮細胞への分化誘導方法。
項2、前記培養が、無血清培地中で行なわれる、項1に記載の分化誘導方法。
項3、前記無血清培地が、少なくとも培養開始から培養開始後1~2週間の間BMP4を実質的に含まない、項2に記載の分化誘導方法。
項4、前記多能性幹細胞が、胚性幹細胞(ES細胞)または誘導多能性幹細胞(iPS細胞)である、項1~3のいずれかに記載の分化誘導方法。
項5、前記誘導多能性幹細胞が、眼表面上皮由来の細胞に、多能性幹細胞に誘導可能な再プログラム因子を導入することにより得られる誘導多能性幹細胞である、項4に記載の方法。
項6、下記の工程を含む、角膜上皮幹細胞および/または角膜上皮細胞の製造方法:
(1)多能性幹細胞をストローマ細胞または羊膜由来因子の存在下で培養する工程、および
(2)前記培養した細胞から、角膜上皮幹細胞および/または角膜上皮細胞を選択する工程。
項7、前記角膜上皮幹細胞を選択する工程が、自己増殖能を有し、かつ、角膜上皮幹細胞特異的マーカーを発現している細胞を選択する工程である、項6に記載の製造方法。
項8、項6または7に記載の製造方法により得られる角膜上皮幹細胞および/または角膜上皮細胞に由来する、角膜上皮細胞シート。
項9、眼表面上皮由来の細胞に対して、多能性幹細胞に誘導可能な再プログラム因子を導入することにより得られる誘導多能性幹細胞。
項10、角膜上皮幹細胞および/または角膜上皮細胞への分化誘導に用いられる、項9に記載の誘導多能性幹細胞。
項11、項9または10に記載の誘導多能性幹細胞を含む、角膜上皮幹細胞および/または角膜上皮細胞への分化誘導のための細胞材料。
本発明の、多能性幹細胞の角膜上皮幹細胞および/または角膜上皮細胞への分化誘導方法は、多能性幹細胞をストローマ細胞の存在下または羊膜由来因子の存在下において培養する工程含む分化誘導方法である。以下、本発明の分化誘導方法について詳述するが、この説明は後述する角膜上皮幹細胞及び/又は角膜上皮細胞の製造方法についても共通する。
本発明の角膜上皮幹細胞および/または角膜上皮細胞の製造方法は、下記の工程を含む:
(1)多能性幹細胞を培養する工程、および
(2)前記培養した細胞から、角膜上皮幹細胞および/または角膜上皮細胞を選択する工程。
(a)工程(1)の分化誘導後、角膜上皮幹細胞マーカーを発現する細胞を確認し、その後、細胞を酵素処理等により回収し、角膜上皮幹細胞を選択的に培養することができる系(例えば、NIH/3T3細胞をフィーダー細胞とした共培養系など)に再播種し、コロニーが形成されることを指標として増殖する細胞を選択することで、角膜上皮幹細胞を純化する。
(b)工程(1)の分化誘導後、角膜上皮特異的マーカーを発現する細胞を確認し、その後、セルソーター等を用いて角膜上皮幹細胞に特異的に発現している細胞表面マーカーを発現する細胞を選択することで、角膜上皮幹細胞を純化する。
本発明の角膜上皮細胞シートは、上記「2.製造方法」欄に記載の方法により得られる角膜上皮幹細胞および/または角膜上皮細胞に由来する、角膜上皮細胞シートである。上記角膜上皮細胞シートは、角膜上皮細胞シートの重層化シートであってもよい。
眼表面上皮由来の細胞に対して、多能性幹細胞に誘導可能な再プログラム因子を導入することにより得られる誘導多能性幹細胞は、線維芽細胞などに再プログラム因子を導入して得られる誘導多能性幹細胞と比較して、より早く、より効率良く角膜上皮細胞及び/又は角膜上皮細胞に分化誘導され得るため有益である。
前述の通り、本発明の誘導多能性幹細胞は角膜上皮幹細胞へと分化誘導される高い指向性を有する。従って、本発明は、前述「4.誘導多能性幹細胞」欄に記載の誘導多能性幹細胞を含む、角膜上皮幹細胞への分化誘導のための細胞材料をも提供する。
研究用の輸入強角膜片より、ヒト由来角膜内皮細胞、周辺の結合組織等を取り除いた上で、ディスパーゼ処理により眼表面の角結膜上皮層のみを単離した。得られた上皮細胞をトリプシン処理により単一細胞として、NIH/3T3細胞の培養上清と新鮮KCM培地との1:1混合培地中で培養した。
樹立したいずれのヒトiPS細胞株も公知の繊維芽細胞由来ヒトiPS細胞株と同様にMEFフィーダー細胞上で、多能性幹細胞に特徴的な敷石状のコロニーを形成した。
上記(I)で製造したiPS細胞(B41株)、および公知のヒト線維芽細胞由来253G1株および201B7株について、MC3T3-G2/PA6細胞(PA6細胞)をフィーダーとして培養を行なった。
(1)上記3種のヒトiPS細胞それぞれについて、MEF(mouse embryonic fibroblast)フィーダー細胞上にて下記培地中を用いて継代培養を行なった。
ヒトiPS用培地
DMEM/F12 (Invitrogen)
25% KSR (Invitrogen)
2mM L-Glutamine (Invitrogen)
1% nonessential amino acids (Invitrogen, 100x)
0.1% 2-mercaptoethanol (Invitrogen 1000x)。
角膜誘導培地
G-MEM (Invitrogen)
10% KSR(Invitrogen)
2mM L-Glutamine (Invitrogen)
1% Pyruvate (Invitrogen, 100x)
1% nonessential amino acids (Invitrogen, 100x)
0.1% 2-mercaptoethanol (Invitrogen, 1000x)。
用いたプローブは、下記の通りである:
<TaqManプローブ>
Pax6: Hs00240871_m1
K12: Hs00165015_m1
GAPDH: Hs99999905_m1。
KCM培地組成
69% Dulbecco's Modified Eagle's Medium(DMEM) (Sigma-Aldrich)
23% Nutrient Mixture F-12 Ham (Sigma-Aldrich)
5% Fetus Bovine Serum(FBS) (Japan Bio Serum)
1% Penicillin-Streptomycin (Invitrogen,100x)
0.4 μg/ml Hydrocortisone Succinate (Wako)
2 nM 3,3',5-Triiodo-L-thyronine sodium salt (MP BIOMEDICALS)
100 nM Cholera Toxin (Calbiochem)
2mM L-Glutamine (Invitrogen)
0.5% Insuline Transferrin Selenium (GIBCO, 200x)
10 ng/ml Recombinant Human EGF (R&D Systems)。
Claims (11)
- 多能性幹細胞をストローマ細胞または羊膜由来因子の存在下において培養する工程を含む、多能性幹細胞の角膜上皮幹細胞および/または角膜上皮細胞への分化誘導方法。
- 前記培養が、無血清培地中で行なわれる、請求項1に記載の分化誘導方法。
- 前記無血清培地が、少なくとも培養開始から培養開始後1~2週間の間、BMP4を実質的に含まない、請求項2に記載の分化誘導方法。
- 前記多能性幹細胞が、胚性幹細胞(ES細胞)または誘導多能性幹細胞(iPS細胞)である、請求項1~3のいずれかに記載の分化誘導方法。
- 前記誘導多能性幹細胞が、眼表面上皮由来の細胞に、多能性幹細胞に誘導可能な再プログラム因子を導入することにより得られる誘導多能性幹細胞である、請求項4に記載の分化誘導方法。
- 下記の工程を含む、角膜上皮幹細胞および/または角膜上皮細胞の製造方法:
(1)多能性幹細胞をストローマ細胞又は羊膜由来因子の存在下で培養する工程、および
(2)前記培養した細胞から、角膜上皮幹細胞および/または角膜上皮細胞を選択する工程。 - 前記角膜上皮幹細胞を選択する工程が、増殖能を有し、かつ、角膜上皮幹細胞特異的マーカーを発現している細胞を選択する工程である、請求項6に記載の製造方法。
- 請求項6または7に記載の製造方法により得られる角膜上皮幹細胞および/または角膜上皮細胞に由来する、角膜上皮細胞シート。
- 眼表面上皮由来の細胞に対して、多能性幹細胞に誘導可能な再プログラム因子を導入することにより得られる誘導多能性幹細胞。
- 角膜上皮幹細胞および/または角膜上皮細胞への分化誘導に用いられる、請求項9に記載の誘導多能性幹細胞。
- 請求項9または10に記載の誘導多能性幹細胞を含む、角膜上皮幹細胞および/または角膜上皮細胞への分化誘導のための細胞材料。
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201280019381.7A CN103492555A (zh) | 2011-04-20 | 2012-04-20 | 角膜上皮分化取向性iPS细胞 |
US14/112,572 US20140127803A1 (en) | 2011-04-20 | 2012-04-20 | iPS CELL HAVING DIFFERENTIATION PROPENSITY FOR CORNEAL EPITHELIUM |
JP2013511044A JP5759536B2 (ja) | 2011-04-20 | 2012-04-20 | 角膜上皮分化指向性iPS細胞 |
EP12774375.5A EP2700709A4 (en) | 2011-04-20 | 2012-04-20 | IPS CELL HAVING PREDISPOSITION FOR CORNEAL EPITHELIUM DIFFERENTIATION |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2011093846 | 2011-04-20 | ||
JP2011-093846 | 2011-04-20 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2012144582A1 true WO2012144582A1 (ja) | 2012-10-26 |
Family
ID=47041686
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2012/060667 WO2012144582A1 (ja) | 2011-04-20 | 2012-04-20 | 角膜上皮分化指向性iPS細胞 |
Country Status (5)
Country | Link |
---|---|
US (1) | US20140127803A1 (ja) |
EP (1) | EP2700709A4 (ja) |
JP (1) | JP5759536B2 (ja) |
CN (1) | CN103492555A (ja) |
WO (1) | WO2012144582A1 (ja) |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2016114285A1 (ja) * | 2015-01-15 | 2016-07-21 | 国立大学法人大阪大学 | 多能性幹細胞からの角膜上皮細胞の分化誘導 |
WO2018131491A1 (ja) * | 2017-01-13 | 2018-07-19 | 国立大学法人大阪大学 | 角膜上皮細胞集団の製造方法 |
KR20180135015A (ko) * | 2016-04-20 | 2018-12-19 | 교토후고리츠다이가쿠호진 | 배양 상피 시트의 제조 방법 |
JPWO2018143312A1 (ja) * | 2017-01-31 | 2019-12-12 | 国立大学法人大阪大学 | 多能性幹細胞の分化制御方法 |
JPWO2018139618A1 (ja) * | 2017-01-27 | 2019-12-19 | 国立大学法人大阪大学 | 体表組織の解剖学的部位判定方法 |
US11357798B2 (en) | 2015-08-03 | 2022-06-14 | Osaka University | Mesenchymal stem cell-derived exosome |
Families Citing this family (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2765188B1 (en) * | 2011-10-06 | 2017-12-06 | Keio University | Method for producing corneal endothelial cell |
CN105518123B (zh) * | 2013-08-06 | 2021-01-15 | 国立研究开发法人理化学研究所 | 制备眼前段组织的方法 |
CN104862271A (zh) * | 2014-02-22 | 2015-08-26 | 山西医科大学 | 一种简易角膜缘干细胞分离和体外培养试剂盒及方法 |
WO2015171663A2 (en) * | 2014-05-05 | 2015-11-12 | Indiana University Research And Technology Corporation | Method for producing retinal ganglion cells from pluripotent cells |
SG10201806498RA (en) | 2014-06-27 | 2018-08-30 | Univ California | Cultured mammalian limbal stem cells, methods for generating the same, and uses thereof |
US10501725B2 (en) | 2014-12-11 | 2019-12-10 | Keio University | Method for producing therapeutic corneal endothelial substitute cell sphere |
KR102015815B1 (ko) * | 2016-08-10 | 2019-08-29 | 가톨릭대학교 산학협력단 | 유도만능줄기세포의 분화를 유도하여 각막 상피 세포를 배양하는 방법 및 시스템 |
CN113811316A (zh) | 2019-05-15 | 2021-12-17 | 味之素株式会社 | 神经嵴细胞或角膜上皮细胞的纯化方法 |
Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001088100A1 (fr) | 2000-05-16 | 2001-11-22 | Kyowa Hakko Kogyo Co., Ltd. | Nouvelle methode permettant de declencher une differentiation de cellules embryonnaires dans des cellules de l'ectoderme et leurs applications |
JP2004261533A (ja) | 2003-02-14 | 2004-09-24 | Mitsuo Okano | 高生着性角膜上皮代替細胞シート、製造方法及びその利用方法 |
JP2005333904A (ja) | 2004-05-27 | 2005-12-08 | Institute Of Physical & Chemical Research | 羊膜由来因子による胚性幹細胞の培養方法 |
WO2007069666A1 (ja) | 2005-12-13 | 2007-06-21 | Kyoto University | 核初期化因子 |
JP2007524411A (ja) * | 2004-01-27 | 2007-08-30 | リライアンス ライフ サイエンシーズ ピーヴィーティー. リミテッド | 角膜輪部由来の未分化幹細胞を有する組織系 |
WO2010134619A1 (ja) * | 2009-05-18 | 2010-11-25 | 国立大学法人東北大学 | 人工多能性幹細胞からの上皮系前駆細胞・幹細胞群及び角膜上皮細胞群の分化誘導方法 |
WO2012073238A1 (en) * | 2010-12-02 | 2012-06-07 | Technion Research & Development Foundation Ltd. | Methods of generating corneal cells and cell populations comprising same |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1289156C (zh) * | 2003-07-25 | 2006-12-13 | 吕伟光 | 组织工程自体角膜上皮及其制备方法 |
JP5409359B2 (ja) * | 2007-07-13 | 2014-02-05 | 田辺三菱製薬株式会社 | 細胞の単離方法、細胞用無血清培養培地および細胞の培養方法 |
-
2012
- 2012-04-20 US US14/112,572 patent/US20140127803A1/en not_active Abandoned
- 2012-04-20 EP EP12774375.5A patent/EP2700709A4/en not_active Withdrawn
- 2012-04-20 WO PCT/JP2012/060667 patent/WO2012144582A1/ja active Application Filing
- 2012-04-20 JP JP2013511044A patent/JP5759536B2/ja active Active
- 2012-04-20 CN CN201280019381.7A patent/CN103492555A/zh active Pending
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001088100A1 (fr) | 2000-05-16 | 2001-11-22 | Kyowa Hakko Kogyo Co., Ltd. | Nouvelle methode permettant de declencher une differentiation de cellules embryonnaires dans des cellules de l'ectoderme et leurs applications |
JP2004261533A (ja) | 2003-02-14 | 2004-09-24 | Mitsuo Okano | 高生着性角膜上皮代替細胞シート、製造方法及びその利用方法 |
JP2007524411A (ja) * | 2004-01-27 | 2007-08-30 | リライアンス ライフ サイエンシーズ ピーヴィーティー. リミテッド | 角膜輪部由来の未分化幹細胞を有する組織系 |
JP2005333904A (ja) | 2004-05-27 | 2005-12-08 | Institute Of Physical & Chemical Research | 羊膜由来因子による胚性幹細胞の培養方法 |
WO2007069666A1 (ja) | 2005-12-13 | 2007-06-21 | Kyoto University | 核初期化因子 |
WO2010134619A1 (ja) * | 2009-05-18 | 2010-11-25 | 国立大学法人東北大学 | 人工多能性幹細胞からの上皮系前駆細胞・幹細胞群及び角膜上皮細胞群の分化誘導方法 |
WO2012073238A1 (en) * | 2010-12-02 | 2012-06-07 | Technion Research & Development Foundation Ltd. | Methods of generating corneal cells and cell populations comprising same |
Non-Patent Citations (9)
Title |
---|
HOMMA,R. ET AL.: "Induction of epithelial progenitors in vitro from mouse embryonic stem cells and application for reconstruction of damaged cornea in mice", INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE, vol. 45, no. 12, 2004, pages 4320 - 6, XP055082514 * |
KAWASAKI H. ET AL., NEURON, vol. 28, 2000, pages 31 - 40 |
KAZUHIRO SUDO ET AL.: "Kansaibo o Sasaeru Kankyo II. Feeder no Yakuwari to Feederless-ka eno Challenge, Amnion as a promising source of feeder cells for regenerative medicine", REGENERATIVE MEDICINE, vol. 8, no. 2, 2009, pages 195 - 200, XP008171942 * |
NISHIDA K. ET AL., N ENGL J MED, vol. 351, 2004, pages 1187 - 1195 |
NISHIDA K. ET AL., TRANSPLANTATION, vol. 77, 2004, pages 379 - 385 |
OKANO,H. ET AL.: "Differentiation of induced pluripotent stem cells into corneal epithelial cells", DIFFERENTIATION, vol. 80, 2010, pages S44 - S45, XP055137049 * |
RYUHEI HAYASHI ET AL.: "'Ishoku Iryo ni Okeru iPS Saibo Kenkyu no Impact' iPS Saibo o Mochiita Kakumaku Johi Saisei Chiryoho no Kaihatsu", JAPANESE JOURNAL OF TRANSPLANTATION, vol. 44, no. 3, 2009, pages 226 - 230, XP008171630 * |
See also references of EP2700709A4 |
WANG,Z. ET AL.: "Differentiation of embryonic stem cells into corneal epithelium", SCIENCE IN CHINA. SERIES C, LIFE SCIENCES / CHINESE ACADEMY OF SCIENCES, vol. 48, no. 5, 2005, pages 471 - 480, XP009122403 * |
Cited By (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11066641B2 (en) | 2015-01-15 | 2021-07-20 | Osaka University | Method for inducing differentiation of corneal epithelial cells from pluripotent stem cells |
KR20170092694A (ko) | 2015-01-15 | 2017-08-11 | 고꾸리쯔 다이가꾸 호우징 오사까 다이가꾸 | 다능성 줄기세포로부터의 각막 상피 세포의 분화 유도 방법 |
JPWO2016114285A1 (ja) * | 2015-01-15 | 2017-09-14 | 国立大学法人大阪大学 | 多能性幹細胞からの角膜上皮細胞の分化誘導 |
US20180010093A1 (en) * | 2015-01-15 | 2018-01-11 | Osaka University | Method for inducing differentiation of corneal epithelial cells from pluripotent stem cells |
WO2016114285A1 (ja) * | 2015-01-15 | 2016-07-21 | 国立大学法人大阪大学 | 多能性幹細胞からの角膜上皮細胞の分化誘導 |
US11357798B2 (en) | 2015-08-03 | 2022-06-14 | Osaka University | Mesenchymal stem cell-derived exosome |
KR20180135015A (ko) * | 2016-04-20 | 2018-12-19 | 교토후고리츠다이가쿠호진 | 배양 상피 시트의 제조 방법 |
KR102311121B1 (ko) | 2016-04-20 | 2021-10-13 | 교토후고리츠다이가쿠호진 | 배양 상피 시트의 제조 방법 |
WO2018131491A1 (ja) * | 2017-01-13 | 2018-07-19 | 国立大学法人大阪大学 | 角膜上皮細胞集団の製造方法 |
JPWO2018131491A1 (ja) * | 2017-01-13 | 2019-11-21 | 国立大学法人大阪大学 | 角膜上皮細胞集団の製造方法 |
JPWO2018139618A1 (ja) * | 2017-01-27 | 2019-12-19 | 国立大学法人大阪大学 | 体表組織の解剖学的部位判定方法 |
JPWO2018143312A1 (ja) * | 2017-01-31 | 2019-12-12 | 国立大学法人大阪大学 | 多能性幹細胞の分化制御方法 |
US11649433B2 (en) | 2017-01-31 | 2023-05-16 | Osaka University | Method for controlling differentiation of pluripotent stem cells |
Also Published As
Publication number | Publication date |
---|---|
JP5759536B2 (ja) | 2015-08-05 |
JPWO2012144582A1 (ja) | 2014-07-28 |
US20140127803A1 (en) | 2014-05-08 |
CN103492555A (zh) | 2014-01-01 |
EP2700709A4 (en) | 2014-11-19 |
EP2700709A1 (en) | 2014-02-26 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP5759536B2 (ja) | 角膜上皮分化指向性iPS細胞 | |
TWI810142B (zh) | 神經組織的製造方法 | |
EP3246394B1 (en) | Method for inducing differentiation of corneal epithelial cells from pluripotent stem cells | |
JP6873438B2 (ja) | 前眼部組織の製造方法 | |
WO2017183732A1 (ja) | 網膜組織の製造法 | |
CN107002040A (zh) | 视网膜组织的制备方法 | |
WO2017043605A1 (ja) | 網膜色素上皮細胞の製造方法 | |
EP3683306A1 (en) | Method for producing retinal tissues | |
KR102210180B1 (ko) | 모양체 주연부형 구조체의 제조 방법 | |
JP6758631B2 (ja) | 角膜内皮細胞マーカー及びその利用 | |
WO2020184720A1 (ja) | 移植用神経網膜の品質を評価する方法及び移植用神経網膜シート | |
WO2018143312A1 (ja) | 多能性幹細胞の分化制御方法 | |
US20230138022A1 (en) | Methods of making pluripotent stem cells and uses thereof | |
WO2019103125A1 (ja) | 神経系細胞又は神経組織と非神経上皮組織とを含む細胞塊の製造方法及びその細胞塊 | |
JPWO2018199142A1 (ja) | 神経堤細胞および交感神経細胞の製造方法 | |
CN113528441B (zh) | 快速高效的临床级色素上皮细胞诱导方法、试剂盒及应用 | |
CN109852587B (zh) | 一种人科凯恩氏综合征特异性成体干细胞的制备方法 | |
Ahmadiankia et al. | Effects of different extracellular matrices and co-cultures on human limbal stem cell expansion in vitro | |
WO2009146098A2 (en) | Stem cells and uses thereof | |
US20220135940A1 (en) | Method for producing kidney structure having dendritically branched collecting duct from pluripotent stem cells | |
Chichagova | Investigating retinal pathology in patients carrying m. 3243A> G mutation using human induced pluripotent stem cells | |
JP2016067243A (ja) | 多能性幹細胞由来心筋細胞の取得方法 | |
CN111440771A (zh) | 一种含有clcn2纯合突变的白质脑病患者特异诱导多能干细胞系 | |
Progenitor | AlphaMed Press |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 12774375 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 2013511044 Country of ref document: JP Kind code of ref document: A |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2012774375 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 14112572 Country of ref document: US |