WO2016153027A1 - Es細胞及びその他の幹細胞から、涙腺上皮細胞を誘導する方法 - Google Patents
Es細胞及びその他の幹細胞から、涙腺上皮細胞を誘導する方法 Download PDFInfo
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Definitions
- the present invention relates to a method for producing lacrimal gland epithelial cells from pluripotent stem cells, a lacrimal gland epithelial cell produced by the method, and a reagent kit for inducing differentiation from pluripotent stem cells to lacrimal gland epithelial cells.
- Dry eye also known as dry eye, causes tears quantitative and / or qualitative abnormalities due to reduced or impaired lacrimal secretion function of the lacrimal gland.
- the surface of the eyeball that is, the keratoconjunctiva It is a disease that causes damage.
- Known causes of decreased lacrimal secretion function and disorders of the lacrimal gland include VDT work (video screen terminal work), aging, diseases associated with inflammation in the lacrimal gland, such as Sjogren's syndrome Autoimmune abnormalities, dry keratoconjunctivitis, Steven-Jenson syndrome, eyelid margin inflammation and the like are known.
- a method for improving dry eye a method of instilling artificial tears is generally used, but it is only a symptomatic treatment and the effect is temporary, so 10-20 times a day It was cumbersome for the patient to apply.
- drugs such as steroids and cyclosporine are used to reduce the inflammatory response, but it is possible to improve the secretion of tears to a normal state. Can not.
- tissue stem cells adult stem cells
- stem cell transfer therapy capable of repairing the damaged lacrimal gland tissue and realizing the fundamental recovery of lacrimal gland function.
- tissue stem cells of the lacrimal gland that have the ability to form the lacrimal gland have not yet been isolated.
- Non-patent document 1 Nat. Methods 4, 227-230 (2007) from lacrimal gland epithelial cells and lacrimal gland mesenchymal cells derived from mouse fetuses.
- Non-patent document 2 Nature Communications 4, 2497 (2013), doi: 10.1038 / ncomms3497.
- ES cells embryonic stem cells
- iPS cells induced pluripotent stem cells; induced pluripotent stem cells; induced pluripotent
- a technique for inducing lacrimal gland from a cell source that can be applied clinically is necessary. Since it is such a technical situation, it has not yet been reported that it has been successfully induced from ES cells or iPS cells to lacrimal gland epithelial cells or lacrimal gland mesenchymal cells.
- An object of the present invention is to provide a method for producing lacrimal epithelial cells from pluripotent stem cells, a lacrimal epithelial cell produced by the method, and a reagent kit for inducing differentiation from pluripotent stem cells to lacrimal epithelial cells. It is in.
- the present inventors analyzed the expression of various mRNAs in eight types of cells including lacrimal gland epithelial cells using a microarray, and identified genes that are specifically highly expressed in lacrimal gland epithelial cells.
- the present inventors paid attention to two types of transcription factors (Six2, Foxc1) among those genes identified.
- the present inventors also paid attention to Six1, Six4, and Foxc2 as transcription factor genes that show the same expression tendency as the Six2 gene in the eight types of cells.
- the present inventors also focused on the Foxp1 gene, the Runx1 gene, and the ILF2 gene as transcription factor genes showing the same expression tendency as the Pax6 gene and the Foxc1 gene.
- the present inventors artificially synthesize each mRNA of these candidate genes using these nine genes of interest as candidate genes.
- Those mRNAs were introduced into human ES cells in various combinations, further cultured, and the expression of the marker gene group of lacrimal gland epithelial cells was confirmed.
- the mRNA of Pax6 gene and the mRNA of Foxc1 gene were introduced, and Pax6
- gene mRNA and Foxp1 gene mRNA were introduced, it was found that expression of marker gene groups of lacrimal epithelial cells was confirmed.
- the mRNA of the Pax6 gene and the mRNA of the Foxp1 gene were introduced, the increase in the expression of the Foxc1 gene was confirmed.
- the expression of the Pax6 gene and the Foxc1 gene should be increased in pluripotent stem cells.
- the present inventors have found that it is essential for inducing differentiation into lacrimal epithelial cells. It was also surprising to those skilled in the art that differentiation of pluripotent stem cells into lacrimal gland epithelial cells can be induced by increasing the expression of only two genes.
- the present inventors introduced the mRNA of the Six1 gene in addition to the mRNA of the Pax6 gene and the mRNA of the Foxc1 gene, the expression of the Barx2 gene, which is one of the lacrimal gland epithelial cell marker genes, is further improved. It has been found that the morphological change to cells becomes clearer and the direction of differentiation to the lacrimal epithelial cell line can be made clearer.
- the present inventors also determined whether the lacrimal gland epithelial cells produced by introducing mRNA of the above-mentioned specific two or three kinds of genes have the ability to regenerate the three-dimensional structure of the lacrimal gland organs. To confirm, the lacrimal gland epithelial cells were co-cultured with the lacrimal gland primordium of the mouse fetus, and a three-dimensional structure similar to the mature lacrimal gland was formed. From this, it was shown that the lacrimal epithelial cells produced by the method of the present invention have the ability to regenerate the three-dimensional structure of the lacrimal gland organ.
- a method for producing lacrimal gland epithelial cells comprising the following step A; Step A: A step of increasing Pax6 gene expression or introducing PAX6 protein and increasing Foxc1 gene or Foxp1 gene expression or introducing FOXC1 protein or FOXP1 protein in pluripotent stem cells.
- the step of increasing the expression of the Pax6 gene is a step of introducing a polynucleotide encoding the PAX6 protein into a pluripotent stem cell, and the step of increasing the expression of the Foxc1 gene is a pluripotent polynucleotide encoding the FOXC1 protein.
- the method of introducing into a stem cell wherein the step of increasing the expression of Foxp1 gene is a step of introducing a polynucleotide encoding FOXP1 protein into a pluripotent stem cell.
- Production method 3. 3. The method for producing lacrimal gland epithelial cells according to item 1 or 2, wherein the pluripotent stem cells of step A further increase the expression of Six1 gene or introduce SIX1 protein. 4). 4. The method for producing lacrimal gland epithelial cells according to item 3 above, wherein increasing the expression of the Six1 gene is introducing a polynucleotide encoding SIX1 protein into a pluripotent stem cell. 5. 5. 5.
- Reagent kit for inducing differentiation from pluripotent stem cells to lacrimal gland epithelial cells including the following (a) and (b): (A) a polynucleotide encoding PAX6 protein or PAX6 protein: (B) A polynucleotide or FOXC1 protein encoding a FOXC1 protein, or a polynucleotide or FOXP1 protein encoding a FOXP1 protein. 12 (C) The reagent kit for inducing differentiation according to item 11 above, further comprising (c) a polynucleotide encoding SIX1 protein or SIX1 protein.
- lacrimal gland epithelial cells and lacrimal gland mesenchymal cells which are the materials, from embryonic mammals.
- lacrimal gland epithelial cells can be efficiently produced in large quantities in a short period of time from pluripotent stem cells (for example, ES cells and iPS cells) that can be applied clinically.
- pluripotent stem cells for example, ES cells and iPS cells
- the lacrimal gland epithelial cells produced according to the present invention are lacrimal epithelial cells having the ability to regenerate the three-dimensional structure of the lacrimal gland organ.
- HES p-fc1 day2 represents the result of cells on the second day after the start of mRNA introduction
- hES p-fc1 day5 represents the result of cells on the fifth day after the start of mRNA introduction.
- hES ⁇ day0 represents the result of cells before mRNA introduction on the day of introduction of mRNA (1 day after the start of culture)
- “ hES DKSFM day6 ” is the same except that no mRNA was introduced.
- HES p-fp1 day2 represents the result of the cell on the second day after the start of the introduction of mRNA
- hES p-fp1 day5 represents the result of the cell on the fifth day after the start of the introduction of the mRNA.
- FIG. 4 shows the results of measuring the expression level of a lacrimal gland epithelial cell marker gene different from that shown in FIG. 3 in the same cells as in FIG.
- HES P / S / Fc1 day2 represents the results of cells on the second day after the start of mRNA introduction
- hES P / S / Fc1 day5 represents the results of cells on the fifth day after the start of mRNA introduction.
- FIG. 6 shows the results obtained by measuring the expression level of a lacrimal gland epithelial cell marker gene different from that shown in FIG. 5 in the same cells as in FIG.
- FIG. 6 is a result of measuring the expression level of a lacrimal gland epithelial cell marker gene different from that shown in FIGS. 5 and 6 in the same cells as in FIG.
- FIG. 1 The results of immunohistological staining of cells in the case where mRNA of specific three types of transcription factors (PAX6, FOXC1, SIX1) was introduced into human ES cells and then cultured in a culture medium for keratinocyte proliferation are shown.
- the upper left panel shows the morphology of the cells immediately before the introduction of mRNA
- the upper right panel shows the morphology of the cells of Day 1 (1 day after the start of the introduction of mRNA)
- the lower left panel of Day 2 (the second day after the start of the introduction of mRNA).
- the cell morphology is shown
- the lower right panel shows the cell morphology of Day 5 (5 days after the start of mRNA introduction).
- the result of having observed the cell mass obtained by three-dimensionally coculturing the lacrimal gland epithelial cell and the lacrimal gland primordium-derived cell in the present invention is shown.
- the upper left panel shows the result of observation of the cell mass as it is with an optical microscope
- the other panels show the result of observation of the frozen section of the cell mass with an optical microscope after staining with hematoxylin and eosin.
- Step A As a method for producing lacrimal gland epithelial cells of the present invention (hereinafter also referred to as “production method of the present invention”), expression of Pax6 gene is increased or PAX6 protein is introduced into Foxc1 in pluripotent stem cells.
- Step B is cultured in a growth medium.
- the “medium” means a state in which water is added to a “medium component” capable of culturing cells.
- Pax6 gene and “Foxc1 gene or Foxp1 gene”, preferably “Six1 gene” are also collectively referred to as “gene in the present invention”
- PAX6 protein and “FOXC1” “Protein or FOXP1 protein”, preferably “SIX1 protein” is also collectively referred to as “protein in the present invention”.
- Step A instead of Foxc1 gene or together with Foxc1 gene, Foxp1 gene Alternatively, the FOXP1 protein may be introduced instead of or together with the FOXC1 protein.
- PAX6 protein and FOXC1 protein are present in a pluripotent stem cell, more of both of the aforementioned proteins are converted into the pluripotent stem cell. It is considered that the pluripotent stem cells are induced to differentiate into lacrimal gland epithelial cells.
- step A in the pluripotent stem cell of step A, it is possible to further increase the expression of Six1 gene or introduce SIX1 protein.
- “increase the expression of Pax6 gene or introduce PAX6 protein” and “increase the expression of Foxc1 gene or Foxp1 gene or “Introducing FOXC1 protein or FOXP1 protein” also increases the expression of the Six1 gene, so it is not essential to the present invention to increase the expression of the Six1 gene or introduce the SIX1 protein, but the form into the lacrimal epithelial cells This is preferable in that the change can be made clearer and the direction of differentiation into the lacrimal epithelial cell line can be made clearer.
- the production method of the present invention is a method for producing lacrimal gland epithelial cells in vitro, and step A and step B can be performed in vitro.
- “increasing the expression of the gene in the present invention” means finally increasing the expression of the gene at the protein level.
- Increasing the expression of the gene transcription level (mRNA level) usually increases the expression at the protein level. Therefore, the increase in gene expression in the present invention may be confirmed at the protein level or at the transcription level. You may confirm by (mRNA level).
- the presence / absence or degree of increase in expression of the gene at the protein level in the present invention can be confirmed by a known method such as immunohistological staining.
- a known method such as immunohistological staining.
- the labeled antibody used for the immunohistological staining method a commercially available one may be used, or a known method may be used.
- the presence / absence or degree of increase in the transcription level of the gene in the present invention can be confirmed by a known method such as quantitative RT-PCR.
- a person skilled in the art can appropriately design and prepare a primer sequence used for quantitative RT-PCR based on sequence information of a target gene.
- accession number of the sequence information of human Pax6 is NM_000280.4
- accession number of the sequence information of human Foxc1 is NM_001453.2
- accession number of the sequence information of human Foxp1 is NM_032682.5
- human Six1 The accession number of the sequence information is NM_005982.3.
- SEQ ID NOs: 9 and 10 primer set for human Pax6
- SEQ ID NOs: 11 and 12 primer set for human Foxc1
- SEQ ID NO: 13 and Preferable examples include a primer set consisting of nucleotide sequences shown in No. 14 (primer set for human Foxp1) and SEQ ID NOs: 15 and 16 (primer set for human Six1).
- (A-1) a polynucleotide encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 2;
- (B-1) a polynucleotide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted and / or added in the amino acid sequence shown in SEQ ID NO: 2 and encoding a protein having PAX6 activity;
- (C-1) a polynucleotide comprising an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 2 and encoding a protein having PAX6 activity;
- (D-1) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1;
- (E-1) a polynucleotide comprising a nucleotide sequence in which one or several nucleotides are deleted, substituted and / or added in the nucleotide sequence represented by SEQ ID NO: 1, and encoding
- the above SEQ ID NO: 1 represents the nucleotide sequence of cDNA of the human Pax6 gene
- the above SEQ ID NO: 2 represents the amino acid sequence of the human PAX6 protein.
- Pax6 sequence data is already known in various vertebrates other than humans, and is disclosed in public data banks such as GenBank. For example, BC036957.1 for mice, BC128741.1 for rats, EF141016.1 for dogs, BC116038.1 for cattle, BC075551.1 for Xenopus, and Pax6 gene and PAX6 protein sequence data Is disclosed.
- (A-2) a polynucleotide encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 4;
- (B-2) a polynucleotide encoding a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted and / or added in the amino acid sequence shown in SEQ ID NO: 4 and having FOXC1 activity;
- (C-2) a polynucleotide comprising an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 4 and encoding a protein having FOXC1 activity;
- (D-2) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 3;
- (E-2) a polynucleotide comprising a nucleotide sequence in which one or several nucleotides are deleted, substituted and / or added in the nucleotide sequence represented by SEQ ID NO: 3 and en
- A-2) a protein comprising the amino acid sequence represented by SEQ ID NO: 4;
- B-2) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted and / or added in the amino acid sequence shown in SEQ ID NO: 4 and having FOXC1 activity;
- C-2) a protein comprising an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 4 and having FOXC1 activity;
- the protein is not particularly limited as long as it is any of the above proteins, and “the protein having FOXC1 activity” refers to the protein or the polynucleotide encoding the protein, the polynucleotide encoding the PAX6 protein, or the PAX6 protein.
- it means a protein capable of producing lacrimal epithelial cells when introduced into pluripotent stem cells and then cultured in a medium.
- the above SEQ ID NO: 3 represents the nucleotide sequence of the cDNA of the human Foxc1 gene
- the above SEQ ID NO: 4 represents the amino acid sequence of the human FOXC1 protein.
- the sequence data of Foxc1 is already known in various vertebrates other than humans, and is disclosed in public data banks such as GenBank. For example, NM_008592.2 for mice, NM_134338.1 for rats, NM_001088214.1 and NM_001096377.1 for Xenopus laevis, EU196406.1 for red tiger sharks, and Foxc1 gene and FOXC1 protein sequence data are disclosed. Yes.
- (A-3) a polynucleotide encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 6;
- (B-3) a polynucleotide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted and / or added in the amino acid sequence represented by SEQ ID NO: 6 and encoding a protein having FOXP1 activity;
- (C-3) a polynucleotide comprising an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 6 and encoding a protein having FOXP1 activity;
- (D-3) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 5;
- (E-3) a polynucleotide comprising a nucleotide sequence obtained by deleting, substituting and / or adding one or several nucleotides in the nucleotide sequence represented by SEQ ID NO: 5 and en
- the above SEQ ID NO: 5 represents the nucleotide sequence of the cDNA of the human Foxp1 gene
- the above SEQ ID NO: 6 represents the amino acid sequence of the human FOXP1 protein.
- the sequence data of Foxp1 is already known in various vertebrates other than humans and is disclosed in public data banks such as GenBank.
- the sequence data of Foxp1 gene and FOXP1 protein is disclosed in NM_053202.2 for mice, NM_001034131.1 for rats, and NM_001095533.1 for Xenopus.
- (A-4) a polynucleotide encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 8;
- (B-4) a polynucleotide comprising an amino acid sequence in which one or several amino acids are deleted, substituted and / or added in the amino acid sequence represented by SEQ ID NO: 8, and encoding a protein having SIX1 activity;
- (C-4) a polynucleotide comprising an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 8 and encoding a protein having SIX1 activity;
- (D-4) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 7;
- (E-4) a polynucleotide comprising a nucleotide sequence obtained by deleting, substituting and / or adding one or several nucleotides in the nucleotide sequence represented by SEQ ID NO: 7 and encoding a protein
- A-4) a protein comprising the amino acid sequence represented by SEQ ID NO: 8;
- B-4) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted and / or added in the amino acid sequence represented by SEQ ID NO: 8, and having SIX1 activity;
- C-4) a protein comprising an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 8 and having SIX1 activity; If it is any protein of these, it will not restrict
- a protein having SIX1 activity means a polynucleotide encoding the protein (P) or the protein (P), a polynucleotide encoding the PAX6 protein or a PAX6 protein, and a polynucleotide encoding the FOXC1 protein.
- “Stringent conditions” refers to conditions in which only specific hybridization occurs and non-specific hybridization does not occur. Such conditions are usually hybridization at 37 ° C. in a buffer containing 5 ⁇ SSC and 1% SDS and washing treatment at 37 ° C. with a buffer containing 1 ⁇ SSC and 0.1% SDS. Preferably, conditions such as hybridization at 42 ° C. in a buffer containing 5 ⁇ SSC and 1% SDS and washing treatment at 42 ° C. with a buffer containing 0.5 ⁇ SSC and 0.1% SDS, more preferably , Hybridization at 65 ° C. in a buffer containing 5 ⁇ SSC and 1% SDS, and washing treatment at 65 ° C.
- DNA obtained by using hybridization encodes an active polypeptide is determined by, for example, introducing the DNA into Escherichia coli or the like and expressing it, and the Escherichia coli or the like producing the target protein. You can find out if you can do it.
- DNA obtained by hybridization usually has high identity with each gene. High identity refers to 90% or more identity, preferably 95% or more identity, more preferably 98% or more identity.
- the above SEQ ID NO: 7 represents the nucleotide sequence of cDNA of the human Six1 gene
- the above SEQ ID NO: 8 represents the amino acid sequence of the human SIX1 protein.
- the sequence data of Six1 is already known in various vertebrates other than humans, and is disclosed in public data banks such as GenBank. For example, NM_009189.3 for mice, NM_053759.1 for rats, KF381338.1 for dogs, BC169929.1 for Xenopus, and sequence data of Six1 gene and SIX1 protein are disclosed.
- amino acid sequence in which one or several amino acids are deleted, substituted and / or added is, for example, in the range of 1 to 30, preferably in the range of 1 to 20, and more preferably in the range of 1 to 15.
- Is a deleted, substituted or added amino acid sequence, and the above-mentioned “nucleotide sequence in which one or several nucleotides are deleted, substituted and / or added” means, for example, 1 to 40 amino acid sequences.
- range preferably within the range of 1-30, more preferably within the range of 1-20, even more preferably within the range of 1-15, more preferably within the range of 1-10, more preferably 1 Within 5 ranges, and Mashiku within 1-3 range, more preferably the number of nucleotides in the 1-2 range deletions, means a substituted and / or added in the nucleotide sequence.
- a polynucleotide consisting of a nucleotide sequence in which one or several nucleotides are deleted, substituted and / or added is known to those skilled in the art such as chemical synthesis, genetic engineering techniques, mutagenesis, etc. It can also be produced by any method. Specifically, a method of contacting a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1, 3, 5 or 7 with a mutagen agent, a method of irradiating ultraviolet rays, a genetic engineering method, etc. In use, mutation polynucleotides can be obtained by introducing mutations into these polynucleotides.
- Site-directed mutagenesis which is one of the genetic engineering methods, is useful because it can introduce a specific mutation at a specific position.
- Molecular Cloning Alaboratory Mannual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY., 1989. (hereinafter abbreviated as “Molecular Cloning Second Edition”), Current Protocols in Molecular Biology, Supplement 1-38, John Wiley & Sons (1987-1997), etc. It can be carried out.
- Molecular Cloning Second Edition By expressing this mutant polynucleotide using an appropriate expression system, a protein having an amino acid sequence in which one or several amino acids are deleted, substituted and / or added can be obtained.
- nucleotide sequence having at least 80% identity with the nucleotide sequence shown in SEQ ID NO: 1, 3, 5 or 7 refers to the nucleotide sequence shown in SEQ ID NO: 1, 3, 5 or 7, respectively.
- the identity is not particularly limited as long as it is 80% or more, preferably 85% or more, more preferably 88% or more, still more preferably 90% or more, still more preferably 93% or more, particularly preferably 95%. As mentioned above, it contains that it is 98% or more most preferably.
- amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 2, 4, 6 or 8 refers to the amino acid sequence shown in SEQ ID NO: 2, 4, 6 or 8, respectively.
- the identity is not particularly limited as long as it is 80% or more, preferably 85% or more, more preferably 88% or more, still more preferably 90% or more, still more preferably 93% or more, particularly preferably 95%. As mentioned above, it contains that it is 98% or more most preferably.
- the method for obtaining and preparing the gene according to the present invention is not particularly limited, and the sequence information of the human gene according to the present invention (Pax6 gene: SEQ ID NO: 1; Foxc1 gene: SEQ ID NO: 3; Foxp1 gene: SEQ ID NO: 5; Six1 gene: SEQ ID NO: 7) and other oligonucleotides based on the known sequence information of those genes, synthesized with appropriate oligonucleotides as probes or primers, derived from vertebrate cells / tissues such as humans From the mRNA, cDNA or cDNA library, the cDNA of the gene of the present invention of vertebrates such as humans is cloned using the hybridization method or (RT-) PCR method (the method described in Molecular Cloning 2nd edition, etc.)
- the method for obtaining a gene in the present invention of vertebrates such as humans It can also be prepared by chemical synthesis according to a conventional method.
- the gene of the present invention can be obtained from a vertebrate of a different type from the vertebrate from which the gene of the present invention is derived by a hybridization method using the obtained gene of the present invention or a part of those genes as a probe. It can also be acquired.
- the method for obtaining and preparing the protein in the present invention is not particularly limited, and may be a naturally derived protein, a chemically synthesized protein, or a recombinant protein produced by a gene recombination technique.
- a naturally derived protein is obtained, the protein in the present invention can be obtained by appropriately combining protein isolation / purification methods from cells or tissues expressing such protein.
- a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted and / or added in the amino acid sequence shown in SEQ ID NO: 2, 4, 6 or 8, or shown in SEQ ID NO: 2, 4, 6 or 8
- the protein consisting of an amino acid sequence having 80% or more identity with the amino acid sequence to be identified is the sequence information (SEQ ID NO: 2, 4, 6, 8) of the human protein of the present invention, or the gene sequence information of the human present invention.
- sequence information SEQ ID NO: 2, 4, 6, 8
- Those skilled in the art can appropriately prepare or obtain (SEQ ID NOs: 1, 3, 5, 7) and sequence information of known proteins and genes of other biological species.
- human Pax6 SEQ ID NO: 1, 2), Foxc1 (SEQ ID NO: 3, 4), Foxp1 (SEQ ID NO: 5, 6) or Six1 (SEQ ID NO: 7, 8) sequence information or other known species
- appropriate oligonucleotides are synthesized as probes or primers, and hybridization or RT (RT) is performed from mRNA, cDNA or cDNA library derived from human vertebrate cells / tissues.
- a polynucleotide encoding the protein of the present invention of a vertebrate such as a human by cloning the cDNA of the gene of the present invention of a vertebrate such as a human by using the PCR method (the method described in Molecular Cloning 2nd edition, etc.) Can be obtained.
- a polynucleotide encoding the protein of the present invention can be obtained from a vertebrate of a different type from the vertebrate from which the polynucleotide is derived by a hybridization method using such a polynucleotide as a probe.
- the polynucleotide encoding the protein of the present invention thus obtained is incorporated into an expression vector, introduced into an appropriate host cell, cultured, and the recombinant protein is recovered from the cultured cell or its conditioned medium.
- the protein in the present invention derived from vertebrates such as humans can be obtained.
- the method for increasing the gene expression in the present invention in the pluripotent stem cell is not particularly limited, and the present invention is performed without introducing the polynucleotide encoding the protein in the present invention into the pluripotent stem cell.
- a method for increasing the expression of a gene in can be mentioned, but a method for introducing a polynucleotide encoding a protein in the present invention into a pluripotent stem cell can be preferably mentioned.
- Such a polynucleotide may be DNA or RNA, may be single-stranded or double-stranded, may be linear or circular, Mention may be made of genomic DNA, cDNA, mRNA or a polynucleotide containing them encoding the protein in the invention.
- sequence number 1, 3, 5, 7 is described as a DNA sequence, when the polynucleotide of this invention is RNA, T in the nucleotide sequence of the above-mentioned sequence number represents U.
- Examples of the polynucleotide encoding the protein in the present invention include, for example, “an expression vector containing a polynucleotide encoding the protein in the present invention” (hereinafter also referred to as “expression vector in the present invention”), and “in the present invention.
- “MRNA encoding a protein” (hereinafter also referred to as “mRNA in the present invention”) can be mentioned, and among these, “mRNA in the present invention” is preferred because it is safer in clinical application. be able to. Since "mRNA in the present invention” is directly translated from the mRNA into the protein in the present invention when introduced into pluripotent stem cells, the polynucleotide of the gene in the present invention having increased expression is pluripotent. There is no concern of being integrated into the stem cell genome.
- a poly A sequence is added to the 3 ′ end of any polynucleotide selected from the group consisting of the polynucleotides shown in the above (a-1) to (g-1). And polyribonucleotides.
- a poly A sequence is added to the 3 ′ end of any polynucleotide selected from the group consisting of the polynucleotides shown in the above (a-2) to (g-2). And polyribonucleotides.
- a polyA sequence is added to the 3 ′ end of any polynucleotide selected from the group consisting of the polynucleotides shown in the above (a-3) to (g-3). And polyribonucleotides.
- a polyA sequence is added to the 3 ′ end of any polynucleotide selected from the group consisting of the polynucleotides shown in the above (a-4) to (g-4). And polyribonucleotides.
- the number of nucleotides of the aforementioned poly A sequence is not particularly limited as long as the polyribonucleotide can be translated into a protein encoded by the polyribonucleotide when the polyribonucleotide is introduced into a pluripotent stem cell. Within a range of 50 to 250, preferably within a range of 50 to 250.
- the mRNA in the present invention preferably has a cap structure at the 5 ′ end from the viewpoint of obtaining higher translation efficiency in the introduced cell.
- An example of such a cap structure is preferably 7-methylguanosine.
- the mRNA in the present invention can be prepared by a conventional method based on the gene sequence information in the present invention. For example, it can be prepared by chemical synthesis or by in vitro transcription. In vitro transcription can be performed by a conventional method using, for example, the expression vector of the present invention and a commercially available kit for in vitro transcription reaction.
- the polynucleotide As a method for introducing a polynucleotide encoding the protein in the present invention (for example, “expression vector in the present invention”, “mRNA in the present invention”, etc.) into a pluripotent stem cell, the polynucleotide is introduced into the pluripotent stem cell.
- it can include lipofection method, liposome method, electroporation method, calcium phosphate coprecipitation method, DEAE (diethylaminoethyl) dextran method, microinjection method, gene gun method, etc.
- the lipofection method is preferable because the operation is simple and the introduction efficiency of the polynucleotide is high.
- the length of time for contacting the polynucleotide and the pluripotent stem cell is, for example, within a range of 0.5 to 4 hours, preferably 1 to Within 3.5 hours, more preferably within 1.5 to 3 hours.
- the expression vector in the present invention is a viral vector
- the vector can be collected and pluripotent stem cells can be infected with the vector by an appropriate method according to each viral vector.
- the expression of the gene of the present invention may be increased in pluripotent stem cells, or the protein of the present invention may be introduced into pluripotent stem cells. Further, among the genes in the present invention, expression of some types of genes may be increased in pluripotent stem cells, and proteins corresponding to the remaining types of genes may be introduced into pluripotent stem cells. Among the genes described above, the expression of some or all types of genes may be increased in the pluripotent stem cells, and a protein corresponding to the gene may be introduced into the pluripotent stem cells.
- the method for introducing the protein in the present invention into pluripotent stem cells is not particularly limited as long as it is a method capable of introducing the protein in the present invention into pluripotent stem cells.
- a method using a protein introduction reagent, a protein introduction domain examples thereof include a method using a PTD)-or cell membrane permeation peptide (CPP) -fusion protein, and a microinjection method.
- PTD protein introduction reagent
- CPP cell membrane permeation peptide
- Protein introduction reagents include cationic lipid-based BioPOTER Protein Delivery Reagent (Gene Therapy Systmes), Pro-Ject TM Protein Transfection Reagent (PIERCE) and ProVectin (IMGENEX), lipid-based GenomONE (Ishihara) using Profect-1 (Targeting Systems), Penetrain Peptide (Q biogene) and Chariot Kit (Active Motif) based on a membrane-permeable peptide, and HVJ envelope (inactivated Sendai virus) Etc.) are commercially available.
- the introduction can be carried out according to the protocol attached to these reagents, but the general procedure is as follows.
- the protein in the present invention is diluted in an appropriate solvent (for example, a buffer solution such as PBS, HEPES, etc.), an introduction reagent is added and incubated at room temperature for about 5 to 15 minutes to form a complex, which is then added to a serum-free medium. It can be added to the exchanged cells and incubated at 37 ° C. for 1 to several hours.
- an appropriate solvent for example, a buffer solution such as PBS, HEPES, etc.
- the above microinjection is a method in which a protein solution is put into a glass needle having a tip diameter of about 1 ⁇ m and puncture is introduced into a cell, and the protein can be reliably introduced into the cell.
- the degree of increasing the gene expression in the present invention is not particularly limited as long as lacrimal gland epithelial cells can be produced from pluripotent stem cells, but at the transcription level (mRNA level) and / or protein level. For example, 10 times or more (for example, 10 times or more and less than 50 times) compared to before increase, at least until a certain time point (preferably, within 5 days after the operation for increasing expression is performed on pluripotent stem cells) Can be reached at least once.
- the degree to which gene expression in the present invention is increased may be the same or different for each gene in the present invention, and the transcription level (mRNA level) and At the protein level, for example, Pax6 gene: Foxc1 gene is within the range of 1.0: 0.1 to 0.1: 1.0, preferably 1.0: 0.5 to 0.5: 1. Within the range of 0, more preferably within the range of 1: 0.8 to 0.8: 1.0.
- the degree of increase in expression when the expression of the Six1 gene is also increased is such that Pax6 gene: Six1 gene is within the range of 1.0: 0.1 to 0.1: 1.0, preferably 1.0.
- Foxp1 gene is 1.0: 0.1 to 0.1: 1.
- 0.0 preferably within the range of 1.0: 0.5 to 0.5: 1.0, more preferably within the range of 1.0: 0.8 to 0.8: 1.0. be able to.
- the amount of the protein of the present invention or the polynucleotide encoding the protein of the present invention introduced into the pluripotent stem cell is not particularly limited as long as lacrimal gland epithelial cells can be produced from the pluripotent stem cell.
- the amount of introduction can be within the range of, for example, 0.001 pg to 100 ng per protein in the present invention with respect to 1 cell of pluripotent stem cells.
- the amount of the polynucleotide encoding the protein in the present invention depends on whether the expression vector is included or not, and whether the polynucleotide is single-stranded or double-stranded.
- the polynucleotide encoding the protein in the present invention is mRNA
- the amount introduced is, for example, within a range of 0.1 pg to 10 ng per mRNA per type of pluripotent stem cell, preferably It can be in the range of 1 pg to 1 ng, more preferably in the range of 3 pg to 300 pg.
- the amount of protein introduced in the present invention may be the same or different for each type of protein in the present invention.
- PAX6 protein: FOXC1 protein is The weight ratio is in the range of 1.0: 0.1 to 0.1: 1.0, preferably in the range of 1.0: 0.5 to 0.5: 1.0, more preferably 1: 0. It can be mentioned that it is in the range of .8 to 0.8: 1.0.
- the introduction ratio when SIX1 protein is also introduced is such that the weight ratio of PAX6 protein: SIX1 protein is 1.0: 0.1 to 0.1: 1.0, preferably 1.0: 0.
- the introduction ratio in the case of introducing the FOXP1 protein instead of the FOXC1 protein or together with the FOXC1 protein is 1.0: 0.1 to 0.1: 1.0 by weight ratio of PAX6 protein: FOXP1 protein.
- the range of 1.0 preferably in the range of 1.0: 0.5 to 0.5: 1.0, more preferably in the range of 1: 0.8 to 0.8: 1.0. Can do.
- the amount of the polynucleotide encoding the protein in the present invention may be the same or different depending on the type of the polynucleotide.
- Pax6: Foxc1 is in the range of 1.0: 0.1 to 0.1: 1.0, preferably in the range of 1.0: 0.5 to 0.5: 1.0, more preferably 1 by weight ratio. : In the range of 0.8 to 0.8: 1.0.
- Pax6: Six1 is within a range of 1.0: 0.1 to 0.1: 1.0, preferably 1.0: 0.
- Pax6: Foxp1 is 1. Within the range of 0: 0.1 to 0.1: 1.0, preferably within the range of 1.0: 0.5 to 0.5: 1.0, more preferably 1: 0.8 to 0.8. : Within the range of 1.0.
- each polynucleotide or protein into the pluripotent stem cell may be simultaneous or sequential. Alternatively, some may be simultaneous and sequential with respect to the other.
- the order of introduction when the introduction is sequential is not particularly limited as long as lacrimal gland epithelial cells can be produced from pluripotent stem cells, [1] “Polynucleotide encoding PAX6 protein or PAX6 protein”: [2] “Polynucleotide encoding FOXC1 protein or FOXP1 protein, or FOXC1 protein or FOXP1 protein”: In addition, in the case of sequentially introducing [3] “polynucleotide encoding SIX1 protein or SIX1 protein”, or between [1] and [2] above and [2] above After, after [1], or simultaneously with [2].
- the interval between the introduction of [1] and the introduction of [2] is preferably within 1 to 24 hours, more preferably within 1 to 10 hours, and even more preferably within 1 to 5 hours.
- the interval between the introduction of the above [1] and the introduction of the above [3] is preferably within 1 to 24 hours, more preferably within 1 to 10 hours. More preferably within 5 hours.
- the interval between the introduction of the above [2] and the introduction of the above [3] is preferably within 1 to 24 hours, more preferably within 1 to 10 hours. Within 5 hours is more preferred, and within 1 to 3 hours is more preferred.
- the number of introductions of each polynucleotide or each protein into the pluripotent stem cell when the protein encoding the protein according to the present invention or the protein according to the present invention is introduced into the pluripotent stem cell is from the pluripotent stem cell to the lacrimal gland epithelium It is not particularly limited as long as cells can be produced, and may be once for each polynucleotide or protein, but twice for any or all of the introduced polynucleotide or any or all of the introduced protein. The number of times can be increased (preferably 2 to 4 times, more preferably 2 or 3 times, still more preferably 2 times).
- each polynucleotide or each protein twice or more, particularly when the introduction is mRNA or protein.
- the expression vector of the present invention when the mRNA of the present invention or the protein of the present invention is introduced, the protein generated from the mRNA or the introduced protein is released from the cell within about 24 hours from the introduction. Since it almost disappears, it may be preferable to introduce it more than once in order to produce lacrimal epithelial cells from pluripotent stem cells.
- each mRNA or protein of the present invention is more than twice (preferably 2 to 4 times, more preferably every 12 to 36 hours (preferably every 18 to 30 hours). 2 or 3 times, more preferably 2 times), an embodiment in which the same kind of mRNA or the same kind of protein is introduced can be preferably mentioned.
- a preferred embodiment when sequentially introducing the mRNA in the present invention or the protein in the present invention is, for example, within 1 to 10 hours (preferably from 1 to 10 hours) after the introduction of the “polynucleotide encoding PAX6 protein or PAX6 protein”.
- a step of introducing “a polynucleotide encoding FOXC1 protein or FOXP1 protein or FOXC1 protein or FOXP1 protein”, preferably further “polynucleotide encoding SIX1 protein or SIX1 protein” An embodiment in which the process is repeated again within 12 to 36 hours (preferably within 18 to 30 hours) can be given.
- step A in the case of increasing the expression of the gene in the present invention or introducing the protein in the present invention, medium components other than the components particularly required for the increasing method or the introducing method are particularly limited.
- a commercially available pluripotent stem cell growth medium can be used.
- pluripotent stem cell growth media include STEM FIT (registered trademark in Japan) (manufactured by Ajinomoto Co., Inc.), Essential 8 TM Medium (manufactured by Life Technologies), HyCell-STEM TM Media (manufactured by GE Healthcare), and the like.
- a ROCK (Rho-associated coiled-coil forming kinase / Rho-binding kinase) inhibitor such as Y27632 and / or a B18R protein
- a pluripotent stem cell growth medium can be preferably mentioned, ROCK inhibitors such as Y27632 and B18R protein can be added, or a pluripotent stem cell growth medium can be more preferably mentioned, More preferable examples include STEM FIT (Japan registered trademark) (manufactured by Ajinomoto Co., Inc.) to which Y27632 and B18R protein are added.
- the culture medium for pluripotent stem cells described in this paragraph and the culture medium of a preferred embodiment thereof increase the gene expression in the present invention or introduce the protein in the present invention in the above step A. It can also be suitably used when culturing pluripotent stem cells before.
- ROCK inhibitors such as Y27632 are known to have the activity of suppressing cell death during cell dispersion of pluripotent stem cells and the activity of maintaining the undifferentiated state of pluripotent stem cells for a longer time. It is often added to the medium for proliferating sex stem cells.
- concentration of the ROCK inhibitor such as Y27632 in the medium used in Step A is, for example, in the range of 0.1 to 1000 ⁇ M, preferably in the range of 1 to 100 ⁇ M, more preferably in the range of 5 to 20 ⁇ M. be able to.
- a commercially available ROCK inhibitor such as Y27632 can be used.
- Y27632 manufactured by Wako Pure Chemical Industries, Ltd.
- the B18R protein is a protein having an activity to strongly neutralize type I interferon, and is known to suppress cell death after introduction of a polynucleotide or protein.
- concentration of B18R protein in the medium used in Step A is, for example, in the range of 2.5 ng / mL to 25 ⁇ g / mL, preferably in the range of 25 ng / mL to 2.5 ⁇ g / mL, more preferably 0.125. Within the range of ⁇ 0.5 ⁇ g / mL.
- a commercially available B18R protein can be used, for example, those manufactured by Affymetrix Japan KK.
- a culture dish usually used for culturing pluripotent stem cells
- Preferred examples include culture dishes coated with an outer matrix.
- Examples of the extracellular matrix include laminin, collagen, fibronectin, or a combination thereof.
- the type of pluripotent stem cell used in the production method of the present invention is not particularly limited as long as it is a cell that can produce lacrimal gland epithelial cells by the production method of the present invention.
- ES cells, iPS cells, etc. are preferably mentioned.
- the biological species from which the pluripotent stem cells are derived is not particularly limited as long as it is a vertebrate, and examples of the vertebrate include mammals, birds, reptiles, amphibians, fish, etc. Preferred examples include mammals such as mice, rats, guinea pigs, rabbits, cats, dogs, horses, cows, monkeys, sheep, goats, and pigs, with human beings being particularly preferred.
- ES cells and iPS cells used in the production method of the present invention can be obtained from RIKEN Bioresource Center CELL BANK, JCRB Cell Bank, National Institute of Medical Science.
- ES cells and iPS cells may be prepared.
- the ES cell production method is not particularly limited, and a currently known production method may be used, or a newly developed method may be used.
- blastocyst of a fertilized egg of the target vertebrate It can be established by removing the inner cell mass and culturing the inner cell mass on a fibroblast feeder.
- the production method of iPS cells is not particularly limited, and currently known production methods (re-table 2009/075119, special table 2011-529329, special table 2011-529330, special table 2012-507258, special table 2013-501505, special table Table 2013-519371, special table 2013-54069) may be used, or a production method newly developed in the future may be used.
- the production method of the present invention preferably has a step B in which the cells obtained in the step A are cultured in a medium.
- a keratinocyte corneal epithelial cell
- lacrimal gland epithelial cells can be produced.
- the medium for keratinocyte proliferation means a medium that can proliferate and maintain keratinocytes, but for convenience includes any medium that can produce lacrimal epithelial cells when the cells obtained in step A are cultured.
- a keratinocyte growth medium a medium obtained by adding an additive factor to a keratinocyte basic medium capable of allowing keratinocytes to survive for a certain period can be preferably exemplified.
- EGF epidermal growth factor
- cholera toxin cholera toxin
- the concentration of the epidermal growth factor used is not particularly limited as long as lacrimal gland epithelial cells can be produced. For example, it is within the range of 0.1 to 1000 ng / mL, preferably within the range of 1 to 100 ng / mL, and more preferably 5 Within the range of ⁇ 20 ng / mL.
- the use concentration of the cholera toxin is not particularly limited as long as lacrimal gland epithelial cells can be produced, but it is preferably in the range of 1 ⁇ g / mL to 10 mg / mL, preferably in the range of 10 ⁇ g / mL to 1 mg / mL, and more preferably. Can be in the range of 50 ⁇ g / mL to 200 ⁇ g / mL.
- the upper limit of the calcium concentration of the keratinocyte growth medium in the present invention is preferably 0.15 mM or less, and more preferably 0.10 mM or less. .
- the calcium concentration may be 0 mM, but is preferably 0.03 mM or more.
- the above-mentioned keratinocyte basic medium means a medium capable of allowing keratinocytes to survive for a certain period of time.
- keratinocyte basal medium include one or two or more types of sugar (s), one or more types of inorganic salts (s), one or more types of amino acids (s), and 1 or 2 It is preferred to include one or more types of vitamin (s) and one or more other components.
- saccharide examples include monosaccharides such as glucose, mannose, fructose, and galactose, and disaccharides such as sucrose, maltose, and lactose.
- glucose is particularly preferable.
- two or more can be added in combination.
- the inorganic salts include calcium chloride, calcium nitrate, copper sulfate pentahydrate, iron (III) nitrate nonahydrate, iron (II) sulfate heptahydrate, magnesium chloride hexahydrate.
- examples include one or more inorganic salts (s) selected from sodium dihydrate, sodium selenite pentahydrate, and zinc sulfate heptahydrate, but the lacrimal gland from pluripotent stem cells Any inorganic salt or combination thereof can be used as long as it is a component that advantageously acts on the production of epithelial cells.
- amino acids include alanine, arginine, asparagine, aspartic acid, cystine, cysteine, glutamine, glycine, histidine, glutamic acid, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine.
- amino acid (s) selected from, tryptophan, tyrosine, valine, etc., preferably L-amino acids and their derivatives and their salts and their hydrates Can do.
- vitamins include one or two selected from biotin, choline, folic acid, inositol, niacin, pantothenic acid, pyridoxine, riboflavin, thiamine, vitamin B12, paraaminobenzoic acid (PABA), and ascorbic acid. Mention may be made of more than one type of vitamin (s) and derivatives of each of these components and their salts and their hydrates.
- buffers such as HEPES
- nucleic acids such as nucleotides, pyruvate, derivatives thereof and salts thereof, derivatives such as hydrates thereof, phenol red, and the like.
- Can preferably include ATP, UTP, GTP, CTP, preferably an equimolar mixture of these four, and the derivative of pyruvic acid can preferably include sodium pyruvate.
- the keratinocyte basal medium examples include DKSFM medium (defined keratinocyte serum-free medium) (manufactured by Life Technologies) and EpiLife (registered trademark in Japan). Among them, the DKSFM medium is preferably mentioned. Can do. Note that the calcium concentration of the DKSFM medium is 0.10 mM or less.
- the above-mentioned DKSFM medium contains 0.1 to 1000 ng / mL, preferably 1 to 100 ng / mL, more preferably 5 to Epidermal growth factor in the range of 20 ng / mL and cholera in the range of 1 ⁇ g / mL to 10 mg / mL, preferably in the range of 10 ⁇ g / mL to 1 mg / mL, more preferably in the range of 50 to 200 ⁇ g / mL
- a medium supplemented with toxins can be mentioned.
- the time to start culturing in a medium is not particularly limited as long as lacrimal gland epithelial cells can be produced.
- a medium particularly, a keratinocyte growth medium
- the start time of the above culture is given from another aspect, it can be mentioned within the range of 8 to 48 hours after the start of the first introduction of the gene or protein in the present invention in Step A. From the viewpoint of obtaining better production efficiency of cells, the range after 12 to 36 hours can be preferably mentioned.
- the period of culturing the cells in a medium is not particularly limited as long as lacrimal gland epithelial cells can be produced. In order to decrease, it can be in the range of 2 to 6 days, and preferably in the range of 2 to 3 days.
- “Lacrimal epithelial cells” produced in the present invention are those in which expression of one or more (preferably three or more) lacrimal epithelial cell marker genes is increased, and the lacrimal gland mesenchymal cells and lacrimal gland organs By cell that can be induced.
- the above lacrimal gland epithelial cell marker genes include FGF5 (fibroblast growth factor 5), LEFTY2 (left / right determinant 2), FGF10 (fibroblast growth factor 10), Barx2 (Barx homeobox 2), Krt15 (cytokeratin) 15), AQP5 (Aquaporin 5), LTF (Lactoferrin).
- a cell having “elevated lacrimal epithelial cell marker gene expression” refers to the mRNA or protein of the lacrimal epithelial cell marker gene in the cell before increasing the gene in the present invention or before introducing the protein in the present invention. It means a cell in which the expression level of mRNA or protein of the lacrimal gland epithelial cell marker gene is increased compared to the expression level.
- Non-Patent Document 1 the method described in Non-Patent Document 1 can be preferably exemplified.
- the lacrimal epithelial cells of the present invention are not particularly limited as long as they are lacrimal epithelial cells produced by the production method of the present invention.
- the description and preferred embodiments of the production method of the present invention are also applicable to the lacrimal epithelial cells of the present invention.
- the reagent kit for inducing differentiation from pluripotent stem cells to lacrimal gland epithelial cells in the present invention includes the following (a) and (b): As long as it is, it is not particularly limited.
- the reagent kit for inducing differentiation of the present invention is a use invention in which the following (a) and (b) are used for specific uses (reagent kit for inducing differentiation from pluripotent stem cells to lacrimal gland epithelial cells). It is not a mere combination invention of (a) and (b).
- A a polynucleotide encoding PAX6 protein or PAX6 protein:
- B A polynucleotide or FOXC1 protein encoding a FOXC1 protein, or a polynucleotide or FOXP1 protein encoding a FOXP1 protein.
- the differentiation-inducing reagent kit of the present invention preferably further comprises (c) a polynucleotide encoding SIX1 protein or SIX1 protein.
- Preferred examples of the polynucleotide in the reagent kit for inducing differentiation of the present invention include mRNA.
- Examples of the weight ratio of the polynucleotide or protein contained in the differentiation-inducing reagent kit of the present invention include the weight ratio of the aforementioned polynucleotide or protein introduced in the production method of the present invention.
- the expression pattern obtained by the analysis was the lacrimal gland epithelium and lacrimal gland mesenchyme derived from the lacrimal gland primordium, the Harder gland epithelium and Harder gland mesenchyme derived from the Harder gland primordia, fetal eyelid conjunctival epithelium and mesenchyme, adult lacrimal gland and adult Compared with the expression pattern of Harder's gland, we searched for genes that seem to be specific to lacrimal gland epithelial cells. The present inventors have found, among these genes, the Six2 gene and the Foxc1 gene as transcription factor genes that are particularly highly expressed in lacrimal gland epithelial cells.
- the inventors found the Six1 gene, Six4 gene and Foxc2 gene.
- the present inventors also focused on the Pax6 gene.
- the present inventors also paid attention to the Foxp1 gene, the Runx1 gene, and the ILF2 gene as transcription factor genes that show the same expression tendency as the Foxc1 gene.
- the present inventors as candidates for transcription factors that can induce differentiation of pluripotent stem cells into lacrimal gland epithelial cells, are nine types of genes, namely, the Six2 gene, the Foxc1 gene, the Six1 gene, the Six4 gene, the Foxc2 Gene, Pax6 gene, Foxp1 gene, Runx1 gene and ILF2 gene were found.
- Example 2 Search for transcription factors capable of inducing differentiation of pluripotent stem cells into lacrimal epithelial cells
- Nine candidate genes found in Example 1 to search for transcription factors capable of inducing differentiation of pluripotent stem cells into lacrimal epithelial cells (Six2 gene, Foxc1 gene, Six1 gene, Six4 gene, Foxc2 gene, Pax6 gene, Foxp1 gene, Runx1 gene, ILF2 gene) mRNA is prepared, and the mRNA is introduced into pluripotent stem cells to lacrimal gland epithelial cells It was confirmed whether differentiation could be induced.
- the sequence information of the above 9 types of candidate genes in humans was obtained from the NCBI website, and a primer set capable of amplifying each gene was prepared based on the sequence information.
- the accession number of the sequence information of human Pax6 is NM_000280.4
- the accession number of the sequence information of human Foxc1 is NM_001453.2
- the accession number of the sequence information of human Foxp1 is NM_032682
- the accession number of the sequence information of human Six1 is NM_005982.3.
- Table 9 below shows the nucleotide sequence numbers of primer sets that can amplify these four candidate genes among the nine candidate genes.
- PCR was performed using a human genome sequence as a template and each of the above primer sets, and DNA fragments of each target gene were amplified. After each amplified DNA fragment was incorporated into a pCR2-UTR-R1R2 plasmid vector, it was confirmed that the DNA fragment was of the target gene.
- the target gene was purified from each plasmid vector containing each gene and used as a template for in vitro transcription. To this template, GTP, ATP, Me-CTP, Pseudo UTP, and tail sequence were added to perform an in vitro transcription reaction (AMBION IV TKIT, manufactured by Life Technologies). Each of the artificial mRNAs generated by the in vitro transcription reaction was purified, and then the sequence was confirmed with a sequencer to obtain the mRNAs of the nine types of candidate genes described above.
- Human ES cells that are pluripotent stem cells were prepared. A culture medium was added to a culture dish (Corning) coated with laminin, and ES cells were seeded at 1 ⁇ 10 4 cells / cm 2 to start culture. As this medium, a medium in which 10 ⁇ M Y27632 (manufactured by Wako Pure Chemical Industries, Ltd.) was added to STEM FIT (Japan registered trademark) (manufactured by Ajinomoto Co., Inc.), which is a culture solution for pluripotent stem cells, was used.
- STEM FIT Japanese registered trademark
- the day after the start of culture (the first day after the start of culture), the medium was replaced with STEM FIT (Japan registered trademark) containing 0.25 ⁇ g / mL B18R protein (Affymetrix Japan KK). Then, mRNA of candidate genes, Lipofectamin (Japan registered trademark) 2000 (Life Technologies, Inc., Opti-MEM (Japan registered trademark) (Life Technologies, Inc.), respectively 1 ⁇ g / 3cm 2, 2 ⁇ L / 3cm 2, 200 ⁇ L / 3cm 2
- the candidate gene mRNA was introduced into the ES cells by adding to the medium, mixing, and continuing the culture for 2 hours (ie, the first introduction), and the number of cells per 1 cm 2 of the culture dish was about Since it was 10,000, the amount of the above candidate gene mRNA added to the medium is 33.33 pg / 1 cell.
- the same kind of candidate gene mRNA is added to the medium (1 ⁇ g / 3 cm 2 ) and mixed. By continuing the culture for 2 hours, the mRNA was introduced into the ES cell for the second time.
- candidate gene mRNAs when two or three candidate gene mRNAs are used as candidate gene mRNAs, three hours after the completion of the introduction of the first candidate gene mRNA (first introduction),
- the candidate gene mRNA (1 ⁇ g / 3 cm 2 ) ”or“ mRNA of the second and third candidate genes (1 ⁇ g / 3 cm 2 per one candidate gene mRNA) ” is added to the medium and mixed,
- “mRNA of the second candidate gene” or “mRNA of the second and third candidate genes” was introduced into ES cells.
- the same operation as the mRNA introduction operation performed on the first day of introduction (total of two introductions) is performed on the next day (the first day after the start of introduction, ie, The second day after the start of culture) was also repeated.
- the expression level of the marker gene in the cells before the introduction of mRNA on the day of introduction of mRNA (the first day after the start of culture), and the same except that no mRNA was introduced
- the expression level of the marker gene (“hES DKSFM day6") in the cells of day6 cultured by treatment with the above was also measured. Note that Pax6, Six1, Foxc1, Foxp1, FGF5, LEFTY2, FGF10, Barx2, Krt15, and AQP5 were used as the aforementioned lacrimal gland epithelial cell marker genes.
- the mRNA expression level of the lacrimal gland epithelial cell marker gene was determined by extracting total RNA from the cells, Takara SYBR (Japan registered trademark) Premix Ex Taq TM II (Takara Bio), Thermal Cycler Dice (Japan registered trademark) Real Time System (Takara Bio) was used to perform the real-time PCR method.
- one of the above 9 types of candidate gene mRNAs is introduced into each pluripotent stem cell, and then the above [Introduction of artificial mRNA]
- the cells were cultured in a keratinocyte growth medium according to the method described later.
- expression of the lacrimal gland epithelial cell marker gene was not increased. From this, it was shown that these candidate genes cannot induce differentiation into lacrimal gland epithelial cells even if the expression of only one kind is increased.
- the gene name described on the left is a candidate gene into which mRNA has been introduced on the first day of the introduction
- the gene name described on the right is the second time on the first day of introduction. It is a candidate gene into which mRNA has been introduced. The same operation as the introduction operation on the first introduction day was repeated on the next day after introduction (the first day after the start of introduction).
- the cells were cultured in a keratinocyte growth medium according to the above-mentioned method of [culture of cells after artificial mRNA introduction in keratinocyte growth medium]. .
- Cells were separated on the fifth day after the introduction of mRNA, and the expression of lacrimal gland epithelial cell marker protein was confirmed by immunohistological staining. As a result, the expression of each protein corresponding to the expression of mRNA of the lacrimal gland epithelial cell marker gene was confirmed.
- mRNA of the third transcription factor As the mRNA of the third transcription factor, mRNA such as Six1, Runx1, or ILF2 was used. According to the method of [Introduction of artificial mRNA into pluripotent stem cells], mRNAs of three types of transcription factors are introduced into ES cells, and then [keratinocyte proliferation of cells after introduction of artificial mRNA described above] In the culture medium for keratinocytes, the cells were cultured in the medium for keratinocyte proliferation. On the fifth day after the start of the introduction of mRNA, the cells were separated, and the expression level of the lacrimal epithelial cell marker gene was measured in accordance with the above-mentioned method [Confirmation of presence or absence of increased expression of lacrimal epithelial cell marker gene].
- lacrimal gland epithelial cell marker protein when mRNA of three specific transcription factors is introduced.
- the expression of lacrimal epithelial cell markers is increased in cells that have been introduced into ES cells after mRNA of specific three types of transcription factors (PAX6, FOXC1, and SIX1). As described above. Whether or not the lacrimal gland epithelial cell marker protein was actually expressed in the cells on the 5th day after the introduction of mRNA was confirmed by immunohistological staining.
- the lacrimal epithelial cell marker proteins to be confirmed were LTF (lactoferrin), KRT15 (cytokeratin 15), AQP5 (aquaporin 5), FGF10 (fibroblast growth factor 10), and BARX2. The results of such immunohistological staining are shown in FIG.
- Example 2 After introducing mRNA of specific two or three types of transcription factors into pluripotent stem cells, keratinization It was described that the expression of the lacrimal gland epithelial cell marker gene is increased by culturing in the cell growth medium. Results obtained by observing, using a phase-contrast microscope, the morphological changes of cells when culturing in a culture medium for keratinocyte proliferation after introducing mRNAs of three specific transcription factors (Pax6, Foxc1, and Six1) into pluripotent stem cells. Is shown in FIG. As can be seen from FIG.
- the ES cells have a morphology characteristic of lacrimal epithelial cells, ie, a rectangular shape extending elongated outward. It changed to the form of cells.
- mRNAs of specific three types of transcription factors are introduced into ES cells, and then in the above Example
- the cells were cultured in a keratinocyte growth medium according to the method of 2 [Culturing of cells after introduction of artificial mRNA in keratinocyte growth medium].
- Cells on day 5 after the start of mRNA introduction were isolated as lacrimal gland epithelial cells.
- both lacrimal gland epithelial cells and lacrimal gland mesenchymal cells are required for the formation of lacrimal gland organs.
- the lacrimal gland primordium of a 16.5-day-old mouse fetus was collected, and the cells of the lacrimal gland primordium were separated for each cell.
- these cells and the aforementioned lacrimal gland epithelial cells were three-dimensionally co-cultured, a three-dimensional cell cluster similar to the mature lacrimal gland (tear gland organ) was formed. The result of observing the cell mass with an optical microscope is shown in the upper left panel of FIG. 10.
- the frozen section of the cell mass is stained with hematoxylin and eosin, and the result of observation with an optical microscope is shown in the other panel of FIG.
- the cell mass produced by the above-described method was shown to have a three-dimensional structure similar to an acinus or duct that is a characteristic structure of the mature lacrimal gland. From this, it was shown that the lacrimal gland epithelial cells produced by the method of the present invention actually have the ability to regenerate the three-dimensional structure of the lacrimal gland organ.
- lacrimal gland epithelial cells can be efficiently produced in a short period of time from pluripotent stem cells (for example, ES cells and iPS cells) that can be clinically applied.
- the lacrimal gland epithelial cells produced according to the present invention are lacrimal epithelial cells having the ability to regenerate the three-dimensional structure of the lacrimal gland organ.
- lacrimal gland epithelial cells having the ability to regenerate the three-dimensional structure of the lacrimal gland organ.
- lacrimal gland organs The feasibility of regenerative medicine is further increased.
- the method for producing lacrimal epithelial cells in the present invention can be said to be a great step toward that possibility.
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Abstract
Description
本出願は、参照によりここに援用されるところの日本特願2015-062726号優先権を請求する。
すなわち、本発明は、以下の通りです。
1.以下の工程Aを有することを特徴とする、涙腺上皮細胞の製造方法;
工程A:多能性幹細胞において、Pax6遺伝子の発現を増加させるか又はPAX6タンパク質を導入し、かつ、Foxc1遺伝子若しくはFoxp1遺伝子の発現を増加させるか又はFOXC1タンパク質若しくはFOXP1タンパク質を導入する工程。
2.Pax6遺伝子の発現を増加させる工程が、PAX6タンパク質をコードするポリヌクレオチドを多能性幹細胞に導入する工程であり、Foxc1遺伝子の発現を増加させる工程が、FOXC1タンパク質をコードするポリヌクレオチドを多能性幹細胞に導入する工程であり、Foxp1遺伝子の発現を増加させる工程が、FOXP1タンパク質をコードするポリヌクレオチドを多能性幹細胞に導入する工程であることを特徴とする前項1に記載の涙腺上皮細胞の製造方法。
3.工程Aの多能性幹細胞において、さらに、Six1遺伝子の発現を増加させるか又はSIX1タンパク質を導入することを特徴とする前項1又は2に記載の涙腺上皮細胞の製造方法。
4.Six1遺伝子の発現を増加させることが、SIX1タンパク質をコードするポリヌクレオチドを多能性幹細胞に導入することであることを特徴とする前項3に記載の涙腺上皮細胞の製造方法。
5.さらに、前記工程Aで得られた細胞を角化細胞増殖用培地で培養する工程を含む前項1~4のいずれか1に記載の涙腺上皮細胞の製造方法。
6.角化細胞増殖用培地が、上皮成長因子及び/又はコレラトキシンを含有することを特徴とする前項5に記載の涙腺上皮細胞の製造方法。
7.角化細胞増殖用培地中のカルシウム濃度が0.15mM以下であることを特徴とする前項5又は6に記載の涙腺上皮細胞の製造方法。
8.ポリヌクレオチドがmRNAであることを特徴とする前項2~7のいずれか1に記載の涙腺上皮細胞の製造方法。
9.前項1~8のいずれか1に記載の涙腺上皮細胞の製造方法により製造される涙腺上皮細胞。
10.涙腺器官の立体構造を再生する能力を有する前項9に記載の涙腺上皮細胞。
11.以下の(a)及び(b)を含む、多能性幹細胞から涙腺上皮細胞への分化誘導用試薬キット:
(a)PAX6タンパク質をコードするポリヌクレオチド又はPAX6タンパク質:
(b)FOXC1タンパク質をコードするポリヌクレオチド若しくはFOXC1タンパク質、又は、FOXP1タンパク質をコードするポリヌクレオチド若しくはFOXP1タンパク質。
12.さらに、(c)SIX1タンパク質をコードするポリヌクレオチド又はSIX1タンパク質を含む、前項11に記載の分化誘導用試薬キット。
本発明の涙腺上皮細胞の製造方法(以下、「本発明の製造方法」とも表示する)としては、多能性幹細胞において、Pax6遺伝子の発現を増加させるか又はPAX6タンパク質を導入し、かつ、Foxc1遺伝子若しくはFoxp1遺伝子の発現を増加させるか又はFOXC1タンパク質若しくはFOXP1タンパク質を導入する工程Aを有し、さらに、必要に応じて、前記工程Aで得られた細胞を、角化細胞(角膜上皮細胞)増殖用培地で培養する工程Bを有する。
ここで「培地」とは、細胞を培養できる「培地成分」に水を添加した状態のものをいう。本明細書において、「Pax6遺伝子」、及び、「Foxc1遺伝子又はFoxp1遺伝子」、好ましくはさらに「Six1遺伝子」、をまとめて「本発明における遺伝子」とも表示し、「PAX6タンパク質」、及び、「FOXC1タンパク質又はFOXP1タンパク質」、好ましくはさらに「SIX1タンパク質」、をまとめて「本発明におけるタンパク質」とも表示する。
(a-1)配列番号2に示されるアミノ酸配列からなるタンパク質をコードするポリヌクレオチド;
(b-1)配列番号2に示されるアミノ酸配列において、1若しくは数個のアミノ酸が欠失、置換及び/又は付加されたアミノ酸配列からなり、かつPAX6活性を有するタンパク質をコードするポリヌクレオチド;
(c-1)配列番号2に示されるアミノ酸配列と少なくとも80%以上の同一性を有するアミノ酸配列からなり、かつPAX6活性を有するタンパク質をコードするポリヌクレオチド;
(d-1)配列番号1に示されるヌクレオチド配列からなるポリヌクレオチド;
(e-1)配列番号1に示されるヌクレオチド配列において、1若しくは数個のヌクレオチドが欠失、置換及び/又は付加されたヌクレオチド配列からなり、かつPAX6活性を有するタンパク質をコードするポリヌクレオチド;
(f-1)配列番号1に示されるヌクレオチド配列と少なくとも80%以上の同一性を有するヌクレオチド配列からなり、かつPAX6活性を有するタンパク質をコードするポリヌクレオチド;
(g-1)配列番号1に示されるヌクレオチド配列と相補的な塩基配列とストリンジェントな条件下でハイブリダイズし、かつPAX6活性を有するタンパク質をコードするポリヌクレオチド;
のいずれかのポリヌクレオチドからなるPax6遺伝子であれば特に制限されず、また、本発明におけるPAX6タンパク質としては、
(A-1)配列番号2に示されるアミノ酸配列からなるタンパク質;
(B-1)配列番号2に示されるアミノ酸配列において、1若しくは数個のアミノ酸が欠失、置換及び/又は付加されたアミノ酸配列からなり、かつPAX6活性を有するタンパク質;
(C-1)配列番号2に示されるアミノ酸配列と少なくとも80%以上の同一性を有するアミノ酸配列からなり、かつPAX6活性を有するタンパク質;
のいずれかのタンパク質であれば特に制限されず、ここで、「PAX6活性を有するタンパク質」とは、そのタンパク質、又はそのタンパク質をコードするポリヌクレオチドを、FOXC1タンパク質をコードするポリヌクレオチド、又はFOXP1タンパク質と共に多能性幹細胞に導入し、次いで、該細胞を培養した場合に、涙腺上皮細胞を製造し得るタンパク質を意味する。
(a-2)配列番号4に示されるアミノ酸配列からなるタンパク質をコードするポリヌクレオチド;
(b-2)配列番号4に示されるアミノ酸配列において、1若しくは数個のアミノ酸が欠失、置換及び/又は付加されたアミノ酸配列からなり、かつFOXC1活性を有するタンパク質をコードするポリヌクレオチド;
(c-2)配列番号4に示されるアミノ酸配列と少なくとも80%以上の同一性を有するアミノ酸配列からなり、かつFOXC1活性を有するタンパク質をコードするポリヌクレオチド;
(d-2)配列番号3に示されるヌクレオチド配列からなるポリヌクレオチド;
(e-2)配列番号3に示されるヌクレオチド配列において、1若しくは数個のヌクレオチドが欠失、置換及び/又は付加されたヌクレオチド配列からなり、かつFOXC1活性を有するタンパク質をコードするポリヌクレオチド;
(f-2)配列番号3に示されるヌクレオチド配列と少なくとも80%以上の同一性を有するヌクレオチド配列からなり、かつFOXC1活性を有するタンパク質をコードするポリヌクレオチド;
(g-2)配列番号3に示されるヌクレオチド配列と相補的な塩基配列とストリンジェントな条件下でハイブリダイズし、かつFOXC1活性を有するタンパク質をコードするポリヌクレオチド;
のいずれかのポリヌクレオチドからなるFoxc1遺伝子であれば特に制限されず、また、本発明におけるFOXC1タンパク質としては、
(A-2)配列番号4に示されるアミノ酸配列からなるタンパク質;
(B-2)配列番号4に示されるアミノ酸配列において、1若しくは数個のアミノ酸が欠失、置換及び/又は付加されたアミノ酸配列からなり、かつFOXC1活性を有するタンパク質;
(C-2)配列番号4に示されるアミノ酸配列と少なくとも80%以上の同一性を有するアミノ酸配列からなり、かつFOXC1活性を有するタンパク質;
のいずれかのタンパク質であれば特に制限されず、ここで、「FOXC1活性を有するタンパク質」とは、そのタンパク質、又はそのタンパク質をコードするポリヌクレオチドを、PAX6タンパク質をコードするポリヌクレオチド、又はPAX6タンパク質と共に多能性幹細胞に導入し、次いで、該細胞を培地で培養した場合に、涙腺上皮細胞を製造し得るタンパク質を意味する。
(a-3)配列番号6に示されるアミノ酸配列からなるタンパク質をコードするポリヌクレオチド;
(b-3)配列番号6に示されるアミノ酸配列において、1若しくは数個のアミノ酸が欠失、置換及び/又は付加されたアミノ酸配列からなり、かつFOXP1活性を有するタンパク質をコードするポリヌクレオチド;
(c-3)配列番号6に示されるアミノ酸配列と少なくとも80%以上の同一性を有するアミノ酸配列からなり、かつFOXP1活性を有するタンパク質をコードするポリヌクレオチド;
(d-3)配列番号5に示されるヌクレオチド配列からなるポリヌクレオチド;
(e-3)配列番号5に示されるヌクレオチド配列において、1若しくは数個のヌクレオチドが欠失、置換及び/又は付加されたヌクレオチド配列からなり、かつFOXP1活性を有するタンパク質をコードするポリヌクレオチド;
(f-3)配列番号5に示されるヌクレオチド配列と少なくとも80%以上の同一性を有するヌクレオチド配列からなり、かつFOXP1活性を有するタンパク質をコードするポリヌクレオチド;
(g-3)配列番号5に示されるヌクレオチド配列と相補的な塩基配列とストリンジェントな条件下でハイブリダイズし、かつFOXP1活性を有するタンパク質をコードするポリヌクレオチド;
のいずれかのポリヌクレオチドからなるFoxp1遺伝子であれば特に制限されず、また、本発明におけるFOXP1タンパク質としては、
(A-3)配列番号6に示されるアミノ酸配列からなるタンパク質;
(B-3)配列番号6に示されるアミノ酸配列において、1若しくは数個のアミノ酸が欠失、置換及び/又は付加されたアミノ酸配列からなり、かつFOXP1活性を有するタンパク質;
(C-3)配列番号6に示されるアミノ酸配列と少なくとも80%以上の同一性を有するアミノ酸配列からなり、かつFOXP1活性を有するタンパク質;
のいずれかのタンパク質であれば特に制限されず、ここで、「FOXP1活性を有するタンパク質」とは、そのタンパク質、又はそのタンパク質をコードするポリヌクレオチドを、PAX6タンパク質をコードするポリヌクレオチド、又はPAX6タンパク質と共に多能性幹細胞に導入し、次いで、該細胞を培地で培養した場合に、涙腺上皮細胞を製造し得るタンパク質を意味する。
(a-4)配列番号8に示されるアミノ酸配列からなるタンパク質をコードするポリヌクレオチド;
(b-4)配列番号8に示されるアミノ酸配列において、1若しくは数個のアミノ酸が欠失、置換及び/又は付加されたアミノ酸配列からなり、かつSIX1活性を有するタンパク質をコードするポリヌクレオチド;
(c-4)配列番号8に示されるアミノ酸配列と少なくとも80%以上の同一性を有するアミノ酸配列からなり、かつSIX1活性を有するタンパク質をコードするポリヌクレオチド;
(d-4)配列番号7に示されるヌクレオチド配列からなるポリヌクレオチド;
(e-4)配列番号7に示されるヌクレオチド配列において、1若しくは数個のヌクレオチドが欠失、置換及び/又は付加されたヌクレオチド配列からなり、かつSIX1活性を有するタンパク質をコードするポリヌクレオチド;
(f-4)配列番号7に示されるヌクレオチド配列と少なくとも80%以上の同一性を有するヌクレオチド配列からなり、かつSIX1活性を有するタンパク質をコードするポリヌクレオチド;
(g-4)配列番号7に示されるヌクレオチド配列と相補的な塩基配列とストリンジェントな条件下でハイブリダイズし、かつSIX1活性を有するタンパク質をコードするポリヌクレオチド;
のいずれかのポリヌクレオチドからなるSix1遺伝子であれば特に制限されず、また、本発明におけるSIX1タンパク質としては、
(A-4)配列番号8に示されるアミノ酸配列からなるタンパク質;
(B-4)配列番号8に示されるアミノ酸配列において、1若しくは数個のアミノ酸が欠失、置換及び/又は付加されたアミノ酸配列からなり、かつSIX1活性を有するタンパク質;
(C-4)配列番号8に示されるアミノ酸配列と少なくとも80%以上の同一性を有するアミノ酸配列からなり、かつSIX1活性を有するタンパク質;
のいずれかのタンパク質であれば特に制限されない。
ここで、「SIX1活性を有するタンパク質」とは、そのタンパク質(P)又はそのタンパク質(P)をコードするポリヌクレオチドを、PAX6タンパク質をコードするポリヌクレオチド又はPAX6タンパク質と、FOXC1タンパク質をコードするポリヌクレオチド又はFOXC1タンパク質と共に多能性幹細胞に導入し、次いで、該細胞を培地で培養した場合(ケースA)と、PAX6タンパク質をコードするポリヌクレオチド又はPAX6タンパク質と、FOXC1タンパク質をコードするポリヌクレオチド又はFOXC1タンパク質と共に多能性幹細胞に導入し、次いで、該細胞を培地で培養した場合(ケースB)とを比較した際に、ケースAの方がBarx2遺伝子のmRNAレベルの発現量が高く、かつ、涙腺上皮細胞への形態変化がより明確であるタンパク質(P)を意味する。
本発明におけるmRNAは、本発明における遺伝子の配列情報に基づいて、常法により作製することができる。例えば化学合成により作製することもできるし、インビトロ転写により作製することもできる。インビトロ転写は、例えば本発明における発現ベクターと、インビトロ転写反応用の市販のキットとを用いて、常法により行うことができる。
[1]「PAX6タンパク質をコードするポリヌクレオチド、又は、PAX6タンパク質」:
[2]「FOXC1タンパク質若しくはFOXP1タンパク質をコードするポリヌクレオチド、又は、FOXC1タンパク質若しくはFOXP1タンパク質」:
の順であることが好ましく、またさらに
[3]「SIX1タンパク質をコードするポリヌクレオチド、又は、SIX1タンパク質」を逐次導入する場合は、上記[1]と[2]の間や、上記[2]の後や、上記[1]と同時や、上記[2]と同時であることを挙げることができる。
角化細胞増殖用培地とは、角化細胞を増殖及び維持できる培地を意味するが、便宜上、工程Aで得られた細胞を培養した場合に涙腺上皮細胞を製造し得るいかなる培地をも含む。かかる角化細胞増殖用培地としては、角化細胞を一定期間生存させ得る角化細胞基本培地に、添加因子が添加された培地を好ましく挙げることができる。かかる添加因子としては、上皮成長因子(EGF)、コレラトキシン等を好ましく挙げることができる。上皮成長因子やコレラトキシンは市販のものを用いることができ、例えばそれぞれ、Peprotech社製、List biological社製のものを用いることができる。上記上皮成長因子の使用濃度としては、涙腺上皮細胞を製造し得る限り特に制限されないが、例えば0.1~1000ng/mLの範囲内、好ましくは1~100ng/mLの範囲内、より好ましくは5~20ng/mLの範囲内を挙げることができる。また、上記コレラトキシンの使用濃度としては、涙腺上皮細胞を製造し得る限り特に制限されないが、1μg/mL~10mg/mLの範囲内、好ましくは10μg/mL~1mg/mLの範囲内、より好ましくは50μg/mL~200μg/mLの範囲内を挙げることができる。
本発明の涙腺上皮細胞としては、本発明の製造方法により製造される涙腺上皮細胞である限り特に制限されない。本発明の製造方法における記載や好適な態様は、本発明の涙腺上皮細胞においても適用される。
本発明における多能性幹細胞から涙腺上皮細胞への分化誘導用試薬キット(以下、「本発明の分化誘導用試薬キット」とも表示する。)としては、以下の(a)及び(b)を含んでいる限り特に制限されない。本発明の分化誘導用試薬キットは、以下の(a)及び(b)を、特定の用途(多能性幹細胞から涙腺上皮細胞への分化誘導用の試薬キット)に用いる用途発明であり、以下の(a)及び(b)の単なる組合せの発明ではない。
(a)PAX6タンパク質をコードするポリヌクレオチド又はPAX6タンパク質:
(b)FOXC1タンパク質をコードするポリヌクレオチド若しくはFOXC1タンパク質、又は、FOXP1タンパク質をコードするポリヌクレオチド若しくはFOXP1タンパク質。
本発明の分化誘導用試薬キットにおけるポリヌクレオチドとしてはmRNAを好ましく挙げることができる。
本発明の分化誘導用試薬キットに含まれるポリヌクレオチドや、タンパク質の重量比としては、本発明の製造方法における前述のポリヌクレオチドやタンパク質の導入量の重量比を挙げることができる。
幹細胞から涙腺上皮細胞への分化誘導に関連する因子を特定するために、マウスの涙腺原基から分離した涙腺上皮細胞における遺伝子発現を、市販のDNAマイクロアレイにより網羅的に解析した。解析により得られたその発現様式を、涙腺原基由来の涙腺上皮と涙腺間葉、ハーダー腺原基由来のハーダー腺上皮とハーダー腺間葉、胎生期眼瞼結膜上皮と間葉、成体涙腺と成体ハーダー腺の発現様式と比較し、涙腺上皮細胞に特異的と思われる遺伝子を探索した。本発明者らは、これらの遺伝子の中から、涙腺上皮細胞で特に高発現している転写因子遺伝子として、Six2遺伝子とFoxc1遺伝子を見いだした。
多能性幹細胞を涙腺上皮細胞へ分化誘導し得る転写因子を探索するために、実施例1で見いだされた9種類の候補遺伝子(Six2遺伝子、Foxc1遺伝子、Six1遺伝子、Six4遺伝子、Foxc2遺伝子、Pax6遺伝子、Foxp1遺伝子、Runx1遺伝子、ILF2遺伝子)のmRNAを作製し、そのmRNAを多能性幹細胞に導入して、涙腺上皮細胞へ分化誘導できるかどうかを確認した。
ヒトにおける上記の9種類の候補遺伝子の配列情報を、NCBIのウェブサイトから入手し、それぞれの配列情報に基づいて、それぞれの遺伝子を増幅し得るプライマーセットを作製した。なお、候補遺伝子のうち、ヒトPax6の配列情報のアクセッション番号はNM_000280.4であり、ヒトFoxc1の配列情報のアクセッション番号はNM_001453.2であり、ヒトFoxp1の配列情報のアクセッション番号はNM_032682.5であり、ヒトSix1の配列情報のアクセッション番号はNM_005982.3である。また、9種の候補遺伝子のうち、これら4種の候補遺伝子を増幅し得るプライマーセットのヌクレオチド配列の配列番号を以下の表1に示す。
多能性幹細胞であるヒトES細胞を用意した。ラミニンでコーティングした培養ディッシュ(Corning社製)に培地を添加し、ES細胞を1×104cells/cm2となるように播種して培養を開始した。この培地としては、多能性幹細胞用培養液であるSTEM FIT(日本登録商標)(味の素社製)に、10μMのY27632(和光純薬工業社製)を添加した培地を用いた。培養開始の翌日(培養開始後1日目)に、0.25μg/mLのB18Rタンパク質(アフィメトリクス・ジャパン株式会社製)を含むSTEM FIT(日本登録商標)へ培地を交換した。その後、候補遺伝子のmRNA、Lipofectamin(日本登録商標)2000(Life Technologies社製、Opti-MEM(日本登録商標)(Life Technologies社製)がそれぞれ1μg/3cm2、2μL/3cm2、200μL/3cm2となるように培地へ添加、混合し、2時間培養を続けることで、候補遺伝子のmRNAをES細胞に導入した(すなわち、1回目の導入)。なお、培養ディッシュ1cm2当たりの細胞数は約1万であったため、前述の候補遺伝子のmRNAの培地への添加量は33.33pg/1cellとなる。
mRNAの導入開始後2日目(培養開始後3日目)に、DKSFM培地(Life Technologies社製)に10ng/mLの上皮成長因子(EGF)(Peprotech社製)及び100μg/mLのコレラトキシン(Listbiological社製)を添加した培地へ、培地を交換し、培養を継続した。
候補遺伝子のmRNAを導入した後、上記の[人工mRNA導入後の細胞の、角化細胞増殖用培地での培養]の方法にしたがって、角化細胞増殖用培地で培養した。mRNAの導入開始後2日目(day2)(培養開始後3日目)又は5日目(day5)(培養開始後6日目)に細胞を分離し、かかる細胞内で涙腺上皮細胞マーカー遺伝子の発現が増加しているかをmRNAレベル(転写レベル)で確認した。また、陰性対照として、mRNAの導入日(培養開始後1日目)の、mRNA導入前の細胞における前記マーカー遺伝子の発現レベル(「hES day0」)や、mRNAを導入しなかったこと以外は同様に処理して培養したday6の細胞における前記マーカー遺伝子の発現レベル(「hES DKSFM day6」)も測定した。なお、前述の涙腺上皮細胞マーカー遺伝子としては、Pax6、Six1、Foxc1、Foxp1、FGF5、LEFTY2、FGF10、Barx2、Krt15、AQP5を用いた。
前述の実験により、多能性幹細胞に導入することによって、涙腺上皮細胞へ分化誘導させるのに必須の転写因子は、PAX6とFOXC1であることが示されたが、その2種の転写因子のmRNAに加えて、別のもう1種(3種目)の転写因子のmRNAを導入した場合に、よりよい結果が得られる転写因子があるかどうかを探索した。
特定の3種類の転写因子(PAX6、FOXC1、SIX1)のmRNAをES細胞に導入し、次いで、角化細胞増殖用培地で培養した細胞において、涙腺上皮細胞マーカーの発現が増加していることは先に述べたとおりである。mRNAの導入開始後5日目の細胞において涙腺上皮細胞マーカータンパク質が実際に発現しているかを免疫組織学的染色法により確認した。確認する涙腺上皮細胞マーカータンパク質としては、LTF(ラクトフェリン)、KRT15(サイトケラチン15)、AQP5(アクアポリン5)、FGF10(線維芽細胞増殖因子10)、BARX2を対象とした。かかる免疫組織学的染色の結果を図8に示す。
実施例2において、特定の2種又は3種の転写因子のmRNAを多能性幹細胞に導入した後、角化細胞増殖用培地で培養することにより、涙腺上皮細胞マーカー遺伝子の発現が増加することを述べた。特定の3種の転写因子(Pax6、Foxc1、Six1)のmRNAを多能性幹細胞に導入した後、角化細胞増殖用培地で培養した場合の細胞の形態変化を、位相差顕微鏡により観察した結果を図9に示す。図9から分かるように、mRNAの導入開始後5日目(Day5)(培養開始後6日目)に、ES細胞は、涙腺上皮細胞に特徴的な形態、すなわち、細長く外側に向かって伸びる長方形の形態の細胞へと変化を起こした。
転写因子のmRNAを導入することにより誘導した涙腺上皮細胞が、涙腺器官の立体構造を再生する能力を実際に有しているかどうかを確認することとした。
Claims (12)
- 以下の工程Aを有することを特徴とする、涙腺上皮細胞の製造方法;
工程A:多能性幹細胞において、Pax6遺伝子の発現を増加させるか又はPAX6タンパク質を導入し、かつ、Foxc1遺伝子若しくはFoxp1遺伝子の発現を増加させるか又はFOXC1タンパク質若しくはFOXP1タンパク質を導入する工程。
- Pax6遺伝子の発現を増加させる工程が、PAX6タンパク質をコードするポリヌクレオチドを多能性幹細胞に導入する工程であり、Foxc1遺伝子の発現を増加させる工程が、FOXC1タンパク質をコードするポリヌクレオチドを多能性幹細胞に導入する工程であり、Foxp1遺伝子の発現を増加させる工程が、FOXP1タンパク質をコードするポリヌクレオチドを多能性幹細胞に導入する工程であることを特徴とする請求項1に記載の涙腺上皮細胞の製造方法。
- 工程Aの多能性幹細胞において、さらに、Six1遺伝子の発現を増加させるか又はSIX1タンパク質を導入することを特徴とする請求項1又は2に記載の涙腺上皮細胞の製造方法。
- Six1遺伝子の発現を増加させることが、SIX1タンパク質をコードするポリヌクレオチドを多能性幹細胞に導入することであることを特徴とする請求項3に記載の涙腺上皮細胞の製造方法。
- さらに、前記工程Aで得られた細胞を角化細胞増殖用培地で培養する工程を含む請求項1~4のいずれか1に記載の涙腺上皮細胞の製造方法。
- 角化細胞増殖用培地が、上皮成長因子及び/又はコレラトキシンを含有することを特徴とする請求項5に記載の涙腺上皮細胞の製造方法。
- 角化細胞増殖用培地中のカルシウム濃度が0.15mM以下であることを特徴とする請求項5又は6に記載の涙腺上皮細胞の製造方法。
- ポリヌクレオチドがmRNAであることを特徴とする請求項2~7のいずれか1に記載の涙腺上皮細胞の製造方法。
- 請求項1~8のいずれか1に記載の涙腺上皮細胞の製造方法により製造される涙腺上皮細胞。
- 涙腺器官の立体構造を再生する能力を有する請求項9に記載の涙腺上皮細胞。
- 以下の(a)及び(b)を含む、多能性幹細胞から涙腺上皮細胞への分化誘導用試薬キット:
(a)PAX6タンパク質をコードするポリヌクレオチド又はPAX6タンパク質:
(b)FOXC1タンパク質をコードするポリヌクレオチド若しくはFOXC1タンパク質、又は、FOXP1タンパク質をコードするポリヌクレオチド若しくはFOXP1タンパク質。
- さらに、(c)SIX1タンパク質をコードするポリヌクレオチド又はSIX1タンパク質を含む、請求項11に記載の分化誘導用試薬キット。
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WO2019189640A1 (ja) * | 2018-03-28 | 2019-10-03 | 国立大学法人大阪大学 | 幹細胞由来涙腺組織の作製方法 |
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Cited By (3)
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WO2019189640A1 (ja) * | 2018-03-28 | 2019-10-03 | 国立大学法人大阪大学 | 幹細胞由来涙腺組織の作製方法 |
JPWO2019189640A1 (ja) * | 2018-03-28 | 2021-02-25 | 国立大学法人大阪大学 | 幹細胞由来涙腺組織の作製方法 |
JP7016185B2 (ja) | 2018-03-28 | 2022-02-04 | 国立大学法人大阪大学 | 幹細胞由来涙腺組織の作製方法 |
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US10392602B2 (en) | 2019-08-27 |
JP6749595B2 (ja) | 2020-09-02 |
US20180230425A1 (en) | 2018-08-16 |
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