WO2013176249A1 - 膵臓ホルモン産生細胞の生産方法及び膵臓ホルモン産生細胞、並びに分化誘導促進剤 - Google Patents
膵臓ホルモン産生細胞の生産方法及び膵臓ホルモン産生細胞、並びに分化誘導促進剤 Download PDFInfo
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Definitions
- the present invention relates to pluripotent stem cells such as induced pluripotent stem cells (hereinafter also referred to as “iPS cells”) and embryonic stem cells (hereinafter also referred to as “ES cells”), or pancreatic tissue stem / progenitor cells.
- iPS cells induced pluripotent stem cells
- ES cells embryonic stem cells
- pancreatic tissue stem / progenitor cells pancreatic tissue stem / progenitor cells.
- the present invention relates to a method for producing pancreatic hormone-producing cells for producing pancreatic hormone-producing cells, the produced pancreatic hormone-producing cells, and a differentiation induction promoter used in the production method.
- the pancreas has endocrine cells and exocrine cells, and is an organ that plays an important role in both endocrine and exocrine.
- Endocrine cells play a role in producing and secreting pancreatic hormones.
- Glucagon is secreted from ⁇ cells
- insulin is secreted from ⁇ cells
- somatostatin is secreted from ⁇ cells
- pancreatic polypeptide is secreted from PP cells. It has been.
- insulin has a blood glucose lowering effect and plays an important role in maintaining blood glucose at the correct concentration.
- pancreatic hormone-producing cells In recent years, many methods for inducing differentiation from pluripotent stem cells or pancreatic tissue stem / progenitor cells into pancreatic hormone-producing cells have been reported (see Non-Patent Documents 1 to 4, Patent Documents 1 to 6, etc.). If pancreatic hormone-producing cells can be obtained efficiently by such a differentiation induction method, it is expected to lead to a method for treating diabetes that is an alternative to islet transplantation. Furthermore, it is considered that the problem of rejection can be solved by obtaining pancreatic hormone-producing cells from pluripotent stem cells or pancreatic tissue stem / progenitor cells derived from the patient himself.
- Non-Patent Document 1 the efficiency of inducing differentiation into insulin-producing cells in Non-Patent Document 1 is about 12%. Therefore, a differentiation induction method capable of highly efficiently inducing differentiation from pluripotent stem cells or pancreatic tissue stem / progenitor cells to pancreatic hormone-producing cells is desired.
- Non-patent Document 3 differentiation is induced into insulin-producing cells by introducing and culturing the pdx1 gene into mouse ES cells. From the viewpoint of safety, a differentiation induction method that does not involve gene introduction is used. Preferably there is.
- the present invention has been made in view of such conventional circumstances, and can produce pancreatic hormones that can be efficiently induced to differentiate from pluripotent stem cells or pancreatic tissue stem / progenitor cells into pancreatic hormone-producing cells. It aims at providing the production method (differentiation induction method) of a cell, the produced pancreatic hormone production cell, and the differentiation induction promoter used for the production method.
- the inventors of the present invention have made extensive studies to solve the above problems.
- a polypeptide known as human TM4SF20 or a culture supernatant of a cell in which a DNA encoding human TM4SF20 is incorporated as a foreign gene is added to the culture medium, so that the pancreas is transformed from a pluripotent stem cell or pancreatic tissue stem / progenitor cell
- differentiation can be efficiently induced into hormone-producing cells.
- the present invention has been completed based on such findings, and more specifically is as follows.
- a method for producing pancreatic hormone-producing cells which produces pancreatic hormone-producing cells from pluripotent stem cells or pancreatic tissue stem / progenitor cells, comprising: In the process of inducing differentiation from pluripotent stem cells or pancreatic tissue stem / progenitor cells to pancreatic hormone producing cells, adding at least one differentiation induction promoter selected from the following (1) to (3) to the medium:
- a method for producing pancreatic hormone-producing cells (1) a polypeptide comprising an amino acid sequence encoded by DNA comprising the base sequence set forth in SEQ ID NO: 1, (2) A pancreatic hormone-producing cell comprising an amino acid sequence in which one or several amino acids are substituted, deleted, and / or added in the amino acid sequence encoded by the DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1.
- a polypeptide having an action of inducing differentiation into (3) A culture supernatant of a cell in which a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 or a DNA hybridizing with a DNA complementary to this DNA under stringent conditions is incorporated as a foreign gene.
- (A2) at least selected from the group consisting of a growth factor belonging to the TGF- ⁇ superfamily, a growth factor belonging to the Wnt (wingless MMTV integration site) family, and a GSK-3 (glycogen synthase kinase-3) inhibitor Culturing pluripotent stem cells in the presence of one factor; (B2) culturing the cells obtained in the above step (A2) in the presence of a growth factor belonging to the TGF- ⁇ superfamily; (C2) a step of culturing the cells obtained in the step (B2) in the presence of a retinoid, (D2)
- the cell obtained in the step (C2) is at least one selected from the group consisting of a cAMP (cyclic adenosine monophosphate) increasing agent, dexamethasone, a TGF- ⁇ 1 type receptor inhibitor, and nicotinamide. Culturing in the presence of The method for producing pancreatic hormone-producing cells according to [1] above, wherein the
- a differentiation induction promoter that induces differentiation of pluripotent stem cells or pancreatic tissue stem / progenitor cells into pancreatic hormone-producing cells, comprising at least one of the following (1) to (3); (1) a polypeptide comprising an amino acid sequence encoded by DNA comprising the base sequence set forth in SEQ ID NO: 1, (2) A pancreatic hormone-producing cell comprising an amino acid sequence in which one or several amino acids are substituted, deleted, and / or added in the amino acid sequence encoded by the DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1.
- a polypeptide having an action of inducing differentiation into (3) A culture supernatant of a cell in which a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 or a DNA hybridizing with a DNA complementary to this DNA under stringent conditions is incorporated as a foreign gene.
- pancreatic hormone-producing cells differentiated-inducing method
- a pancreatic hormone-producing cell and a differentiation induction promoter used in the production method can be provided.
- the culture supernatant of a cell transfected with an expression vector of a polypeptide (IBCAP) encoded by a DNA having the nucleotide sequence set forth in SEQ ID NO: 1 is used as the final stage of the differentiation induction process from human iPS cells to pancreatic hormone-producing cells. It is a figure which shows the expression level of glucagon (GCG) at the time of adding at (process (D1), (E1)) and somatostatin (SST).
- GCG glucagon
- D1 glucagon
- E1 somatostatin
- FIG. 3 is a diagram showing the expression levels of glucagon (GCG) and somatostatin (SST) when added at (step (A1-1), (A1-1)) or at an intermediate stage (steps (B1), (C1)). .
- the method for producing pancreatic hormone-producing cells comprises adding a specific differentiation-inducing promoter in the medium in the differentiation induction process from pluripotent stem cells or pancreatic tissue stem / progenitor cells to pancreatic hormone-producing cells.
- pluripotent stem cells, pancreatic tissue stem / progenitor cells, and differentiation induction promoters will be described in order, and then a specific method for producing pancreatic hormone-producing cells (differentiation induction method) will be described.
- a pluripotent stem cell is a self-replicating stem cell capable of differentiating into a differentiated cell belonging to at least one type of three germ layers (ectodermal, mesoderm, endoderm), for example, Induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), embryonic germ cells (EG cells), embryonic cancer cells (EC cells), adult pluripotent stem cells (APS cells), etc.
- iPS cells Induced pluripotent stem cells
- ES cells embryonic stem cells
- EG cells embryonic germ cells
- EC cells embryonic cancer cells
- APS cells adult pluripotent stem cells
- IPS cells are pluripotent cells obtained by reprogramming somatic cells.
- the production of iPS cells is carried out by Dr. Kyoto University. Dr. Yamanaka et al., Dr. of Massachusetts Institute of Technology. Jaenisch et al., Dr. of University of Wisconsin. Thomson et al., Dr. Harvard University.
- Several groups have been successful, including the group of Hochedlinger et al.
- somatic cell used for production of iPS cells is not particularly limited, and any somatic cell can be used. That is, all cells other than internal germ cells of the cells constituting the living body can be used, and may be differentiated somatic cells or undifferentiated stem cells.
- the origin of somatic cells may be any of mammals, birds, fishes, reptiles and amphibians, but is not particularly limited, but mammals are preferred, and humans or mice are particularly preferred. When human somatic cells are used, any fetal, neonatal, or adult somatic cells may be used.
- the reprogramming gene is a gene encoding a reprogramming factor that has the action of reprogramming somatic cells to become iPS cells. Examples of combinations of reprogramming genes for producing human iPS cells include (i) to (iv) below, but are not limited to these examples.
- OCT gene (I) OCT gene, KLF gene, SOX gene, MYC gene (ii) OCT gene, SOX gene, NANOG gene, LIN28 gene (iii) OCT gene, KLF gene, SOX gene, MYC gene, hTERT gene, SV40 large T gene (Iv) OCT gene, KLF gene, SOX gene
- ES cells are stem cells having pluripotency and self-renewal ability, which are produced from an inner cell mass belonging to a part of an embryo at the blastocyst stage, which is an early stage of animal development.
- the origin of ES cells is not particularly limited, but mammals are preferred, and humans or mice are particularly preferred.
- a cell having a reporter gene introduced in the vicinity of the PDX1 gene can be used in order to facilitate confirmation of the degree of differentiation.
- the pancreatic tissue stem / progenitor cell is a tissue stem / progenitor cell present in the pancreas of an animal and having multipotency and self-renewal ability.
- the origin of pancreatic tissue stem / progenitor cells is not particularly limited, but mammals are preferable, and humans or mice are particularly preferable.
- As a method for separating tissue stem / progenitor cells from the pancreas a conventionally known method can be arbitrarily employed, and is not particularly limited.
- PDX1 is known as a marker molecule for pancreatic tissue stem / progenitor cells (Jonsson, J.
- pancreatic tissue stem / progenitor cells differentiate into endocrine cells, exocrine cells, and pancreatic duct cells, giving rise to all types of cells present in the adult pancreas. Therefore, pancreatic tissue stem / progenitor cells can be separated using PDX1 as a marker molecule. In addition, this pancreatic tissue stem / progenitor cell may be not established or may be established.
- the differentiation induction promoter used in the method for producing pancreatic hormone-producing cells according to the present invention is at least one selected from the following (1) to (3).
- a pancreatic hormone-producing cell comprising an amino acid sequence in which one or several amino acids are substituted, deleted, and / or added in the amino acid sequence encoded by the DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1.
- a culture supernatant of a cell in which a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 or a DNA hybridizing with a DNA complementary to this DNA under stringent conditions is incorporated as a foreign gene.
- the DNA consisting of the base sequence described in SEQ ID NO: 1 has a length of 2308 bp known as DNA encoding human TM4SF20 (NCBI: LOCUS NM 024795).
- the CDS of this DNA is 38. . 720, and encodes a polypeptide having the 229 amino acid sequence set forth in SEQ ID NO: 3 (see SEQ ID NO: 2).
- this polypeptide can be used as a differentiation induction promoter.
- pancreatic hormone-producing cells in the method for producing pancreatic hormone-producing cells according to the present invention, as long as the action of promoting differentiation induction into pancreatic hormone-producing cells is maintained, one or several amino acids in the amino acid sequence of the above polypeptide are substituted or missing.
- a polypeptide comprising a lost and / or added amino acid sequence (hereinafter also referred to as “modified polypeptide”) can be used as a differentiation induction promoter. It is already known that a polypeptide consisting of an amino acid sequence modified by substitution, deletion and / or addition of one or several amino acids to an amino acid sequence maintains its biological activity (Mark, DF et al., Proc. Natl. Acad. Sci. USA, 81, pp.
- amino acid side chains include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H , K, S, T), an amino acid having an aliphatic side chain (G, A, V, L, I, P), an amino acid having a hydroxyl group-containing side chain (S, T, Y), and a sulfur atom-containing side chain.
- hydrophobic amino acids A, I, L, M, F, P, W, Y, V
- hydrophilic amino acids R, D, N, C, E, Q, G, H , K, S, T
- G, A, V, L, I, P amino acid having a hydroxyl group-containing side chain
- S, T, Y hydroxyl group-containing side chain
- the number may be, for example, 1 to 20, 1 to 15, or 1 to 10.
- the number may be 1 to 5.
- the homology between the modified polypeptide and the original polypeptide is preferably 80% or more, more preferably 90% or more, still more preferably 93% or more, particularly preferably 95% or more, and most preferably 98% or more.
- amino acids 1 to 163 and 179 to 229 of SEQ ID NO: 3 are highly conserved portions between different species. For this reason, it is preferable that the amino acid of the part is preserve
- polypeptides and modified polypeptides may be chemically synthesized, but can also be obtained by genetic engineering.
- a DNA comprising the base sequence set forth in SEQ ID NO: 1 or a DNA hybridizing with a DNA comprising a base sequence complementary to this DNA under stringent conditions is incorporated as a foreign gene into a culturable host cell, By culturing the host cell and expressing the gene, the above-mentioned polypeptide or modified polypeptide can be obtained from the culture supernatant.
- the host cell known cells such as bacteria, yeast, insect cells, animal cells and the like can be appropriately used. Examples of animal cells include HEK293 cells, HEK293T cells, CHO-K1 cells, and COS cells.
- DNA that hybridizes under stringent conditions means that a specific DNA (DNA consisting of a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 1) is used as a probe, and colony high It means DNA that can be obtained by employing a hybridization method, a plaque hybridization method, a Southern blot hybridization method, or the like. For example, after hybridization at 65 ° C. in the presence of 0.7 to 1.0 M sodium chloride using a filter on which DNA derived from colonies or plaques is immobilized, 0.1 to 2 ⁇ SSC solution (1 ⁇ SSC composition: 150 mM sodium chloride, 15 mM sodium citrate) and DNA etc. that can be identified by washing the filter under 65 ° C.
- the homology of the base sequence of the DNA that hybridizes under stringent conditions with the base sequence of the DNA used as the probe is preferably 80% or more, more preferably 90% or more, still more preferably 93% or more, 95 % Or more is particularly preferable, and 98% or more is most preferable.
- polypeptides and modified polypeptides can be separated and purified by methods usually used in peptide chemistry, such as ion exchange resin, partition chromatography, gel chromatography, and reverse phase chromatography.
- a culture supernatant containing the above-mentioned polypeptide or modified polypeptide can be used as a differentiation induction promoter.
- the culture supernatant it is preferable to concentrate the culture supernatant by ultrafiltration or the like.
- dialysis may be performed as necessary to remove unnecessary chemical substances.
- the above-described differentiation induction promoter is added to the medium in the process of inducing differentiation from pluripotent stem cells to pancreatic hormone-producing cells.
- a conventionally known method can be arbitrarily employed and is not particularly limited.
- the concentration is preferably 10 to 200 ng / mL, more preferably 50 to 180 ng / mL, and even more preferably 60 to 150 ng / mL.
- the concentration is preferably 0.5 to 20% (v / v), more preferably 1 to 10% (v / v), and 1.5 to 5% (v / v) is more preferable.
- pancreatic hormone-producing cells two examples of methods for inducing differentiation from pluripotent stem cells to pancreatic hormone-producing cells (methods for producing pancreatic hormone-producing cells) will be described.
- the method for producing pancreatic hormone-producing cells according to the present invention is described below. It is not limited to examples.
- the first differentiation induction method is based on the method described in Non-Patent Document 1. This document is incorporated herein by reference.
- the first differentiation induction method includes the following steps (A1) to (E1). In at least one of these steps, the differentiation induction promoter described above is added to the medium.
- the step of adding the differentiation induction promoter is preferably at least one of steps (A1) to (C1), and more preferably at least one of steps (B1) to (C1).
- (B1) A step of culturing the cells obtained in the step (A1) in the presence of FGF.
- C1 A step of culturing the cells obtained in the step (B1) in the presence of a retinoid.
- (D1) A step of culturing the cells obtained in the step (C1) in the presence of a ⁇ -secretase inhibitor.
- (E1) A step of culturing the cells obtained in the step (D1) in the presence of at least one factor selected from the group consisting of exendin-4, HGF, IGF-1, and nicotinamide.
- pluripotent stem cells are cultured in the presence of a growth factor belonging to the TGF- ⁇ superfamily.
- growth factors belonging to the TGF- ⁇ superfamily include activin, nodal, BMP (bone morphogenetic protein), and among them, activin is preferable.
- Such growth factors belonging to the TGF- ⁇ superfamily are known to promote differentiation from pluripotent stem cells into definitive endoderm cells (see Non-Patent Document 1, Patent Documents 1 to 3, etc.) .
- the activin include activin A, activin B, activin AB and the like. Among these, activin A is preferable.
- the concentration of the growth factor belonging to the TGF- ⁇ superfamily is preferably 5 to 250 ng / mL, more preferably 10 to 200 ng / mL, and further preferably 50 to 150 ng / mL.
- the step (A1) it is preferable to add a growth factor belonging to the Wnt family to the medium.
- a growth factor belonging to the Wnt family By adding a growth factor belonging to the Wnt family together with a growth factor belonging to the TGF- ⁇ superfamily, differentiation efficiency into definitive endoderm cells can be increased.
- the growth factor belonging to the Wnt family include Wnt1, Wnt3a, Wnt5a, Wnt7a and the like. Wnt1 and Wnt3a are preferable, and Wnt3a is more preferable.
- the concentration of the growth factor belonging to the Wnt family is preferably 1 to 1000 ng / mL, more preferably 10 to 100 ng / mL, and even more preferably 10 to 50 ng / mL.
- a GSK-3 inhibitor for example, CHIR
- a growth factor belonging to the Wnt family may be added instead of a growth factor belonging to the Wnt family.
- GSK-3 inhibitors eg, CHIR
- CHIR are known to activate the Wnt signaling pathway (J. Biol. Chem. 277 (34), pp. 30998-31004 (2002)).
- an additional factor that can increase the efficiency of differentiation into definitive endoderm cells may be added to the medium.
- Additional factors include, for example, PDGF (platelet-derived growth factor), EGF (epidermal growth factor), VEGF (vascular endothelial growth factor), KGF (keratinocyte growth factor), HGF, NGF (nerve growth factor), GDF ( Growth and differentiation factor), GLP (glucagon-like peptide), nicotinamide, exendin-4, retinoic acid, ethanolamine, parathyroid hormone, progesterone, aprotinin, hydrocortisone, gastrin, steroid alkaloid, copper chelator (triethylenepentamine, etc.), Forskolin, sodium butyrate, noggin, valproic acid, trichostatin A, Indian hedgehog, sonic hedgehog, proteasome inhibitor, Notch pathway inhibitor, hedgehog pathway inhibitor and the like.
- a culture plate coated with a scaffold using a biocompatible material is preferable from the viewpoint of differentiation inducing ability, function expression ability, viability and the like.
- the scaffold include laminin, fibronectin, collagen, heparan sulfate proteoglycan, gelatin, entactin, polyornithine and the like.
- Examples of commercially available products, Becton Dickinson made of MATRIGEL TM, growth factor reduced MATRIGEL TM, etc. are available.
- the medium used for culturing is prepared by adding various nutrient sources and other components necessary for the maintenance and growth of cells to a basic medium that can be used for culturing animal cells.
- RPMI1640 medium As the basic medium, RPMI1640 medium, DMEM medium, CMRL1066 medium, Ham F12 medium, Eagle MEM medium, Glasgow MEM medium, IMEM Zinc Option medium, IMDM medium, William E medium, Fisher medium, McCoy medium, BME medium, ⁇ MEM medium, Examples thereof include BGJb medium, Medium 199 medium, and mixed media thereof.
- Nutrient sources include carbon sources such as glycerol, glucose, fructose, sucrose, lactose, starch, dextrin; hydrocarbons such as fatty acids, fats and oils, lecithin, alcohol; nitrogen such as ammonium sulfate, ammonium nitrate, ammonium chloride, urea, sodium nitrate Sources: inorganic salts such as sodium salt, potassium salt, magnesium salt, calcium salt and phosphate; various vitamins; various amino acids;
- antibiotics such as penicillin and streptomycin
- cholera toxin insulin
- transferrin selenite
- albumin 2-mercaptoethanol
- serum or serum substitute commercially available products such as ITS-X, ITS-A, and ITS-G manufactured by Invitrogen are available.
- ITS-X, ITS-A, and ITS-G commercially available products
- B-27 TM supplement, N-2 supplement, Knockout TM serum substitute, etc., manufactured by Invitrogen are commercially available.
- the step (A1) it is known that in order to increase the differentiation efficiency in the step (A1), it is important to sufficiently reduce the content of insulin, IGF, and the like in the medium (International Publication No. 2006/020919). See). For this reason, in the step (A1), it is preferable to use a serum-free medium or a low serum medium (see Non-Patent Document 1, Patent Documents 1 to 3, etc.).
- the serum concentration is preferably 0 to 2% (v / v), more preferably 0 to 1% (v / v), and still more preferably 0 to 0.5% (v / v).
- a serum-free or low-serum RPMI 1640 medium supplemented with activin A, Wnt3a, antibiotics such as penicillin or streptomycin, L-glutamine or a dipeptide containing L-glutamine is used.
- the culture period in the step (A1) is, for example, 1 to 6 days, and preferably 2 to 4 days.
- the progress of differentiation induction into definitive endoderm cells can be evaluated not only by morphological observation but also by confirming gene expression by RT-PCR.
- stem cell marker genes such as OCT4, NANOG, SOX2, ECAD, etc. decreases, and definitive endoderm cell marker genes SOX17, CER, Expression of FOXA2, CXCR4, etc. is enhanced.
- the step (A1) is preferably divided into a step (A1-1) for culturing in a serum-free first medium and a step (A1-2) for culturing in a low serum second medium.
- the first medium used in step (A1-1) may be the same as above except that it is serum-free. That is, the first medium contains a growth factor belonging to the TGF- ⁇ superfamily, and may contain a growth factor belonging to the Wnt family. The first medium preferably contains a growth factor belonging to the Wnt family.
- the culture period in the step (A1-1) is, for example, 1 to 3 days, and preferably 1 to 2 days.
- differentiation from pluripotent stem cells to mesendoderm cells proceeds.
- the progress of differentiation induction into mesendoderm cells can be evaluated not only by morphological observation but also by confirming gene expression by RT-PCR.
- stem cell marker genes such as OCT4, NANOG, SOX2, ECAD and the like decreases, and mesendoderm cell marker genes BRA, FGF4, Expression of WNT3, NCAD, etc. is enhanced.
- the second medium used in the step (A1-2) may be the same as described above except for low serum. That is, the second medium contains a growth factor belonging to the TGF- ⁇ superfamily, and may contain a growth factor belonging to the Wnt family.
- the serum concentration is preferably 0.05 to 2% (v / v), more preferably 0.05 to 1% (v / v), still more preferably 0.1 to 0.5% (v / v).
- the culture period in the step (A1-2) is, for example, 1 to 3 days, and preferably 1 to 2 days.
- differentiation from mesendoderm cells to definitive endoderm cells proceeds.
- the progression of differentiation induction into definitive endoderm cells can be evaluated not only by morphological observation but also by confirming gene expression by RT-PCR.
- the obtained cells may be concentrated, isolated and / or purified by a known method before proceeding to the next step (B1).
- step (B1) the cells obtained in step (A1) are cultured in the presence of FGF.
- FGF examples include FGF-1, FGF-2 (bFGF), FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, and FGF-11.
- FGF-2 (bFGF), FGF-5, FGF-7, and FGF-10 are preferable.
- the concentration of FGF is preferably 5 to 150 ng / mL, more preferably 10 to 100 ng / mL, and even more preferably 20 to 80 ng / mL.
- Hedgehog pathway inhibitors include KAAD-cyclopamine (28- [2-[[6-[(3-phenylpropanoyl) amino] hexanoyl] amino] ethyl] -17 ⁇ , 23 ⁇ -epoxyveratraman-3-one) , KAAD-cyclopamine analogs, jervin (17,23 ⁇ -epoxy-3 ⁇ -hydroxyveratraman-11-one), gerbin analogs, hedgehog pathway blocking antibodies, and the like, among which KAAD-cyclopamine is preferred.
- the concentration of the hedgehog pathway inhibitor is preferably 0.01 to 5 ⁇ M, more preferably 0.02 to 2 ⁇ M, and even more preferably 0.1 to 0.5 ⁇ m.
- the container used for culture may be the same as in step (A1).
- the medium may be the same as in step (A1) except for the above-described factors and the serum concentration of the medium.
- the serum concentration of the medium is preferably 0.1 to 5% (v / v), more preferably 0.5 to 5% (v / v), and even more preferably 1 to 5% (v / v).
- low serum RPMI 1640 medium supplemented with FGF-10, KAAD-cyclopamine, antibiotics such as penicillin and streptomycin, L-glutamine or a dipeptide containing L-glutamine is used.
- the culture period in the step (B1) is, for example, 1 to 6 days, and preferably 2 to 4 days.
- the progress of differentiation induction can be evaluated not only by morphological observation but also by confirming gene expression by RT-PCR. As differentiation progresses, the expression of genes such as HNF1B and HNF4A increases.
- the obtained cells may be concentrated, isolated, and / or purified by a known method before proceeding to the next step (C1).
- step (C1) the cells obtained in step (B1) are cultured in the presence of a retinoid.
- retinoids examples include retinol, retinal, retinoic acid, etc. Among them, retinoic acid is preferable.
- the concentration of retinoid is preferably 0.2 to 10 ⁇ M, more preferably 0.4 to 8 ⁇ M, and even more preferably 1 to 4 ⁇ M.
- a hedgehog pathway inhibitor it is preferable to add a hedgehog pathway inhibitor to the medium. Differentiation efficiency can be enhanced by adding a hedgehog pathway inhibitor together with the retinoid.
- the hedgehog pathway inhibitor include KAAD-cyclopamine, an analog of KAAD-cyclopamine, jervin, an analog of jervin, a hedgehog pathway blocking antibody, and among them, KAAD-cyclopamine is preferable.
- the concentration of the hedgehog pathway inhibitor is preferably 0.01 to 5 ⁇ M, more preferably 0.02 to 2 ⁇ M, and even more preferably 0.1 to 0.5 ⁇ M.
- FGF FGF-1, FGF-2 (bFGF), FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, and FGF-11.
- FGF-2 (bFGF), FGF-5, FGF-7, and FGF-10 are preferable.
- the concentration of FGF is preferably 0.5 to 50 ng / mL, more preferably 1 to 25 ng / mL, and even more preferably 2 to 10 ng / mL.
- a growth factor belonging to the TGF- ⁇ superfamily may be added to the medium.
- the concentration of the growth factor belonging to the TGF- ⁇ superfamily is preferably 5 to 250 ng / mL, more preferably 10 to 200 ng / mL, and further preferably 20 to 150 ng / mL.
- the container used for culture may be the same as in step (B1).
- the medium may be basically the same as in the step (B1) except for the above-described factors.
- a serum substitute is preferably added to the medium instead of serum.
- examples of commercially available serum substitutes include B-27 TM supplement, N-2 supplement, Knockout TM serum substitute, etc. manufactured by Invitrogen, and among them, B-27 TM supplement is preferable.
- the concentration of B-27 TM supplement is preferably 0.1 to 10% (v / v), more preferably 0.2 to 5% (v / v), and 0.4 to 2.5% (v / v). Is more preferable.
- this B-27 TM supplement is commercially available as a 50-fold stock solution
- the B-27 TM supplement concentration is 5- to 500-fold diluted to achieve a concentration of 0.1 to 10% (v / v). As long as it is added to the medium.
- serum-free DMEM / ham F12 medium supplemented with retinoic acid, KAAD-cyclopamine, FGF-10, antibiotics such as penicillin and streptomycin, and B-27 TM supplement is used.
- the culture period in the step (C1) is, for example, 1 to 6 days, and preferably 2 to 4 days.
- the progress of differentiation induction can be evaluated not only by morphological observation but also by confirming gene expression by RT-PCR. As differentiation progresses, the expression of genes such as PDX1, HNF6, and HLXB9 increases.
- the obtained cells may be concentrated, isolated and / or purified by a known method before proceeding to the next step (D1).
- step (D1) the cells obtained in step (C1) are cultured in the presence of a ⁇ -secretase inhibitor.
- DAPT N- [N- (3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine-tert-butyl ester
- L-658458 [1S-benzyl- 4R- [1- (1S-carbamoyl-2-phenethylcarbamoyl) -1S-3-methylbutylcarbamoyl] -2R-hydroxy-5-phenethylpentyl] carbamic acid tert-butyl ester
- concentration of the ⁇ -secretase inhibitor is preferably 1 to 50 ⁇ M, more preferably 2 to 40 ⁇ M, and even more preferably 5 to 20 ⁇ M.
- exendin-4 is preferably added to the medium. Differentiation efficiency can be increased by adding exendin-4 together with a ⁇ -secretase inhibitor.
- concentration of exendin-4 is preferably 5 to 150 ng / mL, more preferably 10 to 100 ng / mL, and further preferably 20 to 80 ng / mL.
- the container and medium used for the culture may be the same as in step (C1). That is, it is preferable to add a serum substitute to the medium.
- serum-free DMEM / Ham F12 medium supplemented with DAPT, exendin-4, antibiotics such as penicillin and streptomycin, and B-27 TM supplement is used.
- the culture period in the step (D1) is, for example, 1 to 6 days, and preferably 2 to 3 days.
- the progress of differentiation induction can be evaluated not only by morphological observation but also by confirming gene expression by RT-PCR. As differentiation proceeds, the expression of genes such as NKX6-1, NGN3, PAX4, and NKX2-2 increases.
- the obtained cells may be concentrated, isolated and / or purified by a known method before proceeding to the next step (E1).
- step (E1) the cells obtained in step (D1) are cultured in the presence of at least one factor selected from the group consisting of exendin-4, HGF, IGF-1, and nicotinamide.
- exendin-4 As exendin-4, HGF, IGF-1, and nicotinamide, it is preferable to add two or more of them, and it is more preferable to add three or more.
- the concentration of exendin-4 is preferably 5 to 150 nM, more preferably 10 to 100 nM, and even more preferably 20 to 80 nM.
- the concentration of HGF is preferably 5 to 150 ng / mL, more preferably 10 to 100 ng / mL, and further preferably 20 to 80 ng / mL.
- the concentration of IGF-1 is preferably 5 to 150 ng / mL, more preferably 10 to 100 ng / mL, and further preferably 20 to 80 ng / mL.
- the concentration of nicotinamide is preferably 1 to 30 mM, more preferably 3 to 20 mM, and even more preferably 5 to 15 mM.
- the container and medium used for the culture may be the same as in step (D1). That is, it is preferable to add a serum substitute to the medium.
- serum-free CMRL 1066 medium supplemented with antibiotics such as exendin-4, HGF, IGF-1, penicillin, streptomycin, B-27 TM supplement is used.
- the culture period in the step (E1) is, for example, 3 to 20 days, and preferably 3 to 10 days.
- pancreatic hormone-producing cells are obtained.
- the progress of differentiation induction into pancreatic hormone-producing cells can be evaluated by confirming gene expression by RT-PCR in addition to confirming production of pancreatic hormones such as insulin, glucagon and somatostatin.
- pancreatic hormones such as insulin, glucagon and somatostatin.
- the expression of at least one gene among INS, GCG, GHRL, SST, PPY, etc. increases.
- the second differentiation induction method is based on the method described in Non-Patent Document 4. This document is incorporated herein by reference.
- the second differentiation induction method includes the following steps (A2) to (D2). In at least one of these steps, the differentiation induction promoter described above is added to the medium.
- the step of adding the differentiation induction promoter is preferably at least one of steps (C2) to (D2), and particularly preferably step (D2).
- (A2) culturing pluripotent stem cells in the presence of a growth factor belonging to the TGF- ⁇ superfamily and at least one factor selected from the group consisting of a growth factor belonging to the Wnt family and a GSK-3 inhibitor Process.
- (B2) A step of culturing the cells obtained in the step (A2) in the presence of a growth factor belonging to the TGF- ⁇ superfamily.
- (C2) A step of culturing the cells obtained in the step (B2) in the presence of a retinoid.
- the cells obtained in the above step (C2) are cultured in the presence of at least one factor selected from the group consisting of a cAMP increasing agent, dexamethasone, a TGF- ⁇ 1 type receptor inhibitor, and nicotinamide. Process.
- Step (A2) pluripotent stem cells in the presence of a growth factor belonging to the TGF- ⁇ superfamily and at least one factor selected from the group consisting of a growth factor belonging to the Wnt family and a GSK-3 inhibitor Is cultured.
- Examples of the growth factor belonging to the TGF- ⁇ superfamily include activin, nodal, BMP and the like, among which activin is preferable.
- Examples of the activin include activin A, activin B, activin AB and the like. Among these, activin A is preferable.
- the concentration of the growth factor belonging to the TGF- ⁇ superfamily is preferably 5 to 250 ng / mL, more preferably 10 to 200 ng / mL, and further preferably 50 to 150 ng / mL.
- Examples of the growth factor belonging to the Wnt family include Wnt1, Wnt3a, Wnt5a, Wnt7a and the like. Wnt1 and Wnt3a are preferable, and Wnt3a is more preferable.
- the concentration of the growth factor belonging to the Wnt family is preferably 1 to 1000 ng / mL, more preferably 10 to 100 ng / mL, and even more preferably 10 to 50 ng / mL.
- GSK-3 inhibitor either a GSK-3 ⁇ inhibitor or a GSK-3 ⁇ inhibitor may be used, but a GSK-3 ⁇ inhibitor is preferably used.
- Specific examples include CHIR99021 (6-[[2-[[4- (2,4-dichlorophenyl) -5- (5-methyl-1H-imidazol-2-yl) -2-pyrimidinyl] amino] ethyl] amino) ) -3-pyridinecarbonitrile), SB415286 (3-[(3-chloro-4-hydroxyphenyl) amino] -4- (2-nitrophenyl) -1H-pyrrole-2,5-dione), SB216673 (3 -(2,4-dichlorophenyl) -4- (1-methyl-1H-indol-3-yl) -1H-pyrrole-2,5-dione), indirubin-3'-monooxime (3-[(3E)- 3- (hydroxyimino) -2,3-dihydro-1H-ind
- an additional factor that can increase differentiation efficiency may be added to the medium.
- Additional factors include, for example, PDGF, EGF, VEGF, KGF, HGF, NGF, GDF, GLP, nicotinamide, exendin-4, retinoic acid, ethanolamine, parathyroid hormone, progesterone, aprotinin, hydrocortisone, gastrin, steroids Alkaloids, copper chelators (such as triethylenepentamine), forskolin, sodium butyrate, noggin, valproic acid, trichostatin A, Indian hedgehog, sonic hedgehog, proteasome inhibitor, Notch pathway inhibitor, hedgehog pathway inhibitor, etc. Is mentioned.
- a culture plate coated with a scaffold using a biocompatible material is preferable from the viewpoint of differentiation inducing ability, function expression ability, viability and the like.
- the scaffold include laminin, fibronectin, collagen, heparan sulfate proteoglycan, gelatin, entactin, polyornithine and the like.
- Examples of commercially available products, Becton Dickinson made of MATRIGEL TM, growth factor reduced MATRIGEL TM, etc. are available.
- the medium used for culturing is prepared by adding various nutrient sources and other components necessary for the maintenance and growth of cells to a basic medium that can be used for culturing animal cells.
- RPMI1640 medium As the basic medium, RPMI1640 medium, DMEM medium, CMRL1066 medium, Ham F12 medium, Eagle MEM medium, Glasgow MEM medium, IMEM Zinc Option medium, IMDM medium, William E medium, Fisher medium, McCoy medium, BME medium, ⁇ MEM medium, Examples thereof include BGJb medium, Medium 199 medium, and mixed media thereof.
- Nutrient sources include carbon sources such as glycerol, glucose, fructose, sucrose, lactose, starch, dextrin; hydrocarbons such as fatty acids, fats and oils, lecithin, alcohol; nitrogen such as ammonium sulfate, ammonium nitrate, ammonium chloride, urea, sodium nitrate Sources: inorganic salts such as sodium salt, potassium salt, magnesium salt, calcium salt and phosphate; various vitamins; various amino acids;
- antibiotics such as penicillin and streptomycin
- cholera toxin insulin
- transferrin selenite
- albumin 2-mercaptoethanol
- serum or serum substitute commercially available products such as ITS-X, ITS-A, and ITS-G manufactured by Invitrogen are available.
- ITS-X, ITS-A, and ITS-G commercially available products
- B-27 TM supplement, N-2 supplement, Knockout TM serum substitute, etc., manufactured by Invitrogen are commercially available.
- the serum concentration is preferably 0 to 3% (v / v), more preferably 0 to 2% (v / v).
- low serum RPMI 1640 medium supplemented with activin A, CHIR99021 is used.
- the culture period in the step (A2) is, for example, 1 to 3 days, and preferably 1 to 2 days.
- step (B2) the cells obtained in step (A2) are cultured in the presence of a growth factor belonging to the TGF- ⁇ superfamily.
- Examples of the growth factor belonging to the TGF- ⁇ superfamily include activin, nodal, BMP and the like, among which activin is preferable.
- Examples of the activin include activin A, activin B, activin AB and the like. Among these, activin A is preferable.
- the concentration of the growth factor belonging to the TGF- ⁇ superfamily is preferably 5 to 250 ng / mL, more preferably 10 to 200 ng / mL, and further preferably 50 to 150 ng / mL.
- the container and medium used for culture may be the same as in step (A2). That is, in a preferred embodiment, low serum RPMI 1640 medium supplemented with activin A is used.
- the culture period in the step (B2) is, for example, 1 to 4 days, and preferably 1 to 3 days.
- the cells obtained in the step (B2) are cultured in the presence of a retinoid.
- retinoids examples include retinol, retinal, retinoic acid, etc. Among them, retinoic acid is preferable.
- the concentration of retinoid is preferably 0.2 to 10 ⁇ M, more preferably 0.4 to 8 ⁇ M, and even more preferably 1 to 4 ⁇ M.
- BMP receptor inhibitors include dorsomorphin (6- [4- [2- (1-piperidinyl) ethoxy] phenyl] -3- (4-pyridyl) pyrazolo [1,5-a] pyrimidine), LDN-193189 (4- (6- (4- (piperazin-1-yl) phenyl) pyrazolo [1,5-a] pyrimidin-3-yl) quinoline) and the like, among which dorsomorphin is preferable.
- the concentration of the BMP receptor inhibitor is preferably 0.2 to 5 ⁇ M, more preferably 0.3 to 3 ⁇ M, and even more preferably 0.5 to 2 ⁇ M.
- TGF- ⁇ 1 type receptor inhibitors include SB431542 (4- [4- (1,3-benzodioxol-5-yl) -5- (2-pyridinyl) -1H-imidazol-2-yl] benzamide), SB525334. (6- [2- (1,1-dimethylethyl) -5- (6-methyl-1,2-pyridinyl) -1H-imidazol-4-yl] quinoxaline), LY364947 (4- [3- (2- Pyridinyl) -1H-pyrazol-4-yl] quinoline), among which SB431542 is preferred.
- TGF- ⁇ 1 type receptor inhibitor Alk5 inhibitor II manufactured by Calbiochem can also be used.
- concentration of the TGF- ⁇ 1 type receptor inhibitor is preferably 1 to 50 ⁇ M, more preferably 2 to 30 ⁇ M, and even more preferably 5 to 20 ⁇ M.
- the container used for culture may be the same as in step (B2).
- the medium may be basically the same as in the step (B2) except for the above-described factors.
- a serum substitute is preferably added to the medium instead of serum.
- examples of commercially available serum substitutes include B-27 TM supplement, N-2 supplement, Knockout TM serum substitute, etc. manufactured by Invitrogen, and among them, B-27 TM supplement is preferable.
- the concentration of B-27 TM supplement is preferably 0.1 to 10% (v / v), more preferably 0.2 to 5% (v / v), and 0.4 to 2.5% (v / v). Is more preferable.
- this B-27 TM supplement is commercially available as a 50-fold stock solution
- the B-27 TM supplement concentration is 5- to 500-fold diluted to achieve a concentration of 0.1 to 10% (v / v). As long as it is added to the medium.
- serum-free IMEM Zinc Option medium supplemented with retinoic acid, dorsomorphin, SB431542, B-27 TM supplement is used.
- the culture period in the step (C2) is, for example, 5 to 9 days, and preferably 6 to 8 days.
- step (D2) the cells obtained in step (C2) are treated in the presence of at least one factor selected from the group consisting of a cAMP increasing agent, dexamethasone, a TGF- ⁇ 1 type receptor inhibitor, and nicotinamide. Incubate at
- cAMP increasing agent dexamethasone, TGF- ⁇ 1 type receptor inhibitor, and nicotinamide, it is preferable to add two or more of them, and it is more preferable to add three or more.
- the cAMP increasing agent examples include an adenylate cyclase activator such as forskolin; a phosphodiesterase inhibitor such as 3-isobutyl-1-methylxanthine; a cAMP analog such as dibutyryl cAMP; among them, forskolin is preferable.
- the concentration of the cAMP increasing agent is preferably 1 to 50 ⁇ M, more preferably 2 to 30 ⁇ M, and even more preferably 5 to 20 ⁇ M.
- the concentration of dexamethasone is preferably 1 to 50 ⁇ M, more preferably 2 to 30 ⁇ M, and even more preferably 5 to 20 ⁇ M.
- TGF- ⁇ 1 type receptor inhibitor examples include SB431542, SB525334, LY364947 and the like, among which SB431542 is preferable.
- TGF- ⁇ 1 type receptor inhibitor Alk5 inhibitor II manufactured by Calbiochem can also be used.
- concentration of the TGF- ⁇ 1 type receptor inhibitor is preferably 1 to 50 ⁇ M, more preferably 2 to 30 ⁇ M, and even more preferably 5 to 20 ⁇ M.
- the concentration of nicotinamide is preferably 1 to 30 mM, more preferably 3 to 20 mM, and even more preferably 5 to 15 mM.
- the container and medium used for culture may be the same as in step (C2). That is, it is preferable to add a serum substitute to the medium.
- serum-free IMEM Zinc Option medium supplemented with forskolin, dexamethasone, Alk5 inhibitor II, nicotinamide, B-27 TM supplement is used.
- the culture period in the step (D2) is, for example, 9 to 13 days, and preferably 10 to 12 days.
- pancreatic hormone-producing cells are obtained.
- the progress of differentiation induction into pancreatic hormone-producing cells can be evaluated by confirming gene expression by RT-PCR in addition to confirming production of pancreatic hormones such as insulin, glucagon and somatostatin.
- pancreatic hormones such as insulin, glucagon and somatostatin.
- the expression of at least one of the marker genes of pancreatic hormone-producing cells increases.
- the differentiation-inducing promoter described above is added to the medium in the process of inducing differentiation from pancreatic tissue stem / progenitor cells to pancreatic hormone-producing cells.
- a conventionally known method can be arbitrarily employed and is not particularly limited.
- a polypeptide or a modified polypeptide When a polypeptide or a modified polypeptide is added as a differentiation induction promoter, its concentration is preferably 10 to 200 ng / mL, more preferably 50 to 150 ng / mL, and even more preferably 60 to 120 ng / mL. In addition, when a culture supernatant is added as a differentiation induction promoter, the concentration is preferably 0.5 to 20% (v / v), more preferably 1 to 10% (v / v), and 1.5 to 5% (v / v) is more preferable.
- pancreatic hormone-producing cells a method for producing pancreatic hormone-producing cells
- the method for producing pancreatic hormone-producing cells according to the present invention is limited to this example. Is not to be done.
- This differentiation induction method includes the following steps (A3) to (E3). In at least one of these steps, the differentiation induction promoter described above is added to the medium.
- the step of adding a differentiation induction promoter is preferably at least one of steps (D3) to (E3), and particularly preferably step (E3).
- A3 A step of culturing pancreatic tissue stem / progenitor cells in the absence of growth factors belonging to the TGF- ⁇ superfamily, retinoids, FGF, and nicotinamide.
- B3 A step of culturing the cells obtained in the step (A3) in the presence of a growth factor belonging to the TGF- ⁇ superfamily.
- C3 A step of culturing the cells obtained in the step (B3) in the presence of a retinoid.
- D3) A step of culturing the cells obtained in the step (C3) in the presence of FGF.
- E3) A step of culturing the cells obtained in the step (D3) in the presence of nicotinamide.
- step (A3) pancreatic tissue stem / progenitor cells are cultured in the absence of growth factors belonging to the TGF- ⁇ superfamily, retinoids, FGF, and nicotinamide.
- a culture plate coated with a scaffold using a biocompatible material is preferable from the viewpoint of differentiation inducing ability, function expression ability, viability and the like.
- the scaffold include laminin, fibronectin, collagen, heparan sulfate proteoglycan, gelatin, entactin, polyornithine and the like.
- Examples of commercially available products, Becton Dickinson made of MATRIGEL TM, growth factor reduced MATRIGEL TM, etc. are available.
- the medium used for culturing is prepared by adding various nutrient sources and other components necessary for the maintenance and growth of cells to a basic medium that can be used for culturing animal cells.
- RPMI1640 medium As the basic medium, RPMI1640 medium, DMEM medium, CMRL1066 medium, Ham F12 medium, Eagle MEM medium, Glasgow MEM medium, IMEM Zinc Option medium, IMDM medium, William E medium, Fisher medium, McCoy medium, BME medium, ⁇ MEM medium, Examples thereof include BGJb medium, Medium 199 medium, and mixed media thereof.
- Nutrient sources include carbon sources such as glycerol, glucose, fructose, sucrose, lactose, starch, dextrin; hydrocarbons such as fatty acids, fats and oils, lecithin, alcohol; nitrogen such as ammonium sulfate, ammonium nitrate, ammonium chloride, urea, sodium nitrate Sources: inorganic salts such as sodium salt, potassium salt, magnesium salt, calcium salt and phosphate; various vitamins; various amino acids;
- antibiotics such as penicillin and streptomycin
- cholera toxin insulin
- transferrin selenite
- 2-mercaptoethanol albumin
- serum or serum substitutes As insulin, transferrin, and selenite, commercially available products such as ITS-X, ITS-A, and ITS-G manufactured by Invitrogen are available.
- ITS-X, ITS-A, and ITS-G commercially available products
- B-27 TM supplement, N-2 supplement, Knockout TM serum substitute, etc., manufactured by Invitrogen are commercially available.
- serum-free DMEM / Ham F12 supplemented with antibiotics such as penicillin, streptomycin, insulin, transferrin, selenite, 2-mercaptoethanol, albumin is used.
- concentration of insulin is preferably 2 to 30 ⁇ g / mL, more preferably 5 to 20 ⁇ g / mL.
- the transferrin concentration is preferably 1 to 20 ⁇ g / mL, more preferably 3 to 10 ⁇ g / mL.
- the concentration of selenious acid is preferably 1 to 20 ng / mL, and more preferably 5 to 20 ng / mL.
- the concentration of 2-mercaptoethanol is preferably 50 to 200 ⁇ M, more preferably 50 to 100 ⁇ M.
- concentration of albumin is preferably 1 to 10 ng / mL, and more preferably 2 to 5 ng / mL.
- the culture period in the step (A3) is, for example, 1 to 3 days, and preferably 1 to 2 days.
- step (B3) the cells obtained in step (A3) are cultured in the presence of a growth factor belonging to the TGF- ⁇ superfamily.
- Examples of the growth factor belonging to the TGF- ⁇ superfamily include activin, nodal, BMP and the like, among which activin is preferable.
- Examples of the activin include activin A, activin B, activin AB and the like. Among these, activin A is preferable.
- the concentration of the growth factor belonging to the TGF- ⁇ superfamily is preferably 5 to 250 ng / mL, more preferably 10 to 200 ng / mL, and further preferably 50 to 150 ng / mL.
- the container used for culture may be the same as in step (A3).
- the medium may be the same as in step (A3) except that a growth factor belonging to the TGF- ⁇ superfamily is added. That is, in a preferred embodiment, serum-free DMEM / ham F12 supplemented with antibiotics such as penicillin and streptomycin, insulin, transferrin, selenite, 2-mercaptoethanol, and albumin is used.
- the culture period in the step (B3) is, for example, 2 to 6 days, and preferably 3 to 5 days.
- the cells obtained in the step (B3) are cultured in the presence of a retinoid.
- retinoids examples include retinol, retinal, retinoic acid and the like, and among them, all-trans retinoic acid is preferable.
- concentration of retinoid is preferably 0.2 to 10 ⁇ M, more preferably 0.4 to 8 ⁇ M, and even more preferably 1 to 4 ⁇ M.
- the container used for culture may be the same as in step (A3).
- the medium may be the same as in step (A3) except that a growth factor belonging to the TGF- ⁇ superfamily is added.
- serum-free DMEM / Ham F12 supplemented with antibiotics such as all-trans retinoic acid, penicillin, streptomycin, insulin, transferrin, selenite, 2-mercaptoethanol, albumin is used.
- the culture period in the step (C3) is, for example, 2 to 6 days, and preferably 3 to 5 days.
- Step (D3) the cells obtained in the step (C3) are cultured in the presence of FGF.
- FGF examples include FGF-1, FGF-2 (bFGF), FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, and FGF-11.
- FGF-2 (bFGF), FGF-5, FGF-7, and FGF-10 are preferable.
- the concentration of FGF is preferably 1 to 30 ng / mL, more preferably 2 to 20 ng / mL, and even more preferably 5 to 15 ng / mL.
- the container used for culture may be the same as in step (A3).
- the medium may be basically the same as in step (C3) except that FGF is added.
- FGF FGF-2
- serum-free DMEM / ham F12 supplemented with antibiotics such as FGF-2 (bFGF), penicillin, streptomycin, insulin, transferrin, selenite, albumin is used.
- the culture period in the step (D3) is, for example, 1 to 5 days, and preferably 2 to 4 days.
- step (E3) the cells obtained in step (D3) are cultured in the presence of nicotinamide.
- concentration of nicotinamide is preferably 1 to 30 mM, more preferably 3 to 20 mM, and even more preferably 5 to 15 mM.
- FGF FGF-1, FGF-2 (bFGF), FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, and FGF-11.
- FGF-2 (bFGF), FGF-5, FGF-7, and FGF-10 are preferable.
- the concentration of FGF is preferably 1 to 30 ng / mL, more preferably 2 to 20 ng / mL, and even more preferably 5 to 15 ng / mL.
- the container used for culture may be the same as in step (A3).
- the medium may be basically the same as in step (D3) except that nicotinamide is added.
- nicotinamide is added.
- serum-free DMEM / ham F12 supplemented with antibiotics such as nicotinamide, FGF-2 (bFGF), penicillin, streptomycin, insulin, transferrin, selenite, albumin is used.
- the culture period in the step (E3) is, for example, 3 to 20 days, and preferably 3 to 10 days.
- pancreatic hormone-producing cells are obtained.
- the progress of differentiation induction into pancreatic hormone-producing cells can be evaluated by confirming gene expression by RT-PCR in addition to confirming production of pancreatic hormones such as insulin, glucagon and somatostatin.
- pancreatic hormones such as insulin, glucagon and somatostatin.
- the expression of at least one gene among marker genes of pancreatic hormone-producing cells such as INS, GCG, GHRL, SST, PPY, increases. .
- pancreatic hormone-producing cells obtained as described above can be applied to therapeutic agents such as diabetes.
- a cell mass such as a pellet obtained by concentrating the insulin-producing cells as it is or by filtering can be used as a therapeutic agent for diabetes.
- This therapeutic agent for diabetes can be cryopreserved by adding a protective agent such as DMSO.
- DMSO protective agent
- a small incision is made in the right lower abdomen of a human patient, a thin blood vessel in the mesentery is exposed, and a catheter is directly viewed. Transplant the cells by inserting a cell, identify the portal vein of the liver by echo, transplant the cells by puncturing the catheter, or transplant to the spleen by puncturing the spleen directly under the abdominal echo guide A method is mentioned.
- Dose (implantation amount), 1 ⁇ 10 8 ⁇ is preferably 1 ⁇ 10 10 cells / animal, and more preferably 5 ⁇ 10 8 ⁇ 1 ⁇ 10 10 cells / animal.
- the dose (transplant amount) can be appropriately changed depending on the age, weight, symptoms, etc. of the patient to be administered.
- pancreatic hormone-producing cells obtained as described above can be used as a research reagent.
- a new drug can be screened by adding the new drug to a culture vessel in which pancreatic hormone-producing cells are cultured or a bioreactor containing the pancreatic hormone-producing cells.
- bioartificial pancreas using the pancreatic hormone-producing cells obtained as described above.
- the bioartificial pancreas include a hybrid type artificial pancreas in which a hollow fiber type bioreactor (device) and pancreatic hormone-producing cells are combined.
- Bioartificial pancreas is attached outside the body and connected to the blood vessel, placed in the body and connected to the blood vessel, placed in the abdominal cavity without connecting to the blood vessel, placed subcutaneously without connecting to the blood vessel It can be applied to any form of bioartificial pancreas.
- a differentiation induction promoter was prepared as follows. 10 cm of CHO-K1 cells subcultured in Ham's F12 medium (Sigma, N6658) supplemented with 10% FBS (fetal bovine serum) (Nichirei, 171012), 1% penicillin / streptomycin (Life Technologies Japan, 15140-122) 5 ⁇ 10 5 plates were plated on the dish. On the next day, using FuGENE6 (Roche), an expression vector (pCAGGS-IBCAP) of a polypeptide (hereinafter referred to as “IBCAP”) encoded by a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 was selected as CHO-K1.
- IBCAP-expressing stable CHO cells were transfected and IBCAP was forced to be expressed. After 48 hours, the cells were diluted to 1/20 concentration and re-plated into 10 cm dishes. The next day, G418 (Nacalai Tesque, 09380-44) having a final concentration of 400 ⁇ g / mL was added, and thereafter, the medium was changed every 3 to 5 days to form colonies. Colonies cloned by limiting dilution are isolated, and after growth, gene expression is confirmed by Southern blotting and Northern blotting. A stable IBCAP-expressing CHO-K1 cell line (hereinafter referred to as “IBCAP-expressing stable CHO cells”). Was made.
- the IBCAP-expressing Stable CHO cells were supplemented with 1% GLUTAMAX I (Life Technologies Japan, 35050-061) and 1% penicillin / streptomycin (Life Technologies Japan, 15140-122). 011). Further, the conditioned IBCAP-expressing stable CHO cells were subjected to forced aeration CO 2 incubator (Tytech, CO 2 -BR-43FL, temperature: 37 ° C., shaking speed: 120 rpm, gas conditions: 5% CO 2 , 20 mL / min. / Flask), and culture supernatant was prepared using these cells.
- forced aeration CO 2 incubator Teytech, CO 2 -BR-43FL, temperature: 37 ° C., shaking speed: 120 rpm, gas conditions: 5% CO 2 , 20 mL / min. / Flask
- the culture supernatant was confirmed to have a survival rate of 90% or more, and subcultured so that the number of cells was 5 ⁇ 10 5 cells / mL (culture solution volume: 150 mL culture solution / 500 mL flask), and collected after 5 days. (The number of cells was about 4-5 ⁇ 10 5 cells / mL).
- the collected culture supernatant is then concentrated about 10 times using Centriprep (Millipore, 4302, YM-3) (300 mL is concentrated to about 30 mL), and further against 2 L of 30 mM HEPES (pH 7.6). And dialyzed 3 times. Then, the culture supernatant after dialysis (hereinafter referred to as “IBCAP culture supernatant”) was prepared as a differentiation induction promoter.
- pCAGGS a culture supernatant after dialysis
- TIG3 / KOSM cells consist of 1% (v / v) penicillin / streptomycin (Gibco), 20% (v / v) Knockout TM serum replacement (Gibco), 1% (v / v) nonessential amino acids (Gibco), Maintained in DMEM / Ham F12 medium supplemented with 2.5 mM L-glutamine, 0.1 mM 2-mercaptoethanol (Gibco), 5 ng / mL FGF-2 (R & D Systems), 5 mM sodium chloride.
- TIG3 / KOSM cells increased to 10 cm dish were plated at a cell density of 1 ⁇ 10 9 cells / well on a 6-well plate coated with MATRIGEL TM (Becton Dickinson), and STO feeder cells were used. Incubated overnight in conditioned medium. Then, the medium was removed, 1 mL of CTK (0.25% trypsin, 1 mg / mL Collagenase IV, 20% KSR, 1 mM CaCl 2 in PBS) was added, treated at 37 ° C. for 5 minutes, and STO feeder cells were removed. Later, TIG3 / KOSM cells were detached by suspending by pipetting.
- CTK 0.25% trypsin, 1 mg / mL Collagenase IV, 20% KSR, 1 mM CaCl 2 in PBS
- the peeled TIG3 / KOSM cells are taken into a 15 mL tube, centrifuged at 1000 rpm (150 ⁇ g) for 5 minutes, the supernatant is removed, and the cell density is 1 ⁇ 10 9 cells / well in a Matrigel TM coated 6-well plate. Plated with.
- said conditioned medium was prepared as follows. Specifically, 7.5 ⁇ 10 6 mitomycin-treated STO cells were plated on a 15 cm dish, and the next day, the medium was replaced with human iPS medium (without FGF-2), treated for 1 to 3 hours, and then again into human iPS medium. The culture was changed and cultured for 24 hours. The next day, the supernatant was collected and the cells were removed by centrifugation at 1500 rpm (330 ⁇ g) for 10 minutes and stocked at ⁇ 20 to ⁇ 30 ° C.
- 1% (v / v) penicillin / streptomycin (Gibco), 0.2% (v / v) FBS (fetal bovine serum), 2 mM L-glutamine (Gibco), 100 ng / mL activin A (Gibco) are added.
- the medium was replaced with the Advanced RPMI 1640 medium (Gibco) and cultured for 2 days (step (A1-2)).
- step (D1) 1% (v / v) penicillin / streptomycin (Gibco), 2% (v / v) B-27 TM supplement (Gibco), 10 ⁇ M DAPT (Sigma), 55 nM exendin-4 (Phoenix Pharmaceuticals). Further, the medium was changed to DMEM / Ham F12 medium (Gibco) supplemented with 2% (v / v) IBCAP culture supernatant or Mock culture supernatant, and cultured for 3 days (step (D1)).
- GIGAGON GIGAGON
- SST somatostatin gene expression of TIG3 / KOSM cells before differentiation induction and cells obtained through the step (E1) by quantitative RT-PCR confirmed.
- RNA was first extracted from cells using NucleoSpin TM RNA II (Takara Bio), and quantitative RT-PCR analysis was performed using Fast SYBR TM Green PCR Master Mix (Applied Biosystems). Primer sequences are shown below.
- HsGCG — 264F GCATTTACTTTGTGGCTGGA (SEQ ID NO: 4)
- HsGCG — 368R CCTGGGAAGCTGAGAATGAT (SEQ ID NO: 5)
- HsSST — 206F CCCCAGACTCCGTCAGTTTTC (SEQ ID NO: 6)
- HsSST — 313R TCCGTCTGGTTGGGTTCAG (SEQ ID NO: 7)
- PCR products were separated by 3% agarose gel electrophoresis and visualized with ethidium bromide, BioDoc-It Imaging System (BMbio).
- FIGS. 1 (a) and 1 (b) show relative values (induction ratio) where the expression levels of glucagon (GCG) and somatostatin (SST) in TIG3 / KOSM cells before differentiation induction are taken as 1.
- Example 2 In addition to adding IBCAP culture supernatant or Mock culture supernatant in steps (A1-1), (A1-1), or steps (B1), (C1), the culture period in step (E1) is 3 days In the same manner as in Example 1, TIG3 / KOSM cells were cultured, and gene expression of glucagon (GCG) and somatostatin (SST) was confirmed by quantitative RT-PCR.
- GCG glucagon
- SST somatostatin
- FIGS. 2 (a) and 2 (b) show relative values with the expression levels of glucagon (GCG) and somatostatin (SST) in TIG3 / KOSM cells before differentiation induction as 1.
- the glucagon (GCG) induction magnification was about 120.1 times in the case of no addition, whereas in the steps (A1-1) and (A1-2), the mocking rate When the culture supernatant was added, it was about 100.7 times, and when the IBCAP culture supernatant was added, it was about 193.8 times.
- the Mock culture supernatant was added in steps (B1) and (C1), the ratio was about 31.2 times, and when the IBCAP culture supernatant was added, the ratio was about 218.5 times.
- FIG. 2 (a) the glucagon (GCG) induction magnification was about 120.1 times in the case of no addition, whereas in the steps (A1-1) and (A1-2), the mocking rate When the culture supernatant was added, it was about 100.7 times, and when the IBCAP culture supernatant was added, it was about 193.8 times.
- the Mock culture supernatant was added in steps (B1) and (C1), the ratio was about 31.2
- the induction ratio of somatostatin (SST) was about 117.6 times, whereas steps (A1-1) and (A1-2)
- SST somatostatin
- steps (A1-1) and (A1-2) When the Mock culture supernatant was added in step 1, it was about 16.6 times, and when the IBCAP culture supernatant was added in steps (A1-1) and (A1-2), it was about 65.2 times.
- the ratio was about 8.8 times, and when IBCAP culture supernatant was added, the ratio was about 164.1 times.
- mouse pancreatic tissue stem / progenitor cells include Dr. Tec3DR cells donated by Matsumoto were used. This cell was established by cloning tissue stem / progenitor cells isolated from mouse fetal pancreas. Tec3DR cells were maintained in DMEM medium supplemented with 1% (v / v) penicillin / streptomycin (Gibco), 15% (v / v) FBS, 50 ⁇ M 2-mercaptoethanol (Gibco).
- Tec3DR cells were plated at a cell density of 1 ⁇ 10 5 cells / well in a 24-well plate, and 1% (v / v) penicillin / streptomycin (Gibco), 0.1% (v / v) Cultured in DMEM / Ham F12 medium (Gibco) supplemented with BSA (bovine serum albumin) (Sigma), 1% (v / v) ITS-X (Gibco), 55 ⁇ M 2-mercaptoethanol (Gibco) for 2 days (Step (A3)).
- Cells in culture were then collected by treatment with trypsin-EDTA, plated at a cell density of 1 ⁇ 10 5 cells / well in a 24-well plate, and 1% (v / v) penicillin / streptomycin (Gibco)
- the cells were cultured for 3 days in DMEM / Ham F12 medium (Gibco) supplemented with 2 mg / mL BSA (Sigma), 1% (v / v) ITS-X (Gibco), 10 ng / mL FGF-2 (Nacalai Tesque) ( Step (D3)).
- PCR products were separated by 3% agarose gel electrophoresis, stained with ethidium bromide, and visualized by BioDoc-It Imaging System (BMbio).
- FIG. 3 shows the expression level of insulin-1 (Ins1) in the cells obtained through the step (E3).
- FIG. 3 shows a relative value with the expression level of insulin-1 (Ins1) in Tec3DR cells before differentiation induction as 1.
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Abstract
Description
多能性幹細胞又は膵臓組織幹/前駆細胞から膵臓ホルモン産生細胞を生産する膵臓ホルモン産生細胞の生産方法であって、
多能性幹細胞又は膵臓組織幹/前駆細胞から膵臓ホルモン産生細胞への分化誘導過程で、下記(1)~(3)から選ばれる少なくとも1種の分化誘導促進剤を培地中に添加することを特徴とする膵臓ホルモン産生細胞の生産方法、
(1)配列番号1に記載の塩基配列からなるDNAによりコードされるアミノ酸配列からなるポリペプチド、
(2)配列番号1に記載の塩基配列からなるDNAによりコード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置換、欠失、及び/又は付加されたアミノ酸配列からなり、かつ、膵臓ホルモン産生細胞への分化誘導促進作用を持つポリペプチド、
(3)配列番号1に記載の塩基配列からなるDNA、又はこのDNAと相補的な塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズするDNAを外来遺伝子として組み込んだ細胞の培養上清。
(A1)TGF-β(トランスフォーミング増殖因子β)スーパーファミリーに属する増殖因子の存在下で多能性幹細胞を培養する工程、
(B1)上記工程(A1)で得られた細胞をFGF(線維芽細胞増殖因子)の存在下で培養する工程、
(C1)上記工程(B1)で得られた細胞をレチノイドの存在下で培養する工程、
(D1)上記工程(C1)で得られた細胞をγ-セクレターゼ阻害剤の存在下で培養する工程、及び
(E1)上記工程(D1)で得られた細胞を、エキセンジン-4、HGF(肝細胞増殖因子)、IGF-1(インスリン様増殖因子-1)、及びニコチンアミドからなる群から選択される少なくとも1種の因子の存在下で培養する工程、を含み、
上記工程(A1)~(E1)の少なくとも1つの工程で上記分化誘導促進剤を培地中に添加する上記[1]記載の膵臓ホルモン産生細胞の生産方法。
(A2)TGF-βスーパーファミリーに属する増殖因子と、Wnt(ウィングレス型MMTV組み込み部位)ファミリーに属する増殖因子及びGSK-3(グリコーゲン合成酵素キナーゼ-3)阻害剤からなる群から選択される少なくとも1種の因子との存在下で多能性幹細胞を培養する工程、
(B2)上記工程(A2)で得られた細胞をTGF-βスーパーファミリーに属する増殖因子の存在下で培養する工程、
(C2)上記工程(B2)で得られた細胞をレチノイドの存在下で培養する工程、
(D2)上記工程(C2)で得られた細胞を、cAMP(環状アデノシン一リン酸)増加剤、デキサメタゾン、TGF-β1型受容体阻害剤、及びニコチンアミドからなる群から選択される少なくとも1種の因子の存在下で培養する工程、を含み、
上記工程(A2)~(D2)の少なくとも1つの工程で上記分化誘導促進剤を培地中に添加する上記[1]記載の膵臓ホルモン産生細胞の生産方法。
(A3)TGF-βスーパーファミリーに属する増殖因子、レチノイド、FGF、及びニコチンアミドの非存在下で膵臓組織幹/前駆細胞を培養する工程、
(B3)上記工程(A3)で得られた細胞をTGF-βスーパーファミリーに属する増殖因子の存在下で培養する工程、
(C3)上記工程(B3)で得られた細胞をレチノイドの存在下で培養する工程、
(D3)上記工程(C3)で得られた細胞をFGFの存在下で培養する工程、及び
(E3)上記工程(D3)で得られた細胞をニコチンアミドの存在下で培養する工程
を含み、
上記工程(A3)~(E3)の少なくとも1つの工程で上記分化誘導促進剤を培地中に添加する上記[1]記載の膵臓ホルモン産生細胞の生産方法。
上記[1]~[4]のいずれか1項記載の膵臓ホルモン産生細胞の生産方法によって人工的に生産された膵臓ホルモン産生細胞。
次の(1)~(3)の少なくとも1種を含み、多能性幹細胞又は膵臓組織幹/前駆細胞から膵臓ホルモン産生細胞への分化を誘導する分化誘導促進剤;
(1)配列番号1に記載の塩基配列からなるDNAによりコードされるアミノ酸配列からなるポリペプチド、
(2)配列番号1に記載の塩基配列からなるDNAによりコード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置換、欠失、及び/又は付加されたアミノ酸配列からなり、かつ、膵臓ホルモン産生細胞への分化誘導促進作用を持つポリペプチド、
(3)配列番号1に記載の塩基配列からなるDNA、又はこのDNAと相補的な塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズするDNAを外来遺伝子として組み込んだ細胞の培養上清。
多能性幹細胞とは、少なくとも一種類ずつの三胚葉(外胚葉、中胚葉、内胚葉)に属する分化細胞に分化する能力(多分化能)のある自己複製可能な幹細胞のことをいい、例えば、誘導多能性幹細胞(iPS細胞)、胚性幹細胞(ES細胞)、胚性生殖細胞(EG細胞)、胚性癌細胞(EC細胞)、成体多能性幹細胞(APS細胞)等が包含される。本発明に係る生産方法では、その中でも、誘導多能性幹細胞(iPS細胞)又は胚性幹細胞(ES細胞)を用いることが好ましい。
(i)OCT遺伝子、KLF遺伝子、SOX遺伝子、MYC遺伝子
(ii)OCT遺伝子、SOX遺伝子、NANOG遺伝子、LIN28遺伝子
(iii)OCT遺伝子、KLF遺伝子、SOX遺伝子、MYC遺伝子、hTERT遺伝子、SV40 large T遺伝子
(iv)OCT遺伝子、KLF遺伝子、SOX遺伝子
膵臓組織幹/前駆細胞とは、動物の膵臓に存在する、多分化能、自己複製能を有する組織幹/前駆細胞である。膵臓組織幹/前駆細胞の由来は、特に限定されないが、哺乳動物が好ましく、ヒト又はマウスが特に好ましい。膵臓から組織幹/前駆細胞を分離する方法としては、従来公知の方法を任意に採用することができ、特に限定されない。例えば、胎児膵臓では、PDX1が膵臓組織幹/前駆細胞のマーカー分子として既知である(Jonsson,J. et al., Nature, 371, pp.606-609(1994);Offield,M.F. et al., Development, 22, pp.983-995(1996))。胎児のPDX1発現細胞は、内分泌細胞、外分泌細胞、及び膵管細胞に分化し、成体膵に存在するあらゆる種類の細胞を生じる。そこで、PDX1をマーカー分子として、膵臓組織幹/前駆細胞を分離することができる。
なお、この膵臓組織幹/前駆細胞は、株化されていないものであってもよく、株化されたものであってもよい。
本発明に係る膵臓ホルモン産生細胞の生産方法で用いられる分化誘導促進剤は、下記(1)~(3)から選ばれる少なくとも1種である。
(1)配列番号1に記載の塩基配列からなるDNAによりコードされるアミノ酸配列からなるポリペプチド。
(2)配列番号1に記載の塩基配列からなるDNAによりコード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置換、欠失、及び/又は付加されたアミノ酸配列からなり、かつ、膵臓ホルモン産生細胞への分化誘導促進作用を持つポリペプチド。
(3)配列番号1に記載の塩基配列からなるDNA、又はこのDNAと相補的な塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズするDNAを外来遺伝子として組み込んだ細胞の培養上清。
また、改変ポリペプチドと元のポリペプチドとの相同性は、80%以上が好ましく、90%以上がより好ましく、93%以上がさらに好ましく、95%以上が特に好ましく、98%以上が最も好ましい。
宿主細胞としては、細菌、酵母、昆虫細胞、動物細胞等の公知の細胞を適宜使用することができる。動物細胞としては、HEK293細胞、HEK293T細胞、CHO-K1細胞、COS細胞等が挙げられる。
本発明に係る膵臓ホルモン産生細胞の生産方法では、多能性幹細胞から膵臓ホルモン産生細胞への分化誘導過程で、上述した分化誘導促進剤を培地中に添加する。多能性幹細胞から膵臓ホルモン産生細胞への分化誘導方法としては、従来公知の方法を任意に採用することができ、特に限定されない。分化誘導促進剤としてポリペプチド又は改変ポリペプチドを添加する場合、その濃度は、10~200ng/mLが好ましく、50~180ng/mLがより好ましく、60~150ng/mLがさらに好ましい。また、分化誘導促進剤として培養上清を添加する場合、その濃度は、0.5~20%(v/v)が好ましく、1~10%(v/v)がより好ましく、1.5~5%(v/v)がさらに好ましい。
第1の分化誘導方法は、非特許文献1に記載の方法に準じたものである。この文献は参照により本願に援用する。
第1の分化誘導方法は、下記の工程(A1)~(E1)を含む。このうち少なくとも1つの工程で、上述した分化誘導促進剤が培地中に添加される。分化誘導促進剤を添加する工程は、工程(A1)~(C1)の少なくとも1つの工程であることが好ましく、工程(B1)~(C1)の少なくとも1つの工程であることがより好ましい。なお、ある工程に分化誘導促進剤を添加する場合、その工程の最初から添加してもよく、工程の途中から添加してもよい。
(A1)TGF-βスーパーファミリーに属する増殖因子の存在下で多能性幹細胞を培養する工程。
(B1)上記工程(A1)で得られた細胞をFGFの存在下で培養する工程。
(C1)上記工程(B1)で得られた細胞をレチノイドの存在下で培養する工程。
(D1)上記工程(C1)で得られた細胞をγ-セクレターゼ阻害剤の存在下で培養する工程。
(E1)上記工程(D1)で得られた細胞を、エキセンジン-4、HGF、IGF-1、及びニコチンアミドからなる群から選択される少なくとも1種の因子の存在下で培養する工程。
工程(A1)では、TGF-βスーパーファミリーに属する増殖因子の存在下で多能性幹細胞を培養する。
TGF-βスーパーファミリーに属する増殖因子の濃度は、5~250ng/mLが好ましく、10~200ng/mLがより好ましく、50~150ng/mLがさらに好ましい。
Wntファミリーに属する増殖因子としては、Wnt1、Wnt3a、Wnt5a、Wnt7a等が挙げられ、Wnt1、Wnt3aが好ましく、Wnt3aがより好ましい。
Wntファミリーに属する増殖因子の濃度は、1~1000ng/mLが好ましく、10~100ng/mLがより好ましく、10~50ng/mLがさらに好ましい。
胚体内胚葉細胞への分化誘導の進行は、形態学的観察によるほか、RT-PCRにより遺伝子発現を確認することによっても評価することができる。多能性幹細胞から胚体内胚葉細胞への分化が進行するに従って、幹細胞のマーカー遺伝子であるOCT4、NANOG、SOX2、ECAD等の発現が減少し、胚体内胚葉細胞のマーカー遺伝子であるSOX17、CER、FOXA2、CXCR4等の発現が亢進する。
そこで、工程(A1)を、無血清の第1の培地で培養する工程(A1-1)と、低血清の第2の培地で培養する工程(A1-2)とに分けることが好ましい。
中内胚葉細胞への分化誘導の進行は、形態学的観察によるほか、RT-PCRにより遺伝子発現を確認することによっても評価することができる。多能性幹細胞から中内胚葉細胞への分化が進行するに従って、幹細胞のマーカー遺伝子であるOCT4、NANOG、SOX2、ECAD等の発現が減少し、中内胚葉細胞のマーカー遺伝子であるBRA、FGF4、WNT3、NCAD等の発現が亢進する。
上述したとおり、胚体内胚葉細胞への分化誘導の進行は、形態学的観察によるほか、RT-PCRにより遺伝子発現を確認することによっても評価することができる。
工程(B1)では、工程(A1)で得られた細胞をFGFの存在下で培養する。
FGFの濃度は、5~150ng/mLが好ましく、10~100ng/mLがより好ましく、20~80ng/mLがさらに好ましい。
ヘッジホッグ経路阻害剤としては、KAAD-シクロパミン(28-[2-[[6-[(3-フェニルプロパノイル)アミノ]ヘキサノイル]アミノ]エチル]-17β,23β-エポキシベラトラマン-3-オン)、KAAD-シクロパミンの類似体、ジェルビン(17,23β-エポキシ-3β-ヒドロキシベラトラマン-11-オン)、ジェルビンの類似体、ヘッジホッグ経路遮断抗体等が挙げられ、その中でもKAAD-シクロパミンが好ましい。
ヘッジホッグ経路阻害剤の濃度は、0.01~5μMが好ましく、0.02~2μMがより好ましく、0.1~0.5μmがさらに好ましい。
なお、工程(A1)で低血清培地が用いられる場合、工程(B1)では、工程(A1)よりも高い血清濃度の培地を用いることが好ましい。
分化誘導の進行は、形態学的観察によるほか、RT-PCRにより遺伝子発現を確認することによっても評価することができる。分化が進行するに従って、HNF1B、HNF4A等の遺伝子の発現が亢進する。
工程(C1)では、工程(B1)で得られた細胞をレチノイドの存在下で培養する。
レチノイドの濃度は、0.2~10μMが好ましく、0.4~8μMがより好ましく、1~4μMがさらに好ましい。
ヘッジホッグ経路阻害剤としては、KAAD-シクロパミン、KAAD-シクロパミンの類似体、ジェルビン、ジェルビンの類似体、ヘッジホッグ経路遮断抗体等が挙げられ、その中でもKAAD-シクロパミンが好ましい。
ヘッジホッグ経路阻害剤の濃度は、0.01~5μMが好ましく、0.02~2μMがより好ましく、0.1~0.5μMがさらに好ましい。
FGFとしては、FGF-1、FGF-2(bFGF)、FGF-3、FGF-4、FGF-5、FGF-6、FGF-7、FGF-8、FGF-9、FGF-10、FGF-11、FGF-12、FGF-13、FGF-14、FGF-15、FGF-16、FGF-17、FGF-18、FGF-19、FGF-20、FGF-21、FGF-22、FGF-23等が挙げられ、FGF-2(bFGF)、FGF-5、FGF-7、FGF-10が好ましい。
FGFの濃度は、0.5~50ng/mLが好ましく、1~25ng/mLがより好ましく、2~10ng/mLがさらに好ましい。
TGF-βスーパーファミリーに属する増殖因子の濃度は、5~250ng/mLが好ましく、10~200ng/mLがより好ましく、20~150ng/mLがさらに好ましい。
B-27TMサプリメントの濃度は、0.1~10%(v/v)が好ましく、0.2~5%(v/v)がより好ましく、0.4~2.5%(v/v)がさらに好ましい。なお、このB-27TMサプリメントは、50倍ストック溶液として市販されているため、B-27TMサプリメントの濃度を0.1~10%(v/v)とするには、5~500倍希釈されるように培地中に添加すればよい。
分化誘導の進行は、形態学的観察によるほか、RT-PCRにより遺伝子発現を確認することによっても評価することができる。分化が進行するに従って、PDX1、HNF6、HLXB9等の遺伝子の発現が亢進する。
工程(D1)では、工程(C1)で得られた細胞をγ-セクレターゼ阻害剤の存在下で培養する。
γ-セクレターゼ阻害剤の濃度は、1~50μMが好ましく、2~40μMがより好ましく、5~20μMがさらに好ましい。
エキセンジン-4の濃度は、5~150ng/mLが好ましく、10~100ng/mLがより好ましく、20~80ng/mLがさらに好ましい。
分化誘導の進行は、形態学的観察によるほか、RT-PCRにより遺伝子発現を確認することによっても評価することができる。分化が進行するに従って、NKX6-1、NGN3、PAX4、NKX2-2等の遺伝子の発現が亢進する。
工程(E1)では、工程(D1)で得られた細胞を、エキセンジン-4、HGF、IGF-1、及びニコチンアミドからなる群から選択される少なくとも1種の因子の存在下で培養する。
エキセンジン-4の濃度は、5~150nMが好ましく、10~100nMがより好ましく、20~80nMがさらに好ましい。
HGFの濃度は、5~150ng/mLが好ましく、10~100ng/mLがより好ましく、20~80ng/mLがさらに好ましい。
IGF-1の濃度は、5~150ng/mLが好ましく、10~100ng/mLがより好ましく、20~80ng/mLがさらに好ましい。
ニコチンアミドの濃度は、1~30mMが好ましく、3~20mMがより好ましく、5~15mMがさらに好ましい。
この工程(E1)により、膵臓ホルモン産生細胞が得られる。
膵臓ホルモン産生細胞への分化誘導の進行は、インスリン、グルカゴン、ソマトスタチン等の膵臓ホルモンの産生を確認するほか、RT-PCRにより遺伝子発現を確認することによっても評価することができる。分化が進行するに従って、INS、GCG、GHRL、SST、PPY等のうち、少なくとも1つの遺伝子の発現が亢進する。
第2の分化誘導方法は、非特許文献4に記載の方法に準じたものである。この文献は参照により本願に援用する。
第2の分化誘導方法は、下記の工程(A2)~(D2)を含む。このうち少なくとも1つの工程で、上述した分化誘導促進剤が培地中に添加される。分化誘導促進剤を添加する工程は、工程(C2)~(D2)の少なくとも1つの工程であることが好ましく、工程(D2)であることが特に好ましい。なお、ある工程に分化誘導促進剤を添加する場合、その工程の最初から添加してもよく、工程の途中から添加してもよい。
(A2)TGF-βスーパーファミリーに属する増殖因子と、Wntファミリーに属する増殖因子及びGSK-3阻害剤からなる群から選択される少なくとも1種の因子との存在下で多能性幹細胞を培養する工程。
(B2)上記工程(A2)で得られた細胞をTGF-βスーパーファミリーに属する増殖因子の存在下で培養する工程。
(C2)上記工程(B2)で得られた細胞をレチノイドの存在下で培養する工程。
(D2)上記工程(C2)で得られた細胞を、cAMP増加剤、デキサメタゾン、TGF-β1型受容体阻害剤、及びニコチンアミドからなる群から選択される少なくとも1種の因子の存在下で培養する工程。
工程(A2)では、TGF-βスーパーファミリーに属する増殖因子と、Wntファミリーに属する増殖因子及びGSK-3阻害剤からなる群から選択される少なくとも1種の因子との存在下で多能性幹細胞を培養する。
TGF-βスーパーファミリーに属する増殖因子の濃度は、5~250ng/mLが好ましく、10~200ng/mLがより好ましく、50~150ng/mLがさらに好ましい。
Wntファミリーに属する増殖因子の濃度は、1~1000ng/mLが好ましく、10~100ng/mLがより好ましく、10~50ng/mLがさらに好ましい。
GSK-3阻害剤の濃度は、0.01~20μMが好ましく、0.1~20μMがより好ましく、1~5μMがさらに好ましい。
工程(A2)の培養期間は例えば1~3日であり、1~2日が好ましい。
工程(B2)では、工程(A2)で得られた細胞をTGF-βスーパーファミリーに属する増殖因子の存在下で培養する。
TGF-βスーパーファミリーに属する増殖因子の濃度は、5~250ng/mLが好ましく、10~200ng/mLがより好ましく、50~150ng/mLがさらに好ましい。
工程(B2)の培養期間は例えば1~4日であり、1~3日が好ましい。
工程(C2)では、工程(B2)で得られた細胞をレチノイドの存在下で培養する。
レチノイドの濃度は、0.2~10μMが好ましく、0.4~8μMがより好ましく、1~4μMがさらに好ましい。
BMP受容体阻害剤としては、ドルソモルフィン(6-[4-[2-(1-ピペリジニル)エトキシ]フェニル]-3-(4-ピリジル)ピラゾロ[1,5-a]ピリミジン)、LDN-193189(4-(6-(4-(ピペラジン-1-イル)フェニル)ピラゾロ[1,5-a]ピリミジン-3-イル)キノリン)等が挙げられ、その中でもドルソモルフィンが好ましい。
BMP受容体阻害剤の濃度は、0.2~5μMが好ましく、0.3~3μMがより好ましく、0.5~2μMがさらに好ましい。
TGF-β1型受容体阻害剤としては、SB431542(4-[4-(1,3-ベンゾジオキソル-5-イル)-5-(2-ピリジニル)-1H-イミダゾール-2-イル]ベンズアミド)、SB525334(6-[2-(1,1-ジメチルエチル)-5-(6-メチル-1,2-ピリジニル)-1H-イミダゾール-4-イル]キノキサリン)、LY364947(4-[3-(2-ピリジニル)-1H-ピラゾール-4-イル]キノリン)等が挙げられ、その中でもSB431542が好ましい。また、TGF-β1型受容体阻害剤としては、Calbiochem製のAlk5インヒビターIIを用いることも可能である。
TGF-β1型受容体阻害剤の濃度は、1~50μMが好ましく、2~30μMがより好ましく、5~20μMがさらに好ましい。
B-27TMサプリメントの濃度は、0.1~10%(v/v)が好ましく、0.2~5%(v/v)がより好ましく、0.4~2.5%(v/v)がさらに好ましい。なお、このB-27TMサプリメントは、50倍ストック溶液として市販されているため、B-27TMサプリメントの濃度を0.1~10%(v/v)とするには、5~500倍希釈されるように培地中に添加すればよい。
工程(C2)の培養期間は例えば5~9日であり、6~8日が好ましい。
工程(D2)では、工程(C2)で得られた細胞を、cAMP増加剤、デキサメタゾン、TGF-β1型受容体阻害剤、及びニコチンアミドからなる群から選択される少なくとも1種の因子の存在下で培養する。
cAMP増加剤の濃度は、1~50μMが好ましく、2~30μMがより好ましく、5~20μMがさらに好ましい。
TGF-β1型受容体阻害剤の濃度は、1~50μMが好ましく、2~30μMがより好ましく、5~20μMがさらに好ましい。
工程(D2)の培養期間は例えば9~13日であり、10~12日が好ましい。
膵臓ホルモン産生細胞への分化誘導の進行は、インスリン、グルカゴン、ソマトスタチン等の膵臓ホルモンの産生を確認するほか、RT-PCRにより遺伝子発現を確認することによっても評価することができる。多能性幹細胞から膵臓ホルモン産生細胞への分化が進行するに従って、膵臓ホルモン産生細胞のマーカー遺伝子であるINS、GCG、GHRL、SST、PPY等のうち、少なくとも1つの遺伝子の発現が亢進する。
本発明に係る膵臓ホルモン産生細胞の生産方法では、膵臓組織幹/前駆細胞から膵臓ホルモン産生細胞への分化誘導過程で、上述した分化誘導促進剤を培地中に添加する。膵臓組織幹/前駆細胞から膵臓ホルモン産生細胞への分化誘導方法としては、従来公知の方法を任意に採用することができ、特に限定されない。分化誘導促進剤としてポリペプチド又は改変ポリペプチドを添加する場合、その濃度は、10~200ng/mLが好ましく、50~150ng/mLがより好ましく、60~120ng/mLがさらに好ましい。また、分化誘導促進剤として培養上清を添加する場合、その濃度は、0.5~20%(v/v)が好ましく、1~10%(v/v)がより好ましく、1.5~5%(v/v)がさらに好ましい。
この分化誘導方法は、下記の工程(A3)~(E3)を含む。このうち少なくとも1つの工程で、上述した分化誘導促進剤が培地中に添加される。分化誘導促進剤を添加する工程は、工程(D3)~(E3)の少なくとも1つの工程であることが好ましく、工程(E3)であることが特に好ましい。なお、ある工程に分化誘導促進剤を添加する場合、その工程の最初から添加してもよく、工程の途中から添加してもよい。
(A3)TGF-βスーパーファミリーに属する増殖因子、レチノイド、FGF、及びニコチンアミドの非存在下で膵臓組織幹/前駆細胞を培養する工程。
(B3)上記工程(A3)で得られた細胞をTGF-βスーパーファミリーに属する増殖因子の存在下で培養する工程。
(C3)上記工程(B3)で得られた細胞をレチノイドの存在下で培養する工程。
(D3)上記工程(C3)で得られた細胞をFGFの存在下で培養する工程。
(E3)上記工程(D3)で得られた細胞をニコチンアミドの存在下で培養する工程。
工程(A3)では、TGF-βスーパーファミリーに属する増殖因子、レチノイド、FGF、ニコチンアミドの非存在下で膵臓組織幹/前駆細胞を培養する。
インスリンの濃度は、2~30μg/mLが好ましく、5~20μg/mLがより好ましい。トランスフェリンの濃度は、1~20μg/mLが好ましく、3~10μg/mLがより好ましい。亜セレン酸の濃度は、1~20ng/mLが好ましく、5~20ng/mLがより好ましい。2-メルカプトエタノールの濃度は、50~200μMが好ましく、50~100μMがより好ましい。アルブミンの濃度は、1~10ng/mLが好ましく、2~5ng/mLがより好ましい。
工程(A3)の培養期間は例えば1~3日であり、1~2日が好ましい。
工程(B3)では、工程(A3)で得られた細胞をTGF-βスーパーファミリーに属する増殖因子の存在下で培養する。
TGF-βスーパーファミリーに属する増殖因子の濃度は、5~250ng/mLが好ましく、10~200ng/mLがより好ましく、50~150ng/mLがさらに好ましい。
工程(B3)の培養期間は例えば2~6日であり、3~5日が好ましい。
工程(C3)では、工程(B3)で得られた細胞をレチノイドの存在下で培養する。
レチノイドの濃度は、0.2~10μMが好ましく、0.4~8μMがより好ましく、1~4μMがさらに好ましい。
好適な実施形態では、全トランス型レチノイン酸、ペニシリン、ストレプトマイシン等の抗生物質、インスリン、トランスフェリン、亜セレン酸、2-メルカプトエタノール、アルブミンを添加した、無血清のDMEM/ハムF12が用いられる。
工程(C3)の培養期間は例えば2~6日であり、3~5日が好ましい。
工程(D3)では、工程(C3)で得られた細胞をFGFの存在下で培養する。
FGFの濃度は、1~30ng/mLが好ましく、2~20ng/mLがより好ましく、5~15ng/mLがさらに好ましい。
好適な実施形態では、FGF-2(bFGF)、ペニシリン、ストレプトマイシン等の抗生物質、インスリン、トランスフェリン、亜セレン酸、アルブミンを添加した、無血清のDMEM/ハムF12が用いられる。
工程(D3)の培養期間は例えば1~5日であり、2~4日が好ましい。
工程(E3)では、工程(D3)で得られた細胞をニコチンアミドの存在下で培養する。
ニコチンアミドの濃度は、1~30mMが好ましく、3~20mMがより好ましく、5~15mMがさらに好ましい。
FGFとしては、FGF-1、FGF-2(bFGF)、FGF-3、FGF-4、FGF-5、FGF-6、FGF-7、FGF-8、FGF-9、FGF-10、FGF-11、FGF-12、FGF-13、FGF-14、FGF-15、FGF-16、FGF-17、FGF-18、FGF-19、FGF-20、FGF-21、FGF-22、FGF-23等が挙げられ、FGF-2(bFGF)、FGF-5、FGF-7、FGF-10が好ましい。
FGFの濃度は、1~30ng/mLが好ましく、2~20ng/mLがより好ましく、5~15ng/mLがさらに好ましい。
好適な実施形態では、ニコチンアミド、FGF-2(bFGF)、ペニシリン、ストレプトマイシン等の抗生物質、インスリン、トランスフェリン、亜セレン酸、アルブミンを添加した、無血清のDMEM/ハムF12が用いられる。
工程(E3)の培養期間は例えば3~20日であり、3~10日が好ましい。
膵臓ホルモン産生細胞への分化誘導の進行は、インスリン、グルカゴン、ソマトスタチン等の膵臓ホルモンの産生を確認するほか、RT-PCRにより遺伝子発現を確認することによっても評価することができる。膵臓組織幹/前駆細胞から膵臓ホルモン産生細胞への分化が進行するに従って、膵臓ホルモン産生細胞のマーカー遺伝子であるINS、GCG、GHRL、SST、PPY等のうち、少なくとも1つの遺伝子の発現が亢進する。
上述のようにして得られた膵臓ホルモン産生細胞は、糖尿病等の治療薬に応用することができる。例えば、膵臓ホルモン産生細胞がインスリンを産生・分泌する場合には、そのインスリン産生細胞をそのまま、あるいはフィルター濾過により濃縮したペレット等の細胞塊を糖尿病治療薬として用いることができる。この糖尿病治療薬は、DMSO等の保護剤を加え、凍結保存することもできる。なお、より安全に利用するためには、加熱処理、放射線処理、マイトマイシンC処理など、糖尿病治療薬としての機能を残しつつ、病原体のタンパク質が変性する程度の条件下で処理をすることが好ましい。
(1)分化誘導促進剤の調製
分化誘導促進剤は以下のようにして調製した。10% FBS(ウシ胎児血清)(ニチレイ、171012)、1% ペニシリン/ストレプトマイシン(Life Technologies Japan、15140-122)を添加したハムF12培地(Sigma、N6658)で継代培養したCHO-K1細胞を10cmディッシュに5×105個プレーティングした。その翌日、FuGENE6(Roche)を使用して、配列番号1に記載の塩基配列からなるDNAによりコードされるポリペプチド(以下、「IBCAP」という。)の発現ベクター(pCAGGS-IBCAP)をCHO-K1細胞にトランスフェクトし、IBCAPを強制発現させた。その48時間後に細胞を1/20濃度に希釈し、10cmディッシュに再度プレーティングした。その翌日、最終濃度400μg/mLのG418(ナカライテスク、09380-44)を添加し、以後、3~5日おきに培地交換を行い、コロニー形成させた。限界希釈法でクローン化したコロニーを単離し、増殖後、サザンブロット法及びノザンブロット法で遺伝子発現を確認し、安定型IBCAP発現CHO-K1細胞株(以下、「IBCAP発現Stable CHO細胞」という。)を作製した。
ヒトiPS細胞としては、埼玉医科大学のDr.Mitaniから供与されたTIG3/KOSM細胞を用いた。この細胞は、センダイウイルスを用いてTIG-3細胞に4因子(OCT遺伝子、KLF遺伝子、SOX遺伝子、MYC遺伝子)を導入することにより、産業技術総合研究所にて樹立されたものである(Nishimura,K. et al., J. Biol. Chem., 286, pp.4760-4771(2011))。TIG3/KOSM細胞は、1%(v/v) ペニシリン/ストレプトマイシン(Gibco)、20%(v/v) KnockoutTM血清代替物(Gibco)、1% (v/v) 非必須アミノ酸(Gibco)、2.5mM L-グルタミン、0.1mM 2-メルカプトエタノール(Gibco)、5ng/mL FGF-2(R&D Systems)、5mM 塩化ナトリウムを添加したDMEM/ハムF12培地中で維持した。
分化誘導前のTIG3/KOSM細胞、及び工程(E1)を経て得られた細胞について、グルカゴン(GCG)及びソマトスタチン(SST)の遺伝子発現を定量的RT-PCRで確認した。具体的には、まず、NucleoSpinTM RNA II(タカラバイオ)を用いて細胞からRNAを抽出し、Fast SYBRTM Green PCR Master Mix(Applied Biosystems)を用いて定量的RT-PCR分析を行った。プライマー配列を以下に示す。
HsGCG_264F:GCATTTACTTTGTGGCTGGA(配列番号4)
HsGCG_368R:CCTGGGAAGCTGAGAATGAT(配列番号5)
HsSST_206F:CCCCAGACTCCGTCAGTTTC(配列番号6)
HsSST_313R:TCCGTCTGGTTGGGTTCAG(配列番号7)
また、図1(b)に示すように、無添加の場合にはソマトスタチン(SST)の誘導倍率がほぼ0倍であったのに対し、工程(D1)、(E1)でMock培養上清を添加した場合には約0.4倍であり、IBCAP培養上清を添加した場合には約9.5倍であった。
この結果から、工程(D1)、(E1)でIBCAP培養上清を添加することにより、ヒトiPS細胞から膵臓ホルモン産生細胞への分化誘導効率が向上することが分かる。
IBCAP培養上清又はMock培養上清を工程(A1-1)、(A1-1)、あるいは工程(B1)、(C1)で添加するとともに、工程(E1)の培養期間を3日間とするほかは、実施例1と同様にしてTIG3/KOSM細胞を培養し、グルカゴン(GCG)及びソマトスタチン(SST)の遺伝子発現を定量的RT-PCRで確認した。
また、図2(b)に示すように、無添加の場合にはソマトスタチン(SST)の誘導倍率が約117.6倍であったのに対し、工程(A1-1)、(A1-2)でMock培養上清を添加した場合には約16.6倍であり、工程(A1-1)、(A1-2)でIBCAP培養上清を添加した場合には約65.2倍であった。また、工程(B1)、(C1)でMock培養上清を添加した場合には約8.8倍であり、IBCAP培養上清を添加した場合には約164.1倍であった。
この結果から、工程(A1-1)、(A1-2)でIBCAP培養上清を添加する場合と、工程(B1)、(C1)でIBCAP培養上清を添加する場合とのいずれも、ヒトiPS細胞から膵臓ホルモン産生細胞への分化誘導効率が向上することが分かる。
(1)マウス膵臓組織幹/前駆細胞から膵臓ホルモン産生細胞への分化誘導
マウス膵臓組織幹/前駆細胞としては、埼玉医科大学のDr.Matsumotoから供与されたTec3DR細胞を用いた。この細胞は、マウス胎児期の膵臓から分離された組織幹/前駆細胞をクローン化することにより樹立されたものである。Tec3DR細胞は、1%(v/v) ペニシリン/ストレプトマイシン(Gibco)、15%(v/v) FBS、50μM 2-メルカプトエタノール(Gibco)を添加したDMEM培地中で維持した。
分化誘導前のTec3DR細胞、及び工程(E3)を経て得られた細胞について、マウスインスリン-1(Ins1)の遺伝子発現を定量的RT-PCRで確認した。具体的には、まず、NucleoSpinTM RNA II(タカラバイオ)を用いて細胞からRNAを抽出し、Power SYBRTM Green PCR Master Mix(Applied Biosystems)を用いて定量的RT-PCR分析を行った。プライマー配列を以下に示す。
MnIns1_qPCR_Fw:CACTTCCTACCCCTGCTGG(配列番号8)
MnIns1_qPCR_Rv:ACGCCAAGGTCTGAAGGTC(配列番号9)
この結果から、工程(E3)でIBCAP培養上清を添加することにより、マウス膵臓組織幹/前駆細胞から膵臓ホルモン産生細胞への分化誘導効率が向上することが分かる。
Claims (6)
- 多能性幹細胞又は膵臓組織幹/前駆細胞から膵臓ホルモン産生細胞を生産する膵臓ホルモン産生細胞の生産方法であって、
多能性幹細胞又は膵臓組織幹/前駆細胞から膵臓ホルモン産生細胞への分化誘導過程で、下記(1)~(3)から選ばれる少なくとも1種の分化誘導促進剤を培地中に添加することを特徴とする膵臓ホルモン産生細胞の生産方法、
(1)配列番号1に記載の塩基配列からなるDNAによりコードされるアミノ酸配列からなるポリペプチド、
(2)配列番号1に記載の塩基配列からなるDNAによりコード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置換、欠失、及び/又は付加されたアミノ酸配列からなり、かつ、膵臓ホルモン産生細胞への分化誘導促進作用を持つポリペプチド、
(3)配列番号1に記載の塩基配列からなるDNA、又はこのDNAと相補的な塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズするDNAを外来遺伝子として組み込んだ細胞の培養上清。 - (A1)TGF-β(トランスフォーミング増殖因子β)スーパーファミリーに属する増殖因子の存在下で多能性幹細胞を培養する工程、
(B1)前記工程(A1)で得られた細胞をFGF(線維芽細胞増殖因子)の存在下で培養する工程、
(C1)前記工程(B1)で得られた細胞をレチノイドの存在下で培養する工程、
(D1)前記工程(C1)で得られた細胞をγ-セクレターゼ阻害剤の存在下で培養する工程、及び
(E1)前記工程(D1)で得られた細胞を、エキセンジン-4、HGF(肝細胞増殖因子)、IGF-1(インスリン様増殖因子-1)、及びニコチンアミドからなる群から選択される少なくとも1種の因子の存在下で培養する工程、を含み、
前記工程(A1)~(E1)の少なくとも1つの工程で前記分化誘導促進剤を培地中に添加する請求項1記載の膵臓ホルモン産生細胞の生産方法。 - (A2)TGF-β(トランスフォーミング増殖因子β)スーパーファミリーに属する増殖因子と、Wnt(ウィングレス型MMTV組み込み部位)ファミリーに属する増殖因子及びGSK-3(グリコーゲン合成酵素キナーゼ-3)阻害剤からなる群から選択される少なくとも1種の因子との存在下で多能性幹細胞を培養する工程、
(B2)前記工程(A2)で得られた細胞をTGF-βスーパーファミリーに属する増殖因子の存在下で培養する工程、
(C2)前記工程(B2)で得られた細胞をレチノイドの存在下で培養する工程、
(D2)前記工程(C2)で得られた細胞を、cAMP(環状アデノシン一リン酸)増加剤、デキサメタゾン、TGF-β1型受容体阻害剤、及びニコチンアミドからなる群から選択される少なくとも1種の因子の存在下で培養する工程、を含み、
前記工程(A2)~(D2)の少なくとも1つの工程で前記分化誘導促進剤を培地中に添加する請求項1記載の膵臓ホルモン産生細胞の生産方法。 - (A3)TGF-β(トランスフォーミング増殖因子β)スーパーファミリーに属する増殖因子、レチノイド、FGF(線維芽細胞増殖因子)、及びニコチンアミドの非存在下で膵臓組織幹/前駆細胞を培養する工程、
(B3)前記工程(A3)で得られた細胞をTGF-βスーパーファミリーに属する増殖因子の存在下で培養する工程、
(C3)前記工程(B3)で得られた細胞をレチノイドの存在下で培養する工程、
(D3)前記工程(C3)で得られた細胞をFGFの存在下で培養する工程、及び
(E3)前記工程(D3)で得られた細胞をニコチンアミドの存在下で培養する工程
を含み、
前記工程(A3)~(E3)の少なくとも1つの工程で前記分化誘導促進剤を培地中に添加する請求項1記載の膵臓ホルモン産生細胞の生産方法。 - 請求項1~4のいずれか1項記載の膵臓ホルモン産生細胞の生産方法によって人工的に生産された膵臓ホルモン産生細胞。
- 次の(1)~(3)の少なくとも1種を含み、多能性幹細胞又は膵臓組織幹/前駆細胞から膵臓ホルモン産生細胞への分化を誘導する分化誘導促進剤;
(1)配列番号1に記載の塩基配列からなるDNAによりコードされるアミノ酸配列からなるポリペプチド、
(2)配列番号1に記載の塩基配列からなるDNAによりコード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置換、欠失、及び/又は付加されたアミノ酸配列からなり、かつ、膵臓ホルモン産生細胞への分化誘導促進作用を持つポリペプチド、
(3)配列番号1に記載の塩基配列からなるDNA、又はこのDNAと相補的な塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズするDNAを外来遺伝子として組み込んだ細胞の培養上清。
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WO2015178397A1 (ja) * | 2014-05-20 | 2015-11-26 | 国立大学法人熊本大学 | インスリン産生細胞の分化誘導方法 |
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CN104988111A (zh) * | 2015-07-08 | 2015-10-21 | 深圳爱生再生医学科技有限公司 | 用于将uc-msc转化为胰岛细胞的诱导液及其应用 |
WO2017188378A1 (ja) * | 2016-04-28 | 2017-11-02 | 武田薬品工業株式会社 | 多能性幹細胞由来膵前駆細胞の純化法とその増幅法 |
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JP7369346B2 (ja) * | 2018-11-14 | 2023-10-26 | 株式会社片岡製作所 | インスリン産生細胞の製造方法、及び組成物 |
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CN111607556B (zh) * | 2019-01-25 | 2022-06-07 | 中国科学院广州生物医药与健康研究院 | 一种培养扩增人肝祖细胞的培养基及其应用 |
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EP2857500B1 (en) | 2018-01-31 |
US20150140661A1 (en) | 2015-05-21 |
JP6161603B2 (ja) | 2017-07-12 |
CN104428410B (zh) | 2016-08-31 |
CN104428410A (zh) | 2015-03-18 |
JPWO2013176249A1 (ja) | 2016-01-14 |
EP2857500A4 (en) | 2015-11-11 |
EP2857500A1 (en) | 2015-04-08 |
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