WO2012150707A1 - 幹細胞からインスリン産生細胞への分化誘導を促進する低分子化合物および該化合物を用いた幹細胞からインスリン産生細胞への分化誘導方法 - Google Patents
幹細胞からインスリン産生細胞への分化誘導を促進する低分子化合物および該化合物を用いた幹細胞からインスリン産生細胞への分化誘導方法 Download PDFInfo
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- WO2012150707A1 WO2012150707A1 PCT/JP2012/061549 JP2012061549W WO2012150707A1 WO 2012150707 A1 WO2012150707 A1 WO 2012150707A1 JP 2012061549 W JP2012061549 W JP 2012061549W WO 2012150707 A1 WO2012150707 A1 WO 2012150707A1
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- A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
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- C12N2501/30—Hormones
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- C12N2510/00—Genetically modified cells
Definitions
- the present invention relates to a low-molecular compound that promotes differentiation induction from stem cells to insulin-producing cells, and an agent for promoting differentiation induction from stem cells to insulin-producing cells containing the compound.
- the present invention also relates to a method for inducing differentiation from a stem cell to an insulin-producing cell using the compound, and an insulin-producing cell induced by the method.
- the present invention relates to a compound that promotes differentiation induction from ES cells into insulin-producing cells, an agent for promoting differentiation induction from ES cells containing the compound into insulin-producing cells, and ES cells using the compounds.
- the present invention relates to a method for inducing differentiation into insulin-producing cells and insulin-producing cells induced by the method.
- Islet transplantation is a useful treatment for patients with insulin-dependent diabetes.
- the pancreas is composed of exocrine and endocrine glands. Insulin, the only hormone in the body that acts to lower blood sugar, is secreted from the beta cells of the islet. Islet transplantation aims to replace this islet from the pancreas and transplant it to a patient with insulin-dependent diabetes to replace and regenerate the hypoglycemic system.
- Diabetes is roughly classified into two. One is that pancreatic ⁇ cells are destroyed or damaged due to some cause and insulin secretion is lost, and the other is that insulin is present in the blood normally or more than normal, but insulin resistance exists in peripheral tissues The action of insulin is reduced.
- the former is called type 1 diabetes or juvenile type diabetes, and the latter is called type 2 diabetes.
- the indication for islet transplantation is type 1 diabetes.
- Type 1 diabetes is caused by the specific destruction of pancreatic ⁇ -cells that produce insulin due to abnormal autoimmunity.
- islet transplantation which is one of the regenerative replacement therapies for pancreatic ⁇ cells, can be considered, but current islet transplantation has a serious problem that its supply amount is insufficient. In general, it takes several to a dozen years to benefit from transplantation. Therefore, transplantation is not mentioned as a treatment option presented to patients with type 1 diabetes in daily clinical practice.
- Non-Patent Documents 1 to 3 the induction of differentiation of insulin-secreting cells from embryonic stem cells (ES cells), tissue stem cells or human induced pluripotent stem cells (iPS cells) has attracted attention as a source of cells that replace human mature islet ⁇ cells. Many reports have been made so far.
- ES cells are pluripotent stem cells derived from the inner cell mass of blastocysts. It has been shown that when ES cells are cultured with embryonic mesenchymal cells, ES cells are directed to the pancreas and liver lineage. The present inventors have reported that differentiation into the liver can be induced with high efficiency by culturing ES cells on M15 cells, which are a feeder cell line (Non-patent Document 4). The present inventors have established a high-efficiency differentiation induction technique from ES cells to embryonic endoderm respiratory and digestive organs by planar culture by a method using feeder cells (Patent Documents 1 and 2).
- ES cells can be induced in vitro over time to organs derived from mesendoderm, definitive endoderm, and finally region-specific definitive endoderm.
- M15 feeder cells are useful tools for generating lineage-specific cell types derived from ES cells belonging to three types of germ layers (neural ectoderm, mesoderm and definitive endoderm) (Non-patent Document 6).
- Non-patent Document 6 The present inventors have further reported an efficient differentiation induction method from ES cells to pancreas and liver using M15M cells.
- Patent Document 3 the above method is a method using living support cells, and when screening low molecular weight compounds that affect differentiation induction of ES cells, it is not possible to exclude the influence of support cells, and the target compound is efficiently obtained. There was a problem with screening.
- An object of the present invention is to provide a substance that promotes differentiation induction of insulin-producing cells from stem cells.
- an object of the present invention is to provide a low-molecular compound that promotes differentiation induction from ES cells into insulin-producing cells.
- Another object of the present invention is to provide a method for inducing differentiation of insulin-producing cells from ES cells using such a compound and a differentiation-inducing promoter for insulin-producing cells.
- the object of the present invention is also to provide insulin-producing cells so induced.
- the present inventors differentiated into endoderm, mesoderm, and ectoderm by culturing ES cells using a synthetic nanofiber (sNF) matrix without using supporting cells. I found out that it can be guided. That is, it was found that ES cells grown on a synthetic nanofiber matrix can be induced into the endoderm and into the pancreas.
- the induction efficiency in this method is high enough to obtain candidate compounds that increase the proportion of insulin-expressing cells by using Pdx1 / GFP ES cell lines or Ins1 / GFP ES cell lines in high-throughput screening of small molecules be able to.
- a differentiation induction promoter for inducing differentiation of insulin-producing cells from mammalian stem cells comprising at least one compound selected from the group consisting of dopamine metabolism inhibitors and serotonin metabolism inhibitors.
- the compound is a vesicular monoamine transporter (VMAT2) inhibitor, a dopamine or serotonin synthesis inhibitor, a dopamine D2 receptor inhibitor, an agonist of dopamine or serotonin, a monoamine oxidase inhibitor, a MAO-A inhibitor,
- VMAT2 vesicular monoamine transporter
- H1 antagonists at least one compound selected from the group consisting of antihistamines, H1 antagonists, histamine H2 receptor inhibitors, histamine H2 receptor agonists, selective serotonin reuptake inhibitors, dopamine antagonists, and 5-HT3 receptor antagonists
- the differentiation induction promoter according to 1).
- VMAT2 vesicular monoamine transporter
- the compound is reserpine, tetrabenazine, carbidopa, ⁇ -methyltyrosine, 5HTP, bromocriptine, quinacrine dihydrochloride dihydrate, pirulindol mesylate, pargyline hydrochloride, fexofenadine hydrochloride, hydroxyzine dihydrochloride
- Differentiation induction promoter (9) The differentiation induction promoter according to (8) above, wherein the compound is a compound selected from the group consisting of acetylcotine, etheroline fumarate, palmatin chloride, carbachol, and gingolide A.
- the mammalian stem cell is an embryonic stem cell (ES cell), a tissue stem cell, or an induced pluripotent stem cell (iPS cell). Accelerator.
- Insulin-producing cells from stem cells derived from mammals comprising culturing or treating stem cells derived from mammals in a medium containing at least one compound selected from the group consisting of dopamine metabolism inhibitors and serotonin metabolism inhibitors. To induce differentiation.
- the compound is a vesicular monoamine transporter (VMAT2) inhibitor, a dopamine or serotonin synthesis inhibitor, a dopamine D2 receptor inhibitor, an agonist of dopamine or serotonin, a monoamine oxidase inhibitor, a MAO-A inhibitor,
- VMAT2 vesicular monoamine transporter
- the above-mentioned at least one compound selected from the group consisting of antihistamines, H1 antagonists, histamine H2 receptor inhibitors, histamine H2 receptor agonists, selective serotonin reuptake inhibitors, dopamine antagonists, and 5-HT3 receptor antagonists
- the differentiation induction method according to 14).
- VMAT2 vesicle monoamine transporter
- the compound is reserpine, tetrabenazine, carbidopa, ⁇ -methyltyrosine, 5HTP, bromocriptine, quinacrine dihydrochloride dihydrate, pirrindol mesylate, pargyline hydrochloride, fexofenadine hydrochloride, hydroxyzine dihydrochloride
- (21) Culturing or treating stem cells derived from mammals in a medium containing at least one compound selected from the group consisting of acetylcotine, an acetylcholine degrading enzyme inhibitor acetylcholine receptor activator, and a cholinergic antagonist.
- a method for inducing differentiation of insulin-producing cells from stem cells derived from mammals. (22) The differentiation-inducing method according to (21), wherein the compound is selected from the group consisting of acetylcotine, etheroline fumarate, palmatin chloride, carbachol, and gingolide A.
- a method for inducing differentiation of an insulin-producing cell from a stem cell derived from a mammal comprising culturing or treating the stem cell derived from a mammal with a medium containing folic acid.
- ES cell embryonic stem cell
- iPS cell induced pluripotent stem cell
- the differentiation inducing method includes (1) a medium containing activin and bFGF, (2) a medium containing retinoic acid, FGF-10, B27 SUPPLEMENT and Shh signaling antagonist, and (3) a medium containing nicotinamide, at least one medium condition selected
- a pharmaceutical composition comprising the differentiation induction promoter according to any one of (1) to (13).
- stem cells derived from mammals can be efficiently induced to differentiate into insulin-producing cells.
- the insulin-producing cells thus induced to induce differentiation and the differentiation induction promoter of the present invention are useful not only for the study of differentiation induction of stem cells into pancreatic cells but also as therapeutic agents for type 1 diabetes. It is.
- FIG. 1 is a diagram showing an outline of a low molecular weight compound screening method for promoting mouse ES cell differentiation using a nanofiber plate.
- FIG. 2 is a diagram showing an outline of a scheme for determining a compound candidate in quantitative image analysis in compound screening.
- FIG. 2A shows that, in image analysis, in addition to nuclear staining with DAPI, Pdx1 is stained with Alexa 488 and Insulin is stained with Alexa 568 for fluorescence observation.
- FIG. 2B shows that the area occupied by triple positive cells of DAPI, Pdx1, and Insulin is calculated by image analysis using MetaXpress software, and this is a numerical value that reflects the number of insulin positive cells.
- FIG. 3 is a diagram showing a screening process.
- FIG. 3 is a diagram showing a screening process.
- FIG. 4 is a diagram showing the results of examining the concentration-dependent effect of reserpine, which is one of the present invention. The results of fluorescence observation after staining Pdx1 with Alexa 488 and insulin with Alexa 568 are shown.
- FIG. 5 is a diagram comparing the differentiation promoting effects of each compound selected by compound screening into insulin-expressing cells. The results of fluorescence observation after staining Pdx1 with Alexa 488 and insulin with Alexa 568 are shown.
- FIG. 6 is a graph showing the influence of a monoamine oxidase inhibitor (MAOBi) on differentiation into insulin-expressing cells and the influence of reserpine on the differentiation promoting effect.
- MAOBi monoamine oxidase inhibitor
- FIG. 9 shows the effect of reserpine on the differentiation of dopamine D2 receptor knockdown cell line.
- FIG. 9A shows the expression of Drd2.
- FIG. 9B is a graph showing an increase in differentiation of normal ES cells (NS) and dopamine D2 receptor knockdown cell line (KD) into insulin-producing cells in a ratio to the control. Res shows the case where reserpine is added.
- NS normal ES cells
- KD dopamine D2 receptor knockdown cell line
- FIG. 10 shows the results of examining the effect of cAMP analogs on differentiation of normal ES cells (NS) and vesicular monoamine transporter knockdown cell lines (VMAT2KD) into insulin-producing cells.
- FIG. 11A is a diagram showing the result of FIG. 10 as a ratio to the control (normal cells not added with cAMP analog are taken as 1).
- FIG. 11B shows the synergistic effect of reserpine, dopamine and dBu-cAMP.
- FIG. 12A is a diagram showing the results of examining the amount of insulin in cells differentiated using the compound of the present invention.
- FIG. 12B is a graph showing the results of examining the glucose concentration-dependent insulin secretion amount of cells differentiated using the compound of the present invention.
- FIG. 13 shows the results of examining the expression of response molecules to glucose sensing and secretion in cells differentiated with the compound of the present invention using real-time PCR.
- FIG. 14 is a graph showing changes in the number of Pdx1 / GFP positive cells and the number of insulin positive cells by addition of serotonin and histamine.
- FIG. 15 is a diagram showing changes in the expression levels of Ngn3, Nkx6.1, MafA and Insulin1 genes when cultured under the conditions of addition of four compounds.
- FIG. 16 is a diagram showing the effect of addition of TBZ or dBu-cAMP on the Ngn3-positive cell induction rate.
- A is a schematic diagram showing the addition period of TBZ or dBu-cAMP.
- FIG. 17 is a diagram showing the effect of addition of dBu-cAMP on day 11 to day 17 of culture on secretion of sugar-responsive C-peptide. The upper part is a schematic diagram showing the addition period.
- FIG. 18 is a diagram showing the results of measuring GFP-positive cells with a cell sorter and measuring the C-peptide content and glucose concentration-dependent insulin secretion when each compound was added.
- the present invention is a compound that induces differentiation of stem cells derived from mammals into insulin-producing cells, or promotes the differentiation of these cells into insulin-producing cells, and a differentiation induction promoter containing these compounds.
- the present invention further relates to a pharmaceutical composition for promoting insulin production comprising these compounds.
- the present invention is also a method for inducing differentiation of stem cells derived from mammals into insulin-producing cells, or a method for promoting the differentiation of these cells into insulin-producing cells.
- the present invention is further an insulin producing cell differentiated by the above method of the present invention.
- the stem cell derived from a mammal used in the present invention may be a stem cell derived from a mammal, and the kind thereof is not particularly limited.
- a mouse, rat, pig, cow, monkey or human-derived cell is used.
- it is preferably derived from mouse or human.
- the mammalian stem cell used in the present invention is an embryonic stem cell (ES cell), tissue stem cell or induced pluripotent stem cell (iPS cell: induced pluripotent stem cell) derived from a mammal.
- ES cell embryonic stem cell
- iPS cell induced pluripotent stem cell
- ES cell embryonic stem cell
- iPS cell induced pluripotent stem cell
- Mammal-derived ES cells can be cultured by a conventional method, for example, 1000 unit / ml leukemia inhibitory factor (LIF) in the presence of mitomycin C-treated mouse embryonic fibroblasts (MEF) as feeder cells if necessary.
- LIF unit / ml leukemia inhibitory factor
- IPS cells are pluripotent cells obtained by reprogramming somatic cells.
- Artificial pluripotent stem cells are produced by a group of Professor Shinya Yamanaka at Kyoto University, a group of RudolfudoJaenisch et al. At Massachusetts Institute of Technology, a group of James Thomson et al. At University of Wisconsin, Harvard University Several groups have been successful, including the group by Konrad Hochedlinger et al. Artificial pluripotent stem cells are highly expected as ideal pluripotent cells without rejection and ethical problems. For example, International Publication No.
- WO2007 / 069666 discloses somatic cell nuclear reprogramming factors including gene products of Oct family gene, Klf family gene, and Myc family gene, and Oct family gene, Klf family gene, Sox family gene and A somatic cell nuclear reprogramming factor containing a gene product of a Myc family gene is described, and further, a pluripotent stem cell induced by somatic cell nuclear reprogramming, comprising a step of contacting the nuclear reprogramming factor with the somatic cell. A method of manufacturing is described.
- IPS cells used in the present invention can be produced by reprogramming somatic cells.
- the type of somatic cell used here is not particularly limited, and any somatic cell can be used. That is, the somatic cell referred to in the present invention includes all cells other than the internal germ cells of the cells constituting the living body, and may be a differentiated somatic cell or an undifferentiated stem cell.
- the somatic cell is derived from a mammal (for example, a rodent such as a mouse or a primate such as a human), particularly preferably a mouse or a human.
- any fetal, neonatal or adult somatic cells may be used.
- the iPS cells referred to in the present invention have a self-replicating ability over a long period of time under predetermined culture conditions (for example, conditions under which ES cells are cultured), and under the predetermined differentiation-inducing conditions, ectoderm, mesoderm and endoderm
- predetermined culture conditions for example, conditions under which ES cells are cultured
- differentiation-inducing conditions ectoderm, mesoderm and endoderm
- the induced pluripotent stem cell in the present invention may be a stem cell capable of forming a teratoma when transplanted to a test animal such as a mouse.
- the reprogramming factor is a substance (group) capable of inducing iPS cells by introduction into somatic cells or by contacting with somatic cells together with a factor for improving iPS cell establishment efficiency.
- the reprogramming factor is a substance (group) capable of inducing iPS cells by introduction into somatic cells or by contacting with somatic cells together with a factor for improving iPS cell establishment efficiency.
- it is a protein factor or a gene encoding it, or a low molecular weight compound.
- Specific examples of the combination of initialization factors include the following combinations, but are not limited thereto.
- the family shown here is a group of proteins identified by homology on the primary structure, that is, having a similar structure.
- the Oct family includes Oct3 / 4, which is a typical reprogramming factor of iPS cells.
- Klf family includes Klf4, Klf1, Klf5 etc.
- Sox family includes Sox2, Sox1, Sox3, Sox7 etc.
- Myc family includes c-Myc, L-Myc etc.
- the LIN28 family includes Lin28, Lin28B and the like
- the Glis family includes Glis1, Glis2, and Glis3.
- inducing differentiation-inducing insulin-producing cells from stem cells means other inducement of differentiation from stem cells to insulin-producing cells in addition to inducing differentiation from stem cells to insulin-producing cells. It means to include both in the case of promoting differentiation induction by using in combination with a substance.
- the differentiation induction promoter of the present invention includes at least one compound selected from the group consisting of a dopamine metabolism inhibitor and a serotonin metabolism inhibitor.
- a compound selected from a dopamine metabolism inhibitor or a serotonin metabolism inhibitor is an inhibitor of dopamine or a serotonin transporter, such as a vesicular monoamine transporter (VMAT2) inhibitor, a dopamine or a serotonin synthesis inhibitor.
- VMAT2 vesicular monoamine transporter
- Dopamine D2 receptor inhibitor agonist of dopamine or serotonin, monoamine oxidase inhibitor, MAO-A inhibitor, antihistamine, H1 antagonist, histamine H2 receptor inhibitor, histamine H2 receptor agonist, selective serotonin reuptake inhibitor, A dopamine antagonist or a 5-HT3 receptor antagonist can be mentioned.
- the synthesis inhibitor means a compound that inhibits synthesis, and includes, for example, an inhibitor of an enzyme involved in synthesis, and a synthetic product that inhibits the synthesis system by a feedback action.
- the dopamine metabolism inhibitor or serotonin metabolism inhibitor is not limited thereto, but for example, VMAT2 inhibitor reserpine or tetrabenazine, levodopa decarboxylase inhibitor carbidopa, serotonin synthesis inhibitor 5-hydroxytryptophan (5-HTP), dopamine synthesis inhibitor ⁇ -methyltyrosine, dopamine agonist bromocriptine, monoamine oxidase inhibitor quinacrine dihydrochloride dihydrate or pargyline hydrochloride, MAO-A inhibition Pirulindol mesylate as a drug, fexofenadine hydrochloride as an antihistamine, hydroxyzine dihydrochloride as an H1 antagonist or chlorpheniramine maleate, cimetidine as a histamine H2 receptor inhibitor, histamine H2 receptor jaw
- dimaprit dihydrochloride as a nyst
- fluvoxamine maleate as a selective serotonin
- the differentiation induction promoter of the present invention is a compound selected from the group consisting of a dopamine metabolism inhibitor and a serotonin metabolism inhibitor, and further contains cAMP, cAMP analog, G ⁇ s protein couple receptor. Body agonists, or antagonists of G ⁇ i protein coupled receptors. Thereby, a stronger differentiation induction or promotion effect can be obtained.
- the dopamine metabolism inhibitor or serotonin metabolism inhibitor has the same definition as above.
- the cAMP analog referred to in the present invention is a substance having cell membrane permeability by changing the structure of the base part or sugar part of cAMP. Specifically, for example, dibutyrate-cAMP and 8-CPT-2 -Can give me-cAMP.
- the differentiation induction promoter of the present invention contains at least one of reserpine or tetrabenazine and cAMP or cAMP analog.
- the differentiation induction promoter of the present invention comprises a compound selected from the group consisting of acetylcholine, acetylcholine degrading enzyme inhibitor, acetylcholine receptor activator, and cholinergic antagonist.
- acetylcholine degrading enzyme inhibitor include, but are not limited to, etheroline fumarate and palmatin chloride.
- acetylcholine receptor activator include, but are not limited to, carbachol.
- cholinergic antagonists include, but are not limited to, gingolide A.
- the differentiation induction promoter of the present invention can also be used in combination with other substances that induce differentiation of stem cells into insulin-producing cells. Thereby, differentiation induction of insulin-producing cells can be achieved more efficiently, and insulin secretion can be brought about.
- substances that induce differentiation of other insulin-producing cells include nicotinamide and glucagon-like peptide-1, but are not limited thereto.
- the differentiation induction promoter of the present invention can also be used as a pharmaceutical composition, which can be expected to improve pancreatic function of patients transplanted with type 1 diabetes appreciation and differentiation-induced pancreatic cells / tissues.
- it may be a pharmaceutical composition comprising the differentiation induction promoter of the present invention containing the above compound having differentiation-inducing activity and any known pharmaceutically acceptable carrier.
- the pharmaceutical composition containing the differentiation induction promoter of the present invention can be administered orally or parenterally.
- oral administration it can be administered in the form of capsules, tablets, granules, liquids and the like.
- parenteral administration it can be administered in the form of injection solution, infusion preparation or the like.
- the content of the compound of the present invention varies depending on the type of compound, the subject of administration, symptoms, and the administration method.
- the content of the compound of the present invention varies depending on the type of compound, the subject of administration, symptoms, and the administration method.
- for tetrabenazine, carbidopa, or cereroline fumarate 1 mg to 1000 mg per day, preferably 10 mg to 500 mg
- In bromocriptine it is 1 mg to 1000 mg, preferably 1 mg to 100 mg per day.
- the method of the present invention comprises culturing or treating stem cells derived from a mammal in a medium containing a compound selected from the group consisting of a dopamine metabolism inhibitor and a serotonin metabolism inhibitor.
- a method for inducing differentiation of insulin-producing cells from stem cells is a concentration that promotes differentiation induction from stem cells to insulin-producing cells, and affects the differentiation promotion that is added depending on the substance added and at the same time. Depending on the concentration of other substances to be changed, it can be appropriately changed.
- the concentration in the medium is 0.15 to 10.0 ⁇ M, preferably 0.15 to 2.5 ⁇ M, more preferably 0.63 ⁇ M to 1.25 ⁇ M.
- the concentration in the medium is 0.15 to 10.0 ⁇ M, preferably 1.25 to 5.0 ⁇ M.
- a known medium suitable for culturing or maintaining stem cells can be used, and examples thereof include Glasgow minimum essential medium and Dulbecco's modified Eagle medium.
- a compound selected from the group consisting of a dopamine metabolism inhibitor or a serotonin metabolism inhibitor cAMP, a cAMP analog, an agonist of a G ⁇ s protein couple receptor, or a G ⁇ i protein couple receptor.
- Antagonists can be included in the media.
- the concentration of cAMP or cAMP analog in the medium is 0.15 ⁇ M to 5.0 ⁇ M, preferably 0.3 ⁇ M to 0.6 ⁇ M. Thereby, a stronger differentiation induction or promotion effect can be obtained.
- the differentiation induction promoter of the present invention contains folic acid
- the differentiation induction method of the present invention comprises culturing or treating stem cells derived from mammals in a medium containing folic acid.
- the concentration of folic acid in the medium is 0.08 to 10.0 ⁇ M, preferably 0.15 to 0.63 ⁇ M.
- the method of the present invention is a stem cell derived from a mammal selected from the group consisting of acetylcotine, acetylcholine degrading enzyme inhibitor, acetylcholine receptor activator and cholinergic antagonist.
- a method for inducing differentiation of an insulin-producing cell from a stem cell comprising culturing or treating in a medium containing a compound.
- the acetylcholine degrading enzyme inhibitor, acetylcholine receptor activator or cholinergic antagonist is as defined above.
- the concentration of acetylcotine, acetylcholine degrading enzyme inhibitor, acetylcholine receptor activator or cholinergic antagonist added to the medium is a concentration that promotes differentiation induction from stem cells to insulin-producing cells. Depending on the concentration of other substances that affect the differentiation promotion added simultaneously.
- the concentration in the medium is 0.08 to 10.0 ⁇ M, preferably 0.15 to 0.63 ⁇ M, and in the case of eseroline fumarate, the concentration in the medium is 0.15 to 10.0.
- the concentration in the medium is 0.15-5.0 ⁇ M, preferably 0.63-2.5 ⁇ M.
- the timing of adding the compound of the present invention in the method for inducing differentiation of insulin-producing cells from the stem cells of the present invention is not particularly limited as long as it can effectively induce differentiation into insulin-producing cells, but preferably Pdx1-positive cells It is time before or before.
- insulin-producing cells derived from stem cells using the differentiation induction promoter of the present invention or the differentiation induction method of the present invention are provided.
- the insulin-producing cells of the present invention are useful as a source of cells in place of human mature islet ⁇ cells in islet transplantation therapy for diabetic patients.
- ES cell lines Two ES cell lines were used. One is a cell that expresses green fluorescent protein (GFP) under the control of the insulin 1 promoter and is named ING [Higuchi Y., Shiraki N., Yamane K, Qin Z., Mochitate K., Araki K ., Senokuchi K., Yamagata K., Hara M., Kume K., and Kume S., Synthesized basement membranes direct the differentiation of mouse embryonic stem cells into pancreatic lineages.J. Cell Science, 123, 2733-2742, 2010 ].
- GFP green fluorescent protein
- the other is a cell that expresses GFP under the control of the Pdx1 promoter and is named SK7 [Shiraki N, Yoshida T, Araki K, et al. Guided differentiation of embryonic stem cells into Pdx1-expressing regional-specific definitive endoderm Stem Cells. 2008; 26: 874-885].
- Both cell lines are ES maintenance medium, 1000 units / ml leukemia inhibitory factor (LIF; Chemicon), 15% knockout serum replacement (KSR; Gibco), 1% fetal bovine serum (FBS; Hyclone), 100 ⁇ M non-essential amino acids (NEAA; Invitrogen), 2 mM L-glutamine (L-Gln; Invitrogen), 1 mM sodium pyruvate (Invitrogen), 50 units / ml penicillin and 50 ⁇ g / ml streptomycin (PS; Invitrogen), and 100 ⁇ M ⁇ -mercaptoethanol ( ⁇ Maintained on mouse embryonic fibroblast (MEF) feeder in Glasgow minimal essential medium (Invitrogen) with -ME; Sigma).
- LIF leukemia inhibitory factor
- KSR knockout serum replacement
- FBS Hyclone
- NEAA non-essential amino acids
- L-Gln L-glutamine
- PS 50 ⁇ g / ml str
- Synthetic nanofiber (sNF) matrices were fabricated by electrospinning using an industrial electrospinning process with a 30 kV field strength.
- sNF matrix consists of two kinds of polyamide polymer A (C 28 O 4 N 4 H 47) n and B (C 28 O 4.4 N 4 H 47) n , which is crosslinked in the presence of an acid catalyst.
- the sNF matrix has a diameter of 200-400 nm and an average diameter of 280 nm.
- the pore size is about 700 nm, which is similar to the cell basement membrane.
- ES cells were transformed into 96-well plates with an ultraweb synthetic polyamine surface (Ultra-Web Synthetic Polyamine Surface (# 3873XX1, Corning Coster, Cambridge, MS) ) In a flat culture at 5,000 cells per well.
- Ultraweb synthetic polyamine surface Ultra-Web Synthetic Polyamine Surface (# 3873XX1, Corning Coster, Cambridge, MS)
- Cells contain 4500 mg / L glucose, NEAA, L-Gln, PS, ⁇ -ME, 10 ⁇ g / ml insulin (Sigma-Aldrich), 5.5 ⁇ g / ml transferrin (Sigma-Aldrich), 6.7 pg / ml selenium ( Sigma-Aldrich), and 0.25% Albmax (Invitrogen), 10 ng / mL recombinant human activin-A (R & D Systems, Minneapolis, MN), 5 ng / ml; Dulbecco's modified Eagle medium (DMEM: Invitrogen, Glasgow, (UK)) for 7 days (d1-d7) (I in FIG. 1).
- DMEM Dulbecco's modified Eagle medium
- the medium is then 2000 mg / L glucose (Sigma, St Louis, MO), 1 ⁇ M retinoic acid (Sigma-Aldrich), 50 ng / ml human recombinant fibroblast growth factor-10 (Peprotech, Rocky Hill, NJ), B27 SUPPLEMENT (Invitrogen) and d7 in RPMI 1640 medium (Invitrogen) containing 0.25 ⁇ M 3-keto-N- (aminoethyl-aminocaproyl-dihydrocinnamoyl) cyclopamine Shh signaling antagonist II (Calbiochem, San Diego, Calif.) The exchange was performed during ⁇ d11 (II in FIG. 1).
- the medium contains 1000 mg / L glucose, NEAA, L-Gln, PS, ⁇ -ME, 10 ⁇ g / ml insulin (Sigma-Aldrich), 5.5 ⁇ g / ml transferrin (Sigma-Aldrich), 6.7 pg DMEM containing 10 ml / ml selenium (Sigma-Aldrich), 0.25% Albmax (Invitrogen), and 10 mM nicotinamide (Sigma-Aldrich) was switched from day 11 (d11) to day 17 (d17) (in FIG. 1) III). The medium was changed every 2 days with fresh medium and growth factors until d17.
- DAPI 6-diamidino-2-phenylindole
- FIG. 1 shows an outline of the screening method.
- Mouse ES cells (Pdx1 / GFP) were induced to differentiate for 17 days to differentiate insulin-positive cells.
- Pdx1-positive pancreatic progenitor cells are induced, and the gene expression level reaches the maximum value.
- Test compounds dissolved in DMSO were added on days 11, 13, and 15 of culture.
- One hundred percent of the compound library / DMSO solution was added to each well. This resulted in a DMSO concentration of 1% final.
- the cells were fixed with 4% PFA, and then analyzed by immunohistological techniques.
- FIG. 3 shows, as an example, the effect of reserpine, which is a component of the differentiation induction promoter that is one embodiment of the present invention. In this case, 0.63 ⁇ M showed the maximum effect, and a concentration-dependent effect was observed. In this experiment, four hit compounds were finally obtained.
- Vmat2-knockdown and Drd2-knockdown assays Expression Arrest non-silencing control shRNA (Open Biosystems, # RHS4080), Vmat2 hRNA (Open Biosystems, # RMM3981-97058457 and # RMM3981-97058458) or Drd2 hRNA (Open Biosystems, # RMM3981-9594258) lentiviral vector was used.
- HEK293-FT cells Invitrogen
- the lentiviral vector and ViraPower TM Lentiviral Packaging Mix were introduced into the cells using FuGENE6 Transfection Reagent (Roche) according to the manufacturer's protocol.
- the cells were cultured in ES maintenance medium for 24 hours, and the virus supernatant was collected.
- SK7 cells were infected with virus supernatant.
- the virus-containing medium was replaced with fresh ES maintenance medium.
- the cells were further cultured for 24 hours, and infected cells were selected using 1.5 ⁇ g / ml puromycin. Viable cells were cloned as knockdown cell lines.
- RNA extraction, reverse transcription reaction, PCR analysis and real-time PCR analysis were reported by Shiraki et al. (Shiraki et al., Biochem Biophys Res Commun. 2009; 381: 694-699; Shiraki et al., Genes Cells. 2008; 13: 731-746; Shiraki et al., Stem Cells. 2008; 26: 874-885: non-patent documents 4 to 6).
- Table 1 shows the primer sequences of each primer.
- PCR conditions for each cycle were as follows: denaturation at 96 ° C for 30 seconds, annealing at 60 ° C for 2 seconds and extension at 72 ° C for 45 seconds.
- RT-PCR products were separated by non-denaturing polyacrylamide gel electrophoresis, stained with SYBR Green I (Molecular Probes), and then confirmed using Gel Logic 200 Imaging System (Kodak).
- Real-time PCR conditions were as follows: denaturation at 95 ° C for 3 seconds, and annealing and extension at 60 ° C for 30 seconds up to 40 cycles.
- Target mRNA levels expressed as arbitrary units were determined using a standard curve.
- the library was diluted 1: 100 with DMSO, added to the medium according to the experimental method described above, and replaced with medium containing the compound twice at d13 and d15.
- cells were stained with anti-insulin antibody and analyzed.
- Compounds that increased both the number and proportion of Ins1 / GFP staining or insulin staining with antibodies were selected as primary hits.
- the primary hit compounds were then assayed using SK7 ES cells at various concentrations ranging from 0.08 to 10 ⁇ M. Hit compounds were confirmed by dose response. As a result, five compounds (reserpine, ellipticine, palmatin chloride, etheroline fumarate, and folic acid) were identified as positive.
- the results of measuring the concentration-dependent activity for reserpine, which is one of the hit compounds, are shown in FIG.
- Reserpine is an antipsychotic and antihypertensive agent of indole alkaloids, showing strong effects and increased insulin positive staining It was.
- the antihypertensive effect of reserpine is known as a result of the action of depleting catecholamines from peripheral synaptic nerve endings by blocking the vesicular monoamine transporter (VMAT2).
- VMAT2 vesicular monoamine transporter
- terabenazine another VMAT2 inhibitor (TBZ) was examined.
- addition of telabenazine also showed the same effect and promoted differentiation into insulin-expressing cells.
- the addition of telabenazine resulted in an increase in both strength and number of insulin-expressing cells.
- ⁇ -methyltyrosine ( ⁇ -MT), carbidopa, 5HTP and bromocriptine are compounds that inhibit dopamine or serotonin metabolism as shown in Table 2. It is.
- ⁇ -MT is an inhibitor of tyrosine 3-monooxygenase, an enzyme that leads to the synthesis of catecholamines.
- dopamine suppressed pancreatic differentiation in a dose-dependent manner, and completely inhibited ⁇ -cell differentiation at a concentration of 100 nM or more.
- Cyclic AMP the second messenger that positively controls the differentiation of beta cells Drd2 is known as a G protein-coupled receptor that activates G ⁇ i / o protein and has an inhibitory activity on adenyl cyclase.
- Drd2 the effect of cAMP was examined.
- Addition of a cell-permeable cAMP analog, 0.63 ⁇ M dibutyrate-cAMP (dBu-cAMP) to the differentiation medium at d11-d17 enhanced differentiation of insulin-expressing cells (FIG. 10). This enhancement was stronger when dBu-cAMP was added to VMAT2KD ES cells (FIG. 10).
- the respective addition concentrations are reserpine (0.6 ⁇ M), dopamine (0.1 ⁇ M), and dBu-cAMP (0.6 ⁇ M).
- the addition of dopamine decreased insulin positive cells.
- the increase in insulin-expressing cells mediated by reserpine was completely inhibited by the addition of dopamine (FIG. 11B). This is thought to be related to the fact that dopamine is the main downstream molecule of VMAT2. This result suggested that VMAT2 performs signal transduction by incorporating dopamine into intracellular granules.
- insulin positive cells increased to about 7 times by cAMP analog (dBu-cAMP), but the enhancement effect of dBu-cAMP was hardly influenced by dopamine.
- ES cells differentiated by simultaneous addition of TBZ (2.5 ⁇ M) and dBu-cAMP (0.6 ⁇ M), or VMAT2KD ES cells to which dBu-cAMP (0.6 ⁇ M) was added showed a large increase in the amount of c-peptide, The value corresponded to one islet (FIG. 12A).
- induced insulin-expressing cells obtained in the presence of TBZ and dBu-cAMP showed glucose concentration-dependent insulin secretion (FIG. 12B). From these results, it was found that monoamine release inhibition and cAMP activation have a synergistic effect.
- Abcc8 and Glut2 are genes that have been identified as causative genes for type 2 diabetes in large-scale SNP analysis, and are classified as important genes responsible for pancreatic ⁇ -cell function among the same causative genes. It is an important gene for fulfilling the functions of sugar sensitivity and insulin secretion. It was shown that the expression levels of these genes were increased by the compounds and methods of the present invention.
- Ngn3, Nkx6.1, MafA and Insulin1 genes Specific to Ngn3, a differentiation marker for pancreatic endocrine cells, Nkx6.1, a differentiation marker for pancreatic ⁇ cells, and pancreatic islets of Langerhans
- the effects of TBZ, dBu-cAMP, and TBZ and dBu-cAMP on the expression of MafA and Insulin1 genes expressed in RNA was extracted from cells at 0, 9, and 11 days in culture.
- Nkx6.1 The expression level of Nkx6.1 was strongly promoted by TBZ after the 15th day, and the effect was enhanced by dBu-cAMP.
- MafA was promoted by TBZ and dBu-cAMP after the 13th day, and the addition of both increased synergistically.
- Insulin1 was promoted by TBZ and dBu-cAMP after the 13th day, and a synergistic increase effect by both was observed after the 15th day.
- FIG. 16A is a diagram schematically showing the addition period of TBZ or dBu-cAMP.
- the addition periods are respectively a) 9th to 11th day of culture, b) 11th to 13th day of culture, c ) From the 9th day to the 13th day of culture.
- TBZ was added in the absence of the compound or on the 9th to 13th days (period c)), and visible and fluorescent photographs of Ngn3 / GFP mouse ES cells on the 13th day of culture were observed.
- the results are shown in FIG. 16B.
- the left is a visible light photograph and the right is a fluorescent photograph.
- An increase in Ngn3-positive cells was confirmed by the addition of TBZ.
- the ratio of GFP positive cells on the 13th day of culture when no compound was added and TBZ or dBu-cAMP was added under the conditions a), b) and c) was confirmed.
- the ratio of the ratio of GFP positive cells to Ngn3 positive cells is shown in FIG.
- the result was an increase in the number of GFP positive cells under all conditions. The effects were in the order of a), c), and b) from the strongest.
- FIG. 17 is a diagram schematically showing the addition period of dBu-cAMP.
- the addition periods are respectively represented by a) the 11th to 17th day of culture, and b) the 11th to 15th day of culture. c) Day 13 to day 17 of culture.
- FIG. 18A is a diagram schematically showing the process.
- FIG. 18B shows the result of measuring the distribution of GFP fluorescence intensity of the cultured cells on the 17th day of culture.
- FIG. 18C shows a visible light photograph (left) and a fluorescent photograph (right) of the GFP positive cells and negative cells of the obtained cultured cells on the 17th day of culture.
- the upper row shows GFP-negative cells, and the lower row shows GFP-positive cells.
- the result is shown in FIG. 18D.
- C-peptide content was measured using islets and undifferentiated mouse ES cells purified from 6-week-old ICR male mice.
- TBZ and TBZ and dBu-cAMP increased the C-peptide content.
- Glucose concentration is 5.5 mM or 27.5 mM using cells cultured under 4 conditions without compound, TBZ, dBu-cAMP, TBZ and dBu-cAMP added every 2 days from day 11 to day 17 of culture. In this case, the amount of secreted C-peptide was measured. The result is shown in FIG. 18E.
- C-peptide content was measured using islets and undifferentiated mouse ES cells purified from 6-week-old ICR male mice. As a result, secretion of C-peptide was observed when glucose concentration was 27.5 mM by addition of dBu-cAMP.
- the differentiation induction promoter and method of the present invention are useful in the field using stem cells, for example, in the field of medical regeneration.
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Abstract
Description
(1)ドーパミン代謝阻害剤およびセロトニン代謝阻害剤からなる群より選ばれる少なくとも一つの化合物を含む、哺乳類動物由来の幹細胞からインスリン産生細胞を分化誘導する分化誘導促進剤。
(2)前記化合物が、小胞モノアミントランスポーター(VMAT2)阻害剤、ドーパミンまたはセロトニンの合成阻害剤、ドーパミンD2受容体阻害剤、ドーパミンまたはセロトニンのアゴニスト、モノアミンオキシダーゼ阻害剤、MAO-A阻害剤、抗ヒスタミン、H1アンタゴニスト、ヒスタミンH2レセプター阻害剤、ヒスタミンH2レセプターアゴニスト、選択的セロトニン再取り込み阻害剤、ドーパミンアンタゴニスト、および5-HT3受容体アンタゴニストからなる群より選ばれる少なくともひとつの化合物である、上記(1)に記載の分化誘導促進剤。
(3)前記化合物が、小胞モノアミントランスポーター(VMAT2)阻害剤である、上記(2)に記載の分化誘導促進剤。
(4)前記化合物が、レセルピン、テトラベナジン、カルビドパ、α-メチルチロシン、5HTP、ブロモクリプチン、キナクリン二塩酸塩二水和物、ピルリンドールメシル酸塩、塩酸パルギリン、フェキソフェナジン塩酸塩、ヒドロキシジン二塩酸塩、マレイン酸クロルフェニラミン、シメチジン、ジマプリット二塩酸塩、マレイン酸フルボキサミン、アザペロン、および塩酸オンダンセトロンからなる群より選ばれる化合物である、上記(1)に記載の分化誘導促進剤。
(5)前記化合物が、レセルピンまたはテトラベナジンである上記(1)に記載の分化誘導促進剤。
(6)さらに、cAMP、cAMPアナログ、Gαsタンパク質カップル受容体アゴニスト、およびGαiタンパク質カップル受容体アンタゴニストからなる群より選ばれる少なくとも一つを含む、上記(1)から(5)のいずれかひとつに記載の分化誘導促進剤。
(7)レセルピンまたはテトラベナジンの少なくとも一つ、およびcAMPまたはcAMPアナログの少なくとも一つを含む、上記(6)に記載の分化誘導促進剤。
(8)アセチルコチン、アセチルコリン分解酵素阻害剤、アセチルコリン受容体活性化剤、およびコリン作動性アンタゴニストからなる群より選ばれる少なくとも一つの化合物を含む、哺乳類動物由来の幹細胞からインスリン産生細胞を分化誘導する分化誘導促進剤。
(9)前記化合物が、アセチルコチン、エセロリンフマル酸塩、塩化パルマチン、カルバコール、およびギンゴライドAからなる群より選ばれる化合物である、上記(8)に記載の分化誘導促進剤。
(10)葉酸を含む、哺乳類動物由来の幹細胞からインスリン産生細胞を分化誘導する分化誘導促進剤。
(11)前記哺乳類動物由来の幹細胞が、胚性幹細胞(ES細胞)、組織幹細胞または人工多能性幹細胞(iPS細胞)である上記(1)から(10)のいずれかひとつに記載の分化誘導促進剤。
(12)前記哺乳類動物由来の幹細胞が、胚性幹細胞(ES細胞)である上記(11)に記載の分化誘導促進剤。
(13)前記哺乳類動物由来の幹細胞が、多能性幹細胞から誘導されたpdx1陽性細胞である請求項11に記載の分化誘導促進剤。
(15)前記化合物が、小胞モノアミントランスポーター(VMAT2)阻害剤、ドーパミンまたはセロトニンの合成阻害剤、ドーパミンD2受容体阻害剤、ドーパミンまたはセロトニンのアゴニスト、モノアミンオキシダーゼ阻害剤、MAO-A阻害剤、抗ヒスタミン、H1アンタゴニスト、ヒスタミンH2レセプター阻害剤、ヒスタミンH2レセプターアゴニスト、選択的セロトニン再取り込み阻害剤、ドーパミンアンタゴニスト、および5-HT3受容体アンタゴニストからなる群より選ばれる少なくともひとつの化合物である、上記(14)に記載の分化誘導方法。
(16)前記化合物が、小胞モノアミントランスポーター(VMAT2)阻害剤である、上記(15)に記載の分化誘導方法。
(17)前記化合物が、レセルピン、テトラベナジン、カルビドパ、α-メチルチロシン、5HTP、ブロモクリプチン、キナクリン二塩酸塩二水和物、ピルリンドールメシル酸塩、塩酸パルギリン、フェキソフェナジン塩酸塩、ヒドロキシジン二塩酸塩、マレイン酸クロルフェニラミン、シメチジン、ジマプリット二塩酸塩、マレイン酸フルボキサミン、アザペロン、および塩酸オンダンセトロンからなる群より選ばれる化合物である、上記(14)に記載の分化誘導方法。
(18)前記化合物が、レセルピンまたはテトラベナジンである、上記(14)に記載の分化誘導方法。
(19)前記培地がさらに、cAMP、cAMPアナログ、Gαsタンパク質カップル受容体アゴニスト、およびGαiタンパク質カップル受容体アンタゴニストからなる群より選ばれる少なくとも一つを含む培地である、上記(14)から(18)のいずれかひとつに記載の分化誘導方法。
(20)前記培地が、レセルピンまたはテトラベナジンの少なくとも一つ、および、cAMPまたはcAMPアナログの少なくとも一つを含む、上記(19)に記載の分化誘導方法。
(21)哺乳類動物由来の幹細胞を、アセチルコチン、アセチルコリン分解酵素阻害剤アセチルコリン受容体活性化剤、およびコリン作動性アンタゴニストからなる群より選ばれる少なくとも一つの化合物を含む培地で培養または処理することを含む、哺乳類動物由来の幹細胞からインスリン産生細胞を分化誘導する方法。
(22)前記化合物が、アセチルコチン、エセロリンフマル酸塩、塩化パルマチン、カルバコール、およびギンゴライドAからなる群より選ばれる、上記(21)に記載の分化誘導方法。
(23)哺乳類動物由来の幹細胞を、葉酸を含む培地で培養または処理することを含む、哺乳類動物由来の幹細胞からインスリン産生細胞を分化誘導する方法。
(24)前記哺乳類動物由来の幹細胞が、胚性幹細胞(ES細胞)、組織幹細胞または人工多能性幹細胞(iPS細胞)である上記(14)から(23)のいずれかひとつに記載の分化誘導方法。
(25)前記哺乳類動物由来の幹細胞がES細胞である上記(24)に記載の分化誘導方法。
(26)前記分化誘導方法は、
(1)アクチビンおよびbFGFを含有する培地、
(2)レチノイン酸、FGF-10、B27 SUPPLEMENTおよびShhシグナル伝達アンタゴニストを含有する培地、および
(3)ニコチン酸アミドを含有する培地、の培地条件の中から選択される少なくとも一つの培地条件下において幹細胞を培養する工程を含む前記(14)から(25)に記載の分化誘導方法。
(27)合成ナノ繊維存在環境下において前記培地条件が選択されることを特徴とする前記(26)に記載の分化誘導方法。
(28)前記(14)から(27)のいずれか一つに記載の方法により分化誘導されたインスリン産生細胞。
(29)前記(1)から(13)のいずれか一つに記載の分化誘導促進剤を含む医薬組成物。
本発明は、哺乳類動物由来の幹細胞をインスリン産生細胞に分化誘導する、またはそれらの細胞のインスリン産生細胞への分化を促進する化合物、ならびにそれらの化合物を含む分化誘導促進剤である。本発明はさらには、それらの化合物を含むインスリン産生を促進するための医薬組成物に関する。
本発明はまた、哺乳類動物由来の幹細胞をインスリン産生細胞に分化誘導する方法、またはそれらの細胞のインスリン産生細胞への分化を促進する方法である。
本発明はさらには、本発明の上記方法によって分化したインスリン産生細胞である。
本発明で用いる哺乳類動物由来の幹細胞とは、哺乳類動物由来の、胚性幹細胞(ES細胞)、組織幹細胞または人工多能性幹細胞(iPS細胞:人工多能性幹細胞:induced pluripotent stem cell)であり、好ましくは、ES細胞である。これらの幹細胞は、本技術分野で公知の方法で調製または培養することができる。
(i)Octファミリー、Klfファミリー、Soxファミリー、Mycファミリー、
(ii)Octファミリー、Soxファミリー、Nanogファミリー、Lin28ファミリー、
(iii)Octファミリー、Klfファミリー、Soxファミリー、Mycファミリー、hTERTファミリー、SV40 large Tファミリー、
(iv)Octファミリー、Klfファミリー、Soxファミリー、
(v)Octファミリー、Klfファミリー、Soxファミリー、Mycファミリー、Glisファミリー、または
(vi)Octファミリー、Klfファミリー、Soxファミリー、Glisファミリー。
ここで示すファミリーとは、一次構造上の相同性により同定された、即ち類似構造を有するタンパク質群のことであり、例えばOctファミリーとしてはiPS細胞の代表的な初期化因子であるOct3/4の他にOct1A, Oct6等を含み、KlfファミリーとしてはKlf4, Klf1, Klf5等を含み、SoxファミリーとしてはSox2, Sox1, Sox3, Sox7等を含み、Mycファミリーとしてはc-Myc, L-Myc等、LIN28ファミリーとしてはLin28, Lin28B等を含み、GlisファミリーとしてはGlis1, Glis2, Glis3等を含む。また、初期化因子の体細胞への導入にあたっては、Takahashi, K. and Yamanaka, S., Cell, 126: 663-676 (2006)、Okita, K. et al., Nature, 448: 313-317 (2007)、Okita, K. et al., Nature Method, 8(5): 409-412 (2011)に記載の導入方法に従って、ウイルス系ベクターあるいは非ウイルス系ベクターを用いて各種初期化因子の導入を行うことができる。
本発明において、ドーパミン代謝阻害剤またはセロトニン代謝阻害剤から選ばれる化合物とは、ドーパミンまたはセロトニンのトランスポーターの阻害剤、例えば、小胞モノアミントランスポーター(VMAT2)阻害剤、ドーパミンまたはセロトニンの合成阻害剤、ドーパミンD2受容体阻害剤、ドーパミンまたはセロトニンのアゴニスト、モノアミンオキシダーゼ阻害剤、MAO-A阻害剤、抗ヒスタミン、H1アンタゴニスト、ヒスタミンH2レセプター阻害剤、ヒスタミンH2レセプターアゴニスト、選択的セロトニン再取り込み阻害剤、ドーパミンアンタゴニスト、または、5-HT3受容体アンタゴニストをあげることができる。合成阻害剤は、合成を阻害する化合物を意味するが、例えば、合成に関与する酵素の阻害剤、フィードバック作用により合成系を阻害する合成産物を含む。
本発明における、ドーパミン代謝阻害剤またはセロトニン代謝阻害剤とは、これには限定されないが、例えば、VMAT2阻害剤であるレセルピンまたはテトラベナジン、レボドパの脱炭酸酵素阻害剤であるカルビドパ、セロトニン合成阻害剤である5-ヒドロキシトリプトファン(5-HTP)、ドーパミン合成阻害剤であるα-メチルチロシン、ドーパミンアゴニストであるブロモクリプチン、モノアミンオキシダーゼ阻害剤であるキナクリン二塩酸塩二水和物または塩酸パルギリン、MAO-A阻害剤であるピルリンドールメシル酸塩、抗ヒスタミンであるフェキソフェナジン塩酸塩、H1アンタゴニストであるヒドロキシジン二塩酸塩またはマレイン酸クロルフェニラミン、ヒスタミンH2レセプター阻害剤であるシメチジン、ヒスタミンH2レセプターアゴニストであるジマプリット二塩酸塩、選択的セロトニン再取り込み阻害剤であるマレイン酸フルボキサミン、ドーパミンアンタゴニストであるアザペロン、5-HT3受容体のアンタゴニストである塩酸オンダンセトロン等をあげることができる。好ましくは、レセルピンまたはテトラベナジンである。
本発明で言う、cAMPアナログとは、cAMPの塩基部分または糖部分の構造を変化させることで細胞膜透過性を有する物質であり、具体的には、例えば、ジブチレート-cAMPおよび8-CPT-2-Me-cAMPをあげることができる。
好ましくは、本発明の分化誘導促進剤は、レセルピンまたはテトラベナジンの少なくとも一つ、および、cAMPまたはcAMPアナログを含む。
本発明の分化誘導促進剤を医薬組成物として用いる場合は、本発明の化合物の含有量は、化合物の種類、投与対象、症状および投与方法によって異なる。例えば、レセルピンでは、1日あたり0.01mg~100mg、好ましくは0.1mg~10mgであり、テトラベナジン、カルビドパ、またはエセロリンフマル酸塩では、1日あたり1mg~1000mg、好ましくは、10mg~500mgであり、ブロモクリプチンでは、1日あたり1mg~1000mg、好ましくは1mg~100mgである。
培地中に添加する、ドーパミン代謝阻害剤またはセロトニン代謝阻害剤の濃度は、幹細胞からインスリン産生細胞への分化誘導を促進する濃度であり、添加する物質に応じて、また同時に添加する分化促進に影響する他の物質の濃度に応じて適宜変更できる。特に制限されるものではないが、レセルピンでは、培地中の濃度は、0.15~10.0μM、好ましくは0.15~2.5μM、さらに好ましくは0.63μM~1.25μMであり、テラベナジンでは、培地中の濃度は、0.15~10.0μM、好ましくは1.25~5.0μMである。
培地は、幹細胞の培養または維持に適した公知の培地を使用でき、例えば、グラスゴー最小必須培地やダルベッコ改変イーグル培地をあげることができる。
培地中に添加する、アセチルコチン、アセチルコリン分解酵素阻害剤、アセチルコリン受容体活性化剤またはコリン作動性アンタゴニストの濃度は、幹細胞からインスリン産生細胞への分化誘導を促進する濃度であり、添加する物質に応じて、また同時に添加する分化促進に影響をする他の物質の濃度に応じて適宜変更できる。特に制限されるものではないが、例えば、塩化パルマチンでは、培地中の濃度は、0.08~10.0μM、好ましくは0.15~0.63μMであり、エセロリンフマル酸塩では培地中の濃度は、0.15~10.0μM、好ましくは0.32~2.5μMであり、アセチルコリンでは、培地中の濃度は、0.15~5.0μM、好ましくは0.63~2.5μMである。
(1)ES細胞株
2つのES細胞株を使用した。一方は、インスリン1プロモーターの制御下で緑色蛍光タンパク質(GFP)を発現し、INGと命名される細胞である[Higuchi Y., Shiraki N., Yamane K, Qin Z., Mochitate K., Araki K., Senokuchi K., Yamagata K., Hara M., Kume K., and Kume S., Synthesized basement membranes direct the differentiation of mouse embryonic stem cells into pancreatic lineages. J. Cell Science, 123, 2733-2742, 2010]。他方は、Pdx1プロモーターの制御下でGFPを発現し、SK7と命名される細胞である[Shiraki N, Yoshida T, Araki K, et al. Guided differentiation of embryonic stem cells into Pdx1-expressing regional-specific definitive endoderm. Stem Cells. 2008;26:874-885]。両細胞株は、ES維持培地である、1000ユニット/ml白血病抑制因子(LIF;Chemicon)、15%ノックアウト血清リプレースメント(KSR;Gibco)、1%ウシ胎児血清(FBS;Hyclone)、100μM非必須アミノ酸(NEAA;Invitrogen)、2mM L-グルタミン(L-Gln;Invitrogen)、1mMピルビン酸ナトリウム(Invitrogen)、50ユニット/mlペニシリンおよび50μg/mlストレプトマイシン(PS;Invitrogen)、および100μM β-メルカプトエタノール(β-ME;Sigma)を含むグラスゴー最小必須培地(Invitrogen)中のマウス胚線維芽細胞(MEF)フィーダー上で維持した。
合成ナノ繊維(sNF)マトリックスは、30kVの電界強度を用いて、工業用エレクトロスピニングプロセスを使用してエレクトロスピニングによって作製した。sNFマトリックスは、酸触媒の存在下で架橋結合された2種類のポリアミドポリマーA(C28O4N4H47)nおよびB(C28O4.4N4H47)nからなる。sNFマトリックスは、直径200~400nmを有し、平均直径は280nmである。ポアサイズは約700nmであり、これは細胞の基底膜に類似している。
分化試験のために、ES細胞を、ウルトラウェブ合成ポリアミン表面をもつ96ウェルプレート(Ultra-Web Synthetic Polyamine Surface(#3873XX1、Corning Coster、Cambridge、MS))中で、ウェル当たり5,000細胞で平面培養した。細胞は、4500mg/Lグルコースを含有し、NEAA、L-Gln、PS、β-ME、10μg/mlインスリン(Sigma-Aldrich)、5.5μg/mlトランスフェリン(Sigma-Aldrich)、6.7pg/mlセレン(Sigma-Aldrich)、および0.25%Albmax(Invitrogen)、10ng/mL組み換えヒトアクチビン-A(R&D Systems、Minneapolis、MN)、5ng/ml;組み換えヒトbFGFを含むダルベッコ改変イーグル培地(DMEM:Invitrogen、Glasgow、UK)中で7日間培養(d1~d7)した(図1中のI)。次いで、培地は、2000mg/Lグルコース(Sigma、St Louis、MO)、1μMレチノイン酸(Sigma-Aldrich)、50ng/mlヒト組み換え線維芽細胞成長因子-10(Peprotech、Rocky Hill、NJ)、B27 SUPPLEMENT(Invitrogen)、および0.25μM 3-ケト-N-(アミノエチル-アミノカプロイル-ジヒドロシンナモイル)シクロパミンShhシグナル伝達アンタゴニストII(Calbiochem、San Diego、CA)を含有するRPMI 1640培地(Invitrogen)にd7~d11の間交換した(図1中のII)。最終的に、培地は、1000mg/Lグルコースを含有し、NEAA、L-Gln、PS、β-ME、10μg/mlインスリン(Sigma-Aldrich)、5.5μg/mlトランスフェリン(Sigma-Aldrich)、6.7pg/mlセレン(Sigma-Aldrich)、0.25%Albmax(Invitrogen)、および10mMニコチン酸アミド(Sigma-Aldrich)を含むDMEMに11日目(d11)~17日目(d17)に切り替えた(図1中のIII)。培地は、d17まで、新しい培地および成長因子を用いて2日ごとに交換した。
すべての試験化合物は、任意の濃度になるようにしてジメチルスルホキシド(DMSO)に溶解し、化合物の溶液を、d11、d13およびd15に培地に添加した。培地中のDMSOの最終濃度は、すべての化合物の実験において、1.0%になるように調製した。
17日間の細胞培養後、培養ES細胞を室温で30分間、4%パラホルムアルデヒドPBS溶液で固定し、PBSで数回すすいだ。次に、細胞を加湿チャンバー中で、PBST(PBS中0.1% Tween-20)で20%としたBlocking One(Nacalai tesque、Tokyo、Japan)を用いて希釈した一次抗体を添加し、4℃で一晩インキュベートした。PBST中で細胞を洗浄した後に、暗中、室温で2時間、20% Blocking Oneで希釈した二次抗体と共に細胞をインキュベートした。PBST中で二次抗体を洗い流した後に、6-ジアミジノ-2-フェニルインドール(DAPI)(Roche Diagnostics、 Basel、Switzerland)を用いて細胞を対比染色した。以下の抗体を一次抗体として使用した;ウサギ抗GFP(MBL International Corp、Woburn、MA)、マウス抗インスリン(Sigma)。以下の抗体を二次抗体として使用した;Alexa568結合抗体およびAlexa488結合抗体(Invitrogen)。
図1にスクリーニング方法の概略を示す。マウスES細胞(Pdx1/GFP)を17日間分化誘導し、インスリン陽性細胞を分化させた。培養11日目でPdx1陽性の膵前駆細胞が誘導され遺伝子発現量が最大値に達する。培養11、13、15日目にDMSOに溶解した試験化合物を添加した。化合物ライブラリー/DMSO溶液は各ウェルに百分の1量添加することとした。これによりDMSO濃度は最終濃度1%となった。培養17日目に細胞を4%PFAで固定した後、免疫組織学的手法により解析した。
サンプル画像の撮影を共焦点レーザースキャン型ハイスループットコンフォーカルシステム(ImageXpress Ultra; Molecular Device社製)により行った(図2A)。さらにMetaXpressソフトウエアを用いた画像解析によりDAPI、Pdx1、Insulinの三重陽性の細胞の占める面積を算出し、これをインスリン陽性細胞の数を反映する数値とした(図2B)。スクリーニングのプロセスを図3に示した。一次スクリーニング行い、次いで、用量反応性試験を行った。具体的には、全ウェルの平均値より1.7倍以上インスリン陽性細胞の占める面積が増加したものをヒット化合物とみなした。試験化合物の中から1.7倍以上の増加を示したものをヒット化合物候補とし、さらにそれらの化合物の濃度依存的効果を調べるために0~10μMの濃度で再度効果を検討した。
Vmat2-ノックダウンおよびDrd2-ノックダウンアッセイにおいて、Expression Arrest non-silencing control shRNA(Open Biosystems, #RHS4080)、Vmat2 hRNA (Open Biosystems, #RMM3981-97058457 および #RMM3981-97058458)または Drd2 hRNA (Open Biosystems, #RMM3981-9594258)レンチウィルスベクターを用いた。ウィルスの調製は、HEK293-FT細胞(Invitrogen)を、トランスフェクションの前日に平板培地に播種した。一晩培養した後、メーカーのプロトコルに従い、FuGENE6 Transfection Reagent(Roche)を用いて細胞に、レンチウィルスベクターおよびViraPowerTM Lentiviral Packaging Mix(Invitrogen)を導入した。細胞を、ES維持培地で24時間培養し、ウィルス上清を回収した。次いで、SK7細胞に、ウィルス上清液を感染させた。24時間培養後、ウィルス含有培地を、新鮮なES維持培地に交換した。さらに24時間培養し、1.5μg/mlピューロマイシンを用いて、感染細胞を選択した。生存細胞を、ノックダウンセルラインとしてクローン化した。
RNA抽出、逆転写反応、PCR解析およびリアルタイムPCR解析は、白木らの報告(Shiraki et al., Biochem Biophys Res Commun. 2009;381:694-699; Shiraki et al., Genes Cells. 2008;13:731-746; Shiraki et al., Stem Cells. 2008;26:874-885:非特許文献4~6)に記載の方法に従って行った。各プライマーのプライマー配列を表1に示す。各サイクルのPCR条件は以下の通りとした: 96℃で30秒間の変性、60℃で2秒間のアニーリングおよび72℃で45秒間の伸長。RT-PCR産物を、非変性ポリアクリルアミドゲル電気泳動により分離し、SYBR Green I (Molecular Probes)で染色した後、Gel Logic 200 Imaging System (Kodak)を用いて確認した。リアルタイムPCR条件は以下の通りとした: 95℃で3秒間の変性、ならびに60℃で30秒間のアニーリングおよび伸長を40サイクルまで。任意単位として表される標的mRNAレベルは、標準曲線を用いて決定した。
分化したES細胞を、低グルコース濃度(5.5mM)のダルベッコ改変最小必須培地(1%牛胎児血清含有)で、4時間前培養した。細胞を、2回PBSで洗浄し、次いで、低(5.5mM)または高(27.5mM)グルコース濃度のダルベッコ改変最小必須培地(1%牛胎児血清含有)で2時間培養した。培養液を回収し、細胞を溶解緩衝液(0.1% Trioton X-100、1/100(v/v)プロテアーゼ阻害剤を混合したPBS)で溶解した。培地中へのインスリンの分泌および細胞溶解物のインスリン量を、マウスCペプチドELISAキット(Shibayagi. Co. Ltd., Japan)を用いて測定した。
(1)ES細胞のインスリン発現細胞への分化を促進する化合物候補のハイスループットスクリーニング
再現可能な結果がハイスループットアッセイで得られるように培養条件を改変した。アッセイ手順の概要を図1に示す。SK7 Pdx1/GFP ES 細胞または ING Ins1/GFP ES 細胞を用いた。ES細胞を、0日目(d0)に、sNF(96ウェルマイクロタイタープレート)上に接種し、低分子化合物の誘導活性をアッセイした。個々の化合物は、11日目(d11)に各ウェルに添加した。DMSO中に溶解した各化合物(アレイ上に配列した1120の生物活性化合物のライブラリー)をスクリーニングした。ライブラリーは、DMSOで1:100に希釈し、上記実験方法に従って培地中に添加し、d13とd15において2回化合物を含有した培地に交換した。d17に、細胞を、抗インスリン抗体で染色して分析した。抗体によるIns1/GFP染色またはインスリン染色の、数および割合の両者を増加させる化合物を、一次ヒットとして選択した。次いで、一次ヒットした化合物を、SK7 ES 細胞を用いて、0.08~10μMになるように希釈した種々の濃度の範囲でアッセイを行った。ヒット化合物を用量反応性により確認した。その結果、5つの化合物(レセルピン、エリプチシン、塩化パルマチン、エセロリンフマル酸塩、および葉酸)が陽性として同定された。
ヒット化合物の一つであるレセルピンについての濃度依存活性を測定した結果を図4に示す。
レセルピンは、インドールアルカロイドの抗精神病薬および抗高血圧剤であり、強い効果および増加したインスリン陽性染色を示した。レセルピンによる降圧作用は、小胞モノアミントランスポーター(VMAT2)を遮断することにより、末梢シナプス神経終末からカテコールアミンを枯渇させる作用の結果として知られているので、次いで、他のVMAT2阻害剤である、テラベナジン(TBZ)を検討した。図5に示すように、テラベナジンの添加も、同様の効果を示し、インスリン発現細胞への分化を促進した。テラベナジンの添加は、インスリン発現細胞の強さと数がともに増加をもたらした。これらの結果は、VMAT2が、Pdx1陽性細胞のインスリン産生細胞への分化を負に制御していることを強く示唆している。
レセルピンはVMAT2の阻害剤であり、またTBZも同様にVMAT2阻害剤である。そこでさらに、VMAT2により細胞内顆粒に取り込まれるモノアミンについて着目し、ドーパミン、セロトニンの生合成と分解に関わる、カルビドパ、α-メチルチロシン、5HTP、ブロモクリプチンを検討した結果、同様の効果を示した。さらに、エセロリンフマル酸塩と塩化パルマチンを調べたところ、どちらもインスリン陽性細胞を増やした(図5)。エセロリンフマル酸塩と塩化パルマチンはアセチルコリン分解酵素の阻害剤であることからアセチルコリン(図5)とアセチルコリン受容体の活性化剤であるカルバコール(図示せず)を試したところどちらもインスリン陽性細胞を増やした。
ドーパミンシグナルが、その受容体の活性化を通じてインスリン発現細胞への分化を抑制することを確認するために、ドーパミンD2受容体ノックダウン細胞セルライン、Drd2KDを確立した(図9A)。図9Bに示すように、Drd2KD細胞(ドーパミンD2受容体の阻害)は、インスリン産生細胞へ分化する能力が、2.5倍にまで増加したことを示した。興味深いことに、0.63μMレセルピンの添加は、Drd2KD細胞においては、分化を促進しなかった。
上記の結果は、モノアミン、特にはドーパミンが、そのレセプターの活性化を介して、インスリン発現細胞への分化を負に制御(分化抑制)していることを示している。
Drd2は、Gαi/oタンパク質を活性化し、アデニルシクラーゼに対して阻害活性をもつ、Gタンパク質共役型受容体として知られている。Drd2による細胞内シグナル伝達現象を同定するために、cAMPの効果を調べた。細胞透過性cAMPアナログである、0.63μMジブチレート-cAMP(dBu-cAMP)の分化培地へのd11-d17における添加は、インスリン発現細胞の分化を高めた(図10)。この増強は、dBu-cAMPをVMAT2KD ES 細胞に添加したときにはより強かった(図10)。定量的分析は、NS細胞において、dBu-cAMPを加えたときには、インスリン発現細胞の数が2.9倍にまで増加したことを明らかにした。VMAT2KDのみでは、8倍の増加を示し、さらに、dBu-cAMP の添加は、31.4倍の増加をもたらした。すなわち、cAMPアナログの添加はVMAT2のノックダウンと相乗効果を示した。これらの結果は、cAMPおよびVMAT2KDが、インスリン生産細胞の数の増加において、相乗的に働いていることを示している(図11A)。
次いで、レセルピン、ドーパミンおよびdBu-cAMPの相乗効果を確かめた。それぞれの添加濃度は、レセルピン(0.6μM)、ドーパミン(0.1μM)、およびdBu-cAMP(0.6μM)である。ドーパミンの添加はインスリン陽性細胞を減少させた。レセルピンにより仲介されるインスリン発現細胞の増加は、ドーパミンの添加により完全に阻害された(図11B)。このことは、ドーパミンがVMAT2の主要な下流分子であることと関係していると考えられる。この結果はVMAT2がドーパミンを細胞内顆粒に取り込むことでシグナル伝達を行っていることを示唆するものであった。一方、cAMPアナログ(dBu-cAMP)によってもインスリン陽性細胞は約7倍まで増加したが、dBu-cAMPの増強効果は、ドーパミンによってほとんど影響されなかった。さらに、レセルピンおよびdBu-cAMPの同時添加は、インスリン発現細胞への分化促進において著しい相乗効果を示し、β細胞の成熟化マーカーの発現が亢進した(図11B)。これらの結果は、cAMPの産生を刺激する他の平行経路の存在を示唆した。
セロトニンまたはヒスタミンの添加による、Pdx1/GFP陽性細胞数とインスリン陽性細胞数の変化に対する影響を確認するために、培養11日目(d11)に、セロトニンまたはヒスタミンを0、0.08、0.16、0.31、0.63、1.25、2.5、5.0、10μMの濃度で添加した。以降17日目まで2日おきに培地交換をした。17日目に培養を停止し、抗GFP抗体および抗インスリン抗体を用いた抗体免疫染色により細胞を染色し、細胞数を計測した。
結果を図14に示す。セトロニンまたはヒスタミンの添加により、濃度依存的にインスリン陽性細胞数が減少した。一方、Pdx1/GFP陽性細胞数には変化がなかった。
膵内分泌細胞への分化マーカーであるNgn3、膵β細胞への分化マーカーであるNkx6.1、膵臓ランゲルハンス島β細胞に特異的に発現するMafA、及びInsulin1遺伝子の発現に対する、TBZ、dBu-cAMP、およびTBZとdBu-cAMPの影響を確認した。
培養0、9、11日の細胞からRNAを抽出した。さらに11日から17日目まで2日おきに化合物なし、TBZ(2.5μM)、dBu-cAMP(0.6μM)、TBZとdBu-cAMP(それぞれ、2.5μMと0.6μM)を添加した4条件において13、15、17日目にRNAを回収した。それぞれのRNA試料をもとにRT-PCR法によりmRNA発現量を測定した。発現量は化合物を添加しない場合での培養13日目の発現量を1とした比率で示している。
結果は、Ngn3の発現量は13日目と15日目にTBZにより著しく増加し、dBu-cAMPによっても増加した。Nkx6.1の発現量は、15日目以降TBZにより強く促進され、その効果がdBu-cAMPによって増強された。MafAは13日目以降TBZとdBu-cAMPにより促進され、その両方の添加により相乗的に発現量が増加した。Insulin1は13日目以降TBZとdBu-cAMPにより促進され、15日目以降はその両方による相乗的な増加効果がみられた。
TBZ(2.5μM)またはdBu-cAMP(0.6μM)の添加によるNgn3陽性細胞誘導率に与える影響を確認した。図16Aは、TBZまたはdBu-cAMPの添加期間を模式的に示した図であり、それぞれ添加期間を、a)培養9日目から11日目、b)培養11日目から13日目、c)培養9日目から13日目とした。
まず、化合物無添加または培養9日目から13日目(期間c))にTBZを添加し、培養13日目におけるNgn3/GFPマウスES細胞の可視光写真と蛍光写真を観察した。結果を図16Bに示す。左が可視光写真であり、右が蛍光写真である。TBZの添加により、Ngn3陽性細胞の増加が確認できた。
次いで、化合物無添加、およびTBZまたはdBu-cAMPを上記a)、b)、c)の条件で添加した場合の培養13日目におけるGFP陽性細胞の割合を確認した。無添加の場合を1として、GFP陽性細胞とNgn3陽性細胞の割合比率を図16Cに示す。結果は、すべての条件においてGFP陽性細胞数が増加した。その効果は強いものからa)、c)、 b)の順であった。
培養11日目から17日目にdBu-cAMP(0.6μM)を添加し(TBZ無添加)、糖応答性C-peptide分泌に与える影響を確認した。図17の上図が、dBu-cAMPの添加期間を模式的に示した図であり、それぞれ添加期間を、a)培養11日目から17日目、b)培養11日目から15日目、c)培養13日目から17日目とした。dBu-cAMPを上記a)、b)、c)の条件で添加した場合の、培養17日目における糖応答性C-peptide分泌量を上記(材料及び方法)の(9)に従い確認した。結果は、すべての条件においてGFP陽性細胞数が増加した。その効果は強いものからc)、a)、 b)の順であった。
化合物無添加、およびTBZ(2.5μM)、dBu-cAMP(0.6μM)、TBZとdBu-cAMP(それぞれ2.5μMと0.6μM)を添加した4条件で培養した後、17日目におけるGFP陽性細胞をセルソーターにより分取し、上記(材料と方法)の(9)に従い、C-peptide含量とグルコース濃度依存的なインスリン分泌量を測定した。
図18Aは、その工程を模式的に示した図である。培養17日目における培養細胞のGFPの蛍光強度の分布を測定した結果を図18Bに示す。得られた培養17日目における培養細胞のGFP陽性細胞と陰性細胞の可視光写真(左)と蛍光写真(右)を図18Cに示す。上段は、GFP陰性細胞を示しており、下段は、GFP陽性細胞を示している。
培養11日から17日目まで2日おきに化合物なし、TBZ、dBu-cAMP、TBZとdBu-cAMPを添加した4条件で培養を行い、17日目におけるGFP陽性細胞のC-peptide含量を測定した。結果を図18Dに示す。コントロールとして、6週令のICRオスマウスから精製した膵島および未分化マウスES細胞を用いてC-peptide含量を測定した。TBZ、およびTBZとdBu-cAMPの添加により、C-peptide含量が増加した。
培養11日から17日目まで2日おきに化合物なし、TBZ、dBu-cAMP、TBZとdBu-cAMPを添加した4条件で培養を行った細胞を用いて、グルコース濃度を5.5 mM または27.5 mMとした場合におけるC-peptide分泌量を測定した。結果を図18Eに示す。コントロールとして、6週令のICRオスマウスから精製した膵島および未分化マウスES細胞を用いてC-peptide含量を測定した。結果は、dBu-cAMPの添加によりグルコース濃度が27.5 mMの場合にC-peptideの分泌がみられた。
Claims (29)
- ドーパミン代謝阻害剤およびセロトニン代謝阻害剤からなる群より選ばれる少なくとも一つの化合物を含む、哺乳類動物由来の幹細胞からインスリン産生細胞を分化誘導する分化誘導促進剤。
- 前記化合物が、小胞モノアミントランスポーター(VMAT2)阻害剤、ドーパミンまたはセロトニンの合成阻害剤、ドーパミンD2受容体阻害剤、ドーパミンまたはセロトニンのアゴニスト、モノアミンオキシダーゼ阻害剤、MAO-A阻害剤、抗ヒスタミン、H1アンタゴニスト、ヒスタミンH2レセプター阻害剤、ヒスタミンH2レセプターアゴニスト、選択的セロトニン再取り込み阻害剤、ドーパミンアンタゴニスト、および5-HT3受容体アンタゴニストからなる群より選ばれる少なくともひとつの化合物である、請求項1に記載の分化誘導促進剤。
- 前記化合物が、小胞モノアミントランスポーター(VMAT2)阻害剤である、請求項2に記載の分化誘導促進剤。
- 前記化合物が、レセルピン、テトラベナジン、カルビドパ、α-メチルチロシン、5HTP、ブロモクリプチン、キナクリン二塩酸塩二水和物、ピルリンドールメシル酸塩、塩酸パルギリン、フェキソフェナジン塩酸塩、ヒドロキシジン二塩酸塩、マレイン酸クロルフェニラミン、シメチジン、ジマプリット二塩酸塩、マレイン酸フルボキサミン、アザペロン、および塩酸オンダンセトロンからなる群より選ばれる、請求項1に記載の分化誘導促進剤。
- 前記化合物が、レセルピンまたはテトラベナジンである請求項1に記載の分化誘導促進剤。
- さらに、cAMP、cAMPアナログ、Gαsタンパク質カップル受容体アゴニスト、およびGαiタンパク質カップル受容体アンタゴニストからなる群より選ばれる少なくとも一つを含む、請求項1から5のいずれかひとつに記載の分化誘導促進剤。
- レセルピンまたはテトラベナジンの少なくとも一つ、および、cAMPまたはcAMPアナログの少なくとも一つを含む、請求項5に記載の分化誘導促進剤。
- アセチルコチン、アセチルコリン分解酵素阻害剤、アセチルコリン受容体活性化剤およびコリン作動性アンタゴニストからなる群より選ばれる少なくとも一つの化合物を含む、哺乳類動物由来の幹細胞からインスリン産生細胞を分化誘導する分化誘導促進剤。
- 前記化合物が、アセチルコチン、エセロリンフマル酸塩、塩化パルマチン、およびギンゴライドAからなる群より選ばれる化合物である、請求項8に記載の分化誘導促進剤。
- 葉酸を含む、哺乳類動物由来の幹細胞からインスリン産生細胞を分化誘導する分化誘導促進剤。
- 前記哺乳類動物由来の幹細胞が、胚性幹細胞(ES細胞)、組織幹細胞または人工多能性幹細胞(iPS細胞)である請求項1から10のいずれかに記載の分化誘導促進剤。
- 前記哺乳類動物由来の幹細胞が、胚性幹細胞(ES細胞)である請求項11に記載の分化誘導促進剤。
- 前記哺乳類動物由来の幹細胞が、多能性幹細胞から誘導されたpdx1陽性細胞である請求項11に記載の分化誘導促進剤。
- 哺乳類動物由来の幹細胞を、ドーパミン代謝阻害剤およびセロトニン代謝阻害剤からなる群より選ばれる少なくとも一つの化合物を含む培地で培養することを含む、哺乳類動物由来の幹細胞からインスリン産生細胞を分化誘導する方法。
- 前記化合物が、小胞モノアミントランスポーター(VMAT2)阻害剤、ドーパミンまたはセロトニンの合成阻害剤、ドーパミンD2受容体阻害剤、ドーパミンまたはセロトニンのアゴニスト、モノアミンオキシダーゼ阻害剤、MAO-A阻害剤、抗ヒスタミン、H1アンタゴニスト、ヒスタミンH2レセプター阻害剤、ヒスタミンH2レセプターアゴニスト、選択的セロトニン再取り込み阻害剤、ドーパミンアンタゴニスト、および5-HT3受容体アンタゴニストからなる群より選ばれる少なくともひとつの化合物である、請求項14に記載の分化誘導方法。
- 前記化合物が、小胞モノアミントランスポーター(VMAT2)阻害剤である、請求項15に記載の分化誘導方法。
- 前記化合物が、レセルピン、テトラベナジン、カルビドパ、α-メチルチロシン、5HTP、ブロモクリプチン、キナクリン二塩酸塩二水和物、ピルリンドールメシル酸塩、塩酸パルギリン、フェキソフェナジン塩酸塩、ヒドロキシジン二塩酸塩、マレイン酸クロルフェニラミン、シメチジン、ジマプリット二塩酸塩、マレイン酸フルボキサミン、アザペロン、および塩酸オンダンセトロンからなる群より選ばれる化合物である、請求項14に記載の分化誘導方法。
- 前記化合物が、レセルピンまたはテトラベナジンである請求項14に記載の分化誘導方法。
- 前記培地がさらに、cAMP、cAMPアナログ、Gαsタンパク質カップル受容体アゴニスト、およびGαiタンパク質カップル受容体アンタゴニストからなる群より選ばれる少なくとも一つを含む培地である、請求項14から18のいずれかひとつに記載の分化誘導方法。
- 前記培地が、レセルピンまたはテトラベナジンの少なくとも一つ、および、cAMPまたはcAMPアナログの少なくとも一つを含む、請求項19に記載の分化誘導方法。
- 哺乳類動物由来の幹細胞を、アセチルコチン、アセチルコリン分解酵素阻害剤、アセチルコリン受容体活性化剤、およびコリン作動性アンタゴニストからなる群より選ばれる少なくとも一つの化合物を含む培地で培養することを含む、哺乳類動物由来の幹細胞からインスリン産生細胞を分化誘導する方法。
- 前記化合物が、アセチルコチン、エセロリンフマル酸塩、塩化パルマチン、カルバコール、ギンゴライドAからなる群より選ばれる、請求項21に記載の分化誘導方法。
- 哺乳類動物由来の幹細胞を、葉酸を含む培地で培養することを含む、哺乳類動物由来の幹細胞からインスリン産生細胞を分化誘導する方法。
- 前記哺乳類動物由来の幹細胞が、胚性幹細胞(ES細胞)、組織幹細胞または人工多能性幹細胞(iPS細胞)である請求項14から23のいずれかひとつに記載の分化誘導方法。
- 前記哺乳類動物由来の幹細胞がES細胞である請求項24に記載の分化誘導方法。
- 前記分化誘導方法は、
(1)アクチビンおよびbFGFを含有する培地、
(2)レチノイン酸、FGF-10、B27 SUPPLEMENTおよびShhシグナル伝達アンタゴニストを含有する培地、および
(3)ニコチン酸アミドを含有する培地、の培地条件の中から選択される少なくとも一つの培地条件下において幹細胞を培養する工程を含む請求項14から25に記載の分化誘導方法。 - 合成ナノ繊維存在環境下において前記培地条件が選択されることを特徴とする請求項26に記載の分化誘導方法。
- 請求項14から27のいずれか一つに記載の方法により分化誘導されたインスリン産生細胞。
- 請求項1から13のいずれか一つに記載の分化誘導促進剤を含む医薬組成物。
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JPWO2012150707A1 (ja) | 2014-07-28 |
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