WO2016104614A1 - 新規成熟心筋細胞マーカー - Google Patents
新規成熟心筋細胞マーカー Download PDFInfo
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Definitions
- the present invention relates to a method for producing cardiomyocytes using a novel cardiomyocyte marker.
- the present invention also relates to a method for testing drug toxicity using cardiomyocytes produced by the method.
- the present invention relates to a cardiomyocyte extraction kit containing a reagent for detecting the cardiomyocyte marker.
- Patent Document 1 Patent Document 2, Non-Patent Document 1, Non-Patent Document 2, and Non-Patent Document 3).
- human pluripotent stem cell-derived cardiomyocytes generally remain in an immature stage similar to fetal cardiomyocytes, and are said to have insufficient ion channel function compared to adult cardiomyocytes. Therefore, in order to screen for drug toxicity and therapeutic agents related to ion channels, it is necessary to use mature cardiomyocytes.
- An object of the present invention is to extract cardiomyocytes from a cell population containing cardiomyocytes. Accordingly, an object of the present invention is to provide a marker specific for cardiomyocytes.
- CORIN CORIN, NCAM1 (Neural cell adhesion molecule 1), CRYAB (crystallin, alpha B), HBEGF (heparin binding-epidermal growth factor-like growth factor), DMD (dystrophin), ATPIF1 (ATPase inhibitory factor 1), CAV2 (Caveolin-2), ITGAV (Integrin alpha-V), DCBLD2 (discoidin, CUB and LCCL domain containing 2), CLIC4 (Chloride intracellular channel) ), BMPR2 (Bone morphogenetic protein receptor type II), CTSB (cathepsin B), TMEM123 (transmembrane protein 123), USP14 (Ubiquitin carboxyl-terminal hydrolase 14) and MIR761 as an index.
- cardiomyocytes can be obtained at a high rate from a cell population containing cardiomyocytes. Based on this finding, the present inventors have at least one selected from the group consisting of CORIN, NCAM1, CRYAB, HBEGF, DMD, ATPIF1, CAV2, ITGAV, DCBLD2, CLIC4, BMPR2, CTSB, TMEM123, USP14 and MIR761. By using positive as an index, cardiomyocytes were successfully isolated and purified, and the present invention was completed.
- the present invention is as follows.
- An indicator that at least one marker selected from the group consisting of CORIN, NCAM1, CRYAB, HBEGF, DMD, ATPIF1, CAV2, ITGAV, DCBLD2, CLIC4, BMPR2, CTSB, TMEM123, USP14 and MIR761 is positive
- a method for producing cardiomyocytes comprising a step of extracting mature cardiomyocytes from a cell population containing cardiomyocytes.
- the marker is CORIN.
- the cytokine is at least one cytokine selected from the group consisting of activin A, BMP4 (bone morphogenetic protein 4), bFGF (basic fibroblast growth factor), VEGF (vascular endothelial growth factor) and Wnt inhibitor.
- the method according to [9]. [11] The method according to [10], wherein the differentiation induction into cardiomyocytes includes the following steps. (1) forming an embryoid body from pluripotent stem cells; (2) culturing the embryoid body obtained in the step (1) in a culture medium containing activin A, BMP4 and bFGF, (3) A step of dissociating the embryoid body obtained in the step (2), (4) A step of culturing the cells obtained in the step (3) in a culture solution containing VEGF and a Wnt inhibitor to reaggregate them into embryoid bodies, and (5) obtained in the step of (4). Culturing the embryoid body in a culture medium containing VEGF and bFGF.
- a method for producing cardiomyocytes comprising: (1) forming an embryoid body from pluripotent stem cells; (2) culturing the embryoid body obtained in the step (1) in a culture medium containing activin A, BMP4 and bFGF, (3) A step of dissociating the embryoid body obtained in the step (2), (4) A step of culturing the cells obtained in the step (3) in a culture solution containing VEGF and a Wnt inhibitor to reaggregate them into embryoid
- the present invention can provide mature cardiomyocytes more closely resembling the cardiomyocytes present in the living body, which is useful in more accurate drug toxicity tests and heart disease model studies.
- FIG. 1 shows the result of comparison of cells with high and low CORIN expression levels for cardiomyocyte maturity.
- A shows the results of FACS analysis in which a cell group on the 30th day after induction of differentiation into cardiomyocytes was selected by GFP and CORIN.
- B shows the results of comparing the relative expression levels of cardiomyocyte markers (MYH7, MYL2, MYL7, TCAP, SCN5A and RYR2).
- “High” indicates a cell group in which both the expression levels of GFP and CORIN are high
- “Low” indicates a cell group in which the expression level of GFP is high but the expression level of CORIN is low.
- FIG. 2 shows the results of testing the function of cardiomyocytes by an electrophysiological method.
- FIG. 3 shows the result of comparison of cells with high and low CORIN expression levels for cardiomyocyte maturity.
- A is a photograph showing the morphology of a cell group on the 30th day after induction of differentiation into cardiomyocytes.
- FIG. 1 shows the results of FACS analysis in which a cell group on the 30th day after induction of differentiation into cardiomyocytes was selected using a known cardiomyocyte marker (TNT).
- TNT cardiomyocyte marker
- C shows the result of FACS analysis in which the cell group that is positive for the cardiomyocyte marker (TNT) selected in (B) is selected by CORIN.
- D shows the results of comparing the relative expression levels of cardiomyocyte markers (MYH7, MYL2, MYL7, TCAP, SCN5A and RYR2).
- FIG. 4 shows a micrograph (a) showing the cell size in the CORIN-High group and the CORIN-Low group, and a graph (b) showing the comparison result of the cell size.
- red indicates actin
- blue indicates Hoechst33342
- the scale bar indicates 20 ⁇ m.
- the value is mean ⁇ SD
- “**” indicates that p ⁇ 0.01 by unpaired t test.
- FIG. 5 is an electron micrograph showing myocardial sarcomere structures in the CORIN-High group (left) and the CORIN-Low group (right).
- FIG. 6 is a graph showing a comparison of the width of sarcomere in the CORIN-High group (left) and the CORIN-Low group (right) determined from the data of the electron micrograph of FIG. Values are mean ⁇ SD, and “**” indicates unpaired t-test with p ⁇ 0.01.
- FIG. 7 is a diagram showing an evaluation of mitochondrial respiration in the CORIN-High group (black) and the CORIN-Low group (gray).
- (A) is a graph showing the time change of oxygen consumption rate (OCR) using a mitochondrial function inhibitor (Oligo: oligomycin, FCCP: carbonyl cyanide-4-phenylhydrazone, A / R: antimycine + rotenone), Data are shown as mean ⁇ SD.
- (B) is a graph showing the values of basal respiration, preliminary respiration ability, proton leak, and ATP production related respiration calculated from the data of (a). The value is an average value ⁇ SD, and “*” and “**” indicate that p ⁇ 0.05 and p ⁇ 0.001, respectively, in an unpaired t-test.
- At least one selected from the group consisting of CORIN, NCAM1, CRYAB, HBEGF, DMD, ATPIF1, CAV2, ITGAV, DCBLD2, CLIC4, BMPR2, CTSB, TMEM123, USP14 and MIR761 is positive.
- a method for producing cardiomyocytes including a step of extracting cardiomyocytes from a cell population containing cardiomyocytes, a method for testing the toxicity of a drug using cardiomyocytes produced by the method, and the cardiomyocytes
- the present invention relates to a cardiomyocyte extraction kit containing a reagent for detecting a marker.
- the “cardiomyocyte” means a cell expressing at least one marker gene selected from the group consisting of at least cardiac troponin (cTNT), ⁇ MHC ( ⁇ myosin heavy chain) and MYH6.
- cTNT cardiac troponin
- ⁇ MHC ⁇ myosin heavy chain
- MYH6 is exemplified by NCBI accession number NM_002471 for humans and NM_001164171 for mice.
- mature cardiomyocytes mean cardiomyocytes that are more functionally and morphologically similar to adult cardiomyocytes present in the living body. That is, the mature cardiomyocyte in the present invention is a cardiomyocyte in which the ion channel function is sufficiently developed as compared with an immature fetal cardiomyocyte, for example, an adult myocardium having a resting membrane potential, a peak voltage, an amplitude, etc. It is a cell similar to a cell.
- the mature cardiomyocytes in the present invention have a resting membrane potential of ⁇ 55 mV or less, preferably ⁇ 60 mV or less, more preferably ⁇ 65 mV or less, and a peak voltage of 15 mV or more, preferably 20 mV or more, more preferably 25 mV or more. And the amplitude is characterized by 70 mV or more, preferably 80 mV or more, more preferably 90 mV or more.
- “Mature cardiomyocytes” are also characterized by the expression of one or more markers selected from MYH7, MYL2, MYL7, TCAP, SCN5A and RYR2.
- Table 1 below illustrates the sequences of the genes encoded for the novel mature cardiomyocyte markers in the present invention.
- sequence of each marker is not limited to the following sequence, and any marker may be used as long as it is expressed in the cell to be extracted.
- each marker protein When extracting mature cardiomyocytes using the novel mature cardiomyocyte marker in the present invention, the expression of each marker protein may be used as an index, and the gene encoding each marker protein is expressed (mRNA). May be used as an index.
- mRNA messenger RNA
- the isoform is also included in the range of each gene.
- the “cell population containing cardiomyocytes” is not particularly limited as long as it is an aggregate of cells containing mature cardiomyocytes.
- it may be a cell population contained in peripheral blood, heart, bone marrow tissue, adipose tissue, skeletal muscle tissue, amniotic tissue, placenta tissue, umbilical cord blood, etc. obtained by any method, and pluripotent It may be a cell population containing cardiomyocytes differentiated from stem cells.
- “extracting mature cardiomyocytes” means increasing the ratio of mature cardiomyocytes compared to before extraction, and preferably 50%, 60%, 70%, 80% of mature cardiomyocytes. % Or 90% or more. More preferably, cells comprising 100% mature cardiomyocytes are obtained.
- mature cardiomyocytes are extracted from a cell population containing cardiomyocytes using CORIN, NCAM1, CRYAB, HBEGF, DMD, ATPIF1, CAV2, ITGAV, DCBLD2, CLIC4, BMPR2, CTSB, TMEM123, USP14 or MIR761 as an index.
- these genes or proteins encoded by the genes can be used alone or in any combination thereof.
- CORIN, NCAM1, CRYAB, HBEGF, DMD, ATPIF1, CAV2, ITGAV, DCBLD2, CLIC4, BMPR2, CTSB, TMEM123, USP14 or MIR761 preferably compared to using alone
- the concentration rate of cardiomyocytes is increased.
- NCAM1 is preferably used in combination with the other cardiomyocyte markers described above, rather than NCAM1 alone.
- the pluripotent stem cell that can be used for obtaining a cell population containing cardiomyocytes in the present invention has pluripotency that can be differentiated into all cells existing in the living body, and also has proliferative ability.
- Stem cells including but not limited to, embryonic stem (ES) cells, sperm stem cells (“GS cells”), embryonic germ cells (“EG cells”), induced pluripotent stems (iPS) cells, embryonic stem (ntES) cells derived from cloned embryos obtained by nuclear transfer, and the like are included.
- Preferred pluripotent stem cells are ES cells, iPS cells and ntES cells.
- Embryonic stem cells ES cells are stem cells established from the inner cell mass of early embryos (for example, blastocysts) of mammals such as humans and mice, and having pluripotency and proliferation ability by self-replication.
- ES cells are embryonic stem cells derived from the inner cell mass of the blastocyst, the embryo after the morula, in the 8-cell stage of a fertilized egg, and have the ability to differentiate into any cell that constitutes an adult, so-called differentiation. And ability to proliferate by self-replication.
- ES cells were discovered in mice in 1981 (MJ Evans and MH Kaufman (1981), Nature 292: 154-156), and then ES cell lines were established in primates such as humans and monkeys (JA Thomson et al. (1998), Science 282: 1145-1147; JA Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92: 7844-7848; JA Thomson et al. (1996), Biol. Reprod 55: 254-259; JA JA Thomson and VS Marshall (1998), Curr. Top. Dev. Biol., 38: 133-165).
- ES cells can be established by taking an inner cell mass from a blastocyst of a fertilized egg of a target animal and culturing the inner cell mass on a fibroblast feeder. In addition, maintenance of cells by subculture is performed using a culture solution to which substances such as leukemia inhibitory factor (LIF) and basic fibroblast growth factor (basic fibroblast growth factor (bFGF)) are added. It can be carried out.
- LIF leukemia inhibitory factor
- bFGF basic fibroblast growth factor
- DMEM / F-12 culture medium supplemented with 0.1 mM 2-mercaptoethanol, 0.1 mM non-essential amino acid, 2 mM L-glutamic acid, 20% KSR and 4 ng / ml bFGF is used as the culture medium for ES cell production.
- Human ES cells can be maintained in a humid atmosphere of 37 ° C., 2% CO 2 /98% air (O. Fumitaka et al. (2008), Nat. Biotechnol., 26: 215-224).
- ES cells need to be passaged every 3-4 days, where passage is eg 0.25% trypsin and 0.1 mg / ml collagenase IV in PBS containing 1 mM CaCl 2 and 20% KSR. Can be used.
- ES cells can be generally selected by Real-Time PCR using the expression of gene markers such as alkaline phosphatase, Oct-3 / 4, Nanog as an index.
- gene markers such as alkaline phosphatase, Oct-3 / 4, Nanog
- OCT-3 / 4, NANOG, and ECAD can be used as an index (E. Kroon et al. (2008), Nat. Biotechnol., 26: 443). -452).
- Human ES cell lines for example, WA01 (H1) and WA09 (H9) are obtained from WiCell Research Institute, and KhES-1, KhES-2 and KhES-3 are obtained from Research Institute for Regenerative Medicine (Kyoto, Japan), Kyoto University Is possible.
- sperm stem cells are testis-derived pluripotent stem cells that are the origin of spermatogenesis. Like ES cells, these cells can be induced to differentiate into various types of cells, and have characteristics such as the ability to create chimeric mice when transplanted into mouse blastocysts (M. Kanatsu-Shinohara et al. ( 2003) Biol. Reprod., 69: 612-616; K. Shinohara et al. (2004), Cell, 119: 1001-1012).
- GDNF glial cell line-derived neurotrophic factor
- Embryonic germ cells are cells that are established from embryonic primordial germ cells and have the same pluripotency as ES cells, such as LIF, bFGF, stem cell factor, etc. It can be established by culturing primordial germ cells in the presence of these substances (Y. Matsui et al. (1992), Cell, 70: 841-847; JL Resnick et al. (1992), Nature, 359: 550 -551).
- iPS Artificial pluripotent stem cells
- somatic cells in the form of DNA or protein, which is almost equivalent to ES cells
- It is an artificial stem cell derived from a somatic cell having the characteristics of, for example, differentiation pluripotency and proliferation ability by self-replication (K. Takahashi and S. Yamanaka (2006) Cell, 126: 663-676; K. Takahashi et al. (2007), Cell, 131: 861-872; J. Yu et al. (2007), Science, 318: 1917-1920; Nakagawa, M. et al., Nat. Biotechnol.
- the reprogramming factor is a gene that is specifically expressed in ES cells, its gene product or non-coding RNA, a gene that plays an important role in maintaining undifferentiation of ES cells, its gene product or non-coding RNA, or It may be constituted by a low molecular compound.
- genes included in the reprogramming factor include Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15 -2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, etc. are exemplified, and these reprogramming factors may be used alone or in combination.
- the reprogramming factors include histone deacetylase (HDAC) inhibitors [for example, small molecule inhibitors such as valproate (VPA), trichostatin A, sodium butyrate, MC 1293, M344, siRNA and shRNA against HDAC (eg , Nucleic acid expression inhibitors such as HDAC1 siRNApoolSmartpool (registered trademark) (Millipore), HuSH 29mer shRNA Constructs against HDAC1 (OriGene), etc.], MEK inhibitors (eg, PD184352, PD98059, U0126, SL327 and PD0325901), Glycogen synthase kinase-3 inhibitors (eg, Bio and CHIR99021), DNA methyltransferase inhibitors (eg, 5-azacytidine), histone methyltransferase inhibitors (eg, small molecule inhibitors such as BIX-01294, Suv39hl, Suv39h2, Nucleic acid expression inhibitors such as
- the reprogramming factor may be introduced into a somatic cell by a technique such as lipofection, fusion with a cell membrane-permeable peptide (for example, TAT and polyarginine derived from HIV), or microinjection.
- a cell membrane-permeable peptide for example, TAT and polyarginine derived from HIV
- DNA it can be introduced into somatic cells by techniques such as vectors such as viruses, plasmids, artificial chromosomes, lipofection, liposomes, and microinjection.
- viral vectors examples include retroviral vectors and lentiviral vectors (above, Cell, 126, pp.663-676, 2006; Cell, 131, pp.861-872, 2007; Science, 318, pp.1917-1920, 2007 ), Adenovirus vectors (Science, 322, 945-949, 2008), adeno-associated virus vectors, Sendai virus vectors (WO 2010/008054) and the like.
- artificial chromosome vectors examples include human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), and bacterial artificial chromosomes (BAC, PAC).
- a plasmid for mammalian cells can be used (Science, 322: 949-953, 2008).
- the vector can contain regulatory sequences such as a promoter, enhancer, ribosome binding sequence, terminator, polyadenylation site, etc. so that a nuclear reprogramming substance can be expressed.
- Selective marker sequences such as kanamycin resistance gene, ampicillin resistance gene, puromycin resistance gene, thymidine kinase gene, diphtheria toxin gene, reporter gene sequences such as green fluorescent protein (GFP), ⁇ -glucuronidase (GUS), FLAG, etc. Can be included.
- the above vector has a LoxP sequence before and after the introduction of the gene into a somatic cell in order to excise the gene or promoter encoding the reprogramming factor and the gene encoding the reprogramming factor that binds to it. May be.
- RNA it may be introduced into somatic cells by techniques such as lipofection and microinjection, and in order to suppress degradation, RNA incorporating 5-methylcytidine and pseudouridine® (TriLink® Biotechnologies) is used. Yes (Warren L, (2010) Cell Stem Cell. 7: 618-630).
- Examples of the culture medium for inducing iPS cells include DMEM, DMEM / F12 or DME culture medium containing 10 to 15% FBS (these culture media include LIF, penicillin / streptomycin, puromycin, L-glutamine). , Non-essential amino acids, ⁇ -mercaptoethanol, etc.) or commercially available culture media (eg, culture media for mouse ES cell culture (TX-WES culture solution, Thrombo X), primate ES cells) Culture medium for culture (primate ES / iPS cell culture medium, Reprocell), serum-free medium (mTeSR, Stemcell Technology).
- DMEM DMEM / F12 or DME culture medium containing 10 to 15% FBS
- these culture media include LIF, penicillin / streptomycin, puromycin, L-glutamine). , Non-essential amino acids, ⁇ -mercaptoethanol, etc.
- commercially available culture media eg, culture media for mouse ES cell culture (TX
- the somatic cell and the reprogramming factor are brought into contact with DMEM or DMEM / F12 containing 10% FBS for about 4 to 7 days. Then, re-spread the cells on feeder cells (e.g., mitomycin C-treated STO cells, SNL cells, etc.), and use bFGF-containing primate ES cell culture medium about 10 days after contact of the somatic cells with the reprogramming factor. Culturing and generating iPS-like colonies about 30 to about 45 days or more after the contact.
- feeder cells e.g., mitomycin C-treated STO cells, SNL cells, etc.
- 10% FBS-containing DMEM culture medium including LIF, penicillin / streptomycin, etc.
- feeder cells eg, mitomycin C-treated STO cells, SNL cells, etc.
- 5% CO 2 at 37 ° C. can be suitably included with puromycin, L-glutamine, non-essential amino acids, ⁇ -mercaptoethanol, etc.
- ES-like colonies after about 25 to about 30 days or more .
- somatic cells to be reprogrammed themselves are used (Takahashi K, et al. (2009), PLoS One.
- extracellular matrix eg, Laminin- 5 (WO2009 / 123349) and Matrigel (BD)
- a method of culturing using a medium not containing serum is also exemplified (Sun N, et al. (2009), Proc Natl Acad Sci USA 106.15720-15725).
- iPS cells may be established under hypoxic conditions (oxygen concentration of 0.1% or more and 15% or less) (Yoshida Y, et al. (2009), Cell Stem Cell. 5: 237 -241 or WO2010 / 013845).
- the culture medium is exchanged with a fresh culture medium once a day from the second day onward.
- the number of somatic cells used for nuclear reprogramming is not limited, but ranges from about 5 ⁇ 10 3 to about 5 ⁇ 10 6 cells per 100 cm 2 of culture dish.
- IPS cells can be selected according to the shape of the formed colonies.
- a drug resistance gene that is expressed in conjunction with a gene that is expressed when somatic cells are initialized for example, Oct3 / 4, Nanog
- a culture solution containing the corresponding drug selection The established iPS cells can be selected by culturing with the culture medium.
- the marker gene is a fluorescent protein gene
- iPS cells are selected by observing with a fluorescence microscope, in the case of a luminescent enzyme gene, by adding a luminescent substrate, and in the case of a chromogenic enzyme gene, by adding a chromogenic substrate can do.
- the term “somatic cell” refers to any animal cell (preferably, a mammalian cell including a human) except a germ line cell such as an egg, oocyte, ES cell, or totipotent cell.
- Somatic cells include, but are not limited to, fetal (pup) somatic cells, neonatal (pup) somatic cells, and mature healthy or diseased somatic cells, as well as primary cultured cells. , Passage cells, and established cell lines are all included.
- somatic cells include, for example, (1) neural stem cells, hematopoietic stem cells, mesenchymal stem cells, tissue stem cells such as dental pulp stem cells (somatic stem cells), (2) tissue progenitor cells, (3) lymphocytes, epithelium Cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, hepatocytes, gastric mucosal cells, enterocytes, spleen cells, pancreatic cells (exocrine pancreas cells, etc.), brain cells, lung cells, kidney cells Examples thereof include differentiated cells such as fat cells.
- the mammal individual from which somatic cells are collected is not particularly limited, but is preferably a human.
- somatic cells should be collected from the patient or another person with the same or substantially the same HLA type from the viewpoint that no rejection occurs.
- the type of HLA is “substantially the same” means that when the cells obtained by inducing differentiation from iPS cells derived from the somatic cells are transplanted into a patient by using an immunosuppressant or the like, the transplanted cells are This means that the HLA types match to the extent that they can be engrafted.
- the origin of somatic cells that are the source of iPS cells is not particularly limited.
- somatic cells from the patient or others with the same genetic polymorphism that correlates with drug sensitivity or side effects It is desirable to collect.
- E Cloned embryo-derived ES cells obtained by nuclear transfer nt ES cells are cloned embryo-derived ES cells produced by nuclear transfer technology and have almost the same characteristics as ES cells derived from fertilized eggs (T. Wakayama et al. (2001), Science, 292: 740-743; S. Wakayama et al. (2005), Biol. Reprod., 72: 932-936; J. Byrne et al. (2007) , Nature, 450: 497-502).
- an ES cell established from an inner cell mass of a clonal embryo-derived blastocyst obtained by replacing the nucleus of an unfertilized egg with the nucleus of a somatic cell is an nt ES (nuclear transfer ES) cell.
- nt ES nuclear transfer ES
- nuclear transfer technology JB Cibelli et al. (1998), Nature Biotechnol., 16: 642-646) and ES cell production technology (above) is used (Wakayama). Seika et al. (2008), Experimental Medicine, Vol. 26, No. 5 (extra number), 47-52).
- Nuclear transfer can be initialized by injecting a somatic cell nucleus into a mammal's enucleated unfertilized egg and culturing for several hours.
- cardiomyocytes can be produced from pluripotent stem cells by, for example, the method reported by Laflamme MA et al. (Laflamme MA & Murry CE, Nature 2011, Review).
- a method of producing cardiomyocytes by forming a cell mass (embryoid body) by suspension culture of induced pluripotent stem cells, myocardium in the presence of a substance that suppresses BMP signaling A method for producing cells (WO2005 / 033298), a method for producing cardiomyocytes by sequentially adding Activin A and BMP (WO2007 / 002136), and producing cardiomyocytes in the presence of a substance that promotes activation of the canonical Wnt signaling pathway (WO2007 / 126077), a method of isolating Flk / KDR positive cells from induced pluripotent stem cells and producing cardiomyocytes in the presence of cyclosporin A (WO2009 / 118928).
- the method for inducing differentiation from a pluripotent stem cell to a cell population containing cardiomyocytes is not particularly limited, and for example, the following method is used.
- Step (1) it is preferable to form embryoid bodies after dissociating the pluripotent stem cells that have formed colonies into single cells.
- the step of dissociating pluripotent stem cells cells that are adhered to each other to form a population are dissociated (separated) into individual cells.
- a method for dissociating pluripotent stem cells for example, a method for mechanically dissociating, a dissociation solution having protease activity and collagenase activity (eg, Accutase TM and Accumax TM), or dissociation having only collagenase activity Examples include a dissociation method using a solution.
- a method of dissociating pluripotent stem cells using a dissociation solution having protease activity and collagenase activity is used.
- dissociated pluripotent stem cells are artificially treated on the surface of a culture dish for the purpose of improving adhesion to the cells (for example, Matrigel (BD), Not treated with collagen, gelatin, laminin, heparan sulfate proteoglycan, or entactin), or treated artificially (eg, coated with polyhydroxyethyl methacrylic acid (poly-HEMA))
- BD Matrigel
- poly-HEMA polyhydroxyethyl methacrylic acid
- the culture solution used in this step can be prepared by adding activin A, BMP4 and bFGF to the basal medium from the medium used for culturing animal cells.
- the basal medium include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, ⁇ MEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, Neurobasal Medium (Life Technologies) ), StemPro34 (Invitrogen) and mixed media thereof.
- the medium may contain serum or may be serum-free.
- the medium can be, for example, albumin, transferrin, Knockout Serum Replacement (KSR) (serum substitute for FBS during ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acid, insulin, collagen May contain one or more serum replacements such as precursors, trace elements, 2-mercaptoethanol, 1-thiolglycerol, lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, growth It may also contain one or more substances such as factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts and the like.
- a preferred basal medium is StemPro34 containing transferrin, 1-thiolglycerol, L-glutamine, ascorbic acid.
- the concentration of activin A used in this step is preferably 1 ng / ml to 100 ng / ml, 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml ml, 9ng / ml, 10ng / ml, 11ng / ml, 12ng / ml, 13ng / ml, 14ng / ml, 15ng / ml, 16ng / ml, 17ng / ml, 18ng / ml, 19ng / ml, 20ng / ml, Examples are 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng /
- the concentration of BMP4 used in this step is preferably 1 ng / ml to 100 ng / ml, 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml 9ng / ml, 10ng / ml, 11ng / ml, 12ng / ml, 13ng / ml, 14ng / ml, 15ng / ml, 16ng / ml, 17ng / ml, 18ng / ml, 19ng / ml, 20ng / ml, 30ng Examples include / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90
- the concentration of bFGF used in this step is preferably 1 ng / ml to 100 ng / ml, 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml 9ng / ml, 10ng / ml, 11ng / ml, 12ng / ml, 13ng / ml, 14ng / ml, 15ng / ml, 16ng / ml, 17ng / ml, 18ng / ml, 19ng / ml, 20ng / ml, 30ng Examples include / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml,
- the culture temperature is not limited to the following, but is about 30 to 40 ° C., preferably about 37 ° C., and it is desirable to carry out under low oxygen conditions.
- the low oxygen condition is a condition of an oxygen partial pressure lower than the oxygen partial pressure (20%) in the atmosphere, and examples thereof include an oxygen partial pressure of 1% to 15%. Examples are%, 8%, 7%, 6%, 5%, 4%, 3%, 2% and 1%. More preferably, it is 5%. Culturing is performed in an atmosphere of CO 2 -containing air, and the CO 2 concentration is preferably about 2 to 5%.
- the culture period is exemplified by 1 day or more and 7 days or less, and considering the establishment efficiency of cardiomyocytes, it is exemplified by 1 day or more and 5 days or less, 1.5 days or more and 5 days or less, and 2 days or more and 4 days or less.
- the culture period of the present invention is, for example, 1 day, 1.5 days, 2 days, 2.5 days, 3 days, 3.5 days, 4 days, 4.5 days, 5 days, 5.5 days, 6 days, 6.5 days, 7 days, etc. It may be, preferably 2 days.
- Step (3) ⁇ Step of dissociating the embryoid body during the manufacturing process to step (3)>
- the method for dissociating the embryoid body can be the same as described in the step (1).
- Step (4) in which an embryoid body is formed by re-aggregation after culturing in a culture medium containing VEGF and a Wnt inhibitor>
- the surface of the culture vessel is not artificially treated for the purpose of improving cell adhesion, or artificially suppressed for adhesion. It is preferable to carry out suspension culture using a culture vessel.
- the culture solution used in this step can be prepared by adding VEGF and a Wnt inhibitor to a basal medium that is used for animal cell culture.
- basal media include IMDM medium, MediumMedi199 medium, Eagle's Minimum Essential Medium (EMEM) medium, ⁇ MEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer Life's medium, Neurosal's medium ), StemPro34 (Invitrogen) and mixed media thereof.
- the medium may contain serum or may be serum-free.
- the medium can be, for example, albumin, transferrin, Knockout Serum Replacement (KSR) (serum substitute for FBS during ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acid, insulin, collagen May contain one or more serum replacements such as precursors, trace elements, 2-mercaptoethanol, 1-thiolglycerol, lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, growth It may also contain one or more substances such as factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts and the like.
- a preferred basal medium is StemPro34 containing transferrin, 1-thiolglycerol, L-glutamine, ascorbic acid.
- a Wnt inhibitor is a substance that inhibits signal transduction from binding of Wnt to a receptor to accumulation of ⁇ -catenin, a substance that inhibits binding to the receptor Frizzled family, or ⁇
- the substance is not particularly limited as long as it is a substance that promotes the degradation of catenin.
- DKK1 protein for example, NCBI accession number: NM_012242 for humans
- sclerostin for example, NCBI accession number for humans: NM_025237
- IWR-1 Merck Millipore
- IWP-2 Sigma-Aldrich
- IWP-3 Sigma-Aldrich
- IWP-4 Sigma-Aldrich
- PNU-74654 Sigma-Aldrich
- XAV939 Sigma- Aldrich
- the Wnt inhibitor used in this step can be preferably IWP-3 or IWP-4.
- the concentration of a Wnt inhibitor such as IWP-3 or IWP-4 in the culture solution is not particularly limited as long as it is a concentration that inhibits Wnt, but is preferably 1 nM to 50 ⁇ M, for example, 1 nM, 10 nM, 50 nM, 100 nM, 500 nM, 750 nM, 1 ⁇ M, 2 ⁇ M, 3 ⁇ M, 4 ⁇ M, 5 ⁇ M, 6 ⁇ M, 7 ⁇ M, 8 ⁇ M, 9 ⁇ M, 10 ⁇ M, 15 ⁇ M, 20 ⁇ M, 25 ⁇ M, 30 ⁇ M, 40 ⁇ M, 50 ⁇ M, but are not limited thereto. More preferably, it is 1 ⁇ M.
- the concentration of VEGF used in this step is preferably 1 to 100 ng / ml, 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, 10ng / ml, 11ng / ml, 12ng / ml, 13ng / ml, 14ng / ml, 15ng / ml, 16ng / ml, 17ng / ml, 18ng / ml, 19ng / ml, 20ng / ml, 30ng / ml 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml and
- BMP inhibitors include proteinaceous inhibitors such as Chordin, Noggin, Follistatin, Dorsomorphin (ie 6- [4- (2-piperidin-1-yl-ethoxy) phenyl] -3-pyridin-4-yl- pyrazolo [1,5-a] pyrimidine) and its derivatives (PB Yu et al. (2007), Circulation, 116: II_60; PB Yu et al. (2008), Nat. Chem. Biol., 4: 33-41 J. Hao et al.
- TGF ⁇ inhibitor a substance that inhibits signal transduction from binding of TGF ⁇ to the receptor to SMAD, a substance that inhibits binding to the ALK family of receptors, or phosphorylation of SMAD by the ALK family.
- inhibitors such as Lefty-1 (NCBI Accession No., mouse: NM_010094, human: NM_020997 is exemplified), SB431542, SB202190 (above, RKLindemann et al., Mol. Cancer, 2003, 2 : 20), SB505124 (GlaxoSmithKline), NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, LY580276 (Lilly Research Laboratories), A83-01 (WO 2009146408) and derivatives thereof.
- Lefty-1 NCBI Accession No., mouse: NM_010094, human: NM_020997 is exemplified
- SB431542 SB202190 (above, RKLindemann et al., Mol. Cancer, 2003, 2 : 20)
- SB505124 GaxoSmithKline
- NPC30345 SD093, SD908, SD208 (Scios)
- LY2109761 LY36
- the culture temperature is not limited to the following, but is about 30 to 40 ° C., preferably about 37 ° C., and it is desirable to carry out under low oxygen conditions.
- the low oxygen condition is a condition of an oxygen partial pressure lower than the oxygen partial pressure (20%) in the atmosphere, and examples thereof include an oxygen partial pressure of 1% to 15%. Examples are%, 8%, 7%, 6%, 5%, 4%, 3%, 2% and 1%. More preferably, it is 5%. Culturing is performed in an atmosphere of CO 2 -containing air, and the CO 2 concentration is preferably about 2 to 5%.
- the culture period does not affect the establishment of cardiomyocytes by culturing for a long period of time, there is no particular upper limit, but it is preferable to culture for 4 days or more.
- the 4th, 5th, 6th, 7th, 8th, 9th and 10th may be mentioned. More preferably, it is cultured for 4 days.
- embryoid bodies formed by reaggregation differentiate into cardiomyocytes.
- the culture solution used in this step can be prepared by adding VEGF and bFGF to a basal medium from a medium used for animal cell culture.
- the basal medium include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, ⁇ MEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, Neurobasal Medium (Life Technologies) ), StemPro34 (Invitrogen) and mixed media thereof.
- the medium may contain serum or may be serum-free.
- the medium can be, for example, albumin, transferrin, Knockout Serum Replacement (KSR) (serum substitute for FBS during ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acid, insulin, collagen May contain one or more serum replacements such as precursors, trace elements, 2-mercaptoethanol, 1-thiolglycerol, lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, growth It may also contain one or more substances such as factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts and the like.
- a preferred basal medium is StemPro34 containing transferrin, 1-thiolglycerol, L-glutamine, ascorbic acid.
- the concentration of VEGF used in this step is preferably 1 to 100 ng / ml, 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, 10ng / ml, 11ng / ml, 12ng / ml, 13ng / ml, 14ng / ml, 15ng / ml, 16ng / ml, 17ng / ml, 18ng / ml, 19ng / ml, 20ng / ml, 30ng / ml 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml and
- the concentration of bFGF used in this step is preferably 1 to 100 ng / ml, 1 ng / ml, 2 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, 10ng / ml, 11ng / ml, 12ng / ml, 13ng / ml, 14ng / ml, 15ng / ml, 16ng / ml, 17ng / ml, 18ng / ml, 19ng / ml, 20ng / ml, 30ng / ml 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml
- the culture temperature is not limited to the following, but is about 30 to 40 ° C., preferably about 37 ° C., and it is desirable to carry out under low oxygen conditions.
- the low oxygen condition is a condition of an oxygen partial pressure lower than the oxygen partial pressure (20%) in the atmosphere, and examples thereof include an oxygen partial pressure of 1% to 15%. Examples are%, 8%, 7%, 6%, 5%, 4%, 3%, 2% and 1%. More preferably, it is 5%.
- the partial pressure of oxygen may be performed in the middle of the step at the same level as in the atmosphere.
- the upper limit is not particularly set because there is no change in the induction efficiency of cardiomyocytes, particularly when performed under hypoxic conditions. Culturing is performed in an atmosphere of CO 2 -containing air, and the CO 2 concentration is preferably about 2 to 5%.
- the culture period does not affect the establishment of cardiomyocytes by culturing for a long time, there is no particular upper limit, but it is preferable to culture for 12 days or longer. For example, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th or more. More preferably, it is cultured for 23 days.
- the efficiency of differentiation into cardiomyocytes is improved.
- the present invention can include a step of removing cells other than the cardiomyocytes during the step (5) in order to increase the purity of the finally obtained cardiomyocytes.
- this step for example, the method described in JP-A-2013-143968 can be used, but is not limited thereto.
- This step may be anything as long as it is a culture under conditions that can select cells other than cardiomyocytes and non-cardiomyocytes. For example, when cells other than cardiomyocytes die, The culture can be performed under such conditions that the cells can be selected.
- Examples of culture conditions in which cells other than cardiomyocytes die are low-sugar (for example, sugar concentration of 1% or less, or sugars contained 55.60 to 111.20 ⁇ M) and lactic acid added, but are not limited thereto.
- the culture can be performed under the condition of sugar-free and lactic acid added.
- the concentration of lactic acid to be added is not particularly limited, but is, for example, in the range of 0.1 to 5 mM, and preferably 1 mM.
- the culture conditions in which sugar-free and lactic acid are added can be performed by removing saccharides from the components of the culture solution in step (5) and adding lactic acid.
- lactic acid is added directly to the culture solution in step (5), or the culture solution in step (5) is replaced with a fresh culture solution in step (5) to which lactic acid is added. It is not limited to.
- the culture period of the step of removing cells other than cardiomyocytes is preferably cultured for 2 days or more during the step (5).
- the culture start time in the step of removing cells other than cardiomyocytes is preferably 10 days or more after the start of step (5).
- ⁇ Drug reactivity test> it is possible to examine whether or not a drug candidate substance has cardiotoxicity using mature cardiomyocytes extracted using the marker of the present invention.
- the cardiotoxicity test can be performed using any method based on the common general technical knowledge in this field.
- the test in the present invention can be performed based on, for example, an electrophysiological technique or an expression analysis of a toxicity marker gene. Examples of electrophysiological techniques include, but are not limited to, patch clamp tests.
- Examples of the toxic marker gene used for the expression analysis of the toxic marker gene include, for example, ABHD2, CCR1, GJA1, NEXN, PSMD7, TGFB2, VIM, ABRA, CD14, GPM6A, NFIB, PUM2, THRAP3, WIPI1, ACTA1, and CFD.
- Toxicity marker genes can be measured and analyzed by targeting the above gene group using, for example, TruSeq target RNA
- cardiotoxicity caused by a drug candidate substance is exemplified by drug-induced arrhythmia
- the arrhythmia is exemplified by tachycardia, bradycardia, extrasystole, QT prolongation, etc., but is detected by the method of the present invention.
- QT prolongation is preferable. Therefore, it can be said that the cardiotoxicity to be tested in the present invention is drug-induced QT prolongation. Since QT prolongation is caused by inhibition of potassium channels, the method of the present invention can also be said to be a method for examining the ability to inhibit potassium channels that cause arrhythmias caused by drug candidates.
- QT prolongation can be confirmed by confirming the persistence of the potential change of mature cardiomyocytes obtained by the patch clamp test.
- “continuation of potential change of mature cardiomyocytes” means a period of time from the resting membrane potential to the plus side and then to the resting membrane potential again.
- the compound can be identified as a drug having no cardiotoxicity.
- the duration of the potential change in the mature cardiomyocytes contacted with the drug candidate substance is compared with the duration of the potential change in the mature cardiomyocytes not contacted. When the duration of the potential change is prolonged, it can be determined that the drug candidate substance has cardiotoxicity.
- the drug candidate substance is not particularly limited as long as it is a substance presumed to be effective against a disease.
- a cell extract, a cell culture supernatant, a microbial fermentation product, a marine organism-derived extract examples include plant extracts, purified or crude proteins, peptides, non-peptide compounds, synthetic low molecular compounds, natural compounds, and the like.
- the disease targeted by the drug candidate substance is not limited to heart disease.
- the cell used in the drug reactivity test of the present invention may be a single cell, a cell mass or a cell sheet.
- the cell mass is preferably composed only of mature cardiomyocytes, but may be a mixed group of mature cardiomyocytes and a small amount of other cells.
- the cell sheet is obtained by treating a mature cardiomyocyte obtained by the above-described method of the present invention with a (meth) acrylamide compound, an N- (or N, N-di) alkyl-substituted (meth) acrylamide derivative (JP 2010-2010). 255001), or a culture device coated with a temperature-responsive polymer obtained by polymerizing a vinyl ether derivative.
- the main culture equipment can be purchased from Cell Seed as UpCell.
- Reagents used to extract or detect cardiomyocytes from cell populations containing cardiomyocytes include CORIN, NCAM1, CRYAB, HBEGF, DMD, ATPIF1, CAV2, ITGAV, DCBLD2, CLIC4, BMPR2, CTSB, TMEM123 Any reagent having specific affinity for USP14 or MIR761 may be used, and an antibody, aptamer, peptide or a compound that specifically recognizes can be used, and preferably an antibody or a fragment thereof. In addition, when examining the gene expression of these markers, primers and probes that hybridize to these marker genes can be used.
- the antibody may be a polyclonal or monoclonal antibody.
- These antibodies can be made using techniques well known to those skilled in the art (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons. Section 11.12-11.13).
- the antibody of the present invention is a polyclonal antibody, CORIN, NCAM1, CRYAB, HBEGF, DMD, ATPIF1, CAV2, ITGAV, DCBLD2, CLIC4, BMPR2, CTSB, expressed and purified in Escherichia coli according to a conventional method It is possible to synthesize a TMEM123, USP14 or MIR761 protein, or an oligopeptide having a partial amino acid sequence, immunize a non-human animal such as a rabbit, and obtain it from the sera of the immunized animal according to a conventional method.
- a monoclonal antibody in the case of a monoclonal antibody, it can be obtained from hybridoma cells prepared by cell fusion of spleen cells and myeloma cells obtained from the immunized non-human animal described above (Current protocols in Molecular Biology). edit.Ausubel et al. (1987) Publish.John Wiley and Sons.Section 11.4-11.11).
- the antibody fragment include a part of an antibody (eg, Fab fragment) or a synthetic antibody fragment (eg, single-chain Fv fragment “ScFv”).
- Antibody fragments such as Fab and F (ab) 2 fragments can also be prepared by methods well known in genetic engineering.
- the marker is a membrane protein, it is preferably an antibody against the extracellular domain.
- an antibody disclosed in WO2006 / 009241 is exemplified as an antibody against CORIN.
- reagents having such affinity include, for example, detectable substances such as fluorescent labels, radioactive labels, chemiluminescent labels, enzymes, biotin, streptavidin, or protein A, protein G, It may be bound or conjugated to a substance that enables isolation and extraction such as beads and magnetic beads.
- the reagent having the affinity may be indirectly labeled.
- Various methods known to those skilled in the art can be used, e.g. for antibodies against CORIN, NCAM1, CRYAB, HBEGF, DMD, ATPIF1, CAV2, ITGAV, DCBLD2, CLIC4, BMPR2, CTSB, TMEM123, USP14 or MIR761 Examples include a method using a pre-labeled antibody (secondary antibody) that specifically binds.
- the method for detecting cardiomyocytes includes, for example, using a flow cytometer or a method for detecting cells separately after isolation and purification (for example, a protein chip).
- Methods for extracting cardiomyocytes include a method in which large particles are joined and precipitated to a reagent having the affinity, a method in which cells are sorted by magnetism using magnetic beads (for example, MACS), and a cell sorter using a fluorescent label. Or a method using a carrier on which an antibody or the like is immobilized (for example, a cell concentration column).
- a cell population containing cardiomyocytes at least selected from the group consisting of CORIN, NCAM1, CRYAB, HBEGF, DMD, ATPIF1, CAV2, ITGAV, DCBLD2, CLIC4, BMPR2, CTSB, TMEM123, USP14 and MIR761
- selectively collecting (extracting) cells that are positive for the marker may be collecting (extracting) all cells that are positive for the marker. It may be collected (extracted).
- the expression level of the marker is within the top 50%, top 40%, top 33%, top 30%, top 20% Cells or the top 10% of cells can be selectively collected.
- the present invention also contains a reagent for detecting at least one selected from the group consisting of CORIN, CRYAB, HBEGF, DMD, ATPIF1, CAV2, ITGAV, DCBLD2, CLIC4, BMPR2, CTSB, TMEM123, USP14 and MIR761.
- a cardiomyocyte extraction kit is provided.
- the present invention further provides a cardiomyocyte extraction kit containing a reagent for detecting NCAM1.
- the detection reagents contained in this extraction kit are as described above.
- the extraction kit in the present invention describes how to use the detection reagent together with detection reagents for CORIN, NCAM1, CRYAB, HBEGF, DMD, ATPIF1, CAV2, ITGAV, DCBLD2, CLIC4, BMPR2, CTSB, TMEM123, USP14 or MIR761. Instructions can also be included.
- MYH6-GFP reporter strain As pluripotent stem cells , experiments were performed using the following iPS cell line, 201B7 strain, and 201B7 strain having MYH6-GFP reporter (hereinafter referred to as MYH6-GFP reporter strain).
- the 201B7 strain was prepared by the method described in Takahashi K, et al. Cell. 131: 861-72, 2007.
- the MYH6-GFP reporter strain is introduced into the 201B7 strain by introducing a vector operably linked to the EGFP cassette downstream of the MYH6 (myosin heavy chain 6) promoter. It was conducted.
- the 201B7 strain and the MYH6-GFP reporter strain were cultured in a conventional manner (Takahashi K, et al. Cell. 131: 861-72, 2007 and Nakagawa M, et al. Nat Biotechnol. 26: 101-6 , 2008).
- Cardiomyocyte induction method MYH6-GFP reporter strain was treated with CTK solution (ReproCELL) for 2 minutes, then the solution was removed, subsequently treated with Accumax (Innovative Cell Technologies) for 5 minutes, and dissociated into single cells by pipetting.
- the medium was removed by centrifugation, and the resulting cells were seeded in a low-adhesion 96-well dish (Corning) at 2500 cells / well per well, and 1% L-Glutamine, 150 ⁇ g / mL Transferrin, 50 ⁇ g / mL Ascorbic Acid (sigma ), Cultured in STEMPRO 34 supplemented with 3.9 ⁇ 10 ⁇ 3 % MTG, 10 ⁇ M Rock inhibitor and 2 ng / mL BMP4 (R & D), 0.50% Matrigel (Growth Factor Reduced) at 37 ° C. and 5% oxygen An embryoid body (hereinafter referred to as EB) was formed (day 0).
- EB Matrigel
- the obtained EBs were collected and transferred to a 24-well dish so that the number of EBs per well did not exceed 10.
- the medium was replaced once every two days with the same conditions.
- the top 15 genes are shown in Table 2 below.
- Example 1 Extraction of mature cardiomyocytes using CORIN It was examined in detail whether CORIN identified as a novel mature cardiomyocyte marker in Example 1 was actually effective for extraction of mature cardiomyocytes. Briefly, the day30 MYH6-GFP reporter strain differentiated by the cardiomyocyte induction method of Example 1 was analyzed by FACS using GFP and CORIN as alternatives to the expression of MYH6, which is a known cardiomyocyte marker. It was. FACS analysis was performed using a FACS Arial II cell sorter (BD Biosciences). As a result, a cell group having a high expression level of both GFP and CORIN (High group) and a cell group having a high expression level of GFP but a low expression level of CORIN (Low group) were confirmed.
- BD Biosciences FACS Arial II cell sorter
- the High group and the Low group were sorted using a FACS Arial II cell sorter (FIG. 1 (A)).
- the High group was selected as the cells with the highest 33% CORIN expression
- the Low group was selected as the cells with the lowest 33% CORIN expression.
- the maturity degree of the cardiomyocyte was investigated by measuring the relative expression level of the cardiomyocyte marker (MYH7, MYL2, MYL7, TCAP, SCN5A, and RYR2). The expression level was measured by quantitative RT-PCR. Briefly, mRNA was isolated from cells using the RNeasy Mini Kit (Qiagen).
- CDNA was synthesized by reverse transcription using Superscript III reverse transcriptase (Invitrogen) using 1 ⁇ g of the obtained total RNA as a template. Quantitative RT-PCR analysis was measured by StepOne real-time PCR system (ABI) using Power SYBR Green qPCR mastermix (Invitrogen). As a result, it was found that any of the examined cardiomyocyte markers showed a higher expression level in the High group than in the Low group (FIG. 1 (B)). This suggests that the High group is a relatively mature cardiomyocyte cell group.
- Extraction of mature cardiomyocytes using CORIN CORIN identified as a novel mature cardiomyocyte marker in Example 1 can be used to extract mature cardiomyocytes from a group of cardiomyocytes obtained by a cardiomyocyte induction method with partial modification.
- the partial modification is simply to add a step of culturing in a culture solution containing no glucose and lactic acid in day 23-27 of the cardiomyocyte induction method of Example 1.
- a FACS analysis of the day30 MYH6-GFP reporter strain using a known cardiomyocyte marker (TNT) as an index showed that 94.7% of the cells were TNT-positive and differentiated into cardiomyocytes (Fig. 3 (B)).
- FACS analysis was performed using a FACS Arial II cell sorter (BD Biosciences) in the same manner as in Example 2.
- the TNT positive cell group was analyzed by FACS using CORIN as an index.
- CORIN-High group a cell group with a high CORIN expression level
- CORIN-Low group a cell group with a low CORIN expression level
- the CORIN-High group and the CORIN-Low group could be selected using a FACS Arial II cell sorter (Fig. 3 (C)).
- the CORIN-High group was selected as the cells with the highest 33% CORIN expression level, and the CORIN-Low group was selected as the cells with the lowest CORIN expression level of 33%.
- cardiomyocytes were examined by measuring the relative expression level of cardiomyocyte markers (MYH7, MYL2, MYL7, TCAP, SCN5A and RYR2) in the CORIN-High and CORIN-Low groups. .
- the expression level was measured by RT-PCR in the same manner as described above.
- any of the examined cardiomyocyte markers showed a higher expression level in the CORIN-High group than in the CORIN-Low group (FIG. 3 (D)). This suggests that the CORIN-High group is a relatively mature cardiomyocyte cell group.
- iPS cells were induced to differentiate and seeded with cardiomyocytes (20,000) on the 30th day in fibronectin-coated assay wells (Seahorse Bioscience), and 3 days after sowing, the oxygen consumption rate (OCR) was determined. Measurements were taken with an XF24 extracellular flux analyzer from Seahorse Bioscience. As a result, as shown in FIG. 7, it was found that the CORIN-High group showed a high oxygen consumption rate and the activity of the electron transfer system was also high.
- the cardiomyocyte population obtained by differentiation induction from pluripotent stem cells contains cells of various maturity levels. According to the present invention, among such cell populations, they exist in vivo. It is possible to provide a cell group enriched with high-maturity cardiomyocytes that are closer to the cardiomyocytes. By using a more uniformly purified mature cardiomyocyte group obtained by the present invention, it becomes possible to conduct drug toxicity tests and heart disease model studies with higher accuracy than before, and shorten the drug development period. This is useful in the search for new disease targets.
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Abstract
Description
一方、分化誘導された細胞が心筋細胞であるか否かを見極めるための指標として、これまで複数のマーカー遺伝子が同定されてきた。例えば、心筋細胞に対する表面マーカーとして、CD166(ALCM)(非特許文献4)、N-cadherin(特許文献3または非特許文献5)、VCAM1(特許文献4)などが報告されているが、成熟心筋細胞に特異的なマーカー遺伝子については報告がなされていない。
[1]CORIN、NCAM1、CRYAB、HBEGF、DMD、ATPIF1、CAV2、ITGAV、DCBLD2、CLIC4、BMPR2、CTSB、TMEM123、USP14およびMIR761からなる群から選択される少なくとも一つのマーカーが陽性であることを指標として、心筋細胞を含有する細胞集団から成熟心筋細胞を抽出する工程を含む、心筋細胞の製造方法。
[2]前記マーカーが、CORINである、[1]に記載の方法。
[3]心筋細胞を含有する細胞集団に含まれる細胞のうち、前記マーカーの発現量が上位50%以内である細胞が成熟心筋細胞として抽出される、[1]または[2]に記載の方法。
[4]心筋細胞を含有する細胞集団に含まれる細胞のうち、前記マーカーの発現量が上位33%以内の細胞が成熟心筋細胞として抽出される、[3]に記載の方法。
[5]前記心筋細胞がヒト心筋細胞である、[1]から[4]のいずれかに記載の方法。
[6]前記心筋細胞を含有する細胞集団が、多能性幹細胞から分化誘導された心筋細胞を含有する細胞集団である、[1]から[5]のいずれかに記載の方法。
[7]前記多能性幹細胞が、ES細胞またはiPS細胞である、[6]に記載の方法。
[8]前記心筋細胞への分化誘導が、胚様体を形成する工程を含む、[6]または[7]に記載の方法。
[9]前記心筋細胞への分化誘導が、サイトカインを含有する培地で胚様体を培養する工程を含む、[8]に記載の方法。
[10]前記サイトカインが、アクチビンA、BMP4(bone morphogenetic protein 4)、bFGF (basic fibroblast growth factor)、VEGF (vascular endothelial growth factor)およびWnt阻害剤からなる群から選択される少なくとも一つのサイトカインである、[9]に記載の方法。
[11]前記心筋細胞への分化誘導が、下記の工程を含む、[10]に記載の方法。
(1)多能性幹細胞から胚様体を形成する工程、
(2)(1)の工程で得られた胚様体をアクチビンA、BMP4およびbFGFを含有する培養液中で培養する工程、
(3)(2)の工程で得られた胚様体を解離する工程、
(4)(3)の工程で得られた細胞をVEGFおよびWnt阻害剤を含有する培養液中で培養して胚様体に再凝集させる工程、および
(5)(4)の工程で得られた胚様体をVEGFおよびbFGFを含有する培養液中で培養する工程。
[12]前記Wnt阻害剤が、IWP-3またはIWP-4である、[11]に記載の方法。
[13]前記心筋細胞への分化誘導の工程(5)において、乳酸を含み、グルコースを含まない培養液中で培養する期間を含む、[11]または[12]に記載の方法。
[14]薬剤候補物質の心毒性を試験する方法であって、
(A)[1]から[13]のいずれかに記載の方法により作製された成熟心筋細胞と候補物質とを接触させる工程、および
(B)前記工程(A)の接触後に心筋細胞が正常な機能を維持した場合、当該候補物質を心毒性を有さない薬剤として同定する工程
を含む、方法。
[15]前記心筋細胞の正常な機能が、電気生理学的に正常な機能である、[14]に記載の方法。
[16]CORIN、NCAM1、CRYAB、HBEGF、DMD、ATPIF1、CAV2、ITGAV、DCBLD2、CLIC4、BMPR2、CTSB、TMEM123、USP14およびMIR761からなる群から選択される少なくとも一つを検出する試薬を含む、成熟心筋細胞抽出キット。
[17]CORINを検出する試薬を含む、[16]に記載のキット。
[18]前記心筋細胞がヒト心筋細胞である、[16]または[17]に記載のキット。
[19]下記の工程(1)~(5)によって多能性幹細胞から心筋細胞を含有する細胞集団を得ること、および得られた細胞群からCORIN、NCAM1、CRYAB、HBEGF、DMD、ATPIF1、CAV2、ITGAV、DCBLD2、CLIC4、BMPR2、CTSB、TMEM123、USP14およびMIR761からなる群から選択される少なくとも一つのマーカーが陽性であることを指標として心筋細胞を含有する細胞集団から成熟心筋細胞を抽出することを含む、心筋細胞の製造方法。
(1)多能性幹細胞から胚様体を形成する工程、
(2)(1)の工程で得られた胚様体をアクチビンA、BMP4およびbFGFを含有する培養液中で培養する工程、
(3)(2)の工程で得られた胚様体を解離する工程、
(4)(3)の工程で得られた細胞をVEGFおよびWnt阻害剤を含有する培養液中で培養して胚様体に再凝集させる工程、および
(5)(4)の工程で得られた胚様体をVEGFおよびbFGFを含有する培養液中で培養する工程。
また、「成熟心筋細胞」はMYH7、MYL2、MYL7、TCAP、SCN5AおよびRYR2から選択される1以上のマーカーの発現によって特徴づけられる。
本発明で心筋細胞を含有する細胞集団を得るために使用可能な多能性幹細胞は、生体に存在するすべての細胞に分化可能である多能性を有し、かつ、増殖能をも併せもつ幹細胞であり、それには、以下のものに限定されないが、例えば、胚性幹(ES)細胞、精子幹細胞(「GS細胞」)、胚性生殖細胞(「EG細胞」)、人工多能性幹(iPS)細胞、核移植により得られるクローン胚由来の胚性幹(ntES)細胞などが含まれる。好ましい多能性幹細胞は、ES細胞、iPS細胞およびntES細胞である。
ES細胞は、ヒトやマウスなどの哺乳動物の初期胚(例えば胚盤胞)の内部細胞塊から樹立された、多能性と自己複製による増殖能を有する幹細胞である。
精子幹細胞は、精巣由来の多能性幹細胞であり、精子形成のための起源となる細胞である。この細胞は、ES細胞と同様に、種々の系列の細胞に分化誘導可能であり、例えばマウス胚盤胞に移植するとキメラマウスを作出できるなどの性質をもつ(M. Kanatsu-Shinohara et al. (2003) Biol. Reprod., 69:612-616; K. Shinohara et al. (2004), Cell, 119:1001-1012)。神経膠細胞系由来神経栄養因子(glial cell line-derived neurotrophic factor (GDNF))を含む培養液で自己複製可能であるし、またES細胞と同様の培養条件下で継代を繰り返すことによって、精子幹細胞を得ることができる(竹林正則ら(2008),実験医学,26巻,5号(増刊),41~46頁,羊土社(東京、日本))。
胚性生殖細胞は、胎生期の始原生殖細胞から樹立される、ES細胞と同様な多能性をもつ細胞であり、LIF、bFGF、幹細胞因子(stem cell factor)などの物質の存在下で始原生殖細胞を培養することによって樹立しうる(Y. Matsui et al. (1992), Cell, 70:841-847; J.L. Resnick et al. (1992), Nature, 359:550-551)。
人工多能性幹(iPS)細胞は、特定の初期化因子を、DNA又はタンパク質の形態で体細胞に導入することによって作製することができる、ES細胞とほぼ同等の特性、例えば分化多能性と自己複製による増殖能を有する体細胞由来の人工の幹細胞である(K. Takahashi and S. Yamanaka (2006) Cell, 126:663-676; K. Takahashi et al. (2007), Cell, 131:861-872; J. Yu et al. (2007), Science, 318:1917-1920; Nakagawa, M.ら,Nat. Biotechnol. 26:101-106 (2008);国際公開WO 2007/069666)。初期化因子は、ES細胞に特異的に発現している遺伝子、その遺伝子産物もしくはnon-coding RNAまたはES細胞の未分化維持に重要な役割を果たす遺伝子、その遺伝子産物もしくはnon-coding RNA、あるいは低分子化合物によって構成されてもよい。初期化因子に含まれる遺伝子として、例えば、Oct3/4、Sox2、Sox1、Sox3、Sox15、Sox17、Klf4、Klf2、c-Myc、N-Myc、L-Myc、Nanog、Lin28、Fbx15、ERas、ECAT15-2、Tcl1、beta-catenin、Lin28b、Sall1、Sall4、Esrrb、Nr5a2、Tbx3等が例示され、これらの初期化因子は、単独で用いても良く、組み合わせて用いても良い。初期化因子の組み合わせとしては、WO2007/069666、WO2008/118820、WO2009/007852、WO2009/032194、WO2009/058413、WO2009/057831、WO2009/075119、WO2009/079007、WO2009/091659、WO2009/101084、WO2009/101407、WO2009/102983、WO2009/114949、WO2009/117439、WO2009/126250、WO2009/126251、WO2009/126655、WO2009/157593、WO2010/009015、WO2010/033906、WO2010/033920、WO2010/042800、WO2010/050626、WO 2010/056831、WO2010/068955、WO2010/098419、WO2010/102267、WO 2010/111409、WO 2010/111422、WO2010/115050、WO2010/124290、WO2010/147395、WO2010/147612、Huangfu D, et al. (2008), Nat. Biotechnol., 26: 795-797、Shi Y, et al. (2008), Cell Stem Cell, 2: 525-528、Eminli S, et al. (2008), Stem Cells. 26:2467-2474、Huangfu D, et al. (2008), Nat Biotechnol. 26:1269-1275、Shi Y, et al. (2008), Cell Stem Cell, 3, 568-574、Zhao Y, et al. (2008), Cell Stem Cell, 3:475-479、Marson A, (2008), Cell Stem Cell, 3, 132-135、Feng B, et al. (2009), Nat Cell Biol. 11:197-203、R.L. Judson et al., (2009), Nat. Biotech., 27:459-461、Lyssiotis CA, et al. (2009), Proc Natl Acad Sci U S A. 106:8912-8917、Kim JB, et al. (2009), Nature. 461:649-643、Ichida JK, et al. (2009), Cell Stem Cell. 5:491-503、Heng JC, et al. (2010), Cell Stem Cell. 6:167-74、Han J, et al. (2010), Nature. 463:1096-100、Mali P, et al. (2010), Stem Cells. 28:713-720に記載の組み合わせが例示される。
一方、DNAの形態の場合、例えば、ウイルス、プラスミド、人工染色体などのベクター、リポフェクション、リポソーム、マイクロインジェクションなどの手法によって体細胞内に導入することができる。ウイルスベクターとしては、レトロウイルスベクター、レンチウイルスベクター(以上、Cell, 126, pp.663-676, 2006; Cell, 131, pp.861-872, 2007; Science, 318, pp.1917-1920, 2007)、アデノウイルスベクター(Science, 322, 945-949, 2008)、アデノ随伴ウイルスベクター、センダイウイルスベクター(WO 2010/008054)などが例示される。また、人工染色体ベクターとしては、例えばヒト人工染色体(HAC)、酵母人工染色体(YAC)、細菌人工染色体(BAC、PAC)などが含まれる。プラスミドとしては、哺乳動物細胞用プラスミドを使用しうる(Science, 322:949-953, 2008)。ベクターには、核初期化物質が発現可能なように、プロモーター、エンハンサー、リボゾーム結合配列、ターミネーター、ポリアデニル化サイトなどの制御配列を含むことができるし、さらに、必要に応じて、薬剤耐性遺伝子(例えばカナマイシン耐性遺伝子、アンピシリン耐性遺伝子、ピューロマイシン耐性遺伝子など)、チミジンキナーゼ遺伝子、ジフテリアトキシン遺伝子などの選択マーカー配列、緑色蛍光タンパク質(GFP)、βグルクロニダーゼ(GUS)、FLAGなどのレポーター遺伝子配列などを含むことができる。また、上記ベクターには、体細胞への導入後、初期化因子をコードする遺伝子もしくはプロモーターとそれに結合する初期化因子をコードする遺伝子を共に切除するために、それらの前後にLoxP配列を有してもよい。
この他にも、血清を含有しない培地を用いて培養する方法も例示される(Sun N, et al. (2009), Proc Natl Acad Sci U S A. 106:15720-15725)。さらに、樹立効率を上げるため、低酸素条件(0.1%以上、15%以下の酸素濃度)によりiPS細胞を樹立しても良い(Yoshida Y, et al. (2009), Cell Stem Cell. 5:237-241またはWO2010/013845)。
nt ES細胞は、核移植技術によって作製されたクローン胚由来のES細胞であり、受精卵由来のES細胞とほぼ同じ特性を有している(T. Wakayama et al. (2001), Science, 292:740-743; S. Wakayama et al. (2005), Biol. Reprod., 72:932-936; J. Byrne et al. (2007), Nature, 450:497-502)。すなわち、未受精卵の核を体細胞の核と置換することによって得られたクローン胚由来の胚盤胞の内部細胞塊から樹立されたES細胞がnt ES(nuclear transfer ES)細胞である。nt ES細胞の作製のためには、核移植技術(J.B. Cibelli et al. (1998), Nature Biotechnol., 16:642-646)とES細胞作製技術(上記)との組み合わせが利用される(若山清香ら(2008),実験医学,26巻,5号(増刊), 47~52頁)。核移植においては、哺乳動物の除核した未受精卵に、体細胞の核を注入し、数時間培養することで初期化することができる。
本発明における心筋細胞を含む細胞集団への分化誘導方法として、例えばLaflamme MAらにより報告された方法により、多能性幹細胞から心筋細胞を製造することができる(Laflamme MA & Murry CE, Nature 2011, Review)。この他にも特に特定されないが、例えば、人工多能性幹細胞を浮遊培養により細胞塊(胚様体)を形成させて心筋細胞を製造する方法、BMPシグナル伝達を抑制する物質の存在下で心筋細胞を製造する方法(WO2005/033298)、Activin AとBMPを順に添加させて心筋細胞を製造する方法(WO2007/002136)、カノニカルWntシグナル経路の活性化を促す物質の存在下で心筋細胞を製造する方法(WO2007/126077)、人工多能性幹細胞からFlk/KDR陽性細胞を単離し、シクロスポリンAの存在下で心筋細胞を製造する方法(WO2009/118928)などが例示される。
この工程においては、コロニーを形成した多能性幹細胞を解離して単細胞にしたのちに胚様体を形成させることが好ましい。多能性幹細胞を解離させる工程においては、互いに接着して集団を形成している細胞を個々の細胞に解離(分離)させる。多能性幹細胞を解離させる方法としては、例えば、力学的に解離する方法、プロテアーゼ活性とコラゲナーゼ活性を有する解離溶液(例えば、Accutase(商標)およびAccumax(商標)など)またはコラゲナーゼ活性のみを有する解離溶液を用いた解離方法が挙げられる。好ましくは、プロテアーゼ活性とコラゲナーゼ活性を有する解離溶液(特に好ましくは、Accumax)を用いて多能性幹細胞を解離する方法が用いられる。
本工程において用いる培養液は、動物細胞の培養に用いられる培地を基礎培地へアクチビンA、BMP4およびbFGFを添加することにより調製することができる。基礎培地としては、例えばIMDM培地、Medium 199培地、Eagle's Minimum Essential Medium (EMEM)培地、αMEM培地、Dulbecco's modified Eagle's Medium (DMEM)培地、Ham's F12培地、RPMI 1640培地、Fischer's培地、Neurobasal Medium(ライフテクノロジーズ)、StemPro34(Invitrogen)およびこれらの混合培地などが包含される。培地には、血清が含有されていてもよいし、あるいは無血清でもよい。必要に応じて、培地は、例えば、アルブミン、トランスフェリン、Knockout Serum Replacement(KSR)(ES細胞培養時のFBSの血清代替物)、N2サプリメント(Invitrogen)、B27サプリメント(Invitrogen)、脂肪酸、インスリン、コラーゲン前駆体、微量元素、2-メルカプトエタノール、1-チオールグリセロールなどの1つ以上の血清代替物を含んでもよいし、脂質、アミノ酸、L-グルタミン、Glutamax(Invitrogen)、非必須アミノ酸、ビタミン、増殖因子、低分子化合物、抗生物質、抗酸化剤、ピルビン酸、緩衝剤、無機塩類などの1つ以上の物質も含有し得る。好ましい基礎培地は、トランスフェリン、1-チオールグリセロール、L-グルタミン、アスコルビン酸を含有するStemPro34である。
本発明において、胚様体を解離する方法は工程(1)で説明したのと同様の方法を用いることができる。
本工程の培養においては、工程(1)で説明したのと同様に、表面が細胞接着性を向上させる目的で人工的に処理されていない培養容器、もしくは、人工的に接着を抑制する処理した培養容器を用いて浮遊培養させることが好ましい。
BMP阻害剤としては、Chordin、Noggin、Follistatin、などのタンパク質性阻害剤、Dorsomorphin (すなわち、6-[4-(2-piperidin-1-yl-ethoxy)phenyl]-3-pyridin-4-yl-pyrazolo[1,5-a]pyrimidine)、その誘導体 (P. B. Yu et al. (2007), Circulation, 116:II_60; P.B. Yu et al. (2008), Nat. Chem. Biol., 4:33-41; J. Hao et al. (2008), PLoS ONE, 3(8):e2904)およびLDN193189(すなわち、4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline)が例示される。
TGFβ阻害剤としては、TGFβの受容体への結合からSMADへと続くシグナル伝達を阻害する物質であり、受容体であるALKファミリーへの結合を阻害する物質、またはALKファミリーによるSMADのリン酸化を阻害する物質が挙げられ、例えば、Lefty-1(NCBI Accession No.として、マウス:NM_010094、ヒト:NM_020997が例示される)、SB431542、SB202190(以上、R.K.Lindemann et al., Mol. Cancer, 2003, 2:20)、SB505124 (GlaxoSmithKline)、 NPC30345、SD093、 SD908、SD208 (Scios)、LY2109761、LY364947、 LY580276 (Lilly Research Laboratories)、A83-01(WO 2009146408) およびこれらの誘導体などが例示される。
本工程において用いる培養液は、動物細胞の培養に用いられる培地を基礎培地へVEGFおよびbFGFを添加することにより調製することができる。基礎培地としては、例えばIMDM培地、Medium 199培地、Eagle's Minimum Essential Medium (EMEM)培地、αMEM培地、Dulbecco's modified Eagle's Medium (DMEM)培地、Ham's F12培地、RPMI 1640培地、Fischer's培地、Neurobasal Medium(ライフテクノロジーズ)、StemPro34(Invitrogen)およびこれらの混合培地などが包含される。培地には、血清が含有されていてもよいし、あるいは無血清でもよい。必要に応じて、培地は、例えば、アルブミン、トランスフェリン、Knockout Serum Replacement(KSR)(ES細胞培養時のFBSの血清代替物)、N2サプリメント(Invitrogen)、B27サプリメント(Invitrogen)、脂肪酸、インスリン、コラーゲン前駆体、微量元素、2-メルカプトエタノール、1-チオールグリセロールなどの1つ以上の血清代替物を含んでもよいし、脂質、アミノ酸、L-グルタミン、Glutamax(Invitrogen)、非必須アミノ酸、ビタミン、増殖因子、低分子化合物、抗生物質、抗酸化剤、ピルビン酸、緩衝剤、無機塩類などの1つ以上の物質も含有し得る。好ましい基礎培地は、トランスフェリン、1-チオールグリセロール、L-グルタミン、アスコルビン酸を含有するStemPro34である。
本発明においては、本発明のマーカーを用いて抽出された成熟心筋細胞を用いて、薬剤候補物質が心毒性を有するか否かを調べることができる。心毒性の試験は、当分野における技術常識に基づいて、任意の方法を用いて行うことができる。本発明における試験は、例えば、電気生理学的手法または毒性マーカー遺伝子の発現解析などに基づいて行うことができる。電気生理学的な手法としては、例えば、パッチクランプ試験などが挙げられるがこれらに限定されない。また、毒性マーカー遺伝子の発現解析に用いられる毒性マーカー遺伝子としては、例えば、ABHD2、CCR1、GJA1、NEXN、PSMD7、TGFB2、VIM、ABRA、CD14、GPM6A、NFIB、PUM2、THRAP3、WIPI1、ACTA1、CFD、HAMP、PDK4、PVR、TIAM1、ZNF148、AIFM1、COL15A1、HSPA2、PKN2、RBM3、TIMP1、ZNF23、AK3、COL3A1、HSPH1、PLA2G4A、REG3G、TUBB6、 ASH1L、CREM、IFT20、PLAU、RND1、TXNIP、ATP5J、CSNK2A2、IGFBP5、PLN、RPS6KB1、UBA5、BCAT1、DUSP8、IL6、POSTN SERPINE1 UBXN2A、BGN、EGR1、ITPR2、PPBP、SIK1、UCK2、 BSN、FCGR2B、KBTBD10、PPP1R14C、SLC4A3、UCP1、BTG2、FHL1、KBTBD5、PRKAB2、SPP1、VCAN、CCL7、FOSL1、MCM6、PSMA2、TCF4、VEGFAなどが挙げられる。毒性マーカー遺伝子の測定および解析は、例えば、TruSeq ターゲットRNA発現解析-Cardiotoxityパネル(イルミナ社、Catalog ID:RT-202-1009)を用いて、上記遺伝子群を標的とすることにより行うことができる。
薬剤候補物質を接触させた成熟心筋細胞において、正常な機能、好ましくは電気生理的に正常な機能が維持された場合に、当該化合物が心毒性を有さない薬剤として同定することができる。一方、例えば、薬剤候補物質を接触させた成熟心筋細胞における電位変化の持続時間と接触させなかった成熟心筋細胞における電位変化の持続時間を比較して、薬剤候補物質を接触させた成熟心筋細胞における電位変化の持続時間が延長した場合に、当該薬剤候補物質は心毒性を有すると判定することができる。
心筋細胞を含有する細胞集団より心筋細胞の抽出または検出を行うために使用される試薬としては、CORIN、NCAM1、CRYAB、HBEGF、DMD、ATPIF1、CAV2、ITGAV、DCBLD2、CLIC4、BMPR2、CTSB、TMEM123、USP14またはMIR761に特異的親和性を有する試薬であれば何でもよく、抗体、アプタマー、ペプチドまたは特異的に認識する化合物などを用いることができ、好ましくは、抗体もしくはその断片である。
また、これらのマーカーの遺伝子発現を調べる場合は、これらのマーカー遺伝子にハイブリダイズするプライマーやプローブを使用することができる。
以下に実施例を挙げて本発明をより具体的に説明するが、本発明がこれらに限定されないことは言うまでもない。
多能性幹細胞
多能性幹細胞として、次のiPS細胞株、201B7株およびMYH6-GFPレポーターを有する201B7株(以下、MYH6-GFPレポーター株と言う。)を用いて実験を行った。201B7株は、Takahashi K, et al. Cell. 131: 861-72, 2007に記載の方法により作製された。MYH6-GFPレポーター株は、PiggyBacトランスポゾンシステム(System Biosciences, Inc.)を用いて、201B7株に、MYH6(myosin heavy chain 6)プロモーターの下流にEGFPカセットを作動可能に連結したベクターを導入することにより行われた。201B7株およびMYH6-GFPレポーター株の培養は、従来の方法で行われた (Takahashi K, et al. Cell. 131: 861-72, 2007およびNakagawa M, et al. Nat Biotechnol. 26: 101-6, 2008)。
MYH6-GFPレポーター株をCTK solution(ReproCELL)で2分処理後、溶液を除去し、続いてAccumax(Innovative Cell Technologies)で5分処理し、ピペッティングによりシングルセルへと解離した。遠心分離により培地を除去し、得られた細胞を1wellあたり2500 cells/wellにて低接着96wellディッシュ(Corning)へ播種し、1% L-Glutamine、150μg/mL Transferrin、50μg/mL Ascorbic Acid(sigma)、3.9×10-3%MTG、10μM Rock inhibitorおよび2ng/mL BMP4(R&D)、0.50% Matrigel(Growth Factor Reduced)を添加したSTEMPRO 34中、37℃・5%酸素条件下にて培養して、胚様体(以下、EBと言う。)を形成させた(day0)。
上記の心筋細胞誘導法により、MYH6-GFPレポーター株をday9、day21およびday30までそれぞれ分化誘導させた。day9、day21およびday30のそれぞれの細胞と分化誘導前のMYH6-GFPレポーター株について、シングルセルRNA seq解析を行い、それぞれの細胞集団についてそれらを平均したものをバルクデータとした。続いて、下記の4つの基準を元に、すべての基準を満たす遺伝子を同定した。
1.Day 21,30の心筋細胞 > Day 9の心筋細胞、Fold Change (FC) > 2.0
2.Day21,30の心筋細胞 > iPS細胞(MYH6-GFPレポーター株)、FC>5.0
3.成体心臓 > 胎児心臓、FC > 2.0
4.成体心臓 > 他の組織(肝臓、腎臓および脳) 、FC>5.0
実施例1において新規な成熟心筋細胞マーカーとして同定されたCORINが、実際に成熟心筋細胞の抽出に有効であるかについて詳細に調べた。簡潔には、実施例1の心筋細胞誘導法により分化させたday30のMYH6-GFPレポーター株について、既知の心筋細胞マーカーであるMYH6の発現を代替するGFPおよびCORINを指標として、FACSにより解析を行った。FACS解析は、FACS Arial II cell sorter (BD Biosciences)を用いて行った。その結果、GFPおよびCORINのいずれの発現量も高い細胞群(High群)とGFPの発現量は高いが、CORINの発現量が低い細胞群(Low群)を確認した。さらに、High群およびLow群をFACS Arial II cell sorterを用いて選別した(図1(A))。High群は、CORINの発現量が上位33%の細胞とし、Low群は、CORINの発現量が下位33%の細胞として選別した。
得られたHigh群およびLow群について、心筋細胞マーカー(MYH7、MYL2、MYL7、TCAP、SCN5AおよびRYR2)の相対的な発現量を測定することにより、心筋細胞の成熟度を調べた。発現量の測定は、定量的RT-PCRにより行った。簡潔には、RNeasy Mini Kit (Qiagen)を用いて、細胞からmRNAを単離した。得られた全RNAのうち1μgを鋳型として、Superscript III reverse transcriptase (Invitrogen)を用いて逆転写によりcDNAを合成した。定量的RT-PCR解析は、Power SYBR Green qPCR mastermix (Invitrogen)を用いて、StepOne real-time PCR system (ABI)により測定した。その結果、調べられたいずれの心筋細胞マーカーについても、Low群よりもHigh群において高い発現量を示すことが見出された(図1(B))。これは、High群が相対的に成熟した心筋細胞の細胞群であることを示唆している。
実施例1において新規な成熟心筋細胞マーカーとして同定されたCORINが、一部改変を行った心筋細胞誘導法により得られた心筋細胞群から成熟心筋細胞の抽出が可能であるか否かを検討した。当該一部改変とは、簡潔には、実施例1の心筋細胞誘導法のday23-27において、グルコース不含、乳酸添加した培養液中で培養する工程を加えたことである。day30のMYH6-GFPレポーター株を既知の心筋細胞マーカー(TNT)を指標としてFACS解析を行ったところ、94.7%の細胞がTNT陽性であり、心筋細胞へ分化していることが示された(図3(B))。なお、FACS解析は、実施例2と同様にFACS Arial II cell sorter (BD Biosciences)を用いて行った。
<形態解析>
iPS細胞から分化誘導して30日目の心筋細胞を2次元培養ディッシュに再播種した後、顕微鏡を用いて観察したところ、図4のように、CORIN-High群はCORIN-Low群と比較して大きい細胞サイズを示した。
iPS細胞から分化誘導して30日目の心筋細胞をフィブロネクチンコートディッシュに再播種し、その3日後に細胞切片を作製し、透過型電子顕微鏡(H-7650, Hitachi)で解析した結果、図5および6に示すように、CORIN-High群は明確に配列されたサルコメア構造を示し、CORIN-Low群と比較してサルコメアの幅は顕著に長かった。
ミトコンドリア機能を調べるため、iPS細胞から分化誘導して30日目の心筋細胞(20000個)をフィブロネクチンでコートされたアッセイウェル(Seahorse Bioscience)に播種し、播種3日後、酸素消費速度(OCR)をSeahorse BioscienceのXF24細胞外フラックスアナライザーで計測した。その結果、図7に示すように、CORIN-High群は高い酸素消費速度を示し、電子伝達系の活性も高いことが分かった。
Claims (19)
- CORIN、NCAM1、CRYAB、HBEGF、DMD、ATPIF1、CAV2、ITGAV、DCBLD2、CLIC4、BMPR2、CTSB、TMEM123、USP14およびMIR761からなる群から選択される少なくとも一つのマーカーが陽性であることを指標として、心筋細胞を含有する細胞集団から成熟心筋細胞を抽出する工程を含む、心筋細胞の製造方法。
- 前記マーカーが、CORINである、請求項1に記載の方法。
- 心筋細胞を含有する細胞集団に含まれる細胞のうち、前記マーカーの発現量が上位50%以内の細胞が成熟心筋細胞として抽出される、請求項1または2に記載の方法。
- 心筋細胞を含有する細胞集団に含まれる細胞のうち、前記マーカーの発現量が上位33%以内の細胞が成熟心筋細胞として抽出される、請求項3に記載の方法。
- 前記心筋細胞がヒト心筋細胞である、請求項1から4のいずれか1項に記載の方法。
- 前記心筋細胞を含有する細胞集団が、多能性幹細胞から分化誘導された心筋細胞を含有する細胞集団である、請求項1から5のいずれか1項に記載の方法。
- 前記多能性幹細胞が、ES細胞またはiPS細胞である、請求項6に記載の方法。
- 前記心筋細胞への分化誘導が、胚様体を形成する工程を含む、請求項6または7に記載の方法。
- 前記心筋細胞への分化誘導が、サイトカインを含有する培地で胚様体を培養する工程を含む、請求項8に記載の方法。
- 前記サイトカインが、アクチビンA、BMP4、bFGF、VEGFおよびWnt阻害剤からなる群から選択される少なくとも一つのサイトカインである、請求項9に記載の方法。
- 前記心筋細胞への分化誘導が、下記の工程を含む、請求項10に記載の方法。
(1)多能性幹細胞から胚様体を形成する工程、
(2)(1)の工程で得られた胚様体をアクチビンA、BMP4およびbFGFを含有する培養液中で培養する工程、
(3)(2)の工程で得られた胚様体を解離する工程、
(4)(3)の工程で得られた細胞をVEGFおよびWnt阻害剤を含有する培養液中で培養して胚様体に再凝集させる工程、および
(5)(4)の工程で得られた胚様体をVEGFおよびbFGFを含有する培養液中で培養する工程。 - 前記Wnt阻害剤が、IWP-3またはIWP-4である、請求項11に記載の方法。
- 前記心筋細胞への分化誘導の工程(5)において、乳酸を含み、グルコースを含まない培養液中で培養する期間を含む、請求項11または12に記載の方法。
- 薬剤候補物質の心毒性を試験する方法であって、
(A)請求項1から13のいずれか1項に記載の方法により作製された成熟心筋細胞と候補物質とを接触させる工程、および
(B)前記工程(A)の接触後に心筋細胞が正常な機能を維持した場合、当該候補物質を心毒性を有さない薬剤として同定する工程
を含む、方法。 - 前記心筋細胞の正常な機能が、電気生理学的に正常な機能である、請求項14に記載の方法。
- CORIN、NCAM1、CRYAB、HBEGF、DMD、ATPIF1、CAV2、ITGAV、DCBLD2、CLIC4、BMPR2、CTSB、TMEM123、USP14およびMIR761からなる群から選択される少なくとも一つを検出する試薬を含む、成熟心筋細胞抽出キット。
- CORINを検出する試薬を含む、請求項16に記載のキット。
- 前記心筋細胞がヒト心筋細胞である、請求項16または17に記載のキット。
- 下記の工程(1)~(5)によって多能性幹細胞から心筋細胞を含有する細胞集団を得ること、および得られた細胞群からCORIN、NCAM1、CRYAB、HBEGF、DMD、ATPIF1、CAV2、ITGAV、DCBLD2、CLIC4、BMPR2、CTSB、TMEM123、USP14およびMIR761からなる群から選択される少なくとも一つのマーカーが陽性であることを指標として心筋細胞を含有する細胞集団から成熟心筋細胞を抽出することを含む、心筋細胞の製造方法。
(1)多能性幹細胞から胚様体を形成する工程、
(2)(1)の工程で得られた胚様体をアクチビンA、BMP4およびbFGFを含有する培養液中で培養する工程、
(3)(2)の工程で得られた胚様体を解離する工程、
(4)(3)の工程で得られた細胞をVEGFおよびWnt阻害剤を含有する培養液中で培養して胚様体に再凝集させる工程、および
(5)(4)の工程で得られた胚様体をVEGFおよびbFGFを含有する培養液中で培養する工程。
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JP2018143144A (ja) * | 2017-03-03 | 2018-09-20 | パナソニック株式会社 | ヒト由来iPS細胞から分化した心筋細胞においてβミオシン重鎖を効率的に産生させる方法 |
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WO2018139548A1 (ja) * | 2017-01-26 | 2018-08-02 | 国立大学法人大阪大学 | 幹細胞の中胚葉系細胞への分化誘導用培地および中胚葉系細胞の製造方法 |
JPWO2018139548A1 (ja) * | 2017-01-26 | 2019-11-14 | 国立大学法人大阪大学 | 幹細胞の中胚葉系細胞への分化誘導用培地および中胚葉系細胞の製造方法 |
JP7162537B2 (ja) | 2017-01-26 | 2022-10-28 | 国立大学法人大阪大学 | 幹細胞の中胚葉系細胞への分化誘導用培地および中胚葉系細胞の製造方法 |
JP2018143144A (ja) * | 2017-03-03 | 2018-09-20 | パナソニック株式会社 | ヒト由来iPS細胞から分化した心筋細胞においてβミオシン重鎖を効率的に産生させる方法 |
US10988518B2 (en) | 2017-03-03 | 2021-04-27 | Panasonic Corporation | Method for efficiently producing β myosin heavy chain in cardiac muscle cells differentiated from induced pluripotent stem cells derived from Homo sapiens |
WO2018190305A1 (ja) * | 2017-04-12 | 2018-10-18 | Agc株式会社 | 分化細胞スフェロイドの製造方法 |
Also Published As
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JP6780197B2 (ja) | 2020-11-04 |
EP3252149A1 (en) | 2017-12-06 |
EP3252149A4 (en) | 2018-09-12 |
JPWO2016104614A1 (ja) | 2017-10-05 |
US20170349883A1 (en) | 2017-12-07 |
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