WO2014185358A1 - 効率的な心筋細胞の誘導方法 - Google Patents
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Definitions
- the present invention relates to a method for producing cardiomyocytes from pluripotent stem cells.
- ES cells embryonic stem cells
- artificial cells are often used against injured heart tissues caused by myocardial infarction, myocarditis, or aging. Attention has been focused on replacement therapy in which cardiomyocytes obtained by inducing differentiation of pluripotent cells (patent document 1) such as pluripotent stem cells (iPS cells) are transplanted.
- Patent Document 2 Patent Document 3, Non-Patent Document 1, Non-Patent Document 2, and Non-Patent Document 3. In this case, myocardial differentiation efficiency is not sufficiently high.
- An object of the present invention is to provide a robust and highly efficient method for inducing myocardial differentiation that is not affected by differences in sensitivity due to cell lineage in the step of inducing cardiomyocytes from pluripotent stem cells. .
- the present inventors can efficiently induce cardiomyocytes even under the same culture conditions by dissociating and reaggregating the embryoid bodies formed in the myocardial induction process. As a result, the present invention has been completed.
- the present invention includes the following.
- a method for producing cardiomyocytes from pluripotent stem cells comprising the following steps (1) to (4); (1) forming an embryoid body from pluripotent stem cells; (2) culturing the embryoid body in a culture solution containing activin A, bone morphogenetic protein (BMP) 4 and basic fibroblast growth factor (bFGF), (3) The step of dissociating the embryoid body obtained in the step (2), and (4) the cell obtained in the step (3) contains a vascular endothelial growth factor (VEGF) and a Wnt inhibitor. The process of culturing in a culture medium and reaggregating into embryoid bodies.
- BMP bone morphogenetic protein
- bFGF basic fibroblast growth factor
- VEGF vascular endothelial growth factor
- Wnt inhibitor vascular endothelial growth factor
- FIG. 1A shows a protocol for inducing differentiation of cardiomyocytes from pluripotent stem cells.
- FIG. 1B shows the results of evaluating the content of TNT positive cells in cells obtained from iPS cells (201B7) by a conventional method by flow cytometry.
- FIG. 2A shows a modified protocol for inducing differentiation of cardiomyocytes from pluripotent stem cells.
- Fig. 2B shows the content of TNT-positive cells in cells obtained using the new protocol when day 4 dissociation and re-aggregation were not performed (left diagram) and when performed (right diagram) by flow cytometry. The evaluation results are shown.
- FIG. 1A shows a protocol for inducing differentiation of cardiomyocytes from pluripotent stem cells.
- FIG. 1B shows the results of evaluating the content of TNT positive cells in cells obtained from iPS cells (201B7) by a conventional method by flow cytometry.
- FIG. 2A shows a modified protocol for inducing differentiation of cardiomyocytes from pluripot
- FIG. 3 shows TNT-positive cells in cells derived from iPS cells (201B7) (20 days after differentiation induction) when dissociation and reaggregation in the new protocol was performed from day 2 (d2) to day 6 (d6). The result of having evaluated the content rate of by flow cytometry is shown.
- FIG. 4 shows TNT-positive cells in cells derived from ES cells (KhES1) (20 days after differentiation induction) when dissociation and reaggregation in the new protocol were performed from day 2 (d2) to day 6 (d6). The result of having evaluated the content rate of by flow cytometry is shown.
- FIG. 1 shows TNT-positive cells in cells derived from ES cells
- FIG. 5 shows TNT-positive cells in cells derived from iPS cells (610B1) (20 days after differentiation induction) when dissociation and reaggregation in the new protocol was performed from day 2 (d2) to day 6 (d6). The result of having evaluated the content rate of by flow cytometry is shown.
- FIG. 6 shows TNT-positive cells in cells derived from iPS cells (409B2-2) when dissociation and reaggregation in the new protocol (IW) was performed from day 2 (d2) to day 6 (d6). The result of having evaluated the content rate by flow cytometry is shown.
- FIG. 6 shows TNT-positive cells in cells derived from iPS cells (409B2-2) when dissociation and reaggregation in the new protocol (IW) was performed from day 2 (d2) to day 6 (d6). The result of having evaluated the content rate by flow cytometry is shown.
- FIG. 7 shows induction from iPS cells (409B2-2) when dissociation and reaggregation in the method (IWDS) in which Dorsomorphin and SB431542 are added to the new protocol was performed from day 2 (d2) to day 6 (d6).
- the result of having evaluated the content rate of the TNT positive cell in the obtained cell by flow cytometry is shown.
- Fig. 8 shows dissociation and reaggregation in the new protocol (IW) and the method of adding Dorsomorphin and SB431542 to the new protocol (IWDS) every 6 hours for each day from day2.5 (d2.5) to day6 (d6).
- FIG. 3 shows the results of evaluation by flow cytometry of the content of TNT-positive cells in cells derived from various iPS cells and ES cells.
- MYH, 409B2 and 427F1 are derived from fibroblasts
- 606A1 and 610B1 are derived from cord blood
- 457C1 is derived from dental pulp
- 604A1 and 648A1 are derived from blood cells.
- the present invention includes (1) a step of forming an embryoid body from pluripotent stem cells, (2) a step of culturing the embryoid body in a culture medium containing activin A, BMP4 and bFGF, (3) (2) A step of dissociating the embryoid body obtained in the step (4), and the cells obtained in the steps (4) and (3) are cultured in a culture solution containing VEGF and a Wnt inhibitor to reaggregate into the embryoid body.
- a method of inducing cardiomyocytes from pluripotent stem cells comprising a step of additionally culturing the embryoid body obtained in steps (5) and (4) in a culture medium containing VEGF and bFGF provide.
- the cardiomyocyte means a myocardial cell having the characteristics of self-pulsation.
- the cardiomyocytes may include myocardial progenitor cells, and may include cells having the ability to generate cardiac muscle cells and vascular smooth muscles that form pulsating muscles and electrically conductive tissues.
- the myocardial cells and myocardial progenitor cells may be mixed with each other, or may be isolated myocardial progenitor cells.
- Cardiomyocytes and myocardial progenitor cells are characterized by being positive for cardiac troponin (cTNT or troponin T type 2) and / or positive for ⁇ MHC ( ⁇ myosin heavy chain).
- the cardiomyocytes obtained in the present invention may be a cell population containing a larger proportion of cardiomyocytes than other cell types, preferably 50%, 60%, 70% cardiomyocytes, A cell population containing 80% or 90% or more.
- the pluripotent stem cell that can be used in the present invention is a stem cell that has pluripotency that can be differentiated into all cells existing in a living body and also has proliferative ability.
- ES embryonic stem
- ntES embryonic stem
- GS cells sperm stem cells
- EG cells embryonic germ cells
- iPS induced pluripotent stems
- Muse cells bone marrow stem cell-derived pluripotent cells
- iPS cells or Muse cells are preferably used in the sense that they can be obtained without destroying the embryo.
- the pluripotent stem cell preferably used in the method of the present invention can be a cell highly adaptable to the myocardial differentiation induction protocol of the present invention.
- “High adaptability to the myocardial differentiation induction protocol of the present invention” means that when differentiation induction is performed using the myocardial differentiation induction protocol of the present invention, the efficiency is 40% or more, for example, 40%, 45% , 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100% efficiency.
- Preferred pluripotent stem cells in the method of the present invention are cells having a cardiomyocyte production efficiency of 70% or more, and more preferably cells having a cardiomyocyte production efficiency of 80% or more.
- Cells having high adaptability to the myocardial differentiation induction protocol of the present invention are not particularly limited.
- ES cells KhES1, KhES3, etc.
- iPS cells 201B7, 610B1, MYH, 409B2, 427F1
- Cell lines such as 606A1, 610B1, 457C1, 604A1, and 648A1.
- the pluripotent stem cell in the present invention may be a cell having high cardiomyocyte induction efficiency regardless of the timing of dissociation and reaggregation, and is a cell whose cardiomyocyte induction efficiency varies depending on the timing of dissociation and reaggregation. May be. In the latter case, any cell having high cardiomyocyte induction efficiency when dissociated and reaggregated at least at one point of the differentiation induction step can be used as a pluripotent stem cell in the method of the present invention.
- 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).
- 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 at 37 ° C., 5% CO 2 (H. Suemori et al. (2006), Biochem. Biophys. Res. Commun., 345: 926-932).
- ES cells also 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 the WiCell Research Institute, and KhES-1, KhES-2 and KhES-3 are obtained from the Institute of 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 specifically expressed in ES cells, its gene product or non-cording 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.
- 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 siRNA Smartpool (registered trademark) (Millipore), HuSH 29mershRNA 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, SetDBl And 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, HIV-derived TAT and polyarginine), or microinjection.
- a cell membrane-permeable peptide for example, HIV-derived TAT and polyarginine
- Virus vectors include retrovirus vectors, lentivirus vectors (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.
- 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.
- GFP green fluorescent protein
- GUS ⁇ -glucuronidase
- FLAG FLAG
- 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
- 10% FBS-containing DMEM 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. 4: e8067 or WO2010 / 137746), or extracellular matrix (eg, Laminin- 5 (WO2009 / 123349) and Matrigel (BD)) are exemplified.
- 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.
- somatic cells having the same or substantially the same HLA genotype as the transplant destination individual from the viewpoint that rejection does not occur.
- substantially the same means that the HLA genotype matches the transplanted cells to such an extent that an immune response can be suppressed by an immunosuppressive agent.
- HLA-A, HLA-B And somatic cells having an HLA type in which 3 loci of HLA-DR or 4 loci plus HLA-C are matched.
- 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).
- Muse cells are pluripotent stem cells produced by the method described in WO2011 / 007900. Specifically, fibroblasts or bone marrow stromal cells are treated with trypsin for a long time, preferably 8 or 16 hours. It is a pluripotent cell obtained by suspension culture after treatment, and is positive for SSEA-3 and CD105.
- Step (1) it is preferable to form embryoid bodies after dissociating the pluripotent stem cells that have formed colonies into single cells.
- 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 of dissociating pluripotent stem cells for example, a method of dynamically dissociating, a dissociation solution having protease activity and collagenase activity (for example, Accutase (TM) and Accumax (TM), etc.) 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 number of pluripotent stem cells suitably used for forming embryoid bodies for the purpose of inducing cardiomyocytes is 1000 to 4000, preferably 2000 to 4000. .
- 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 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,
- 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 number of cells used to form an embryoid body by reaggregation is not particularly limited as long as the cells can adhere to each other and produce a cell mass, but are 1000 or more and 20000 or less cells. Preferably, 10,000 are exemplified.
- a culture vessel whose surface is not artificially treated for the purpose of improving cell adhesion, or a culture vessel treated artificially to suppress adhesion is used. It is preferable to use the suspension culture.
- 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 TGF ⁇ inhibitor used in this step is preferably IWP-3 or IWP-4.
- 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 BMP inhibitor refers to proteinaceous inhibitors such as Chordin, Noggin, Follistatin, Dorsomorphin (that is, 6- [4- (2-piperidin-1-yl-ethoxy) phenyl] -3-pyridin-4 -yl-pyrazolo [1,5-a] pyrimidine), its derivatives (P. B. Yu et al. (2007), Circulation, 116: II_60; PB Yu et al. (2008), Nat. Chem. Biol. , 4: 33-41; J. Hao et al. (2008), PLoS ONE, 3 (8): e2904) and LDN-193189 (i.e.
- the BMP inhibitor used in this step is preferably Dorsomorphin.
- the concentration of a BMP inhibitor such as Dorsomorphin in the culture solution is not particularly limited as long as it is a concentration that inhibits BMP, but is preferably 1 nM to 50 ⁇ M, for example, 1 nM, 10 nM, 50 nM, 100 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 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 600 nM.
- the TGF ⁇ inhibitor is a substance that inhibits signal transduction from binding of TGF ⁇ to the receptor to SMAD, and a substance that inhibits binding to the receptor ALK family, or SMAD by the ALK family.
- SMAD signal transduction from binding of TGF ⁇ to the receptor to SMAD
- ALK family a substance that inhibits binding to the receptor ALK family, or SMAD by the ALK family.
- the concentration of a TGF ⁇ inhibitor such as SB431542 in the culture solution is not particularly limited as long as it inhibits ALK5, 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, 5.2 ⁇ M, 5.4 ⁇ M, 5.6 ⁇ M, 5.8 ⁇ 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 5.4 ⁇ M.
- 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 time, there is no particular upper limit, but it is preferable to culture for 4 days or more.
- 4th, 5th, 6th, 7th, 8th, 9th, and 10th can be mentioned.
- 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.
- basal media 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 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, but it is preferable that the hypoxic conditions are performed for 4 days or more at the initial stage of the process. 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 or more It is done.
- the efficiency of differentiation into cardiomyocytes is improved.
- the cardiomyocytes obtained in the present invention can be used as a therapeutic agent for animal (preferably human) heart disease.
- the obtained myocardial cells may be suspended in physiological saline and administered directly to the myocardium of the patient's heart, or the obtained myocardial cells may be formed into a sheet and the patient It may be performed by applying to the heart of the patient. In the former case, it may be administered as a single cell, preferably with a scaffold that promotes engraftment.
- the scaffold include, but are not limited to, biologically derived components such as collagen and synthetic polymers such as polylactic acid that are substituted for them.
- a myocardial sheet When a myocardial sheet is administered, it is achieved by placing it so as to cover a desired portion.
- the arrangement so as to cover a desired portion can be performed using a technique well known in the art. In the case of placement, if the desired part is large, it may be placed so as to surround the tissue.
- administration can be performed several times on the same part in order to obtain a desired effect. When arranging several times, it is desirable to allow sufficient time for a desired cell to be engrafted in a tissue and to undergo angiogenesis.
- the mechanism of treatment of such heart diseases may be the effect caused by the engraftment of the myocardial sheet, or an indirect action that does not depend on the engraftment of cells (for example, recipients by secreting attractants).
- Heart diseases that can be treated in the present invention include heart failure, ischemic heart disease, myocardial infarction, cardiomyopathy, myocarditis, hypertrophic cardiomyopathy, dilated phase hypertrophic cardiomyopathy, deficiency due to dilated cardiomyopathy, etc. However, it is not limited to these.
- the number of cardiomyocytes used for the treatment of heart disease is not particularly limited as long as the administered cardiomyocytes or myocardial sheet is an amount that exerts an effect in the treatment of heart disease, It may be prepared by appropriately increasing or decreasing according to the size of the affected part or the size of the body.
- strains below pluripotent stem cells were used.
- the strain 201B7 was prepared by the method described in Takahashi K, et al. Cell. 131: 861-72, 2007.
- an episomal vector pCXLE-hOCT3 / 4-shp53-F , PCXLE-hSK, pCXLE-hUL
- pCXLE-hOCT3 / 4-shp53-F obtained from RIKEN BRC
- an episomal vector pCXLE-hOCT3 / 4-shp53-F , PCXLE-hSK, pCXLE-hUL
- HDF1388 was transformed into pCXLE-hOCT3 / 4-shp53-F, pCXLE-hSK, pCXLE- hUL was introduced and produced.
- the culture was performed in the same manner as described above.
- pCXLE-hOCT3 / 4-shp53-F, pCXLE-hSK, and pCXLE-hUL were introduced into 427F1 strain HDF-1437.
- the culture was performed in the same manner as described above.
- CB CD34 # 1 contains pCXLE-hOCT3 / 4-shp53-F, pCXLE-hSK, pCXLE-hUL was introduced and prepared. The culture was performed in the same manner as described above.
- CB CD34 # 2 contains pCXLE-hOCT3 / 4-shp53-F, pCXLE-hSK, pCXLE-hUL was introduced and prepared. The culture was performed in the same manner as described above.
- the culture was performed in the same manner as described above. (10) Based on the method described in 648A1 strain Okita et al., Stem Cells. 31 (3): 458-66 (2013), pCXLE-hOCT3 / 4-shp53- was added to PBMN # 2 non-T, non-B. F, pCXLE-hSK, and pCXLE-hUL were introduced and prepared. The culture was performed in the same manner as described above. (11) KhES1 strain and KhES3 strain Human ES cells were cultured by the conventional method using the KhES1 strain and the KhES3 strain established by the Research Center for Stem Cell Medicine, Institute of Regenerative Medicine, Kyoto University (Suemori H, et al. Biochem Biophys Res Commun. 345: 926-32, 2006).
- Cardiomyocyte induction method (comparative example: FIG. 1A) Three days before differentiation induction (day-3), the 201B7 cell line was treated with Collagenase type B solution (Roche) for 5 minutes, then the solution was removed, followed by treatment with 0.25% Trypsin-EDTA (invitrogen) for 2-3 minutes. . After washing with PBS solution (Nacalai Tesque), the obtained iPS cells were collected by peeling with a cell scraper, seeded in 1 well of a Matrigel-coated 6-well plate, and conditioned medium (MEF-CM) for MEF (mouse fetal fibroblasts) For 3 days.
- MEF-CM conditioned medium
- EB was collected by centrifugation, transferred to 4 wells of a low adhesion 24-well plate, 1% L-Glutamine, 150 ⁇ g / mL Transferrin, 50 ⁇ g / mL Ascorbic Acid, 3.9 ⁇ 10 ⁇ 3 % MTG, 10 ng /
- the cells were cultured in STEMPRO 34 supplemented with mL BMP4, 5 ng / mL bFGF (R & D) and 6 ng / mL Activin A (R & D) for 3 days at 37 ° C. and 5% oxygen.
- the cells were transferred to an incubator with a normal oxygen concentration and cultured for 8 days. At this time, the medium was replaced once every two days.
- the modified cardiomyocyte induction method 201B7 cell line was treated with CTK solution (ReproCELL) for 2 minutes, then the solution was removed, then treated with Accumax (Innovative Cell Technologies) for 5 minutes, and then dissociated into single cells by pipetting.
- CTK solution ReproCELL
- Accumax Innovative Cell Technologies
- 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 cells were transferred to an incubator with a normal oxygen concentration and cultured for 8 days. At this time, the medium was replaced once every two days.
- the 201B7 strain was dissociated from the 3rd day of induction (d3) to the 4.75th day (d4.75), and reaggregation culture was performed, so that the cardiomyocyte content was 78.5% -87.6%. High efficiency was confirmed (FIG. 3).
- the content of cardiomyocytes is 81.9% -94.0 by dissociating from day 2.5 (d2.5) to 4.75 day (d4.75) and reaggregating culture.
- the content of cardiomyocytes was 80.3% -90.4% by dissociation and reaggregation from the second day (d2) to the third day (d3) (FIG. 6). .
- IWDS method differentiation induction of the 409B2-2 strain was performed using the same method (hereinafter referred to as IWDS method) except for the culture step after the dissociation operation.
- the culture process after the dissociation operation in the IWDS method is as follows.
- the 409B2-2 strain was dissociated from the 5th day (d5) to the 6th day (d6) of induction, and reaggregation culture was performed, so that the content of cardiomyocytes was as high as 59.4% -74.7%. (FIG. 7).
- the cells were dissociated into single cells between the second day of induction (d2) and the sixth day of induction (d6). It was shown that cardiomyocytes can be obtained more efficiently by performing agglutination culture.
- this method does not require seeding on the Matrigel coat in advance, and cardiomyocytes can be induced easily and efficiently by determining the number of cells by dissociating into single cells and forming EB cells. It was suggested that this is a very useful method.
- STEMPRO supplemented with 1% L-Glutamine, 150 ⁇ g / mL Transferrin, 50 ⁇ g / mL Ascorbic Acid, 3.9 ⁇ 10 ⁇ 3 % MTG, 10 ng / mL VEGF and 5 ng / mL bFGF under normal oxygen concentration, the final step After culturing up to d15 in 34, each cell was evaluated by the content of cTNT positive cells.
- MYH, 409B2, 606A1, 457C1 and KhES1 strains were highly efficient when disaggregated and dissociated in almost all steps (d2.5 to d5) in the IW method.
- high efficiency was observed when reaggregation culture was carried out by dissociating from the middle stage to the relatively late stage (d3.5 to d6) (FIG. 8).
- the 427F1, 610B1, 604A1, 648A1 and KhES3 strains were highly efficient when disaggregated and disaggregated at a relatively early stage (d2.5 to d4) in the IW method.
- high efficiency was observed when re-aggregation culture was performed after dissociation from a relatively early stage to a middle stage (d2.5 to d4.75) (FIG. 8).
- the cells are converted to single cells between the induction day 2.5 (d2.5) and the induction day 6 (d6). It was shown that cardiomyocytes can be obtained more efficiently by dissociating and reaggregating culture.
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Abstract
Description
[1]多能性幹細胞から心筋細胞を製造する方法であって、次の(1)から(4)の工程を含む方法;
(1)多能性幹細胞から胚様体を形成する工程、
(2)胚様体をアクチビンA、骨形成タンパク質(BMP)4および塩基性線維芽細胞増殖因子(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、および
(4)(3)の工程で得られた細胞を血管内皮細胞増殖因子(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。
[2]さらに以下の工程を含む、[1]に記載された方法。
(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。
[3]前記工程(5)が、前記胚様体を12日以上の期間培養する工程である、[2]に記載された方法。
[4]前記工程(2)、(4)および(5)において、培養が低酸素条件で行われる、[1]から[3]のいずれか1つに記載された方法。
[5]前記工程(2)が、胚様体を1日以上5日以内の期間培養する工程である、[1]から[4]のいずれか1つに記載された方法。
[6]前記工程(4)が、前記細胞を4日以上の期間培養する工程である、[1]から[5]のいずれか1つに記載された方法。
[7]前記Wnt阻害剤が、IWP-3またはIWP-4である、[1]から[6]のいずれか1つに記載された方法。
[8]前記工程(4)に用いられる培養液が、BMP阻害剤および/またはTGFβ阻害剤をさらに含む、[1]から[7]のいずれか1つに記載された方法。
[9]前記BMP阻害剤が、Dorsomorphinであり、前記TGFβ阻害剤が、SB431542である、[8]に記載された方法。
[10]前記心筋細胞が、ヒト心筋細胞である、[1]から[9]のいずれか1つに記載された方法。
[11][1]から[10]のいずれか1つに記載の方法で製造された心筋細胞を含む、心疾患治療剤。
心筋細胞および心筋前駆細胞は、心筋マーカーである心筋トロポニン(cTNTまたはtroponin T type 2)陽性および/またはαMHC(α myosin heavy chain)陽性であることによって特徴づけられる。また、本発明において得られる心筋細胞は、他の細胞種と比して心筋細胞の割合を多く含有した細胞集団であっても良く、好ましくは、心筋細胞を50%、60%、70%、80%または90%以上含有する細胞集団である。
本発明で使用可能な多能性幹細胞は、生体に存在する全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、例えば胚性幹(ES)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、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、培養線維芽細胞や骨髄幹細胞由来の多能性細胞(Muse細胞)などが含まれる。本発明では、胚の破壊を行わずに得られるという意味では、iPS細胞またはMuse細胞を用いることが好ましい。本発明の方法において好ましく使用される多能性幹細胞は、本発明の心筋分化誘導プロトコールに対して適応性が高い細胞であり得る。「本発明の心筋分化誘導プロトコールに対して適応性が高い」とは、本発明の心筋分化誘導プロトコールを用いて分化誘導を行った際に、40%以上の効率、例えば、40%、45%、50%、55%、60%、65%、70%、75%、80%、85%、90%、95%、100%の効率で心筋細胞が生成されることを意味する。本発明の方法において好ましい多能性幹細胞は、70%以上の心筋細胞生成効率を有する細胞であり、より好ましくは、80%以上の心筋細胞生成効率を有する細胞であり得る。本発明の心筋分化誘導プロトコールに対して適応性が高い細胞は、別段限定されることはないが、例えば、ES細胞について、KhES1、KhES3等、iPS細胞について、201B7、610B1、MYH、409B2、427F1、606A1、610B1、457C1、604A1、648A1等の細胞株が挙げられる。本発明における多能性幹細胞は、解離再凝集の時期に関わらず高い心筋細胞誘導効率を有する細胞であってもよく、また解離再凝集の時期に応じて心筋細胞誘導効率が変動する細胞であってもよい。後者の場合、少なくとも分化誘導工程の一点において解離再凝集を行った場合に高い心筋細胞誘導効率を有する細胞であれば、本発明の方法において多能性幹細胞として使用可能である。
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-cording 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またはGlis1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、Maekawa M, et al. (2011), Nature. 474:225-9.に記載の組み合わせが例示される。
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。
Muse細胞は、WO2011/007900に記載された方法にて製造された多能性幹細胞であり、詳細には、線維芽細胞または骨髄間質細胞を長時間トリプシン処理、好ましくは8時間または16時間トリプシン処理した後、浮遊培養することで得られる多能性を有した細胞であり、SSEA-3およびCD105が陽性である。
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(TM)およびAccumax(TM)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。
再凝集により胚様体を形成させるにあたり、使用する細胞数は、同細胞が互いに接着し、細胞塊を作製できる細胞数であれば、特に限定されないが1000個以上、20000個以下の細胞であり、好ましくは、10000個が例示される。培養に際しては、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。
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Wnt阻害剤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。
より好ましくは、600nMである。
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。
本発明で得られた心筋細胞は、動物(好ましくはヒト)の心疾患の治療剤として用いることができる。心疾患の治療方法として、例えば、得られた心筋細胞を生理食塩水等に懸濁させ、患者の心臓の心筋層へ直接投与してもよく、あるいは得られた心筋細胞をシート化して、患者の心臓に貼付することによって行われてもよい。前者の場合、細胞単体で投与してもよく、好ましくは、生着を促すような足場材と共に投与され得る。ここで足場材とは、コラーゲンなどの生体由来の成分やこれに代替するポリ乳酸などの合成ポリマーが例示されるが、これらに限定されない。心筋シートを投与する場合、所望の部分を覆うように配置することによって達成される。ここで、所望の部分を覆うように配置することは、当該分野において周知技術を用いて行うことができる。配置に際し、所望の部分が大きい場合は、組織を取り巻くように配置してもよい。また、投与は、所望の効果を得るため、同部分へ数回の配置を行うこともできる。数回の配置を行う場合、所望の細胞が組織へ生着し、血管新生を行うために十分な時間をおいて行うことが望ましい。このような心疾患の治療の機序は、心筋シートの生着により生じる効果であってもよく、あるいは細胞の生着によらない間接的な作用(例えば、誘引物質を分泌することによるレシピエント由来細胞の損傷部位への動員による効果)であってもよい。心疾患の治療において、心筋シートを用いる場合には、心筋細胞に加えて、コラーゲン、フィブロネクチン、ラミニン等の細胞足場材料(スキャホールド)を含んでいてもよい。あるいは、心筋細胞の他に、任意の細胞種(複数も可)を含んでいることも可能である。本発明において治療され得る心疾患は、心不全、虚血性心疾患、心筋梗塞、心筋症、心筋炎、肥大型心筋症、拡張相肥大型心筋症、拡張型心筋症などの疾患または障害による欠損等が挙げられるがこれらに限定されない。
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。
以下の株を用いた。
(1)201B7株
Takahashi K, et al. Cell. 131: 861-72, 2007に記載の方法で作製された。
(2)610B1株
Okita. K, et al., Stem Cells. 2012 Nov 29.に記載の方法に基づき、ヒト臍帯血(理研BRCより入手)にエピソーマルベクター(pCXLE-hOCT3/4-shp53-F、pCXLE-hSK、pCXLE-hUL)を電気穿孔法で遺伝子導入し、マイトマイシン処理したマウス胎仔線維芽細胞フィーダー上で培養することにより、iPS細胞株を作製した。培養は、従来の方法で行った (Takahashi K, et al. Cell. 131: 861-72, 2007およびNakagawa M, et al. Nat Biotechnol. 26: 101-6, 2008)。
(3)MYH株
PiggyBacトランスポゾンシステム(System Biosciences, Inc.)を用いて、上記201B7株に、MYH(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して、作製した。培養は、上記と同様の方法で行った。
(4)409B2株
Okita et al., Nat Methods. 8(5):409-12.(2011)に記載の方法に基づき、HDF1388にpCXLE-hOCT3/4-shp53-F、pCXLE-hSK、pCXLE-hULを導入し、作製した。培養は、上記と同様の方法で行った。
(5)427F1株
HDF-1437にpCXLE-hOCT3/4-shp53-F、pCXLE-hSK、pCXLE-hULを導入し、作製した。培養は、上記と同様の方法で行った。
(6)606A1株
Okita et al., Stem Cells. 31(3):458-66 (2013)に記載の方法に基づき、CB CD34#1にpCXLE-hOCT3/4-shp53-F、pCXLE-hSK、pCXLE-hULを導入し、作製した。培養は、上記と同様の方法で行った。
(7)610B1株
Okita et al., Stem Cells. 31(3):458-66 (2013)に記載の方法に基づき、CB CD34#2にpCXLE-hOCT3/4-shp53-F、pCXLE-hSK、pCXLE-hULを導入し、作製した。培養は、上記と同様の方法で行った。
(8)457C1株
Okita et al., Nat Methods. 8(5):409-12.(2011)に記載の方法に基づき、DP74にpCXLE-hOCT3/4-shp53-F、pCXLE-hSK、pCXLE-hULを導入し、作製した。培養は、上記と同様の方法で行った。
(9)604A1株
Kajiwara et al. Proc Natl Acad Sci U S A. 109(31):12538-43 (2012) に記載の方法に基づき、PBMNαβTにpCXLE-hOCT3/4-shp53-F、pCXLE-hSK、pCXLE-hULを導入し、作製した。培養は、上記と同様の方法で行った。
(10)648A1株
Okita et al., Stem Cells. 31(3):458-66 (2013)に記載の方法に基づき、PBMN #2 non-T, non-BにpCXLE-hOCT3/4-shp53-F、pCXLE-hSK、pCXLE-hULを導入し、作製した。培養は、上記と同様の方法で行った。
(11)KhES1株およびKhES3株
ヒトES細胞は、京都大学再生医科学研究所附属幹細胞医学研究センターによって樹立されたKhES1株およびKhES3株を用い、従来の方法で培養した(Suemori H, et al. Biochem Biophys Res Commun. 345:926-32, 2006)。
分化誘導の3日前(day-3)に201B7細胞株をCollagenase type B溶液(Roche)で5分処理後、溶液を除去し、続いて0.25% Trypsin-EDTA(invitrogen)で2~3分処理した。PBS溶液(ナカライテスク)で洗浄後、得られたiPS細胞をセルスクレーパーによって剥離させて回収し、マトリゲルコート6well プレートの1wellに播種し、MEF(マウス胎仔線維芽細胞)馴化培地(MEF-CM)で3日間培養した。
201B7細胞株をCTK solution(ReproCELL)で2分処理後、溶液を除去し、続いてAccumax(Innovative Cell Technologies)で5分処理後、ピペッティングによりシングルセルへと解離した。遠心分離により細胞を回収し、1wellあたり2500 cells/wellを低接着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、EBを形成させた(day0)。この時、1wellあたり1000cells以下または4000cells以上によりEB細胞を形成させると心筋細胞の誘導効率が下がることが確認された。
上述した実施例1の改変心筋細胞誘導法において、day4にて行われたシングルセルへの解離と再凝集の日程を変更してその効果を各多能性幹細胞株(201B7株、610B1株および409B2-2株、ならびにKhES1株)で確認した(図3から6)。day1にて、1% L-Glutamine、150μg/mL Transferrin、50μg/mL Ascorbic Acid、3.9×10-3%MTG 、18ng/mL BMP4、10ng/mL bFGFおよび12ng/mLActivin Aを添加したSTEMPRO 34中での培養を1日間から5日間まで変更し、得られた細胞の解離・再凝集を行った。つまり、それぞれ、誘導2日目(d2)、2.25日目(d2.25)、2.5日目(d2.5)、2.75日目(d2.75)、3日目(d3)、3.25日目(d3)、3.5日目(d3.5)、3.75日目(d3.75)、4.25日目(d4.25)、4.5日目(d4.5)、4.75日目(d4.75)、5日目(d5)又は6日目(d6)にて解離させ、再凝集培養を行った。
(day2~6)解離操作後、培地を除去し、1% L-Glutamine、150μg/mL Transferrin、50μg/mL Ascorbic Acid、3.9×10-3%MTG 、10ng/mL VEGF(R&D)、1μM IWP-3(Stemolecule)、600nM Dorsomorphin及び5.4μM SB431542を添加したSTEMPRO 34中で、4日間、37℃・5%酸素条件下で培養した。
上述した改変心筋細胞誘導法(IW)およびIWDS法において、day4にて行われたシングルセルへの解離と再凝集の日程を変更して、その効果を各多能性幹細胞株(KhES1株、KhES3株、MYH株、409B2株、427F1株、606A1株、610B1株、457C1株、604A1株および648A1株)で確認した(図8)。day1にて、1% L-Glutamine、150μg/mL Transferrin、50μg/mL Ascorbic Acid、3.9×10-3%MTG 、18ng/mL BMP4、10ng/mL bFGFおよび12ng/mLActivin Aを添加したSTEMPRO 34中での培養を1.5日間から5日間まで変更し、得られた細胞の解離・再凝集を行った。すなわち、各細胞株について、誘導2.5日目(d2.5)から6日目(d6)までの工程の間、6時間おきに解離させ、再凝集培養を行った。
Claims (11)
- 多能性幹細胞から心筋細胞を製造する方法であって、次の(1)から(4)の工程を含む方法;
(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、および
(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。 - さらに以下の工程を含む、請求項1に記載された方法。
(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。 - 前記工程(5)が、前記胚様体を12日以上の期間培養する工程である、請求項2に記載された方法。
- 前記工程(2)、(4)および(5)において、培養が低酸素条件で行われる、請求項1から3のいずれか1項に記載された方法。
- 前記工程(2)が、胚様体を1日以上5日以内の期間培養する工程である、請求項1から4のいずれか1項に記載された方法。
- 前記工程(4)が、前記細胞を4日以上の期間培養する工程である、請求項1から5のいずれか1項に記載された方法。
- 前記Wnt阻害剤が、IWP-3またはIWP-4である、請求項1から6のいずれか1項に記載された方法。
- 前記工程(4)に用いられる培養液が、BMP阻害剤および/またはTGFβ阻害剤をさらに含む、請求項1から7のいずれか1項に記載された方法。
- 前記BMP阻害剤が、Dorsomorphinであり、前記TGFβ阻害剤が、SB431542である、請求項8に記載された方法。
- 前記心筋細胞が、ヒト心筋細胞である、請求項1から9のいずれか1項に記載された方法。
- 請求項1から10のいずれか1項に記載の方法で製造された心筋細胞を含む、心疾患治療剤。
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