WO2013111875A1 - 多能性幹細胞の心筋分化誘導法 - Google Patents
多能性幹細胞の心筋分化誘導法 Download PDFInfo
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- CEOHSXRCDBHISA-UHFFFAOYSA-N COc(ccc(CCCCC(Nc([s]c1c2)nc1ccc2Cl)=O)c1)c1OC Chemical compound COc(ccc(CCCCC(Nc([s]c1c2)nc1ccc2Cl)=O)c1)c1OC CEOHSXRCDBHISA-UHFFFAOYSA-N 0.000 description 1
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- C07D277/60—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings condensed with carbocyclic rings or ring systems
- C07D277/62—Benzothiazoles
- C07D277/68—Benzothiazoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached in position 2
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- C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
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Definitions
- the present invention relates to a method for inducing myocardial differentiation of pluripotent stem cells.
- hPSC Human pluripotent stem cell sputum
- hESC human embryonic stem cell sputum
- hiPSC human induced pluripotent stem cell sputum
- Non-patent Documents 13 and 15-17 The most common methods currently in use are suspension cultures with embryoid body cytokines (eg, DKK1, bFGF, Activin A and BMP4), or mouse END2 (proximal endoderm) co-culture with endoderm-like cells) (Non-patent Documents 13 and 15-17).
- these methods have low efficiency (cardiomyocyte production is 10-50%) (Non-patent Document 15), and animal cells or fetal bovine serum sputum (FBS) are used, so contamination of heterogeneous components is inevitable (non-patent document 15).
- Patent Document 18 Patent Document 18), and the use of recombinant cytokines is not cost effective for large scale production.
- Non-Patent Document 19 a universal myocardial differentiation method that does not depend on the hPSC strain has been reported, but this method requires growth factors such as FBS and bFGF and BMP4 for efficient differentiation.
- Non-Patent Document 21 Low molecular weight compounds are promising candidates for recombinant cytokines and substitutes for unknown factors in FBS (Non-Patent Document 21), and are suitable for the preparation of known composition media for large-scale culture.
- Non-Patent Documents 22 and 23 to activate or inhibit signal transduction pathways such as WNT and TGF- ⁇ signals (Non-Patent Documents 22 and 23), or to regulate gene expression in place of transcription factors (non- Patent Documents 24 and 25) and low molecular weight compounds are used.
- a number of small molecule compounds have been tested or screened for the promotion of differentiation.
- Non-patent Documents 26-32 BMP signal inhibitor (Dorsomorphin), p38MAPK signal inhibitor (SB203580), WNT signal activator (BIO), and WNT signal inhibitor (XAV939, IWR-1, IWP-1, and IWP-3) has been reported (Non-patent Documents 26-32). However, with these treatments, only 10-60% myocardial differentiation can be obtained even under serum-containing conditions (Non-patent Documents 26-32). For large-scale clinical applications, low molecular weight compounds that provide more efficient differentiation are needed.
- An object of the present invention is to provide an efficient method for inducing myocardial differentiation of pluripotent stem cells.
- the present invention is a method for inducing myocardial differentiation of pluripotent stem cells, comprising the following steps: (1) culturing pluripotent stem cells in a medium containing one or more WNT signal activators; and (2) A step of culturing the cells obtained in step (1) in a medium containing one or more WNT signal inhibitors.
- a method comprising:
- the present invention provides a kit for promoting myocardial differentiation comprising one or more WNT signal activators and / or one or more WNT signal inhibitors, which is used in the above method.
- differentiation of pluripotent stem cells into cardiomyocytes can be induced with high efficiency and low cost compared to known methods.
- hESC strains KhES-1 and KhES-3
- monkey ESC strains CMK6.4
- mouse ESC strains R1
- hiPSC strains IMR90-1, IMR90-4, 253G1, and RCHIPC0003
- the primers used for qPCR are shown in Table 1.
- KY-CM Functionality of KY02111-induced cardiomyocytes
- KY-CM Spontaneous ventricular-like or pacemaker-like action potential (AP) from KY02111-induced cardiomyocytes in the patch clamp method.
- AP pacemaker-like action potential
- AP Prolonged action potential duration induced by 100 nM E4031 (HERG channel blocker) and 4 ⁇ M chromanol 293B (Chromanol 293B) (KCNQ1 channel blocker).
- IMR90-1 hiPSCs were treated with KY02111 or DMSO for 12 or 24 hours (KY12h, KY24h, DMSO12h, or DMSO24h), and 26 genes were identified. A heat map was generated using 22 down-regulated genes. The four up-regulated genes are shown in Table 2. Table 2 shows the target genes of the WNT signaling pathway.
- the low molecular weight WNT inhibitors were KY02111 (10 ⁇ M), IWP-2 (10 ⁇ M), XAV939 (10 ⁇ M), and IWR-1 (10 ⁇ M).
- the proteinaceous factor was IGFBP4 (1 ⁇ g ml ⁇ 1 ), Dkk1 (300 ng ml ⁇ 1 ), and a mixture (Cytokines) of bFGF, BMP4, VEGF, DKK1, and activin A (Non-patent Document 16).
- the concentrations of both inhibitors were optimized for myocardial differentiation (data not shown).
- (c, d) GSK3 ⁇ inhibitor did not abolish the effect of KY02111 on myocardial differentiation.
- Colonies having a GFP signal induced by the human ⁇ MHC promoter are treated with KY02111 (KY), XAV939 (XAV), IWP-2 (IWP) in the presence or absence of 5 ⁇ M BIO (GSK3 ⁇ inhibitor). Induced.
- KY KY02111
- XAV939 XAV
- IWP-2 IWP-2
- BIO GSK3 ⁇ inhibitor
- the scale bar is 100 ⁇ m.
- e Number of beating colonies (left bar) and total number of cardiomyocytes (right bar) under serum-free known composition culture conditions. On differentiation day 0, 6 ⁇ 10 6 IMR90-1 hiPSC cells per well were added to 6-well plates.
- the right bar shows the number of cardiomyocytes in each well calculated by the product of the total number of cells of all colonies and the ratio of cTnT positive cells.
- the number of beating colonies and total number of cardiomyocytes induced by KY02111 or KY + XAV were both increased more in the known composition medium (Alb) than in the serum-containing medium (FBS).
- Mean ⁇ SEM, n 4; * P ⁇ 0.05, ** P ⁇ 0.001 (Student t test).
- KY02111 and related compounds (a) Synthesis scheme of KY02111. Other related compounds were also synthesized using the same synthesis method.
- INa Voltage-dependent Na + channel current during depolarization from -90 mV to 40 mV.
- ICa Voltage-dependent Ca2 + channel current during depolarization from -40 mV to 10 mV.
- IKs Voltage-dependent K + channel current after a 500 ms depolarization pulse from -40 mV to 40 mV suppressed by chromanol 293B.
- IKr Voltage-dependent K + channel current after 500 ms depolarization pulse from -40 mV to 40 mV suppressed by E4031. If: HCN channel current after 500 ms hyperpolarization pulse from -50 mV to -140 mV suppressed by Zatebradine.
- E4031-APDinc APD90 increase rate induced by E4031.
- C293B-APDinc APD90 induced by chromanol 293B.
- QT interval extension test using KY-CM (a) A single KY-CM colony on a microelectrode array (MEA) dish. (b) ECG-like waves released from KY-CM colonies. Top: Waveform before processing. Bottom: Waveform after treatment with 10 ⁇ M Astemizole. The Na + -K + wave peak interval was extended by astemizole. Astemizole is an antihistamine, but has been reported to inhibit the HERG channel (Non-patent Document 34). (c) Graph of Na + -K + interval extension.
- Wnt3a (W) or BIO (B) was added to increase WNT signaling pathway and TCF promoter activity.
- KY02111 and XAV939 inhibited TCF promoter activity by Wnt3a and BIO.
- IWP-2 did not inhibit TCF promoter activity, probably because IWP-2 is an inhibitor of WNT ligand secretion.
- n 14, mean ⁇ SEM, * P ⁇ 0.05, ** P ⁇ 0.01 (unpaired two-tailed Student ’s t-test).
- n 4 mean ⁇ SEM, * P ⁇ 0.05, ** P ⁇ 0.01 (independent two-sided student t-test). Synergistic effect of KY02111 and XAV939, IWP-2, and XAV939 + IWP-2 on total cell count and cardiomyocyte rate.
- IMR90-1 hiPSC is KY02111 (KY), XAV939 (XAV), IWP-2 (IWP), KY + XAV, KY + IWP, or KY + XAV + IWP as described in “1. Processed by. Total cell counts were measured on day 14 of myocardial differentiation using NucleoCounter® (Chemometec). cTnT positive cardiomyocytes were examined as described in “1. Method” in the Examples. On the first day of differentiation, 6 million IMR90-1 hiPSC cells were used per well of a 6-well plate.
- n 4, average ⁇ SEM, * P ⁇ 0.001 (compared with KY02111 alone). Strong myocardial differentiation in known compositional conditions without cytokines, serum, and xenogeneic components by small molecules that modulate the WNT signaling pathway.
- hESC stock H1, H9, and KhES-3) hi and hiPSC stock (IMR90-1 and 253G1) ⁇ were transferred to cardiomyocytes with high efficiency by treatment with BIO + CHIR99021 in the early stage and KY02111 + XAV939 in the late stage. Differentiated. As shown in FIG. 4a, 0.4% human serum albumin and human laminin 211 were used.
- pluripotent stem cell means pluripotency capable of differentiating into all cells constituting an adult, and pluripotency even after cell division. It means a cell having a self-replicating ability capable of maintaining its ability.
- the “pluripotent stem cell” may be described as an embryonic stem cell (sometimes referred to as ES cell or ESC), an embryonic germ cell (EG cell), or an induced pluripotent stem cell (iPS cell or iPSC). ) Is included.
- the species of “pluripotent stem cells” is not particularly limited, but is preferably a mammal, more preferably a rodent or a primate.
- the present invention is particularly suitable for monkey or human pluripotent stem cells.
- ES cells are pluripotent stem cells derived from early embryos, and can be established from the inner cell mass of blastocysts or epiblasts of early embryos after implantation.
- ES cells include humans (Thomson J. A. et al., Science 282: 1145-1147 (1998), Biochem Biophys Res Commun. 345 (3), 926-32 (2006); primates such as rhesus monkeys and marmosets (Thomson J. A. et al., Proc. Natl. Acad. Sci. USA 92: 7844-7848 (1995); Thomson J. A. et al., Biol. Reprod.
- EG cells are pluripotent stem cells derived from primordial germ cells.
- human EG cells (Shamblott, et al., Proc. Natl. Acad. Sci USA 92: 7844-7848 (1995)) (this book by reference) Included in the specification).
- iPS cell means a pluripotent stem cell derived from a cell other than a pluripotent stem cell such as a somatic cell or a tissue stem cell.
- the method for producing iPS cells is, for example, WO2007 / 069666, WO2009 / 006930, WO2009 / 006997, WO2009 / 007852, WO2008 / 118820, Cell Stem Cell 3 (5): 568-574 (2008), Cell Stem Cell 4 (5 ): 381-384 (2009), Nature 454: 646-650 (2008), Cell 136 (3): 411-419 (2009), Nature Biotechnology 26: 1269-1275 (2008), Cell Stem Cell 3: 475- 479 (2008), Nature Cell Biology 11: 197-203 (2009), Cell 133 (2): 250-264 (2008), Cell 131 (5): 861-72 (2007), Science 318 (5858): 1917 -20 (2007), which are hereby incorporated by reference.
- cells produced by any method are included in the “i
- the “Wnt signal activator” in the present invention means a substance that activates the Wnt signal pathway.
- Wnt signal activators include GSK3 ⁇ inhibitors such as BIO and CHIR99021.
- GSK3 ⁇ inhibitors such as BIO and CHIR99021.
- two or more, for example 2, 3, or 4 types of Wnt signal activators may be used in combination.
- Wnt signal inhibitor in the present invention means a substance that inhibits the Wnt signal pathway.
- Wnt signal inhibitors include, for example, the following compounds of formula (I) or salts thereof, compounds such as IWP2, XAV939, and IWR1, and proteins such as IGFBP4 and Dkk1.
- the Wnt signal inhibitor in the present invention is a compound.
- two or more, for example 2, 3, or 4 types of Wnt signal inhibitors may be used in combination.
- Suitable Wnt signal inhibitors for the present invention are the following compounds of formula (I) or salts thereof: Formula (I): [Where: R 1 to R 5 each independently represent a hydrogen atom; a halogen atom; a hydroxyl group; a straight or branched alkoxy group having 1 to 5 carbon atoms; a straight chain having 1 to 5 carbon atoms that is unsubstituted or substituted with a halogen atom.
- R 12 and R 13 are each independently a straight chain of 1 to 5 carbon atoms substituted with a hydrogen atom, an oxygen atom, or an unsubstituted or halogen atom, or Where two adjacent R 1 —R 5 together form —O—CH 2 —O— or —O— (CH 2 ) 2 —O—.
- R 6 -R 9 is independently a hydrogen atom; the group -C (O) carbon atoms which is substituted by A 1 to 5; a halogen atom, a hydroxyl group, a straight chain of 1 to 5 carbon atoms or a branched alkoxy group
- a straight-chain or branched alkoxy group (A is a saturated or unsaturated 5- or 6-membered ring which is unsubstituted or substituted with a straight-chain or branched alkyl group having 1 to 5 carbon atoms, and the ring is a nitrogen atom, an oxygen atom, And 1 or 2 atoms independently selected from a sulfur atom); a straight-chain or branched alkyl group of 1 to 5 carbon atoms which is unsubstituted or substituted with a halogen atom; or a group —NR 12 R 13 (R 12 and R 13 each independently represents a hydrogen atom, an oxygen atom, or a linear or branched alkyl group
- linear or branched alkoxy group having 1 to 5 carbon atoms examples include methoxy group, ethoxy group, propoxy group, isopropoxy group, butoxy group, isobutoxy group, sec-butoxy group, tert-butoxy group, and pentyloxy group. Can be mentioned.
- Examples of the linear or branched alkyl group having 1 to 5 carbon atoms include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, and a pentyl group. .
- linear or branched acyl group having 1 to 5 carbon atoms examples include formyl group, acetyl group, propionyl group, butyryl group, isobutyryl group, valeryl group, and isovaleryl group.
- R 1 -R 5 are each independently a hydrogen atom, a halogen atom, a hydroxyl group, a linear or branched alkoxy group having 1 to 5 carbon atoms; carbon atoms is substituted with or unsubstituted or halogen atoms 1 To 5 linear or branched alkyl groups, wherein two adjacent R 1 —R 5 are taken together to form —O—CH 2 —O— or —O— (CH 2 ) 2 —O—. It may be formed.
- R 2 and R 3 are preferably straight-chain or branched alkoxy groups having 1 to 5 carbon atoms, or together, —O—CH 2 —O— or —O— (CH 2 ) 2 —. O- is formed. More preferably, R 2 and R 3 are a methoxy group, an ethoxy group, or a propoxy group, and most preferably a methoxy group or an ethoxy group.
- R 1 , R 4 and R 5 are preferably hydrogen atoms.
- R 6 to R 9 each independently represent a hydrogen atom; a halogen atom; a hydroxyl group; a linear or branched alkoxy group having 1 to 5 carbon atoms; an unsubstituted or substituted carbon atom having 1 to 5 carbon atoms; Or a group —NR 12 R 13 (wherein R 12 and R 13 each independently represents a hydrogen atom, an oxygen atom, or an unsubstituted or substituted carbon atom having 1 to 5 carbon atoms) a of a linear or branched alkyl group), wherein -O-CH 2 -O- or -O- (CH 2 two adjacent together among R 6 -R 9) 2 -O - May be formed.
- R 6 and R 9 are preferably each independently a hydrogen atom; a halogen atom; a hydroxyl group; a linear or branched alkoxy group having 1 to 5 carbon atoms; or an unsubstituted or substituted carbon atom having 1 to 5 carbon atoms.
- 5 is a linear or branched alkyl group, and more preferably a hydrogen atom.
- R 7 is a hydrogen atom, a halogen atom, a hydroxyl group, a linear or branched group -C (O) carbon atoms which is substituted by A 1 to 5; linear or branched alkoxy group having 1 to 5 carbon atoms
- An alkoxy group (A is a saturated or unsaturated 5- or 6-membered ring which is unsubstituted or substituted by a linear or branched alkyl group having 1 to 5 carbon atoms, and the ring is formed from a nitrogen atom, an oxygen atom, and a sulfur atom; 1 or 2 atoms independently selected); a linear or branched alkyl group of 1 to 5 carbon atoms which is unsubstituted or substituted with a halogen atom; or a group —NR 12 R 13 (R 12 and R 13 are each independently a hydrogen atom, an oxygen atom, or a linear or branched alkyl group having 1 to 5 carbon atoms which is
- R 7 is a linear alkoxy group having 1 to 5 carbon atoms substituted with a group —C (O) A, and the group —C (O) A is bonded to the terminal carbon atom of the alkoxy group. is doing.
- A contains at least one nitrogen atom, and such A includes pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolyl, imidazolyl, which is unsubstituted or substituted with a linear or branched alkyl group having 1 to 5 carbon atoms.
- Pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, piperidinyl, piperazinyl, morpholinyl, pyridyl, pyrimidinyl, pyrazinyl, and pyridazinyl groups are examples of the group.
- A is a piperidinyl group, piperazinyl group, or morpholinyl group that is unsubstituted or substituted with a linear or branched alkyl group having 1 to 5 carbon atoms.
- A is a piperidin-1-yl group, piperazin-1-yl group, or morpholin-4-yl group which is unsubstituted or substituted with a linear or branched alkyl group having 1 to 5 carbon atoms. is there.
- R 10 and R 11 are preferably a hydrogen atom.
- X is an oxygen atom; a sulfur atom; a group —NR 15 (wherein R 15 is a hydrogen atom, a linear or branched alkyl group having 1 to 5 carbon atoms, a linear or branched acyl group having 1 to 5 carbon atoms). Is). X is preferably a sulfur atom.
- n is an integer from 0 to 4. In a further preferred embodiment, n is 2 or 3.
- Wnt signal inhibitors in the present invention are compounds selected from the following or salts thereof: KY02111 KY010104 T61164 KY02114 KY01045 KY01040 KY02109 KY010104 KY01043 KY01046 PB2852 N11474 PB2572 PB2570 KY02104 SO087 SO102 SO096 SO094
- the step (1) “the step of culturing pluripotent stem cells in a medium containing one or more WNT signal activators” and the step (2) “the cells obtained in the step (1)
- the medium in “the step of culturing in a medium containing one or more WNT signal inhibitors” may be any medium that is generally used for myocardial differentiation of pluripotent stem cells, and the composition thereof is not particularly limited.
- a myocardial differentiation medium based on IMDM medium for example, a medium used in Examples
- a myocardial differentiation medium based on DMEM for example, DMEM / F12 medium (Sigma)
- DMEM for example, DMEM / F12 medium (Sigma)
- GEBCO fetal bovine serum
- MEM non-essential amino acid solution Sigma
- penicillin-streptomycin GBCO
- StemPro A medium such as -34 SFM (GIBCO) + BMP4 (10 ng / ml) is exemplified.
- a culture method generally suitable for myocardial differentiation of pluripotent stem cells can be used.
- the culture method include an adhesion culture method, a suspension culture method, and a suspension culture method.
- the method of the present invention does not require the use of feeder cells such as END2 cells.
- myocardial differentiation of pluripotent stem cells can be efficiently induced even in a serum-free medium (serum-free medium).
- the medium preferably contains albumin
- the culture method is preferably an adhesion culture method.
- albumin include bovine serum albumin and human serum albumin.
- a culture dish coated with gelatin or laminin for example, human laminin 211) can be used.
- serum-free medium containing albumin When a serum-free medium containing albumin is used in the method of the present invention, serum, cytokines, supporting cells, etc., proteins other than albumin, and components derived from biological species different from the pluripotent stem cells used (that is, heterogeneous Pluripotent stem cell myocardial differentiation can be induced in the absence of component).
- the period from the start of culture (myocardial differentiation culture) in the myocardial differentiation medium to the start of steps (1) and (2) and the culture period of steps (1) and (2) can be changed as appropriate.
- Step (2) may be started immediately after the end of step (1), or may be started after a certain period from the end of step (1).
- the WNT signal activator and the WNT signal inhibitor may be added to the early stage and the middle stage of myocardial differentiation of pluripotent stem cells, respectively.
- the initial stage of myocardial differentiation of pluripotent stem cells means the stage of induction of differentiation from pluripotent stem cells to mesoderm, where expression of the mesoderm marker gene occurs, and the mid-stage is from mesoderm It means the differentiation induction period to the myocardium.
- mesoderm marker genes include T, MIXL1, NODAL and the like.
- step (1) is performed on days 0 to 2 or 0 to 3 of myocardial differentiation culture, that is, 2 or 3 days from the start of myocardial differentiation culture, 2 days or more of the myocardial differentiation culture up to day 14 (specifically 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days), preferably 3-10
- the step (2) may be performed for a day, more preferably 4 to 10 days, still more preferably 4 to 8 days, and even more preferably 4 to 6 days.
- step (2) 4 to 6 days out of the 10th day of the myocardial differentiation culture, for example, 3rd to 9th day, 3 to 8th day, 3 to 7th day, 4 to 10th of the myocardial differentiation culture It is preferably carried out on the 4th to 9th day or the 4th to 8th day.
- the concentration of the WNT signal activator and the WNT signal inhibitor is not particularly limited.
- BIO or CHIR99021 is used as the WNT signal activator, it may be used at a final concentration of 100 nM to 100 ⁇ M, preferably 1 ⁇ M to 10 ⁇ M.
- a compound of formula (I) or IWP2, XAV939, or IWR1 is used as a WNT signal inhibitor, it may be used at a final concentration of 0.5 to 20 ⁇ M, preferably 1 to 10 ⁇ M.
- the method of the present invention can be used for the production of cardiomyocytes. Differentiation into cardiomyocytes can be confirmed by, for example, the number of beating cardiomyocytes, expression of myocardial markers, expression of ion channels, response to electrophysiological stimulation, and the like.
- Myocardial markers include ⁇ MHC, ⁇ MHC, cTnT, ⁇ -actinin, and NKX2.5.
- Examples of the ion channel include HCN4, Nav1.5, Cav1.2, Cav3.2, HERG1b, and KCNQ1.
- the cardiomyocytes obtained by the method of the present invention can be used for in vitro drug safety tests or as transplanted cardiomyocytes for heart disease and the like.
- the present invention also provides a kit for promoting myocardial differentiation comprising one or more WNT signal activators and / or one or more WNT signal inhibitors, which is used in the myocardial differentiation inducing method of the present invention.
- Method (1) Cultivation of hESC, hiPSC, and monkey ESC All PSC strains on mitomycin C-treated mouse fetal fibroblasts (MEF) in the Prime ES Cell Culture Medium (ReproCELL Inc., Japan) Along with 5 ng ml -1 bFGF, monkey ESCs were maintained without bFGF.
- human ESC strains KhES-1, KhES-3, H1, and H9 (Non-patent Documents 1, 41) were used.
- human iPSC strains 253G1, IMR90-1, IMR90-4 (Non-patent Documents 42 and 43), and RCHIPC0003 were used.
- RCHIPC0003 was established from human fibroblasts by ReproCELL.
- CMK6.4 Non-patent Documents 1, 44, 45
- the hESC strain was used in accordance with the guidelines on the establishment and use of human ES cells (Ministry of Education, Culture, Sports, Science and Technology).
- IMDM myocardial differentiation medium (20% FBS (Gibco), 1% MEM Non-essential amino acid solution (Sigma), 1% penicillin-streptomycin (Gibco), 2 mM L-glutamine (Sigma), 0.001% 2-mercaptoethanol (Gibco), and 0.005N NaOH, and 10 ng ml -1 BMP4 ( R & D Systems)).
- the medium was replaced with myocardial differentiation medium supplemented with 10 ⁇ M KY02111 and / or other WNT inhibitors: XAV939 (Wako, Japan), IWP -2 (Santa Cruz Biotechnology), IWR-1 (Merck4Bioscience), IGFBP4 (R & D Systems), Dkk1 (R & D Systems), and bFGF (R & D Systems), BMP4 (R & D Systems), VEGF (R & D Systems), DKK1 (R & D) Systems) and Activin A (R & D Systems) (Non-patent Document 16). The medium was changed every 2-3 days.
- KY02111 and other WNT inhibitors were added up to day 9 for hiPSC and monkey ESC strains and up to day 14 for hESC strains.
- the cardiomyocyte colonies on the culture dish were washed with protease solution (0.1% collagenase type I, 0.25% trypsin, 1 U ml -1 DNase I, 116 mM NaCl, 20 mM HEPES, 12.5 mM NaH2PO4, 5.6 mM glucose , 5.4 mM KCl, and 0.8 mM MgSO4, pH 7.35) for 0.5-2 hours to dissociate all myocardial colonies from the bottom of the wells.
- protease solution (0.1% collagenase type I, 0.25% trypsin, 1 U ml -1 DNase I, 116 mM NaCl, 20 mM HEPES, 12.5 mM NaH2PO4, 5.6 mM glucose , 5.4 mM KCl, and 0.8 mM M
- the dissociated myocardial colonies were transferred to a 15 ml tube containing a new myocardial differentiation medium, precipitated by gravity, and the supernatant was removed by aspiration.
- the myocardial colonies were transferred to an ultra-low adhesion culture dish (Corning, 3261) or a 6-well plate (Corning, YO-01835-24) in myocardial differentiation medium containing neither serum nor NaOH. This floating myocardial colony was maintained for over 1 month, and the medium was changed every 5 days.
- the mouse ESC (R1 strain) differentiation method was carried out as previously reported (Non-patent Document 46), and KY02111 was added on days 3-6. Beating colonies were counted on day 9.
- NKX2.5 rabbit polyclonal, 1: 250; Abcam
- ⁇ -actinin mouse monoclonal, 1: 800; Sigma
- cardiac TnT mouse monoclonal, 1: 100; Santa Cruz Biotechnology
- vimentin mouse monoclonal, 1: 800; Sigma V6630
- HCN4 humane monoclonal, 1: 400; Abcam
- Microarray experiment IMR90-1 iPSC was cultured in serum-containing myocardial differentiation medium. On day 3, cells were treated with 10 ⁇ M KY02111 or 0.1% DMSO for 0, 12, or 24 hours. Non-treated cells (0 h), cells treated with KY02111 for 12 or 24 hours (KY12hr or KY24hr), and cells treated with 0.1% DMSO for 12 or 24 hours (DMSO12hr or DMSO24hr) were used for microarray analysis. Total RNA was prepared using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions.
- DiRE Distal Regulating Elements of co-regulated genes
- all genes or the top 10 down-regulated genes can be downloaded from the web program (http://dire.dcode.org/ ) (Non-Patent Document 35).
- TOPFlash assay For TCF reporter assay, IMR90-1 hiPSC was cultured in serum-containing differentiation culture as described above. On the second day, TOPflash plasmid (Millipore 21-170) and pRL-SV40 plasmid (Renilla luciferase) Transfection control, Promega E1980) was transfected using FuGENE HD transfection Reagenet (Promega E2311). After 24 hours, 10 ⁇ M KY02111, 10 ⁇ M XAV939, or 10 ⁇ M IWP-2 was added to the medium along with either 60 ng ml ⁇ 1 mice Wnt3a (Wako 232-02421) or 3 ⁇ M BIO.
- luciferase activity was measured using Dual-Luciferase Reporter 1000 Assay System kit (Promega E1980) and 2030 ARVO X3 plate reader (PerkinElmer). The luciferase activity was normalized to the luminescence of Renilla luciferase, a transfection control, according to the kit protocol.
- HEK293 cells cells were cultured in 10% FBS DMEM medium and transfected with TOPflash and pRL-SV40 plasmids using FuGENE HD.
- transfected cells were seeded in 96-well plates in medium containing 10 ⁇ M KY02111, 10 ⁇ M XAV939, or 10 ⁇ M IWP-2 with 3 ⁇ M BIO or 60 ng ml -1 mouse Wnt3a (10,000 cells / well) . Luciferase activity was measured 48 hours after transfection by the method described above.
- Patch clamp method Cells were isolated from beating hPSC-derived cardiomyocytes using a protease solution. Dissociated cells were cultured on gelatin-coated coverslips for 3-7 days. The patch clamp method was performed in a thermostatic chamber at about 25 ° C. From spontaneous beating colonies, data from a whole cell patch clamp configuration was recorded using a HEKA EPC10 amplifier (HEKA Instruments Inc.). The external solution (containing 140 mM NaCl, 5 mM KCl, 10 mM HEPES, 2 mM CaCl 2 , 1 mM MgCl 2 , and 10 mM glucose) was adjusted to pH 7.2 with NaOH.
- Pipette solution (80 mM potassium aspartate, 50 mM KCl, 1 mM MgCl 2 , 10 mM EGTA, 3 mM ATP-Mg, and 10 mM HEPES) was adjusted to pH 7.2 with KOH.
- cells were treated with 0.1 ⁇ M E4031, 4 ⁇ M chromanol 293B, 4 ⁇ M zatebradine, 1 ⁇ M tetrodotoxin (TTX), or 4 ⁇ M nifedipine for 5 minutes. These inhibitors were obtained from Sigma-Aldrich.
- a 1 mM stock solution of E4031 and TTX was prepared with H 2 O.
- a 20 mM stock solution of chromanol 293B, zatebradine hydrochloride, and nifedipine was prepared in DMSO. All stock solutions were stored at ⁇ 20 ° C. until use.
- the current-voltage curve maintains the membrane potential from -40 mV to -60 mV, depolarizing pulses for 500 milliseconds, -60 mV to 60 mV for INa, -40 mV to 40 mV for ICaL, IKs And IKr were obtained by gradual delivery from -40 mV to 40 mV and for Ih from -50 mV to -140 mV.
- the MEA dish was connected to an MEA amplifier (Multi Channel Systems, Reutlingen, Germany) and an electrocardiogram (ECG) -like wave was recorded.
- ECG electrocardiogram
- 300 pM astemizole (Sigma) or 0.1% DMSO was added and the concentration of astemizole (300 pM-30 ⁇ M) was increased every 4 minutes.
- ECG-like waves were recorded 2 minutes after each astemizole treatment. Beat rate, Na + amplitude, K + amplitude, and Na + -K + interval were analyzed by LabChart TM software v7 (ADInstruments, Sydney, Australia).
- N11474 analogs were chemically synthesized and their ability to promote myocardial differentiation of monkey ESCs was evaluated.
- a structure-activity relationship study reveals that bioactivity is greatly improved by replacing the methoxy group of the benzothiazole ring with an electron withdrawing group and adjusting the length of the methylene linker, resulting in the novel molecule KY02111.
- This low molecular weight compound efficiently promoted myocardial differentiation about 73 times that of the control (DMSO) and 7.4 times that of N11474 (FIG. 1d).
- KY02111 has a myocardial beating colony ratio of 2 human ESC strains (KhES-1 and KhES-3), 4 human iPSC strains (253G1, IMR90-1, IMR90-4, and RCHIPC0003), and 1 There was a 60-94% increase in cell clumps of different mouse ESC strains (R1) (FIG. 1e).
- a time course experiment using hiPSCs (IMR90-1) revealed that beating colonies appeared on day 10 and increased in number until day 25 (FIG. 6).
- Subculture of pulsatile colonies increased the proportion of pulsatile colonies thereafter to 90% (FIG. 6), which is thought to be due to a decrease in mechanical inhibition (Non-patent Document 33).
- Myocardial colonies continued to beat until at least day 50 (FIG. 6).
- hPSC-derived cardiomyocytes can be concentrated simply by collecting KY02111-derived cell colonies without performing cell sorting operations.
- Real-time PCR analysis on days 15 and 30 revealed that KY02111-induced cardiomyocytes (KY-CM) expressed myocardial markers ⁇ MHC, NKH2.5, and HCN4, and all ion channel genes examined were adults. It was shown to be expressed at a level similar to heart tissue (FIG. 2c).
- KY-CM is a functional cell using electrophysiological analysis by whole cell patch clamp method.
- the action potential characteristics suggested that the KY-CM population contains ventricular cells and pacemaker cells (FIG. 2d).
- the characteristics of voltage-dependent Ca 2+ channel, Na + channel, and HCN channel current were examined using the ion channel blockers nifedipine, lidocaine, and zatebradine, respectively (FIGS. 7a-c).
- the relationship between current density and voltage indicates that KY-CM is an electrophysiologically functional cell.
- Treatment of KY-CM with HERG channel blocker E4031 and KCNQ1 channel blocker chromanol 293B increased action potential duration (APD) corresponding to QT prolongation detected by electrocardiogram (ECG) (FIG. 2e).
- APD90 APD at 90% repolarization
- the increase in duration of APD90 was 37.0% ⁇ 11.2 for E4031 treatment and 42.1% ⁇ 8.8 for chromanol 293B treatment (FIGS. 2e, 7e).
- the voltage-dependent K + current was suppressed by treatment with E4031 and chromanol 293B (FIG. 7d, e).
- TCF4 When 22 genes that were down-regulated were examined for a common transcription factor binding site using DiRE (Non-patent Document 35) that can predict a distant regulatory element, TCF4 was predicted as a common transcription factor (FIG. 9b). ). When the top 10 genes that were most down-regulated in the treated cells were examined, LEF1 and TCF4 were predicted as common transcription factors (FIG. 9c). Of the 22 genes down-regulated, 16 genes (70%) were known as target genes of the classical WNT signaling pathway (Table 2). Furthermore, the effect of KY02111 on the WNT target gene was very similar to that of other WNT inhibitors such as XAV939 and IWP-2, but not the effect of BIO, a WNT activator. (FIG. 9d). These results suggest that KY02111 inhibits the classical WNT signaling pathway in hPSC.
- the number of cardiomyocytes produced was 80 times higher in the KY02111 treatment and 130-220 times higher in the combination treatment (KY + XAV, KY + IWP, and KY + XAV + IWP) compared to the control (FIG. 3e). .
- the differentiation efficiency by treatment with KY02111 and serum was lower than the differentiation efficiency by treatment with KY02111 and albumin (93.3% ⁇ 4.4) (Fig. 4b).
- the number of beating colonies and cardiomyocytes was about 3 times higher in the KY02111 treatment in the absence of serum than in the KY02111 treatment in the presence of serum (FIG. 4e).
- treatment with KY + XAV in the presence of serum enhanced the number of beating colonies and cardiomyocytes by about 1.5 times that in the absence of serum (FIG. 4e).
- KY02111 Reaction was performed in the same manner as described above using 3- (3,4-dimethoxyphenyl) propanoyl chloride (100 mg, 0.42 mmol) and 2-amino-6-chlorobenzothiazole (78 mg, 0.42 mmol) as substrates. 113 mg of 2- (3- (3,4-dimethoxyphenyl) propanamide) -6-chlorobenzothiazole was obtained in a yield of 72%.
- KY02109 2- (3,4-Dimethoxyphenyl) acetyl chloride (100 mg, 0.51 mmol) and 2-amino-6-chlorobenzothiazole (94 mg, 0.51 mmol) were used as substrates and reacted in the same manner as above.
- 153 mg of-(2- (3,4-dimethoxyphenyl) acetamido) -6-chlorobenzothiazole was obtained in a yield of 83%.
- KY010104 Reaction was performed in the same manner as described above using 3- (3,4-dimethoxyphenyl) propanoyl chloride (100 mg, 0.5 mmol) and 2-amino-6-nitrobenzothiazole (105 mg, 0.57 mmol) as substrates. 138 mg of 2- (3- (3,4-dimethoxyphenyl) propanamide) -6-nitrobenzothiazole was obtained in a yield of 71%.
- KY01046 Reaction was performed in the same manner as described above using 5- (3,4-dimethoxyphenyl) pentanoyl chloride (30 mg, 0.13 mmol) and 2-amino-6-nitrobenzothiazole (25 mg, 0.13 mmol) as substrates.
- 2- (5- (3,4-dimethoxyphenyl) pentanamide) -6-nitrobenzothiazole 38 mg, yield 70% was obtained.
- KY02104 2- (3,4-Dimethoxyphenyl) acetyl chloride (100 mg, 0.51 mmol) and 2-amino-6-fluorobenzothiazole (86 mg, 0.51 mmol) were used as substrates and reacted in the same manner as above. 157 mg of-(2- (3,4-dimethoxyphenyl) acetamido) -6-fluorobenzothiazole was obtained in a yield of 89%.
- reaction solution was diluted with ethyl acetate and washed with saturated aqueous sodium hydrogen carbonate solution, distilled water, and saturated aqueous sodium chloride solution. After drying with sodium sulfate, the solvent was distilled off. Ethanol was added to the residue and refluxed, and recrystallization was performed to obtain 320 mg (yield 35%) of 2- (3- (3,4-dimethoxyphenyl) propanamide) -6-bromobenzothiazole.
- reaction solution was diluted with ethyl acetate and washed with saturated aqueous sodium hydrogen carbonate solution, distilled water, and saturated aqueous sodium chloride solution. After drying with sodium sulfate, the solvent was distilled off. Ethanol was added to the residue and the mixture was refluxed and recrystallized to obtain 447 mg of ethyl 4-((2- (3- (3,4-dimethoxyphenyl) propanamido) benzothiazol-6-yl) oxy) butanoate, yield Obtained at 76%.
- reaction solution was diluted with ethyl acetate and washed with saturated aqueous sodium hydrogen carbonate solution, distilled water, and saturated aqueous sodium chloride solution. After drying with sodium sulfate, the solvent was distilled off. Ethanol was added to the residue and the mixture was refluxed and recrystallized to give 3- (3,4-dimethoxyphenyl) -N- (6- (4-morpholino-4-oxobutoxy) benzothiazol-2-yl) propanamide. Obtained in 50 mg, 43% yield.
- Non-patent literature Suemori, H. et al. Efficient establishment of human embryonic stem cell lines and long-term maintenance with stable karyotype by laboratory bulk passage.Biochem Biophys Res Commun 345, 926-932 (2006). 2. Smith, KP, Luong, MX & Stein, GS Pluripotency: toward a gold standard for human ES and iPS cells.J Cell Physiol 220, 21-29 (2009). 3. Yamashita, JK ES and iPS cell research for cardiovascular regeneration.Exp Cell Res 316, 2555-2559 (2010). 4. Yoshida, Y. & Yamanaka, S. iPS cells: a source of cardiac regeneration.J Mol Cell Cardiol 50, 327-332 (2011). 5.
- Primer sequence number 1 Primer sequence number 2: Primer sequence number 3: Primer sequence number 4: Primer sequence number 5: Primer sequence number 6: Primer sequence number 7: Primer sequence number 8: Primer sequence number 9: Primer sequence number 10: Primer Sequence number 11: Primer sequence number 12: Primer sequence number 13: Primer sequence number 14: Primer sequence number 15: Primer sequence number 16: Primer sequence number 17: Primer sequence number 18: Primer sequence number 19: Primer sequence number 20: Primer Sequence number 21: Primer sequence number 22: Primer sequence number 23: Primer sequence number 24: Primer sequence number 25: Primer sequence number 26: Primer sequence number 27: Primer sequence number 28: Primer sequence number 29 Primer SEQ ID NO: 30: primer SEQ ID NO 31: Primer SEQ ID NO 32: Primer SEQ ID NO 33: Primer SEQ ID NO 34: Primer SEQ ID NO 35: Primer SEQ ID NO: 36: Primer
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Abstract
Description
(1)多能性幹細胞を1以上のWNTシグナル活性化剤を含む培地中で培養する工程、および;
(2)工程(1)で得られた細胞を1以上のWNTシグナル阻害剤を含む培地中で培養する工程。
を含む方法を提供する。
式(I):
R1-R5は、各々独立して、水素原子;ハロゲン原子;水酸基;炭素数1~5の直鎖又は分岐アルコキシ基;非置換又はハロゲン原子で置換された炭素数1~5の直鎖又は分岐アルキル基;又は基-NR12R13(R12及びR13は、各々独立して、水素原子、酸素原子、又は非置換又はハロゲン原子で置換された炭素数1~5の直鎖または分岐アルキル基である)である、ここでR1-R5のうち隣接する2つが一緒になって-O-CH2-O-または-O-(CH2)2-O-を形成していてもよい、
R6-R9は、各々独立して、水素原子;ハロゲン原子;水酸基;炭素数1~5の直鎖又は分岐アルコキシ基;基-C(O)Aで置換された炭素数1~5の直鎖又は分岐アルコキシ基(Aは、非置換又は炭素数1~5の直鎖または分岐アルキル基で置換された飽和または不飽和5または6員環であり、該環は窒素原子、酸素原子、及び硫黄原子から独立に選択される1または2個の原子を含んでいてもよい);非置換又はハロゲン原子で置換された炭素数1~5の直鎖又は分岐アルキル基;又は基-NR12R13(R12及びR13は、各々独立して、水素原子、酸素原子、又は非置換又はハロゲン原子で置換された炭素数1~5の直鎖または分岐アルキル基である)である、ここでR6-R9のうち隣接する2つが一緒になって-O-CH2-O-または-O-(CH2)2-O-を形成していてもよい、
R10-R11は、各々独立して、水素原子;又は炭素数1~5の直鎖又は分岐アルキル基である、
Xは、-CR14(R14は、水素原子、ハロゲン原子、水酸基、炭素数1~5の直鎖又は分岐アルコキシ基、非置換又はハロゲン原子で置換された炭素数1~5の直鎖又は分岐アルキル基である);酸素原子;硫黄原子;セレン原子;又は基-NR15(R15は、水素原子、炭素数1~5の直鎖又は分岐アルキル基、又は炭素数1~5の直鎖又は分岐アシル基である)である、および
nは、0から6の整数である]。
KY02111
(1)hESC, hiPSC、およびサルESCの培養
PSC株はいずれも、マイトマイシンC処理マウス胎児線維芽細胞 (MEF)上、Primate ES Cell Culture Medium (ReproCELL Inc., Japan)中で、hPSCについては 5 ng ml-1 bFGFとともに、サルESCについてはbFGFなしで、維持した。ヒトESC株は、KhES-1、KhES-3、H1、およびH9(非特許文献1,41)を用いた。ヒトiPSC株は、253G1、IMR90-1、IMR90-4(非特許文献42,43)、およびRCHIPC0003を用いた。RCHIPC0003は、ReproCELLによりヒト線維芽細胞から樹立された。サルESC株は、CMK6.4(非特許文献1,44,45)を用いた。hESC株は、ヒトES細胞の樹立及び使用に関する指針(文部科学省)にしたがい使用した。
ヒトαMHCプロモーターの制御下でEGFPを発現するサルESCを作成するため、転写開始部位の上流からオープンリーディングフレーム直前の第3エキソンまでの918 bpを、KhES-1 hESCのゲノムDNAからPCRにより、プライマーセット(フォワードプライマー, 5’- CCAGGCACCTGCACCCTCTGG-3’; リバースプライマー, 5’- ( SalI リンカーを含む) 22 TAGTCGACCTTGGTGCTTCCCCTGGGTCAGAG-3’)を用いて増幅し、pGEM-T Easy Vector (Promega)にライゲートした。クローニングベクター由来のHindIII/SalI フラグメントをpEGFP-1 (Clontech)に結合した。直鎖化したαMHC-EFGP ベクターを CMK6.4 サルESCにエレクトロポレーションにより導入した。トランスジェニックESCクローンは、G418 (Sigma-Aldrich)により選択的に増殖させた。EGEPの発現は拍動コロニーにおいて確認した。
コンフルエント (継代4-5日後) のαMHC-GFPトランスジェニックサルESCをCTK溶液(非特許文献1)により酵素的に解離させ、ESC塊 (直径40-100 μm) を40μmおよび100 μmのCell Strainer (BD Biosciences)を用いて得た。このESC塊 (約5.0×103 細胞/ウェル) を96ウェルプレート (Greiner Bio-One, 96-well black plate, Cat. No. 655090) に移し、下記(4)の心筋分化培地中でインキュベートした。心筋分化6-14日目に、化合物ライブラリー由来の9600化合物 (DMSOに溶解) を、96ウェルプレートを用いてスクリーニングした。16ウェルを対照 (0.1% DMSO)に、80ウェルを化合物(1ウェルにつき1化合物)に使用した。各化合物の最終濃度は約1-5μMであった。低分子化合物を含む培地は、4日毎に交換した。14日目に、Metamorphイメージングシステム (Molecular Device)を用いたプレート全体のスキャニングにより、ESCのGFP蛍光を測定した。Metamorphソフトウェアを使用してGFP蛍光データを解析し、各ウェルについて、蛍光領域、全蛍光強度、平均蛍光強度、およびGFP陽性コロニーの数を計算した。ヒット化合物は以下の基準により決定した:その化合物のウェルの平均蛍光強度が対照の平均蛍光強度(16ウェルの平均)+6 SDよりも大きく、その化合物の全蛍光強度が対照の全蛍光強度+0 SD よりも大きい;または、その化合物の全蛍光強度が対照の全蛍光強度+4 SD よりも大きく、その化合物の平均蛍光強度が対照の平均蛍光強度+0 SD よりも大きい。120化合物がこの基準を満たし、各化合物につき8ウェルを用いて再試験した。N11474という分子が、DMSOと比較して有意にESCのGFP蛍光を増加させた (スチューデントt検定t, P=0.015)。
コンフルエントのhESCおよびhiPSCをCTK溶液を用いて穏やかに解離させ、Primate ES Cell Culture Medium (ReproCELL, Japan)を用いてペトリ皿 (BD BioSciences) に移した。細胞を懸濁培養にて8-24時間維持し、凝集させた。hPSC凝集体の大きさ (直径0.3-1 mm) は、効率的な分化に重要である。懸濁培養に続き、細胞凝集体を培養ディッシュに以下の心筋分化培地中で付着させた (3-10×105 細胞/cm2):IMDM (Sigma) (20% FBS (Gibco)、1% MEM 非必須アミノ酸溶液 (Sigma)、1% ペニシリン-ストレプトマイシン (Gibco)、2 mM L-グルタミン (Sigma)、0.001% 2-メルカプトエタノール (Gibco)、および0.005N NaOH、並びに10 ng ml-1 BMP4 (R&D Systems)含有)。3日目(hiPSC株)または4日目(ヒトまたはサルESC株)に、培地を10μM KY02111および/または他のWNT阻害剤を添加した心筋分化培地に交換した:XAV939 (Wako, Japan)、IWP-2 (Santa Cruz Biotechnology)、IWR-1 (Merck4Bioscience)、IGFBP4 (R&D Systems)、Dkk1 (R&D Systems)、並びに、bFGF (R&D Systems)、BMP4 (R&D Systems)、VEGF (R&D Systems)、DKK1 (R&D Systems)、およびActivin A (R&D Systems)の混合物(非特許文献16)。培地は2-3日毎に交換した。KY02111および他のWNT阻害剤は、hiPSC株およびサルESC株については9日目まで、hESC株については14日目まで添加した。15日目に、培養ディッシュ上の心筋細胞コロニーを、プロテアーゼ溶液 (0.1% コラゲナーゼタイプI、0.25% トリプシン、1 U ml-1 DNase I、116 mM NaCl、20 mM HEPES、12.5 mM NaH2PO4、5.6 mM グルコース、5.4 mM KCl、および0.8 mM MgSO4、pH 7.35)とともに0.5-2時間インキュベートし、ウェルの底から全ての心筋コロニーを解離させた。解離した心筋コロニーを新しい心筋分化培地を含む15 mlチューブに移し、重力により沈澱させ、上清を吸引により除去した。次いで、心筋コロニーを、超低接着培養ディッシュ (Corning, 3261) または6ウェルプレート (Corning, YO-01835-24) に、血清およびNaOHをいずれも含まない心筋分化培地中にて移した。この浮遊心筋コロニーを1ヵ月以上維持し、培地を5日毎に交換した。マウスESC (R1株)の分化方法は既報のとおり実施し(非特許文献46)、KY02111は3-6日目に添加した。拍動コロニーは9日目に計測した。
上記のペトリ皿での前培養後、細胞凝集体を、ゼラチン (Sigma G2625) またはヒトラミニン211 (BioLamina, Sweden) をコートした培養ディッシュに、以下の血清不含心筋分化培地中、3-10 x 105 細胞/cm2で付着させた:IMDM (Sigma)(1% MEM 非必須アミノ酸溶液 (Sigma)、1% ペニシリン-ストレプトマイシン (Gibco)、2 mM L-グルタミン (Sigma)、0.5 mM L-カルニチン (Sigma)、0.001% 2-メルカプトエタノール (Gibco)、および1-2% ウシ血清アルブミン (Wako, Japan)、または0.4% ヒト血清アルブミン (Sigma)、並びに5μM CHIR99021 (Axon)および2μM BIO (Calbiochem)を含有))。3-9日目に、10μM KY02111および/または他のWNT阻害剤 (XAV939および/またはIWP-2)を細胞培養に添加し、培地を2日毎に交換した。15日目に、心筋コロニーをプロテアーゼ溶液と5-10分間インキュベートし、全てのコロニーをウェルの底から解離させた。この心筋コロニーを、0.1% アルブミンを含む心筋分化培地ともに15 mlチューブに移し、重力により沈澱させ、上清を回収し、心筋コロニーを超低接着培養6ウェルプレート(Corning, YO-01835-24) に0.1% アルブミンを含む心筋分化培地ともに移した。この浮遊心筋コロニーを1ヵ月以上維持し、培地を5日毎に交換した。
心筋コロニーを、前述のプロテアーゼ溶液とともに1-2時間37℃で撹拌することにより解離させ、ゼラチン (Sigma)でコートしたカバーガラス上で1日培養し、4% パラホルムアルデヒドで固定した。Triton Xで透過処理後、細胞を2% スキムミルクで1-2時間処理し、以下の一次抗体と2時間インキュベートした: NKX2.5 (ウサギポリクローナル, 1:250; Abcam)、αアクチニン (マウスモノクローナル, 1:800;Sigma)、心筋TnT (マウスモノクローナル, 1:100; Santa Cruz Biotechnology)、ビメンチン (マウスモノクローナル, 1:800; Sigma V6630)、およびHCN4 (マウスモノクローナル, 1:400; Abcam)。使用した二次抗体は、Alexa546結合抗マウスIgG、Alexa488結合抗マウスIgG、およびAlexa488結合抗ウサギIgG (1:1000; いずれもMolecular Probes, Invitrogenより入手)。核はDAPIにより可視化した。画像は蛍光顕微鏡 (Olympus)下に取得した。cTnT、αアクチニン、NKX2.5、HCN4、およびビメンチン陽性細胞の数を3つのランダムな領域から、Metamorphイメージングシステム (Molecular Devices)を用いて計測した。約1000個のDAPI染色細胞を用いて免疫陽性細胞の割合を計算した (免疫陽性細胞数/DAPI染色細胞数)。フローサイトメトリーのため、hPSC由来心筋コロニーからの単一細胞を抗cTnT抗体により染色し、Alexa488結合抗マウスIgG二次抗体により染色した。細胞は、FACS CantoII フローサイトメーター (BD Biosciences)を用いて解析した。データは、FACADiva ソフトウェア (BD Biosciences)を使用して解析した。
IMR90-1 iPSCを血清含有心筋分化培地中で培養した。3日目に、細胞を10μM KY02111または0.1% DMSOにて0、12、または24時間処理した。非処理細胞(0 h)、KY02111で12時間または24時間処理した細胞 (KY12hrまたはKY24hr)、0.1% DMSOで12時間または24時間処理した細胞 (DMSO12hrまたはDMSO24hr)をマイクロアレイ解析に使用した。全RNAをRNeasy Mini Kit (Qiagen)を用いて製造元の説明にしたがい調製した。cDNAの合成、インビトロ転写、およびビオチンラベル化cRNA、およびHuman Gene 1.0 ST アレイ (Affymetrix)へのハイブリダイゼーションは、Affymetrixのプロトコールにしたがい実施した。ハイブリダイズ後のアレイは、Affymetrix GeneChip Scannerを用いてスキャンした。データの正規化および更なる解析は、GeneSpring GX (Agilent Technologies)を用いて行った。遺伝子を発現する処理細胞の対照に対する割合 (KY02111/DMSO)を、各時点で計算した。KY02111の処理後12時間および24時間の両方の時点でKY/DMSO比において25%を超える変化を示す遺伝子を、KY02111に応答してダウンレギュレーションまたはアップレギュレーションされる遺伝子として同定した(ダウンレギュレーションされる遺伝子=KY12h/DMSO12h <0.75 およびKY24h/DMSO24h <0.75; アップレギュレーションされる遺伝子=KY12h/DMSO12h >1.25およびKY24h/DMSO24h >1.25)。マイクロアレイデータは、NCBIのGene Expression Omnibus (GEO) パブリックデータベースへ提出した (GEO accession number, GSE33622)。DiRE(Distant Regulating Elements of co-regulated genes)解析のため、全ての遺伝子またはダウンレギュレーションされた上位10遺伝子 (KY12h/DMSO12h比に基づく)を、ウェブのプログラム (http://dire.dcode.org/)(非特許文献35)用いて解析した。
全RNAをRNeasy Mini Kit (QIAGEN)を用いて単離した。ゲノムDNAは、DNase I (Invitrogen)を用いて完全に消化した。全RNA (0.5μg)を、SuperScript III Reverse Transcriptase (Invitrogen)を用いたcDNA合成に使用した。qRT-PCRは、7500 Real Time PCR System (Applied Biosystems)にてSYBER GREEN PCR Master Mixを用いてトリプリケートで実施した。サイクル条件は以下のとおりである:95℃10分; 95℃15秒40サイクル; 60℃1分。ヒト成人心臓組織由来の全RNA (BioChain) を用いて、hPSC由来の心筋細胞の心筋マーカー遺伝子の発現レベルを評価した。全ての値をGAPDH発現レベルに対してノーマライズし、ヒト成人心臓における対応する値 (100%)と比較して表した。プライマーセットは表1に示す。
TCFレポーターアッセイのため、IMR90-1 hiPSCを前述のとおり血清含有分化培養にて培養し、2日目に、TOPflashプラスミド(Millipore 21-170)およびpRL-SV40プラスミド (ウミシイタケルシフェラーゼトランスフェクションコントロール, Promega E1980) をFuGENE HD transfection Reagenet (Promega E2311)を用いてトランスフェクトした。24時間後、10μM KY02111、10μM XAV939、または10μM IWP-2を、60 ng ml-1 マウスWnt3a (Wako 232-02421)または3μM BIOのいずれかとともに、培地に添加した。トランスフェクションの48時間後 (4日目)、ルシフェラーゼ活性をDual-Luciferase Reporter 1000 Assay System キット (Promega E1980)および2030 ARVO X3 プレートリーダー (PerkinElmer)を用いて測定した。ルシフェラーゼ活性は、キットのプロトコールにしたがい、トランスフェクションコントロールであるウミシイタケルシフェラーゼの発光に対してノーマライズした。HEK293細胞を用いた実験では、細胞を10% FBS DMEM培地中で培養し、FuGENE HDを用いてTOPflashおよびpRL-SV40プラスミドをトランスフェクトした。24時間後、トランスフェクトした細胞を、10μM KY02111、10μM XAV939、または10μM IWP-2を、3μM BIOまたは60 ng ml-1 マウスWnt3aとともに含む培地中、96ウェルプレートに播種した (10,000 細胞/ウェル)。ルシフェラーゼ活性は、前述の方法によりトランスフェクションの48時間後に測定した。
拍動hPSC由来心筋細胞より、プロテアーゼ溶液を用いて細胞を単離した。解離した細胞を、ゼラチンをコートしたカバーガラス上で3-7日間培養した。パッチクランプ法は、約25℃の恒温室にて実施した。自発的拍動コロニーから、ホールセルパッチクランプの構成から得られるデータをHEKA EPC10 増幅器 (HEKA Instruments Inc.)を用いて記録した。外部溶液(140 mM NaCl、5 mM KCl、10 mM HEPES、2 mM CaCl2、1 mM MgCl2、および10 mMグルコースを含有)は、NaOHによりpH 7.2 に調節した。ピペット溶液(80mM アスパラギン酸カリウム、50 mM KCl、1 mM MgCl2、10 mM EGTA、3 mM ATP-Mg、および10 mM HEPES)は、KOHによりpH 7.2 に調節した。一部の実験では、細胞を、0.1μM E4031、4μM クロマノール 293B、4μM ザテブラジン、1μM テトロドトキシン (TTX)、または4μM ニフェジピンで5分間処理した。これら阻害剤は、Sigma-Aldrichから入手した。E4031およびTTXの1 mM ストック溶液は、H2Oにより調製した。クロマノール 293B、ザテブラジン塩酸塩、およびニフェジピンの20 mM ストック溶液は、DMSOにより調製した。ストック溶液は全て、使用まで-20℃で保存した。電流電圧曲線は、膜電位を-40 mVから-60 mVに維持し、脱分極パルスを500ミリ秒間、INaについては-60 mVから60 mVまで、ICaL については-40 mVから40 mVまで、IKsおよびIKrについては-40 mVから40 mVまで、およびIhについては-50 mVから-140 mV まで、段階的に送達することで取得した。
微小電極アレイ (MEA)ディッシュ (Multi Channel Systems, Reutlingen, Germany)を、ReproCoat (RCESD006, ReproCELL, Japan)により、5% CO2下にて37℃で1時間コーティングした。コーティング後、30日目のKY02111誘導心筋細胞を、前記MAEディッシュ上の1 mlのCardiomyocyte Culture Medium (RCESD006, ReproCELL, Japan)に移した。ピペットチップまたはガラス棒を用いて、心筋細胞を穏やかに電極上へ移動させ、一晩培養してMEAディッシュに付着させた。翌日、培地をCardiomyocyte Test Medium (RCESD003, ReproCELL, Japan)に交換した。拍動コロニーの存在を確認した後、MEAディッシュをMEA 増幅器 (Multi Channel Systems, Reutlingen, Germany)に接続し、心電図(ECG)様波を記録した。記録の2分後、300 pM アステミゾール (Sigma) または0.1% DMSO を添加し、アステミゾールの濃度(300 pM-30μM)を4分毎に増加させた。各アステミゾール処理の2分後にECG様波を記録した。拍動率、Na+振幅、K+振幅、およびNa+-K+間隔は、LabChart TM ソフトウェア v7 (ADInstruments, Adelaide, Australia)により解析した。
結果はいずれも平均±SEMで表した。独立両側スチューデントt検定(Unpaired two-tailed student t-test)を用いて、異なる処理からの測定値の平均値を比較した。P <0.05のときに有意差ありと判断した。
KY02111、KY01041、KY02114、KY01045、KY01040、KY02109、KY01042、KY01043、KY01046、およびKY02104は、「3.製造例」に記載のとおり合成した。PB2852、PB2572、およびPB2570: PB285238764、PB257239592、およびPB257074412はUkrOrgSynthesisから購入した。T61164: T6116478はENAMINEから購入した。
(1)KY02111の発見および特性決定
hPSCからの心筋分化を効率的に促進する低分子化合物を同定するため、ヒトαMHCプロモーターの制御下でEGFPを発現するサルES細胞(ESC)を用いるハイコンテント解析(HCA)システムを構築した(図1a)。増強されたGFPシグナルの化学スクリーニングは「(1)方法」に説明のとおりである。このシステムにより、GFP強度および蛍光領域を含むHCAパラメータの値を対照と比較して有意に増強する分子として、N11474が同定された (スチューデントt検定, P=0.015)。
免疫細胞化学により、KY02111により処理したhiPSC(IMR90-1)では約73-85%が心筋マーカーである心筋トロポニンT(cTnT)、α-アクチン、またはNKX2.5を発現するが、これらマーカーが陽性であるDMSO処理細胞は少数であることが示された(図2a、b)。心臓ペースメーカーマーカーであるHCN4は16%のKY02111処理細胞において発現しており、ビメンチン陽性細胞(線維芽細胞)の比率は3.3倍減少した(図2b)。平滑筋マーカーであるSMA(Smooth Muscle Actin)はKY02111処理細胞ではほとんど検出されなかった(データ非提示)。これらの結果から、hPSC由来心筋細胞は、細胞ソーティングの操作を行うことなく、単にKY02111誘導細胞コロニーを回収することによって濃縮できることが示唆される。15日目および30日目のリアルタイムPCR解析により、KY02111誘導心筋細胞(KY-CM)は、心筋マーカーであるαMHC、NKH2.5、およびHCN4を発現すること、検討したイオンチャネル遺伝子はいずれも成人心臓組織と同様のレベルで発現していることが示された(図2c)。
KY02111が如何にしてhPSCの心筋分化を促進するのかを調べるため、KY02111処理したhiPSC (IMR90-1)の遺伝子発現プロファイルをマイクロアレイ技術を用いて解析した。KY02111またはDMSOを心筋分化培養3日目(IMR90-1細胞にとって最適な開始点)に添加し、12および24時間後に細胞集団を回収した。KY02111処理後、12時間および24時間の両方で、22種類のダウンレギュレーションされる遺伝子および4種類のアップレギュレーションされる遺伝子が同定された(図3a、表2)。マイクロアレイ解析では少数の遺伝子しか抽出されなかった(細胞集団が高度に不均質のためと考えられる)が、マイクロアレイデータをqPCRにより確認した(図9a)。ダウンレギュレーションされた22遺伝子について、遠位の調節要素を予測可能なDiRE(非特許文献35)を用いて共通の転写因子結合部位を調べると、TCF4が共通の転写因子として予測された(図9b)。処理細胞において最もダウンレギュレーションされた上位10遺伝子を調べると、LEF1およびTCF4 が共通の転写因子として予測された(図9c)。ダウンレギュレーションされた22遺伝子のうち16遺伝子 (70%) は、古典的WNTシグナル経路の標的遺伝子として知られていた(表2)。さらに、WNT標的遺伝子に対するKY02111の効果は、XAV939およびIWP-2などの他のWNT阻害剤の効果と非常に類似していたが、WNT活性化剤であるBIOの効果とは類似していなかった(図9d)。これらの結果は、KY02111がhPSCにおける古典的WNTシグナル経路を阻害することを示唆する。
次に、KY02111、数種の化学性またはタンパク性WNT阻害剤(IWP-2、IWR-1、XAV939、DKK1、およびIGFBP4(非特許文献36))、および数種のサイトカインの組合せによるトランスジェニックサルESCにおける心筋分化の促進を比較した。予想どおり、これらはいずれもαMHCプロモーターにより誘導されるGFPシグナル強度を増強したが、KY02111が最も効果的であり、また、他の化学性WNT阻害剤はタンパク性阻害剤やサイトカインよりも効果が高かった(図3c、d)。KY02111の処理により、対照と比較して約80倍分化が増加した。KY02111の処理は、hiPSCの心筋分化の促進においても最も効果的であった(図3d)。得られた分化細胞の全細胞数はWNT阻害剤の間で類似していたが(図10a)、拍動コロニーの割合は相違しており、KY02111、IWP-2、およびXAV939のそれぞれについて87、37、および7.5%であった(図3d)。
cTnT陽性細胞の割合は拍動コロニーの割合と同様の傾向を示した(図10b)。
血清不含培地における分化効率を調べると、ゼラチンまたはヒトラミニン211によるコーティングと、0.4% ヒト血清アルブミンまたは1-2% ウシ血清アルブミンの添加という、2つの要件が明らかとなった。非接着細胞はほとんど心筋細胞へ分化せず、アルブミンを含まない血清不含培地では細胞死が起こった(データ非提示)。
KY01041
3,4-ジメトキシベンゾイルイルクロリド(100mg,0.55mmol)とトリエチルアミン(83,0μl.6mmol)を塩化メチレン(500μl)に溶かし、2-アミノ-6-クロロベンゾチアゾール(105mg,0.57mmol)を加え一時間、室温にて撹拌した。反応終了後、塩化メチレンに希釈し、飽和食塩水にて洗浄した。硫酸マグネシウムで乾燥した後に溶媒を留去した。残渣にエタノールを加え、70度に加熱し、溶解させた後に室温に戻すことで再結晶を行い、2-(3,4-ジメトキシベンズアミド)-6-クロロベンゾチアゾールを130mg、収率68%で得た。
1H NMR (CDCl3): δ10.15(s, 1H), 7.83 (d, J= 1.8 Hz, 1H), 7.63-7.45 (m, 3H), 7.36 (dd, J= 1.8, 8.7 Hz, 1H), 6.91 (d, J= 8.2 Hz, 1H), 3.96 (s, 3H), 3.94 (s, 3H)
MS(ESI) Found: 349 [M+H]+
3-(3,4-ジメトキシフェニル)プロパノイルクロリド(100mg,0.42mmol)と2-アミノ-6-クロロベンゾチアゾール(78mg,0.42mmol)を基質に用いて上記と同様に反応を行い、2-(3-(3,4-ジメトキシフェニル)プロパンアミド)-6-クロロベンゾチアゾールを113mg、収率72%で得た。
1H NMR (CDCl3): δ9.41(s, 1H), 7.79 (d, J= 2.9 Hz, 1H), 7.62 (d, J= 11.7 Hz, 1H), 7.37 (dd, J= 2.6, 11.4 Hz, 1H), 6.80-6.67 (m, 3H), 3.85 (s, 3H), 3.82 (s, 3H), 3.03 (t, J= 9.9 Hz, 2H), 2.77 (t, J= 9.9 Hz, 2H)
MS(ESI) Found: 399 [M+H]+
4-(3,4-ジメトキシフェニル)ブタノイルクロリド(100mg,0.41mmol)と2-アミノ-6-クロロベンゾチアゾール(76mg,0.41mmol)を基質に用いて上記と同様に反応を行い、2-(4-(3,4-ジメトキシフェニル)ブタンアミド)-6-クロロベンゾチアゾールを121mg、収率75%で得た。
1H NMR (CDCl3): δ9.15(s, 1H), 7.79 (d, J= 2.9 Hz, 1H), 7.64 (d, J= 11.3 Hz, 1H), 7.39 (dd, J= 2.6, 11.4 Hz, 1H), 6.80-6.68 (m, 3H), 3.87 (s, 3H), 3.84 (s, 3H), 2.67 (t, J= 9.9 Hz, 2H), 2.48 (t, J= 9.9 Hz, 2H), 2.09 (m, 2H)
MS(ESI) Found: 413 [M+H]+
5-(3,4-ジメトキシフェニル)ペンタノイルクロリド(30mg,0.13mmol)と2-アミノ-6-クロロベンゾチアゾール(23mg,0.13mmol)を基質に用いて上記と同様に反応を行い、2-(5-(3,4-ジメトキシフェニル)ペンタンアミド)-6-クロロベンゾチアゾールを39mg、収率75%で得た。
1H NMR (CDCl3): δ8.91 (s, 1H), 7.79 (d, J= 8.7 Hz, 1H), 7.66 (d, J= 8.7 Hz, 1H), 7.49 (dd, J= 2.3, 8.7 Hz, 1H), 6.79 (d, J= 7.8 Hz, 1H), 6.71 (d, J= 7.8 Hz, 1H) 6.70 (s, 1H), 3.87(s, 3H), 3.86 (s, 3H), 2.62 (t, J= 7.4 Hz, 2H), 2.52 (t, J= 7.3 Hz, 2H), 1.80 (m, 2H), 1.72 (m, 2H)
MS(ESI) Found: 405 [M+H]+
3,4-ジメトキシベンゾイルイルクロリド(100mg,0.5mmol)と2-アミノ-6-ニトロベンゾチアゾール(105mg,0.57mmol)を基質に用いて上記と同様に反応を行い、2-(3,4-ジメトキシベンズアミド)-6-ニトロベンゾチアゾールを100mg、収率56%で得た。
1H NMR (CDCl3): δ10.15(s, 1H), 8.80 (d, J= 2.3 Hz, 1H), 8.31 (dd, J= 2.3, 9.2 Hz, 1H), 7.73 (d, J= 9.2 Hz, 1H), 7.63-7.47 (m, 2H), 6.95 (d, J= 8.7 Hz, 1H), 3.98 (s, 3H), 3.97 (s, 3H)
MS(ESI) Found: 360 [M+H]+
2-(3,4-ジメトキシフェニル)アセチルクロリド(100mg,0.51mmol)と2-アミノ-6-クロロベンゾチアゾール(94mg,0.51mmol)を基質に用いて上記と同様に反応を行い、2-(2-(3,4-ジメトキシフェニル)アセトアミド)-6-クロロベンゾチアゾールを153mg、収率83%で得た。
1H NMR (CDCl3): δ8.91(s, 1H), 8.75 (s, 1H), 8.31 (dd, J= 12.1 Hz, 1H), 7.77 (d, J= 11.7 Hz, 1H), 7.00-6.70 (m, 3H), 3.92 (s, 3H), 3.90 (s, 3H), 3.86 (s, 2H)
MS(ESI) Found: 396 [M+H]+
3-(3,4-ジメトキシフェニル)プロパノイルクロリド(100mg,0.5mmol)と2-アミノ-6-ニトロベンゾチアゾール(105mg,0.57mmol)を基質に用いて上記と同様に反応を行い、2-(3-(3,4-ジメトキシフェニル)プロパンアミド)-6-ニトロベンゾチアゾールを138mg、収率71%で得た。
1H NMR (CDCl3): δ9.29(s, 1H), 8.75 (d, J= 1.8 Hz, 1H), 8.31 (dd, J= 2.3, 9.2 Hz, 1H), 7.77 (d, J= 9.2 Hz, 1H), 6.80 (d, J= 8.7 Hz, 1H), 6.75 (d, J= 8.7 Hz, 1H), 6.74 (s, 1H), 3.85 (s, 3H), 3.84 (s, 3H), 3.06 (t, J= 7.3 Hz, 2H), 2.83 (t, J= 7.3 Hz, 2H)
MS(ESI) Found: 388 [M+H]+
4-(3,4-ジメトキシフェニル)ブタノイルクロリド(55mg,0.25mmol)と2-アミノ-6-ニトロベンゾチアゾール(50mg,0.25mmol)を基質に用いて上記と同様に反応を行い、2-(4-(3,4-ジメトキシフェニル)ブタンアミド)-6-ニトロベンゾチアゾールを65mg、収率66%で得た。
1H NMR (CDCl3): δ8.75 (d, J= 2.3 Hz, 1H), 8.29 (dd, J= 2.3, 8.7 Hz, 1H), 7.70 (d, J= 8.7 Hz, 1H), 6.75 (d, J= 8.7 Hz, 1H), 6.67 (s, 1H), 6.66 (d, J= 8.7 Hz, 1H), 3.83 (s, 3H), 3.83 (s, 3H), 2.66 (t, J= 7.4 Hz, 2H), 2.54 (t, J= 7.3 Hz, 2H), 2.11 (m, 2H)
MS(ESI) Found: 402 [M+H]+
5-(3,4-ジメトキシフェニル)ペンタノイルクロリド(30mg,0.13mmol)と2-アミノ-6-ニトロベンゾチアゾール(25mg,0.13mmol)を基質に用いて上記と同様に反応を行い、2-(5-(3,4-ジメトキシフェニル)ペンタンアミド)-6-ニトロベンゾチアゾールを38mg、収率70%で得た。
1H NMR (CDCl3): δ8.94 (s, 1H), 8.75 (d, J= 2.3 Hz, 1H), 8.32 (dd, J= 2.3, 9.2 Hz, 1H), 7.81 (d, J= 9.2 Hz, 1H), 6.80 (d, J= 7.8 Hz, 1H), 6.72 (d, J= 7.8 Hz, 1H) 6.71 (s, 1H), 3.88 (s, 3H), 3.86 (s, 3H), 2.63 (t, J= 7.4 Hz, 2H), 2.56 (t, J= 7.3 Hz, 2H), 1.82 (m, 2H), 1.73 (m, 2H)
MS(ESI) Found: 416 [M+H]+
2-(3,4-ジメトキシフェニル)アセチルクロリド(100mg,0.51mmol)と2-アミノ-6-フルオロベンゾチアゾール(86mg,0.51mmol)を基質に用いて上記と同様に反応を行い、2-(2-(3,4-ジメトキシフェニル)アセトアミド)-6-フルオロベンゾチアゾールを157mg、収率89%で得た。
1H NMR (CDCl3): δ9.14(s, 1H), 7.64 (dd, J= 6.2, 12.1 Hz, 1H), 7.50 (dd, J= 3.6, 11.0 Hz, 1H), 7.14 (ddt, J= 3.7, 12.1 Hz, 1H), 6.90-6.78 (m, 3H), 3.90 (s, 3H), 3.87 (s, 3H), 3.80 (s, 2H)
MS(ESI) Found: 369 [M+H]+
2-アミノ-6-ブロモベンゾチアゾール(500mg,2.18mmol)、3-(3,4-ジメトキシフェニル)プロピオン酸(505mg,2.40mmol)、O-(6-クロロベンゾトリアゾール-1-イル)-N,N,N’,N’-テトラメチルウロニウムヘキサフルオロホスファート(1.09g,2.63mmol)、およびN,N’-ジイソプロピルエチルアミン(419μl,2.41mmol)のN,N’-ジメチルホルムアミド(5ml)溶液を室温にて終夜攪拌した。反応終了後、反応溶液を酢酸エチルにて希釈し、飽和炭酸水素ナトリウム水溶液、蒸留水、飽和塩化ナトリウム水溶液にて洗浄した。硫酸ナトリウムで乾燥した後に溶媒を留去した。残渣にエタノールを加えて還流し、再結晶を行い、2-(3-(3,4-ジメトキシフェニル)プロパンアミド)-6-ブロモベンゾチアゾールを320mg、収率35%で得た。
1H NMR (DMSO-d6): δ12.45 (s, 1H), 8.25 (d, J = 1.8 Hz, 1H), 7.66 (d, J = 8.4 Hz, 1H), 7.56 (dd, J = 1.8, 8.4 Hz, 1H), 6.87-6.83 (m, 2H), 6.77-6.73 (m, 1H), 3.71 (s, 3H), 3.70 (s, 3H), 2.88 (t, J = 7.0 Hz, 2H), 2.79 (t, J = 7.0 Hz, 2H)
2-アミノ-6-クロロベンゾチアゾール(55mg,0.298mmol)および3-(3,4-ジメトキシフェニル)プロピオン酸(80mg,0.357mmol)を基質に用いてSO087と同様に反応を行い,N-(6-クロロベンゾチアゾール-2-イル)-3-(4-エトキシ-3-メトキシフェニル)プロパンアミドを40mg、収率34%で得た。
1H NMR (DMSO-d6): δ12.44 (s, 1H), 8.11 (d, J = 2.2 Hz, 1H), 7.71 (d, J = 8.8 Hz, 1H), 7.44 (dd, J = 2.2, 8.8 Hz, 1H), 6.90-6.82 (m, 2H), 6.72 (dd, J = 1.8, 7.0 Hz), 3.94 (q, J = 7.0 Hz, 2H), 3.72 (s, 3H), 2.91-2.85 (m, 2H), 2.82-2.75 (m, 2H), 1.28 (t, J = 7.0 Hz, 3H)
2-アミノ-6-ヒドロキシベンゾチアゾール(400mg,2.41mmol)のN,N’-ジメチルホルムアミド(7ml)溶液をアルゴン雰囲気下、氷冷攪拌中、水素化ナトリウム(60%)(106mg,2.65mmol)を加えて30分攪拌後、4-ブロモ酪酸エチル(521μl,3.62mmol)を加えて室温にて終夜攪拌した。反応終了後、反応溶液を酢酸エチルにて希釈し、飽和塩化アンモニウム水溶液、飽和塩化ナトリウム水溶液にて洗浄した。硫酸ナトリウムで乾燥した後に溶媒を留去し、エチル 4-((2-アミノベンゾチアゾール-6-イル)オキシ)ブタノエートを372mg、収率55%で得た。
1H NMR (DMSO-d6): δ12.20 (s, 1H), 7.60 (d, J = 8.8 Hz, 1H), 7.55 (d, J = 2.6 Hz, 1H), 7.01 (dd, J = 2.6, 8.8 Hz, 1H), 6.86-6.83 (m, 2H), 6.75 (dd, J = 1.8, 8.1 Hz), 4.03 (t, J = 6.2 Hz, 2H), 3.71 (s, 3H), 3.70 (s, 3H), 3.56-3.53 (m, 4H), 3.46-3.42 (m, 4H), 2.87 (t, J = 7.0 Hz), 2H), 2.75 (t, J = 7.0 Hz, 2H), 2.51-2.46 (m, 2H), 1.96 (t, J = 7.0 Hz, 2H)
4-((2-(3-(3,4-ジメトキシフェニル)プロパンアミド)ベンゾチアゾール-6-イル)オキシ)ブタン酸(80mg,0.180mmol)および1-メチルピペラジン(21.8μl,0.198mmol)を基質に用いてSO094と同様に反応を行い、3-(3,4-ジメトキシフェニル)-N-(6-(4-(4-メチルピペラジン-1-イル)-4-オキソブトキシ)ベンゾチアゾール-2-イル)プロパンアミドを39mg、収率41%で得た。
1H NMR (DMSO-d6): δ12.20 (br s, 1H), 7.54 (d, J = 8.8 Hz, 1H), 7.48 (d, J = 2.6Hz, 1H), 6.96 (dd, J = 2.6, 8.8 Hz, 1H), 6.86-6.82 (m, 2H), 6.73 (dd, J = 1.8, 8.1 Hz, 1H), 4.01 (t, J = 6.2 Hz, 2H), 3.71 (s, 3H), 3.69 (s, 3H), 3.45-3.41 (m, 4H), 2.86 (t, J = 7.7 Hz, 2H), 2.70 (t, J = 7.7 Hz, 2H), 2.50-2.45 (m, 2H), 2.29-2.20 (m, 4H), 2.15 (s, 3H), 1,94 (t, J = 7.0 Hz, 2H)
非特許文献
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(上記文献はいずれも引用により本明細書に含まれる。)
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Claims (15)
- 多能性幹細胞の心筋分化誘導法であって、以下の工程を含む方法:
(1)多能性幹細胞を1以上のWNTシグナル活性化剤を含む培地中で培養する工程、および;
(2)工程(1)で得られた細胞を1以上のWNTシグナル阻害剤を含む培地中で培養する工程。 - 1以上のWNTシグナル阻害剤が、以下の式(I)の化合物またはその塩を含む、請求項1に記載の方法:
式(I):
R1-R5は、各々独立して、水素原子;ハロゲン原子;水酸基;炭素数1~5の直鎖又は分岐アルコキシ基;非置換又はハロゲン原子で置換された炭素数1~5の直鎖又は分岐アルキル基;又は基-NR12R13(R12及びR13は、各々独立して、水素原子、酸素原子、又は非置換又はハロゲン原子で置換された炭素数1~5の直鎖または分岐アルキル基である)である、ここでR1-R5のうち隣接する2つが一緒になって-O-CH2-O-または-O-(CH2)2-O-を形成していてもよい、
R6-R9は、各々独立して、水素原子;ハロゲン原子;水酸基;炭素数1~5の直鎖又は分岐アルコキシ基;基-C(O)Aで置換された炭素数1~5の直鎖又は分岐アルコキシ基(Aは、非置換又は炭素数1~5の直鎖または分岐アルキル基で置換された飽和または不飽和5または6員環であり、該環は窒素原子、酸素原子、及び硫黄原子から独立に選択される1または2個の原子を含んでいてもよい);非置換又はハロゲン原子で置換された炭素数1~5の直鎖又は分岐アルキル基;又は基-NR12R13(R12及びR13は、各々独立して、水素原子、酸素原子、又は非置換又はハロゲン原子で置換された炭素数1~5の直鎖または分岐アルキル基である)である、ここでR6-R9のうち隣接する2つが一緒になって-O-CH2-O-または-O-(CH2)2-O-を形成していてもよい、
R10-R11は、各々独立して、水素原子;又は炭素数1~5の直鎖又は分岐アルキル基である、
Xは、-CR14(R14は、水素原子、ハロゲン原子、水酸基、炭素数1~5の直鎖又は分岐アルコキシ基、又は非置換又はハロゲン原子で置換された炭素数1~5の直鎖又は分岐アルキル基である);酸素原子;硫黄原子;セレン原子;又は基-NR15(R15は、水素原子、炭素数1~5の直鎖又は分岐アルキル基、又は炭素数1~5の直鎖又は分岐アシル基である)である、および
nは、0から6の整数である]。 - 1以上のWNTシグナル阻害剤が、XAV939およびIWP-2からなる群から選択されるWNTシグナル阻害剤を含む、請求項1~4のいずれかに記載の方法。
- 1以上のWNTシグナル活性化剤が、GSK3β阻害剤を含む、請求項1~5のいずれかに記載の方法。
- 1以上のWNTシグナル活性化剤が、BIOおよびCHIR99021からなる群から選択されるWNTシグナル活性化剤を含む、請求項6記載の方法。
- 工程(1)および(2)における培地が血清を含まない、請求項1~7のいずれかに記載の方法。
- 工程(1)および(2)における培地がサイトカインを含まない、請求項1~8のいずれかに記載の方法。
- 工程(1)および(2)における培地がアルブミンを含む、請求項1~9のいずれかに記載の方法。
- 工程(1)および(2)における培地がアルブミン以外のタンパク質を含まない、請求項10に記載の方法。
- 工程(1)および(2)が異種成分非存在下で行われる、請求項1~11のいずれかに記載の方法。
- 多能性幹細胞がサルまたはヒト多能性幹細胞である、請求項1~12のいずれかに記載の方法。
- 心筋細胞の製造方法である、請求項1~13のいずれかに記載の方法。
- 1以上のWNTシグナル活性化剤および/または1以上のWNTシグナル阻害剤を含む心筋分化促進用キットであって、請求項1~14のいずれかに記載の方法に使用されるキット。
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