Cell differntiation inhibiting agent, cell culture method using the same, culture medium, and cultured cell line

Abstract

The object of the present invention is to provide a differentiation inhibiting agent which allows culture of a stem cell or an embryonic stem cell in an undifferentiated state without use of any feeder cell, a method for culturing using the same, a cell culture liquid using the same, and a cell prepared by culturing using this differentiation inhibiting agent. The present invention provides a differentiation inhibiting agent which comprises a low molecular weight compound, especially a tetrahydroisoquinoline derivative, as an active ingredient; a method for safely culturing a stem cell in large scale in undifferentiated state in the absence of feeder cell which comprises culturing a stem cell by using a tetrahydroisoquinoline derivative; a culture liquid for stem cells comprising a tetrahydroisoquinoline derivative; and a cell which is obtained by culture using a tetrahydroisoquinoline derivative as a differentiation inhibiting agent.

1 . A stem cell differentiation inhibiting agent which comprises a low molecular weight compound or a salt thereof as an active ingredient.  
     
     
         2 . The stem cell differentiation inhibiting agent according to  claim 1 , wherein the low molecular weight compound is a compound represented by the formula (1):  
       
         
           
           
               
               
           
         
       
       wherein, R 1 , R 2 , R 3  and R 4  may be the same or different, each representing an electron-withdrawing group, an electron-donating group or a hydrogen atom; ring A represents 5- to 8-member ring containing at least 1 hetero atom in the ring; X represents an alkylene group of which atomic number of a main chain ranges from 0 to 10; the alkylene group having the atomic number of 0 represents a single bond; one or more ethylene groups constituting the alkylene group may be replaced by —C═C— group, and/or —N═N— group, and/or —CONH— group, and further the group may have a double bond bonding to the ring A; the alkylene group has 1 or more electron-withdrawing groups, electron-donating groups or hydrogen atoms as a substituent; G represents an aromatic group which may have an electron-withdrawing group, an electron-donating group or a hydrogen atom; the ring A may have 1 or more electron-withdrawing group and/or an electron-donating group as a substituent other than an —XG group.  
     
     
         3 . The stem cell differentiation inhibiting agent according to  claim 2 , wherein the ring A is a 5- or 6-member ring containing at least 1 hetero atom selected from a group consisting of a nitrogen atom, an oxygen atom and a sulfur atom, in the ring.  
     
     
         4 . The stem cell differentiation inhibiting agent according to  claim 2 , wherein the ring A is a 5- or 6-member ring containing 1 nitrogen atom in the ring.  
     
     
         5 . The stem cell differentiation inhibiting agent according to  claim 2 , wherein the alkylene group represented by X includes 1 or more groups and/or atoms selected from the group consisting of an alkyl group, an acyl group, an alkoxy group, a nitro group, a hydroxycarbonyl group, an alkoxycarbonyl group, an aminocarbonyl group, a nitrile group and a halogen atom as a substituent.  
     
     
         6 . The stem cell differentiation inhibiting agent according to  claim 2 , wherein the low molecular weight compound is a compound represented by the formula (2):  
       
         
           
           
               
               
           
         
       
       wherein R 1 , R 2 , R 3 , R 4 , Xand G are the same as those defined in  claim 2 , and R 5 , R 6 , R 7 , R 6  and R 9  may be the same or different, each representing an electron-withdrawing group, an electron-donating group or a hydrogen atom.  
     
     
         7 . The stem cell differentiation inhibiting agent according to  claim 2 , wherein the low molecular weight compound is a compound represented by the formula (3):  
       
         
           
           
               
               
           
         
       
       wherein R 1 , R 2 , R 3 , R 4 , X and G are the same as those defined in  claim 2;  R 5  and R 6  may be the same or different, each representing an electron-withdrawing group, an electron-donating group or a hydrogen atom.  
     
     
         8 . The stem cell differentiation inhibiting agent according to  claim 6 , wherein the low molecular weight compound is a compound represented by the formula (4):  
       
         
           
           
               
               
           
         
       
       wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8  and R 9  are the same as those defined in  claim 6;  R 10 , R 11 , R 12  and R 13  may be the same or different, each representing an electron-withdrawing group, an electron-donating group or a hydrogen atom; the double one-side broken line represents a single bond or a double bond; when the double one-side broken line represents a double bond, a wavy portion represents the presence of a geometric isomer; arrangement of these geometric isomers is not specially restricted, but may be any of an E isomer or a Z isomer each independently.  
     
     
         9 . The stem cell differentiation inhibiting agent according to  claim 8 , wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12  and R 13  are each independently groups or atoms selected from the group consisting of an alkyl group, an acyl group, an alkoxy group, a nitro group, a hydroxycarbonyl group, a nitrile group, an alkoxycarbonyl group, an aminocarbonyl group, a halogen atom and a hydrogen atom.  
     
     
         10 . The stem cell differentiation inhibiting agent according to  claim 6 , wherein the low molecular weight compound is a compound represented by the formula (5):  
       
         
           
           
               
               
           
         
       
       wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8  and R 9  are the same as those defined in  claim 6;  R 10 , R 11  and R 12  may be the same or different, each representing an electron-withdrawing group, an electron-donating group or a hydrogen atom.  
     
     
         11 . The stem cell differentiation inhibiting agent according to  claim 10 , wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11  and R 12  are each independently groups or atoms selected from the group consisting of an alkyl group, an acyl group, an alkoxy group, a nitro group, a hydroxycarbonyl group, a nitrile group, an alkoxycarbonyl group, an aminocarbonyl group, a halogen atom and a hydrogen atom.  
     
     
         12 . The stem cell differentiation inhibiting agent according to  claim 7 , wherein the low molecular weight compound is a compound represented by the formula (6):  
       
         
           
           
               
               
           
         
       
       wherein R 1 , R 2 , R 3 , R 4 , R 5  and R 6  are the same as those defined in  claim 7;  R 7 , R 8  and R 9  may be the same or different, each representing an electron-withdrawing group, an electron-donating group or a hydrogen atom.  
     
     
         13 . The stem cell differentiation inhibiting agent according to  claim 12 , wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8  and R 9  are each independently groups or atoms selected from the group consisting of an alkyl group, an alkoxy group, a hydroxyl group, a nitro group, a nitrile group, an acetoxy group, an acetoxy alkyl group, a cyclic alkylaminoalkyl group which may contain an oxygen atom, a dialkylaminoalkyl group, a dialkylaminovinyl group, a hydroxyalkylaminoalkyl group, an arylaminovinyl group and a hydrogen atom.  
     
     
         14 . The stem cell differentiation inhibiting agent according to  claim 8 , wherein R 1 , R 2 , R 3 , R 4 , R 8 , R 9  and R 10  are hydrogen atoms; R 5  is a hydrogen atom, a lower acyl group or a lower alkyl group; R 6  and R 7  are lower alkyl groups which may be the same or different; R 11  is a hydrogen atom, a halogen atom, a nitro group or a lower alkoxy group; R 12  is a hydrogen atom, a lower alkyl group, a lower acyl group or a lower alkoxy group; and R 13  is a hydroxycarbonyl group, a nitrile group, an aminocarbonyl group or a lower alkoxycarbonyl group.  
     
     
         15 . The stem cell differentiation inhibiting agent according to  claim 10 , wherein R 1 , R 2 , R 3 , R 4 , R 8  and R 9  are hydrogen atoms; R 5  is a hydrogen atom, a lower acyl group or a lower alkyl group; R 6  and R 7  are lower alkyl groups which may be the same or different; R 10  is a hydrogen atom or a lower alkyl group; R 11  is a hydrogen atom, a halogen atom, a nitro group, a lower alkoxy group or a lower alkoxycarbonyl group; and R 12  is a hydrogen atom, a lower alkyl group, a lower acyl group or a lower alkoxy group.  
     
     
         16 . The stem cell differentiation inhibiting agent according to  claim 12 , wherein R 1  and R 2  are hydrogen atoms; R 3  is a hydroxyl group or an acetoxy group; R 4  is an acetoxy alkyl group, a cyclic alkylaminoalkyl group which may contain an oxygen atom, a dialkylaminoalkyl group, a hydroxyalkylaminoalkyl group or a hydrogen atom; R 5  is a lower alkyl group or an arylaminovinyl group; R 6  is a nitro group; R 7 , R 3  and R 9  may be the same or different, each representing a lower alkyl group, a lower alkoxy group or a hydrogen atom.  
     
     
         17 . A compound represented by the formula (9), or a salt thereof:  
       
         
           
           
               
               
           
         
       
       wherein R 1 , R 2 , R 3  and R 4  may be the same or different, each representing a hydrogen atom, a lower alkyl group, a hydroxyl group, a lower alkoxy group or a halogen atom; R 5  is a hydrogen atom, a lower acyl group or a lower alkyl group; R 6  and R 7  may be the same or different, each representing a hydrogen atom, a lower alkyl group or a lower alkyl group which may form a ring structure; R 8  and R 9  are hydrogen atoms; R 10 , R 11  and R 12  may be the same or different, each representing a hydrogen atom, a halogen atom, a nitro group, a lower alkyl group, a lower acyl group or a lower alkoxy group; R 13  represents a hydroxycarbonyl group; a lower alkoxycarbonyl group, a secondary aminocarbonyl group or a tertiary aminocarbonyl group; the wavy lined portion represents the presence of the geometric isomer; arrangement of these geometric isomers is not specially restricted, but may be any of the E isomer or the Z isomer each independently.  
     
     
         18 . The compound represented by the formula (9), or a salt thereof:  
       
         
           
           
               
               
           
         
       
       wherein R 1 , R 2 , R 3  and R 4  may be the same or different, each representing a hydrogen atom, a lower alkyl group, a hydroxyl group, a lower alkoxy group or a halogen atom; R 5  is a hydrogen atom, a lower acyl group or a lower alkyl group; R 6  and R 7  may be the same or different, each representing a hydrogen atom, a lower alkyl group or a lower alkyl group which may form a ring structure; R 8  and R 9  are hydrogen atoms; R 10  and R 12  are a hydrogen atom, a halogen atom, a nitro group, a lower alkyl group, a lower acyl group or a lower alkoxy group; R 11  is a lower acyl group; R 13  represents a hydroxycarbonyl group, a lower alkoxycarbonyl group or an aminocarbonyl group; the wavy lined portion represents the presence of the geometric isomer; arrangement of these geometric isomers is not specially restricted, but may be any of the E isomer or the Z isomer each independently.  
     
     
         19 . The compound represented by the formula (9), or a salt thereof:  
       
         
           
           
               
               
           
         
       
       wherein R 1 , R 2 , R 3  and R 4  may be the same or different, each representing a hydrogen atom, a lower alkyl group, a hydroxyl group, a lower alkoxy group or a halogen group; R 5  is a lower acyl group or a lower alkyl group; R 6  and R 7  may be the same or different, each representing a hydrogen atom, a lower alkyl group or a lower alkyl group which may form a ring structure; R 8  and R 9  are hydrogen atoms; R 10 , R 11  and R 12  may be the same or different, each representing a hydrogen atom, a halogen atom, a nitro group, a lower alkyl group, a lower acyl group or a lower alkoxy group; R 13  represents a hydroxycarbonyl group, a lower alkoxycarbonyl group or an aminocarbonyl group; the wavy lined portion represents the presence of the geometric isomer; arrangement of these geometric isomers is not specially restricted, but may be any of the E isomer or the Z isomer each independently.  
     
     
         20 . The compound represented by the formula (9), or a salt thereof:  
       
         
           
           
               
               
           
         
       
       wherein R 1 , R 2 , R 3  and R 4  may be the same or different, each representing a hydrogen atom, a lower alkyl group, a hydroxyl group, a lower alkoxy group or a halogen atom; R 5  is a hydrogen atom, a lower acyl group or a lower alkyl group; R 6  and R 7  may be the same or different, each representing a lower alkyl group or a lower alkyl group which may form a ring structure; R 8  and R 9  are hydrogen atoms; R 10 , R 11  and R 12  may be the same or different, each representing a hydrogen atom, a halogen atom, a nitro group, a lower alkyl group, a lower acyl group or a lower alkoxy group; R 13  represents a nitrile group; the wavy lined portion represents the presence of the geometric isomer; arrangement of these geometric isomers is not specially restricted, but may be any of the E isomer or the Z isomer each independently.  
     
     
         21 . The compound represented by the formula (10), or a salt thereof:  
       
         
           
           
               
               
           
         
       
       wherein R 1 , R 2 , R 3  and R 4  may be the same or different, each representing a hydrogen atom, a lower alkyl group, a hydroxyl group, a lower alkoxy group or a halogen atom; R 5  is a hydrogen atom, a lower acyl group or a lower alkyl group; R 6  and R 7  may be the same or different, each representing a hydrogen atom, a lower alkyl group or a lower alkyl group which may form a ring structure; R 8  and R 9  are hydrogen atoms; R 10 , R 11  and R 12  may be the same or different, each representing a hydrogen atom, a halogen atom, a nitro group, a lower alkyl group, a lower acyl group or a lower alkoxy group; and R 13  represents a hydroxycarbonyl group, a lower alkoxycarbonyl group, an aminocarbonyl group or a nitrile group.  
     
     
         22 . The compound according to  claim 17  or a salt thereof, wherein R 1 , R 2 , R 3  and R 4  are hydrogen atoms; R 5  is a hydrogen atom, a lower acyl group or a lower alkyl group; R 6  and R 7  are lower alkyl groups which may be same or different from each other; R 8 , R 9  and R 10  are hydrogen atoms; R 11  and R 12  may be the same or different, each representing a hydrogen atom, a halogen atom, a nitro group, a lower alkyl group, a lower acyl group or a lower alkoxy group; and R 13  is a hydroxycarbonyl group, a lower alkoxycarbonyl group, a mono-lower alkylaminocarbonyl group, a di-lower alkylaminocarbonyl group or a phenyl aminocarbonyl group.  
     
     
         23 . The compound according to  claim 18  or a salt thereof, wherein R 1 , R 2 , R 3  and R 4  are hydrogen atoms; R 5  is a hydrogen atom, a lower acyl group or a lower alkyl group; R 6  and R 7  are lower alkyl groups which may be the same or different; R 8 , R 9  and R 10  are hydrogen atoms; R 11  is a lower acyl group; R 12  is a hydrogen atom, a halogen atom, a nitro group, a lower alkyl group, a lower acyl group or a lower alkoxy group; and R 13  is a hydroxycarbonyl group, a lower alkoxycarbonyl group, a carbamoyl group, a mono-lower alkylaminocarbonyl group, a di-lower alkylaminocarbonyl group or a phenyl aminocarbonyl group, or a salt thereof.  
     
     
         24 . The compound according to  claim 19  or a salt thereof, wherein R 1 , R 2 , R 3  and R 4  are hydrogen atoms; R 5  is a lower acyl group or a lower alkyl group; R 6  and R 7  are lower alkyl groups which may be the same or different; R 8 , R 9  and R 10  are hydrogen atoms; R 11  and R 12  may be the same or different, each representing a hydrogen atom, a halogen atom, a nitro group, a lower alkyl group, a lower acyl group or a lower alkoxy group; and R 13  is a hydroxycarbonyl group, a lower alkoxycarbonyl group, a carbamoyl group, a mono-lower alkylaminocarbonyl group, a di-lower alkylaminocarbonyl group or a phenyl aminocarbonyl group.  
     
     
         25 . The compound according to  claim 20  or a salt thereof, wherein R 1 , R 2 , R 3  and R 4  are hydrogen atoms; R 5  is a hydrogen atom, a lower acyl group or a lower alkyl group; R 6  and R 7  are lower alkyl groups which may be the same or different; R 8 , R 9  and R 10  are hydrogen atoms; R 11  and R 12  may be the same or different, each representing a hydrogen atom, a halogen atom, a nitro group, a lower alkyl group, a lower acyl group or a lower alkoxy group; and R 13  is a nitrile group.  
     
     
         26 . The compound according to  claim 21  or a salt thereof, wherein R 1 , R 2 , R 3  and R 4  are hydrogen atoms; R 5  is a hydrogen atom, a lower acyl group or a lower alkyl group; R 6  and R 7  are lower alkyl groups which may be the same or different; R 8 , R 9  and R 10  are hydrogen atoms; R 11  and R 12  may be the same or different, each representing a hydrogen atom, a halogen atom, a nitro group, a lower alkyl group, a lower acyl group or a lower alkoxy group; and R 13  is a hydroxycarbonyl group, a lower alkoxycarbonyl group, a carbamoyl group, a mono-lower alkylaminocarbonyl group, a di-lower alkylaminocarbonyl group, a phenyl aminocarbonyl group or a nitrile group.  
     
     
         27 . The compound according to  claim 17  or a salt thereof, which is 
 (4-acetyl-phenylazo)-(3,3-dimethyl-3,4-dihydro-2H-isoquinoline-1-ylidene)-ethyl acetate ester,    (4-acetyl-phenylazo)-(3,3-dimethyl-3,4-dihydro-2H-isoquinoline-1-ylidene)-acetic acid,    2-(4-acetyl-phenylazo)-2-(3,3-dimethyl-3,4-dihydro-2H-isoquinoline-1-ylidene)-N-methyl-acetamide,    2-(4-acetyl-phenylazo)-2-(3,3-dimethyl-3,4-dihydro-2H-isoquinoline-1-ylidene)-N,N-dimethyl-acetamide, or    2-(4-acetyl-phenylazo)-2-(3,3-dimethyl-3,4-dihydro-2H-isoquinoline-1-ylidene)-N-phenyl-acetamide.    
     
     
         28 . The compound according to  claim 18  or a salt thereof, which is 2-(3-acetyl-phenylazo)-2-(3,3-dimethyl-3,4-dihydro-2H-isoquinoline-1-ylidene)-acetamide.  
     
     
         29 . The compound according to  claim 19  or a salt thereof, which is 2-(4-acetyl-phenylazo)-2-(2,3,3-trimethyl-3,4-dihydro-2H-isoquinoline-1-ylidene)-acetamide, or 
 2-(2-acetyl-3,3-dimethyl-3,4-dihydro-2H-isoquinoline-1-ylidene)-2-(4-acetyl-phenylazo)-acetamide.    
     
     
         30 . The compound according to  claim 20  or a salt thereof, which is (4-acetyl-phenylazo)-(3,3-dimethyl-3,4-dihydro-2H-isoquinoline-1-ylidene)-acetonitrile.  
     
     
         31 . The compound according to  claim 21  or a salt thereof, which is 2-(4-acetyl-phenylazo)-2-(3,3-dimethyl-1,2,3,4-tetrahydro-isoquinoline-1-yl)-acetamide.  
     
     
         32 . A stem cell differentiation inhibiting agent which comprises the compound according to any of  claims 17  to  21  or a salt thereof as an active ingredient.  
     
     
         33 . The stem cell differentiation inhibiting agent according to  claim 1  wherein the low molecular weight compound is the compound having an action of keeping or increasing an activity of alkaline phosphatase.  
     
     
         34 . The stem cell differentiation inhibiting agent according to  claim 1  wherein the low molecular weight compound is a compound which does not activate STAT3 (signal transducer and activator of transcription 3).  
     
     
         35 . The stem cell differentiation inhibiting agent according to  claim 1  wherein the low molecular weight compound is a compound having an action of increasing an SSEA-1 antigen expression level.  
     
     
         36 . The stem cell differentiation inhibiting agent according to  claim 1  wherein the low molecular weight compound is a compound having an action of increasing an Nanog gene expression level.  
     
     
         37 . The stem cell differentiation inhibiting agent according to  claim 36  wherein the low molecular weight compound is a bicyclic compound having an action of increasing the Nanog gene expression level.  
     
     
         38 . The stem cell differentiation inhibiting agent according to  claim 37  wherein the low molecular weight compound is a bicyclic compound containing an azo group which has an action of increasing the Nanog gene expression level.  
     
     
         39 . A method for culturing a stem cell, wherein the stem cell is cultured in an undifferentiated state by using the stem cell differentiation inhibiting agent according to  claim 1 .  
     
     
         40 . A method for culturing an embryonic stem cell, wherein the embryonic stem cell is cultured in the undifferentiated state by using the stem cell differentiation inhibiting agent according to  claim 1 .  
     
     
         41 . A culture medium containing the stem cell differentiation inhibiting agent according to  claim 1  as an active ingredient.  
     
     
         42 . A culture medium containing the compound according to any of  claims 17  to  21 , or a salt thereof.  
     
     
         43 . The culture medium according to  claim 41 , wherein the concentration of the differentiation inhibiting agent, the compound, or the salt thereof ranges from 10 ng/mL to 100 μg/mL.  
     
     
         44 . A stem cell cultured in the undifferentiated state by using the differentiation inhibiting agent according to  claim 1 .  
     
     
         45 . A cell or a tissue which is obtained by differentiating a stem cell cultured in the undifferentiated state by using the differentiation inhibiting agent according to  claim 1 .  
     
     
         46 . The cell or the tissue according to  claim 44 , wherein the cell or the tissue is used for in vivo transplantation.  
     
     
         47 . A therapeutic method wherein the cell or/and the tissue according to  claim 44  is subjected to in vivo transplantation.  
     
     
         48 . A prodrug of the compound according to any of  claims 17  to  21  or a salt thereof.  
     
     
         49 . A pharmaceutical composition comprising the compound according to any of  claims 17  to  21  or a salt thereof or a prodrug thereof.