Method for culturing pluripotent stem cell, and polypeptide to be used therefor

Abstract

A polypeptide including: (1) a first region containing at least one selected from the group consisting of an amino acid sequence represented by CSYYQSC (SEQ ID NO:1) and an amino acid sequence represented by RGD; and (2) a second region containing (2-i) an amino acid sequence represented by PRPSLAKKQRFRHRNRKGYRSQRGHSRGRNQN (SEQ ID NO:2), (2-ii) an amino acid sequence having an identity of not less than 50% to the amino acid sequence represented by SEQ ID NO:2 and having an adsorption ability to a cultivation container, or (2-iii) an amino acid sequence that is the amino acid sequence represented by SEQ ID NO:2 in which from 1 to 30 amino acid residues are added, substituted, or deleted, and has an adsorption ability to a cultivation container, in which the polypeptide includes from 40 to 450 amino acid residues.

1 . A polypeptide consisting of from 40 to 450 amino acid residues and comprising:
 (1) a first region comprising at least one selected from the group consisting of an amino acid sequence represented by CSYYQSC (SEQ ID NO:1) and an amino acid sequence represented by RGD; and   (2) a second region comprising
 (2-i) an amino acid sequence represented by PRPSLAKKQRFRHRNRKGYRSQRGHSRGRNQN (SEQ ID NO:2), 
 (2-ii) an amino acid sequence having an identity of not less than 50% to an amino acid sequence represented by SEQ ID NO:2 and having an adsorption ability to a cultivation container, or 
 (2-iii) an amino acid sequence that is the amino acid sequence represented by SEQ ID NO:2, in which from 1 to 30 amino acid residues are added, substituted, or deleted, and that has an adsorption ability to a cultivation container. 
   
     
     
         2 . The polypeptide according to  claim 1 , wherein a GRAVY value is from −2.0 to −0.95. 
     
     
         3 . The polypeptide according to  claim 1 , wherein the first region comprises both of the amino acid sequence represented by CSYYQSC (SEQ ID NO:1) and the amino acid sequence represented by RGD. 
     
     
         4 . The polypeptide according to  claim 1 , wherein the polypeptide consists of from 40 to 400 amino acid residues. 
     
     
         5 . The polypeptide according to  claim 1 , further comprising a third region consisting of any one amino acid sequence of the following (3-i) to (3-iii):
 (3-i) an amino acid sequence consisting of the 56th to 341st amino acid residues in an amino acid sequence represented by SEQ ID NO:3, or a partial amino acid sequence thereof;   (3-ii) an amino acid sequence having an identity of not less than 50% to the (3-i) amino acid sequence or partial amino acid sequence thereof; or   (3-iii) an amino acid sequence that is the (3-i) amino acid sequence or partial amino acid sequence thereof in which from 1 to 30 amino acid residues are added, substituted, or deleted.   
     
     
         6 . The polypeptide according to  claim 1 , further comprising a third region consisting of any one amino acid sequence of the following (3a-i) to (3a-iii):
 (3a-i) an amino acid sequence consisting of the 132th to 341st amino acid residues in an amino acid sequence represented by SEQ ID NO:3, or a partial amino acid sequence thereof;   (3a-ii) an amino acid sequence having an identity of not less than 50% to the (3a-i) amino acid sequence or partial amino acid sequence thereof; or   (3a-iii) an amino acid sequence that is the (3a-i) amino acid sequence or partial amino acid sequence thereof in which from 1 to 30 amino acid residues are added, substituted, or deleted.   
     
     
         7 . The polypeptide according to  claim 1 , further comprising a third region consisting of any one amino acid sequence of the following (3b-i) to (3b-iii):
 (3b-i) an amino acid sequence consisting of the 269th to 341st amino acid residues in an amino acid sequence represented by SEQ ID NO:3, or a partial amino acid sequence thereof;   (3b-ii) an amino acid sequence having an identity of not less than 50% to the (3b-i) amino acid sequence or partial amino acid sequence thereof; or   (3b-iii) an amino acid sequence that is the (3b-i) amino acid sequence or partial amino acid sequence thereof in which from 1 to 30 amino acid residues are added, substituted, or deleted.   
     
     
         8 . The polypeptide according to  claim 1 , further comprising a fourth region consisting of any one amino acid sequence of the following (4-i) to (4-iii):
 (4-i) an amino acid sequence consisting of the 374th to 459th amino acid residues in an amino acid sequence represented by SEQ ID NO:3, or a partial amino acid sequence thereof;   (4-ii) an amino acid sequence having an identity of not less than 50% to the (4-i) amino acid sequence or partial amino acid sequence thereof; or   (4-iii) an amino acid sequence that is the (4-i) amino acid sequence or partial amino acid sequence thereof in which from 1 to 30 amino acid residues are added, substituted, or deleted.   
     
     
         9 . The polypeptide according to  claim 1 , wherein the (2-ii) amino acid sequence has an identity of not less than 80% to the amino acid sequence represented by SEQ ID NO:2. 
     
     
         10 . The polypeptide according to  claim 1 , wherein the (2-iii) amino acid sequence is the amino acid sequence represented by SEQ ID NO:2 in which from 1 to 15 amino acid residues are added, substituted, or deleted. 
     
     
         11 . A polypeptide consisting of from 80 to 450 amino acid residues and comprising:
 (1) a first region consisting of an amino acid sequence consisting of the 25th to 47th amino acid residues in an amino acid sequence represented by SEQ ID NO:3;   (2) a second region consisting of an amino acid sequence consisting of the 342nd to 373rd amino acid residues in the amino acid sequence represented by SEQ ID NO:3; and   at least one selected from the group consisting of the following third and fourth regions:   (3) the third region consisting of an amino acid sequence consisting of the 269th to 341st amino acid residues in the amino acid sequence represented by SEQ ID NO:3, or a partial amino acid sequence thereof; and   (4) the fourth region consisting of an amino acid sequence consisting of the 374th to 459th amino acid residues in the amino acid sequence represented by SEQ ID NO:3, or a partial amino acid sequence thereof.   
     
     
         12 . A polypeptide consisting of from 100 to 450 amino acid residues and comprising:
 (1) a first region consisting of an amino acid sequence consisting of the 1st to 55th amino acid residues in an amino acid sequence represented by SEQ ID NO:3;   (2) a second region consisting of an amino acid sequence consisting of the 342nd to 373rd amino acid residues in the amino acid sequence represented by SEQ ID NO:3; and   at least one selected from the group consisting of the following third and fourth regions:   (3) the third region consisting of an amino acid sequence consisting of the 269th to 341st amino acid residues in the amino acid sequence represented by SEQ ID NO:3, or a partial amino acid sequence thereof; and   (4) the fourth region consisting of an amino acid sequence consisting of the 374th to 459th amino acid residues in the amino acid sequence represented by SEQ ID NO:3, or a partial amino acid sequence thereof.   
     
     
         13 . The polypeptide according to  claim 11 , wherein the polypeptide has a GRAVY value of from −2.0 to −0.95. 
     
     
         14 . The polypeptide according to  claim 1 , the number of amino acid residues of which being 250 or less. 
     
     
         15 . The polypeptide according to  claim 5 , wherein the polypeptide comprises the third region, and the third region comprises an amino acid residue other than a cysteine residue at a position corresponding to a cysteine residue in the amino acid sequence represented by SEQ ID NO:3. 
     
     
         16 . The polypeptide according to  claim 5 , wherein the polypeptide comprises the third region, and the third region comprises a serine residue, an alanine residue, or a glycine residue at a position corresponding to a cysteine residue in the amino acid sequence represented by SEQ ID NO:3. 
     
     
         17 . The polypeptide according to  claim 1 , wherein the first region is located at an N-terminal side of the second region. 
     
     
         18 . The polypeptide according to  claim 1 , wherein two cysteine residues in the amino acid sequence represented by SEQ ID NO:1 are cross-linked to each other. 
     
     
         19 . A polypeptide comprising an amino acid sequence represented by any of SEQ ID NO:4 to SEQ ID NO:23, SEQ ID NO:38, or SEQ ID NO:39. 
     
     
         20 . A method of culturing a pluripotent stem cell, comprising:
 applying the polypeptide according to  claim 1  to a cell culture surface of a support, to obtain a polypeptide-coated culture surface; and   seeding a pluripotent stem cell on the polypeptide-coated culture surface and culturing the pluripotent stem cell.   
     
     
         21 . The method of culturing a pluripotent stem cell according to  claim 20 , wherein the pluripotent stem cell is at least one selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, somatic stem cells, fertilized egg inner cell mass cells, and early embryonic cells. 
     
     
         22 . The method of culturing a pluripotent stem cell according to  claim 20 , wherein the pluripotent stem cell is cultured in the absence of a component derived from a heterologous animal and a component derived from serum.