Peptide Conjugated Hydrogel Substrate for the Maintenance and Expansion of Human Pluripotent Stem Cells

Abstract

Compositions useful for propagation of pluripotent stem cells are provided. The compositions comprise a polysaccharide hydrogel linked to a peptide fragment of the extracellular domain of epithelial cadherin. Methods of making the composition, and culturing pluripotent stem cells also are provided.

We claim: 
     
         1 . A composition comprising at least one pluripotent stem cell encapsulated within a three-dimensional biocompatible hydrogel linked to a polypeptide comprising a cell-binding sequence of an epithelial cadherin extracellular domain. 
     
     
         2 . The composition of  claim 1 , wherein the hydrogel is a synthetic or naturally derived polysaccharide hydrogel. 
     
     
         3 . The composition of  claim 2 , wherein the polysaccharide is a carboxylated polysaccharide. 
     
     
         4 . The composition of  claim 2 , wherein the polysaccharide hydrogel is alginate. 
     
     
         5 . The composition of  claim 1 , wherein the pluripotent stem cell is an induced pluripotent stem cell or an embryonic stem cell and/or is a human pluripotent stem cell. 
     
     
         6 . The composition of  claim 1 , wherein the polypeptide comprises:
 the sequence LFSHAVSSNG (SEQ ID NO: 2), SHAVSS (SEQ ID NO: 3), QGADTPPVGV (SEQ ID NO: 4), and/or ADTPPV (SEQ ID NO: 5);   a sequence comprising the amino acid sequence HAV and/or ADT;   a sequence having at least five consecutive amino acids of SHAVSS (SEQ ID NO: 3) and/or ADTPPV (SEQ ID NO: 5); or   a sequence having at least 80% sequence identity with SHAVSS (SEQ ID NO: 3) and ADTPPV (SEQ ID NO: 5).   
     
     
         7 . The composition of  claim 1 , wherein the polypeptide is chosen from: 
       
         
           
                 
                 
               
                     
                   (HAV10, SEQ ID NO: 2) 
                 
                     
                   LFSHAVSSNG; 
                 
                     
                     
                 
                     
                   (HAV6, SEQ ID NO: 3) 
                 
                     
                   SHAVSS; 
                 
                     
                     
                 
                     
                   (ADT10, SEQ ID NO: 4) 
                 
                     
                   QGADTPPVGV, 
                 
                     
                   and 
                 
                     
                     
                 
                     
                   (ADT6, SEQ ID NO: 5) 
                 
                     
                   ADTPPV. 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         8 . The composition of  claim 1 , wherein the polypeptide is linked directly to the hydrogel by an amide bond. 
     
     
         9 . The composition of  claim 1 , wherein the hydrogel comprises pendant free carboxyl groups, wherein 99% or less, 95% or less, 90% or less, 80% or less, 75% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 25% or less, 20% or less, 10% or less, 5% or less, or 1% or less of monomers of the hydrogel have the peptide linked thereto. 
     
     
         10 . The composition of  claim 1 , wherein the composition is anionic and is in the form of capsules formed with a divalent cation. 
     
     
         11 . A method of preparing an encapsulated pluripotent stem cell, comprising:
 mixing one or more pluripotent stem cells with a hydrogel monomer to form a first mixture, wherein the hydrogel monomer is linked to a polypeptide comprising a cell-binding sequence of an epithelial cadherin extracellular domain; and   adding a divalent cation to the mixture or adding the mixture to a divalent cation, thereby encapsulating the one or more pluripotent stem cells in a three-dimensional hydrogel.   
     
     
         12 . The method of  claim 11 , wherein the divalent cation is calcium chloride or barium chloride. 
     
     
         13 . The method of  claim 11 , wherein the pluripotent stem cells are induced pluripotent stem cells. 
     
     
         14 . The method of  claim 13 , wherein the pluripotent stem cells are human. 
     
     
         15 . The method of  claim 11 , wherein the polypeptide is linked directly to the hydrogel monomer by an amide bond. 
     
     
         16 . The method of  claim 11 , wherein the hydrogel monomer comprises a polysaccharide. 
     
     
         17 . The method of  claim 11 , wherein the hydrogel monomer is a synthetic or naturally derived monomer. 
     
     
         18 . The method of  claim 11 , wherein the hydrogel monomer comprises alginate. 
     
     
         19 . The method of  claim 11 , wherein the polypeptide comprising a cell-binding sequence of an epithelial cadherin extracellular domain comprises:
 the sequence LFSHAVSSNG (SEQ ID NO: 2), SHAVSS (SEQ ID NO: 3), QGADTPPVGV (SEQ ID NO: 4), and/or ADTPPV (SEQ ID NO: 5);   a sequence comprising the amino acid sequence HAV and/or ADT;   a sequence having at least five consecutive amino acids of SHAVSS (SEQ ID NO: 3) and/or ADTPPV (SEQ ID NO: 5); or   a sequence having at least 80% sequence identity with SHAVSS (SEQ ID NO: 3) and ADTPPV (SEQ ID NO: 5).   
     
     
         20 . The method of  claim 11 , wherein the polypeptide is chosen from: 
       
         
           
                 
                 
               
                     
                   (HAV10, SEQ ID NO: 2) 
                 
                     
                   LFSHAVSSNG; 
                 
                     
                     
                 
                     
                   (HAV6, SEQ ID NO: 3) 
                 
                     
                   SHAVSS; 
                 
                     
                     
                 
                     
                   (ADT10, SEQ ID NO: 4) 
                 
                     
                   QGADTPPVGV, 
                 
                     
                   and 
                 
                     
                     
                 
                     
                   (ADT6, SEQ ID NO: 5) 
                 
                     
                   ADTPPV.