Methods for isolation of osteoclast precursor cells and inducing their differentiation into osteoclasts

Abstract

This invention relates to: a method for differentiating osteoclast precursor cells (preosteoclasts) into osteoclasts, which comprises culturing the preosteoclasts in the absence of accessory cells; a method for isolating preosteoclasts, which comprises culturing peripheral blood or joint fluid in the absence of cytokine for 1 to 3 weeks; an preosteoclasts, which is obtainable by the above method; a method for differentiating the preosteoclasts obtained by the above method into osteoclasts, which comprises culturing the preosteoclasts in the absence of accessory cell; an osteoclast, which is obtainable by the above method; a method for screening agents for treating metabolic bone diseases, which comprises using the preosteoclasts or the osteoclasts as described above; and agents for treating metabolic bone diseases, which is obtainable by the above screening method.

1 - 20 . (Canceled).  
     
     
         21 . A method for producing an osteoclast precursor cell, which comprises: 
 culturing a hematopoietic stem cell-derived cell obtained from peripheral blood or joint fluid in an essential medium for mammalian cells, optionally with added serum, in the absence of any additional cytokine(s) for at least 1 to 3 weeks to obtain an osteoclast precursor cell.    
     
     
         22 . The method of  claim 21 , wherein said essential medium for mammalian cells contains serum.  
     
     
         23 . The method of  claim 21 , wherein the hematopoietic stem cell-derived cell is obtained from the mononuclear fraction of peripheral blood.  
     
     
         24 . The method of  claim 21 , wherein said hematopoietic stem cell-derived cell is obtained from the cellular fraction of joint fluid.  
     
     
         25 . The method of  claim 21 , wherein said hematopoietic stem cell-derived cell is cultured at a temperature ranging from 35-37° C. in 5-7% CO 2 -containing air for 1-3 weeks.  
     
     
         26 . An osteoclast precursor cell, which is obtainable by the method of  claim 21 .  
     
     
         27 . A method for producing an osteoclast, comprising: 
 culturing an osteoclast precursor cell in a culture medium in the absence of accessory cells.    
     
     
         28 . The method of  claim 27 , wherein said culture medium comprises one or more compound(s) selected from the group consisting of IL-3, IL-7, GM-CSF, eotaxin, eotaxin-2, and eotaxin-3.  
     
     
         29 . The method of  claim 28 , wherein said culture medium comprises IL-3.  
     
     
         30 . The method of  claim 28 , wherein said culture medium comprises IL-7.  
     
     
         31 . The method of  claim 28 , wherein said culture medium comprises GM-CSF.  
     
     
         32 . The method of  claim 28 , wherein said culture medium comprises eotaxin.  
     
     
         33 . The method of  claim 28 , wherein said culture medium comprises eotaxin 2.  
     
     
         34 . The method of  claim 28 , wherein said culture medium comprises eotaxin-3.  
     
     
         35 . The method of  claim 28 , wherein said culture medium comprises a culture supernatant of nitrogen-stimulated peripheral blood mononuclear cells.  
     
     
         36 . The method of  claim 28 , wherein said culture supernatant comprises a supernatant of phytohemagglutinin-stimulated human peripheral blood mononuclear cells.  
     
     
         37 . An osteoclast, which is obtainable by the method of  claim 27 .  
     
     
         38 . A method for screening an agent for treating or preventing a metabolic bone disease, which comprises: 
 contacting an osteoclast precursor cell isolated by the method of  claim 21 , with an agent to be tested, and    measuring the inhibitory activity of said agent on differentiation of the osteoclast precursor into an osteoclast.    
     
     
         39 . A method for screening an agent for treating or preventing a metabolic bone disease, which comprises using the osteoclast precursor cell of  claim 21 .  
     
     
         40 . A method for screening an agent for the treatment or prevention of a metabolic bone disease, which comprises: 
 contacting the osteoclast of  claim 37  with an agent to be tested and measuring inhibitory activity of said agent on the bone resorption activity of said osteoclast.    
     
     
         41 . An agent for treating or preventing a metabolic bone disease which is obtainable by the method of  claim 40.