Production of cultured human mast cells and basophils for high throughput small molecule drug discovery

Abstract

Provided are methods for producing and screening proliferated populations of CD34-negative progenitor cells, mucosal mast cells, connective tissue-type mast cells and basophil cells. The methods generate uniform proliferated populations of cells. The proliferated populations contain a uniform population of a size suitable for use in high throughput screening methods, for example, screening for agents that alter exocytosis. The invention includes screening the proliferated populations with at least one candidate bioactive agent, and evaluating the cells to detect a cell with an altered phenotype. The invention also includes isolating a candidate bioactive agent that causes the altered phenotype. Additionally, cells formed according to the described methods are also encompassed by the invention.

1 - 36 . (canceled)  
     
     
         37 . A method comprising: 
 (a) contacting at least one CD34-positive cell with a flt-3 ligand and a stem cell factor to generate a proliferated population of progenitor cells; and thereafter    (b) contacting said proliferated population of progenitor cells with said stem cell factor and an IL-3 to form a proliferated population of basophil cells.    
     
     
         38 . The method of  claim 37  in which the flt-3 ligand is human flt-3 ligand  
     
     
         39 . The method of  claim 37  in which the stem cell factor is human stem cell factor.  
     
     
         40 . The method of  claim 37  in which the IL-3 is human IL-3.  
     
     
         41 . The method of  claim 37  in which the CD34-positive cell is from umbilical cord blood.  
     
     
         42 . The method of  claim 37  in which the CD34-positive cell is from bone marrow.  
     
     
         43 . A proliferated population of cultured cells prepared by a method comprising: 
 a) contacting at least one CD34-positive cell with a flt-3 ligand and a stem cell factor to generate a proliferated population of progenitor cells of at least 10 7  cells; and thereafter    b) contacting said proliferated population of progenitor cells with said stem cell factor and a cytokine suitable for causing differentiation of the progenitor cells into a proliferated population of cells that degranulate upon binding to IgE.    
     
     
         44 . The proliferated population of cultured cells of  claim 43  in which the proliferated population of progenitor cells is at least about 10 8  cells.  
     
     
         45 . The proliferated population of cultured cells of  claim 43  in which the proliferated population of progenitor cells is at least about 10 9  cells.  
     
     
         46 . The proliferated population of cultured cells of  claim 43  in which the proliferated population of progenitor cells is at least about 10 10  cells.  
     
     
         47 . The proliferated population of cultured cells of  claim 43  in which the proliferated population of progenitor cells is at least about 10 11  cells.  
     
     
         48 . The proliferated population of cultured cells of  claim 43  in which the cytokine is IL-4 or IL-6 and the cells that degranulate upon IgE binding comprise mast cells.  
     
     
         49 . The proliferated population of cultured cells of  claim 48  in which the IL-4 or IL-6 is human IL-4 or IL-6.  
     
     
         50 . The proliferated population of cultured cells of  claim 43  in which the cytokine is IL-3 and the cells that degranulate upon binding to IgE comprise basophil cells.  
     
     
         51 . The proliferated population of cultured cells of  claim 50  in which the IL-3 is human IL-3.  
     
     
         52 . The proliferated population of cultured cells of  claim 43  in which the stem cell factor is human stem cell factor.  
     
     
         53 . The proliferated population of cultured cells of  claim 43  in which the flt-3 ligand is human flt-3 ligand.