Fluorescent compounds specific for pluripotent stem cells and reprogramming-ready cells and methods of using the same

Abstract

Determining, in a sample, the presence and/or amount of pluripotent stem cells. A sample suspected of containing one or more pluripotent stem cells is provided. The sample with a fluorescent compound is contacted under conditions that allow binding of the fluorescent compound to the pluripotent stem cells, where the fluorescent compound is a fluorescent compound of formula (I) or a pharmaceutically acceptable salt thereof. The presence and/or amount of the pluripotent stem cells is determined by measuring the fluorescence of the cells following the contacting.

What is claimed is: 
     
         1 . A method of determining, in a sample, the presence and/or amount of pluripotent stem cells, said method comprising the steps of:
 (i) providing a sample suspected of containing one or more pluripotent stem cells;   (ii) contacting the sample with a fluorescent compound under conditions that allow binding of said fluorescent compound to the pluripotent stem cells, wherein the fluorescent compound is a fluorescent compound of formula (I) or a pharmaceutically acceptable salt thereof   
       
         
           
           
               
               
           
         
       
       wherein:
 R 1 , R 4 , R 5 , R 8 , R 9  and R 10  are each independently selected from the group consisting of H and C 1-6  alkyl; 
 R 2 , R 3 , R 6  and R 7  are each independently selected from C 1-6  alkyl; 
 R 11  is selected from the group consisting of H, (CR 12 R 13 ) o —N(R 14 R 15 ) and C 1-6  alkyl, wherein o is independently 0, 1, 2, 3, 4, or 5, and R 12 -R 15  are each independently selected from the group consisting of H and C 1-6  alkyl; 
 m and n are each independently 0, 1, or 2; 
 p is 0, 1, 2, or 3, 
 q is 1, 2, 3, or 4, with the proviso that p+q≤4; and 
    means that the respective bond can be a single or double bond and, if it is a single bond, the additional valencies are hydrogen; and 
 (iii) determining the presence and/or amount of the pluripotent stem cells by measuring the fluorescence of the cells following said contacting. 
 
     
     
         2 . The method of  claim 1 , wherein the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells. 
     
     
         3 . The method of  claim 1 , wherein the pluripotent stem cells are mammalian cells. 
     
     
         4 . The method of  claim 1 , wherein step (ii) does not comprise a washing step following said contacting. 
     
     
         5 . The method of  claim 1  further comprising a step of:
 (iv) isolating the pluripotent stem cells labelled by the fluorescent compound from the sample. 
 
     
     
         6 . The method of  claim 5 , wherein the labelled cells are isolated by fluorescence-activated cell sorting (FACS). 
     
     
         7 . A method of determining, in a sample, the presence and/or amount of cells undergoing reprogramming to become induced pluripotent stem cells, said method comprising the steps of:
 (i) providing a sample suspected of containing one or more cells undergoing reprogramming to become induced pluripotent stem cells;   (ii) contacting the sample with a fluorescent compound under conditions that allow binding of said fluorescent compound to the cells undergoing reprogramming to become induced pluripotent stem cells, wherein the fluorescent compound is a fluorescent compound of formula (I) or a pharmaceutically acceptable salt thereof   
       
         
           
           
               
               
           
         
         wherein: 
         R 1 , R 4 , R 5 , R 8 , R 9  and R 10  are each independently selected from the group consisting of H and C 1-6  alkyl; 
         R 2 , R 3 , R 6  and R 7  are each independently selected from C 1-6  alkyl; 
         R 11  is selected from the group consisting of H, (CR 12 R 13 ) o —N(R 14 R 15 ) and C 1-6  alkyl, wherein o is independently 0, 1, 2, 3, 4, or 5, and R 12 -R 15  are each independently selected from the group consisting of H and C 1-6  alkyl; 
         m and n are each independently 0, 1, or 2; 
         p is 0, 1, 2, or 3, 
         q is 1, 2, 3, or 4, with the proviso that p+q≤4; and 
            means that the respective bond can be a single or double bond and, if it is a single bond, the additional valencies are hydrogen; and 
         (iii) determining the presence and/or amount of the cells undergoing reprogramming to become induced pluripotent stem cells by measuring the fluorescence of the cells following said contacting. 
       
     
     
         8 . The method of  claim 7 , wherein the cells undergoing reprogramming to become induced pluripotent stem cells are mammalian cells, preferably human, mouse, or rat cells, more preferably human cells. 
     
     
         9 . The method of  claim 7 , wherein step (ii) does not comprise a washing step following said contacting. 
     
     
         10 . The method of  claim 7  further comprising a step of:
 (iv) isolating the cells undergoing reprogramming to become induced pluripotent stem cells labelled by the fluorescent compound from the sample. 
 
     
     
         11 . The method of  claim 10 , wherein the labelled cells are isolated by fluorescence-activated cell sorting (FACS).