Substrates and methods for staining live stem cells

Abstract

The invention relates to novel substrates and methods for staining live stem cells. The stain may be used to identify induced pluripotent stem cell colonies during the process of somatic cell reprogramming.

1 . A composition comprising a substrate capable of identifying a live stem cell of interest, wherein the substrate is cell permeable and is capable of being modified by the stem cell into a modified substrate, wherein the modified substrate is both permeable and detectable such that the live stem cell can be identified by detecting the presence of the modified substrate, and wherein the substrate and the modified substrate are non-toxic; wherein the substrate is a resorufin derivative. 
     
     
         2 . The composition of  claim 1  wherein the live stem cell of interest is selected from the group consisting of an embryonic stem cell (ESC), an induced pluripotent stem cell (iPSC), a pluripotent cell, a progenitor cell, a reprogrammed cell, and a dedifferentiated cell. 
     
     
         3 . The composition of  claim 1  wherein the detecting is of a fluorescent signal. 
     
     
         4 .- 13 . (canceled) 
     
     
         14 . The composition according to  claim 1 , wherein the substrate is reactive with enzymes chosen from acid or alkaline phosphatases. 
     
     
         15 . (canceled) 
     
     
         16 . The composition according to  claim 1 , wherein the substrate is a resorufin derivative of formula VII or formula VIII: 
       
         
           
           
               
               
           
         
       
       where:
 R1, R2, R3, R4, and R7 are individually chosen from alkyl, aryl, aralkyl, heteroaryl, and heteroaralkyl; 
 X and Y are individually chosen from H, halogen, amino, alkoxy, SH, SR, alkyl, aryl, aralkyl, heteroaryl, and heteroaralkyl; 
 R5 is chosen from H, COOH, COOR, OH, amino, alkoxy, halogen, alkyl, aryl, aralkyl, heteroaryl, and heteroaralkyl; 
 R6 is chosen from OR, NHCOR, and SR; and 
 m and n individually range from 1 to 3. 
 
     
     
         17 . A method of identifying a live stem cell in a colony, said method comprising treating the colony with a composition of  claim 1 , wherein the composition comprises a substrate that is cell permeable and is capable of being modified by the stem cell into a modified substrate, wherein the modified substrate is both permeable and detectable such that the live stem cell can be identified by detecting the presence of the modified substrate, and wherein the substrate and the modified substrate are non-toxic. 
     
     
         18 . (canceled) 
     
     
         19 . The method according to  claim 17 , wherein the composition is reactive with an enzyme chosen from either an acid or an alkaline phosphatase. 
     
     
         20 . (canceled) 
     
     
         21 . A method of staining at least one stem cell in a cell culture, said method comprising treating the cell culture with a composition of  claim 1 , wherein the composition is cell permeable, has a detectable signal and is non-toxic to said stem cell and specifically stains the stem cell. 
     
     
         22 . (canceled) 
     
     
         23 . The method according to  claim 21 , wherein the composition is reactive with an enzyme chosen from either an acid or an alkaline phosphatase. 
     
     
         24 . (canceled) 
     
     
         25 . A kit comprising a composition of  claim 1 . 
     
     
         26 . The kit of  claim 25  further comprising at least one vial comprising a stem cell. 
     
     
         27 .- 28 . (canceled) 
     
     
         29 . A method of staining a live stem cell of interest, said method comprises treating said stem cell with a composition of  claim 1 , wherein the composition comprises a substrate that is:
 a. cell permeable;   b. non-toxic to the cell;   c. is processed to a modified substrate by the stem cell of interest,   wherein the modified substrate can be detected, and optionally, the modified substrate can diffuse out of the cell.   
     
     
         30 . The method of  claim 29 , wherein the live stem cell is selected from the group consisting of an embryonic stem cell (ESC), an induced pluripotent stem cell (iPSC), a pluripotent cell, a progenitor cell, a reprogrammed cell, and a dedifferentiated cell. 
     
     
         31 . The method of  claim 29 , wherein the detecting is of a fluorescent signal. 
     
     
         32 . The method of  claim 17 , wherein the substrate is modified by an enzyme specific to the live stem cell of interest. 
     
     
         33 . The composition of  claim 1 , wherein the substrate is modified by an enzyme specific to the live stem cell of interest. 
     
     
         34 . The method of  claim 17 , wherein the substrate is present in an effective amount for the detection of said live stem cell of interest. 
     
     
         35 . The method of  claim 34 , wherein the effective amount of the substrate is 2-1000 mM. 
     
     
         36 . The method of  claim 34 , wherein the effective amount of the substrate is 2-1000 μM. 
     
     
         37 . The method of  claim 17 , wherein a further step of washing the live stem cell, or diluting the live stem cell with buffer and/or culture media removes the modified substrate.