In vitro-differentiated retinal ganglion cells and method for producing same

Abstract

In vitro-differentiated retinal ganglion cells can be produced by exposing a mammalian retinal ganglion cell line to a protein kinase inhibitor. The differentiated retinal ganglion cells can be used to identify agents that protect retinal ganglion cells in vivo or in vitro from cell injury (including cell death) and agents that affect retinal ganglion cell ion channel activity.

1 . In vitro-differentiated mammalian retinal ganglion cells (RGCs) obtained from a mammalian RGC cell line wherein the differentiated RGCs exhibit at least one feature selected from (1) a cell division rate lower than that of the RGC cell line, (2) a higher number of neurite per cell, on average, than that of the RGC cell line, or (3) expression of one or more ion channels higher than that of the RGC cell line.  
     
     
         2 . The differentiated RGCs of  claim 1 , wherein the cell division rate of the differentiated RGCs is about 20% or less of that of the RGC cell line.  
     
     
         3 . The differentiated RGCs of  claim 1 , wherein the cell division rate of the differentiated RGCs is about 10% or less of that of the RGC cell line.  
     
     
         4 . The differentiated RGCs of  claim 1 , wherein the differentiated RGCs lose the capability to divide.  
     
     
         5 . The differentiated RGCs of  claim 1 , wherein the differentiated RGCs have, on average, at least one more neurite per cell than that of the RGC cell line.  
     
     
         6 . The differentiated RGCs of  claim 1 , wherein the differentiated RGCs have, on average, at least two more neurites per cell than that of the RGC cell line.  
     
     
         7 . The differentiated RGCs of  claim 1 , wherein the differentiated RGCs exhibit at least two of the features.  
     
     
         8 . The differentiated RGCs of  claim 7 , wherein the at least two features are the reduced cell division rate and the higher number of neurites per cell.  
     
     
         9 . The differentiated RGCs of  claim 7 , wherein the differentiated RGCs exhibit three of the features.  
     
     
         10 . The differentiated RGCs of  claim 1 , wherein the differentiated RGCs further exhibit upregulated expression of a neuronal marker selected from Thy-1, NMDA-R1, GABA-B receptor, Brn-3C, neuritin, synaptophysin, GFAP, β-Actin, NGF, NT-3, NT-4, TrkA, TrkB, P75, CNTF, BDNF, or GDNF.  
     
     
         11 . The differentiated RGCs of  claim 1 , wherein the differentiated RGCs were obtained by exposing cells of the RGC cell line to an amount of a protein kinase inhibitor sufficient to differentiate the cells.  
     
     
         12 . The differentiated RGCs of  claim 11 , wherein the protein kinase inhibitor is selected from staurosporine, H-1152, or H-89.  
     
     
         13 . In vitro-differentiated RGC-5 cells obtained from a rat RGC-5 cell line characterized by at least one feature selected from (1) a cell division rate lower than that of the RGC-5 cell line, (2) a higher number of neurite per cell, on average, than that of the RGC-5 cell line, or (3) expression of one or more ion channels higher than that of the RGC-5 cell line.  
     
     
         14 . A method for identifying an agent that protects retinal ganglion cells (RGCs) from cell injury, the method comprising the steps of: 
 exposing in vitro-differentiated RGCs of  claim 1  to a candidate agent;    inducing a cell injury in the exposed differentiated RGCs and in control differentiated RGCs not exposed to the candidate agent;    comparing the extent of injury of the exposed RGCs and to the control RGCs; and    identifying the agent as an agent that can protect the RGCs if the injury in the exposed RGCs is less severe than that in the control RGCs.    
     
     
         15 . A method as recited in  claim 14 , wherein the cell injury is cell death.  
     
     
         16 . A method for identifying an agent that protects retinal ganglion cells (RGCs) from cell injury, the method comprising the steps of: 
 exposing in vitro-differentiated RGCs of  claim 13  to a candidate agent;    inducing a cell injury in the exposed differentiated RGCs and in control differentiated RGCs not exposed to the candidate agent;    comparing the extent of injury of the exposed RGCs and to the control RGCs; and    identifying the agent as an agent that can protect the RGCs if the injury in the exposed RGCs is less severe than that in the control RGCs.    
     
     
         17 . A method as recited in  claim 16 , wherein the cell injury is cell death.  
     
     
         18 . A method for identifying an agent that can affect ion channel activity of in vitro-differentiated retinal ganglion cells (RGCs), the method comprising the steps of: 
 comparing an ion channel activity in differentiated RGCs of  claim 1  exposed to a candidate agent and in control differentiated RGCs not exposed to the candidate agent; and    identifying an agent that affects the ion channel activity in the exposed RGCs relative to the control RGCs as an activity-affecting agent.    
     
     
         19 . A method for identifying an agent that can affect ion channel activity of in vitro-differentiated retinal ganglion cells (RGCs), the method comprising the steps of: 
 comparing an ion channel activity in differentiated RGCs of  claim 13  exposed to a candidate agent and in control differentiated RGCs not exposed to the candidate agent; and    identifying an agent that affects the ion channel activity in the exposed RGCs relative to the control RGCs as an activity-affecting agent.