what to do with the differentiated mouse es cells - (Mar/30/2008 )
Hi guys.
I'm trying to grow mouse es cells (D3 line) in the feeder free condition. I'm facing spontaneous differentiation problem. I have read some articles saying that spontaneous differentiation is pretty common in mouse es cells. However, I'm not sure in what degeree of differentiation is considered acceptable and what is considered not. The cells around and on the border of my ES cells colonies look flatten, like those differentiated characteristic. Do we have any criteria to judge whether which condition to keep using the cells or to throw away? For example, the level of Oct4 protein? or the percentage of OCt4 expressing cells? or other methods? Please help! Thanks a lot in advance.
deperate student
I'm trying to grow mouse es cells (D3 line) in the feeder free condition. I'm facing spontaneous differentiation problem. I have read some articles saying that spontaneous differentiation is pretty common in mouse es cells. However, I'm not sure in what degeree of differentiation is considered acceptable and what is considered not. The cells around and on the border of my ES cells colonies look flatten, like those differentiated characteristic. Do we have any criteria to judge whether which condition to keep using the cells or to throw away? For example, the level of Oct4 protein? or the percentage of OCt4 expressing cells? or other methods? Please help! Thanks a lot in advance.
deperate student
You get spontaneous differentiation in human ESCs, also. Differentiated ones, when I view them under microscope, along the edges are darker, bigger size, spread out, and the differentiated ones in the middle growing in 3D are brownish. My PI said that they should not adhere (differentiated) after you passage them, but if so many of your cells are differentiated, then there are not enough left to grow well in the next passage...just my thoughts.
thanks a lot. I think I have to get a new batch of cells cause I'm afraid the differentiated ones will mess up my experiment
I think, you can stain that cells with marker for differentiate cells to see what kind of cells they were forming.
actually, you can still take the neurospheres by trypsinize them at early stage of differentiation.
you medium must be lack of growth factor to support the undifferentiate cells or maybe you put to low amount of medium that will cause the cell attachment.
hope this will help you. good luck.