We are having difficulty maintaining genome integrity in our ipsc in culture. We have seen several mutational changes when the cells are in culture for longer periods. Any suggestions on how to troubleshoot.

Question

Accepted Answer

It is difficult to answer this question without having details of your culturing conditions. Following are the most common reasons for mutational changes and abnormal karyotypes in human pluripotent stem cell cultures-

1.  Overgrowth of cells- if cells are routinely overgrown and not passaged when they are in log phase (actively growing), the stress can cause mutations

2.  Use of a harsh dissociation agent- if a harsh dissociation agent such as trypsin is used for routine passaging and maintenance of human pluripotent stem cells, the cells get dissociated as single cells and will not be able to survive in cultures. IF they do survive, they will adapt under stress and have mutations

3.  In general any culture conditions that will induce inadvertent stress or selective pressure will result in mutations after long-term passaging
    
4.  Also, passaging the cells too soon (every two or three days) can induce stress
    
5.  I would suggest to check culture conditions, dissociation agents, dissociation methods, passaging frequency as possible stressors

Answer

My lab has begun to try and reduce the amount of serum used in our pluripotent cell culture media. Do you have a method you would recommend for weaning the cells or a lowest serum amount you think would work?

What alternative methods exist for reprogramming adult cells into iPS cells, excluding methods that introduce foreign genetic material into the host cells? If there are any, what are the major limitations of these methods?

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