Among these, one of the most important is the quality of the hiPSC lines and the potential presence of epigenetic abnormalities and somatic mutations 152–155. Poor quality hiPSC lines are more likely to fail or give variable outcomes using any differentiation method, complicating the interpretation of the results. A good practice is to differentiate between 2 and 3 different hiPSC lines for each individual in each experiment. The second most important variable is the method of differentiation. Variables that are often associated with different differentiation protocols include initial enrichment and selection of NPCs with different techniques such as manual isolation, FACS-sorting or bead-isolation, and whether NPCs are cultured in a monolayer or in a 3-D environment. For example, monolayer protocols typically involve a lower cell density, decreased cell-to-cell and increased cell-to-matrix signaling; these variables, along with matrix rigidity and stiffness, have all been shown to influence cell fate.
