The last aspect we want to address in this review

The last aspect we want to address in this review is the importance of suitable cell culture conditions for targeting and single cell cloning of human PSCs. This is probably the most challenging aspect since there are significant variances between different human PSC clones concerning proliferation, transfection efficiency and single cell survival during clone generation. Clearly, targeting via designer nuclease is working quite well for many cell types but identification and isolation of targeted human single PSC clones often requires a lot of experience in hPSC handling and practical training. In principle, you can use either feeder-based or feeder-free approaches. Cultivation on feeder cells highly supports single cell survival and, providing that feeder cell lines expressing appropriate perifosine genes are available, is also suitable for antibiotic selection. However, feeder cell preparation is time-consuming and can be impractical for some applications. In addition, the use of murine feeder cells is definitely excluded when aiming at the production of clinical-grade transgenic PSC lines. Dissociation of PSCs growing as colonies on feeder cells for transfection or sorting purposes may lead to higher rates of cell damage and death than dissociation of cells grown under feeder-free conditions. In general, using trypsin for human PSCs as well as multiple pipetting steps causing unnecessary shear stress for the cells should be avoided. For feeder-free cultivation, PSCs can be grown as monolayer cultures or in colonies on matrices (Burridge et al., 2011; Chen et al., 2011b). Enzymatic (e.g. Accutase) or enzyme-free passaging reagents (e.g. EDTA) can be used in combination with feeder cell-conditioned culture medium, mTeSR or E8 medium and one has to test which work best for each individual PSC clone. Most commercially available media work quite well as long as an appropriate cell density is applied and the Rho-associated coiled-coil kinase (ROCK) inhibitor Y-27632 is added. However, the cloning procedure becomes much more difficult if single cell deposition of human PSCs without feeder cells is intended. In this case, E8 media (Chen et al., 2011b) and feeder cell- or PSC-conditioned cultivation medium containing supporting paracrine factors work much better than mTeSR media. Additionally, surface coating matrix proteins such as laminin-521/E-cadherin (Rodin et al., 2014) or vitronectin (Chen et al., 2011b) can also support the cloning efficiency. In general, suitable culture conditions are dependent on the targeting strategy and have to be adjusted individually for each PSC clone. For single cell cloning after FACS or manual picking of clones after antibiotic selection, we recommend seeding onto feeder cells, as the survival rate is often much better compared to feeder-free conditions. In this case, typically the majority of remaining cell clones ought to be targeted and the establishment of only a few clones is sufficient and easy to manage. Based on our experience, the establishment of human PSC clones using standard feeder cell conditions is clearly the easiest way, since it enables the best morphological evaluation and monitoring of the stem cells. However, targeting without any selection via limiting dilution requires PCR screening of hundreds of single cell clones. Therefore, feeder cell based cultivation is often unsuitable since the preparation of plates with feeder cells is time-consuming and monolayer cultivation has clear advantages concerning single cell dissociation and the splitting procedure. For limiting dilution purposes it is also important to determine for each individual PSC line the minimal number of cells per well required for survival after single cell deposition. This typically varies from clone to clone, and it may be required to seed an average of up to 20 cells per well to enable cell survival. On the other hand, this may of course require an additional round of cloning. In general, protocols and culture conditions usually have to be adjusted to individual PSC clones concerning the most appropriate culture conditions, the dissociation procedure, the transfection approach and the cell densities after reseeding. However, despite careful optimisation and individual adjustment of these aspects, some PSC lines will still be more difficult to handle and successful targeted genome engineering will be more demanding than in other lines.