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Recently, there has been a significant shift from culturing human embryonic stem cells (hESC) on feeders to clinically-relevant, feeder-free and xeno-free culture conditions. This change often requires the adaptation of protocols optimized for old hESC culture systems that are not always effective in feeder-free conditions. In this study we aimed at optimizing the protocol for embryoid body (EB) formation from hESC in single cell suspension that were routinely cultured on laminin-521TM (LN521) in NutristemTM medium, providing xeno-free, chemically defined conditions that effectively support hESC self-renewal. However, hESC long-term expansion on LN521 seems to depend more on cell adhesion to the surface than on cell-to-cell contacts. In feeder-dependent cultures, the latter are very important for hESC viability as suspension of single cells results in apoptosis, only preventable by addition of ROCK inhibitor. In our experiments, harvesting of hESC and plating them in ultr a-low at tachment round bottom 96-well plates (5000 cells/well) in the presence of ROCK inhibitor completely failed to induce aggregate formation. A 2-day pre-incubation in differentiation medium before harvesting, however, enabled the aggregation of single hESC. Next, we evaluated the efficiency of EB formation using different methods for single cell dissociation (enzymatic vs non-enzymatic approaches), as well as different differentiation media. The most efficient EB formation and subsequent growth for 21 days was obtained by enzymatic harvesting of 2-day pre-differentiated cells and subsequent differentiation in serum-free medium. Differentiation in the presence of serum caused poor aggregation of EBs and disaggregation after few days. After the 21-day differentiation, expression of genes characteristic of the three germ layers was observed. Our optimized EB formation protocol has been until now successfully tested on three different hESC lines. This protocol allows generation of equal-si zed EBs, thereby minimizing the impact of EB size on the differentiation potential of cells within. |
