Embryoid body (EB) formation is a standard approach to assess differentiation potential of human pluripotent stem cells (hPSC). However, EB size has been shown to influence the differentiation course of the cells. In order to compare functional variability between individual hPSC lines it is therefore mandatory to use equal-sized EBs, using a standardized EB protocol. Our cells are routinely cultured on laminin-521TM (LN521) in NutristemTM (NS) medium and passaged as single cells, providing feeder- and xeno-free conditions. As previously published and commercially available protocols were optimized for different culture systems, we had to develop a new single cell-based EB protocol. We aimed to obtain equal-sized EBs after spinning the single cell suspension in round bottom 96-well plates (5000 cells seeded per well) in the presence of ROCK inhibitor. In a first approach, cells were pre-differentiated for 2 days before harvesting. Results were highly variable, with cells either aggregating, partially aggregating or completely failing to form EBs. We did not observe significant differences between enzymatic vs non-enzymatic cell harvest approaches, but we noticed that differentiation in the presence of serum caused poor aggregation of EBs and early disaggregation, whereas aggregate formation and subsequent growth for 21 days was much improved in KnockoutTM Serum Replacement (KOSR)-based medium. Finally, we observed that the crucial factor influencing the aggregation of single hPSC was the growth phase of the cells. Only cells that were freshly passaged 2-3 days before EB formation were consistently forming aggregates, not only in KOSR-based medium but also in commercially available xeno-free APELTM medium and in NS medium. With this approach we even managed to obtain hanging drop EBs, which was not possible in our previous experiments. Our optimized EB formation protocol using 96-well plates has until now been successful on 6 different hESC lines and 12 hiPSC lines. We are currently quantifying the size of EBs formed by different numbers of cells during a 21-day differentiation course and investigating the effect of EB size on expression of early lineage specification genes.
|Status||Published - 20 apr 2015|
|Evenement||Interuniversity Stem Cell Meeting - Belgium, Leuven, Belgium|
Duur: 20 apr 2015 → …
|Conference||Interuniversity Stem Cell Meeting|
|Periode||20/04/15 → …|