br Materials and methods br Acknowledgments br Introduction
Materials and methods
Introduction Human embryonic stem 4-ap (hESCs) offer the potential to regenerate any cell in the human body. Initially, many studies utilized serum-containing media to develop differentiation protocols for target cell types (D\'Amour et al., 2006; Kyba et al., 2002; Reubinoff et al., 2000; Zambidis et al., 2005). Serum, however, contains unknown and variable concentrations of growth factors and proteins. More recently, serum-free differentiation (SFD) protocols have emerged in order to maximize reproducibility and to gain a more thorough understanding of biological pathways activated during differentiation to specified lineages (Kennedy et al., 2007; Matsumoto et al., 2009; Nakanishi et al., 2009; Park et al., 2004). Numerous combinations of cytokines, growth factors, and siRNA molecules are being evaluated in order to understand signaling pathways utilized during differentiation and to optimize differentiation protocols to target cell types. By precisely activating or inhibiting the TGFβ family, FGF, Wnt, and other pathways at specific times, the developing embryo directs differentiation to all somatic cells of the body (Gadue et al., 2005). In vitro hESC differentiation systems attempt to recapitulate these developmental pathways by utilizing controlled concentrations of cytokines and inhibitors. As differentiation proceeds down specific lineages, the possible number of cytokine combinations dramatically increases. A high-throughput screening method that streamlines the analysis of various growth factor combinations and their effect on differentiation would be extremely beneficial. Only a few groups have developed small-scale ESC differentiation systems in order to probe biological pathways and optimize differentiation media cocktails. Ng et al. (Ng et al., 2005, 2008) have developed a 96-well spin embryoid body (EB) technique in which hESCs are spun down to form a single EB of a defined size within each well. This technique has been utilized to optimize differentiation to various stages in the hematopoietic lineage (Pick et al., 2007; Davis et al., 2008). Koike et al. have developed a 96-well murine ESC (mESC) differentiation system and explored the effects of EB seeding density on differentiation to cardiomyocytes (Koike et al., 2005, 2007). Due to the small seeding density utilized by both methods to generate single EBs and the tendency for cell counts to drop over the first 5days of differentiation (Pick et al., 2007), it is unclear if enough cells would be available for 96-well flow cytometry analyses without pooling wells. Several studies have utilized an adherent 384-well plate format to screen for small molecule inhibitors or enhancers of ESC differentiation (Borowiak et al., 2009; Desbordes et al., 2008). Although these confocal microscopy-based assays have been utilized to screen several thousand small molecules, they are not conducive to multicolor flow cytometry or live cell sorting. RNA interference (RNAi)-mediated gene knockdown has also become a common technique to explore gene involvement in active pathways. siRNA knockdown of reporter genes does not affect pluripotency in hESCs grown on mouse embryonic fibroblasts (MEFs) (Vallier et al., 2004). Knockdown of OCT4 and NANOG has been shown to induce differentiation and loss of pluripotency in hESCs (Hay et al., 2004; Zaehres et al., 2005; Rodriguez et al., 2007). RNAi has been utilized to elucidate signaling pathways that mediate ESC differentiation to multiple lineages (Izumi et al., 2007). Despite the potential that RNAi offers for understanding biology and modifying pathway activation, there are little data available regarding any potential off-target effects this technique may have on targeted hESC differentiation.
Discussion Serum-free and feeder-free hESCs differentiation protocols are essential in order to (1) maximize experimental control and reproducibility and (2) develop clinically applicable regenerative therapies (Chase and Firpo, 2007). Many studies have focused on titrating growth factors and inhibitors within SFD cocktails to optimize target cell differentiation. As lineages are further explored, the possible combinations of factors will inevitably rise. Screening assays are extremely effective in this pursuit. We have developed a high-throughput, multicolor flow cytometry assay of hESC differentiation that can be utilized to assess a range of SFD conditions. This small-scale format requires approximately 92% less reagents and growth factors than 6-well culture conditions and occupies 6% of the incubator space. Additionally, this assay utilizes surface markers to allow for live-cell analysis and sorting.