Differential protocol

The EST has continued to be a popular test subject to further optimization, especially through the addition of alternative pathways of differentiation and the addition of molecular markers of effects. Besides cardiac muscle differentiation, other cell types such as neurons (29-31), osteoblasts (32), adipocytes (33), and hepato-cytes (34) have been generated in dedicated differentiation protocols, providing additional opportunities for testing the interference of differentiation pathways with chemical exposures. The addition of transcriptomics approaches to measure differential gene expression, both as influenced by the differentiation process as well as due to chemical exposure, has proven informative and may enhance the predictability of EST assays (35). The extension of this concept to human embryonic stem cell lines is still in its infancy, but has opened a new realm of options that bypasses the need for interspecies extrapolation (36). 

When a new batch of cells is started up from liquid nitrogen storage, the quality of the cells should be tested by performing a standard 10-day differentiation protocol, as described in Differentiation into cardiomyocytes by performing a test without a compound and in the presence of 5-FU to verify the sensitivity of the assay. 

In this directed cardiac differentiation protocol, combination of activin A and BMP4 induces a primitive streak-like population and mesoderm in hESCs. The addition of Wnt inhibitor DKK1 induces the cardiac mesoderm formation, while VEGF is included to promote the expansion and maturation of KDR+ population. The additional presence of bFGF supports the continuous expansion of the developing cardiovascular lineages. 

Fig. 4 Schematic representation of cardiac differentiation protocol using hESC...

A different approach has been investigated within the EU FP7 ESNATS project (www.esnats.eu FP7 2009-2013) that is the development of alternative tests based only on one single cell model, the embryonic stem cells (ES cells), taking advantage of their ability to mimic early embryo development and to differentiate in all three germ layers under specific differentiation protocols. The aim of the ESNATS project is to develop a novel all-in-one toxicity test platform, preferably based on human ES cells, to overcome interspecies variations. Several differentiation protocols have been optimized and adapted to the purpose of defining robust standard operation... 

The development of differentiation protocols for human iPSC-derived hepatocytes requires benchmarking studies against primary human hepatocytes and commonly used hepatocyte cell lines. A recent study compared human iPSC-derived hepatocytes (hiPSC-Hep) with primary human hepatocytes and the HepG2 cell line... 

Once ES cell clones with a knockdown of the gene of interest have been identified, several experimental routes can be taken. Their ability to differentiate into a particular cell lineage can be assayed in culture, as many ES cell differentiation protocols have been described (43). To investigate the role of the targeted gene in development, the knockdown ES cells can be used to directly... 

Jafary H, Larijani B, Farrokhi A, Pirouz M, Mollamoha- mmadi S, Baharvand H. Differential effect of activin on mouse embryonic stem cell differentiation in insulin-secreting cells under nestin-positive selection and spontaneous differentiation protocols (2008). Cell Biol hit 32 278-286. 

We have built hESC/hIPSC differentiation assays to study cardiac differentiation that have several main advantages over classic differentiation protocols (Fig. 1). They are completely serum-firee, which focuses the small molecule biology on the differentiation of the cells, rather than on effects of serum components. Secondly, we miniaturized the assay to allow simultaneous screening of thousands of small molecules or small RNAs see Note 7). 

The number of replicates necessary to discern hits should be determined during assay development, and a useful discussion is in the reference by Zhang et al. [21]. We screen in triplicate to ascertain hits since dynamic range of the cardiac differentiation protocol is typically insufficient to screen without replicates. 

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