Stem Cells


used in combination with a new generation of kinetic, live-cell assays, for example – is driving the adoption of these alternative assays in an industry increasingly looking to reduce its reliance on ani- mal studies. “Assays run on cell analysis platforms that


employ built-in injection technologies and study biology in real-time are increasingly valued for their ability to model more complex cellular behaviour,” says Dr David Ferrick, Senior Director of


the Cell Analysis Division at Agilent


Technologies. “It’s hoped that the greater transla- tional value and scales of economy these systems bring will facilitate fewer drugs failing in the devel- opment stage, especially after they have shown promising results in animals.”


Tackling the challenge of slow maturation of human iPSCs While stem cells have enormous potential in drug discovery applications and beyond, several chal- lenges remain. One of the most pressing issues is the fact that in many cases, differentiated cells exhibit expression patterns and functional behaviour that are more closely matched with cells of a foetal age than those of an adult. This can be problematic for researchers working with cell types that are difficult to mature in vitro, such as car- diomyocytes and neural cells, as well as for those modelling late-onset diseases. Many engaged in stem cell research are searching for solutions to this issue. One way to accelerate the human developmental


clock in culture dishes is by manipulating the cul- ture environment, and innovations in culture tech- niques and technologies are making this easier. “The challenge of slow maturation can be addressed using 3D cell culture scaffolds and matrices that mimic the adult extracellular envi- ronment, or through co-culture techniques that employ supportive cells and electrical or physical stimulation,” explains Dr Yichen Shi, CEO at Axol Bioscience. Another solution involves direct pro- gramming of iPSCs by driving stem cell differenti- ation through transient expression of cell type spe- cific transcription factors. “Introducing transcrip- tion factors into the cells by viruses, lipid vehicles or directly inserted into specific locations of the genome using CRISPR-Cas9 can also be effective,” Shi adds. In the latter case, when editing the genomes of


iPSCs to generate isogenic pairs or to insert sequences that could act as detectors or reporters of specific biological end-points, the generation of single-cell derived clones is typically required.


Drug Discovery World Fall 2018


However, in the past, this has been problematic. “Historically, it’s been a real challenge to isolate single iPSCs,” says Dr David Piper, Director of Research and Development, Cell Biology and Synthetic Biology at Thermo Fisher Scientific. “There have been some great strides made in the development of media and supplements to support single-cell culture applications. Similarly, some of the latest Cas9 nucleases have exceptional purity and activity to support high efficiency editing in iPSCs.”


Reducing variation in growth and differentiation potential To support pharmaceutical pipelines, large num- bers of high-quality, homogenous cell cultures are required. Currently, stem cells are typically manu- factured in small scale dishes or T-flasks, and scal- ing-up stem cell production while ensuring quality and reproducibility can be challenging. To mitigate this issue, robust culture conditions and validated protocols that are amenable to scale are needed to ensure consistency between conditions and between experiments. Variability between cell lines or sample sources


is a particular hurdle. “Some groups are addressing this issue using gene editing technologies to enable derivation of isogenic lines to control for genetic background differences,” explains Helen Hardiman, Bioengineering Product Manager at STEMCELL Technologies. Another source of vari- ability is within the culture itself, and this can become a larger issue when large numbers of cells are required. An effective solution to this issue is to use a single large-scale suspension culture vessel instead of traditional well plates or T-flasks. “For human pluripotent stem cells (hPSCs), commercial- ly available culture medium and scale-up systems are enabling researchers to generate large numbers of cells in a single, large-scale suspension culture vessel, thereby reducing variability.” Another issue is a historical lack of support for


certain cell lines. In many cases, published proto- cols for the differentiation of iPSCs into disease- relevant cell types are either not available or are so complicated and labour-intensive that the differ- entiation protocol cannot be easily translated into a robust protocol for manufacturing cells at a scale large enough to accommodate drug screen- ing assays. “Until very recently, most of the focus has been on specific disease-relevant cell types, most notably, cardiac, hepatic, pancreatic and neuronal cells. This means that other cell types like lung, endothelial, or intestinal cells tradition- ally haven’t been well defined,” explains Dr Liz


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