Frequently Asked Questions
iPSCs are somatic cells (e.g. skin, blood) that have been genetically reprogrammed to a pluripotent stem cell state through directed expression of pluripotency genes called reprogramming factors. By definition, iPSCs replicate indefinitely and have the potential to differentiate into any cell type in the human body.
Reprogramming factors are the genes introduced into somatic cells that induce a pluripotent stem cell state. Initial reports describing the production of human iPSCs utilized 4 reprogramming factors: OCT4, SOX2, KLF4 and MYC (OSKM) (Takahashi, et al. 2007) or OCT4, SOX2, NANOG and LIN28 (OSNL) (Yu, et al. 2007). Subsequent studies revealed that reprogramming using a specific combination of all 6 of these factors combined with SV40LT and a cocktail of small molecules yields iPSCs at much higher efficiency (Yu, et al. 2009; Yu, et al. 2011).
A variety of small molecules have been identified that can functionally substitute for one or more reprogramming factors and/or improve the efficiency of iPSC reprogramming. However, no
combination of small molecules has been shown to functionally substitute for all 4 reprogramming factors. The use of small molecules in iPSC reprogramming offers some practical advantages including the ability to optimize the chemical structure, fine-tune dose and concentration, and simplify handling and application protocols. However, the use of small molecules presents a number of scientific challenges. Most notably, small molecules may have more than one target which may or may not be known. In addition, unexpected toxicity and other side effects in vivo may interfere with the clinical application of small molecules.
iPSCs are genetically reprogrammed through directed expression of pluripotency genes into somatic cells. The expression of these genes can be accomplished using a variety of different methods. Episomal reprogramming refers to a method of introducing pluripotency genes into a target cell using circular DNA plasmid vectors (i.e. episomes) that replicate autonomously within the cell cytoplasm and do not integrate into the host cell genome, and are eventually lost from the cells as a result of cell division.
Initial methods of iPSC reprogramming utilized retroviral and lentiviral vectors to introduce pluripotency genes into somatic cells. While these methods generally work well, the viral DNA integrates into the genome of the target cell and the resulting iPSCs (and cells differentiated from them) will contain foreign DNA. By contrast, episomal vectors replicate autonomously within the cell cytoplasm and do not integrate into the host genome. In addition, the episomal vectors are released from the target cell at a rate of ~5% per cell cycle resulting in transgene-free or “footprint-free” iPSCs. These features, combined with recent advancements in episomal reprogramming efficiency, have led to a strong preference for this method to alleviate concerns about genome integrity for drug discovery and cell therapy applications.
Integration-free iPSCs have been generated using a variety of methods including adenovirus, Sendai virus, piggyBac, minicircle vectors, and direct introduction of protein or synthesized mRNA. The efficiency and success rate of these methods varies depending on the source of somatic cells and experimental conditions, but in general these approaches are limited by impractically low
reprogramming efficiency, requirement for higher biosafety containment, and/or labor- and cost intensive protocols that require repeated transfection/infection. Compared to these methods, episomal reprogramming is virus-free, safe to use, stable, and inexpensive.
Episomal reprogramming has been successfully achieved from a variety of somatic cells including fibroblasts, lymphoblastoid cells and peripheral blood mononuclear cells. Importantly, CDI has optimized its episomal reprogramming method to achieve high efficiency iPSC generation from small amounts of human peripheral blood. Not only does this enable more streamlined and less invasive collection of donor samples, but ensures increased sterility and lower cost production of iPSCs. In addition, efficient iPSC production from peripheral blood enables access to large banks of apparently healthy and disease-associated clinical samples for disease research and drug screening.
CDI’s suite of MyCell® Products includes includes episomal reprogramming of customer-provided donor samples and subsequent genetic engineering and/or differentiation of the iPSCs. In addition, for researchers who would like to generate their own iPSCs, CDI’s episomal reprogramming technology is available as a kit from Life Technologies. Customer-generated iPSCs may then be transferred to CDI for genetic engineering and/or differentiation.