CDI | iPS Cells – Cellular Dynamics International

Posted: December 19, 2014 at 5:46 am

How does CDI's technology work? A human biological sample, for example blood or skin, is obtained, and the cells within the sample are grown under appropriate cell culture conditions. In the episomal reprogramming method, vectors containing multiple reprogramming genes are introduced into the cells.

While the vectors turn genes in the cell on and off, reprogramming them to a stem cell state, they do not integrate into the genome itself. This method alleviates concerns arising over the potential risks associated with the insertion of foreign DNA to induce reprogramming, which other prior iPS methods use (bottom row in illustration above).

iPS cells are somatic cells (e.g., skin or blood) that have been genetically reprogrammed to a pluripotent stem cell state through forced expression of pluripotency genes.By definition, iPS cells 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 creation of human iPS cells utilized four 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 iPS cells at much higher efficiency (Yu, et al. 2009; Yu, et al. 2011).

iPS cells are genetically reprogrammed through forced expression of pluripotency genes into somatic cells.The expression of these genes can be accomplished using a variety of different methods.The episomal reprogramming method introduces 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.

Initial methods of iPS cell 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 iPS cells (and cells differentiated from them) will contain foreign DNA, which may result in defects and errors. 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 iPS cells.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.

Episomal reprogramming has been reported successful 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 iPS cell 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 iPS cells. In addition, efficient iPS cell production from peripheral blood enables access to large banks of normal and disease-associated clinical samples for disease research and drug screening.

CDIs suite of MyCell Products includes episomal reprogramming of customer-provided donor samples and subsequent genetic engineering and/or differentiation of the iPS cells. In addition, for researchers who would like to generate their own iPS cells, CDIs episomal reprogramming technology is available as a kit from Life Technologies, including Episomal iPSC Reprogramming Vectors, Vitronectin, and Essential 8 Medium. Customer-generated iPS cells using this kit may then be transferred to CDI for genetic engineering and/or differentiation through MyCell Products.

Integration-free iPS cells 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.

A variety of small molecules have been identified that can functionally substitute for one or more reprogramming factors and/or improve the efficiency of iPS cell reprogramming. However, no combination of small molecules has been shown to functionally substitute for all four reprogramming factors. The use of small molecules in iPS cell 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.

Original post:
CDI | iPS Cells - Cellular Dynamics International

Related Post

Comments are closed.

Archives