Quantitation of Rare Undifferentiated hESCs by Methylation-Specific CastPCR (MeS castPCR)

Jason Gioia, David Deng, Jeff Rosner, Caifu Chen Life Technologies, United States of America

Abstract
Human embryonic stem cells (hESCs) retain the remarkable ability to differentiate into multiple different tissues and to self-renew in vitro. These remarkable abilities make hESCs very attractive to both scientific and clinical communities because they are potentially a renewable source of a wide variety of human tissues which can be used for regenerative medicine, drug discovery and toxicity testing. However, during the cell type-specific differentiation of hESCs, a small fraction of undifferentiated hESCs frequently remain mixed with the differentiated cells. These undifferentiated hESCs pose serious risk by forming tumors after transplantation into patients. Therefore, it is important to detect & quantify rare, undifferentiated hESCs in therapeutic stem cell products. Based on distinct methylation patterns between hESCs and their differentiated cells in a set of genes, here we propose a new technology called methylation-specific castPCR (MeS castPCR) for accurate and sensitive quantification of undifferentiated hESCs. This new technology will bridge the gap in hESC product QC and then overcome this major obstacle in stem cell therapy. CastPCR combines allele-specific TaqMan qPCR with allele-specific minor grove binder (MGB) as blockers to suppress effectively non-specific amplification from the wild type allele. We have successfully designed and validated castPCR assays for ~100 SNPs or InDels. Results demonstrate that castPCR not only maintains wide dynamic range, high sensitivity, and reproducibility of TaqMan assays but also is able to detect one mutant in 100,000,000 wild-type molecules. Recently, Laurent et al. (2010) and Lister et al. (2009) presented a whole-genome comparative view of DNA methylation using bisulfite sequencing of hESCs and various differentiated cells. Based on their findings, we have identified genes and genomic DNA regions such as HOXB6 and others which show distinct DNA methylation patterns between hESCs and differentiated cells. These regions could be used as methylation markers to distinguish between hESCs and their differentiated (somatic) cells. It is important to note that DNA regions with 0% methylated in hESC and 100% in differentiated cells are mostly desirable due to incomplete bisulfate conversion. MeS castPCR is aimed to detect converted CpG sites which are used to estimate % of hESC contamination in therapeutic cell products. Our results suggest that MeS castPCR is capable of detecting hESC contamination as little as 0.01% in differentiated stem cell therapy product during clinical trials.