Establishing standard assays for quality control and identity traceability of human embryonic stem cells

Holly A. Cardoso1 & Ronald P. Hart, Ph.D.2

As the clinical use of stem cells in cell therapy and regenerative medicine moves toward transplanting cells into humans, stem cell culturing facilities need to assure the identity and quality of their cultures. It is crucial that standard quality control assays  be established, since FDA approval of therapeutic cells will likely require documentation of genetic identity as well as validation of pathogen-free cultures in the near future. To establish a method of identity traceability, we used Applied Biosystems' AMPFISTR® Identifiler® kit, which amlifies 16 different short tandem repeat (STR) loci, including the full set of FBI CODIS loci. A number of stable and genetically modified BG01V and SA02 stem cells as well as 2102 P100 human embryonal carcinoma cells and BG01V stem cells grown on different feeder layers were genotyped. All cells of the same lineage were found to have the same genotype at all 16 loci, even the cells that had been genetically modified, suggesting that gene insertion does not facilitate wide-scale genome changes. To engineer a method for pathogen detection, we tested techniques for the detection of common cell culture contaminants as well as common viruses that can be spread to cultures from researchers or stem cell donors. Real-time PCR detection of EBV, CMV, HHV-6, and Mycoplasma was used because the assay is fast, sensitive, and quantitative. The primers and Taq-man™ probes that we used proved to be effective in detecting 0.1 copies of EBV per genome, 10 copies of CMV per genome, 1 copy of HHV-6 per genome, adn mycoplasma in contaminated cultures. In collaboration with Applied Biosystems, our results will be used to create a kit that contains pre-packaged PCR detection reactions for up to 12 pathogens. Ultimately, this kit in conjuction with the AMPFISTR® Identifiler® assay will allow for a standard method of validation and quality control of therapeutic cells, as researchers work toward using hESCs to treat diseases and injuries such as diabetes, heart disease, and spinal cord injury.