Focus on stem cell quality: Improving rigor and reproducibility through characterization
As stem cell scientists, the quality of our research is directly related to the quality of the hPSC materials used. Poor quality cells can impact reproducibility, jeopardize results, waste time, and drain resources. In screening materials submitted to the WiCell Stem Cell Bank, we have identified a substantial and concerning variability in cell quality. Further in-depth analysis of a decade of karyotypic data has allowed us to identify specific areas of recurrent karyotypic instability previously unknown. These results highlight the need for improved testing strategies and standards.
As of this abstract, more than 1600 cell lines have been deposited with the WiCell Stem Cell Bank for banking and characterization by 31 providing laboratories. The vast majority of these cell lines were generated through grant-funded projects as a resource for the larger scientific community, and reportedly screened prior to submission. Various testing strategies were used, and available characterization information was provided to WiCell for reference. To date, nearly 800 of these lines have been independently tested by WiCell for thaw viability, genetic stability (karyotype), identity via short tandem repeat (STR) analysis, sterility (bacteria and fungus), and mycoplasma. Of the hPSC lines examined, more than one-third of WiCell screened cell lines failed this routine quality testing. While there were failures across all tests, the majority of cell lines failed due to unexpected abnormal karyotype.
Stem cell lines deposited with the WiCell Stem Cell Bank are karyotyped internally, and WiCell Characterization additionally performs genetic testing for outside organizations. We performed a retrospective analysis on karyotype data collected over the course of a decade, (including more than 15,000 hPSC cultures). This analysis enabled us to identify striking shifts in relative frequencies of recurrent abnormalities; namely, dramatically increasing rates of chromosomes 1 and 20 gains at the expense of chromosome 12 gains. Additionally, we identified the minimal amplicon for all chromosome 1q gains as chromosomal band (segment) 1q32.1, suggesting that this region harbors the driver gene(s) that give this recurrent aberration its advantage in culture.
Overall, these results show that quality screening strategies in use today are variable, and largely insufficient. Based on this data, we can assume that a substantial percentage of materials used in investigator laboratories have unidentified quality issues that will impact research. The reliability and reproducibility of data gained through experimentation is dependent on maintaining normal, consistent cell cultures. Changes in genetic composition can have dramatic impacts on cell function, and therefore experimental results. This underscores the need for routine testing prior to initiating and following studies, particularly genetic analysis to assure cell line stability.
Erik McIntire, CG(ASCP)CM is the former Lead Cytogenetic Technologist at WiCell Research Institute, where he spent ten years performing pluripotent stem cell (PSC) genetic characterization via G-banded karyotyping, fluorescence in situ hybridization (FISH), spectral karyotyping (SKY), and chromosomal microarray (CMA). He received his Bachelor of Science in Cell and Molecular Biology from Winona State University and completed a postbaccalaureate cytogenetics program at Mayo Clinic. His research focuses on recurrent acquired genetic aberrations in PSC, in particular the identification of minimal overlapping regions as well as the mapping of relative frequencies of individual aberrations over time. He has presented research findings at several scientific conferences, including those hosted by the International Society for Stem Cell Research (ISSCR) and the International Stem Cell Initiative (ISCI). He is currently pursuing his PhD in Human Genetics at the University of Chicago and can be reached at firstname.lastname@example.org.