Cell line misidentification and contamination have become important concerns for researchers. A recent Science article indicated that in some cases, laboratories have invested substantial time and effort researching cell lines that were revealed to be misidentified(1)
. The situation has prompted the U.S. National Institutes of Health to issue a notice strongly urging researchers to authenticate cultured cells used in their laboratory(2)
The StemElite™ ID System uses fluorescently labeled short tandem repeat (STR) genotyping, a mainstay of human forensic DNA profiling, to identify cells and detect human cell mixtures. The STR loci amplified by the StemElite™ ID System have been carefully selected to give optimum discrimination between cell lines, generating a random match probability of 1 in 2.92 x 109 for human cell lines not derived from a common donor line. The system also includes a mouse locus to detect contamination from mouse feeder layers. Confirmation of the identity and purity of a cell line using the StemElite™ ID System avoids wasted time and labor performing experiments on contaminated or misidentified cells.
Methods and Results
Cell Line Identification: StemElite™ ID System Results for Human Embryonic Stem Cells
Cell lines can be distinguished from one another by analyzing multiple STR loci. With each additional locus, the probability of the DNA profile of one cell line matching that of another decreases if the cells are not derived from the same parent cell line. The loci used in the StemElite™ ID System are those used in existing databases (e.g., American Type Culture Collection [ATCC]) with the additional locus D21S11 to increase overall resolving power. Genotypes generated using the StemElite™ System can be compared to those in existing databases or previously generated data to confirm cell line identity.
Figure 1 shows a genotype for the H9 Human Embryonic Stem Cells (hESC), generated using the StemElite™ ID System. DNA was isolated from 1 × 104 hESC using the Maxwell® Cell LEV DNA Purification Kit (Cat.# AS1140) and quantitated using the Quant-iT™ PicoGreen® dsDNA Reagent (Invitrogen, Cat.# P7589). Two nanograms of DNA was amplified with the StemElite™ ID System (Cat.# G9530), amplified products were analyzed using a 3130xl Genetic Analyzer (Applied Biosystems), and a genotype was generated.
Using genotyping software (GeneMapper® ID, Applied Biosystems), allele designations were assigned to the sized peaks within the allelic ladder. The software then compared the sample peaks with allelic ladder peaks and assigned an allele to each sample peak. The sample alleles are listed in a genotype table (Figure 2). The StemElite™ ID genotype was compared to a genotype for this cell line published by WICell and the National Stem Cell Bank. The genotypes matched, and the identity of the H9 hESC line confirmed.
Mouse Cell Contamination: Detection of Mouse Feeder Cells in Human Stem Cell Culture
Human embryonic stem cell lines are often grown on mouse feeder cells. During passaging, the mouse feeder cells can be inadvertently transferred with the hES cells. Future applications will require pure lines free from mouse contamination. The StemElite™ ID System gives a qualitative measure of mouse cell contamination, indicated by the presence of a single peak in the electropherogram (Figure 3). Detection of mouse cells is then indicated on the genotype table.
The sensitivity of the StemElite™ ID System to detect mouse contamination was tested by mixing a human immortalized cell line (HEK293) with increasing amounts of mouse cells (NIH 3T3). DNA was isolated using the Maxwell® Cell LEV DNA Purification Kit and quantitated using Quant-iT™ PicoGreen® Assay. DNA was amplified using the StemElite™ ID System, and the products analyzed. The results in Figure 4 show that mouse DNA was detected in cell mixtures contaminated with 1% mouse cells or more.
Human Cell Contamination: Detection of Human Cell Mixtures
Many labs culture multiple cell lines concurrently, creating a risk for potential cross-contamination of one cell line with another. In StemElite™ ID System reactions, contamination is indicated by the presence of additional alleles at multiple loci compared to the pure culture genotype. In the following example, cell line contamination is mimicked by mixing hESC with HeLa human adenocarcinoma cells, then isolating the DNA. Figure 5 shows the contamination can easily be identified by comparing the StemElite™ ID genotype from the contaminated H9 hESC sample with the genotype from a pure H9 hESC population (e.g., previous experiment, Figure 2, or published genotype from the National Stem Cell Bank).
The StemElite™ ID System genotypes human cell lines, including human embryonic stem cells, and detects contamination of those cell lines. Cell line identity can be confirmed by comparing the StemElite™ ID System-generated genotype with a known genotype standard. Mouse cell contamination is also easily detected by the presence of a mouse-specific locus in the genotype. In addition, the StemElite™ ID System is able to detect the presence of human cell mixtures and thus prevent waste of time and resourses working on contaminated cell lines.
Special acknowledgement to Clive Svendsen (University of Wisconsin—Madison) and Soshana Svendsen (Promega Corporation) who provided the human embryonic stem cells for analysis.