Genomic Characterization and Species Identification of Commercial Human Primary Cell Lines Through Multi-Modal Authentication

Background: Accurate determination of cell identity is crucial for the reproducibility and validity of biomedical research. The species identity of two commercially obtained primary cell preparations labeled as human neuronal cells (hNC) and human gallbladder cells (GBC) was investigated. Methods: Cell identity was determined using a multimodal authentication strategy comprising morphological examination, independent short-tandem repeat (STR) profiling, and RNA sequencing (RNA-seq). To further characterize the hNC preparation, RNA-seq reads were mapped to the Cricetulus griseus reference genome (GCF_003668045.3_CriGri-PICRH-1.0), followed by analysis of hamster-associated non-coding RNAs and canonical neuronal markers. Results: Independent STR profiling failed to yield valid human STR profiles for either preparation. RNA-seq revealed that approximately 91–92% of reads from the hNC preparation mapped to Cricetulus griseus, whereas the GBC preparation exhibited genomic signals from multiple species. Transcriptomic reanalysis of the hNC preparation showed abundant expression of the hamster-associated long non-coding RNA Adapt15 (gadd7) and detectable expression of Mir6092. In contrast, canonical neuronal markers, including Map2, Rbfox3, Snap25, Syp, Nefl, Nefm, and Nefh, showed very low or undetectable expression. Conclusions: These findings identified substantial discrepancies between the labeled identities and the independently determined molecular characteristics of commercially obtained primary cell preparations. The findings highlight the limitations of relying solely on supplier-provided authentication documentation and underscore the importance of independent, orthogonal authentication using complementary genomic and transcriptomic approaches to ensure experimental reproducibility and data integrity.