Tracing genomic instability in induced mesenchymal stromal cell manufacture: an integration-free transfection approach.

Here we systematically investigated genomic alterations from the initiation of induced pluripotent stem (iPS) cell generation to induced mesenchymal stromal/stem cell differentiation. We observed a total of ten copy number alterations (CNAs) and five single-nucleotide variations (SNVs) during the phases of reprogramming, differentiation and passaging. We identified a higher frequency of CNAs and SNVs in iPS cells generated using the Sendai virus (SV) method compared with those generated with episomal vectors (Epi). Specifically, all SV-iPS cell lines exhibited CNAs during the reprogramming phase, while only 40% of Epi-iPS cells showed such alterations. Additionally, SNVs were observed exclusively in SV-derived cells during passaging and differentiation, with no SNVs detected in Epi-derived lines. Gene expression analysis revealed upregulation of chromosomal instability-related genes in late-passage SV-iPSCs, further indicating increased genomic instability. Notably, TP53 mutations were identified, underscoring the vulnerability of the gene and the critical need for careful genomic scrutiny when preparing iPS cells and derived cell lines.