Objective: The duplication of chromosome 20q11.21 is the most recurrent genomic alteration in hPSCs, affecting up to 20% of lines during long-term culture. Given that over 115 clinical trials worldwide use hPSC-derived cells, ensuring genomic integrity through precise characterization of such recurrent alterations is critical. This duplication is also observed in various cancers and linked to features of neoplastic progression. Despite its frequency, the structural diversity and functional consequences remain poorly understood. We aim to characterize its architecture and assess its impact on pluripotency and neural differentiation.

Methods: fifteen hPSC lines with a 20q11.21 duplication previously identified by SNP array were analyzed. SNP array data provided initial information on duplication size and breakpoint variability across lines. Fluorescence in situ hybridization (FISH) was used to determine the chromosomal insertion site of the duplicated segment. Optical Genome Mapping (OGM) is currently underway to gain high-resolution structural insights, including duplication orientation and exact breakpoint positions. Pluripotency was assessed by morphology and expression of core markers. Neural differentiation was evaluated through expression of neuronal markers following standard differentiation protocols.

Results: SNP array analysis revealed variability in duplication size and breakpoints. In the literature, the duplicated region is reported to range from 0.5 to 4.6 Mb, encompassing up to ~80 genes and regulatory elements. FISH analysis showed a tandem insertion at 20q11.21 in most lines, with one case presenting an ectopic insertion at 1p36. Mutant hPSCs showed typical morphology and core pluripotency markers. They self-renewed without exogenous FGF2, unlike controls. Upon differentiation, both mutant and control lines generated neurons expressing markers such as Tuj1, MAP2, DARPP32, and Calbindin. Preliminary OGM data confirm tandem head-to-tail structure and will further refine structural interpretation.

Conclusion: The 20q11.21 duplication is a recurrent, structurally diverse alteration that can confer a growth advantage in hPSC culture without impairing pluripotency or neural differentiation. SSNP arrays, FISH, and OGM provide complementary structural insights and should be part of quality control for clinical-grade lines, given >115 clinical trials using hPSC products.