LAUSR

Background & Aim Induced pluripotent stem cell (iPSC) research is rapidly moving towards translational and clinical applications.These applications require robust and consistent workflows that utilize high quality reagents that are xeno-free.When commonly used animal-origin components are replaced with xeno-free alternatives, performance often suffers,thereby necessitating thorough qualification of reagents and the process.Methods that enhance consistency will minimize extra effort and costs associated with clones that fail to expand,or do not meet quality standards for downstream use. Methods, Results & Conclusion Previously,we reported the first off-the-shelf reprogramming kit specifically designed for clinical and translational research.To streamline iPSC generation and ensure the consistent creation of high quality iPSCs,xeno-free workflows for T cells were optimized to minimize the effect of donor to donor variability.Upfront phenotyping of the T-cells prior to reprogramming was performed,along with optimization of a combination of conditions including hypoxia,matrix,and seeding density.These optimizations were shown to offer consistent iPSC generation from difficult to reprogram donor cells,albeit with varying efficiencies.To confirm the parental cell type,T-cell derived iPSC clones were analyzed for immune repertoire,leveraging a next-generation sequencing based assay.The resulting iPSC lines were also confirmed to be foot-print free,authenticated to be derived from the corresponding donor cells,HLA profiled,and were further subjected to comprehensive characterization methods to assess the quality and safety profile.Pluripotency and differentiation potential of iPSC clones was confirmed using ScoreCard,a focused qPCR panel,and PluriTest,an array-based global gene expression platform.Clones qualified to be pluripotent and possess trilineage differentiation potential were thoroughly investigated for genomic stability.Each of the clones tested showed a normal karyotype using both traditional G-banding as well as array-based methods KaryoStat and KaryoStat HD.Further assessment of oncogenic hotspot mutations that are known to occur at a higher frequency in ESC and iPSC,including TP53,indicated the absence of oncogenic structural variants in all the clones tested.The combination of qualified reagents and defined xeno-free workflows, in combination with comprehensive and predictive characterization assays aids in easy transition of early investigational work towards translational and clinical research.