Generation of the RCPCMi014-A and RCPCMi014-B lines from T-lymphocytes of a healthy donor and verification of their monoclonal origin by TCR V(D)J rearrangement analysis

The generation of induced pluripotent stem cells (iPSCs) from peripheral blood T-lymphocytes offers a promising alternative to skin fibroblasts. This approach combines minimally invasive sample collection with rapid isolation of enriched target cell population and provides a lower baseline mutational burden, as T-lymphocytes are shielded from chronic ultraviolet radiation – a major driver of somatic mutations in skin fibroblasts. Objective verification of monoclonality of the resulting iPSC lines is possible due to V(D)J rearrangements in T-cell receptor (TCR) genes, which are formed as a result of somatic recombination in the thymus during the natural maturation of T-lymphocytes. These rearrangements remain unchanged during reprogramming and serve as a unique marker, allowing for reliable identification of monoclonal lines and exclusion of contaminated or polyclonal lines. Reliable verification of the clonal origin of iPSCs can be crucial in experiments that place high demands on genetic homogeneity. This work is dedicated to the generation and comprehensive characterization of monoclonal iPSC lines derived from CD3+ T-lymphocytes of a healthy donor using episomal reprogramming. The resulting lines, RCPCMi014-A (PBM022E5) and RCPCMi014-B (PBM022E7), meet the accepted quality criteria for iPSCs: they demonstrate expression of key pluripotency markers (OCT4, SOX2, SSEA-4, TRA-1-81), the ability to differentiate into derivatives of all three germ layers, and possess a normal karyotype. Both lines showed a high-efficiency capacity to differentiate into definitive endoderm, as assessed by CXCR4 expression, highlighting their potential for developing protocols to generate pancreatic β-cells. The obtained iPSC lines can be used for fundamental research into the mechanisms of pluripotency and differentiation, as well as for creating isogenic iPSC lines with introduced mutations for modeling rare hereditary diseases.

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