Development of a practical GMP-compliant manufacturing process for T cell-derived induced pluripotent stem cells.

We present a practical approach for implementation of a GMP-compliant manufacturing workflow for generation of autologous induced pluripotent stem cells (iPSC). In this method, activated, T cell-enriched peripheral blood mononuclear cells from a healthy male donor were reprogrammed using commercially available GMP-grade Sendai virus vectors. The process yielded 20 iPSC colonies, all exhibiting high expression of pluripotency markers (>85% Oct-4 and TRA-1-60, with less than 1% SSEA-1). T cell origin was confirmed in 18 out of 20 iPS lines through T cell receptor (TCR) β chain gene arrangement analysis. Among these, 14 iPS lines contained unique, clonally distinct TCR β chain gene sequences, indicating that they were derived from individual T cells. Notably, three lines appear to originate from T cells in which allelic exclusion had not occurred due to an initial unsuccessful β-chain rearrangement in one allele, resulting in clones with just 50% productive TCR β chain gene sequences. Cytogenetic analysis via G-banded karyotyping identified the presence of an extra Y chromosome in three lines, all traced back to the same parental clone. Targeted sequencing of approximately 500 cancer-associated genes (OncoPanel assay) revealed a recurrent deletion in the serine protease 1 gene (PRSS1) with loss of at least one allele across all T cell-derived iPSC lines that was also present in the donor's mature, nonreprogrammed T cells. PRSS1 gene is located within the TCR β locus and is frequently deleted during TCR gene rearrangement. After applying all quality control and release criteria, including assessments of sterility, identity, vector clearance and genomic integrity, we obtained 13 unique, T cell-derived iPSC lines deemed suitable for downstream clinical or translational applications. In addition to technical validation, we describe a practical approach to optimize the manufacturing workflow to improve operational efficiency within clinical environments. These included predefined process pauses to manage personnel and expiration-prone materials, thereby enhancing the feasibility and reproducibility of iPSC manufacturing in real-world GMP-compliant work environments.