Safety considerations in the generation of clinical grade autologous ips cell lines

      The Boston Autologous Islet Replacement Therapy (BAIRT) initiative seeks to generate pancreatic ß-cells from induced Pluripotent Stem Cell (iPS) lines for replacement therapy in diabetic patients. Our first goal was to develop a GMP-compliant protocol to manufacture autologous clinical grade iPS cell lines. T cells isolated from whole blood were chosen as the starting material since the T Cell Receptor (TCR) rearrangement in every mature T cell can serve as a unique genetic marker of each individual iPS cell line and all subsequent differentiated cells derived from this line. T cells were stimulated with soluble CD3/CD28 GMP antibodies and expanded in media containing IL-2 for 3 days. Activated T cells were reprogrammed using the cGMP Sendai virus Cytotune 2.1 kit (Thermo Fisher). To identify iPS lines suitable for potential clinical use, several assays were developed and used to monitor transgene elimination, genetic integrity (karyotype, OncoPanel), pluripotency state and clonal TCR rearrangement. First, qRT-PCR assays were used to monitor the kinetics of transgene elimination and revealed that 70% of the iPS lines were transgene-free by passage 16. Transgene-free iPS lines were subsequently tested for chromosomal abnormalities by G-banding karyotype. 75% of these lines were found to have normal cytogenetic profiles. DNA from these lines were then sent for targeted sequencing of 447 cancer-related genes (OncoPanel). Results were compared to donor T cell DNA to identify any mutations that might have arisen during the reprogramming process and subsequent expansion of individual iPS lines. Using this stringent assay to detect both nucleotide and copy number changes, 46% of iPS lines had no evidence of potential oncogenic mutations. TCR sequencing was used to confirm that these lines were generated from individual T cells and to establish the unique TCR sequence for each iPS line. Finally, iPS lines were confirmed to express pluripotency genes (Oct4, Sox2, Lin28, SSEA4, TRA-1-81, TRA-1-60) by qRT-PCR and/or flow cytometry. The process for reprogramming and iPS cell expansion used defined media throughout, without animal products. This rigorous, GMP-compliant protocol for deriving iPS cell lines will now be used to establish individual iPS Master cell banks for potential clinical use to generate autologous islet cells for transplantation in patients with diabetes and other conditions.
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