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

Area of Science:

• Stem Cell Biology
• Cellular Reprogramming
• Good Manufacturing Practice (GMP)

Background:

• Autologous induced pluripotent stem cells (iPSCs) hold promise for regenerative medicine.
• Establishing GMP-compliant manufacturing workflows is crucial for clinical translation.
• Reprogramming peripheral blood mononuclear cells (PBMCs) offers a viable source for iPSC generation.

Purpose of the Study:

• To develop and validate a GMP-compliant manufacturing workflow for autologous iPSC generation from T cells.
• To assess the quality, identity, and genomic integrity of the generated iPSCs.
• To optimize the workflow for enhanced efficiency and reproducibility in clinical settings.

Main Methods:

• Reprogramming of T cell-enriched PBMCs using GMP-grade Sendai virus vectors.
• Characterization of iPSC colonies for pluripotency markers (Oct-4, TRA-1-60, SSEA-1).
• Confirmation of T cell origin via T cell receptor (TCR) β chain gene rearrangement analysis.
• Cytogenetic analysis (G-banded karyotyping) and targeted sequencing (OncoPanel assay) for genomic integrity.
• Implementation of process pauses for workflow optimization.

Main Results:

• Generation of 20 iPSC colonies with high pluripotency marker expression.
• Confirmation of T cell origin in 18/20 iPSC lines, with 14 unique clonally distinct lines.
• Identification of a recurrent PRSS1 gene deletion in T cell-derived iPSCs, also present in donor T cells.
• Obtained 13 unique T cell-derived iPSC lines meeting all quality control and release criteria.
• Demonstrated workflow optimization strategies for improved operational efficiency.

Conclusions:

• A practical GMP-compliant workflow for autologous T cell-derived iPSC generation was successfully established.
• The generated iPSC lines are suitable for downstream clinical and translational applications.
• Workflow optimization enhances the feasibility and reproducibility of iPSC manufacturing in clinical environments.