A High-Throughput Method as a Diagnostic Tool for HIV Detection in Patient-Specific Induced Pluripotent Stem Cells...

Area of Science:

• Stem Cell Biology
• Translational Medicine
• Molecular Virology

Background:

• Induced pluripotent stem cells (iPSCs) offer patient-specific cell generation for therapeutic applications.
• Evaluating reprogramming methods is crucial for efficiency and pluripotency.
• Ensuring virus-free cell lines is essential for Good Manufacturing Practice (GMP) standards.

Purpose of the Study:

• To compare reprogramming efficiencies and pluripotency of iPSCs derived from different cell types (fibroblasts, PBMCs, MSCs) using various methods.
• To assess the suitability of high-throughput PCR-based methods for detecting human pathogenic viruses in iPSCs.
• To validate a specific high-throughput system for HIV-1 detection in iPSC lines.

Main Methods:

• Generation of iPSCs from skin fibroblasts, PBMCs, and MSCs using integrating and non-integrating reprogramming systems (STEMCCA, Sendai virus, episomal plasmids).
• Quantification of reprogramming efficiencies based on colony formation.
• Detection of HIV-1 contamination in iPSCs using the high-throughput cobas® 6800/8800 system.

Main Results:

• Reprogramming efficiencies varied significantly based on parental cell type and reprogramming method, with MSCs showing the highest efficiency using episomal plasmids.
• All generated iPSCs exhibited similar pluripotency characteristics.
• The cobas® 6800/8800 system effectively detected HIV-1 in iPSCs generated by footprint-free methods (episomal plasmids, Sendai virus).

Conclusions:

• Parental cell type and reprogramming strategy critically influence reprogramming efficiency.
• High-throughput PCR-based assays are feasible and effective for detecting pathogenic viruses in iPSCs generated via non-genome integrating methods.
• This approach supports the development of safe, patient-specific iPSC lines for clinical applications.