Research Associate Kalbe Farma Tbk, Jakarta Raya, Indonesia
Abstract: The integration of stable transgenes into induced pluripotent stem cells (iPSCs) is essential for their application in research and therapy. However, conventional methods often result in random transgene insertion, leading to variable expression and instability over prolonged culture. To address this, we generated iPSCs expressing green fluorescent protein (GFP) by using the CRISPR-Cas9 system to target the AAVS1 safe harbor locus. This approach ensures precise integration and long-term transgene stability, providing a robust platform for studying gene expression consistency during extended cell culture. We successfully targeted the AAVS1 locus and inserted a donor plasmid containing a GFP transgene and a puromycin resistance gene using CRISPR-Cas9. Following puromycin selection, the selected clones were expanded and characterized to confirm their pluripotency. The genome-modified iPSCs (GM-iPSCs) displayed compact colonies with a high nucleus-to-cytoplasm ratio, distinct borders, and well-defined edges, consistent with the morphology of pluripotent stem cells. In addition to these morphological characteristics, the selected clones also expressed GFP. Immunocytochemistry analysis confirmed that the GM-iPSCs were positive for pluripotency markers TRA-1-60, OCT3/4, and SSEA4. Additionally, RT-qPCR demonstrated the expression of core pluripotency genes, including OCT4, SOX2, NANOG, LIN28A, REX1, and E-CAD. The potential of GM-iPSCs to differentiate into all three germ layers was confirmed through positive staining and gene expression of FOXA2 (endoderm), TBXT (mesoderm), and PAX6 (ectoderm). Notably, at passage 10, GM-iPSCs continued to express the GFP protein, confirming the stability of the transgene. These results collectively validate the successful establishment of GM-iPSCs with preserved pluripotency and differentiation potential. This work demonstrates a precise and reliable strategy for stable transgene integration, providing a robust platform for long-term gene expression studies and advancing applications in regenerative medicine and genetic research.
Funding Source: This research is fully funded and supported by PT. Kalbe Farma Tbk.
