Abstract
Our previous study demonstrated that a Rho kinase inhibitor (Y-27632), in combination with irradiated fibroblast feeder cells, enables both normal and tumor epithelial cells from various tissues to proliferate indefinitely in vitro, without the need for exogenous viral or cellular gene transduction. These cells are referred to as Conditionally Reprogrammed Cells (CRCs). This approach has shown great promise for applications in regenerative and personalized medicine. In the present study, we first investigated whether CRCs could be genetically manipulated and stably express exogenous genes. Using lentiviral transduction of GFP, we successfully infected human foreskin keratinocytes (HFKs) cultured under CRC conditions. GFP expression was stably maintained over multiple passages and through freeze-thaw cycles. Similar stable GFP expression was also observed in CRCs derived from human tumor specimens, including neuroendocrine cervical carcinoma and prostate cancer. We next explored the use of GFP-labeled CRC-HFKs in co-culture systems with tumor-derived CRCs to evaluate colony formation and cellular heterogeneity. Our data demonstrated that CRCs from different individuals can form heterogeneous colonies, supporting the potential of CRC-based systems for modeling tissue development, regeneration, and tumor heterogeneity. To optimize the CRC platform for translational applications, we further assessed human fibroblasts as an alternative to the mouse feeder layer, using GFP-expressing CRCs as a functional readout. Finally, we demonstrated the capacity for gene knockdown in CRCs by successfully silencing p53 and HPV16 E6 in cervical cancer-derived CRCs using shRNA lentiviral vectors. These results collectively establish that the CRC method supports stable genetic manipulation and underscore its potential for mechanistic studies, disease modeling, and development of cell-based therapeutic strategies.