Conditional reprogramming: next generation cell culture

Abstract

Long-term primary culture of mammalian cells has been always difficult due to unavoidable senescence. Conventional methods for generating immortalized cell lines usually require manipulation of genome which leads to change of important biological and genetic characteristics. Recently, conditional reprogramming (CR) emerges as a novel next generation tool for long-term culture of primary epithelium cells derived from almost all origins without alteration of genetic background of primary cells. CR co-cultures primary cells with inactivated mouse 3T3-J2 fibroblasts in the presence of RHO-related protein kinase (ROCK) inhibitor Y-27632, enabling primary cells to acquire stem-like characteristics while retain their ability to fully differentiate. With only a few years’ development, CR shows broad prospects in applications in varied areas including disease modeling, regenerative medicine, drug evaluation, drug discovery as well as precision medicine. This review is thus to comprehensively summarize and assess current progress in understanding mechanism of CR and its wide applications, highlighting the value of CR in both basic and translational researches and discussing the challenges faced with CR.

Animal and Cell Culture Models for Cystic Fibrosis

“…Cell lines are commonly cancer derived or are immortalized by conventional methods, such as transformation with viral oncogenes, processes that can result in genome instability, karyotypic changes, and alterations in gene expression. 83,84 Immortalized cell lines also lack biochemical and mechanical cues and can lose expression of differentiation features (eg, immortal lung cell lines do not have polarity, tight junctions, mucus secretion, and cilia formation), 80,82 and as a consequence they poorly represent the in vivo situation. 85 Immortalized cells are typically used in monocultures, which does not reflect the heterogeneity of in vivo tissues, where many different cells interact with one another.…”

“…This has now been overcome by improvements in cell expansion techniques, such as conditional reprogramming, which allow cells to be passaged numerous times while still maintaining a normal cellular phenotype, morphology, and disease-specific functional characteristics upon ALI generation. 84,94 Furthermore, normal and CF airway epithelial cells can now be readily purchased from commercial suppliers or obtained from biobank repositories, avoiding the arduous process of gaining ethical clearances and performing patient tissue collection.…”

Sinking Our Teeth in Getting Dental Stem Cells to Clinics for Bone Regeneration

“…Immortalized cell lines have been established from different kinds of human and mouse dental stem cells, with most researchers focusing on immortalizing DPSCs [132][133][134], including SHED [130,135], while less work has been done on dental epithelial stem cells (HERS/ERM) so far [136][137][138]. Most often, viral oncogenes (e.g., simian vacuolating virus 40 (SV40) large T-antigen or E6/E7) are used to immortalize the cells of interest [139]. SV40 immortalization is effective in most cell types; it acts on the tumor suppressors p53 and Rb, while it also promotes the expression of the human telomerase reverse transcriptase (hTERT) to overcome telomer shortening [132,139].…”

“…Most often, viral oncogenes (e.g., simian vacuolating virus 40 (SV40) large T-antigen or E6/E7) are used to immortalize the cells of interest [139]. SV40 immortalization is effective in most cell types; it acts on the tumor suppressors p53 and Rb, while it also promotes the expression of the human telomerase reverse transcriptase (hTERT) to overcome telomer shortening [132,139]. However, viral oncogene immortalization is often associated with chromosome abnormalities, polyploidies, and changes in gene expression [132,139].…”

Application Research of Individualized Conditional Reprogramming System to Guide Treatment of Gastric Cancer

“…The i-CR system can screen out effective individualized drugs in a short time using the innovative technology of high connotation analysis and an associated detection system, which is rapid, efficient and has the capacity for high-throughput drug sensitivity detection in vitro. This system can nominate individualized chemotherapy regimens, which may both improve treatment effectiveness and lower costs (14)(15)(16). Thus, the i-CR system has good prospects in personalized cancer treatment and translational medicine (17), but it has not been applied in GC.…”