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- Title
Generation of induced pluripotent stem cells by lentiviral reprogramming with six transcription factors.
- Authors
Idris, Izyan Mohd; Muhammad, Nur Jannaim; Mohamad, Rosnani; Nordin, Fatimah Diana Amin; Jalil, Julaina Abd; Ripen, Adiratna Mat; Tye Gee Jun; Ng Min Hwei; Nordin, Fazlina
- Abstract
Introduction: Induced pluripotent stem cell (iPSC)-based models using patient-derived samples are useful resources for studying disease mechanisms in-vitro, particularly in personalized medicine. iPSCs can be differentiated into any cell type in the body and be utilized for disease modelling and therapeutic drug screening. iPSCs can be generated using a combination of key transcription factors; OCT4, SOX2, NANOG, LIN28, KLF4 and C-MYC. This study examined the efficiency of generating iPSCs from three human dermal fibroblasts by integrative lentivirus (LV) reprogramming using 6 transcription factors. Methods: Human dermal fibroblasts were transduced with LV encoding transcription factors OSKMNL. Cells grown on inactivated fibroblast feeder layer in an embryonic stem (ES) cell medium for 20-27 days were subjected to daily morphological analysis. In a feeder-free culture system, ES-like cell colonies were picked and expanded by clonal isolation and further characterized by morphology and immunophenotyping for pluripotency and differential potential by embryoid body formation. Results: Transduction with LV cocktail of 6 transcription factors produced ES-like cell colonies. Morphological changes in transduced cells were seen as early as 9 days. ES-like colonies from both systems could expand and maintain ES-like cell morphology. ES-like colonies expressed pluripotent nuclear markers OCT4, SOX2 and NANOG, and surface markers TRA-1-81, TRA-1-61 and SSEA4. Expression of >90% of pluripotent markers SSEA4 and TRA-1-81 and <5% differentiation marker SSEA1 was identified by flow cytometry. IPSCs generated were able to form embryoid bodies and spontaneously differentiate into cells expressing markers of ectoderm (AFP), mesoderm (SMA) and endoderm (BIII-tubulin). Conclusion: We have successfully generated iPSCs using LV reprogramming of human dermal fibroblasts with a combination of 6 transcription factors. Generated iPSC-lines expressed pluripotency markers with minimal differentiation markers and can differentiate into cells from three embryological lineages. Future applications of the iPSC-cell lines include disease modelling and targeted therapeutics for inherited metabolic and genetic diseases.
- Subjects
INDUCED pluripotent stem cells; TRANSCRIPTION factors; CELL morphology
- Publication
Malaysian Journal of Medicine & Health Sciences, 2022, Vol 18, p30
- ISSN
1675-8544
- Publication type
Article