Abstract
The expression of four transcription factors (OCT3/4, SOX2, KLF4, and MYC) can reprogram mouse as well as human somatic cells to induced pluripotent stem (iPS) cells. We generated iPS cells from mesenchymal stromal cells (MSCs) derived from human third molars (wisdom teeth) by retroviral transduction of OCT3/4, SOX2, and KLF4 without MYC, which is considered as oncogene. Interestingly, some of the clonally expanded MSCs could be used for iPS cell generation with 30-100-fold higher efficiency when compared with that of other clonally expanded MSCs and human dermal fibroblasts. Global gene expression profiles demonstrated some up-regulated genes regarding DNA repair/histone conformational change in the efficient clones, suggesting that the processes of chromatin remodeling have important roles in the cascade of iPS cells generation. The generated iPS cells resembled human embryonic stem (ES) cells in many aspects, including morphology, ES marker expression, global gene expression, epigenetic states, and the ability to differentiate into the three germ layers in vitro and in vivo. Because human third molars are discarded as clinical waste, our data indicate that clonally expanded MSCs derived from human third molars are a valuable cell source for the generation of iPS cells.
Highlights
In 2006, groundbreaking research demonstrated that mouse somatic cells could be reprogrammed to a pluripotent state by transduction of four transcription factors (Oct3/4, Sox2, Klf4, and Myc) [1]
Generation of induced pluripotent stem (iPS) cells opened up a new avenue for the generation of patient-specific pluripotent stem cells, which are useful for drug screening, understanding the mechanisms of pathogenesis, and cell transplantation therapies [13,14,15,16]
We and other groups previously reported that mesenchymal stromal cells (MSCs) from teeth or third molars, which were usually discarded as clinical waste, showed high proliferation when compared with MSCs from bone marrow [21,22,23]
Summary
Plasmid Construction—Reading frame cassette A (Invitrogen) was introduced into the EcoRI site of the pMXs retroviral vector [26]. HDF, Plat-A, and SNL feeder cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) containing 10% FBS, 100 units/ml penicillin, and 100 g/ml streptomycin. The day, pMXs retroviral vectors containing the open reading frames of OCT3/4, SOX2, KLF4, and DsRed-Express were transfected into Plat-A cells with FuGENE HD Transfection Reagent (Roche Diagnostics). Proliferation Rate of Parental Cells—Each MSCs and HDF was seeded at 1 ϫ 104 cells/6-well plate and cultured for 5 days. Small clumps of iPS cells were seeded on the PA6 cells in Glasgow minimum essential medium (Invitrogen) containing 10% knock-out serum replacement, 0.1 mM non-essential amino acids, 0.1 mM 2-mercaptoethanol, 100 units/ml penicillin, and 100 g/ml streptomycin. The following TaqMan primers and probes (TaqMan gene expression assays; Applied Biosystems) were used: -actin (Hs99999903_m1), OCT3/4 (Hs03005111_g1), SOX2 (Hs01053049_s1), NANOG (Hs02387400_g1), KLF4 (Hs00358836_m1), and P53 (Hs99999147_m1). Relative expression of genes of interest was estimated using the relative standard curve method
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