원문정보
초록
영어
Induced pluripotent stem (iPS) cells have been generated from mouse and human somatic cells by etopic expression of transcription factors. iPS cells are indistinguishable from ES cells in terms of morphology and stem cell marker expression. Moreover, mouse iPS cells give rise to chimeric mice that are competent for germline transmission. However, mice derived from iPS cells often develop tumors. Furthermore, the low efficiency of iPS cell generation is a big disadvantage for mechanistic studies. Nonviral plasmid‐based vectors are free of many of the drawbacks that constrain viral vectors. The histone deacetylase inhibitor valproic acid (VPA) has been shown to improve the efficiency of mouse and human iPS cell generation, and vitamin C (Vc) accelerates gene expression changes and establishment of the fully reprogrammed state. The MEK inhibitor PD0325901 (Stemgent) has been shown to increase the efficiency of the reprogramming of human primary fibroblasts into iPS cells. In this report, we described the generation of mouse iPS cells devoid of exogenous DNA by the simple transient transfection of a nonviral vector carrying 2A‐peptide‐linked reprogramming factors. We used VPA, Vc, and the MEK inhibitor PD0325901 to increase the reprogramming efficiency. The reprogrammed somatic cells expressed pluripotency markers and formed EBs.
목차
INTRODUCTION
EXPERIMENTAL PROCEDURES
Mouse Embryonic Fibroblast Isolation and iPS Cell Culture
Plasmid Preparation and Infection
AP Staining
Immunocytochemistry
Quantitative Real-Time Polymerase Chain Reaction (Real-Time PCR) Analysis
Embryoid Body Formation Analysis
Statistical Analyses
RESULTS
Induced Pluripotent Stem Cell Generation using a Nonviral Vector
Virus-Free Induced Pluripotent Stem Cells Express Pluripotency Marker Gene
Characterization of iPS Cells Generated using Nonviral Vectors
In Vitro Differentiation
DISCUSSION
REFERENCES