International Journal of Stem Cells : eISSN 2005-5447

Fig. 1.

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Fig. 1. Mbd3 is required for Episomal Reprogramming. (A) Experimental design used to address the kinetics and efficiency of reprogramming in human dermal fibroblasts. Human fibroblasts were transfected with episomal plasmids encoding pCXLE-hOct3/4-shp53, pCXLE-hSK (Sox-2 and Klf4) and pCXLE-hUL (L-Myc and Lin28) (Reprogramming factors-RF) or RF+shpMbd3 or RF+Mbd3b or GFP only. After transfection, the cells were maintained in Essential 8 media supplemented with 0.5mM sodium butyrate until day 12 and thereafter maintained in Essential 8 media only. Transfection efficiency was measured by Flow Cytometry after a minimum of 24 hours of transfection with an average of 32.8±1.68%. (B) Immunoblot analysis for Mbd3 expression transfected with shpMbd3 compared to sham. Densitometry plot shows that cells transfected with shpMbd3 had 50% reduction in expression of Mbd3 compared to sham (p<0.05). (C) iPSC colony formation in fibroblasts was assessed by alkaline phosphatase (AP) staining between day 25~30 and the results showed that shpMbd3 reduced the number of iPSC colonies. (D) Quantification of AP staining further confirmed reduction of iPSC colonies formed in cells treated with RF+shpMbd3 compared with RF in patient fibroblasts RPD1 and PD1 and BJ neonatal cell line (n=3). Colony number is per 1×106 fibroblasts (p<0.05).
International Journal of Stem Cells 2018;11:235-41
© 2018 International Journal of Stem Cells