Figure 1
Sendai Viral Reprogramming in small scale.
A. Flow chart for the optimization from conventional methods to current method. Four main aspects were improved to improve reprogramming efficiency and handling capacity in a regular lab setting. B. Confirmation of the enhancement of butyrate in Sendai Viral reprogramming. Two control fibroblast lines (ATCC: PCS201-012 and CCD1079) were reprogrammed with the CytoTuneTM 2.0 Sendai Viral Reprogramming kit and then reprogrammed in medium with or without 100 uM Sodium Butyrate. The iPSC Colonies were counted after 25 days. Experiments were done in triplicate (PCS201-012, p < 0.0015; CCD1079, p < 0.023). C. Viral activity conservation after freeze/thaw cycles. GFP-control Sendai virus was thawed and then refrozen for three times and a portion of virus was taken at each cycle to apply to fibroblasts (PCS201-012). GFP expression in the population was analyzed two days after transduction and the transduction efficiency was normalized by virus without the freeze/thaw cycle (control). The results were confirmed in another line CCD1079 (data not shown). Experiments were done in triplicate (3x vs control, p < 0.001). D. Comparison of CytoTuneTM 1.0 and CytoTuneTM 2.0 kits. The three patient samples were transduced by two kits respectively with MOI 3 and the cells were then plated and cultured by the same method in 6 well plate (Fig. 1A) and the iPSC cells were stained by APS staining 25 days after transduction. E. Reprogrammed cells plated on Matrigel. The cells transduced in 1D were plated in a 48-well plate and cultured and stained by the same procedure.
