Enhancement of insulin-producing cell differentiation from embryonic stem cells using pax4-nucleofection method

Figure 1 Flow chart of experiments. Culture medium was changed with glucose (25 mmol/L) and LY294002 (10 μv) supplement 24 h before examination and 8, 12 and 18 d after experiment.

Figure 2 Nucleofection of EB-dissociated cells. A: EB-dissociated cells after four days by hanging drop; B: eGFP fluorescent view (Bar= 50 μm in A, B); C: Morphology of EB-dissociated cells 56 h after nucleofection; D: eGFP filter view; E: DsRed view and the merge image (Bar = 5 μm in C-E); F: Efficiency of gene delivery; G: Latency of gene examination in ES cells after nucleofection. The ratio of DsRed-expressed cells was counted by random photograph 8, 12 and 18 d after experiment.

Figure 3 Relative mRNA level of relative gene expression 4 (before nucleofection), 8, 12 and 18 d after experiment. The highest expression was 100%. Genes such as oct-4, sox-17, foxa 2, mixl 1, pdx-1, insulin 1, glucagons, somatostatin and GADPH were detected.

Figure 4 Efficiency of IPC formation. A: Ratio of DsRed- expressed cells 4 (before nucleofection), 8, 12 and 18 d after experiment in the groups of cells with pax-4, mock and pax-4 siRNA plasmid; B: Insulin secretion in medium detected by ELISA; C: Insulin content in cells after 24 h incubation with different concentrations of glucose (5 mmol/L, 15 mmol/L or 30 mmol/L) and with/without 10 μmol/L Tolbutamide treatment (T).

Figure 5 Normal normoglycemia restoration in STZ- pretreated SCID mice after cell transplantation. The concentration of blood glucose was monitored every two days.