For cell differentiation, FAP-specific iPS cells were cultured with serial changes of media as shown in Fig. 3A. To test whether FAP-specific iPS cells can differentiate into hepatocyte-like cells, we analyzed several markers via real-time PCR analysis on Day 5 (D5), D13 and D20 differentiated FAP-specific iPS cells (Fig. 3B). A decrease in expression of the pluripotency marker Oct3/4 was accompanied by differentiation of FAP-specific iPS cells. Expression of the endoderm marker Sox17 was observed on D5 differentiation and decreased gradually after the medium was changed to hepatic differentiation medium on D7 (Fig. 3B). The hepatic progenitor marker AFP and the mature hepatocyte marker ALB were obviously expressed on D13 and D20. In addition, immunocytochemical analyses showed Sox17 expression on D5, both HNF-4α and AFP expression on D13, and ALB cytoplasmic staining on D20 (Fig. 3C). Quantitative imaging analysis revealed that approximately 78 ± 0.6% of cells were Sox17-positive on D5 and approximately 88 ± 1.1% of cells were AFP-positive on D13 and approximately 29 ± 0.9% of cells were ALB-positive on D20 (Fig. 3D). The ALB secretion in the media of differentiated FAP-specific iPS cells on D20 was approximately 20 μg/ml (Fig. 3E). Moreover, these D20 differentiated FAP-specific iPS cells were also periodic acid-Schiff (PAS)-positive, indicating cytoplasmic glycogen storage (Fig. 3F). These results clearly indicated that FAP-specific iPS cells had the potential to differentiate into hepatocyte-like cells.
cells
Sox17-positive on D5
approximately 78 ± 0.6%
%
cells
AFP-positive on D13
approximately 88 ± 1.1%
%
cells
ALB-positive on D20
approximately 29 ± 0.9%
%
ALB secretion
approximately 20 μg/ml
μg/ml