Research Article

Fine Tuning of Hepatocyte Differentiation from Human Embryonic Stem Cells: Growth Factor vs. Small Molecule-Based Approaches

Figure 2

Growth factor-mediated differentiation of hESCs into definitive endoderm (DE). (a) OCT4-positive hESCs (H9) were subjected to DE differentiation for 3 days, using growth factors ( Activin A/ Wnt3a)  sodium butyrate (NaB) in RPMI-1640 media for the first day with the same media composition for additional 2 days except the concentration of NaB was reduced to (growth factors/NaB (3 d)). Same media composition was used for the second protocol except the duration of DE differentiation was 5 days (growth factors/NaB (5 d)). The third protocol (growth factors/NaB/DMSO (5 d)) was carried out for 5 days and had same media composition as above except the addition of 0.5%–0.25% DMSO. After 3 or 5 days of DE differentiation, the cells were fixed and photographed for phase images. The cells were then stained and imaged by a fluorescence microscope using antibodies against OCT4 for pluripotency and FOXA2 and SOX17 as DE markers. DAPI represents nuclear staining. . (b) The cells from all three DE differentiation protocols as explained above were collected on D0 (undifferentiated H9 cells) and D4/6 of DE differentiation and analyzed for mRNA expression by RT-qPCR for the indicated genes using gene-specific primers. The bars represent normalized (18S rRNA) fold mRNA expression with values of undifferentiated cells (D0) set as 1. The data are represented as . and .