Culture of hESCs in a defined medium designed to stimulate IGF1R/IR and ERBB2/3 signaling. (A) Low (4× objective) and high (40× objective) magnification phase contrast images of morphologically undifferentiated BG02 hESCs growing in DC-HAIF. Overlapping fields (dashed black borders) were aligned to image a representative large colony. Scale bar equals 100 and 25 μm, respectively. (B) Colony counting for serial passaging of CyT49 hESCs in different combinations of growth factors. Starter cultures were growing on MEFs in NCM and the proportion of undifferentiated (■) and differentiated (□) colonies at each stage are indicated. A combination of all 4 factors was necessary to enable long-term maintenance of hESCs. Similar results were obtained with repeated experiments and other hESC lines. Panel B and C abbreviations: H indicates 10 ng/mL HRG1β; A, 10 ng/mL ActA; I, 200 ng/mL LR³-IGF1; and F, 8 ng/mL FGF2. (C) Cell-counting analysis of the role of IGF1 and HRG in hESC proliferation using BG02 cells (left panel). The mean cell number/well before the different growth factor combinations were applied on day 1 is indicated (…). Cultures were disaggregated and counted on day 7, and the mean and standard deviation were plotted. Right panel shows OCT4/DAPI immunostaining of a duplicate repeated experiment (4× objective), which demonstrated that IGF1 and HRG significantly increased the proportion of OCT4⁺ cells compared with ActA/FGF2 conditions. Scale bar equals 50 μm. (D) Direct comparison of CM and DC-HAIF growth conditions with serial passaging. BG03 cells growing on MEFs were passaged to CM and DC-HAIF conditions in parallel (p0 plates). Triplicate cell counts were performed at p1 to p3 and the split ratio–corrected total cell number was plotted (mean ± SD). Split ratios were (CM, DC-HAIF): p0 (1:3, 1:3); p1 (1:2, 1:3); and p2 (1:2, 1:2). Similar results were obtained with repeat experiments. (E) Maintenance of markers of undifferentiated cells in BG02 DC-HAIF p5 cells compared with BG02 in CM by RT-PCR. (F) Positive immunofluorescence of hESC markers in BG02 DC-HAIF p5 cells (10× objective). Scale bar equals 50 μm. (G) Representative G-banding of BG02 DC-HAIF p26 cells. (H) RTK blotting analysis of BG01 DC-HAIF hESCs starved of growth factors overnight; starved, then pulsed with DC-HAIF for 15 minutes; or steady-state cultures are shown (left panel). The mean and range of normalized relative intensity is plotted (right panel).