Figure 1
Embryoid body differentiation and conditioned media collection. (A) Embryoid bodies (EBs) were cultured on a rotary orbital shaker at 40 rpm for 4, 7, and 10 days in serum-containing media, at which point the EBs were switched to serum-free media and cultured for an additional 2 days. After 6, 9, or 12 days of differentiation, EB conditioned media were collected for further analysis. (B) EBs cultured with (top row) or without (bottom row) serum were similar in size, shape, and morphology at each of the time points examined. Scale bars = 500 μm (panel) and 100 μm (inset). (C) The number of EBs remained relatively constant per culture (~5 × 103 EBs/plate) independent of the culture media used. (D, E) Similar EB differentiation in both media conditions was confirmed by polymerase chain reaction analysis, which revealed a significant decrease in the pluripotent marker Oct-4 by day 12 compared with day 6 and a significant increase in endoderm marker Afp by day 12 compared with day 6 for EBs cultured under both conditions. Results indicate the mean ± standard error (n = 6). Analysis of variance: *P < 0.05; Student t test: # t < 0.05. AFP, α-fetoprotein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Oct-4, Octamer-4.