Figure 4: Lysophosphatidic acid (LPA) accounts for much of KOSR activity in blocking Wnt-mediated differentiation.
(a) KOSR was filtered through different molecular-weight cutoff (MWCO) filters. The flow-through containing proteins smaller than the MWCO at their original concentration was added to 20% (v/v) in N2B27 media. The ability of Wnt3a to induce morphological changes after 3 days was used to determine which fraction contained the factor(s) that prevent Wnt-mediated differentiation. The >100 kDa is enriched for this size, but still contains all smaller proteins at ¼ of the original concentration. Phase-contrast images of hESCs on N2B27 treated with vehicle (Veh) or Wnt3a plus the indicated fraction of KOSR for 3 days are shown. Scale bar, 200 μm. (b) TCF-GFP hESCs were grown on N2B27 plus 20% PBS, 1.5% fatty acid-free BSA (FAF-BSA), 1.6% AlbuMaxII (Invitrogen) or 10 μM LPA supplemented with 1% BSA. Phase-contrast and GFP fluorescence images are shown for cells treated with Veh or Wnt3a for 3 days. Scale bar, 200 μm. (c) FACS analysis of cells in b showing the fraction of Wnthigh hESCs. (d) hESCs were grown in N2B27 media, except that irradiated feeders were used in lieu of Matrigel, for three passages in the presence of 1% FAF-BSA, 1 μM LPA or 10 μM LPA. Veh or Wnt3a was added fresh daily throughout. Alkaline phosphatase (AP) staining after three passages is shown. Scale bar, 500 μm. (e–g) The cumulative number of AP-positive colonies present after each of three passages in the indicated conditions are shown. The average ±s.d. of three biological replicates is shown.
