Figure 3: The PEL cells provide paracrine support to their clonallyrelated hESCs by activinA-SMAD pathway. (A) Efficient trypsin dissociation and expansion of pluripotent hESCs under the defined culture conditions. hESCs were treated with trypsin and dissociated into single cell suspension. These single cells were then allowed to seed. Undifferentiated hESC colonies, as indicated by Oct-4 (red) expression, appeared 4-7 days after seeding. White arrows point to a single hESC (day 1 after seeding) and a single-cell-derived hESC colony (day 2-4 after seeding), which are shown at higher magnification in the insets. (B) Comparison of the clonal efficiencies (as assayed by the expansion of single hESCs in a miniwell into Oct4+ colonies [CFUs]) under various culture conditions, including previous conditions employing MEF or HF feeders, MEF- or HF-CM, or high dose of bFGF (100 ng/ml) and/or Noggin (500ng/ml) suggests that the defined conditions require the presence of irradiated clonally-related PEL cells or those cell conditioned media (PEL-CM) to support clonal expansion of single undifferentiated hESCs. The undifferentiated state of representative isolated eGFP+ hESC colonies, expanded from a sorted single cell deposited into a single miniwell in the presence of PEL cells or PEL-CM, is indicated by its Oct-4 expression. DAPI (blue) also visualizes the pre-existing irradiated wild type hESC-derived supporting PEL cells that are eGFP and Oct-4 negative. The clonal origin of isolated eGFP+ hESC colony is confirmed by the presence of a single viral insertion site for the transgene (arrow; Southern blot). Unengineered undifferentiated hESCs were used as a control. (C) Western blot analysis of HF- and PEL-CM confirm the expression of a group of proteins specifically detected in PEL-CM but not in HF-CM by proteomic profiling using MudPIT. (D) Clonogenicity analysis indicates that activin-A (50 ng/ml) supports the clonal growth of hESCs in an efficiency comparable to that of PEL-CM in the defined culture system. (E) Addition of specific inhibitors of the activin-A receptors (ActRIIA/B) or its signaling pathway (Smad2/3), including ligand neutralizing antibodies (anti-activin-A), soluble ActrIIA/B–FC receptors, or SB-431542, into PEL-CM inhibits its ability to support the clonal growth of undifferentiated hESCs.