339 INFLUENCE OF bFGF AND ACTIVIN A ON CAT FEEDER AND EMBRYONIC STEM CELLS.

Different feeder cells (FC) influence the isolation, proliferation, and self-renewal of cat embryonic stem cells (cat ESC; Gómez et al. 2010 Theriogenology 74, 498-515) possibly by secretion of growth factors that affect intracellular signalling pathways involved in self-renewal. Supplementation of the culture medium with fibroblast growth factor (FGF) stimulates the secretion of Activin A in mouse and human FC, which enhances undifferentiation in human ESC (Eiselleova et al. 2008 Int. J. Dev. Biol. 52, 353-363). Moreover, the Activin/Nodal pathway plays an important role in maintaining pluripotency of hESC through mechanism(s) in which FGF acts as a competence factor (Vallier et al. 2005 J. Cell Sci. 118, 4495-4509). Little is known about secretion of growth factors by cat FC and whether cat ESC use the activin/nodal pathway for their self-renewal. Our previous work has indicated that culturing cat ESC with bFGF enhances the stem cell replication and self-renewal (Gómez et al. 2010 Theriogenology 74, 498-515). Here we evaluated the effect of bFGF supplementation in the culture medium on the abilities of cat embryonic fibroblast (CEF) and mouse embryonic fibroblast (MEF) FC to: (1) secrete Activin A and (2) support undifferentiated growth of cat ESC. For experiment 1, mitomycin-C-treated CEF (n=2) and MEF (n=2) were, respectively, cultured with ESC medium supplemented with (1) LIF (1000IU), (2) bFGF (10ngmL(-1)), (3) LIF+bFGF, or (4) no factors. The medium for each condition was collected at 24h after culture and Activin A protein concentration was detected with a feline Activin A-ELISA kit. Results showed that supplementation of ESC medium with bFGF with or without LIF significantly increased the secretion of Activin A in MEF (5256 and 7048ngmL(-1), respectively; P<0.001), but not in CEF (150 and 131ngmL(-1), respectively). Moreover, differences in Activin A secretion were observed between both MEF cell lines (10269 v. 2034ngmL(-1); P<0.001). For experiment 2, cat ESC were cultured in CEF or MEF in the ESC medium supplemented with bFGF (10ngmL(-1)), LIF (1000 UI), and an inhibitor of glycogen synthase kinase-3 β (GSK3-b), SB 216763 (2.1µMmL(-1)). Results showed differences in morphology of cat ESC cultured in CEF or MEF, where colonies cultured in CEF had clearly defined borders and a tightly domed shape, with a high nucleus to cytoplasm ratio and prominent nucleoli. In comparison, ESC cultured in MEF had poorly defined borders and a flattened shape. In addition, the mean cell size of colonies at passage 8 (P8) cultured on CEF was larger (612±0.9µm) than that of those cultured on MEF (360±0.5µm; P<0.001). Colonies cultured on MEF differentiated into fibroblast-like cells and other noncharacterised cell types after P8. These results clearly indicated that CEF do not secrete Activin A. The negative effect of Activin A on the morphology of cat ESC cultured on MEF may suggest a synergism between GSK3b inhibitor and Activin A that may induce differentiation, possibly into mesoendodermal cells (Teo et al. 2014 Stem Cell Rep. 3, 5-14). Studies that evaluate the effects of supplementing ESC medium with a lower concentration of Activin A may help to elucidate the importance of the Activin/Nodal pathway in cat ESC.