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

Abstract

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 (1000 IU), (2) bFGF (10 ng mL–1), (3) LIF + bFGF, or (4) no factors. The medium for each condition was collected at 24 h 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 7048 ng mL–1, respectively; P < 0.001), but not in CEF (150 and 131 ng mL–1, respectively). Moreover, differences in Activin A secretion were observed between both MEF cell lines (10 269 v. 2034 ng mL–1; P < 0.001). For experiment 2, cat ESC were cultured in CEF or MEF in the ESC medium supplemented with bFGF (10 ng mL–1), LIF (1000 UI), and an inhibitor of glycogen synthase kinase-3 β (GSK3-b), SB 216763 (2.1 µM mL–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 ± 0.9 µm) than that of those cultured on MEF (360 ± 0.5 µm; 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.