Subculture of Germ Cell-Derived Colonies with GATA4-Positive Feeder Cells from Neonatal Pig Testes

Figure 3

Maintenance of GATA4-positive feeder cells and germ cell-derived colonies (GDCs) in subculture at passages 1 and 10. GDC cells (labeled red) in passages 1 and 10 were maintained in the colonies in passages 2 and 11 ((A) and (C)). Panels (B) and (D) are bright-field images of panels (A) and (C), respectively. GATA4 and PGP 9.5 double staining in neonatal pig testis tissue (E). Panel (F) shows nuclear staining of panel (E). Total testicular cells were stained with antibodies against GATA4 (green) and PGP 9.5 (red) (G). Nuclear staining and bright-field images are shown in panels (H) and (I). Panel (J) shows negative controls on feeder cells attached to the wells. Panels (K) and (L) show GATA4 staining of old feeder cells (OFCs) and fresh feeder cells (FFCs), respectively, in passage 2. White and yellow arrows indicate PGP 9.5- and GATA4-positive cells, respectively; green arrows indicate PGP 9.5- and GATA4-negative cells. Scale bars indicate 50 μm in panels (A)–(F) and 10 μm in panels (G)–(L). Panel (M) shows PCR results for GATA4 in OFCs and FFCs in passage 2.