Human Amniotic Fluid Cells Form Functional Gap Junctions with Cortical Cells

Figure 1

Expression of connexins in human amniotic fluid (AF) cells. Figure 1(A): RT-PCR analysis of Connexin (CX) expression in AF cells at 15 to 35 weeks (wks) gestation. AF cells expressed CX26, CX43, CX45 in all gestation periods examined. GAPDH transcript and human HaCaT, NT2-D1, and NT2-N cells were used as internal and positive controls, respectively. NTC, No Template Control. HaCaT, Human keratinocyte cell line; NT2-D1, (NTera-2) human teratocarcinoma cell line and NT2-derived neurons (NT2-N). Figure 1(B): Western blot analyses confirmed the expression of CX43 protein in AF26 (top panel) and AF30 (middle panel) cells. Embryonic day 18 (E18) mouse brain (Br) and B-ACTIN (ACTB) were used as positive and internal controls, respectively. Figure 1(C) Immunocytochemistry further verified the presence of CX43 and CX26 proteins in AF cultures. CX43 expression (green) was detected as punctate staining at the perinuclear region and the cell membrane. CX43 appeared to be associated with golgi complex in the perinuclear region, as determined by golgin-97 (red) and CX43 double staining (a). The punctate staining pattern ((a), (c), (e)) demonstrated the dynamic translocation of CX43 protein from Golgi complex (a) to the cell membrane ((e), arrowheads). Unlike CX43, CX26 protein expression appeared limited to the perinuclear region (g). Nuclei were stained with Hoechst (blue). Panels (b), (d), (f) and (h) represent the corresponding phase contrast images of (a, c, e and g); respectively. Scale bar: 25 μm. Figure 1(D) Dye coupling assessment in AF cells. AF donor cells (AF26, (a); AF16, (d)) were preloaded with DiI (red) and calcein (green) and plated as single cell suspensions onto confluent monolayers of receiving AF cells. Calcein transferred from the donor AF cell to adjacent receiving cells, indicating the formation of functional gap junctions between individual AF cells within 4 hours. Scale bar: 50 μm.