Pericyte-like differentiation of human adipose-derived mesenchymal stem cells: An in vitro study

Figure 5 Double-labeling experiments of zonula occludens 1 (green immunofluorescence) and alpha smooth muscle actin (red immunofluorescence) in co-cultures of human retinal endothelial cells and human retinal pericyte cells or different groups of human adipose-derived mesenchymal stem cells. A: Photomicrographs show results obtained after 1 d of co-culture; B: Photomicrographs show results obtained after 4 d of co-culture. Photomicrographs A1 and B1 refer to cultures of human retinal endothelial cells (hRECs) alone, which were taken as a reference. In these cultures, only zonula occludens 1 (ZO-1) immunostaining is present showing typical localization at the level of plasma membranes of adjacent cells. On day 1, when hRECs were co-cultured with different groups of human adipose-derived mesenchymal stem cells (A2-A5) or human retinal pericyte cells (hRPCs) (A6), ZO-1 immunostaining appears more intense. A similar trend of ZO-1 immunostaining is recognizable after 4 d of co-culture (B2-B6). On day 1 of co-culture, an alpha smooth muscle actin (α-SMA) typical filamentous pattern is clearly detectable only in hASCs pre-cultured in basal medium (A2), pre-stimulated with transforming growth factor (A3), or pre-cultured in pericyte medium lacking pericyte growth supplement (A4). However, no α-SMA immunostaining is detected in hRPCs (A6) or in hASCs pre-cultured in pericyte medium (A5). On day 4, α-SMA immunostaining is also detectable in these two conditions (B5, B6). No α-SMA immunoreactivity is noticeable in A1 and B1, which refer to cultures of hRECs alone. Blue fluorescence indicates DAPI staining of cell nuclei. Scale bar: 50 μm. α-SMA, alpha smooth muscle actin; hASCs: Human adipose-derived mesenchymal stem cells; hRECs: Human retinal endothelial cells; hRPCs: Human retinal pericyte cells; TGF-hASCs, hASCs pre-stimulated with transforming growth factor; noPGS-hASCs: hASCs pre-cultured in pericyte medium lacking pericyte growth supplement; PM-hASCs: hASCs pre-cultured in complete pericyte medium; ZO-1: Zonula occludens 1.