Journal: Stem Cells International

Article Title: Comparative Analysis of Spontaneous and Stimulus-Evoked Calcium Transients in Proliferating and Differentiating Human Midbrain-Derived Stem Cells

doi: 10.1155/2017/9605432

Figure Lengend Snippet: Immunocytochemical characterization of proliferating (a, b, c) and differentiating human midbrain-derived stem cells (d, e, f, g, h, i). Immunofluorescence staining of cultures propagated for 4 days by exposure to epidermal growth factor and basic fibroblast growth factor was performed. Almost all cells expressed Ki67, a marker of dividing cells (a). Moreover, a large proportion of the cells expressed the intermediate filament protein nestin, a marker of neural progenitor cells (b). Only very few cells had spontaneously differentiated into β -tubulin III- ( β -tub III-) positive neurons (c). Cells were differentiated for 10 days by exposure to fibroblast growth factor 8 for three days followed by exposure to forskolin, sonic hedgehog, and glial cell line-derived neurotrophic factor for seven days and immunostained for neuronal and astroglial markers. At this time point, some cells expressed the early marker of migrating neuronal cell doublecortin (DCX) (d), whereas extensive staining for another early neuronal marker, β -tub III (e), and a mature neuronal marker, microtubule-associated protein 2ab (Map2) (f), was seen. A large proportion of cells expressed tyrosine hydroxylase (TH), a marker of catecholaminergic neurons (i), whereas only few cells were found to express γ -aminobutyric acid (GABA), a marker for GABAergic neurons (h). Very few cells were found to express glial fibrillary acidic protein (GFAP), a marker of astroglial cells (g). Scale bars = 50 μ m.

Article Snippet: In brief, cells were plated in PLL-coated 24-well culture trays (Nunc) at a density of 5000 cells/cm 2 in a HNSC100 medium with sequential addition of 50 ng/ml recombinant fibroblast growth factor 8 (R&D Systems), 25 μ M forskolin (Sigma-Aldrich), 5 ng/ml recombinant glial cell line-derived neurotrophic factor (Promega, Madison, WI, USA), and 25 ng/ml recombinant sonic hedgehog (R&D Systems).

Techniques: Derivative Assay, Immunofluorescence, Staining, Marker