Fig. 2.
eGFP-transduced mMAPCs (28% transduction efficiency) were cultured on fibronectin-coated chamberslides sequentially for 7 days with 100 ng/ml bFGF, 10 ng/ml FGF8 and 100 ng/ml SHH, 10 ng/ml BDNF, and finally with E16 fetal mouse brain astrocytes plated on coverslips that were placed upside down in the chamberslides. After a total of 28 days, cells were fixed and stained. Slides were analyzed for the presence of GFP-positive cells and cells costaining with Cy3- or Cy5-labeled antibodies. (A) Cells labeled with antibodies against GABA and DDC. (A1-A3) Single fluorescence color analysis of cells stained with antibodies against GABA followed by secondary Cy3-coupled antibody, eGFP-labeled cells, and cells stained with antibodies against DDC followed by secondary Cy5-coupled antibody, respectively. (A4-A6) Overlay pictures of GFP/anti-GABA-Cy3, anti-GABA Cy3/anti-DDC-Cy5, and GFP/anti-DDC-Cy5, respectively. Shown is that GFP-positive cells acquired morphological and phenotypic features of GABA-ergic and dopaminergic neurons, whereas a fraction of cells with morphological and phenotypic features of GABA-ergic and dopaminergic neurons was GFP-negative. (B) Cells labeled with antibodies against TrH and dopamine. (B1-B3) Single fluorescence color analysis of cells stained with antibodies against TrH followed by secondary Cy3-coupled antibody, eGFP-labeled cells, and cells stained with antibodies against dopamine followed by secondary Cy5-coupled antibody, respectively. (B4-B6) Overlay pictures of GFP/anti-TrH-Cy3, anti-TrH-Cy3/anti-dopamine-Cy5, and GFP/anti-dopamine-Cy5, respectively. Shown is that GFP-positive cells acquired morphological and phenotypic features of serotonergic and dopaminergic neurons, whereas a fraction of cells with morphological and phenotypic features of serotonergic and dopaminergic neurons was GFP-negative.