Differentiation of ReNcell VM into a dopaminergic neural phenotype. A) Using the preD protocol there is a significant increase in TH and βIII-tubulin protein expression as assayed by western blot (- undifferentiated, + differentiated with α-tubulin as a loading control). B) Following preD (without cAMP/GDNF), the cells are gathered in clusters which are strongly labelled with βIII-tubulin (green). TH (red) positive neurons are also evident but the clustering makes colocalization difficult to differentiate (data not shown). Ci, ii, iii) ReNcell VM preD in the presence of cAMP/GDNF shows a more even distribution of neuronal differentiation positive for βIII-tubulin (Ci, green) and TH (Cii, red), with the overlay showing co-localization of the two antigens (Ciii). Di, ii, iii). A higher resolution image, under the same conditions as C, show individual neurons stained with βIII-tubulin (Di, green) and TH (Dii, red), with the overlay showing co-localization (Diii). Scale bar: 100 μm.