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. 2014 Oct 14;8:316. doi: 10.3389/fncel.2014.00316

FIGURE 5.

FIGURE 5

(A) Immature neuronal cells that were cultured in a Dopaminergic Neuronal Progenitor Medium began to show morphological signs of neuronal maturity between Day 29–50 (a,b,c) and after 6 months these cells displayed further signs of mature network formation (d). Importantly, control tail cells that were not subject to the reprogramming procedures kept their original shape at 6 months (e). As well, during this time, expression of the early immature neuronal cell marker, DCX, was greatly diminished (f), together with increased expression of the mature general neuronal cell marker, microtubule associated protein 2 (MAP2), (g), and specific dopaminergic neuronal marker, tyrosine hydroxylase (TH), (h) was evident. Further confirmation that the reprogrammed neurons were mature dopaminergic neurons was obtained using HPLC (i); wherein tail cells that were re-programmed (Tail-Dopamine cells) displayed significantly increased dopamine levels, compared to non-reprogrammed controls (Tail cells). Furthermore, dopamine levels in non-reprogrammed controls were, as expected, quite low (Tail tissue), whereas freshly dissected adult midbrain tissue had the expected highest concentration of dopamine (Brain tissue). *p < 0.05, relative to control tails. (B) Shows the time course for dopaminergic neuron derivation. The adult mouse (aged 2 months) fibroblasts were reprogrammed by vector 20866 with c-Myc, Klf4, Oct4, and Sox2 genes and an mOrange marker was used to detect gene expression after 2-weeks in cell culture (a). Induced pluripotent stem cells (iPSCs) began to show obvious outgrowths at Days 15 and 20 (b). The undifferentiated iPSCs were transformed into neural progenitor cells following culture in the Neural Induction Medium (see Materials and Methods) during Days 21–28 (c). The neural progenitor cells were further differentiated into dopaminergic neural progenitors with the Dopamine Neuronal Progenitor Medium from Days 29–35 (d). From Days 36 to 50, the cells were subsequently differentiated into mature dopaminergic neurons by culturing in the Dopamine Neuronal Differentiation Medium (e,f,g). The mature dopaminergic neurons were grown for up to 6 months (h), and were visibly easy to distinguish from the original un-reprogrammed adult fibroblasts from parallel control cultures (i).