methods can be designed to induce iN cells of specific neuronal subtypes. However, some bottlenecks remained such as (a) only up to 25% of the total iN cells were TH positive and the culture remained rather heterogeneous, (b) it is unknown whether the protocols will work on adult human fibroblasts and (c) it was left unexplored whether these iN cells can release dopamine upon stimulation. In addition, (d) the exact phenotypic characterization as midbrain-type neurons remained unclear. Another study by Caiazzo et al., published around the same time, reported that transduction of Ascl1, Nurr1 and Lmx1a (ANL) is sufficient to generate functional dopaminergic neurons in mouse and human fibroblasts [31]. This study showed that the three factors can give rise to vesicular monoamine transporter 2 (VMAT2), dopamine transporter (DAT), aldehyde dehydrogenase 1a1 (ALDH1A1) and calbindin positive neurons in TH-GFP knock in mouse embryonic fibroblasts (MEF). Those iDaN cells also have spontaneous pacemaking properties and released dopamine as confirmed by amperometric and dopamine HPLC measurements. The same transcription factor combination was shown to also convert human fetal and adult fibroblasts into iDaN cells albeit with a much lower efficiency. Similar to mouse iDaN cells, human induced dopaminergic neurons are also positive
