Distinct media requirements for dopaminergic neuron reprogramming from human glia and fibroblasts

Abstract

Direct reprogramming of somatic cells into induced dopaminergic neurons (iDANs) is a promising strategy for replacing degenerated dopamine neurons in Parkinson’s disease. One current bottleneck for translation of direct neuronal reprogramming is the low reprogramming efficiency and difficulty to acquire enough tyrosine hydroxylase (TH) positive iDANs. Here we systematically refine the culture conditions used for dopaminergic reprogramming of human stem cell-derived glial progenitor cells (GPCs) and adult dermal fibroblasts (DFs). Starting from a standard reprogramming medium containing base medium, growth factors and small molecules, we systematically compared three base media and removed individual small molecules to determine their specific contribution to reprogramming outcome. The GPCs and DFs were reprogrammed using established transcription factor-based strategies combined with REST inhibition. We found that the two starting cell types benefited from different culture conditions. For GPCs, B27/Neurobasal medium without small molecules yielded the highest neuronal and TH reprogramming efficiency, whereas DFs yielded the highest efficiencies in N2/DMEM/Neurobasal containing small molecules. Further characterization of the GPC-derived iDANs showed that these cells were functional, expressed key dopaminergic markers, and released dopamine upon stimulation. Together, these findings demonstrate that media composition is a key parameter influencing dopaminergic reprogramming efficiency across starting cell types in vitro.