BackgroundDirectly induced neuronal cells from lineage conversion of somatic cells allow bypassing of the pluripotent-cell stage and, consequently, faster generation of neurons and more disease-specific modelling for neurological diseases. However, the efficiency rates of conversion are still too low for clinical application. This study tested and identified the unique contribution of new factors that could improve the conversion rates compared with existing protocols. MethodsHuman fetal lung (HFL) and embryonic fibroblast (EF) cell lines were used. Two cocktails of small molecules were used for conversion to either cortical neurons (MAP2 and vGLUT co-staining) or dopaminergic neurons (MAP2 and TH co-staining). Neurons were identified through immunofluorescence co-staining and morphology. The effect on conversion rates was measured with varying concentrations of astrocyte-conditioned media (ACM) as well as absolute and relative concentrations of the growth factors and SMAD proteins in the small-molecule cocktails (0%, 25%, 50%, 100%), oxygen levels for expansion (21% and 5%), and concentrations of retinoic acid (20 μM, 10μM, 5μM, 1μM). After transduction, all cells were incubated in 5% oxygen chambers. FindingsExpansion of cell lines at physiological oxygen (5% oxygen) led to faster expansion and higher conversion efficiencies to neuronal cells compared with expansion at 21% oxygen. 25–75% ACM promoted conversion to dopaminergic neurons but did not affect conversion to cortical neurons. Retinoic acid increased the purity of conversion to dopaminergic as well as to cortical neurons. However, it had varying effects on the efficiency of conversion: improving efficiency (HFL to dopaminergic neurons), no effect (EF to cortical neurons), or decreasing efficiency (HFL to cortical neurons and EF to dopaminergic neurons). Optimal small-molecule cocktail concentrations were specific to cell lines and to induced cell type (eg, lower absolute concentrations promoted conversion of HFL to dopaminergic neurons and conversion of EF to cortical neurons). InterpretationACM and early physiological oxygen exposure improved efficiencies of conversion from human fibroblasts to directly induced neurons. Retinoic acid consistently improved the final purity but not necessarily the efficiency of conversion to neurons, which suggests that it could be a useful agent to introduce later in the conversion process to optimise both purity and efficiency in future experiments. The results in this study show the first technique, to my knowledge, with clinically viable conversion rates for disease modelling and therapeutics. FundingNone.