To evaluate the reproducibility of the neuronal differentiation and to check if the indirect contact co-cultures produced pure neuronal batches, we performed mass spectrometry analysis. At day 56, we collected whole cell lysates from triplicate samples of two hiPSC-derived differentiations (namely line B1 and line B2). In line B1, 1384 proteins were detected in 3 replicates; in line B2, 1442 proteins were observed in 3 replicates. Overall the protein expression profiles between samples as analyzed by hierarchical clustering (Fig 6A), were very similar indicative of corresponding expression profiles between the different cell cultures. The average coefficient of variation of each sample normalized with respect to the loading was small, ranging between 0.11–0.14 (Fig 6B) indicating similarity of protein expression between replicates. Typical neuronal-specific proteins, such as MAP2, Beta-3 tubulin, Syntaxin, Synapsin, Synaptotagmin and Gephyrin were detected (S1 Table). Proteins were analyzed for differences in expression by using the average IBAQ values of the 3 technical replicates per line. This revealed the abundant presence (high IBAQ values) of presynaptic and growth cone proteins (Fig 6C) such as Synaptophysin, SNAP25 and Neuromodulin.
