Feline Lindhout

Quantitative mapping of transcriptome and proteome dynamics during polarization of human iPSC-derived neurons 2 39 formation of lamellipodia and filopodia as well as axon guidance (Chou and Wang 2016). Clusters 4, 5, and 6 encompass proteins that are downregulated during the differentiation of NSCs into polarized neurons. GO analysis of these clusters reveals that they contain proteins involved in intracellular metabolism and homeostasis, genomic translation, the cell cycle, and biosynthesis of amino acids and peptides (Fig 2E). Downregulation of DNA replication and cell cycle-related proteins is also reported to coincide with terminal differentiation in neuroblastoma cells and with development of cultured rat neurons (Murillo et al. 2017; Frese et al. 2017). These results suggest that the overall proteome dynamics are indicative of cellular processes such as cell cycle exit and neuronal differentiation. Comparison of transcriptomic and proteomic profiles of developing human iPSC-derived neurons To compare the transcriptome and proteome dynamics, we performed correlative analysis of the relative RNA and protein expressions on day 7 compared to day 1 (Table 6). Based on their annotated gene names we were able to compare the expression dynamics of 7,021 factors. Of these, 4,536 followed the same trend for transcriptomic and proteomic expression changes, and overall, we found a significant correlation between the relative transcriptomic and proteomic expression profiles (Fig 2F). In agreement with the observed immunofluorescence, typical stem cell markers (NOTCH1, SOX2, MKI67, LIN28A, OTX2, and NES) showed a downregulation of both RNA and protein levels during neuronal differentiation. RNA levels as well as protein levels of typical neuron markers (DCX, ENO2, SYP, MAP2, STMN1, and TUBB3) and of axonal markers (TRIM46, MAPT, BASP1, ANK3, NCAM1, GAP43, and NFASC) displayed a marked increase during neuronal development (Fig 2G,H). Through quantitative analysis of transcriptomic and proteomic dynamics we were able to characterize human iPSC-derived neuronal differentiation and identify early neurodevelopmental processes in an unbiased manner. This quantitative map of neuronal transcriptome and proteome dynamics provides a rich resource for further analyses and may identify molecular mechanisms involved in neuronal polarity and axon specification. Identification and characterization of intermediate stages during axon specification Transcriptomic and proteomic profiling of developing neurons revealed that axonal components are upregulated after ~7 days, and assembled AIS structures were detected at proximal axons after ~14 days. Next, we studied the process of axon specification in human iPSC-derived neurons in more detail, and investigated the appearance of AIS proteins at different timepoints between day 5 and 11. In stage 2 neurons, in which neurites are unpolarized and have similar lengths, Trim46 and AnkG proteins appeared as punctate structures in one or more neurites in a subset of neurons (Fig 3A,B). Quantification of the relative abundance of stage 2 neurons lacking AIS proteins (referred to as stage 2a), or containing AIS proteins at one or more neurites (referred to as stage 2b), showed a developmental transition over time from stage 2a to stage 2b neurons (Fig 3A,B). Stage