Feline Lindhout

Quantitative mapping of transcriptome and proteome dynamics during polarization of human iPSC-derived neurons 2 37 (Table 3). The dynamic shift in the transcriptome reflects a population-wide transformation from proliferating cells to terminal differentiating cells with intrinsic neuronal properties, and highlights changes in themicrotubule cytoskeleton expression profiles at the onset of stage 3. Proteomic profiling of developing human iPSC-derived neurons In addition to transcriptome analysis, we assessed gene expression on the translational level by performing mass spectrometry based quantitative proteomics. We collected samples for proteome analysis on the same days as described above for RNA sequencing. We identified 7,512 proteins in two replicates and quantified 5,620 proteins across all three time points (Table 5, Fig S2C). Of these, 2,218 proteins showed a changed expression profile during differentiation and neurodevelopment. We assessed the global proteome changes Figure 1. Successful and protracted transition of early developmental stages in human iPSC-derived neurons A. Schematic illustration and timing of neurodevelopmental stages 1, 2 and 3 in human iPSC-derived NSCs/neurons. B. Representative images of stage 1 (day 1), 2 (day 5) and 3 (day 14) hiPSC-derived NSCs/neurons. Cells were subjected to FUGW-GFP lentivirus and immunostained for NSC markers Nestin and Ki67, or neuron markers β3-Tubulin and MAP2, or AIS markers AnkG and Trim46. Outline of cells was defined by the FUGW-GFP signal. Scale bar: 15 µm in overview, 5 µm in zooms. C. Quantifications of percentage of human iPSC-derived NSCs positive for Ki67 or Nestin at 1, 5 or 14 days in culture. N=2, n=100-109 cells. D. Quantifications of percentage of human iPSC-derived neurons positive for β3-Tubulin or MAP2 at 1, 5 or 14 days in culture. N=2, n=100-109 cells. E. Quantifications of percentage of human iPSC-derived neurons positive for AnkG or Trim46 at 1, 5 or 14 days in culture. N=2, n=100-109 cells. F. Representative image of a polarized human iPSC-derived neuron immunostained for MAP2, Trim46 and AnkG. Zoom represents the AIS structure. Scale bar: 20 µm in overview, 5 µm in zoom. G. Quantifications of average normalized fluorescent intensity profiles for Trim46 and AnkG at proximal axons (n=9) of human iPSC-derived neurons (day 15), distances are normalized to Trim46 peak intensities. H. Left: Schematic illustration of the experimental electrophysiology setup. To determine AP frequency, somatic current injections from -10 pA to 50 pA (steps of 5 pA, 400 ms) were applied. Right: Representative example of evoked AP firing in a human iPSC-derived neuron, response to hyperpolarizing and two depolarizing current steps, recorded at day 14. Insert: first AP to minimal (rheobase) current injection. I. Phase plot of a single AP of a human iPSC-derived neuron that fires multiple APs recorded at day 14. NSC: neuronal stem cell, AIS: axon initial segment, AP: action potential. Used tests: Chi-square test (day 1 vs. day 14) ( C-E ); *** p<0.001; error bars are ± SEM.