1. Cell Biology
  2. Neuroscience
Download icon

Quantitative mapping of transcriptome and proteome dynamics during polarization of human iPSC-derived neurons

  1. Feline W Lindhout
  2. Robbelien Kooistra
  3. Sybren Portegies
  4. Lotte J Herstel
  5. Riccardo Stucchi
  6. Basten L Snoek
  7. AF Maarten Altelaar
  8. Harold D MacGillavry
  9. Corette J Wierenga
  10. Casper C Hoogenraad  Is a corresponding author
  1. Cell Biology, Neurobiology and Biophysics, Department of Biology, Faculty of Science, Utrecht University, Netherlands
  2. Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Netherlands
  3. Theoretical Biology and Bioinformatics, Utrecht University, Netherlands
  4. Department of Neuroscience, Genentech, Inc, United States
Research Article
Cite this article as: eLife 2020;9:e58124 doi: 10.7554/eLife.58124
Voice your concerns about research culture and research communication: Have your say in our 7th annual survey.
4 figures, 2 videos and 1 additional file

Figures

Figure 1 with 2 supplements
Successful and protracted transition through 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 transduced with FUGW-GFP lentivirus and immunostained for NSC marker Nestin and proliferation marker Ki67, neuron markers β3-Tubulin and MAP2, or AIS markers AnkG and Trim46. Outlines of cells were defined by the FUGW-GFP signal. Scale bar: 15 µm in overview, 5 µm in zooms. (C,D,E) Quantifications of percentage of human iPSC-derived NSCs positive for Ki67 or Nestin (C), β3-Tubulin or MAP2 (D) and AnkG or Trim46 (E) 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 of human iPSC-derived neurons (day 15) (n = 9). Distances are normalized to Trim46 peak intensities. (H) Left: Schematic illustration of the experimental electrophysiology setup. To determine AP frequency, somatic current injections ranging 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 (day 14) that fires multiple APs. NSC: neuronal stem cell, AIS: axon initial segment, AP: action potential. Used tests: Chi-square test (day one vs. day 14) (C–E); ***p<0.001; graphs represent mean ± SEM.

Figure 1—figure supplement 1
Successful and protracted transition of developmental stages in human iPSC-derived neurons.

(A) Representative images of hiPSC-derived NSCs/neurons at indicated time points. Neurons are immunostained for NSC marker Sox2, neuron marker β3-Tubulin, cortical marker Ctip2 and axon marker Trim46. Arrowheads indicate examples of cortical neurons identified by Ctip2/ß3-Tubulin double-positive neurons. Scale bar: 20 µm. (B) Western blot analysis of cell lysates harvested at indicated time points and immunostained for NSC markers Nestin and Sox2, neuron marker β3-Tubulin, cortical marker Ctip2 and axon markers Trim46 and Gap43. Actin and Gapdh are used as loading control. (C) Representative images of neurite morphology of axonal and dendritic structures at day 7 and 13. Scale bar: 5 µm. (D) Quantifications of neurite width of axonal and dendritic structures at day 7 and 13 (N = 3, n = 20–23). (E,F) Representative images of polarized human iPSC-derived neurons following the induced differentiation #2 and the spontaneous differentiation protocols (E) and derived from donor cell lines #2 and #3 (F). Neurons were immunostained for Trim46 and ß3-Tubulin. Zooms represent AIS structures. Scale bar: 10 µm in overview, 5 µm in zoom. (G) Representative image of a polarized human iPSC-derived neuron (day 13) immunostained for Trim46, PanNaV and AnkG. Zoom represents the AIS structure. Scale bar: 10 µm in overview, 5 µm in zooms. (H,I) Representative examples of evoked AP firing of human iPSC-derived neurons (day 12) differentiated using the induced differentiation #2 and the spontaneous differentiation protocol (H) and in neurons from donor cell lines #2 and #3 (I). Shown is the response to a single depolarizing current step. The offset current (Ihold) was adjusted to keep the baseline membrane potential at approximately −60 mV (H) and −70 mV (I) (dashed lines). In the neurons generated using the induced differentiation #2 and spontaneous differentiation protocols AP firing was recorded in 2/2 and 3/6 cells, respectively (H), and in the neurons from donor #2 and #3 AP firing was recorded in 2/2 and 4/4 cells, respectively (I). NSC: neuronal stem cell. AP: action potential. Used tests: One-way ANOVA with Tukey’s post-hoc analysis (D); ***p<0.001, ns p≥0.05; graphs represent mean ± SEM.

Figure 1—figure supplement 2
Characterizing AP firing of early neurodevelopmental stages in human iPSC-derived neurons.

(A) Number of APs versus input current injection of hiPSC-derived neurons (day 7–14) that fire multiple APs (red, N = 4, n = 20 cells) or fire only one AP (blue, N = 4, n = 32 cells). Two cells in the multiple firing group were excluded due to variation in baseline. (B) Resting membrane potential of human iPSC-derived neurons that fire a single AP (N = 4, n = 32 cells) or multiple APs (N = 4, n = 22 cells). (C) Input resistance of human iPSC-derived neurons that fire a single AP (N = 4, n = 32 cells) or multiple APs (N = 4, n = 21 cells). (D) AP after-hyperpolarization of human iPSC-derived neurons that fire a single AP (N = 4, n = 32 cells) or multiple APs (N = 4, n = 22 cells). (E) Maximum sodium current of human iPSC-derived neurons that fire a single AP (N = 3, n = 22 cells) or multiple APs (N = 3, n = 17 cells). AP: action potential. Used tests: Mann-Whitney U test (B–E); **p<0.01, *p<0.05; graphs represent mean ± SEM.

Figure 2 with 1 supplement
Transcriptomic and proteomic profiling of early developmental stages in human iPSC-derived neurons.

(A) Volcanoplot of differentially expressed transcripts between day 7 and day 1 (false discovery rate (FDR) p<0.05, Benjamini and Hochberg corrected). (B,C) Top 10 most significantly enriched GO terms of downregulated (B) and upregulated (C) genes at day 7. FDR p<0.05, Benjamini and Hochberg corrected. (D,E) Six clusters with distinct protein expression profiles, divided in upregulated (D) and downregulated (E) protein expression, obtained by unsupervised clustering, and the GO enrichment analysis for each cluster. (F) Correlative analysis of relative transcriptomic and proteomic expression levels (day7/day1) (Pearson’s correlation, R = 0.51, p<0.0001). Highlighted are selected typical stem cell (blue), neuron (yellow), and axon (red) markers. (G,H) Heatmaps showing the relative expression of RNA (G) and protein (H) levels of typical stem cell, neuron, and axon markers at different timepoints. CC (Cyan): cellular components, BP (Yellow): biological processes, (MF) (Green): molecular function.

Figure 2—figure supplement 1
Transcriptomic and proteomic profiles of early neurodevelopmental stages in human iPSC-derived neurons.

(A) Correlation matrix of biological and technical replicates used for transcriptome analysis. (B) Volcanoplot of differentially expressed transcripts day3/day1 and day7/day3 (false discovery rate (FDR) cutoff p<0.05, Benjamini and Hochberg corrected). (C) Correlation matrix of technical replicates used for proteome analysis. (D,E) Heatmaps showing the relative expression of RNA (D) and protein (E) levels of semaphorins and Rap1GTPases at different timepoints.

Figure 3 with 1 supplement
Identification of intermediate developmental stages during onset of axon formation.

(A) Schematic illustration and timing of neurodevelopmental stages 2a, 2b, 3a and 3b in human iPSC-derived NSCs/neurons. (B) Representative images of stage 2a, 2b, 3a, and 3b hiPSC-derived neurons. Cells were transduced with FUGW-GFP lentivirus and immunostained for AnkG and Trim46. Arrowheads mark Trim46 and AnkG accumulations. Zooms represent a nonpolarized neurite in a stage 2 neuron or a developing axon in a 3 neuron. Scale bar: 40 µm overview, 5 µm zooms. (C) Quantifications of the relative abundance of stage 2a, 2b, 3a or 3b human iPSC-derived neurons (N = 2, n = 50–55 cells). (D) Quantifications of the total length of Trim46 structures in neurites of stage 2b, 3a and 3b human iPSC-derived neurons (N = 2, n = 20 cells). (E) Quantifications of distance from soma to start of the Trim46 signal in neurites of stage 2b, 3a and 3b human iPSC-derived neurons (N = 2, n = 20 cells). (F) Phase plots of a representative AP recorded of a human iPSC-derived neuron at 10 days and 14 days. (G) Scatter plot of AP amplitude versus AP half-width grouped by days after plating (N = 4; 7 days: n = 7 cells, 10–11 days: n = 15 cells, 13–14 days: n = 36 cells). (H) AP amplitude recorded in human iPSC-derived neurons of 10–11 days (N = 4, n = 15 cells) and 13–14 days (N = 4, n = 36 cells). (I) AP half-width recorded in human iPSC-derived neurons of 10–11 days (N = 4, n = 15 cells) and 13–14 days (N = 4, n = 36 cells). AP: Action potential. Used tests: Chi-square test with Bonferroni post-hoc correction (C), One-way ANOVA with Bonferroni post-hoc correction (D, E), Student’s t-test (H), Mann-Whitney U test (I), ***p<0.001, **p<0.01, *p<0.05, ns p≥0.05; graphs represent mean ± SEM.

Figure 3—figure supplement 1
Extra developmental stage and gradual action potential maturation during axon formation.

(A,B) Representative images of stage 3a human iPSC-derived neurons following the induced differentiation #2 and spontaneous differentiation protocols (A) and in neurons from donor cell lines #2 and #3 (B). Neurons were immunostained for Trim46 and ß3-Tubulin. Zooms represent distal axonal structures. Scale bar: 10 µm in overview, 5 µm in zoom. (C) Quantifications of distance from soma to distal end of Trim46 signal in neurites of stage 2b, 3a and 3b human iPSC-derived neurons (N = 2, n = 20 cells). (D) Quantifications of total length of AnkG structures in neurites of stage 2b, 3a and 3b human iPSC-derived neurons (N = 2, n = 14–20 cells). (E) Quantifications of distance from soma to start of the AnkG signal in neurites of stage 2b, 3a and 3b human iPSC-derived neurons (N = 2, n = 14–20 cells). (F) Quantifications of distance from soma to distal end of AnkG signal in neurites of stage 2b, 3a and 3b human iPSC-derived neurons (N = 2, n = 14–20 cells). (G) Representative images of localization of voltage-gated sodium channels in stage 3a and 3b human iPSC-derived neurons. Neurons were immunostained for Trim46, AnkG, PanNaV and β3-tubulin. Arrowheads mark AIS structures. Scale bar: 20 µm in overview, 5 µm in zoom. Used tests: One-way ANOVA including post-hoc analysis with Bonferroni correction (C–F); ***p<0.001, **p<0.01, *p<0.05, ns p≥0.05; graphs represent mean ± SEM.

Figure 4 with 1 supplement
Axonal microtubule cytoskeleton is reorganized in a distal-to-proximal fashion during development.

(A) Schematic illustration of stage 2 (day 5), 3a (day 7) and 3b (day 14) human iPSC-derived neurons. Different locations of the neurons that are characterized for experiments are outlined and annotated. (B) Stills from a spinning-disk time-lapse recording of specified neurites transfected with MARCKS-tagRFP_IRES_GFP-MACF18 at specific time points. The top panel is a still of a typical neurite in MARCKS-tagRFP. The other panels show moving GFP-MT+TIP comets (GFP-MACF18) moving either anterogradely (green arrowheads) or retrogradely (blue arrowheads). P and D indicate the proximal and distal direction of the neurite, respectively. Scale bar: 5 µm. (C) Kymographs and schematic representations of time-lapse recordings shown in (B). Scale bar: 5 µm. (D) Quantifications of the ratios of comets moving anterogradely (green) or retrogradely (blue) direction (N = 3, n = 8–23 cells). (E, F) Quantifications of the number of comets per minute moving anterogradely (E) and retrogradely (F) (N = 3, n = 8–23 cells). (G, H) Quantifications of the growth speed (G) and run length (H) of comets (N = 3, n = 8–23 cells). (I) Schematic representation of microtubule LS experiments. (J) Kymographs and schematic representations of time-lapse recordings of LS experiments shown in Figure 4—figure supplement 1b. Red arrowheads and dotted lines indicate when LS is performed. Scale bar: 5 µm. (K) Quantifications of the ratios of comets moving anterogradely (green) or retrogradely (blue) direction, 10 µm before and after the LS position (N = 3, n = 20–30 cells). (L, M) Quantifications of the number of comets per minute moving anterogradely (L) and retrogradely (M), 10 µm before and after the LS position (N = 3, n = 20–30 cells). LS: laser-severing. Used tests: One-way ANOVA with Tukey’s post-hoc analysis (E–H, L, M); ***p<0.001, **p<0.005, *p<0.05, ns p≥0.05; graphs represent mean ± SEM.

Figure 4—figure supplement 1
Microtubule remodeling in axons and dendrites during early neuronal development.

(A,B) Quantifications of the growth speed (A) and run length (B) of comets moving anterogradely (green) or retrogradely (blue) (N = 3, n = 8–23 cells). (C) Schematic illustration of stage 2 (day 5), 3a (day 7) and 3b (day 14) hiPSC-derived neurons. Different locations of the neurons that are characterized for experiments are outlined and annotated. (D) Stills from a spinning-disk time-lapse recording of specified neurites transfected with MARCKS-tagRFP_IRES_GFP-MACF18 at specific time points. The top panel is a still of a typical example neurite in MARCKS-tagRFP. The other panels show moving GFP-MT+TIP comets (GFP-MACF18) moving either anterogradely (green arrowheads) or retrogradely (blue arrowheads). Red line indicates location of laser severing, and red arrowhead indicates time of laser severing. P and D indicate the proximal and distal direction of the neurite, respectively. Scale bar: 5 µm. (E) Schematic illustration of the proposed distal to proximal reorganization of the axon during stage 3a and stage 3b. LS: laser-severing. Graphs represent mean ± SEM.

Videos

Video 1
Representative movie of MT+TIP comet dynamics in a dendrite (day 7).

Scale bar: 5 µm. Time in minutes:seconds.

Video 2
Representative movie of MT+TIP comet dynamics following laser severing in a dendrite (day 7).

Scale bar: 5 µm. Time in minutes:seconds.

Additional files

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)