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, 23 (8), 1687-1698

Patient-derived iPSCs Show Premature Neural Differentiation and Neuron Type-Specific Phenotypes Relevant to Neurodevelopment

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Patient-derived iPSCs Show Premature Neural Differentiation and Neuron Type-Specific Phenotypes Relevant to Neurodevelopment

E Yeh et al. Mol Psychiatry.

Abstract

Ras/MAPK pathway signaling is a major participant in neurodevelopment, and evidence suggests that BRAF, a key Ras signal mediator, influences human behavior. We studied the role of the mutation BRAFQ257R, the most common cause of cardiofaciocutaneous syndrome (CFC), in an induced pluripotent stem cell (iPSC)-derived model of human neurodevelopment. In iPSC-derived neuronal cultures from CFC subjects, we observed decreased p-AKT and p-ERK1/2 compared to controls, as well as a depleted neural progenitor pool and rapid neuronal maturation. Pharmacological PI3K/AKT pathway manipulation recapitulated cellular phenotypes in control cells and attenuated them in CFC cells. CFC cultures displayed altered cellular subtype ratios and increased intrinsic excitability. Moreover, in CFC cells, Ras/MAPK pathway activation and morphological abnormalities exhibited cell subtype-specific differences. Our results highlight the importance of exploring specific cellular subtypes and of using iPSC models to reveal relevant human-specific neurodevelopmental events.

Conflict of interest statement

Conflict of interest:

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1. CFC neural cultures show progenitor pool depletion, early maturation, and imbalance of neural cell types
(A) Western blot analysis of the components of the Ras/MAPK pathway and its phosphorylation status, and quantification of the blots (B) from protein lysates extracted from CFC and control NPC cultures. The results were normalized with their corresponding α-tubulin values (assigned a value of 1) and are mean ±SD of two independent experiments (C) Double immunostaining for BrdU (green) and Ki67 (red) of early neuronal progenitors within 48h of differentiation; progenitors that exited the cell cycle, %(BrdU+Ki67)/BrDU+ cells (orange arrows) were quantified. (D) Cleaved caspase 3 staining for cell death of the same NPCs and subsequent quantification. (E) Immunostaining of TUJ1, MAP2, NEUN, GABA, and TBR1 of CFC lines and control lines at week 1 of neural differentiation. Quantification of these stainings are shown as percentage of TUJ1+ cells (F), MAP2+ cells (G), NEUN+ cells (H), average neurite length of TUJ1+ cells (I), average number of neurites per TUJ1+ cell (J), percentage of GABA+ cells (K), and TBR1+ cells (L). (M) mRNA levels of MAP2, deep layer markers TBR1, CTIP2, and FEZF2, upper layer markers CUX1 and SATB2, and GABA precursors GAD1 and GAD2 in one-week old neural cultures. ΔCt values were calculated by normalizing the average Ct value of each cell line by the average Ct value of GUSB of the same cell line; ΔΔCt was calculated by normalizing the ΔCt value of each line to the average ΔCt value of the control lines. (N) Immunostaining of TUJ1, GFAP, CD44, S100, MAP2, FOXP2, and CUX1 at week 5 of neural differentiation. Quantification of these stainings is shown as a percentage of cells expressing GFAP (O), MAP2 (P), FOXP2 (Q) and CUX1 (R). (S) mRNA level analysis of astroglial cell markers GFAP, S100B, CD44, neuronal marker MAP2, deep layer markers TBR1, CTIP2, and FEZF2, upper layer markers CUX1 and SATB2 in five-week old neural cultures. ΔCt values were calculated by normalizing the average Ct value of each cell lines by the average Ct value of GUSB of the same cell line; ΔΔCt was calculated by normalizing the ΔCt value of each line to the average ΔCt value of the control lines. For all immunostainings depicted, at least three fields per coverslip from three independent experiments were counted. For all scatter plots illustrated, values represent mean±SD per condition, calculated with repeated measures ANOVA, accounting for biological and technical replicate values. Number of subclones and independent experiments performed are detailed in Supplementary Table 3. Values for mean and standard deviation of each control and CFC subject for each experiment are detailed in Supplementary Table 7. p≤0.05 (*), p≤0.01 (**), p≤0.001 (***).
Figure 2
Figure 2. Increased intrinsic excitability in CFC-derived neurons
(A) Overlaid dsRFP fluorescence and phase-contrast images depicting a SYN1DsRFP+ neuron. For the patch clamp experiments (B–G), we performed two independent blind experiments, with one subclone per cell line. (B) The average current per individual required to elicit a single AP in neurons from control-derived neurons (blue), CFC-derived neurons (red). (C) The quantified AP response evoked by step currents of increasing amplitude. (D) Example current-clamp recordings from control-derived and CFC-derived neurons. Additional measures of intrinsic excitability: IK (E), Peak INa (F), Rin (G). (H) Immunostaining of Synapsin1+ puncta on MAP2+ control- and CFC-derived neurons at week 5 of differentiation. (I) Quantification of Synapsin1+ puncta per 50 μm neurite length in control- and CFC-derived neurons. For all immunostainings depicted, at least ten MAP2+ neurons per coverslip from three independent experiments were counted. For all scatter plots illustrated, values represent mean±SD per condition, with significance calculated using repeated measures ANOVA, accounting for biological and technical replicates. Number of subclones and independent experiments performed are detailed in Supplementary Table 3. Values for mean and standard deviation of each control and CFC subject for each experiment are detailed in Supplementary Table 7. p≤0.05 (*), p≤0.01 (**), p≤0.001 (***).
Figure 3
Figure 3. Decreased phosphorylation of AKT is associated with progenitor cell pool depletion in CFC
(A) Crosstalk between the Ras/Raf/MEK/ERK and Ras/PI3K/AKT pathway, and the targets of Wortmannin and SC79 (adapted from). (B) Double immunostaining for BrdU (red) and Ki67 (green) of early neuronal progenitors within 48h of differentiation in control NPCs, control NPCs pre-treated with Wortmannin, CFC NPCs, and CFC NPCs pre-treated with SC79; progenitors that exited the cell cycle, %(BrdU+Ki67)/BrdU+ cells (orange arrows) were quantified and graphed in (C). (D) Immunostaining of MAP2 in control one-week old neural cultures, control one-week old neural cultures pre-treated with Wortmannin, CFC one-week old neural cultures, and CFC one-week old neural cultures pre-treated with SC79. (E) Quantification of the percentage of MAP2 positive DAPI in these stainings. (F) Immunostaining of TUJ1 in control one-week old neural cultures, control one-week old neural cultures pre-treated with Wortmannin, CFC one-week neural cultures, and CFC one-week old neural cultures pre-treated with SC79. Quantification of the average number of neurites per TUJ1+ cell (G). (H) Immunostaining of GFAP and TUJ1 in control five-week old neural cultures, control five-week old neural cultures pre-treated with Wortmannin, CFC five-week old neural cultures, and CFC five-week old neural cultures pre-treated with SC79. (I) Quantification of the percentage of GFAP positive DAPI in these stainings. (J) Immunostaining of MAP2 and FOXP2 in control five-week old neural cultures, control five-week old neural cultures pre-treated with Wortmannin, CFC five-week old neural cultures, and CFC five-week old neural cultures pre-treated with SC79. (K) Quantification of the percentage of FOXP2 positive DAPI in these stainings. (L) Immunostaining of MAP2 and CUX1 in control five-week old neural cultures, control five-week old neural cultures pre-treated with Wortmannin, CFC five-week old neural cultures, and CFC five-week old cultures pre-treated with SC79. (M) Quantification of the percentage of CUX1 positive DAPI in these stainings. For all immunostainings depicted, at least three fields per coverslip from three independent experiments were counted. For all scatter plots illustrated, values represent mean±SD per condition, calculated with repeated measures ANOVA, accounting for biological and technical replicates. Number of subclones and independent experiments performed are detailed in Supplementary Table 3. Values for mean and standard deviation of each control and CFC subject for each experiment are detailed in Supplementary Table 7. p≤0.05 (*), p≤0.01 (**), p≤0.001 (***).
Figure 4
Figure 4. CFC cortical glutamatergic neuronal cultures show increased number of neurites per cell and decreased phosphorylation of BRAF
(A) Double immunostaining for BrdU and Ki67 in early neuronal progenitors within 48h of differentiation with and without the addition of Compound E; progenitors that exited the cell cycle, %(BrdU+Ki67)/BrDU+ cells were quantified. (B) Immunostaining of TUJ1 and TBR1 at week 1 of glutamatergic cortical neuron differentiation, and (E) of MAP2, TBR1, CUX1, and FOXP2 at week 5 of glutamatergic cortical neuron differentiation. Quantification of these staining is shown in (C–I). For the above immunostainings depicted, at least three fields per coverslip from three independent experiments were counted. (J) Fluorescent microscopy analysis of GFP-labelled five-week old control-derived and CFC-derived neurons. For the morphometric analysis, 20 GFP+TUJ1+ neurons per coverslip from three independent experiments were counted. Image J analysis of soma size (K), neurites per cell (L), neurite length (M), branches per neurite (N), branch length (O), and Sholl analysis of the number of intersecting neurites (P). Western blot analysis of the components of the Ras/MAPK pathway and its phosphorylation status (Q), and quantification of the blots (R) from protein lysates extracted from five-week old CFC and control glutamatergic cortical neuron cultures. The results were normalized with their corresponding α-tubulin values (assigned a value of 1) and are mean±SD of two independent experiments. For all immunostainings depicted, at least three fields per coverslip from three independent experiments were counted. For all scatter plots illustrated, values represent mean±SD per condition, calculated with repeated measures ANOVA, accounting for biological and technical replicates. Number of subclones and independent experiments performed are detailed in Supplementary Table 3. Values for mean and standard deviation of each control and CFC subject for each experiment are detailed in Supplementary Table 7. p≤0.05 (*), p≤0.01 (**), p≤0.001 (***).
Figure 5
Figure 5. CFC GABAergic interneuron cultures show decreased number of neurites per cell and increased phosphorylation of BRAF
(A) Immunostaining of GABA and NKX2.1 at week 1 of GABAergic interneuron differentiation. Quantification of these stainings is shown as the percentage of cells expressing GABA (B), NKX2.1 (C). (D) Immunostaining of GABA and GAD67 at week 5 of GABAergic interneuron differentiation and quantification of these stainings shown as the percentage of cells expressing GAD65/GAD67. For the above immunostainings depicted, at least three fields per coverslip from three independent experiments were counted. (E) Fluorescent microscopy analysis of GFP-labelled GABA+ cells in five-week old control-derived and CFC-derived neurons. For the morphometric analysis, 20 GFP+GABA+ neurons per coverslip from three independent experiments were counted. Image J analysis of soma size (F), neurites per cell (G), neurite length (H), branches per neurite (I), branch length (J), and Sholl analysis of the number of intersecting neurites (K). Western blot analysis of the components of the Ras pathway and its phosphorylation status (L), and quantification of the blots (M) from protein lysates extracted from five-week old CFC and control GABAergic interneuron cultures. The results were normalized with their corresponding α-tubulin values (assigned a value of 1) and are mean±SD of two independent experiments. For all immunostainings depicted, at least three fields per coverslip from three independent experiments were counted. For all scatter plots illustrated, values represent mean±SD per condition, calculated with repeated measures ANOVA, accounting for biological and technical replicates. Number of subclones and independent experiments performed are detailed in Supplementary Table 3. Values for mean and standard deviation of each control and CFC subject for each experiment are detailed in Supplementary Table 7. p≤0.05 (*), p≤0.01 (**), p≤0.001 (***).

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