Adrenomedullin restores the human cortical interneurons migration defects induced by hypoxia
- Department of Pediatrics, Stanford University, United States
- Department of Psychiatry and Behavioral Sciences, UCSF Weill Institute for Neurosciences, University of California, San Francisco, United States
- Sarafan ChEM-H, Stanford University, United States
- Quantitative Biosciences Institute (QBI), University of California, San Francisco, United States
- Department of Human Genetics, Emory University, United States
Figures
Figure 1 with 1 supplement
Human cellular model to study migration patterns in cortical interneurons under hypoxic stress.
(A) Schematic illustrating overall experimental design: hiPSCs were used to derive human cortical organoids (hCO) and human subpallial organoids (hSO); for direct visualization of migrating interneurons, at ~45–55 days in culture hSO were infected with lentivirus Dlxi1/2b::eGFP and then fused with hCO into human forebrain assembloid (hFA); to study interneuron migration during exposure to hypoxia, hFA were imaged 10–14 days post infection using confocal live-imaging setup focused on the hCO part of the hFA, the movement of the same cells was followed for a total of 48 hr, each condition for 24 hr: 0–24 hr in control and 24–48 hr in hypoxia; Created with BioRender.com.(B) Schematic of migratory pattern of interneurons, focused on number of saltations, average saltation length, and directionality; (C) Example of migration pattern of one cortical interneuron during control and hypoxic conditions; Created with BioRender.com. (D) Quantification of saltations number/24 hr in hypoxia-exposed and non-exposed cortical interneurons by individual cells (paired Wilcoxon test, p<0.0001) and by hiPSC line (two-tailed paired t-test, p=0.003); (E) Quantification of average saltation length for hypoxia-exposed and non-exposed cortical interneurons by individual cells (paired Wilcoxon test, p=0.29) and by hiPSC line (two-tailed paired t-test, p=0.15); (F) Quantification of directionality of migration in hypoxia-exposed and non-exposed cortical interneurons by individual cells (paired Wilcoxon test, p=0.36), and by hiPSC cell line (two-tailed paired t-test, p=0.47); (G) Quantification of saltations number/24 hr in control conditions, in the first 24 hr (0–24 hr) versus the subsequent 24 hr (24–48 hr) of live imaging by individual cells (paired Wilcoxon test, p=0.09) and hiPSC line (two-tailed paired t-test, p=0.2); (H) Quantification of average saltation length in control conditions, in the first 24 hr (0–24 hr) versus the subsequent 24 hr (24–48 hr) of live imaging by individual cells (two-tailed paired t-test, p=0.79) and by hiPSC line (two-tailed paired t-test, p=0.47); (I) Quantification of directionality of migration in control conditions, in the first 24 hr (0–24 hr) versus the subsequent 24 hr (24–48 hr) of live imaging by individual cells (paired Wilcoxon test, p=0.38) and by hiPSC line (two-tailed paired t-test, p=0.62); Bar charts: mean ± s.e.m; scale bar: 50 μm.
Figure 1—figure supplement 1
Example of hFA, oxygen level measurements, qPCR changes in expression of hypoxia-responsive genes and cell death analyses.
(A) Example image of hFA showing cortical interneurons migrated from the hSO side to the hCO side; arrow indicates the direction of migration from hSO to hCO; (B) Oxygen levels (PO2 (mmHg)) in cell culture media under control conditions and following exposure to hypoxia for 24 hr in the confocal microscope environmental chamber (unpaired t-test, p<0.0001); (C) Example of western blot showing the stabilization of HIF1α protein in hSOs from four hiPSC lines upon exposure to hypoxia, and β-actin expression for normalization; (D) Quantification of HIF1α protein, normalized to β-actin (two-tailed paired t-test; p=0.01); (E) Transcriptional upregulation of hypoxia-responsive genes in hSO exposed to 24 hr of hypoxia (<1% O2): PFKP (two-tailed paired t-test, p=0.004), PDK1 (two-tailed paired t-test, p=0.001), VEGFA (two-tailed paired t-test, p=0.003); (F) Quantification of Annexin V levels in Dlx+ interneurons in hSOs in control and hypoxia conditions (unpaired t-test, p=0.362); (G) Representative image of Dlxi1/2b::eGFP and cleaved-CAS3 positive cells in control and hypoxia conditions; (H) Quantification (percentage) of Dlxi1/2b::eGFP and cleaved-CAS3 positive cells in control and hypoxia conditions (unpaired t-test, p=0.125); (I) Table showing the number of non-migratory interneurons from 4 hiPSC lines in control and hypoxia. Bar charts: mean ± s.e.m.; scale bar: 100 μm; Different dot colors represent individual hiPSC lines.
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Figure 1—figure supplement 1—source data 1
Original files for western blot analysis are shown in Figure 1C.
- https://cdn.elifesciences.org/articles/108134/elife-108134-fig1-figsupp1-data1-v1.zip
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Figure 1—figure supplement 1—source data 2
PDF file containing original western blots Figure 1C, including the relevant bands, iPSC lines, and experimental conditions.
- https://cdn.elifesciences.org/articles/108134/elife-108134-fig1-figsupp1-data2-v1.zip
Figure 2 with 1 supplement
Transcriptional changes in hSO exposed to hypoxia.
(A) Schematic of hypoxia exposure of hSO and collection of samples for RNA-Sequencing; Created with BioRender.com. (B) Heatmap of differentially expressed genes in RNA-Seq data showing clear transcriptional changes in hypoxia-exposed samples. Samples (n = 24) from hSO differentiated from four hiPSC lines were collected at 12 hr and 24 hr of exposure to hypoxia, as well as 72 hr after reoxygenation. The union of all differentially expressed genes (n=1473) and samples (n=24) is ordered by hierarchical clustering (complete linkage of Euclidean distance). Z-score normalized expression values are depicted on a continuous scale from lower values (purple) to higher values (orange). Cell lines, treatment, and time point are depicted at the top and represented by different colors; (C) Volcano plots of differentially expressed genes (DEGs) at 12 hr, 24 hr, and 72 hr after reoxygenation. Each dot represents a single gene. DEGs with a padj <0.05 and an absolute fold change >1.5 are shown in red (upregulated) or blue (downregulated) and unchanged genes are shown in gray; (D) Bar plot of the top 10 shared enriched gene pathways across hypoxic conditions. Adjusted p-values are depicted as text and colors represent the length of hypoxic exposure; (E) Dumbbell plot of the top 6 DEGs with the largest positive difference and the largest negative difference in Log2 fold change between 12 and 24 hr of hypoxia exposure; the arrow indicates the direction of change from 12 to 24 hr. (F) Transcriptional upregulation (by qPCR) of ADM gene in hSO samples exposed to 24 hr of hypoxia: ADM (two-tailed paired t-test, p=0.02); (G) Quantitative enzyme immunoassay analysis of adrenomedullin (ADM) peptide in media from hSO exposed and non-exposed to hypoxia (unpaired Mann-Whitney test, p<0.0001); for values below the minimal detection range of <0.01 ng/mL, value was approximated to 0 (we had 9 values approximated to 0 in the control samples). Bar charts: mean ± s.e.m.; Different dot colors represent individual hiPSC lines.
Figure 2—figure supplement 1
Oxygen level measurements for RNA-Sequencing experiments and dendrogram of sample clustering.
(A) Oxygen levels (PO2 (mmHg)) in cell culture media under control conditions and following exposure to hypoxia for 24 hr in the C-chamber hypoxia sub-chamber (Biospherix; unpaired t-test, p<0.0001); (B) Hierarchical clustering, using Ward’s criterion, of the first three principal components of the normalized gene expression profiles of all 17,777 genes passing quality control shows clear separation of 12 and 24 hr hypoxia-exposed samples versus control and from reoxygenated samples. Bar charts: mean ± s.e.m.
Figure 3 with 1 supplement
Single-cell transcriptional profiling in control and hypoxia-exposed hSO and hCO.
Schematic of single-cell RNA-seq of hCO and hSO from hFA; Created with BioRender.com. (B) UMAP visualization of the resolved single-cell RNA-seq data of hSO with assignment of main cell clusters, with control and hypoxia exposure shown by condition (n[total]=16,022 cells, n[control]=8691 cells, n[hypoxia]=7331 cells); (C) UMAP visualization of the resolved single-cell RNA-seq data of hCO with assignment of main cell clusters, with control and hypoxia exposure shown by condition (n[total]=12,567 cells, n[control]=6786 cells, n[hypoxia]=5781 cells); (D) Single cell gene expression level (log) of adrenomedullin (ADM) in main cell clusters of hSO under control and hypoxia conditions; (E) Single-cell gene expression level (log) of RAMP1 in main cell clusters of hSO under control and hypoxia conditions; Single-cell gene expression level (log) of RAMP2 in main cell clusters of hSO under control and hypoxia conditions; Single-cell gene expression level (log) of RAMP3 in main cell clusters of hSO under control and hypoxia conditions; (F) (Left) Representative blot for RAMP1 protein expression in control conditions, (Right) Representative blot for RAMP2 protein expression in control conditions; normalized to ɑ-Tubulin; (G) Quantification of RAMP1 and RAMP2 protein expression by (Left) individual hSO sample (two-tailed paired test, p=0.0004) and (Right) by hiPSC line (two-tailed paired test, p=0.0036) in control conditions; (H) Representative blots for RAMP2 protein expression changes in control and hypoxia conditions; (I) Quantification of RAMP2 protein expression (Left) by individual hSO sample (two-tailed paired test, p=0.0027) and (Right) by hiPSC line (two-tailed paired test, p=0.0235) in control and hypoxia conditions. Bar charts: mean ± s.e.m.; Different dot colors represent individual hiPSC lines.
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Figure 3—source data 1
Original files for western blot analysis are shown in Figure 3F and H.
- https://cdn.elifesciences.org/articles/108134/elife-108134-fig3-data1-v1.zip
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Figure 3—source data 2
PDF file containing original western blots Figure 3F and H, including the relevant bands, iPSC lines, and experimental conditions.
- https://cdn.elifesciences.org/articles/108134/elife-108134-fig3-data2-v1.zip
Figure 3—figure supplement 1
Quality control and robustness analyses for scRNA-sequencing data.
(A) UMAP visualization of main cellular subclusters in hSOs with corresponding gene expression including, astrocytes (SPARC, S100A10), progenitors (SOX9), cycling progenitors (SOX9, TOP2A), glutamatergic neurons (STMN2, NEUROD2), interneuron progenitors (NKX2.1, LHX6, DLX2), and interneurons (STMN2, GAD1, SLC32A1, SCG2); (B) UMAP visualization of main cellular subclusters in hCO with corresponding gene expression including, choroid plexus (TTR), progenitors (SOX2), cycling progenitors (SOX2, TOP2A), glutaminergic neurons (STMN2, NEUROD2), interneuron progenitors (NKX2.1), and interneurons (STMN2, GAD1, SLC32A1, SCG2); (C) Single-cell gene expression level (log) of hypoxia-responsive genes (PDK1 and PFKP) across main cellular subclusters in hSO; (D) Single-cell gene expression level (log) of hypoxia-responsive genes (PDK1 and PFKP) across main cell clusters in hCO; (E) Single-cell gene expression level (log) of well-established interneuron markers of MGE and CGE origin (NKX2.1, CALB2, SST, LHX6) across interneuron subclusters and additional interneuron markers (CALB1 and PVALB) MGE and CGE interneurons across hSOs; (f) Single-cell gene expression level (log) of well-established interneuron markers of MGE and CGE origin (NKX2.1, CALB2, SST, LHX6) across interneuron subclusters and additional interneuron markers (CALB1 and PVALB) MGE and CGE interneurons across hCOs; (F) Example images of interneuron subtype immunostaining in hSOs. Magenta: Somatostatin; Yellow: Calbindin; Cyan: Calretinin. Scale bar: 20 μm; (G) Single-cell gene expression level of ADM in main cell clusters of hCO; (H) Single-cell gene expression level (log) of other RAMP family receptors (RAMP1, RAMP2, RAMP3) in main cell clusters of hCO.
Figure 4 with 1 supplement
Exogenous administration of ADM peptide rescues the migration defects in hypoxia-exposed human cortical interneurons in an ex vivo model using human prenatal cerebral cortex at mid-gestation.
(A) Schematic of experimental design for pharmacological rescue experiments using ADM; 0.5 μM ADM was added to the media at the beginning of hypoxia exposure; Created with BioRender.com. (B) Example of migration pattern for one interneuron in control versus hypoxia +ADM conditions; (C) (Left) Quantification of saltation number/24 hr in control versus hypoxia conditions (paired Wilcoxon test, p<0.0001) and in control versus hypoxia +ADM by individual cells (paired Wilcoxon test, p=0.3); (Right) Quantification of saltation number/24 hr in control versus hypoxia conditions (two-tailed paired t-test, p=0.02) and in control versus hypoxia +ADM conditions by hiPSC line (two-tailed paired t-test, p=0.90); (D) (Left) Quantification of saltation number/24 hr in control versus control +ADM conditions by individual cells (paired Wilcoxon test, p=0.23); (Right) Quantification of saltation number/24 hr in control versus control +ADM conditions by hiPSC line (two-tailed paired t-test, p=0.77); (E) Schematic of denaturing procedure for ADM, including reduction and alkylation using dithiothreitol (DTT) and iodoacetamide (IAM), resulting in carbamidomethylated (CAM) cysteines at Cys16 and Cys21; Created with BioRender.com. (F) (Left) Quantification by individual cells of saltation number/24 hr in control versus hypoxia (paired Wilcoxon test, p<0.0001), control versus hypoxia +ADM (paired Wilcoxon test, p=0.05) and control versus hypoxia +denatured ADM (paired Wilcoxon test, p<0.0001); (Right) Quantification by hiPSC line of saltation number/24 hr in control versus hypoxia (two-tailed paired t-test, p=0.002), control versus hypoxia +ADM (two-tailed paired t-test, p=0.07) and control versus hypoxia +denatured ADM (two-tailed paired t-test, p=0.04); (G) Schematic of experimental design for pharmacological rescue experiments using ADM22-52 receptor blocker; Created with BioRender.com. (H) (Left) Quantification by individual cells of saltation number/24 hr in control versus hypoxia conditions (paired Wilcoxon test, p<0.0001), control versus hypoxia +ADM (paired Wilcoxon test, p=0.06), and control versus hypoxia +ADM + ADM22-52 conditions (paired Wilcoxon test, p<0.0001); (Right) Quantification of saltation number/24 hr by hiPSC line in control versus hypoxia conditions (paired t-test, p=0.0006), control versus hypoxia +ADM (paired t-test, p=0.28), and control versus hypoxia +ADM + ADM22-52 conditions (two-tailed paired t-test, p=0.003). Bar charts: mean ± s.e.m.; scale bar: 50 μm.
Figure 4—figure supplement 1
Additional data to support the inactivation of ADM and the effects of reoxygenation on migration.
(A) LC-MS-based analysis of the non-modified ADM (top) and the denatured ADM form (bottom), displays characteristic M+4 through M+7 peaks with two CAM modifications (+114 Da) in the bottom panel represented by the peak shift (arrow); (B) (Left) Quantification of saltations number/24 hr in control versus hypoxia by individual cells (Friedman test, p<0.0001) and control versus reoxygenation by individual cells (Friedman test, p<0.0001); (Right) Quantification of saltations/24 hr in control versus hypoxia (one-way ANOVA, p=0.003) by hiPSC line and control versus reoxygenation by hiPSC line (one-way ANOVA, p=0.003); (C) (Left) Quantification of saltations number/24 hr in control versus hypoxia +ADM condition by individual cells (Friedman test, p=0.05) and in control versus re-oxygenation after hypoxia +ADM by individual cells (Friedman test, p<0.0001); 35% decrease compared to 65% decrease when ADM not added to hypoxia; (Right) Quantification of saltations number/24 hr in control versus hypoxia +ADM condition by hiPSC line (one-way ANOVA, p=0.84) and in control versus re-oxygenation after hypoxia +ADM condition by hiPSC line (one-way ANOVA, p=0.12); Bar charts: mean ± s.e.m.; Different dot colors represent individual hiPSC lines.
Figure 5 with 1 supplement
Migration defect and rescue by ADM in an ex vivo model using human prenatal cerebral cortex at mid-gestation.
(A) Schematic illustrating the overall experimental design: sections of ex vivo human cerebral cortex were collected and initially sectioned at ~3 mm and subsequently at 400 μm thickness; sections were transferred onto cell culture membrane inserts suspended in culture media; for visualization, tissue was transfected with Dlxi1/2b::eGFP lentivirus and imaged 7–10 days post infection directly on inserts; GFP-tagged ex vivo human prenatal cortical interneurons were monitored for 24 hr in control conditions and 24 hr in hypoxic conditions in the presence or absence of 0.5 μM ADM; Created with BioRender.com. (B) Example of macroscopic view of a 3 mm section of fresh ex vivo human prenatal cerebral cortex; scale bar: 1 cm; (C) Representative image of fluorescently tagged cortical interneurons in a section of ex vivo human prenatal cerebral cortex; (D) Transcriptional increase of ADM gene following 24 hr of exposure to hypoxia of ex vivo human prenatal cerebral cortex samples (two-tailed unpaired t-test, p=0.0005); (E) Quantification of saltation numbers/24 hr in control versus hypoxia conditions (paired Wilcoxon test, p<0.0001); (F) Quantification of average saltation length for control versus hypoxia conditions (paired Wilcoxon test, p=0.88); (G) Quantification of directionality of migration for control versus hypoxia conditions (paired Wilcoxon test, p=0.3); (H) Quantification of saltation numbers/24 hr in control versus hypoxia +ADM conditions by individual cells (paired Wilcoxon test, p=0.09). Bar charts: mean ± s.e.m.; scale bars: 1 cm, 200 μm and 50 μm.
Figure 5—figure supplement 1
Additional data to support molecular mechanisms of rescue by ADM.
(A) UMAP showing three clusters, including a MGE cluster, a CGE cluster, and a third cluster represented by doublets; (B) Single-cell gene expression level (log) of the well-established genes present in the interneurons of MGE and CGE origin (e.g. NKX2.1, CALB2, LHX6, SST) across the interneurons subclusters and Graphical representation of expression of genes associated with GABAergic interneurons subtypes, including CALB1 and PVALB.
Figure 6 with 1 supplement
Molecular mechanism of rescue by ADM.
(A) Schematic of the previously reported main molecular pathways modulated by ADM; Created with BioRender.com. (B) (Left) Quantification of cAMP concentration (pmol/μL) by individual hSOs in control versus hypoxia (one-way ANOVA, p>0.99) and, control versus hypoxia +ADM (one-way ANOVA, p=0.0007), (Right) Quantification of cAMP concentration (pmol/μL) by hiPSC line in control versus hypoxia (one-way ANOVA, p=0.99) and, control versus hypoxia +ADM (one-way ANOVA, p=0.002); (C) (Left) Quantification of PKA activity (O.D. 450 nm) by individual hSOs in control versus hypoxia (one-way ANOVA test, p=0.98) and control versus hypoxia +ADM (one-way ANOVA, p<0.0001); (Right) Quantification of PKA activity (O.D. 450 nm) by hiPSC line in control versus hypoxia (one-way ANOVA, p>0.99) and control versus hypoxia +ADM (one-way ANOVA, p<0.0001); (D) (Left) Quantification of pAKT/AKT by individual hSOs in control versus hypoxia (one-way ANOVA, p<0.0001) and control versus hypoxia +ADM (one-way ANOVA, p<0.0001); (Right) Quantification of pAKT/AKT by hiPSC line in control versus hypoxia (Kruskal-Wallis test, p=0.01) and control versus hypoxia +ADM (Kruskal-Wallis test, p=0.03); (E) (Left) Quantification of pERK/ERK by individual hSOs in control versus hypoxia (one-way ANOVA test, p=0.001) and control versus hypoxia +ADM (one-way ANOVA test, p=0.004); (Right) Quantification of pERK/ERK by hiPSC line in control versus hypoxia (one-way ANOVA test, p=0.02) and control versus hypoxia +ADM (one-way ANOVA test, p=0.04); (F) (Left) Quantification of pCREB/CREB by individual hSOs in control versus hypoxia (one-way ANOVA test, p=0.003) and control versus hypoxia +ADM (one-way ANOVA test, p=0.27); (Right) Quantification of pCREB/CREB by hiPSC line in control versus hypoxia (one-way ANOVA test, p=0.048) and control versus hypoxia +ADM (one-way ANOVA test, p=0.6); (G) Schematic of the proposed molecular pathway activation by exogenous ADM in hSOs, including the most common pentameric structure of the GABAA receptor; Created with BioRender.com. (H) (Left) Quantification (by q-PCR) of GABRA1 in hSOs samples in control versus hypoxia conditions (one-way ANOVA test, p=0.002) and control versus hypoxia +ADM (one-way ANOVA test, p=0.17); (Center) Quantification (by q-PCR) of GABRB2 in hSOs samples in control versus hypoxia (one-way ANOVA test, p=0.08) and control versus hypoxia +ADM (one-way ANOVA test, p=0.25); (Right) Quantification (by q-PCR) of GABRG2 in hSO samples in control versus hypoxia conditions (one-way ANOVA test, p=0.007) and control versus hypoxia +ADM (one-way ANOVA test, p=0.23); (I) (Left) Quantification (by q-PCR) of CXCR4 in hSOs samples in control versus hypoxia conditions (one-way ANOVA test, p=0.0005) and control versus hypoxia +ADM (one-way ANOVA test, p=0.0031); (Center) Quantification (by q-PCR) of CXCR7 in hSOs samples in control versus hypoxia conditions (one-way ANOVA test, p=0.026) and control versus hypoxia +ADM (one-way ANOVA test, p=0.043); (Right) Quantification (by q-PCR) of CXCL12 in hSOs samples in control versus hypoxia conditions (one-way ANOVA test, p=0.024) and control versus hypoxia +ADM (one-way ANOVA test, p=0.0039). Bar charts: mean ± s.e.m.; Different dot colors represent individual hiPSC lines.
Figure 6—figure supplement 1
Additional data to support molecular mechanisms of rescue by ADM.
(A) (Left) Quantification (by q-PCR) of GABRB3 in individual samples in control versus hypoxia conditions (Kruskal-Wallis test, p=0.02) and control versus hypoxia +ADM (Kruskal-Wallis test, p=0.05); (Center) Quantification (by q-PCR) of GABRG3 in individual samples in control versus hypoxia conditions (one-way ANOVA test, p=0.01) and control versus hypoxia +ADM (one-way ANOVA test, p=0.1); (Right) Quantification (by q-PCR) of GABRA2 in individual samples in control versus hypoxia conditions (one-way ANOVA, p=0.8) and control versus hypoxia +ADM (one-way ANOVA, p=0.75). Bar charts: mean ± s.e.m.; Different dot colors represent individual hiPSC lines.
Figure 7
Schematic of the overall proposed mechanism of interneuron migration defect rescue by ADM upon hypoxia exposure.
(A) Based on our findings and existing data from literature, we propose endogenously produced ADM has decreased biological activity by impaired ability to form the necessary disulfide bond in the absence of oxygen in hypoxia. However, exogenous ADM does have biological activity as the disulfide bond is present, and thus it binds efficiently to its receptors, especially RAMP2. This binding initiates an activation of the cAMP/PKA/pCREB pathway, which in turn restores the expression of GABAA receptors and rescues the migration. Created with BioRender.com.
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Homo sapiens) | hiPSC lines derived from fibroblasts | Paşca et al., 2015; Pașca et al., 2019 | No other identifiers available or required for publication based on previous publications with these lines | |
| Transfected construct (Dlx) | Dlxi1/2b-mScarlet lentiviral | Birey et al., 2022 | 254611 | Lentiviral construct to transfect and express the shRNA. |
| Antibody | β-actin (anti-rabbit) Clone 13E5 Rabbit Monoclonal | Cell Signaling Technology | 4970 S RRID:AB_3740851 | WB (1:1000) |
| Antibody | HIF1α (anti-rabbit Clone D2U3T, Rabbit Monoclonal) | Cell Signaling Technology | 14179 S RRID:AB_2622225 | WB (1:1000) |
| Antibody | RAMP1 (anti-rabbit) Rabbit Recombinant Monoclonal | Abcam ab156575 | ab156575 RRID:AB_2801501 | WB (1:500) |
| Antibody | RAMP2 (anti-mouse) Mouse Monoclonal | Santacruz | sc-365240 RRID:AB_10844326 | WB (1:500) |
| Antibody | α-tubulin (anti-rat) Rat Monoclonal | Abcam | ab6160 RRID:AB_305328 | WB (1:1000) |
| Antibody | Anti-rat IgG Polyclonal | Cell Signaling Technology | #7077 RRID:AB_10694715 | WB (1:2000) |
| Antibody | Anti-mouse IgG, HRP-linked | Cell Signaling | #7076 RRID:AB_330924 | WB (1:2000) |
| Antibody | Anti-rabbit IgG | Cell Signaling | #7074 RRID:AB_2099233 | WB (1:2000) |
| Antibody | Annexin V-Alexa Fluor-647 | Invitrogen | A23204 RRID:AB_3740829 | IF (1:1000) |
| Antibody | Alexa Fluor 594 (anti-rabbit) | Invitrogen | A-21207 RRID:AB_141637 | IF (1:1000) |
| Antibody | anti-GFP Chicken Polyclonal | GeneTex | GTX13970 RRID:AB_371416 | IF (1:1000) |
| Antibody | Hoescht 33258 | Life Technologies | 33258 | IF (1:10,000) |
| Antibody | cleaved-caspase 3 (anti-rabbit) Rabbit Polyclonal | Cell Signaling Technology | 9661T RRID:AB_3740831 | IF (1:100) |
| Commercial assay or kit | Chromium Single cell 3′ GEM, Library & Gel Bead Kit v2 | 10 x Genomics | PN: 120237 | |
| Commercial assay or kit | Annexin V kit | Invitrogen | V13246 | FACS (1:20) |
| Commercial assay or kit | The SuperSignal West Femto Maximum Sensitivity Substrate | Thermo Fisher Scientific | 34095 | |
| Commercial assay or kit | RNeasy Mini Kit and RNase-Free DNase set | Qiagen | 74136 | |
| Commercial assay or kit | PowerUp SYBR Green master mix | Life Technologies | A25742 | |
| Commercial assay or kit | Pierce BCA Protein Assay Kits | Thermo Fisher Scientific | 23225 | |
| Commercial assay or kit | Bolt LDS Sample Buffer | Invitrogen | B0007 | |
| Commercial assay or kit | iBlot 2 Transfer Stacks | Invitrogen | IB24002 | |
| Commercial assay or kit | iBlot2 | Thermo Fisher Scientific | IB24002 | |
| Commercial assay or kit | 5% skim milk | BD | 232100 | |
| Commercial assay or kit | TBST (Tris-buffered saline) | Boston BioProducts | BM-301X | |
| Commercial assay or kit | 0.1% Tween 20 | Sigma-Aldrich | P1379 | |
| Commercial assay or kit | blocking solution (TBST) containing 5% BSA | Gendepot | A0100-005 | |
| Commercial assay or kit | Adrenomedullin (ADM) competitive enzyme immunoassay | Phoenix Pharmaceuticals | EK-010–01 | |
| Commercial assay or kit | pAKT:AKT(pS473) AKT (Total/Phospho) InstantOne ELISA Kit | Thermo Fisher Scientific | 85-86046-11 | |
| Commercial assay or kit | ERK1/2 (Total/Phospho) InstantOne ELISA Kit | Thermo Fisher Scientific | 8586013–11 | |
| Commercial assay or kit | cAMP Assay Kit (Competitive ELISA) | Abcam | ab65355 | |
| Commercial assay or kit | PKA (Protein Kinase A) Colorimetric Activity Kit | Thermo Fisher Scientific | EIA PKA | |
| Commercial assay or kit | phosphoELISA CREB (pS133) | Thermo Scientific | KHO0241 | |
| Commercial assay or kit | phosphoELISA CREB (Total) | Thermo Scientific | KHO0231 | |
| Chemical compound, drug | ROCK inhibitor Y-27632 | Selleck Chemicals | S1049 | |
| Chemical compound, drug | dorsomorphin | SigmaAldrich | P5499 | |
| Chemical compound, drug | SB-431542 | Tocris | 1614 | |
| Chemical compound, drug | XAV-939 | Tocris | 3748 | |
| Chemical compound, drug | SHH pathway agonist SAG | Thermo Fisher Scientific | 566660 | |
| Chemical compound, drug | EGF | R&D Systems | 236-EG | |
| Chemical compound, drug | FGF2 | R&D Systems | 233-FB | |
| Chemical compound, drug | BDNF | Peprotech | 50–02 | |
| Chemical compound, drug | NT3 | Peprotech | 450–03 | |
| Chemical compound, drug | Puromycin | Sigma-Aldrich | P8833 | |
| Chemical compound, drug | RIPA lysis buffer containing phosphatase inhibitor cocktail | Santa Cruz Biotechnology | sc-45065 | |
| Chemical compound, drug | protease inhibitor cocktail | Sigma-Aldrich | P1860 | |
| Chemical compound, drug | phenylmethylsulfonyl fluoride | Sigma-Aldrich, | 10837091001 | |
| Chemical compound, drug | Activated Sodium Orthovanadate | Calibiochem, | 567540 | |
| Chemical compound, drug | Adrenomedullin (ADM) | Anaspec | AS-60447 | |
| Chemical compound, drug | ADM22-52 (the RAMP2 receptor blocker) | Cayman Chemical Company | 24892 | |
| Chemical compound, drug | Dithiothreitol (DTT) | Sigma Aldrich | D5545 | |
| Chemical compound, drug | Iodoacetamide (IAM) | Sigma-Aldrich | I1149 | |
| Chemical compound, drug | 0.1% formic acid | Fisher Scientific | A117-50 | |
| Chemical compound, drug | formic acid in acetonitrile | Sigma-Aldrich | 34998 | |
| Software, algorithm | Tecan Infinite M1000 Pro and i-control 1.10 | Tecan | RRID:SCR_025732 | |
| Software, algorithm | Xcalibur FreeStyle 1.6 | Thermo Scientific | RRID:SCR_014593 | |
| Software, algorithm | ImageJ (1.53t) software | NIH | RRID:SCR_003070 | |
| Software, algorithm | BioRad CFX Maestro | BioRad | RRID:SCR_018064 | |
| Software, algorithm | NovoExpress 1.5.6 software | NovoCyte Penteon | RRID:SCR_024676 | |
| Software, algorithm | Cell Ranger software suite v6.1.2 | 10 X Genomics | RRID:SCR_017344 | |
| Software, algorithm | R (v4.1.2) package Seurat (v4.2) | Hao et al., 2021 | RRID:SCR_001905 RRID:SCR_007322 | RNA-seq analysis |
| Software, algorithm | STAR v2.7.3 | Dobin et al., 2013 | RRID:SCR_004463 | GRCh38 human genome reference |
| Software, algorithm | Picard (v 2.21.1) | https://github.com/broadinstitute/picard | RRID:SCR_006525 | Alignment and RNA-Seq quality control |
Additional files
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Supplementary file 1
List of hiPSC lines and number of cells used for each experiment.
- https://cdn.elifesciences.org/articles/108134/elife-108134-supp1-v1.xlsx
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Supplementary file 2
List of primers used for qPCRs.
- https://cdn.elifesciences.org/articles/108134/elife-108134-supp2-v1.docx
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Supplementary file 3
List of differentially expressed genes by RNA-Sequencing in hSOs exposed to hypoxia.
- https://cdn.elifesciences.org/articles/108134/elife-108134-supp3-v1.docx
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Supplementary file 4
List of GSEA terms from RNA-Sequencing.
- https://cdn.elifesciences.org/articles/108134/elife-108134-supp4-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/108134/elife-108134-mdarchecklist1-v1.pdf
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Adrenomedullin restores the human cortical interneurons migration defects induced by hypoxia
eLife 14:RP108134.
https://doi.org/10.7554/eLife.108134.3
