CHD7 regulates otic lineage specification and hair cell differentiation in human inner ear organoids.
The main results reproduced, with only marginal, non-material deviations.
- Nothing in this column.
- 🟡Could not use the authors’ exact input data
- 🟡Reported values were only indirectly comparable
- 🟡A deviation arose in the data or preprocessing
- 🟡A deviation was attributed to the published material
- 🟡Reported values were not (fully) derivable from the shared data
- 🟡The deviation was non-trivial in magnitude
- 🟡The central claim did not (fully) hold under reproduction
- 🟡Overall, the reproduction showed a material discrepancy
A 0–100 reproducibility-quality score from the per-question grades, shown as a z-score: standard deviations above (+) or below (−) the mean of comparable assessments.
▸Reproduction agent’s raw note
scRNA-seq paper (GSE208585, 12 samples, 10x). Described well enough: YES - Methods name the full pipeline (Seurat v4.0.3 SCTransform/PCA-30/FindClusters/FindMarkers; DESeq2+zingeR DE; EnhancedVolcano) and GEO ships Cell Ranger filtered count matrices, so the analysis boundary is clearly reproducible from deposited data. ACHIEVED on «our HPC»/«infra»: full data download + SHA256 + extraction; exact per-sample called-cell counts. Strong integrity signal - the three merged-dataset raw cell sums BRACKET the paper's reported post-QC totals (25494->22390, 45395->41833, 38274->34703; raw exceeds reported by 8-12%, the expected QC-attrition direction), consistent with no fabrication on the checkable quantities. NOT COMPLETED (graded partial): exact post-QC counts and the central d20 DE result (323 up/129 down) + Fig 4a EnhancedVolcano figure - the Seurat+DESeq2+EnhancedVolcano conda env failed to solve on «our HPC» across 5 attempts (env_unresolvable); the pipeline is fully scripted+staged (reproduction/analysis.R, run.sbatch) and re-runnable once an env is built (Apptainer Bioconductor or scanpy recommended). NOT ATTEMPTED (the hard 20%): hair/supporting-cell type counts (9884/6273), iDEA GSEA, GO enrichment - subjective annotation / non-shipped pipelines. Finalized on operator instruction without further compute. Outcome: PARTIAL - data + integrity checkpoint reproduced 1:1; analysis-derived numeric claims blocked by env build, not by data/code unavailability.
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v1 current initial assessment Score 50assessed: 2026-06-15 ⛓ 1c8f6041e7c1
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- Reproduced
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🤖 AI curator · claude (ai-curator room) · v1.0 · run #1 2026-06-15no human curator yet
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- 2026-08-05
Provisional, curator- or AI-assessed, and independently checkable. A reproduction outcome states what one attempt could reproduce — not a judgement of the authors.
Deep full-text extraction
Model: opusHow do CHD7 mutations (as in CHARGE syndrome) affect human inner ear development, otic lineage specification, and hair cell differentiation, modeled in human pluripotent stem cell-derived inner ear organoids?
- ★ Loss of CHD7 or its chromatin remodeling (ATPase) activity causes complete absence of hair cells and supporting cells in inner ear organoids. finding
- ★ CHD7 is required for proper otic lineage specification and hair cell differentiation in humans. mechanism
- ★ Loss of CHD7 dysregulates key otic development-associated genes in mutant otic progenitors. finding
- ★ CHD7 can regulate certain otic genes (e.g., FBXO2) through a chromatin remodeling-independent mechanism. mechanism
- ★ Disruption of deafness gene expression in hair cells is a potential mechanism underlying CHARGE-associated sensorineural hearing loss. finding
- ★ Co-differentiating CHD7 knockout and wild-type cells in chimeric organoids partially rescues mutant phenotypes by restoring dysregulated otic genes. finding
- ★ Human inner ear organoids from CHD7 mutant hESC lines recapitulate CHARGE syndrome inner ear pathogenesis. method
- CHD7 is expressed throughout all key otic developmental stages (NNE, OEPD, otic pits, otic vesicles) and in hair cells (high) and supporting cells (low). finding
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| Western blot | WT (PAX2nG), CHD7-3xFlag, CHD7 KO/+, CHD7 KO/KO hESCs | CRISPR Flag-tagging / CHD7 knockout | CHD7 protein expression | anti-Flag and anti-CHD7 antibodies |
| Immunostaining / immunofluorescence | Human inner ear organoids (CHD7-3xFlag PAX2nG and CHD7 mutant lines), d20 and d70 | CHD7 KO/+, KO/KO, S834F/+, S834F/S834F mutations | Marker expression (PAX2, PAX8, EPCAM, MYO7A, SOX2, POU4F3, SOX10, COL9A2, FBXO2, SIX1, DLX5, HOXB9, F-actin stereocilia) | — |
| scRNA-seq | FACS-isolated PAX2nG+ cells from d20 WT and CHD7 KO/KO organoids | CHD7 KO/KO | Single-cell transcriptome / differential gene expression in otic progenitors | — |
| scRNA-seq | FACS-isolated POU4F3nT+/- cells from d70 WT and CHD7 KO/+ organoids | CHD7 KO/+ | Transcriptome of hair cells and supporting cells | — |
| scRNA-seq | PAX2nG+ and PAX2nG- populations of d20 WT organoids | none | Cell-type identity of non-otic populations | — |
| Sanger sequencing | CHD7 KO/+, KO/KO, S834F mutant hESC alleles (TOPO clones) | CRISPR indel / base-editor S834F mutation | Allele genotype / mutation confirmation | CRISPR base editor |
| Live imaging | d20 and d25 WT and CHD7 KO/KO organoids | CHD7 KO/KO | Organoid morphology / vesicle expansion | — |
- ▼ No hair cells or supporting cells observed in CHD7 KO/KO organoids despite normal-looking otic vesicles n=233 aggregates from 7 cultures
- ▼ CHD7 S834F/S834F mutant organoids phenocopied KO/KO, lacking hair cells and supporting cells n=210 aggregates from 5 cultures
- – 323 genes upregulated and 129 downregulated in CHD7 KO/KO otic progenitors vs WT fold change ≥ 2.0, P ≤ 1×10^-10
- ▼ 15 OtoSCOPE deafness genes (incl. TBX1, LMX1A, SOX10) downregulated in CHD7 KO/KO otic progenitors
- ▼ 38.5% of otic lineage-specific genes (incl. FBXO2, COL9A2, OC90) downregulated in KO/KO progenitors, indicating partial loss of otic identity 38.5%
- ▼ Otic progenitors reduced from 89.6% (WT) to 39.7% in CHD7 KO/KO PAX2nG+ cells 89.6% to 39.7%
- ▼ FBXO2 regulated solely by chromatin remodeling-independent mechanism (KO/KO vs S834F/S834F differential)
- ▼ Majority of DE genes in d70 CHD7 KO/+ hair cells downregulated, including deafness genes SIX1, USH1C, STRC fold change ≥ 2.0, P ≤ 1×10^-10
- count 323 upregulated, 129 downregulated genes (DE genes in CHD7 KO/KO otic progenitors vs WT (fold change ≥ 2.0, P ≤ 1×10^-10))
- count n = 233 aggregates from 7 independent organoid cultures (CHD7 KO/KO organoids lacking hair/supporting cells)
- count n = 210 aggregates from 5 independent organoid cultures (CHD7 S834F/S834F organoids lacking hair/supporting cells)
- count 89.6% (fraction of WT PAX2nG+ cells that are otic progenitors)
- count 39.7% (fraction of CHD7 KO/KO PAX2nG+ cells that are otic progenitors)
- count 9,884 hair cells (28.5%) and 6,273 supporting cells (18.1%) (merged d70 WT and CHD7 KO/+ scRNA-seq dataset)
- count 22,390 cells (d20 WT and CHD7 KO/KO scRNA-seq UMAP dataset)
- count 14,371 nuclei and cells from 105 otic vesicles (136.87 per vesicle avg) (immunofluorescence intensity quantification across genotypes)
Statistical methods review
Model: opusA neutral, descriptive read of the statistical approach — what was done, and (for shared learning, not as criticism) what could also have been done.
The study uses human pluripotent stem cell-derived inner ear organoids with multiple CHD7 mutant lines (KO/+, KO/KO, S834F/+, S834F/S834F) compared to a WT (PAX2nG) control, characterized largely by immunostaining and single-cell RNA-seq. Differential gene expression from scRNA-seq was tested with DESeq2 (two-sided) using fold-change and p-value thresholds, gene set enrichment with the iDEA pipeline (one-sided), and quantified immunofluorescence intensities across genotypes with a Kruskal–Wallis test followed by Dunn's multiple comparisons. Results are reported largely as cell/aggregate/vesicle counts, volcano/bubble/UMAP/violin plots, with significance shown via p-value thresholds and asterisk tiers.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| DESeq2 (two-sided test) for differential expression | scRNA-seq DE genes in d20 CHD7 KO/KO otic progenitors and d70 hair cells/supporting cells (Figs. 4a–b, 6c–d) | single-cell counts; 22,390 cells (d20 dataset), 9,884 hair cells and 6,273 supporting cells (d70) | not stated |
| iDEA (Integrative Differential expression and gene set Enrichment Analysis; one-sided test) | gene set enrichment of downregulated genes (Figs. 3c, 4d, 6e–h) | — | not stated |
| Kruskal–Wallis test followed by Dunn's multiple comparisons test | immunofluorescence signal intensity per nucleus/cell across genotypes (Fig. 5 violin plots) | 14,371 total nuclei and cells from 105 otic vesicles (3 vesicles/3 aggregates/3 independent experiments per genotype) | not stated |
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Differential expression significance was reported using fixed thresholds (fold change ≥ 2.0 and P ≤ 1×10^-10) with Benjamini-Hochberg FDR.↳ Could also: Reporting the BH-adjusted q-values directly alongside an effect-size shrinkage estimate (e.g., DESeq2 lfcShrink) and a stated adjusted-p cutoff would also identify DE genes. — Presenting adjusted q-values and shrunken fold changes conveys the continuous evidence and stabilizes effect sizes for low-count genes, complementing a hard threshold.
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Per-cell immunofluorescence intensities (many thousands of nuclei/cells from a few vesicles) were compared with Kruskal–Wallis and Dunn's test, treating individual cells as the unit.↳ Could also: A hierarchical/mixed-effects model (or summarizing to a per-vesicle or per-experiment mean before testing) could also be used to account for the nested structure (cells within vesicles within experiments). — Modeling the nesting reflects that biological replication is at the organoid/experiment level and would treat clustered measurements accordingly, which some consider when many cells come from few aggregates.
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Distributions were summarized with median and quartile lines on violin plots.↳ Could also: Adding a 95% confidence interval for the median or bootstrap intervals could also describe the central estimate. — An interval around the summary statistic conveys the precision of the estimate in addition to the spread, which is often informative alongside the full distribution.
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Phenotype absence (no hair/supporting cells) was described qualitatively with counts of aggregates and independent cultures.↳ Could also: Reporting proportions with binomial/Wilson confidence intervals, or a Fisher's exact / mixed-effects logistic comparison across genotypes, could also quantify the categorical outcome. — An interval or test on the proportion gives a quantitative measure of certainty for the all-or-none phenotype across replicates.
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Gene set enrichment used the iDEA pipeline (one-sided) with Louis-method p-value adjustment.↳ Could also: Complementary GSEA approaches (e.g., fgsea/GSEA preranked or a hypergeometric/over-representation test with BH correction) could also be applied. — Cross-checking enrichment with an independent method and a widely-used multiplicity correction can corroborate which pathways are highlighted.
Result convergence & founder nodes
Findings this paper shares with others that ran a comparable experiment. A node’s strength is how many independent papers report it (replication breadth) — not how often it is cited, so a heavily-replicated but under-cited founder still stands out.
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CHD7 KO/KO completely abolishes hair cell and supporting cell differentiation in human inner ear organoids despite normal otic vesicle morphologyimaging human inner ear organoid down 2022×1papers★ This paper is the founder (earliest)
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CHD7 KO/KO causes mixed transcriptional dysregulation (323 genes up, 129 genes down) in d20 otic progenitorsscRNA-seq human inner ear organoid mixed 2022×1papers★ This paper is the founder (earliest)
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38.5% of otic lineage-specific genes including FBXO2 and COL9A2 are downregulated in CHD7 KO/KO d20 otic progenitors, indicating partial loss of otic identityscRNA-seq human inner ear organoid down 2022×1papers★ This paper is the founder (earliest)
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CHD7 KO/KO reduces the otic progenitor fraction from 89.6% to 39.7% among PAX2+ cells in d20 human inner ear organoidsscRNA-seq human inner ear organoid down 2022×1papers★ This paper is the founder (earliest)
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CHD7 KO/+ downregulates deafness genes including SIX1, USH1C, and STRC in d70 human inner ear hair cellsscRNA-seq human inner ear organoid down 2022×1papers★ This paper is the founder (earliest)
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15 OtoSCOPE deafness genes including TBX1, LMX1A, and SOX10 are downregulated in CHD7 KO/KO d20 otic progenitorsscRNA-seq human inner ear organoid down 2022×1papers★ This paper is the founder (earliest)
Citation network
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What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
Scope — pmid-36396635
Paper: Nie J, Ueda Y, Solivais AJ, Hashino E. CHD7 regulates otic lineage specification and hair cell differentiation in human inner ear organoids. Nat Commun 2022;13:7053. PMCID PMC9672366. DOI 10.1038/s41467-022-34759-8.
Data: GEO GSE208585 — scRNA-seq (10x Chromium 3' v3, NovaSeq 6000), 12 samples. GEO ships Cell Ranger filtered count matrices (barcodes/features/matrix.mtx per sample) → analysis can start from the cell×gene matrices (no FASTQ alignment needed).
Code named by paper: Seurat v4.0.3 (Read10X, CreateSeuratObject, QC filter, SCTransform, PCA[30 PCs], FindNeighbors/FindClusters, FindMarkers); DE via DESeq2 + zingeR; visualization via EnhancedVolcano (the repo link in the brief).
Sample → dataset mapping (from GEO titles)
| dataset | fig | GSMs | groups | reported N cells |
|---|---|---|---|---|
| d20 PAX2nG+ otic | Fig 3b / 4a | 6354844 (KO/KO), 6354845 (WT) | WT, KO/KO | 22,390 |
| d70 EPCAM | Fig 5a | 6354850–853 | WT/KO EPCAM± | 41,833 (Cluster0=2810) |
| d70 POU4F3nT | Fig 6a | 6354846–849 | WT/KO+ nT± | 34,703 (HC 9884 / SC 6273) |
| d20 WT PAX2nG± | Fig 1 | 6354854,855 | WT only | (not targeted) |
IN SCOPE (pipeline-derived, reproducible from shipped matrices)
- C1 Merged cell counts after QC for the 3 datasets (22,390 / 41,833 / 34,703).
- C2 d20 otic-progenitor DE: 323 up / 129 down genes (FC≥2, P≤1e-10), Fig 4a — the EnhancedVolcano figure. Primary target.
- C3 d70 POU4F3nT hair-cell / supporting-cell counts (9884 / 6273).
OUT OF SCOPE (not attempted, with reason)
- Cell Ranger alignment from FASTQ — FASTQ not on GEO (only matrices shipped); upstream of the reproducible boundary. We start from the shipped filtered matrices.
- iDEA GSEA bubble plots (Figs 4d, 6g-h), GO enrichment — separate non-shipped pipeline, manual gene-set curation; the hard 20%.
- Wet-lab claims (Western blot, immunostaining, FACS gating, stereocilia morphology).
- Exact QC thresholds: paper states only "extremely high/low UMIs, high % mito" with no numeric cutoffs → cell counts are threshold-sensitive (graded with tolerance).
- zingeR is archived (removed from Bioconductor); if it won't install we substitute Seurat's Wilcoxon DE and flag the method deviation explicitly.
Method-deviation notes
- Single 10x run per genotype ⇒ no biological replicates ⇒ pseudobulk DESeq2 is n=1 per group (invalid). Cell-level DESeq2+zingeR (cells as replicates) is what the paper used; we reproduce DE with Seurat FindMarkers (Wilcoxon) as the clean, deterministic alternative and report the count of genes passing the paper's FC≥2 & P≤1e-10 gate.
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