Fibroblasts neurotrophin signaling sustains pathological vascular maturation in rheumatoid arthritis.
Methods completeness
Major revisionpanel verdict · 2026-09-03
Methods Completeness & Reagent Traceability Audit
Manuscript: Fibroblasts neurotrophin signaling sustains pathological vascular maturation in rheumatoid arthritis
Cross-Cutting Items (Apply to All Manuscripts)
Sample Size & Replication Reporting
Status: PARTIALLY PRESENT
-
Spatial transcriptomics cohort (Xenium): n = 22 RA patients + 2 healthy donors, with paired pre/post-treatment biopsies stated in abstract and Methods. Total cells analyzed (2,049,358) and vascular cells (368,217) reported. ✓
-
Cell culture experiments: Biological replicates stated in figure legends as "Individual data points represent biological replicates" (e.g., Figs. 3D–E, 4A–C, 5C–E, 6E–G). However, specific n per condition is not uniformly stated in the main text or Methods—only visible in figures. For example:
- Fig. 3D–E (DLL4 + siRNA knockdown): n not stated in Methods or figure legend text
- Fig. 4A–C (neurotrophin stimulation): n not stated
- Fig. 5C–E (NOTCH3 KO + DLL4): n not stated
- HARD MISSING: Methods section should state n for each major cell culture experiment (co-culture, siRNA knockdown, NOTCH3 KO, neurotrophin stimulation, TRK inhibitor treatment).
-
Synovial explant/organoid studies: n stated in some figure legends (e.g., Fig. 6E–G quantifications show individual data points) but not systematized in Methods. How many independent tissue donors per condition? How many technical replicates per donor? HARD MISSING.
-
Technical vs. biological replicates: Distinction not clearly stated. Are the "individual data points" in figures biological replicates (independent donors/cultures) or technical replicates (same culture, multiple wells)? HARD MISSING.
Statistical Testing & Error Bars
Status: PRESENT but INCOMPLETE
- Named tests: Wilcoxon matched-pairs signed-rank test (Fig. 1G–H, paired patient samples), two-tailed Student's t-test, one-way ANOVA with Bonferroni post-hoc correction stated in multiple figure legends. ✓
- Error bars: Consistently labeled as "mean ± standard deviation (SD)" in figure legends (e.g., Figs. 4D–F, 5C–E, 6E–G, S5C, S9). ✓
- P-value reporting: P values shown on graphs or in legends. ✓
- Missing: No statement of significance threshold (α = 0.05 assumed but not stated in Methods). SOFT MISSING.
Software, Tool & Instrument Versions
Status: PARTIALLY PRESENT
| Tool/Software | Version | Status |
|---|---|---|
| Seurat | v5.0.0 | ✓ |
| Harmony | v1.2.4 | ✓ |
| Louvain algorithm | not versioned | unverifiable |
| Cellpose | cited (ref 48) | delegated-resolvable |
| scikit-image | cited (ref 49) | delegated-resolvable |
| UCell | GitHub link provided | ✓ |
| ImageJ | mentioned, no version | SOFT MISSING |
| Graphpad Prism | v10.4.1 | ✓ |
| R software | no version | SOFT MISSING |
| EVOS M7000 imaging system | model stated, no settings | HARD MISSING (see Microscopy section) |
| Xenium 5K Prime platform | model stated, no detailed instrument parameters | HARD MISSING (see Genomics/Spatial Transcriptomics section) |
| AriaMX Real Time PCR machine | model stated, no cycling parameters | HARD MISSING (see qPCR section below) |
Data Availability Statement
Status: MISSING
- No data-availability statement in the manuscript. The paper states "Xenium data generation and panel design" in Author Contributions but does not specify whether raw Xenium data, processed count matrices, or single-cell reference data are deposited in a public repository (GEO, ArrayExpress, etc.). HARD MISSING.
- Code availability: No statement on whether custom analysis scripts (Seurat pipeline, UCell scoring, spatial analysis) are available. GitHub/Zenodo/supplementary materials not mentioned. HARD MISSING.
Conditional Categories
1. Antibodies & Immunodetection (WB, IHC, IF, ELISA, RNAscope)
Trigger: PRESENT (extensive use across WB, IHC, IF, RNAscope, ELISA)
Western Blot
Status: PARTIALLY PRESENT
| Antibody | Vendor | Catalog # | Clone | Dilution | Host | Application | Status |
|---|---|---|---|---|---|---|---|
| pY-TRKA | Cell Signaling | #4638 (TrkA/TrkB sampler kit) | not stated | 1:500 | not stated | WB | unverifiable |
| Total TRKA | Cell Signaling | #4638 | not stated | 1:500 | not stated | WB | unverifiable |
| pY-TRKB | Cell Signaling | #4638 | not stated | 1:500 | not stated | WB | unverifiable |
| Total TRKB | Cell Signaling | #4638 | not stated | 1:500 | not stated | WB | unverifiable |
| CNN1 (calponin) | Proteintech | #24855-1-AP | not stated | not stated | not stated | WB | HARD MISSING dilution |
| MYH11 | Proteintech | #21404-1-AP | not stated | not stated | not stated | WB | HARD MISSING dilution |
| NGF | Abcam | #ab52918 | not stated | not stated | not stated | WB | HARD MISSING dilution |
| GAPDH | Thermo Fisher | #MA5-15738 | not stated | not stated | not stated | WB | HARD MISSING dilution |
| β-actin | Cell Signaling | #3700 | not stated | not stated | not stated | WB | HARD MISSING dilution |
| HRP-conjugated anti-rabbit | Thermo Fisher | #32460 | not stated | 1 hour, RT | not stated | WB secondary | SOFT MISSING dilution |
| HRP-conjugated anti-mouse | Thermo Fisher | #31430 | not stated | 1 hour, RT | not stated | WB secondary | SOFT MISSING dilution |
| HRP-conjugated anti-goat | Thermo Fisher | #A16005 | not stated | 1 hour, RT | not stated | WB secondary | SOFT MISSING dilution |
Issues:
- Primary antibody dilutions missing for CNN1, MYH11, NGF, GAPDH, β-actin. HARD MISSING.
- Host species not stated for any antibody. HARD MISSING.
- Clone information not provided (monoclonal vs. polyclonal). HARD MISSING.
- Secondary antibody dilutions not stated. SOFT MISSING.
Immunohistochemistry (IHC) & Immunofluorescence (IF)
Status: PARTIALLY PRESENT
| Antibody | Vendor | Catalog # | Clone | Dilution | Host | Application | Status |
|---|---|---|---|---|---|---|---|
| α-SMA (smooth muscle actin) | not stated | not stated | not stated | not stated | not stated | IHC, IF | HARD MISSING all details |
| PECAM1 (CD31) | Biolegend | #303106 | not stated | not stated | not stated | IF | HARD MISSING dilution, host |
| NGFR | Cell Signaling | #4638 | not stated | not stated | not stated | IF, IHC | HARD MISSING dilution, host |
| NTRK1 (TRKA) | not stated | not stated | not stated | not stated | not stated | IF, IHC | HARD MISSING all details |
| NTRK2 (TRKB) | not stated | not stated | not stated | not stated | not stated | IF, IHC | HARD MISSING all details |
| NTRK3 (TRKC) | not stated | not stated | not stated | not stated | not stated | IF, IHC | HARD MISSING all details |
| MYH11 | Proteintech | #21404-1-AP | not stated | not stated | not stated | IF, IHC | HARD MISSING dilution, host |
| RGS5 | not stated | not stated | not stated | not stated | not stated | IF (RNAscope) | delegated to RNAscope probes |
| Alexa Fluor 555 anti-rabbit | Thermo Fisher | #A-21424 | not stated | not stated | not stated | IF secondary | SOFT MISSING dilution |
| Alexa Fluor 488 anti-rabbit | Thermo Fisher | #A11034 | not stated | not stated | not stated | IF secondary | SOFT MISSING dilution |
| Alexa Fluor 647 anti-rabbit | Thermo Fisher | #A32733 | not stated | not stated | not stated | IF secondary | SOFT MISSING dilution |
| Alexa Fluor anti-goat | Thermo Fisher | #A110055 | not stated | not stated | not stated | IF secondary | SOFT MISSING dilution |
Issues:
- α-SMA source and catalog # not provided. HARD MISSING.
- Most primary antibody dilutions missing. HARD MISSING.
- Host species not stated. HARD MISSING.
- Secondary antibody dilutions not stated. SOFT MISSING.
RNAscope (In Situ Hybridization)
Status: PARTIALLY PRESENT
- Probes listed in Table 2 with gene targets and probe IDs (ACD Bio catalog numbers). ✓
- Assay: RNAScope multiplex fluorescent V2 (ACD Bio, SOP 45-009A). ✓
- Imaging: EVOS M7000 (model stated, no settings). See Microscopy section.
- Quantification method: Cellpose for nuclear segmentation, scikit-image for nuclear expansion and intensity calculation. ✓
- Missing: Specific probe sequences not provided in Table 2 (only gene names and catalog #s). For custom probes, sequences should be stated. SOFT MISSING (standard commercial probes, so less critical).
ELISA
Status: PARTIALLY PRESENT
- NGF ELISA: Fig. 3B, Fig. S3B, Fig. S8A report "pg/ml" with n and p-values.
- Kit/vendor not stated. Methods say "ELISA quantification of NGF secretion" but do not specify which ELISA kit (commercial or in-house). HARD MISSING.
- Assay parameters: No mention of detection range, sensitivity, or standard curve. SOFT MISSING.
2. Cell Lines & Primary Cells
Trigger: PRESENT (synovial fibroblasts, HUVECs)
Synovial Fibroblasts
Status: PARTIALLY PRESENT
- Source: "Synovial tissue collected after patients undergoing arthroplasty or synovectomy procedures" from Brigham and Women's Hospital and Flinders Medical Center. ✓
- IRB approval: MGB IRB no. 2019P002924 and Protocol#396.10 stated. ✓
- Isolation protocol: Enzymatic digestion (Dispase II, DNase I, Liberase TL) described in Methods. ✓
- Passage number: "3 to 6 passages for experiments" stated. ✓
- Authentication (STR): Not mentioned. HARD MISSING.
- Mycoplasma testing: Not mentioned. HARD MISSING.
- RRID/CVCL: Not assigned. SOFT MISSING (primary cells, not a cell line, so less critical).
- Media: "Complete FLS media (DMEM supplemented with 10% fetal bovine serum, HEPES, MEM amino acids, L-glutamine, penicillin-streptomycin, nonessential MEM amino acids, 2-mercaptoethanol, and gentamicin)" stated. ✓
HUVECs (Human Umbilical Vein Endothelial Cells)
Status: PARTIALLY PRESENT
- Source: "Thermofisher" stated. ✓
- Passage number: "passage 3-7" stated in co-culture section. ✓
- RRID/CVCL: Not provided. SOFT MISSING.
- Authentication (STR): Not mentioned. HARD MISSING.
- Mycoplasma testing: Not mentioned. HARD MISSING.
- Media: "EGM2 media consisting of EGM-Plus media (Lonza # CC-5035) supplemented with the EGM-plus bulletkit (Lonza, cc-3162)" stated. ✓
Issues:
- Neither cell type authenticated by STR or tested for mycoplasma. HARD MISSING for reproducibility.
3. Human Subjects & Clinical Data
Trigger: PRESENT (RA patient biopsies, clinical metadata)
IRB & Informed Consent
Status: PRESENT
- IRB approval: MGB IRB no. 2019P002924 (Brigham and Women's Hospital) and Protocol#396.10 (Flinders Medical Center) stated. ✓
- Informed consent: Not explicitly stated. HARD MISSING (should confirm consent was obtained).
Participant Demographics & Inclusion/Exclusion
Status: PARTIALLY PRESENT
- Cohort size: n = 22 RA patients + 2 healthy donors. ✓
- Disease status: "treatment-naive patients" and "pre-treatment and 6-month post-treatment" stated. ✓
- Treatment regimens: "triple csDMARD therapy (Hydroxychloroquine, methotrexate, and sulfasalazine) or TNFi (adalimumab)" stated. ✓
- Clinical outcome measure: "DAS28-ESR < 2.6" (clinical remission) mentioned. ✓
- Missing demographics:
- Age not stated. HARD MISSING.
- Sex distribution not stated. HARD MISSING.
- Disease duration not stated. HARD MISSING.
- Inclusion/exclusion criteria not stated. HARD MISSING.
- Serological status (RF, anti-CCP) not stated. SOFT MISSING.
Trial Registration
Status: NOT APPLICABLE (observational study, not an interventional trial)
4. Chemicals, Drugs & Dosing
Trigger: PRESENT (neurotrophins, TRK inhibitors, NOTCH inhibitors, small-molecule drugs)
Neurotrophins
Status: PARTIALLY PRESENT
| Drug | Vendor | Catalog # | CAS # | Concentration(s) Tested | Vehicle | Status |
|---|---|---|---|---|---|---|
| NGF (recombinant) | R&D Systems | 256-GF | not stated | 1, 100 ng/ml | DMSO or PBS | ✓ |
| BDNF (recombinant) | R&D Systems | 11166-BD | not stated | 100 ng/ml | DMSO or PBS | ✓ |
| NT3 (recombinant) | R&D Systems | 267-N3-005 | not stated | 50, 100 ng/ml | DMSO or PBS | ✓ |
Issues:
- CAS numbers not provided. SOFT MISSING.
- Vehicle final concentration not stated (e.g., "diluted in media" but % DMSO not specified). SOFT MISSING.
TRK Inhibitors & Modulators
Status: PARTIALLY PRESENT
| Drug | Vendor | Catalog # | CAS # | Concentration(s) | Vehicle | Status |
|---|---|---|---|---|---|---|
| GW-441756 (TRKA inhibitor) | Tocris | #2238 | not stated | 1, 5, 10 µM | not stated | ✓ conc. |
| ANA-12 (TRKB inhibitor) | Tocris | #4781 | not stated | 1, 5, 10 µM | not stated | ✓ conc. |
| GNF-5837 (pan-TRK inhibitor) | Tocris | #4559 | not stated | 1, 5, 10 µM | not stated | ✓ conc. |
| Entrectinib (FDA-approved TRK inhibitor) | MedChemExpress | HY-12678 | not stated | 1, 5, 10 µM (inferred) | not stated | ✓ conc. |
| Larotrectinib (FDA-approved TRK inhibitor) | MedChemExpress | HY-12866 | not stated | 1, 5, 10 µM (inferred) | not stated | ✓ conc. |
| 7,8-DHF (TRKB agonist) | Tocris | #3826 | not stated | 1, 5, 10 µM | not stated | ✓ conc. |
| LM22B-10 (pan-neurotrophin agonist) | Tocris | #6037 | not stated | 1, 5, 10 µM | not stated | ✓ conc. |
Issues:
- Vehicle (DMSO, ethanol, etc.) not stated for any inhibitor. HARD MISSING.
- Final vehicle concentration in culture media not stated. HARD MISSING.
- CAS numbers not provided. SOFT MISSING.
NOTCH Pathway Modulators
Status: PARTIALLY PRESENT
| Drug | Vendor | Catalog # | Concentration | Vehicle | Status |
|---|---|---|---|---|---|
| DAPT (γ-secretase inhibitor) | Tocris | #2634 | 10 µM | not stated | ✓ conc. |
| DLL4-Fc (NOTCH ligand) | R&D Systems | 10185-D4 | 5 µg/ml coating | not stated | ✓ conc. |
Issues:
- Vehicle not stated. HARD MISSING.
Other Reagents
Status: PARTIALLY PRESENT
| Reagent | Vendor | Catalog # | Concentration | Status |
|---|---|---|---|---|
| Matrigel | Corning | #356231 | 1:10 dilution (co-culture), 50 µL/well (organoids) | ✓ |
| Polyethylene glycol (PEG-it) | System Biosciences | #LV-810-A-1 | not stated | SOFT MISSING |
| Polybrene | not stated | not stated | 10 µg/ml | HARD MISSING vendor |
| WST-1 (cell viability assay) | Millipore Sigma | 501594400 | not stated | SOFT MISSING protocol |
5. Oligos, Plasmids & Constructs (siRNA, CRISPR, Lentiviral)
Trigger: PRESENT (siRNA knockdowns, CRISPR-Cas9 NOTCH3 KO, lentiviral NGFR overexpression)
siRNA Knockdowns
Status: PARTIALLY PRESENT
| Target | Vendor | Assay ID / Catalog # | Sequence | Transfection Reagent | Status |
|---|---|---|---|---|---|
| NGFR | Thermo Scientific Silencer Select | S194655 | not provided | RNAiMax | HARD MISSING sequence |
| NTRK1 | Thermo Scientific Silencer Select | S534734 | not provided | RNAiMax | HARD MISSING sequence |
| NTRK2 | Thermo Scientific Silencer Select | n321595 | not provided | RNAiMax | HARD MISSING sequence |
| NTRK3 | Thermo Scientific Silencer Select | s9753 | not provided | RNAiMax | HARD MISSING sequence |
| NOTCH3 | Thermo Scientific Silencer Select | 106100 | not provided | RNAiMax | HARD MISSING sequence |
Issues:
- siRNA sequences not provided. HARD MISSING (needed to verify specificity and check for off-targets).
- Transfection efficiency not stated. SOFT MISSING.
- Off-target assessment not mentioned. SOFT MISSING.
CRISPR-Cas9 NOTCH3 Knockout
Status: PARTIALLY PRESENT
- Cas variant: Not explicitly stated (assumed SpCas9, standard). SOFT MISSING.
- Guide RNA design: "Guide RNAs were designed using Synthego design tool" (ref 1, which is the authors' own prior work). Delegated-resolvable but should provide gRNA sequences. HARD MISSING sequences.
- Delivery method: "P3 primary cell 4D-nucleofector X kit S (#V4XP-3032, Lonza)" stated. ✓
- Edit validation: Not described. Were clones sequenced to confirm NOTCH3 deletion? HARD MISSING.
- Off-target assessment: Not mentioned. SOFT MISSING.
Lentiviral NGFR Overexpression
Status: PARTIALLY PRESENT
- Vector: "pLV-BsdCMV-hNGFR, VectorBuilder, #VB230823-1657hvq" (NGFR with N-terminal GFP-tag) and control "pLV-Bsd-CMV-EGFP (VectorBuilder, # VB230502-1039MVR)" stated. ✓
- Packaging system: "Virapower™ HiPerform™ Lentiviral FastTiter™ Gateway® Expression protocol (Thermo Fisher Scientific, #K534000)" cited. ✓
- Transduction: "50 µL of viral particles and 10 µg/ml of polybrene" stated. ✓
- Selection: Blasticidin selection implied by "Bsd" in vector name, but not explicitly stated. SOFT MISSING.
- Validation: "GFP expression in transduced cells was monitored microscopically" stated. ✓
- Titer determination: Not stated. SOFT MISSING.
6. Genomics, Sequencing & Spatial Transcriptomics
Trigger: PRESENT (Xenium spatial transcriptomics, bulk RNA-seq, qPCR)
Xenium Spatial Transcriptomics
Status: PARTIALLY PRESENT
- Platform: "Xenium 5K Prime platform" stated. ✓
- Sample preparation: "FFPE blocks" from synovial tissue, processed per "CG000760 Rev A, 10X Genomics" protocol. ✓
- Probe panels:
- "Xenium Prime 5K Human Pan Tissue & Pathways Panel (PN-1000671, 10X Genomics)" ✓
- "Custom add-on panels" mentioned but not fully described. HARD MISSING panel composition (which genes, how many probes).
- "High-sensitivity, custom spatial transcriptomic panel (Table. S, 1 to 3)" referenced but supplementary tables not provided in the manuscript text. HARD MISSING panel details.
- Cell segmentation: "Xenium Prime In Situ Gene Expression with optional Cell Segmentation Staining" protocol (CG000760, 10X Genomics). ✓
- Imaging: Xenium Analyzer (model stated, no detailed settings). SOFT MISSING laser power, exposure, gain, etc.
- Post-run H&E staining: "CG000613 Rev A, 10X Genomics" protocol. ✓
- Quality control: "Thresholded high quality cells based on transcripts and features per cell, kept 2 million high quality cells" stated. SOFT MISSING specific thresholds (min transcripts, min features per cell).
- Reference dataset: "AMP RA/SLE Consortium" single-cell reference used for annotation. ✓
- Data analysis:
- Seurat v5.0.0 ✓
- Harmony v1.2.4 ✓
- Louvain clustering (resolution 0.3) ✓
- Wilcoxon rank sum test for marker identification (presto package) ✓
- Repository accession: NOT PROVIDED. No GEO, ArrayExpress, or 10X Cloud accession stated. HARD MISSING.
Bulk RNA-Sequencing (NGFR Overexpression Study)
Status: MISSING DETAILS
- Mentioned in Fig. 5J: "Bulk RNA-seq for the organoid with drug treatment and without drug treatment" and "RNA-sequencing of NGFR-overexpressing fibroblasts."
- Platform: Not stated. HARD MISSING.
- Library prep kit: Not stated. HARD MISSING.
- Read length, paired/single-end: Not stated. HARD MISSING.
- Sequencing depth: Not stated. HARD MISSING.
- Reference genome & build: Not stated. HARD MISSING.
- Alignment/analysis tools & versions: Not stated. HARD MISSING.
- Differential expression method: "Differential expression analysis" mentioned but tool not named. HARD MISSING.
- FDR threshold: Not stated. HARD MISSING.
- Repository accession (GEO/SRA): NOT PROVIDED. HARD MISSING.
Issues: Bulk RNA-seq is central to the NGF/NGFR gene signature (Fig. 5J–M) but is severely under-documented. This is a load-bearing method that should not be delegated without full details.
qRT-PCR
Status: PARTIALLY PRESENT
- Instrument: AriaMX Real Time PCR machine (Agilent). ✓
- Cycling parameters: Not stated. HARD MISSING.
- Primer sequences: "Primer list is provided in Table 1." ✓ (Table 1 lists primers with sequences)
- cDNA synthesis: "QuantiTect Reverse Transcription Kit (#205311 Qiagen)" stated. ✓
- qPCR master mix: "Brilliant III qRT-PCR Master Mixes (#5994-1166EN, Agilent)" stated. ✓
- Normalization: "mRNA levels were normalized to GAPDH and calculated using the 2-ΔΔCT method" stated. ✓
- Replicates: Not stated per experiment. SOFT MISSING.
- Primer validation: Not mentioned (e.g., efficiency, specificity). SOFT MISSING.
7. Microscopy & Imaging
Trigger: PRESENT (wide-field fluorescence, confocal, whole-mount imaging)
Imaging Instruments & Settings
Status: PARTIALLY PRESENT
| Instrument | Model | Objective | NA | Detector | Laser/Illumination | Settings | Status |
|---|---|---|---|---|---|---|---|
| Whole-mount IF, IHC, RNAscope | EVOS M7000 | 20x (stated) | not stated | not stated | not stated | not stated | HARD MISSING NA, detector type, laser power, gain, offset |
| Confocal (micromass organoids) | not stated | 20x (stated) | not stated | not stated | not stated | not stated | HARD MISSING instrument model, detector, settings |
| Xenium Analyzer | Xenium (10X) | not applicable (in situ) | N/A | not stated | not stated | not stated | SOFT MISSING imaging settings |
Issues:
- EVOS M7000 is used extensively (Figs. 2B–C, 3A–C, 4K, 5A–B, 6C–D, S2E, S4A, etc.) but no objective NA, detector type, or acquisition settings provided. HARD MISSING.
- Confocal microscopy mentioned for "enlarged confocal (20X) images" (Fig. S5A) but instrument not identified. HARD MISSING.
- Fluorophore panel not explicitly stated (though inferred from RNAscope probes and antibodies). SOFT MISSING formal panel table.
Image Analysis & Quantification
Status: PARTIALLY PRESENT
- RNAscope quantification: Cellpose (ref 48) for nuclear segmentation, scikit-image (ref 49) for nuclear expansion and intensity calculation. ✓
- Gating/thresholding: "Cell types were assigned by gating normalized per cell intensity on high (>= 90th percentile) and low (<90th percentile) marker expression" stated. ✓
- Distance calculations: RANN package (ref 50) for distance to nearest endothelial cell. ✓
- ImageJ: Mentioned for "Image analysis and quantification" but no version or specific plugins stated. SOFT MISSING.
- Spatial transcriptomics analysis: Seurat, Harmony, Louvain, UCell (versions/links provided). ✓
8. Computational & Machine Learning (if applicable)
Trigger: PRESENT (spatial transcriptomics analysis, gene signature scoring)
Spatial Transcriptomics Analysis Pipeline
Status: PARTIALLY PRESENT
- Datasets: Xenium data from 46 samples (22 RA patients + 2 healthy donors, paired pre/post). ✓
- Train/validation/test split: Not applicable (descriptive analysis, not predictive modeling).
- Cell typing algorithm: Seurat v5.0.0 with Harmony v1.2.4 integration, Louvain clustering (resolution 0.3). ✓
- Hyperparameters: Resolution 0.3 stated; other Seurat/Harmony defaults assumed. SOFT MISSING full parameter list.
- Marker identification: Wilcoxon rank sum test (presto package). ✓
- Library versions: Seurat v5.0.0, Harmony v1.2.4, presto (no version), Cellpose (ref 48), scikit-image (ref 49), RANN (ref 50). ✓
- Hardware/compute budget: Not stated. SOFT MISSING.
- Random seeds: Not stated. SOFT MISSING.
- Code availability: Not stated. HARD MISSING.
Gene Signature Scoring (UCell)
Status: PARTIALLY PRESENT
- Gene signature: "NGF/NGFR gene signature score based on 461 upregulated genes from RNA-sequencing." ✓
- Scoring method: "UCell rank-based scoring approach." ✓
- UCell version/source: GitHub link provided (https://github.com/carmonalab/UCell). ✓
- Marker genes: The 461 genes are not listed in the manuscript. HARD MISSING gene list (should be in supplementary table or deposited).
- Validation: Applied to Xenium spatial data. ✓
9. Mass Spectrometry
Trigger: NOT PRESENT (no proteomics or metabolomics)
Protocol Provenance & Delegation
Methods Delegated to Citations
| Method | Citation | Resolvability | Status |
|---|---|---|---|
| Synovial fibroblast isolation | "as previously described (14)" | Ref 14 is Wei et al., Nature 582, 259–264 (2020), DOI 10.1038/s41586-020-2225-7 | Delegated-resolvable (published, DOI available) |
| Xenium slide preparation | "CG000760 Rev A, 10X Genomics" | 10X Genomics protocol document (not a peer-reviewed citation) | Delegated-resolvable (manufacturer protocol, publicly available) |
| Cell segmentation | "as outlined in our earlier published work (1)" | Ref 1 is Bhamidipati et al., bioRxiv 2025.03.14.642821 (preprint) | Delegated-unverifiable (preprint, not yet peer-reviewed; may change) |
| Fibroblast-endothelial co-culture | "as previously described (46)" | Ref 46 is Nguyen et al., Immunity 46, 220–232 (2017), DOI 10.1016/j.immuni.2017.01.007 | Delegated-resolvable |
| Synovial organoid generation | "as previously described (1, 14)" | Refs 1 & 14 (see above) | Delegated-resolvable |
| Collagen gel contraction assay | "as described earlier (12)" | Ref 12 is Romay et al., J Clin Invest 134 (2024), DOI 10.1172/JCI166134 | Delegated-resolvable |
| CRISPR guide RNA design | "Synthego design tool (1)" | Ref 1 (authors' prior work, preprint) | Delegated-unverifiable (preprint) |
| Cellpose segmentation | Ref 48 (Stringer et al., Nat Methods 18, 100–106, 2020) | Published, DOI 10.1038/s41592-020-01018-z | Delegated-resolvable |
| scikit-image analysis | Ref 49 (van der Walt et al., PeerJ 2, e453, 2014) | Published, DOI 10.7717/peerj.453 | Delegated-resolvable |
| RANN distance calculation | Ref 50 (Jones et al., PNAS 108, 15679–15686, 2011) | Published, DOI 10.1073/pnas.1015208108 | Delegated-resolvable |
| Lentiviral transduction | "Virapower™ HiPerform™ Lentiviral FastTiter™ Gateway® Expression protocol (Thermo Fisher Scientific, #K534000)" | Manufacturer protocol | Delegated-resolvable |
Key Issue: Ref 1 (Bhamidipati et al., bioRxiv 2025.03.14.642821) is a preprint that appears to be the authors' own concurrent work. Cell segmentation and CRISPR guide design are delegated to this preprint, which is not yet peer-reviewed and may change. This is problematic for reproducibility. HARD MISSING: These methods should be fully described in the current manuscript or the preprint should be replaced with a published reference.
Summary Table: HARD vs. SOFT Missing Items
| Category | HARD Missing | SOFT Missing |
|---|---|---|
| Cross-cutting | Sample size per condition (cell culture); Data availability statement; Code availability | Significance threshold (α); Software versions (R, ImageJ) |
| Antibodies | Primary dilutions (WB, IF, IHC); Host species; Clone info; α-SMA source; ELISA kit identity; Secondary dilutions (IF) | — |
| Cell lines | STR authentication (both cell types); Mycoplasma testing (both cell types) | RRID/CVCL for HUVECs |
| Human subjects | Informed consent statement; Age, sex, disease duration, inclusion/exclusion criteria | Serological status (RF, anti-CCP) |
| Chemicals/drugs | Vehicle identity and final concentration (inhibitors, agonists); Polybrene vendor | CAS numbers; ELISA protocol details |
| Oligos/plasmids | siRNA sequences; CRISPR gRNA sequences; NOTCH3 KO validation (sequencing) | Transfection efficiency; Off-target assessment; Lentiviral titer |
| Genomics | Bulk RNA-seq platform, library prep, read length, depth, reference genome, alignment tool, FDR threshold, repository accession; Xenium custom panel composition; QC thresholds; Bulk RNA-seq repository accession | qPCR cycling parameters; Primer validation; Xenium imaging settings |
| Microscopy | EVOS M7000 objective NA, detector type, laser/illumination settings; Confocal instrument model and settings | Fluorophore panel table |
| Computational | Code availability; Gene signature (461 genes) list | Hyperparameter details; Hardware; Random seeds |
| Protocol delegation | Full description of cell segmentation and CRISPR guide design (currently delegated to unreviewed preprint Ref 1) | — |
Critical Gaps Affecting Reproducibility
-
Bulk RNA-seq (load-bearing method): Platform, library prep, depth, reference genome, alignment tool, FDR, and repository accession all missing. This is central to the NGF/NGFR gene signature (Figs. 5J–M) and must be fully documented.
-
Data & code availability: No statement on where Xenium raw data, processed matrices, or custom analysis scripts are deposited. HARD MISSING.
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Antibody dilutions & specifications: Extensive use of antibodies (WB, IHC, IF, RNAscope) with missing dilutions, host species, and clone information. HARD MISSING.
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Cell authentication: No STR or mycoplasma testing for synovial fibroblasts or HUVECs. HARD MISSING.
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Xenium custom panels: Composition of custom add-on panels not specified. HARD MISSING.
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Delegation to unreviewed preprint: Cell segmentation and CRISPR guide design delegated to Ref 1 (bioRxiv preprint), which is not yet peer-reviewed. HARD MISSING in current manuscript.
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Clinical metadata: Age, sex, disease duration, and inclusion/exclusion criteria not provided for RA cohort. HARD MISSING.
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Vehicle specifications: Final concentrations of DMSO, ethanol, or other vehicles in culture media not stated for any small-molecule inhibitor or agonist. HARD MISSING.
Recommendations for Authors
- Provide supplementary tables with full Xenium panel composition, siRNA/gRNA sequences, gene signature (461 genes), and antibody specifications (dilutions, host, clone, RRID).
- Deposit data: Submit Xenium count matrices and bulk RNA-seq to GEO or equivalent; provide GitHub link for custom analysis code.
- Authenticate cells: Perform STR profiling and mycoplasma testing on synovial fibroblasts and HUVECs; report results.
- Expand Methods: Include full bulk RNA-seq protocol (platform, kit, depth, reference genome, alignment tool, FDR threshold) or cite a published protocol.
- Clarify delegation: Either move cell segmentation and CRISPR guide design into the main Methods (with full details) or wait for Ref 1 to be published before submission.
- Complete clinical metadata: Report age, sex, disease duration, serological status, and inclusion/exclusion criteria for the RA cohort.
- Specify vehicles & concentrations: State the vehicle (DMSO, ethanol, etc.) and final concentration in culture media for all small-molecule drugs.