Deletion of RFX6 impairs iPSC-derived islet organoid development and survival, with no impact on PDX1+/NKX6.1+ progenitors.

{"sci_version": "1.2", "schema": "https://corpus.bntrg.co.uk/sci/1.0/sci.schema.json", "id": "pmid:39080045", "kind": "science-record", "revision": 2, "content_hash": "sha256:8fd0cdb2ca62613d712e61176fb6c0def130d09848adc2c19a10a57b2be02aea", "created_at": "2026-11-01T20:29:09Z", "updated_at": "2026-11-01T21:33:33Z", "generator": {"name": "corpus-assay-engine", "run_id": "reading-sci-2026-11-01"}, "paper": {"state": "complete", "title": "Deletion of RFX6 impairs iPSC-derived islet organoid development and survival, with no impact on PDX1+/NKX6.1+ progenitors.", "doi": "10.1007/s00125-024-06232-2", "pmid": "39080045", "authors": ["Noura Aldous", "Ahmed K Elsayed", "Bushra Memon", "Sadaf Ijaz", "Sikander Hayat", "Essam M Abdelalim"], "journal": "Diabetologia", "year": 2024, "abstract": "AIMS/HYPOTHESIS: Homozygous mutations in RFX6 lead to neonatal diabetes accompanied by a hypoplastic pancreas, whereas heterozygous mutations cause MODY. Recent studies have also shown RFX6 variants to be linked with type 2 diabetes. Despite RFX6's known function in islet development, its specific role in diabetes pathogenesis remains unclear. Here, we aimed to understand the mechanisms underlying the impairment of pancreatic islet development and subsequent hypoplasia due to loss-of-function mutations in RFX6.\nMETHODS: We examined regulatory factor X6 (RFX6) expression during human embryonic stem cell (hESC) differentiation into pancreatic islets and re-analysed a single-cell RNA-seq dataset to identify RFX6-specific cell populations during islet development. Furthermore, induced pluripotent stem cell (iPSC) lines lacking RFX6 were generated using CRISPR/Cas9. Various approaches were then employed to explore the consequences of RFX6 loss across different developmental stages. Subsequently, we evaluated transcriptional changes resulting from RFX6 loss through RNA-seq of pancreatic progenitors (PPs) and endocrine progenitors (EPs).\nRESULTS: RFX6 expression was detected in PDX1+ cells in the hESC-derived posterior foregut (PF). However, in the PPs, RFX6 did not co-localise with pancreatic and duodenal homeobox 1 (PDX1) or NK homeobox 1 (NKX6.1) but instead co-localised with neurogenin 3, NK2 homeobox 2 and islet hormones in the EPs and islets. Single-cell analysis revealed high RFX6 expression levels in endocrine clusters across various hESC-derived pancreatic differentiation stages. Upon differentiating iPSCs lacking RFX6 into pancreatic islets, a significant decrease in PDX1 expression at the PF stage was observed, although this did not affect PPs co-expressing PDX1 and NKX6.1. RNA-seq analysis showed the downregulation of essential genes involved in pancreatic endocrine differentiation, insulin secretion and ion transport due to RFX6 deficiency. Furthermore, RFX6 deficiency resulted in the formation of smaller islet organoids due to increased cellular apoptosis, linked to reduced catalase expression, implying a protective role for RFX6. Overexpression of RFX6 reversed defective phenotypes in RFX6-knockout PPs, EPs and islets.\nCONCLUSIONS/INTERPRETATION: These findings suggest that pancreatic hypoplasia and reduced islet cell formation associated with RFX6 mutations are not due to alterations in PDX1+/NKX6.1+ PPs but instead result from cellular apoptosis and downregulation of pancreatic endocrine genes.\nDATA AVAILABILITY: RNA-seq datasets have been deposited in the Zenodo repository with accession link (DOI: https://doi.org/10.5281/zenodo.10656891 ).", "claims": [{"id": "claim-method", "statement": "At least three biological replicates were used in most experiments while statistical analysis was done using unpaired two-tailed Student’s t test on Prism version 8 (GraphPad Software, Boston, MA, USA, www.graphpad.com), with data represented as mean ± SD.", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:39080045", "locator": "sections[12] \"Statistical analysis\" paragraph 2", "quote": "At least three biological replicates were used in most experiments while statistical analysis was done using unpaired two-tailed Student’s t test on Prism version 8 (GraphPad Software, Boston, MA, USA, www.graphpad.com), with data represented as mean ± SD."}], "state": "complete"}, {"id": "claim-data-access", "statement": "RNA-seq datasets have been deposited in the Zenodo repository with accession link (DOI: 10.5281/zenodo.10656891).", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:39080045", "locator": "sections[25] \"notes\" paragraph 1", "quote": "RNA-seq datasets have been deposited in the Zenodo repository with accession link (DOI: 10.5281/zenodo.10656891)."}], "state": "complete"}, {"id": "claim-target-result", "statement": "RFX6 deficiency causes smaller pancreatic islet organoids through increased cellular apoptosis associated with reduced catalase expression, while disrupting pancreatic endocrine gene expression without preventing PDX1+/NKX6.1+ pancreatic progenitor formation.", "kind": "qualitative", "target": null, "evidence": [{"asset": "source:pmid:39080045", "locator": "sections[0] \"Abstract\" paragraph 3", "quote": "Furthermore, RFX6 deficiency resulted in the formation of smaller islet organoids due to increased cellular apoptosis, linked to reduced catalase expression, implying a protective role for RFX6."}, {"asset": "source:pmid:39080045", "locator": "sections[0] \"Abstract\" paragraph 4", "quote": "These findings suggest that pancreatic hypoplasia and reduced islet cell formation associated with RFX6 mutations are not due to alterations in PDX1+/NKX6.1+ PPs but instead result from cellular apoptosis and downregulation of pancreatic endocrine genes."}], "state": "complete"}], "gaps": []}, "experiment": {"state": "complete", "objective": "RFX6 deficiency causes smaller pancreatic islet organoids through increased cellular apoptosis associated with reduced catalase expression, while disrupting pancreatic endocrine gene expression without preventing PDX1+/NKX6.1+ pancreatic progenitor formation.", "steps": [{"id": "read_published_estimate", "order": 1, "name": "Read the published estimate from the source", "kind": "other", "state": "complete", "blocked_on": null, "description": "The claim, method and evidence span published with the source article.", "duration_hours": null, "parameters": {}, "inputs": ["source:pmid:39080045"], "outputs": ["the published estimate and its evidence"], "instruments": [], "reagents": []}, {"id": "obtain_source_data", "order": 2, "name": "Obtain the source data from the named repository", "kind": "data-preparation", "state": "complete", "blocked_on": null, "description": "The GEO series accession resolves and its sample accessions are listed. This lane did not download the dataset or read its licence.", "duration_hours": null, "parameters": {}, "inputs": ["data:geo:GSE202497"], "outputs": ["the source data with its repository provenance"], "instruments": [], "reagents": []}, {"id": "run_analysis", "order": 3, "name": "Run the published analysis", "kind": "analysis", "state": "complete", "blocked_on": null, "description": "Apply the paper's statistical method to the resolved series and compare the result with the published target.", "duration_hours": null, "parameters": {}, "inputs": ["data:geo:GSE202497"], "outputs": ["the replicated result and its comparison"], "instruments": [], "reagents": []}], "materials": [], "instruments": [], "reagents": [], "conditions": {}, "gaps": []}, "data": {"state": "partial", "assets": [{"id": "source:pmid:39080045", "role": "manuscript", "format": "xml", "media_type": "application/xml", "uri": "https://doi.org/10.1007/s00125-024-06232-2", "sha256": "sha256:1098acfe02298a2b5bcba44a365c12fe9d1ae206559f6d43139b7576ed366c13", "bytes": 42537, "access": "open", "state": "resolved", "reader": null, "reader_status": "reader_required", "identifier": "10.1007/s00125-024-06232-2", "version": null, "note": "Full article fetched from PMC; the checksum covers the rendered section body, not the original XML bytes.", "structure": {"pmcid": "PMC11604831", "source": "europe_pmc_fulltext", "coverage": "whole_body_without_references", "text_hash_scope": "rendered_section_body"}}, {"id": "data:geo:GSE202497", "role": "raw", "format": "geo-series", "media_type": "application/vnd.ncbi.geo-series+json", "uri": "https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE202497", "sha256": null, "bytes": null, "access": "open", "state": "resolved", "reader": null, "reader_status": "reader_required", "identifier": "GSE202497", "version": null, "note": "RNA-seq datasets have been deposited in the Zenodo repository with accession link (DOI: 10.5281/zenodo.10656891).", "structure": {"repository": "GEO", "access_route": "GEO accession URL", "access_statement_verbatim": "RNA-seq datasets have been deposited in the Zenodo repository with accession link (DOI: 10.5281/zenodo.10656891).", "access_statement_kind": "data_availability_statement", "sample_accessions": ["GSM6123264", "GSM6123265", "GSM6123266", "GSM6123267", "GSM6123268"], "sample_count": 5, "group_sizes": null, "group_sizes_basis": null, "platform": "18573", "taxon": "Homo sapiens", "title": "Improving stem cell-derived pancreatic islets using single-cell multiome-inferred regulomes [SC-islet scRNA-seq]", "resolution_checked_at": "2026-11-01T20:16:08Z", "resolution_check": "identifier_resolution_only_no_download_no_licence_read", "downloaded": false, "licence_read": false}}], "gaps": ["asset:data:geo:GSE202497 not downloaded", "asset:data:geo:GSE202497 licence not read"]}, "analysis": {"state": "partial", "methods": [{"id": "method-1", "family": null, "specific": null, "label": "unpaired two-tailed Student’s t test", "dsl_operations": [], "missing_operations": [], "expressible": null, "parameters": {}, "data_shapes": [], "evidence": {"score": 1.0, "band": "verbatim", "quote": "At least three biological replicates were used in most experiments while statistical analysis was done using unpaired two-tailed Student’s t test on Prism version 8 (GraphPad Software, Boston, MA, USA, www.graphpad.com), with data represented as mean ± SD."}}], "targets": [], "gaps": ["method.reader_named_unmapped"]}, "capability": {"state": "complete", "requirements": [{"capability_id": "capability:computational_analysis", "label": "Computational analysis platform", "assay_families": ["meta_analysis", "machine_learning", "survival_analysis", "statistical_regression", "pca", "molecular_docking", "network_analysis", "differential_expression", "gwas"], "availability_state": "owned", "tier_unlocked": "R1", "candidate": "Automated Assays Laravel application and simulated adapters"}], "gaps": []}, "readiness": {"state": "blocked", "tier": "R4", "blocking_axis": "method", "blocked_on": "method:not_determinable", "axes": {"method": {"tier": "R4", "expressible": false, "required_operations": [], "missing_operations": [], "basis": "The reader quoted the method the paper names (unpaired two-tailed Student’s t test), but the corpus's method extractor could not map it onto a DSL operation, so the method is published but not expressible."}, "capability": {"tier": "R1", "capability_id": "capability:computational_analysis", "availability_state": "owned", "basis": "The computational analysis is owned. The physical bench the paper used is not, so a physical validation cannot be run here."}, "data": {"tier": "R1", "reachable": true, "accessions": ["GSE202497"], "resolved_accessions": ["GSE202497"], "statements": ["RNA-seq datasets have been deposited in the Zenodo repository with accession link (DOI: 10.5281/zenodo.10656891)."], "basis": "The paper names GSE202497 in GEO and the accession resolves in the repository. No dataset was downloaded and no licence was read."}}, "can_run": {"dsl_operations": [], "assets": ["source:pmid:39080045", "data:geo:GSE202497"], "steps": ["run_analysis"]}, "must_build": {"dsl_operations": [], "converters": [], "readers": [], "capabilities": [], "data_gaps": ["data:geo:GSE202497"]}, "gaps": ["method:not_determinable"]}, "review": {"state": "partial", "machine_critique": {"kind": "extraction_review", "reader": {"model": "MiniMax-M3.1-Flash", "provider": "minimax", "provider_family": "minimax", "read_at": "2026-11-01T19:25:40Z", "independence": "single_reader"}, "evidence_check": {"method": "verbatim", "availability": "verbatim"}, "calls": 5, "usage": {"prompt_tokens": 13627, "completion_tokens": 1915, "cached_tokens": 264}}, "human_critique": null, "stages": [{"id": "extraction", "state": "complete", "reader": "MiniMax-M3.1-Flash", "provider": "minimax"}], "gaps": ["review.independent_reader", "review.machine_critique", "review.human_critique"]}, "execution": {"state": "absent", "status": "not_run", "mode": null, "runs": [], "gaps": ["execution.not_run"]}, "provenance": {"entities": [{"id": "pmid:39080045", "type": "paper", "label": "pmid:39080045"}, {"id": "source:pmid:39080045", "type": "manuscript", "label": "PMID 39080045 full text"}, {"id": "data:geo:GSE202497", "type": "dataset", "label": "GEO GSE202497"}], "activities": [{"id": "activity-reading-sci-2026-11-01", "type": "classification", "used": [{"id": "pmid:39080045", "type": "paper", "label": "pmid:39080045"}, {"id": "source:pmid:39080045", "type": "manuscript", "label": "PMID 39080045 full text"}, {"id": "data:geo:GSE202497", "type": "dataset", "label": "GEO GSE202497"}], "agent": "agent:corpus-assay-engine"}, {"id": "activity-read-39080045", "type": "extraction", "used": ["source:pmid:39080045"], "agent": "agent:minimax-reader"}, {"id": "activity-resolve-GSE202497", "type": "accession_resolution", "used": ["data:geo:GSE202497"], "agent": "agent:corpus-assay-engine"}], "agents": [{"id": "agent:corpus-assay-engine", "type": "software", "label": "Corpus assay engine"}, {"id": "agent:minimax-reader", "type": "software", "label": "MiniMax literature reader"}], "source_run_id": "reading-sci-2026-11-01", "dsl_catalogue_fingerprint": "343fb30756ba6ec3", "capability_table_fingerprint": "2cb3e42f047d2ecf", "snapshot_id": "ba34b49490d9bfaf"}, "lineage": {"parent_id": "pmid:39080045", "parent_hash": "sha256:df567f2e0900d8feb345096cf85550ba8ebbe96d2658a5268d7d2107774de135", "fork_of": null, "generation": 1}, "gaps": ["asset:data:geo:GSE202497 licence not read", "asset:data:geo:GSE202497 not downloaded", "execution.not_run", "method.reader_named_unmapped", "review.human_critique", "review.independent_reader", "review.machine_critique"], "assays": [{"role": "data_generation", "definition_id": "rna_seq", "definition_version": "99e1195c7d6f", "definition_digest": null, "required_operations": [], "support_state": "partial", "source_hashes": {"assay_lexicon": "99e1195c7d6f"}, "confidence": 0.99, "evidence": ["creatic islets and re-analysed a single-cell RNA-seq dataset to identify RFX6-specific cell popul", "nal changes resulting from RFX6 loss through RNA-seq of pancreatic progenitors (PPs) and endocrin", "ot affect PPs co-expressing PDX1 and NKX6.1. RNA-seq analysis showed the downregulation of essent", "ncreatic endocrine genes. DATA AVAILABILITY: RNA-seq datasets have been deposited in the Zenodo r"], "order_basis": "paper_text_order:765", "id": "assay:1"}, {"role": "data_generation", "definition_id": "transfection", "definition_version": "99e1195c7d6f", "definition_digest": null, "required_operations": [], "support_state": "partial", "source_hashes": {"assay_lexicon": "99e1195c7d6f"}, "confidence": 0.85, "evidence": ["PSC) lines lacking RFX6 were generated using CRISPR/Cas9. Various approaches were then employed"], "order_basis": "paper_text_order:940", "id": "assay:2"}, {"role": "data_generation", "definition_id": "apoptosis_assay", "definition_version": "99e1195c7d6f", "definition_digest": null, "required_operations": [], "support_state": "partial", "source_hashes": {"assay_lexicon": "99e1195c7d6f"}, "confidence": 0.93, "evidence": ["er islet organoids due to increased cellular apoptosis, linked to reduced catalase expression, impl", "NKX6.1+ PPs but instead result from cellular apoptosis and downregulation of pancreatic endocrine g"], "order_basis": "paper_text_order:2168", "id": "assay:3"}]}