Deletion of <i>RFX6</i> impairs iPSC-derived islet organoid development and survival, with no impact on PDX1<sup>+</sup>/NKX6.1<sup>+</sup> progenitors
<h3>Aims/hypothesis</h3><p dir="ltr">Homozygous mutations in <i>RFX6</i> lead to neonatal diabetes accompanied by a hypoplastic pancreas, whereas heterozygous mutations cause MODY. Recent studies have also shown <i>RFX6</i> variants to be linked wi...
محفوظ في:
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| مؤلفون آخرون: | , , , , |
| منشور في: |
2024
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| الموضوعات: | |
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إضافة وسم
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| _version_ | 1864513541454692352 |
|---|---|
| author | Noura Aldous (15429873) |
| author2 | Ahmed K. Elsayed (13275302) Bushra Memon (4792767) Sadaf Ijaz (10992855) Sikander Hayat (35163) Essam M. Abdelalim (5768072) |
| author2_role | author author author author author |
| author_facet | Noura Aldous (15429873) Ahmed K. Elsayed (13275302) Bushra Memon (4792767) Sadaf Ijaz (10992855) Sikander Hayat (35163) Essam M. Abdelalim (5768072) |
| author_role | author |
| dc.creator.none.fl_str_mv | Noura Aldous (15429873) Ahmed K. Elsayed (13275302) Bushra Memon (4792767) Sadaf Ijaz (10992855) Sikander Hayat (35163) Essam M. Abdelalim (5768072) |
| dc.date.none.fl_str_mv | 2024-07-30T09:00:00Z |
| dc.identifier.none.fl_str_mv | 10.1007/s00125-024-06232-2 |
| dc.relation.none.fl_str_mv | https://figshare.com/articles/journal_contribution/Deletion_of_i_RFX6_i_impairs_iPSC-derived_islet_organoid_development_and_survival_with_no_impact_on_PDX1_sup_sup_NKX6_1_sup_sup_progenitors/29900402 |
| dc.rights.none.fl_str_mv | CC BY 4.0 info:eu-repo/semantics/openAccess |
| dc.subject.none.fl_str_mv | Biological sciences Genetics Biomedical and clinical sciences Medical biochemistry and metabolomics Paediatrics Diabetes Endocrine specification Islet organoids Pancreatic hypoplasia Pancreatic progenitors Transcription factors |
| dc.title.none.fl_str_mv | Deletion of <i>RFX6</i> impairs iPSC-derived islet organoid development and survival, with no impact on PDX1<sup>+</sup>/NKX6.1<sup>+</sup> progenitors |
| dc.type.none.fl_str_mv | Text Journal contribution info:eu-repo/semantics/publishedVersion text contribution to journal |
| description | <h3>Aims/hypothesis</h3><p dir="ltr">Homozygous mutations in <i>RFX6</i> lead to neonatal diabetes accompanied by a hypoplastic pancreas, whereas heterozygous mutations cause MODY. Recent studies have also shown <i>RFX6</i> 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 <i>RFX6</i>.</p><h3>Methods</h3><p dir="ltr">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).</p><h3>Results</h3><p dir="ltr">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.</p><h3>Conclusions/interpretation</h3><p dir="ltr">These findings suggest that pancreatic hypoplasia and reduced islet cell formation associated with RFX6 mutations are not due to alterations in PDX1<sup>+</sup>/NKX6.1<sup>+</sup> PPs but instead result from cellular apoptosis and downregulation of pancreatic endocrine genes.</p><h2>Other Information</h2><p dir="ltr">Published in: Diabetologia<br>License: <a href="https://creativecommons.org/licenses/by/4.0" target="_blank">https://creativecommons.org/licenses/by/4.0</a><br>See article on publisher's website: <a href="https://dx.doi.org/10.1007/s00125-024-06232-2" target="_blank">https://dx.doi.org/10.1007/s00125-024-06232-2</a></p> |
| eu_rights_str_mv | openAccess |
| id | Manara2_20444607ce2c1dc8f136a6854e49ec0f |
| identifier_str_mv | 10.1007/s00125-024-06232-2 |
| network_acronym_str | Manara2 |
| network_name_str | Manara2 |
| oai_identifier_str | oai:figshare.com:article/29900402 |
| publishDate | 2024 |
| repository.mail.fl_str_mv | |
| repository.name.fl_str_mv | |
| repository_id_str | |
| rights_invalid_str_mv | CC BY 4.0 |
| spelling | Deletion of <i>RFX6</i> impairs iPSC-derived islet organoid development and survival, with no impact on PDX1<sup>+</sup>/NKX6.1<sup>+</sup> progenitorsNoura Aldous (15429873)Ahmed K. Elsayed (13275302)Bushra Memon (4792767)Sadaf Ijaz (10992855)Sikander Hayat (35163)Essam M. Abdelalim (5768072)Biological sciencesGeneticsBiomedical and clinical sciencesMedical biochemistry and metabolomicsPaediatricsDiabetesEndocrine specificationIslet organoidsPancreatic hypoplasiaPancreatic progenitorsTranscription factors<h3>Aims/hypothesis</h3><p dir="ltr">Homozygous mutations in <i>RFX6</i> lead to neonatal diabetes accompanied by a hypoplastic pancreas, whereas heterozygous mutations cause MODY. Recent studies have also shown <i>RFX6</i> 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 <i>RFX6</i>.</p><h3>Methods</h3><p dir="ltr">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).</p><h3>Results</h3><p dir="ltr">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.</p><h3>Conclusions/interpretation</h3><p dir="ltr">These findings suggest that pancreatic hypoplasia and reduced islet cell formation associated with RFX6 mutations are not due to alterations in PDX1<sup>+</sup>/NKX6.1<sup>+</sup> PPs but instead result from cellular apoptosis and downregulation of pancreatic endocrine genes.</p><h2>Other Information</h2><p dir="ltr">Published in: Diabetologia<br>License: <a href="https://creativecommons.org/licenses/by/4.0" target="_blank">https://creativecommons.org/licenses/by/4.0</a><br>See article on publisher's website: <a href="https://dx.doi.org/10.1007/s00125-024-06232-2" target="_blank">https://dx.doi.org/10.1007/s00125-024-06232-2</a></p>2024-07-30T09:00:00ZTextJournal contributioninfo:eu-repo/semantics/publishedVersiontextcontribution to journal10.1007/s00125-024-06232-2https://figshare.com/articles/journal_contribution/Deletion_of_i_RFX6_i_impairs_iPSC-derived_islet_organoid_development_and_survival_with_no_impact_on_PDX1_sup_sup_NKX6_1_sup_sup_progenitors/29900402CC BY 4.0info:eu-repo/semantics/openAccessoai:figshare.com:article/299004022024-07-30T09:00:00Z |
| spellingShingle | Deletion of <i>RFX6</i> impairs iPSC-derived islet organoid development and survival, with no impact on PDX1<sup>+</sup>/NKX6.1<sup>+</sup> progenitors Noura Aldous (15429873) Biological sciences Genetics Biomedical and clinical sciences Medical biochemistry and metabolomics Paediatrics Diabetes Endocrine specification Islet organoids Pancreatic hypoplasia Pancreatic progenitors Transcription factors |
| status_str | publishedVersion |
| title | Deletion of <i>RFX6</i> impairs iPSC-derived islet organoid development and survival, with no impact on PDX1<sup>+</sup>/NKX6.1<sup>+</sup> progenitors |
| title_full | Deletion of <i>RFX6</i> impairs iPSC-derived islet organoid development and survival, with no impact on PDX1<sup>+</sup>/NKX6.1<sup>+</sup> progenitors |
| title_fullStr | Deletion of <i>RFX6</i> impairs iPSC-derived islet organoid development and survival, with no impact on PDX1<sup>+</sup>/NKX6.1<sup>+</sup> progenitors |
| title_full_unstemmed | Deletion of <i>RFX6</i> impairs iPSC-derived islet organoid development and survival, with no impact on PDX1<sup>+</sup>/NKX6.1<sup>+</sup> progenitors |
| title_short | Deletion of <i>RFX6</i> impairs iPSC-derived islet organoid development and survival, with no impact on PDX1<sup>+</sup>/NKX6.1<sup>+</sup> progenitors |
| title_sort | Deletion of <i>RFX6</i> impairs iPSC-derived islet organoid development and survival, with no impact on PDX1<sup>+</sup>/NKX6.1<sup>+</sup> progenitors |
| topic | Biological sciences Genetics Biomedical and clinical sciences Medical biochemistry and metabolomics Paediatrics Diabetes Endocrine specification Islet organoids Pancreatic hypoplasia Pancreatic progenitors Transcription factors |