RFX3 is essential for the generation of functional human pancreatic islets from stem cells
<h3>Aims/hypothesis</h3><p dir="ltr">The role of regulatory factor X 3 (RFX3) in human pancreatic islet development has not been explored. This study aims to investigate the function of RFX3 in human pancreatic islet development using human islet organoids derived from in...
محفوظ في:
| المؤلف الرئيسي: | |
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| مؤلفون آخرون: | , , , , |
| منشور في: |
2025
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| الموضوعات: | |
| الوسوم: |
إضافة وسم
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| _version_ | 1864513534625316864 |
|---|---|
| author | Bushra Memon (4792767) |
| author2 | Noura Aldous (15429873) Ahmed K. Elsayed (13275302) Sadaf Ijaz (10992855) Sikander Hayat (35163) Essam M. Abdelalim (5768072) |
| author2_role | author author author author author |
| author_facet | Bushra Memon (4792767) Noura Aldous (15429873) Ahmed K. Elsayed (13275302) Sadaf Ijaz (10992855) Sikander Hayat (35163) Essam M. Abdelalim (5768072) |
| author_role | author |
| dc.creator.none.fl_str_mv | Bushra Memon (4792767) Noura Aldous (15429873) Ahmed K. Elsayed (13275302) Sadaf Ijaz (10992855) Sikander Hayat (35163) Essam M. Abdelalim (5768072) |
| dc.date.none.fl_str_mv | 2025-04-23T09:00:00Z |
| dc.identifier.none.fl_str_mv | 10.1007/s00125-025-06424-4 |
| dc.relation.none.fl_str_mv | https://figshare.com/articles/journal_contribution/RFX3_is_essential_for_the_generation_of_functional_human_pancreatic_islets_from_stem_cells/30405370 |
| 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 Differentiation Endocrine pancreas Enterochromaffin cells iPSC model Transcription factor |
| dc.title.none.fl_str_mv | RFX3 is essential for the generation of functional human pancreatic islets from stem cells |
| 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">The role of regulatory factor X 3 (RFX3) in human pancreatic islet development has not been explored. This study aims to investigate the function of RFX3 in human pancreatic islet development using human islet organoids derived from induced pluripotent stem cells (iPSCs), hypothesising that RFX3 regulates human islet cell differentiation.</p><h3>Methods</h3><p dir="ltr">We generated <i>RFX3 </i> knockout (<i>RFX3 KO</i>) iPSC lines using CRISPR/Cas9 and differentiated them into pancreatic islet organoids. Various techniques were employed to assess gene expression, cell markers, apoptosis, proliferation and glucose-stimulated insulin secretion. Single-cell RNA-seq datasets from human embryonic stem cell-derived pancreatic islet differentiation were re-analysed to investigate RFX3 expression in specific cell populations at various developmental stages. Furthermore, bulk RNA-seq was conducted to further assess transcriptomic changes. RFX3<i> </i>overexpression was implemented to reverse dysregulated gene expression.</p><h3>Results</h3><p dir="ltr"><i>RFX3 </i>was found to be highly expressed in pancreatic endocrine cell populations within pancreatic progenitors (PPs), endocrine progenitors (EPs) and mature islet stages derived from iPSCs. Single-cell RNA-seq further confirmed <i>RFX3</i> expression across different endocrine cell clusters during differentiation. The loss of <i>RFX3</i> disrupted pancreatic endocrine gene regulation, reduced the number of hormone-secreting islet cells and impaired beta cell function and insulin secretion. Despite a significant reduction in the expression levels of pancreatic islet hormones, the pan-endocrine marker chromogranin A remained unchanged at both EP and islet stages, likely due to an increase in the abundance of enterochromaffin cells (ECs). This was supported by our findings of high EC marker expression levels in <i>RFX3</i> KO EPs and islets. In addition, RFX3 loss led to smaller islet organoids, elevated thioredoxin-interacting protein levels and increased apoptosis in EPs and islets. Furthermore, <i>RFX3</i> overexpression rescued the expression of dysregulated genes in <i>RFX3</i> KO at the PP and EP stages.</p><h3>Conclusions/interpretation</h3><p dir="ltr">These findings underscore the crucial role of RFX3 in regulating human islet cell differentiation and its role in suppressing EC specification. These insights into RFX3 function have implications for understanding islet biology and potential diabetes susceptibility.</p><h3>Data availability</h3><p dir="ltr">The RNA-seq datasets have been submitted to the Zenodo repository and can be accessed via the following links: DOI https://doi.org/10.5281/zenodo.13647651 (PPs); and DOI https://doi.org/10.5281/zenodo.13762055 (SC-islets).</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/s00125-025-06424-4" target="_blank">https://dx.doi.org/10.1007/s00125-025-06424-4</a></p> |
| eu_rights_str_mv | openAccess |
| id | Manara2_2909cab761c709f3ff7be56ef8f28f42 |
| identifier_str_mv | 10.1007/s00125-025-06424-4 |
| network_acronym_str | Manara2 |
| network_name_str | Manara2 |
| oai_identifier_str | oai:figshare.com:article/30405370 |
| publishDate | 2025 |
| repository.mail.fl_str_mv | |
| repository.name.fl_str_mv | |
| repository_id_str | |
| rights_invalid_str_mv | CC BY 4.0 |
| spelling | RFX3 is essential for the generation of functional human pancreatic islets from stem cellsBushra Memon (4792767)Noura Aldous (15429873)Ahmed K. Elsayed (13275302)Sadaf Ijaz (10992855)Sikander Hayat (35163)Essam M. Abdelalim (5768072)Biological sciencesGeneticsBiomedical and clinical sciencesMedical biochemistry and metabolomicsDifferentiationEndocrine pancreasEnterochromaffin cellsiPSC modelTranscription factor<h3>Aims/hypothesis</h3><p dir="ltr">The role of regulatory factor X 3 (RFX3) in human pancreatic islet development has not been explored. This study aims to investigate the function of RFX3 in human pancreatic islet development using human islet organoids derived from induced pluripotent stem cells (iPSCs), hypothesising that RFX3 regulates human islet cell differentiation.</p><h3>Methods</h3><p dir="ltr">We generated <i>RFX3 </i> knockout (<i>RFX3 KO</i>) iPSC lines using CRISPR/Cas9 and differentiated them into pancreatic islet organoids. Various techniques were employed to assess gene expression, cell markers, apoptosis, proliferation and glucose-stimulated insulin secretion. Single-cell RNA-seq datasets from human embryonic stem cell-derived pancreatic islet differentiation were re-analysed to investigate RFX3 expression in specific cell populations at various developmental stages. Furthermore, bulk RNA-seq was conducted to further assess transcriptomic changes. RFX3<i> </i>overexpression was implemented to reverse dysregulated gene expression.</p><h3>Results</h3><p dir="ltr"><i>RFX3 </i>was found to be highly expressed in pancreatic endocrine cell populations within pancreatic progenitors (PPs), endocrine progenitors (EPs) and mature islet stages derived from iPSCs. Single-cell RNA-seq further confirmed <i>RFX3</i> expression across different endocrine cell clusters during differentiation. The loss of <i>RFX3</i> disrupted pancreatic endocrine gene regulation, reduced the number of hormone-secreting islet cells and impaired beta cell function and insulin secretion. Despite a significant reduction in the expression levels of pancreatic islet hormones, the pan-endocrine marker chromogranin A remained unchanged at both EP and islet stages, likely due to an increase in the abundance of enterochromaffin cells (ECs). This was supported by our findings of high EC marker expression levels in <i>RFX3</i> KO EPs and islets. In addition, RFX3 loss led to smaller islet organoids, elevated thioredoxin-interacting protein levels and increased apoptosis in EPs and islets. Furthermore, <i>RFX3</i> overexpression rescued the expression of dysregulated genes in <i>RFX3</i> KO at the PP and EP stages.</p><h3>Conclusions/interpretation</h3><p dir="ltr">These findings underscore the crucial role of RFX3 in regulating human islet cell differentiation and its role in suppressing EC specification. These insights into RFX3 function have implications for understanding islet biology and potential diabetes susceptibility.</p><h3>Data availability</h3><p dir="ltr">The RNA-seq datasets have been submitted to the Zenodo repository and can be accessed via the following links: DOI https://doi.org/10.5281/zenodo.13647651 (PPs); and DOI https://doi.org/10.5281/zenodo.13762055 (SC-islets).</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/s00125-025-06424-4" target="_blank">https://dx.doi.org/10.1007/s00125-025-06424-4</a></p>2025-04-23T09:00:00ZTextJournal contributioninfo:eu-repo/semantics/publishedVersiontextcontribution to journal10.1007/s00125-025-06424-4https://figshare.com/articles/journal_contribution/RFX3_is_essential_for_the_generation_of_functional_human_pancreatic_islets_from_stem_cells/30405370CC BY 4.0info:eu-repo/semantics/openAccessoai:figshare.com:article/304053702025-04-23T09:00:00Z |
| spellingShingle | RFX3 is essential for the generation of functional human pancreatic islets from stem cells Bushra Memon (4792767) Biological sciences Genetics Biomedical and clinical sciences Medical biochemistry and metabolomics Differentiation Endocrine pancreas Enterochromaffin cells iPSC model Transcription factor |
| status_str | publishedVersion |
| title | RFX3 is essential for the generation of functional human pancreatic islets from stem cells |
| title_full | RFX3 is essential for the generation of functional human pancreatic islets from stem cells |
| title_fullStr | RFX3 is essential for the generation of functional human pancreatic islets from stem cells |
| title_full_unstemmed | RFX3 is essential for the generation of functional human pancreatic islets from stem cells |
| title_short | RFX3 is essential for the generation of functional human pancreatic islets from stem cells |
| title_sort | RFX3 is essential for the generation of functional human pancreatic islets from stem cells |
| topic | Biological sciences Genetics Biomedical and clinical sciences Medical biochemistry and metabolomics Differentiation Endocrine pancreas Enterochromaffin cells iPSC model Transcription factor |