Genome editing of TXNIP in human pluripotent stem cells for the generation of hepatocyte-like cells and insulin-producing islet-like aggregates
Abstract Background Thioredoxin-interacting protein (TXNIP) plays a role in regulating endoplasmic reticulum (ER) and oxidative stress, which disrupt glucose homeostasis in diabetes. However, the impact of TXNIP deficiency on the differentiation and functionality of human stem cell-derived somatic m...
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BMC
2025-05-01
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| Series: | Stem Cell Research & Therapy |
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| Online Access: | https://doi.org/10.1186/s13287-025-04314-5 |
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| author | Leonardo Traini Javier Negueruela Bernat Elvira Wadsen St-Pierre-Wijckmans Valerie Vandenbempt Carlos E. Buss Ao Li Israel Pérez-Chávez Francisco Ribeiro-Costa Mariana Nunes Joris Messens Daria Ezeriņa David C. Hay Mayank Bansal Esteban N. Gurzov |
| author_facet | Leonardo Traini Javier Negueruela Bernat Elvira Wadsen St-Pierre-Wijckmans Valerie Vandenbempt Carlos E. Buss Ao Li Israel Pérez-Chávez Francisco Ribeiro-Costa Mariana Nunes Joris Messens Daria Ezeriņa David C. Hay Mayank Bansal Esteban N. Gurzov |
| author_sort | Leonardo Traini |
| collection | DOAJ |
| description | Abstract Background Thioredoxin-interacting protein (TXNIP) plays a role in regulating endoplasmic reticulum (ER) and oxidative stress, which disrupt glucose homeostasis in diabetes. However, the impact of TXNIP deficiency on the differentiation and functionality of human stem cell-derived somatic metabolic cells remains unclear. Methods We used CRISPR-Cas12a genome editing to generate TXNIP-deficient (TXNIP −/− ) H1 human embryonic stem cells (H1-hESCs). These cells were differentiated into hepatocyte-like cells (HLCs) and stem-cell-derived insulin-producing islets (SC-islets). The maturation and functionality TXNIP −/− and TXNIP +/+ SC-islets were assessed by implantation under the kidney capsule of male or female NOD-SCID mice. Results TXNIP deficiency significantly increased H1-hESC proliferation without affecting pluripotency, viability, or differentiation potential into HLCs and SC-islets. Bulk RNA-sequencing of thapsigargin-treated TXNIP −/− and TXNIP +/+ hESCs revealed differential expression of stress-responsive genes, with enriched apoptosis-related pathways in TXNIP +/+ cells, but minimal transcriptional changes specific to TXNIP deficiency. In HLCs, TXNIP deletion reduced albumin secretion and insulin signalling, as indicated by decreased AKT phosphorylation, while showing no differences in glycolytic activity or lipid metabolism markers. Under thapsigargin-induced ER stress, TXNIP −/− HLCs exhibited transiently reduced eIF2α phosphorylation and lower BiP expression, suggesting compromised adaptive responses to prolonged stress. SC-islets derived from TXNIP −/− hESCs showed comparable viability, endocrine cell composition, and cytokine responses to TXNIP +/+ islets. Following IFNα or IFNγ treatment, STAT1 phosphorylation was increased in TXNIP −/− SC-islets, indicating that IFN signalling remained intact despite TXNIP deficiency. Upon implantation into NOD-SCID mice, both TXNIP −/− and TXNIP +/+ SC-islets produced human C-peptide and responded to glucose stimulation. However, TXNIP −/− SC-islets did not demonstrate enhanced glycaemic control or glucose-stimulated insulin secretion compared to controls. Conclusions Our study demonstrates that TXNIP deficiency does not improve the differentiation or functionality of HLCs and SC-islets. We present the generation and characterisation of TXNIP −/− and TXNIP +/+ H1-hESCs, HLCs, and SC-islets as valuable models for future studies on the role of TXNIP in metabolic cell biology. |
| format | Article |
| id | doaj-art-022a161cd7344fc0b37e43d1aace9ff3 |
| institution | DOAJ |
| issn | 1757-6512 |
| language | English |
| publishDate | 2025-05-01 |
| publisher | BMC |
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| series | Stem Cell Research & Therapy |
| spelling | doaj-art-022a161cd7344fc0b37e43d1aace9ff32025-08-20T03:09:35ZengBMCStem Cell Research & Therapy1757-65122025-05-0116111810.1186/s13287-025-04314-5Genome editing of TXNIP in human pluripotent stem cells for the generation of hepatocyte-like cells and insulin-producing islet-like aggregatesLeonardo Traini0Javier Negueruela1Bernat Elvira2Wadsen St-Pierre-Wijckmans3Valerie Vandenbempt4Carlos E. Buss5Ao Li6Israel Pérez-Chávez7Francisco Ribeiro-Costa8Mariana Nunes9Joris Messens10Daria Ezeriņa11David C. Hay12Mayank Bansal13Esteban N. Gurzov14Signal Transduction and Metabolism Laboratory, Université Libre de BruxellesSignal Transduction and Metabolism Laboratory, Université Libre de BruxellesSignal Transduction and Metabolism Laboratory, Université Libre de BruxellesSignal Transduction and Metabolism Laboratory, Université Libre de BruxellesSignal Transduction and Metabolism Laboratory, Université Libre de BruxellesSignal Transduction and Metabolism Laboratory, Université Libre de BruxellesSignal Transduction and Metabolism Laboratory, Université Libre de BruxellesSignal Transduction and Metabolism Laboratory, Université Libre de BruxellesSignal Transduction and Metabolism Laboratory, Université Libre de BruxellesSignal Transduction and Metabolism Laboratory, Université Libre de BruxellesVIB-VUB Center for Structural Biology, Vlaams Instituut Voor BiotechnologieVIB-VUB Center for Structural Biology, Vlaams Instituut Voor BiotechnologieInstitute for Regeneration and Repair, Centre for Regenerative Medicine, University of EdinburghSignal Transduction and Metabolism Laboratory, Université Libre de BruxellesSignal Transduction and Metabolism Laboratory, Université Libre de BruxellesAbstract Background Thioredoxin-interacting protein (TXNIP) plays a role in regulating endoplasmic reticulum (ER) and oxidative stress, which disrupt glucose homeostasis in diabetes. However, the impact of TXNIP deficiency on the differentiation and functionality of human stem cell-derived somatic metabolic cells remains unclear. Methods We used CRISPR-Cas12a genome editing to generate TXNIP-deficient (TXNIP −/− ) H1 human embryonic stem cells (H1-hESCs). These cells were differentiated into hepatocyte-like cells (HLCs) and stem-cell-derived insulin-producing islets (SC-islets). The maturation and functionality TXNIP −/− and TXNIP +/+ SC-islets were assessed by implantation under the kidney capsule of male or female NOD-SCID mice. Results TXNIP deficiency significantly increased H1-hESC proliferation without affecting pluripotency, viability, or differentiation potential into HLCs and SC-islets. Bulk RNA-sequencing of thapsigargin-treated TXNIP −/− and TXNIP +/+ hESCs revealed differential expression of stress-responsive genes, with enriched apoptosis-related pathways in TXNIP +/+ cells, but minimal transcriptional changes specific to TXNIP deficiency. In HLCs, TXNIP deletion reduced albumin secretion and insulin signalling, as indicated by decreased AKT phosphorylation, while showing no differences in glycolytic activity or lipid metabolism markers. Under thapsigargin-induced ER stress, TXNIP −/− HLCs exhibited transiently reduced eIF2α phosphorylation and lower BiP expression, suggesting compromised adaptive responses to prolonged stress. SC-islets derived from TXNIP −/− hESCs showed comparable viability, endocrine cell composition, and cytokine responses to TXNIP +/+ islets. Following IFNα or IFNγ treatment, STAT1 phosphorylation was increased in TXNIP −/− SC-islets, indicating that IFN signalling remained intact despite TXNIP deficiency. Upon implantation into NOD-SCID mice, both TXNIP −/− and TXNIP +/+ SC-islets produced human C-peptide and responded to glucose stimulation. However, TXNIP −/− SC-islets did not demonstrate enhanced glycaemic control or glucose-stimulated insulin secretion compared to controls. Conclusions Our study demonstrates that TXNIP deficiency does not improve the differentiation or functionality of HLCs and SC-islets. We present the generation and characterisation of TXNIP −/− and TXNIP +/+ H1-hESCs, HLCs, and SC-islets as valuable models for future studies on the role of TXNIP in metabolic cell biology.https://doi.org/10.1186/s13287-025-04314-5TXNIPCRISPR-Cas12aStem cellsHepatocyte-like cellsStem cell-derived islets |
| spellingShingle | Leonardo Traini Javier Negueruela Bernat Elvira Wadsen St-Pierre-Wijckmans Valerie Vandenbempt Carlos E. Buss Ao Li Israel Pérez-Chávez Francisco Ribeiro-Costa Mariana Nunes Joris Messens Daria Ezeriņa David C. Hay Mayank Bansal Esteban N. Gurzov Genome editing of TXNIP in human pluripotent stem cells for the generation of hepatocyte-like cells and insulin-producing islet-like aggregates Stem Cell Research & Therapy TXNIP CRISPR-Cas12a Stem cells Hepatocyte-like cells Stem cell-derived islets |
| title | Genome editing of TXNIP in human pluripotent stem cells for the generation of hepatocyte-like cells and insulin-producing islet-like aggregates |
| title_full | Genome editing of TXNIP in human pluripotent stem cells for the generation of hepatocyte-like cells and insulin-producing islet-like aggregates |
| title_fullStr | Genome editing of TXNIP in human pluripotent stem cells for the generation of hepatocyte-like cells and insulin-producing islet-like aggregates |
| title_full_unstemmed | Genome editing of TXNIP in human pluripotent stem cells for the generation of hepatocyte-like cells and insulin-producing islet-like aggregates |
| title_short | Genome editing of TXNIP in human pluripotent stem cells for the generation of hepatocyte-like cells and insulin-producing islet-like aggregates |
| title_sort | genome editing of txnip in human pluripotent stem cells for the generation of hepatocyte like cells and insulin producing islet like aggregates |
| topic | TXNIP CRISPR-Cas12a Stem cells Hepatocyte-like cells Stem cell-derived islets |
| url | https://doi.org/10.1186/s13287-025-04314-5 |
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