Direct Epigenetic Reprogramming of Human Somatic Cells into Insulin-Producing Cell
Main Article Content
Abstract
Restoring insulin production through replacement of pancreatic β-cells presents a promising strategy for treating individuals with type 1 diabetes. However, current methods involving induced pluripotent stem cell differentiation are often time-consuming, multi-stage, and limited by safety and efficiency concerns. To overcome these challenges, we developed a simplified and direct strategy to convert human somatic cells into insulin-producing cells using an epigenetic activation system. This system combines a multiplex epigenetic engineering vector composed of dCas9.P300core and guide RNAs targeting five key β-cell genes: PDX1, NKX6.1, MAFA, Insulin, and glucose transporter type 2 (Glut2). The resulting Glut2⁺ cells exhibited glucose-responsive insulin secretion and expressed essential β-cell transcription factors including NKX2.2, along with insulin-processing and secretory machinery genes (Cav1.3, GSK3β, KCNJ11, SLC30A8). Absence of α-cell markers (aristaless-related homeobox or glucagon) confirmed lineage specificity and functional fidelity. This reprogramming approach eliminates the need for pluripotent intermediates and significantly reduces the time required to generate functional β-like cells. Our platform offers a rapid, non-integrative, and scalable method for producing insulin-secreting cells, with potential applications in personalized cell therapy, disease modeling, and high-throughput drug screening for diabetes research.
Article Details
How to Cite
NAQVI, Raza Ali et al.
Direct Epigenetic Reprogramming of Human Somatic Cells into Insulin-Producing Cell.
Medical Research Archives, [S.l.], v. 13, n. 9, oct. 2025.
ISSN 2375-1924.
Available at: <https://esmed.org/MRA/mra/article/view/6852>. Date accessed: 05 dec. 2025.
doi: https://doi.org/10.18103/mra.v13i9.6852.
Section
Research Articles
The Medical Research Archives grants authors the right to publish and reproduce the unrevised contribution in whole or in part at any time and in any form for any scholarly non-commercial purpose with the condition that all publications of the contribution include a full citation to the journal as published by the Medical Research Archives.
References
1 Hering, B. J. et al. Phase 3 Trial of Transplantation of Human Islets in Type 1 Diabetes Complicated by Severe Hypoglycemia. Diabetes Care 39, 1230-1240, doi:10.2337/dc15-1988 (2016).
2 Shapiro, A. M. et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 343, 230-238, doi:10.1056/NEJM200007273430401 (2000).
3 Markmann, J. F. et al. Phase 3 trial of human islet-after-kidney transplantation in type 1 diabetes. Am J Transplant 21, 1477-1492, doi:10.1111/ajt.16174 (2021).
4 Hering, B. J. et al. Prolonged diabetes reversal after intraportal xenotransplantation of wild-type porcine islets in immunosuppressed nonhuman primates. Nat Med 12, 301-303, doi:10.1038/nm1369 (2006).
5 Graham, M. L. et al. Clinically available immunosuppression averts rejection but not systemic inflammation after porcine islet xenotransplant in cynomolgus macaques. Am J Transplant 22, 745-760, doi:10.1111/ajt.16876 (2022).
6 Singh, A. et al. Long-term tolerance of islet allografts in nonhuman primates induced by apoptotic donor leukocytes. Nat Commun 10, 3495, doi:10.1038/s41467-019-11338-y (2019).
7 Cardona, K. et al. Long-term survival of neonatal porcine islets in nonhuman primates by targeting costimulation pathways. Nat Med 12, 304-306, doi:10.1038/nm1375 (2006).
8 van der Windt, D. J. et al. Long-term controlled normoglycemia in diabetic non-human primates after transplantation with hCD46 transgenic porcine islets. Am J Transplant 9, 2716-2726, doi:10.1111/j.1600-6143.2009.02850.x (2009).
9 Naqvi, R. A. et al. The future treatment for type 1 diabetes: Pig islet- or stem cell-derived beta cells? Front Endocrinol (Lausanne) 13, 1001041, doi:10.3389/fendo.2022.1001041 (2022).
10 Denner, J. Porcine Endogenous Retroviruses and Xenotransplantation, 2021. Viruses 13, doi:10.3390/v13112156 (2021).
11 Millman, J. R. et al. Generation of stem cell-derived beta-cells from patients with type 1 diabetes. Nat Commun 7, 11463, doi:10.1038/ncomms11463 (2016).
12 Pagliuca, F. W. et al. Generation of functional human pancreatic beta cells in vitro. Cell 159, 428-439, doi:10.1016/j.cell.2014.09.040 (2014).
13 Rezania, A. et al. Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells. Nat Biotechnol 32, 1121-1133, doi:10.1038/nbt.3033 (2014).
14 Watanabe, T. et al. Evaluating teratoma formation risk of pluripotent stem cell-derived cell therapy products: a consensus recommendation from the Health and Environmental Sciences Institute's International Cell Therapy Committee. Cytotherapy 27, 1072-1084, doi:10.1016/j.jcyt.2025.04.062 (2025).
15 Maxwell, K. G. & Millman, J. R. Applications of iPSC-derived beta cells from patients with diabetes. Cell Rep Med 2, 100238, doi:10.1016/j.xcrm.2021.100238 (2021).
16 Pellegrini, S., Zamarian, V. & Sordi, V. Strategies to Improve the Safety of iPSC-Derived beta Cells for beta Cell Replacement in Diabetes. Transpl Int 35, 10575, doi:10.3389/ti.2022.10575 (2022).
17 Tyumentseva, M., Tyumentsev, A. & Akimkin, V. CRISPR/Cas9 Landscape: Current State and Future Perspectives. Int J Mol Sci 24, doi:10.3390/ijms242216077 (2023).
18 Laurent, M., Geoffroy, M., Pavani, G. & Guiraud, S. CRISPR-Based Gene Therapies: From Preclinical to Clinical Treatments. Cells 13, doi:10.3390/cells13100800 (2024).
19 Qi, L. S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173-1183, doi:10.1016/j.cell.2013.02.022 (2013).
20 Hilton, I. B. et al. Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat Biotechnol 33, 510-517, doi:10.1038/nbt.3199 (2015).
21 Riedmayr, L. M. et al. dCas9-VPR-mediated transcriptional activation of functionally equivalent genes for gene therapy. Nat Protoc 17, 781-818, doi:10.1038/s41596-021-00666-3 (2022).
22 Yeo, N. C. et al. An enhanced CRISPR repressor for targeted mammalian gene regulation. Nat Methods 15, 611-616, doi:10.1038/s41592-018-0048-5 (2018).
23 Liu, G., Lin, Q., Jin, S. & Gao, C. The CRISPR-Cas toolbox and gene editing technologies. Mol Cell 82, 333-347, doi:10.1016/j.molcel.2021.12.002 (2022).
24 Nasteska, D. et al. PDX1(LOW) MAFA(LOW) beta-cells contribute to islet function and insulin release. Nat Commun 12, 674, doi:10.1038/s41467-020-20632-z (2021).
25 Taylor, B. L., Liu, F. F. & Sander, M. Nkx6.1 is essential for maintaining the functional state of pancreatic beta cells. Cell Rep 4, 1262-1275, doi:10.1016/j.celrep.2013.08.010 (2013).
26 Ackermann, A. M., Wang, Z., Schug, J., Naji, A. & Kaestner, K. H. Integration of ATAC-seq and RNA-seq identifies human alpha cell and beta cell signature genes. Mol Metab 5, 233-244, doi:10.1016/j.molmet.2016.01.002 (2016).
27 Kang, Y., Kang, J. & Kim, A. Histone H3K4me1 strongly activates the DNase I hypersensitive sites in super-enhancers than those in typical enhancers. Biosci Rep 41, doi:10.1042/BSR20210691 (2021).
28 Bell, O., Tiwari, V. K., Thoma, N. H. & Schubeler, D. Determinants and dynamics of genome accessibility. Nat Rev Genet 12, 554-564, doi:10.1038/nrg3017 (2011).
29 Dorrell, C. et al. Human islets contain four distinct subtypes of beta cells. Nat Commun 7, 11756, doi:10.1038/ncomms11756 (2016).
30 Baumann, K. Stem cells: Insulin-producing beta cells in a dish. Nat Rev Mol Cell Biol 15, 768, doi:10.1038/nrm3907 (2014).
31 Kao, C. Y., Mills, J. A., Burke, C. J., Morse, B. & Marques, B. F. Role of Cytokines and Growth Factors in the Manufacturing of iPSC-Derived Allogeneic Cell Therapy Products. Biology (Basel) 12, doi:10.3390/biology12050677 (2023).
32 Zhou, Q., Brown, J., Kanarek, A., Rajagopal, J. & Melton, D. A. In vivo reprogramming of adult pancreatic exocrine cells to beta-cells. Nature 455, 627-632, doi:10.1038/nature07314 (2008).
33 Kalo, E., Read, S. & Ahlenstiel, G. Reprogramming-Evolving Path to Functional Surrogate beta-Cells. Cells 11, doi:10.3390/cells11182813 (2022).
34 Niu, F. et al. beta-cell neogenesis: A rising star to rescue diabetes mellitus. J Adv Res 62, 71-89, doi:10.1016/j.jare.2023.10.008 (2024).
35 Orive, G. et al. Engineering a Clinically Translatable Bioartificial Pancreas to Treat Type I Diabetes. Trends Biotechnol 36, 445-456, doi:10.1016/j.tibtech.2018.01.007 (2018).
Supplementary References
1. Stoffers DA, Zinkin NT, Stanojevic V, Clarke WL, Habener JF. Pancreatic agenesis attributable to a single nucleotide deletion in the human IPF1 gene coding sequence. Nat Genet. 1997 Jan; 15(1):106-10.
2. Sachdeva MM, Claiborn KC, Khoo C, Yang J, Groff DN, Mirmira RG, Stoffers DA. Pdx1 (MODY4) regulates pancreatic beta cell susceptibility to ER stress. Proc Natl Acad Sci U S A. 2009; 106:19090–19095.
3. Daniella A. Babu, Tye G. Deering, Raghavendra G. Mirmira, A feat of metabolic proportions: Pdx1 orchestrates islet development and function in the maintenance of glucose homeostasis, Molecular Genetics and Metabolism,
4. beta-cell-specific inactivation of the mouse Ipf1/Pdx1 gene results in loss of the beta-cell phenotype and maturity onset diabetes. Volume 92, Issues 1–2, 2007, Pages 43-55,
5. Ahlgren, U. et al. Genes Dev. 1998 Jun 15;12(12):1763-8.
6. Ashleigh E. Schaffer, Brandon L. Taylor, Jacqueline R. Benthuysen, Jingxuan Liu, Fabrizio Thorel, Weiping Yuan, Yang Jiao, Klaus H. Kaestner, Pedro L. Herrera, Mark A. Magnuson, Catherine Lee May, Maike Sander. Nkx6.1 Controls a Gene Regulatory Network Required for Establishing and Maintaining Pancreatic Beta Cell Identity. PLoS Genet. 2013 Jan; 9(1): e1003274.
7. Schisler J. C. et al. The Nkx6.1 homeodomain transcription factor suppresses glucagon expression and regulates glucose-stimulated insulin secretion in islet beta cells. Proc Natl Acad Sci U S A. 2005;102(20):7297–7302.
8. Brandon L. Taylor, Fen-Fen Liu, Maike Sander. Nkx6.1 is essential for maintaining the functional state of pancreatic beta cells. Cell Rep. Author manuscript; available in PMC 2014 Jun 15. Published in final edited form as: Cell Rep. 2013 Sep 26; 4(6): 1262–1275.
9. Fu Z, Gilbert ER, Liu D. Regulation of insulin synthesis and secretion and pancreatic Beta-cell dysfunction in diabetes. Curr Diabetes Rev. 2013;9(1):25–53.
10. Waeber G., Thompson N., Haefliger J.A., Nicod P. Characterization of the murine high Km glucose transporter GLUT2 gene and its transcriptional regulation by glucose in a differentiated insulin-secreting cell line. J. Biol. Chem. 1994; 269:26912–26919.
11. Leturque A., Brot-Laroche E., Le Gall M. GLUT2 mutations, translocation, and receptor function in diet sugar managing. Am. J. Physiol. Endocrinol. Metab. 2009;296: E985–E992.
12. Benzinou M, Creemers JW, Choquet H, et al. Common nonsynonymous variants in PCSK1 confer risk of obesity. Nat Genet. 2008;40(8):943–945.
13. Nicolson TJ et al. Diabetes. 2009 Sep; 58(9):2070-83.
14. Frans C. et al. Diabetes Jan 2001, 50 (1) 1-11.
15. Matschinsky, F.M. et al., 1968. Metabolism of glucose in the islets of Langerhans. J. Biol. Chem. 243:2730-2736
16. Yasuo T, et al., J Clin Invest. 2007;117(1):246-257
17. Li XN et al., J Pharmacol Exp Ther. 2013 Feb;344(2):407-16.
18. Frances M. et al., Nat Rev Endocrinol. 2013 Nov; 9(11): 660–669.
2 Shapiro, A. M. et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 343, 230-238, doi:10.1056/NEJM200007273430401 (2000).
3 Markmann, J. F. et al. Phase 3 trial of human islet-after-kidney transplantation in type 1 diabetes. Am J Transplant 21, 1477-1492, doi:10.1111/ajt.16174 (2021).
4 Hering, B. J. et al. Prolonged diabetes reversal after intraportal xenotransplantation of wild-type porcine islets in immunosuppressed nonhuman primates. Nat Med 12, 301-303, doi:10.1038/nm1369 (2006).
5 Graham, M. L. et al. Clinically available immunosuppression averts rejection but not systemic inflammation after porcine islet xenotransplant in cynomolgus macaques. Am J Transplant 22, 745-760, doi:10.1111/ajt.16876 (2022).
6 Singh, A. et al. Long-term tolerance of islet allografts in nonhuman primates induced by apoptotic donor leukocytes. Nat Commun 10, 3495, doi:10.1038/s41467-019-11338-y (2019).
7 Cardona, K. et al. Long-term survival of neonatal porcine islets in nonhuman primates by targeting costimulation pathways. Nat Med 12, 304-306, doi:10.1038/nm1375 (2006).
8 van der Windt, D. J. et al. Long-term controlled normoglycemia in diabetic non-human primates after transplantation with hCD46 transgenic porcine islets. Am J Transplant 9, 2716-2726, doi:10.1111/j.1600-6143.2009.02850.x (2009).
9 Naqvi, R. A. et al. The future treatment for type 1 diabetes: Pig islet- or stem cell-derived beta cells? Front Endocrinol (Lausanne) 13, 1001041, doi:10.3389/fendo.2022.1001041 (2022).
10 Denner, J. Porcine Endogenous Retroviruses and Xenotransplantation, 2021. Viruses 13, doi:10.3390/v13112156 (2021).
11 Millman, J. R. et al. Generation of stem cell-derived beta-cells from patients with type 1 diabetes. Nat Commun 7, 11463, doi:10.1038/ncomms11463 (2016).
12 Pagliuca, F. W. et al. Generation of functional human pancreatic beta cells in vitro. Cell 159, 428-439, doi:10.1016/j.cell.2014.09.040 (2014).
13 Rezania, A. et al. Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells. Nat Biotechnol 32, 1121-1133, doi:10.1038/nbt.3033 (2014).
14 Watanabe, T. et al. Evaluating teratoma formation risk of pluripotent stem cell-derived cell therapy products: a consensus recommendation from the Health and Environmental Sciences Institute's International Cell Therapy Committee. Cytotherapy 27, 1072-1084, doi:10.1016/j.jcyt.2025.04.062 (2025).
15 Maxwell, K. G. & Millman, J. R. Applications of iPSC-derived beta cells from patients with diabetes. Cell Rep Med 2, 100238, doi:10.1016/j.xcrm.2021.100238 (2021).
16 Pellegrini, S., Zamarian, V. & Sordi, V. Strategies to Improve the Safety of iPSC-Derived beta Cells for beta Cell Replacement in Diabetes. Transpl Int 35, 10575, doi:10.3389/ti.2022.10575 (2022).
17 Tyumentseva, M., Tyumentsev, A. & Akimkin, V. CRISPR/Cas9 Landscape: Current State and Future Perspectives. Int J Mol Sci 24, doi:10.3390/ijms242216077 (2023).
18 Laurent, M., Geoffroy, M., Pavani, G. & Guiraud, S. CRISPR-Based Gene Therapies: From Preclinical to Clinical Treatments. Cells 13, doi:10.3390/cells13100800 (2024).
19 Qi, L. S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173-1183, doi:10.1016/j.cell.2013.02.022 (2013).
20 Hilton, I. B. et al. Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat Biotechnol 33, 510-517, doi:10.1038/nbt.3199 (2015).
21 Riedmayr, L. M. et al. dCas9-VPR-mediated transcriptional activation of functionally equivalent genes for gene therapy. Nat Protoc 17, 781-818, doi:10.1038/s41596-021-00666-3 (2022).
22 Yeo, N. C. et al. An enhanced CRISPR repressor for targeted mammalian gene regulation. Nat Methods 15, 611-616, doi:10.1038/s41592-018-0048-5 (2018).
23 Liu, G., Lin, Q., Jin, S. & Gao, C. The CRISPR-Cas toolbox and gene editing technologies. Mol Cell 82, 333-347, doi:10.1016/j.molcel.2021.12.002 (2022).
24 Nasteska, D. et al. PDX1(LOW) MAFA(LOW) beta-cells contribute to islet function and insulin release. Nat Commun 12, 674, doi:10.1038/s41467-020-20632-z (2021).
25 Taylor, B. L., Liu, F. F. & Sander, M. Nkx6.1 is essential for maintaining the functional state of pancreatic beta cells. Cell Rep 4, 1262-1275, doi:10.1016/j.celrep.2013.08.010 (2013).
26 Ackermann, A. M., Wang, Z., Schug, J., Naji, A. & Kaestner, K. H. Integration of ATAC-seq and RNA-seq identifies human alpha cell and beta cell signature genes. Mol Metab 5, 233-244, doi:10.1016/j.molmet.2016.01.002 (2016).
27 Kang, Y., Kang, J. & Kim, A. Histone H3K4me1 strongly activates the DNase I hypersensitive sites in super-enhancers than those in typical enhancers. Biosci Rep 41, doi:10.1042/BSR20210691 (2021).
28 Bell, O., Tiwari, V. K., Thoma, N. H. & Schubeler, D. Determinants and dynamics of genome accessibility. Nat Rev Genet 12, 554-564, doi:10.1038/nrg3017 (2011).
29 Dorrell, C. et al. Human islets contain four distinct subtypes of beta cells. Nat Commun 7, 11756, doi:10.1038/ncomms11756 (2016).
30 Baumann, K. Stem cells: Insulin-producing beta cells in a dish. Nat Rev Mol Cell Biol 15, 768, doi:10.1038/nrm3907 (2014).
31 Kao, C. Y., Mills, J. A., Burke, C. J., Morse, B. & Marques, B. F. Role of Cytokines and Growth Factors in the Manufacturing of iPSC-Derived Allogeneic Cell Therapy Products. Biology (Basel) 12, doi:10.3390/biology12050677 (2023).
32 Zhou, Q., Brown, J., Kanarek, A., Rajagopal, J. & Melton, D. A. In vivo reprogramming of adult pancreatic exocrine cells to beta-cells. Nature 455, 627-632, doi:10.1038/nature07314 (2008).
33 Kalo, E., Read, S. & Ahlenstiel, G. Reprogramming-Evolving Path to Functional Surrogate beta-Cells. Cells 11, doi:10.3390/cells11182813 (2022).
34 Niu, F. et al. beta-cell neogenesis: A rising star to rescue diabetes mellitus. J Adv Res 62, 71-89, doi:10.1016/j.jare.2023.10.008 (2024).
35 Orive, G. et al. Engineering a Clinically Translatable Bioartificial Pancreas to Treat Type I Diabetes. Trends Biotechnol 36, 445-456, doi:10.1016/j.tibtech.2018.01.007 (2018).
Supplementary References
1. Stoffers DA, Zinkin NT, Stanojevic V, Clarke WL, Habener JF. Pancreatic agenesis attributable to a single nucleotide deletion in the human IPF1 gene coding sequence. Nat Genet. 1997 Jan; 15(1):106-10.
2. Sachdeva MM, Claiborn KC, Khoo C, Yang J, Groff DN, Mirmira RG, Stoffers DA. Pdx1 (MODY4) regulates pancreatic beta cell susceptibility to ER stress. Proc Natl Acad Sci U S A. 2009; 106:19090–19095.
3. Daniella A. Babu, Tye G. Deering, Raghavendra G. Mirmira, A feat of metabolic proportions: Pdx1 orchestrates islet development and function in the maintenance of glucose homeostasis, Molecular Genetics and Metabolism,
4. beta-cell-specific inactivation of the mouse Ipf1/Pdx1 gene results in loss of the beta-cell phenotype and maturity onset diabetes. Volume 92, Issues 1–2, 2007, Pages 43-55,
5. Ahlgren, U. et al. Genes Dev. 1998 Jun 15;12(12):1763-8.
6. Ashleigh E. Schaffer, Brandon L. Taylor, Jacqueline R. Benthuysen, Jingxuan Liu, Fabrizio Thorel, Weiping Yuan, Yang Jiao, Klaus H. Kaestner, Pedro L. Herrera, Mark A. Magnuson, Catherine Lee May, Maike Sander. Nkx6.1 Controls a Gene Regulatory Network Required for Establishing and Maintaining Pancreatic Beta Cell Identity. PLoS Genet. 2013 Jan; 9(1): e1003274.
7. Schisler J. C. et al. The Nkx6.1 homeodomain transcription factor suppresses glucagon expression and regulates glucose-stimulated insulin secretion in islet beta cells. Proc Natl Acad Sci U S A. 2005;102(20):7297–7302.
8. Brandon L. Taylor, Fen-Fen Liu, Maike Sander. Nkx6.1 is essential for maintaining the functional state of pancreatic beta cells. Cell Rep. Author manuscript; available in PMC 2014 Jun 15. Published in final edited form as: Cell Rep. 2013 Sep 26; 4(6): 1262–1275.
9. Fu Z, Gilbert ER, Liu D. Regulation of insulin synthesis and secretion and pancreatic Beta-cell dysfunction in diabetes. Curr Diabetes Rev. 2013;9(1):25–53.
10. Waeber G., Thompson N., Haefliger J.A., Nicod P. Characterization of the murine high Km glucose transporter GLUT2 gene and its transcriptional regulation by glucose in a differentiated insulin-secreting cell line. J. Biol. Chem. 1994; 269:26912–26919.
11. Leturque A., Brot-Laroche E., Le Gall M. GLUT2 mutations, translocation, and receptor function in diet sugar managing. Am. J. Physiol. Endocrinol. Metab. 2009;296: E985–E992.
12. Benzinou M, Creemers JW, Choquet H, et al. Common nonsynonymous variants in PCSK1 confer risk of obesity. Nat Genet. 2008;40(8):943–945.
13. Nicolson TJ et al. Diabetes. 2009 Sep; 58(9):2070-83.
14. Frans C. et al. Diabetes Jan 2001, 50 (1) 1-11.
15. Matschinsky, F.M. et al., 1968. Metabolism of glucose in the islets of Langerhans. J. Biol. Chem. 243:2730-2736
16. Yasuo T, et al., J Clin Invest. 2007;117(1):246-257
17. Li XN et al., J Pharmacol Exp Ther. 2013 Feb;344(2):407-16.
18. Frances M. et al., Nat Rev Endocrinol. 2013 Nov; 9(11): 660–669.