{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T11:40:48Z","timestamp":1788435648389,"version":"build-2803163510"},"reference-count":42,"publisher":"MDPI AG","issue":"15","license":[{"start":{"date-parts":[[2022,8,8]],"date-time":"2022-08-08T00:00:00Z","timestamp":1659916800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Research Project of Russian Foundation for Basic Research (RFBR)","award":["#19-29-04121"],"award-info":[{"award-number":["#19-29-04121"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>One of the causes of diabetes in infants is the defect of the insulin gene (INS). Gene mutations can lead to proinsulin misfolding, an increased endoplasmic reticulum (ER) stress and possible beta-cell apoptosis. In humans, the mechanisms underlying beta-cell failure remain unclear. We generated induced pluripotent stem cells (iPSCs) from a patient diagnosed with neonatal diabetes mellitus carrying the INS mutation in the 2nd intron (c.188-31G&gt;A) and engineered isogenic CRISPR\/Cas9 mutation-corrected cell lines. Differentiation into beta-like cells demonstrated that mutation led to the emergence of an ectopic splice site within the INS and appearance of the abnormal RNA transcript. Isogenic iPSC lines differentiated into beta-like cells showed a clear difference in formation of organoids at pancreatic progenitor stage of differentiation. Moreover, MIN6 insulinoma cell line expressing mutated cDNA demonstrated significant decrease in proliferation capacity and activation of ER stress and unfolded protein response (UPR)-associated genes. These findings shed light on the mechanism underlying the pathogenesis of monogenic diabetes.<\/jats:p>","DOI":"10.3390\/ijms23158824","type":"journal-article","created":{"date-parts":[[2022,8,8]],"date-time":"2022-08-08T21:09:18Z","timestamp":1659992958000},"page":"8824","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":21,"title":["Aberrant Splicing of INS Impairs Beta-Cell Differentiation and Proliferation by ER Stress in the Isogenic iPSC Model of Neonatal Diabetes"],"prefix":"10.3390","volume":"23","author":[{"given":"Alexandra V.","family":"Panova","sequence":"first","affiliation":[{"name":"Vavilov Institute of General Genetics, Russian Academy of Sciences, 119991 Moscow, Russia"},{"name":"Endocrinology Research Centre, 115478 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2742-372X","authenticated-orcid":false,"given":"Natalia V.","family":"Klementieva","sequence":"additional","affiliation":[{"name":"Endocrinology Research Centre, 115478 Moscow, Russia"},{"name":"Institute of Gene Biology, Russian Academy of Sciences, 119334 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anna V.","family":"Sycheva","sequence":"additional","affiliation":[{"name":"Endocrinology Research Centre, 115478 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9752-9714","authenticated-orcid":false,"given":"Elena V.","family":"Korobko","sequence":"additional","affiliation":[{"name":"Vavilov Institute of General Genetics, Russian Academy of Sciences, 119991 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anastasia O.","family":"Sosnovtseva","sequence":"additional","affiliation":[{"name":"Vavilov Institute of General Genetics, Russian Academy of Sciences, 119991 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5931-7959","authenticated-orcid":false,"given":"Tatiana S.","family":"Krasnova","sequence":"additional","affiliation":[{"name":"Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maria R.","family":"Karpova","sequence":"additional","affiliation":[{"name":"Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9867-2344","authenticated-orcid":false,"given":"Petr M.","family":"Rubtsov","sequence":"additional","affiliation":[{"name":"Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yulia V.","family":"Tikhonovich","sequence":"additional","affiliation":[{"name":"Endocrinology Research Centre, 115478 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anatoly N.","family":"Tiulpakov","sequence":"additional","affiliation":[{"name":"Endocrinology Research Centre, 115478 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sergey L.","family":"Kiselev","sequence":"additional","affiliation":[{"name":"Vavilov Institute of General Genetics, Russian Academy of Sciences, 119991 Moscow, Russia"},{"name":"Endocrinology Research Centre, 115478 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,8,8]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"957","DOI":"10.1016\/S0140-6736(15)60098-8","article-title":"The effect of early, comprehensive genomic testing on clinical care in neonatal diabetes: An international cohort study","volume":"386","author":"Flanagan","year":"2015","journal-title":"Lancet"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"732431","DOI":"10.3389\/fendo.2021.732431","article-title":"Advances in Pancreatic Islet Transplantation Sites for the Treatment of Diabetes","volume":"12","author":"Cayabyab","year":"2021","journal-title":"Front. Endocrinol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"562","DOI":"10.1016\/j.ebiom.2018.11.028","article-title":"Medium- to Long-Term Survival and Functional Examination of Human iPSC-Derived Retinas in Rat and Primate Models of Retinal Degeneration","volume":"39","author":"Tu","year":"2019","journal-title":"EBioMedicine"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"729","DOI":"10.1007\/s00109-017-1531-7","article-title":"Stem Cell-Derived Organoids and Their Application for Medical Research and Patient Treatment","volume":"95","author":"Bartfeld","year":"2017","journal-title":"J. Mol. Med."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"3369","DOI":"10.1038\/s41467-020-17165-w","article-title":"Preclinical study of induced pluripotent stem cell-derived dopaminergic progenitor cells for Parkinson\u2019s disease","volume":"11","author":"Doi","year":"2020","journal-title":"Nat. Commun."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"27","DOI":"10.1186\/s13024-016-0092-5","article-title":"Manifestation of Huntington\u2019s Disease Pathology in Human Induced Plu-ripotent Stem Cell-Derived Neurons","volume":"11","author":"Nekrasov","year":"2016","journal-title":"Mol. Neurodegener."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"327","DOI":"10.2217\/rme-2022-0046","article-title":"Industry updates from the field of stem cell research and regenerative medicine in February","volume":"17","author":"Ilic","year":"2022","journal-title":"Regen. Med."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1121","DOI":"10.1038\/nbt.3033","article-title":"Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells","volume":"32","author":"Rezania","year":"2014","journal-title":"Nat. Biotechnol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"428","DOI":"10.1016\/j.cell.2014.09.040","article-title":"Generation of Functional Human Pancreatic \u03b2 Cells In Vitro","volume":"159","author":"Pagliuca","year":"2014","journal-title":"Cell"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"eaax9106","DOI":"10.1126\/scitranslmed.aax9106","article-title":"Gene-edited human stem cell\u2013derived \u03b2 cells from a patient with monogenic diabetes reverse preexisting diabetes in mice","volume":"12","author":"Maxwell","year":"2020","journal-title":"Sci. Transl. Med."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1407","DOI":"10.1016\/j.stemcr.2018.11.006","article-title":"\u03b2 Cell Replacement after Gene Editing of a Neonatal Dia-betes-Causing Mutation at the Insulin Locus","volume":"11","author":"Ma","year":"2018","journal-title":"Stem Cell Rep."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"e38519","DOI":"10.7554\/eLife.38519","article-title":"Insulin mutations impair beta-cell development in a patient-derived iPSC model of neonatal diabetes","volume":"7","author":"Balboa","year":"2018","journal-title":"eLife"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"263","DOI":"10.1038\/s41556-018-0271-4","article-title":"Recapitulating endocrine cell clustering in culture promotes maturation of human stem-cell-derived \u03b2 cells","volume":"21","author":"Nair","year":"2019","journal-title":"Nat. Cell Biol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"100238","DOI":"10.1016\/j.xcrm.2021.100238","article-title":"Applications of iPSC-Derived Beta Cells from Patients with Diabetes","volume":"2","author":"Maxwell","year":"2021","journal-title":"Cell. Rep. Med."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Rouhani, F., Kumasaka, N., de Brito, M.C., Bradley, A., Vallier, L., and Gaffney, D. (2014). Genetic Background Drives Transcrip-tional Variation in Human Induced Pluripotent Stem Cells. PLoS Genet., 10.","DOI":"10.1371\/journal.pgen.1004432"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"102362","DOI":"10.1016\/j.ceca.2021.102362","article-title":"Mitochondrial Dysfunction in Neurodegenerative Diseases: A Focus on iPSC-Derived Neuronal Models","volume":"94","author":"Dolga","year":"2021","journal-title":"Cell Calcium"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"6978303","DOI":"10.1155\/2019\/6978303","article-title":"Induced Pluripotent Stem Cell Derivation and Ex Vivo Gene Correction Using a Mucopolysaccharidosis Type 1 Disease Mouse Model","volume":"2019","author":"Miki","year":"2019","journal-title":"Stem Cells Int."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Lee, M.H., Shin, J.I., Yang, J.W., Lee, K.H., Cha, D.H., Hong, J.B., Park, Y., Choi, E., Tizaoui, K., and Koyanagi, A. (2022). Genome Editing Using CRISPR-Cas9 and Autoimmune Diseases: A Comprehensive Review. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms23031337"},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Klementieva, N., Goliusova, D., Krupinova, J., Yanvarev, V., Panova, A., Mokrysheva, N., and Kiselev, S.L. (2021). A Novel Iso-genic Human Cell-Based System for MEN1 Syndrome Generated by CRISPR\/Cas9 Genome Editing. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms222112054"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Garin, I., De Nanclares, G.P., Gastaldo, E., Harries, L., Rubio-Cabezas, O., and Casta\u00f1o, L. (2012). Permanent Neonatal Diabetes Caused by Creation of an Ectopic Splice Site within the INS Gene. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0029205"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"414","DOI":"10.14341\/DM12737","article-title":"Clinical, hormonal and molecular-genetic characteristics of monogenic diabetes mellitus associated with the mutations in the INS gene","volume":"24","author":"Tikhonovich","year":"2022","journal-title":"Diabetes Mellit."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"101929","DOI":"10.1016\/j.scr.2020.101929","article-title":"Generation of an induced pluripotent stem cell line MNDINSi001-A from a patient with neonatal diabetes caused by a heterozygous INS mutation","volume":"47","author":"Panova","year":"2020","journal-title":"Stem Cell Res."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"184","DOI":"10.1038\/nbt.3437","article-title":"Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9","volume":"34","author":"Doench","year":"2016","journal-title":"Nat. Biotechnol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"827","DOI":"10.1038\/nbt.2647","article-title":"DNA targeting specificity of RNA-guided Cas9 nucleases","volume":"31","author":"Hsu","year":"2013","journal-title":"Nat. Biotechnol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"329","DOI":"10.1038\/nprot.2016.171","article-title":"Precise and efficient scarless genome editing in stem cells using CORRECT","volume":"12","author":"Kwart","year":"2017","journal-title":"Nat. Protoc."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"e64","DOI":"10.1002\/cpsc.64","article-title":"Highly Efficient CRISPR-Cas9-Mediated Genome Editing in Human Pluripotent Stem Cells","volume":"48","author":"Maguire","year":"2019","journal-title":"Curr. Protoc. Stem Cell Biol."},{"key":"ref_27","first-page":"2148","article-title":"Seven mutations in the human insulin gene linked to permanent neonatal\/infancy-onset diabetes mellitus","volume":"118","author":"Colombo","year":"2008","journal-title":"J. Clin. Investig."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"27013","DOI":"10.1016\/S0021-9258(19)61473-0","article-title":"Activation of ATF6 and an ATF6 DNA Binding Site by the Endoplasmic Reticulum Stress Response","volume":"275","author":"Wang","year":"2000","journal-title":"J. Biol. Chem."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1007\/s12192-012-0351-5","article-title":"Gene regulatory network of unfolded protein response genes in endoplasmic reticulum stress","volume":"18","author":"Takayanagi","year":"2013","journal-title":"Cell Stress Chaperon"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"205","DOI":"10.1007\/s11154-010-9151-3","article-title":"Clinical and molecular genetics of neonatal diabetes due to mutations in the insulin gene","volume":"11","author":"Steiner","year":"2010","journal-title":"Rev. Endocr. Metab. Disord."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1449","DOI":"10.1016\/j.bbrc.2009.12.090","article-title":"Mutant proinsulin proteins associated with neonatal diabetes are retained in the endoplasmic reticulum and not efficiently secreted","volume":"391","author":"Park","year":"2010","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"612","DOI":"10.1136\/jmedgenet-2015-103220","article-title":"Continued lessons from theINSgene: An intronic mutation causing diabetes through a novel mechanism","volume":"52","author":"Carmody","year":"2015","journal-title":"J. Med Genet."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"194","DOI":"10.1016\/j.scr.2013.10.003","article-title":"Characterization of polyhormonal insulin-producing cells derived in vitro from human embryonic stem cells","volume":"12","author":"Bruin","year":"2014","journal-title":"Stem Cell Res."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1392","DOI":"10.1038\/nbt1259","article-title":"Production of pancreatic hormone\u2013expressing endocrine cells from human embryonic stem cells","volume":"24","author":"Bang","year":"2006","journal-title":"Nat. Biotechnol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1759","DOI":"10.15252\/embj.201591058","article-title":"Controlled induction of human pancreatic progenitors produces functional beta-like cells in vitro","volume":"34","author":"Russ","year":"2015","journal-title":"EMBO J."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"650158","DOI":"10.3389\/fendo.2021.650158","article-title":"Pathological \u03b2-Cell Endoplasmic Reticulum Stress in Type 2 Diabetes: Current Evidence","volume":"12","author":"Shrestha","year":"2021","journal-title":"Front. Endocrinol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"421","DOI":"10.1038\/s41580-020-0250-z","article-title":"Mechanisms, regulation and functions of the unfolded protein response","volume":"21","author":"Hetz","year":"2020","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"e38472","DOI":"10.7554\/eLife.38472","article-title":"Inhibition of mTORC1 by ER stress impairs neonatal \u03b2-cell expansion and predisposes to diabetes in the Akita mouse","volume":"7","author":"Riahi","year":"2018","journal-title":"eLife"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1268","DOI":"10.2337\/db06-0658","article-title":"Dominant-Negative Effects of a Novel Mutated Ins2 Allele Causes Early-Onset Diabetes and Severe \u03b2-Cell Loss in Munich Ins2C95S Mutant Mice","volume":"56","author":"Herbach","year":"2007","journal-title":"Diabetes"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"2497","DOI":"10.2337\/db14-1412","article-title":"The Basic Helix-Loop-Helix Transcription Factor NEUROG3 Is Required for Development of the Human Endocrine Pancreas","volume":"64","author":"McGrath","year":"2015","journal-title":"Diabetes"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1304","DOI":"10.1038\/cdd.2008.52","article-title":"Insulin-like growth factor-I protects cells from ER stress-induced apoptosis via enhancement of the adaptive capacity of endoplasmic reticulum","volume":"15","author":"Novosyadlyy","year":"2008","journal-title":"Cell Death Differ."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"3649","DOI":"10.1016\/j.febslet.2010.07.040","article-title":"Insulin modulates induction of glucose-regulated protein 78 during endoplasmic reticulum stress via augmentation of ATF4 expression in human neuroblastoma cells","volume":"584","author":"Inageda","year":"2010","journal-title":"FEBS Lett."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/15\/8824\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T00:05:52Z","timestamp":1760141152000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/15\/8824"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,8,8]]},"references-count":42,"journal-issue":{"issue":"15","published-online":{"date-parts":[[2022,8]]}},"alternative-id":["ijms23158824"],"URL":"https:\/\/doi.org\/10.3390\/ijms23158824","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,8,8]]}}}