Transdifferentiation of pancreatic alpha to beta cells using Epi-CRISPR directed DNA methylation
Arambašić Jovanović, Jelena
Jurkowski, Tomasz P.
© University of Belgrade, Faculty of Biology
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Introduction: Since diabetes is characterized by impaired ability of pancreatic betacells to respond and/or produce insulin, new approaches for renewal and replacement of deficient beta-cells are indispensable. The aim of this study is direct pancreatic alpha- to beta-cells transdifferentiation by using a new synthetic epigenetic tool, Epi-CRISPR system. Using Epi-CRISPR system we aim to introduce targeted DNA methylation and subsequent repression of genes responsible for maintaining alpha-cell identity. Methods: AlphaTC1-6 cells (α-cells) were transiently transfected with dCas9-Dnmt3a- Dnmt3L constructs and one or four different vectors containing guide RNA components for specific targeting the promoter region of aristaless-related homeobox gene (Arx). The success of α-cells transdifferentiation into insulinproducing cells was evaluated by measuring Arx and insulin mRNA level, amount of secreted insulin and by immunostaining of insulin/glucagon in the cells. Results: We observed Arx transcriptional repression in α-cell transfected with Epi- CRISPR construct that targets the Arx gene promoter inducing subsequent methylation. At fifth day post-transfection the expression of Arx was decreased in α- cells followed by consequent increase in insulin (mRNA and protein level). At the same time, the glucagon levels remained unchanged. At twelfth day posttransfection the transfected cells start to lose glucagon while still secreting insulin. Conclusion: This study is near to confirm Epi-CRISPR system functionality and to verify the concept of cell transdifferentiation through silencing of genes responsible for maintaining cell phenotype. The obtained results will be valuable for later Epi- CRISPRs use in mouse in vivo models of diabetes and eventually as a future therapy for diabetes attenuation in humans.
- Сигнални молекули у дијабетесу: идентификација потенцијалних биолошких маркера укључених у модификацију и интеграцију сигналних путева у циљу предикције и интервенције у дијабетесу (RS-173020)
- AstraZeneca within the European Foundation for the Study of Diabetes (EFSD): European Diabetes Research Programme in Cellular Plasticity Underlying the Pathophysiology of Type 2 Diabetes