Reprogramming of Pancreatic Exocrine Cells AR42J Into Insulin-producing Cells Using mRNAs for Pdx1, Ngn3, and MafA Transcription Factors

Abstract

Direct reprogramming of pancreatic nonendocrine cells into insulin-producing β-cells represents a promising approach for the treatment of insulin-dependent diabetes. However, its clinical application is limited by the potential for insertional mutagenesis associated with the viral vectors currently used for cell reprogramming. With the aim of developing a nonintegrative reprogramming strategy for derivation of insulin-producing cells, here, we evaluated a new approach utilizing synthetic messenger RNAs encoding reprogramming transcription factors. Administration of synthetic mRNAs encoding three key transcription regulators of β-cell differentiation—Pdx1, Neurogenin3, and MafA—efficiently reprogrammed the pancreatic exocrine cells into insulin-producing cells. In addition to the insulin genes expression, the synthetic mRNAs also induced the expressions of genes important for proper pancreatic β-cell function, including Sur1, Kir6.2, Pcsk1, and Pcsk2. Pretreating cells with the chromatin-modifying agent 5-Aza-2′-deoxycytidine further enhanced reprogramming efficiency, increasing the proportion of insulin-producing cells from 3.5 ± 0.9 to 14.3 ± 1.9% (n = 4). Moreover, 5-Aza-2′-deoxycytidine pretreatment enabled the reprogrammed cells to respond to glucose challenge with increased insulin secretion. In conclusion, our results support that the reprogramming of pancreatic exocrine cells into insulin-producing cells, induced by synthetic mRNAs encoding pancreatic transcription factors, represents a promising approach for cell-based diabetes therapy.

Nanomedicines to Deliver mRNA: State of the Art and Future Perspectives

“…Transdifferentiation of somatic cells with IVT mRNA has been mainly focused on the generation of insulin secreting β-cells for type 1 diabetes patients [247][248][249], and on obtaining cardiomyocytes to regenerate the cardiac tissue after a heart attack [155]. mRNA is a promising tool for the delivery of factors targeting altered signaling pathways in the early hours of infarction, as well as to address experimental and clinical needs to regenerate cardiac tissue and cardiac function in ischemic heart disease.…”

Enzymatic Protein Biopolymers as a Tool to Synthetize Eukaryotic Messenger Ribonucleic Acid (mRNA) with Uses in Vaccination, Immunotherapy and Nanotechnology

“…The regeneration process requires functional proteins including cytokines, transcription and growth factors which control cell growth, cell differentiation, and cell migration, which can be delivered in the mRNA form into cells. One example of this is the generation of insulin secreting B-cells for type 1 diabetes [31]. In addition, passive immunization with mRNA encoding monoclonal antibodies is gaining great biomedical interest.…”

Biomedical applications of mRNA nanomedicine

“…A similar strategy has been applied to reprogram other types of cells. Pancreatic exocrine cells and human pancreatic duct-derived cells could be transdifferentiated into insulin-secreting β cells by transfecting with mRNA encoding pancreatic transcription factors [212, 213]. Human fibroblasts could directly be reprogrammed into hepatocyte-like cells by synthetically modified mRNA encoding HNF1A plus any two of FOXA1, FOXA3, or HNF4A in the presence of an optimized hepatic growth medium [214].…”