Human NEUROG3 mRNA

Reprogramming non-endocrine pancreatic cells into insulin-secreting cells is a promising therapeutic approach to restore endogenous insulin secretion in patients with diabetes. Here, researchers reprogram human organoids derived from pancreatic tissue into insulin-secreting cells (IPCs) by combining in vitro transcribed modified mRNA encoding the transcription factor neuroligin 3 with small molecules that modulate epigenetic states and signaling pathways. After reprogramming, IPCs accounted for 4.6±1.2% of total cells and expressed typical markers (insulin, glucokinase, ABCC8, KCNJ11, SLC2A2, SLC30A8) and transcription factors (PDX1, NEUROD1, MAFA, NKX2.2, NKX6.1, PAX4, PAX6) required for the normal function of pancreatic β cells. In addition, researchers found that the ALK5 inhibitor RepSox had a positive effect on the overall reprogramming efficiency. However, the reprogrammed IPCs had only partial insulin secretory capacity, as they were unable to respond to changes in extracellular glucose concentrations by increasing insulin secretion. Based on the present findings, it can be concluded that IPCs have immature properties due to incomplete reprogramming and possess only partial properties of native human β cells.

qRT-PCR analysis showed that the combination of IVT-modified NEUROG3 mRNA and RAPFIG small molecules induced the expression of MAF bZIP A (MAFA) transcription factors, glucokinase (GCK), islet-specific glucose-6-phosphatase catalytic subunit 2 (G6PC2), and glucagon and insulin genes in IPCs (Figure 1B). In addition, upregulation of ISL1 and PAX6 transcription factors, somatostatin, and functional genes such as SLC2A2, SLC30A8, PCSK1, PCSK2, and ABCC8 was detected. On the other hand, the expression of ghrelin and NKX6.1 transcription factors was downregulated compared to the expression levels of these genes in organoid cells treated with IVT-modified NEUROG3 mRNA alone (i.e., without RAPFIG small molecules for reprogramming). The production of insulin, somatostatin, and ghrelin at the protein level was confirmed by immunostaining for these hormones. In addition, positive co-staining for insulin and C-peptide (CPEP) was detected, indicating that the reprogrammed IPCs were able to process prohormone insulin into fully mature insulin and C-peptide by endopeptidases PCSK1 and PCSK2. Immunostaining for glucagon was negative, confirming the results of qRT-PCR, as glucagon was expressed at a low mRNA level compared with other pancreatic hormones (Figure 1B). In addition, the group that received combined IVT-modified NEUROG3 mRNA and small molecule treatment had a 58.9 ± 12.7% decrease in total cell number compared with the untreated control group.

Figure 1. Reprogramming of pancreatic organoid-derived cells into the IPCs by the combination of IVT NEUROG3 mRNA and small molecules. (Koblas T, et al., 2019)