Human PDX1 mRNA

In vitro synthesized pancreatic duodenal homeobox 1 (PDX1) mRNA was used to induce differentiation of hESCs into insulin-secreting cells. Here, the researchers found that electroporation was the most efficient transfection method and resulted in more than 95% transgene expression in multiple hESC lines. Synthesized mRNAs with a combination of polyA tails, caps, and base analogs were more efficiently translated into protein in hESCs compared to single modified mRNAs. Transfection of mRNA into hESCs by trypsinizing cells into a single-cell suspension did not affect their pluripotency, and multiple types of mRNA could be efficiently transfected into hESCs. The researchers found that PDX-1 mRNA transfection significantly increased the expression levels of genes associated with beta cells and differentiated cells expressing insulin and C-peptide. ELISA analysis validated that the islet-like cell clusters secreted insulin upon glucose stimulation. These results indicate that electroporation of in vitro synthesized mRNA can be used for genetic manipulation of hESCs and differentiation of hESCs into specific cell types, a finding that will pave the way for clinical application of this approach.

To evaluate the role of PDX1 mRNA transfection during hESC differentiation into IPCs, the researchers performed experiments based on published protocols, and the PDX1 mRNA transfection protocol is shown in Figure 1A. Genes associated with β cells were detected at the end of differentiation. The chemically treated group and combination of chemically treated and PDX1 mRNA-transfected group both expressed key beta cell genes, and beta cell gene expression was not observed in the control group (Figure 1B). However, different gene expression levels were observed between the chemical induction group and the combination group, with the combination group having higher gene expression levels than the chemical induction group (Figure 1C).

Figure 1. Differentiation of hESCs into insulin-producing cells. (Li Wang X, et al., 2015)