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

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

Synthetic mRNA is a more reliable tool for the delivery of DNA-targeting proteins into the cell nucleus than fusion with a protein transduction domain

<div><p>Cell reprogramming requires efficient delivery of reprogramming transcription factors into the cell nucleus. Here, we compared the robustness and workload of two protein delivery methods that avoid the risk of genomic integration. The first method is based on fusion of the protein of interest to a protein transduction domain (PTD) for delivery across the membranes of target cells. The second method relies on de novo synthesis of the protein of interest inside the target cells utilizing synthetic mRNA (syn-mRNA) as a template. We established a Cre/lox reporter system in three different cell types derived from human (PANC-1, HEK293) and rat (BRIN-BD11) tissues and used Cre recombinase to model a protein of interest. The system allowed constitutive expression of red fluorescence protein (RFP), while green fluorescence protein (GFP) was expressed only after the genomic action of Cre recombinase. The efficiency of protein delivery into cell nuclei was quantified as the frequency of GFP⁺ cells in the total cell number. The PTD method showed good efficiency only in BRIN-BD11 cells (68%), whereas it failed in PANC-1 and HEK293 cells. By contrast, the syn-mRNA method was highly effective in all three cell types (29–71%). We conclude that using synthetic mRNA is a more robust and less labor-intensive approach than using the PTD-fusion alternative.</p></div

Quantification of intracellular Cre recombinase protein in three cell types at two time points after single administration of either 15 μM PTD-Cre or 2.1 nM syn-mRNA-Cre.

<p>(A) Western blot images are representative of three independent experiments. (B) Relative quantification of Cre recombinase protein, mean±SD, <i>n=3</i>.</p

Verification of engineered clones after their expansion.

<p>No leaky GFP expression was observed.</p

Frequency of GFP⁺ cells after the treatment of Cre-responsive cells with either PTD-Cre2 (A) or syn-mRNA-Cre (B).

<p>Analysis of flow cytometry data from three independent experiments. X-axis: relative dose on the logarithmic scale, (1 is the maximum dose); Asterisk, double administration; Y-axis: efficiency of the delivery of Cre recombinase into nuclei of fl-PANC (red triangles), fl-BRIN (blue squares), fl-HEK (green circles) cells; Error bars: mean±SD, n = 3; GraphPad was used to construct asymmetrical (five-parameter) dose-response curves and to calculate the approximate logEC50.</p

Experimental design.

<p>(A) RFP/GFP expression cassette inserted randomly into the genomes of the target cells. In these Cre-sensitive cells, the GFP expression was dependent on the delivery of functional Cre protein into cell nuclei. Scissors, parallel loxP sequences; Cre, Cre recombinase; stop, two stop codons. (B) The PTD-fusion proteins were produced in bacteria and purified in three steps. (C) The syn-mRNA (cap-NLS-T7-Cre) was synthesized <i>in vitro</i>. aa, number of amino acids; NLS, Nuclear localization signal (9 aa); Cre, Cre recombinase (343 aa); PTD, Protein transduction domain of HIV TAT (11 aa); T7, T7-tag (11 aa). Time frames of the flow cytometry (D) and western blot (E) analyses. Black arrows, cell seeding; white arrows, administration of Cre recombinase; dashed white arrow, second administration of Cre recombinase; grey triangles, harvesting of cells for analysis.</p

Detection of Cre recombinase activity in cell nuclei of the target cells after administration of PTD-Cre (15 μM) or syn-mRNA-Cre (2.1 nM).

<p>Bar 200 μm.</p

Comparison of PTD-fusion and syn-mRNA.

<p>Half maximum effective doses (EC50) and half maximum efficiency in three cell types.</p