NKX2-2 adenovirus

Direct reprogramming of one somatic cell into another, without passing through a progenitor stage, has emerged as a strategy to generate clinically relevant cell types. One of these cell types of interest is the pancreatic insulin-secreting β-cell, whose absence and/or dysfunction leads to diabetes. To date, it has been possible to create β-like cells from related endoderm cell types by forced expression of developmental transcription factors, but not from more distant cell lineages such as fibroblasts. Given the therapeutic benefits of choosing a readily accessible cell type as a starting cell, here, researchers set out to analyze the feasibility of converting human skin fibroblasts into β-like cells. They describe how the timed introduction of five developmental transcription factors (Neurog3, Pdx1, MafA, Pax4, and Nkx2-2) promotes the conversion of fibroblasts into β-cells. The reprogrammed cells exhibited β-cell characteristics, including β-cell gene expression and glucose-responsive intracellular calcium mobilization. Furthermore, the reprogrammed cells exhibited glucose-induced insulin secretion both in vitro and in vivo. This study provides proof-of-concept for the ability to make insulin-secreting cells from human fibroblasts by direct transcription factor-mediated reprogramming.

Since the NKX6-1 gene remained silent upon NPM + Pax4 stimulation (Figure 1), the researchers attempted to add Nkx6-1 directly to the NPM reprogramming mix. However, exogenous Nkx6-1 resulted in extensive cell death regardless of expression level or timing of introduction. As an alternative approach, the researchers added exogenous Nkx2-2, which also regulates early β-cell differentiation and is an upstream activator of Nkx6-1 during mouse islet development. Treatment with adenovirus encoding Nkx2-2 three days after NPM resulted in endogenous activation of NKX6-1 expression without affecting fibroblast viability (Figure 1). Nkx2-2 also induced PAX6, a pan-endocrine gene required for high levels of islet hormone gene expression during mouse pancreatic development. Notably, ectopic Nkx2-2 reduced NPM-induced activation of the GCG gene without affecting expression of the INS gene (Figure 1).

Figure 1. Human fibroblasts (HFF1) were infected with Ad-NPM alone or sequentially with Ad-NPM and adenoviruses encoding the transcription factors Pax4 and Nkx2-2. (Fontcuberta-PiSunyer M, et al., 2023)