Direct reprogramming of human fibroblasts into insulin-producing cells using transcription factors.

Direct lineage reprogramming of one somatic cell into another without transitioning through a progenitor stage has emerged as a strategy to generate clinically relevant cell types. One cell type of interest is the pancreatic insulin-producing ß cell whose loss and/or dysfunction leads to diabetes. To date it has been possible to create ß-like cells from related endodermal cell types by forcing the expression of developmental transcription factors, but not from more distant cell lineages like fibroblasts. In light of the therapeutic benefits of choosing an accessible cell type as the cell of origin, in this study we set out to analyze the feasibility of transforming human skin fibroblasts into ß-like cells. We describe how the timed-introduction of five developmental transcription factors (Neurog3, Pdx1, MafA, Pax4, and Nkx2-2) promotes conversion of fibroblasts toward a ß-cell fate. Reprogrammed cells exhibit ß-cell features including ß-cell gene expression and glucose-responsive intracellular calcium mobilization. Moreover, reprogrammed cells display glucose-induced insulin secretion in vitro and in vivo. This work provides proof-of-concept of the capacity to make insulin-producing cells from human fibroblasts via transcription factor-mediated direct reprogramming.

Wisp1 is a circulating factor that stimulates proliferation of adult mouse and human beta cells.

Expanding the mass of pancreatic insulin-producing beta cells through re-activation of beta cell replication has been proposed as a therapy to prevent or delay the appearance of diabetes. Pancreatic beta cells exhibit an age-dependent decrease in their proliferative activity, partly related to changes in the systemic environment. Here we report the identification of CCN4/Wisp1 as a circulating factor more abundant in pre-weaning than in adult mice. We show that Wisp1 promotes endogenous and transplanted adult beta cell proliferation in vivo. We validate these findings using isolated mouse and human islets and find that the beta cell trophic effect of Wisp1 is dependent on Akt signaling. In summary, our study reveals the role of Wisp1 as an inducer of beta cell replication, supporting the idea that the use of young blood factors may be a useful strategy to expand adult beta cell mass.

Modulation of the endocrine transcriptional program by targeting histone modifiers of the H3K27me3 mark.

Posttranscriptional modifications of histones constitute an epigenetic mechanism that is closely linked to both gene silencing and activation events. Trimethylation of Histone3 at lysine 27 (H3K27me3) is a repressive mark that associates with developmental gene regulation during differentiation programs. In the developing pancreas, expression of the transcription factor Neurogenin3 in multipotent progenitors initiates endocrine differentiation that culminates in the generation of all pancreatic islet cell lineages, including insulin-producing beta cells. Previously, we showed that Neurogenin3 promoted the removal of H3K27me3 marks at target gene promoters in vitro, suggesting a functional connection between this factor and regulators of this chromatin mark. In the present study, we aimed to specifically evaluate whether targeting the activity of these histone modifiers can be used to modulate pancreatic endocrine differentiation. Our data show that chemical inhibition of the H3K27me3 demethylases Jmjd3/Utx blunts Neurogenin3-dependent gene activation in vitro. Conversely, inhibition of the H3K27me3 methyltransferase Ezh2 enhances both the transactivation ability of Neurogenin3 in cultured cells and the formation of insulin-producing cells during directed differentiation from pluripotent cells. These results can help improve current protocols aimed at generating insulin-producing cells for beta cell replacement therapy in diabetes.

Late-stage differentiation of embryonic pancreatic ß-cells requires Jarid2.

Jarid2 is a component of the Polycomb Repressor complex 2 (PRC2), which is responsible for genome-wide H3K27me3 deposition, in embryonic stem cells. However, Jarid2 has also been shown to exert pleiotropic PRC2-independent actions during embryogenesis. Here, we have investigated the role of Jarid2 during pancreas development. Conditional ablation of Jarid2 in pancreatic progenitors results in reduced endocrine cell area at birth due to impaired endocrine cell differentiation and reduced prenatal proliferation. Inactivation of Jarid2 in endocrine progenitors demonstrates that Jarid2 functions after endocrine specification. Furthermore, genome-wide expression analysis reveals that Jarid2 is required for the complete activation of the insulin-producing ß-cell differentiation program. Jarid2-deficient pancreases exhibit impaired deposition of RNAPII-Ser5P, the initiating form of RNAPII, but no changes in H3K27me3, at the promoters of affected endocrine genes. Thus, our study identifies Jarid2 as a fine-tuner of gene expression during late stages of pancreatic endocrine cell development. These findings are relevant for generation of transplantable stem cell-derived ß-cells.