![]() ![]() ![]() We first sought to examine whether the transcription factors Neurog3, Pdx1, and MafA could induce expression of the INSULIN ( INS) gene in human fibroblasts as readout of the capacity of these cells to be transformed toward a β-cell fate. We believe these findings demonstrate the feasibility of this approach and set the basis to further explore this alternative path for generation of β-like cells for disease modeling and cellular therapy.Įxogenous expression of the transcription factors Pdx1, Neurog3, and MafA in human fibroblasts Here we present a protocol based on a cocktail of five endocrine transcription factors that induces human fibroblasts to activate the β-cell transcriptional program while downregulating their native fibroblastic transcriptional program, resulting in the generation of cells that produce and secrete insulin in vitro and in vivo. In light of the expanding number of cell types produced by direct lineage conversion procedures and the therapeutic interest of insulin-producing cells, we chose to thoroughly consider the viability of using fibroblasts as cells of origin in direct reprogramming protocols to generate β-like cells. However, research to date suggests that fibroblasts are resistant to being transformed into β-like cell using lineage-specific transcription factors 4, 9, 18. In this regard, skin fibroblasts have been the preferred cell source for many reprogramming protocols and they have so far been successfully transformed into a variety of somatic cell types including cardiomyocytes 12, chondrocytes 13, neurons 14, oligodendrocyte progenitors 15, hepatocytes 16 or endothelial cells 17. The initial material should ideally be available, simple to handle and to grow in the laboratory. One of the most important aspects of direct reprogramming strategies is the choice of the cell source, especially when taking into account their clinical application. Since then, studies have shown that various combinations of these and other transcription factors can promote conversion toward a β-like fate in other pancreatic cell lineages, including ductal and glucagon-expressing α-cells, and in extra-pancreatic related endodermal cell lineages, such as liver, gallbladder, and gastrointestinal tract cells 6, 7, 8, 9, 10, 11. A major breakthrough in this area was the discovery that three developmental transcription factors, namely Pdx1, Neurog3, and MafA, promoted the in situ conversion of acinar cells into insulin-producing cells in the mouse pancreas 4, 5. One of the strategies pursued to produce replacement β cells has been direct lineage reprogramming. Diabetes is characterized by a relative or total lack of functional β cells, and cell replacement therapy has consequently emerged as a promising therapeutic option to treat and ultimately cure this disease. Pancreatic beta (β) cells produce insulin, which controls whole body glucose homeostasis. As the number of cell types produced through direct conversion has rapidly increased in recent years, this strategy has emerged as a possible method for creating cell types with potential for use in therapeutic settings. ![]() This strategy is often based on the forced expression of cocktails of transcription factors that function as potent fate determinants during development of the cell type of interest 1, 2, 3. This work provides proof-of-concept of the capacity to make insulin-producing cells from human fibroblasts via transcription factor-mediated direct reprogramming.ĭirect lineage reprogramming entails the direct conversion of one differentiated cell type into another bypassing an intermediate pluripotent stage. Moreover, reprogrammed cells display glucose-induced insulin secretion in vitro and in vivo. Reprogrammed cells exhibit β-cell features including β-cell gene expression and glucose-responsive intracellular calcium mobilization. We describe how the timed-introduction of five developmental transcription factors (Neurog3, Pdx1, MafA, Pax4, and Nkx2-2) promotes conversion of fibroblasts toward a β-cell fate. 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 β-like cells. To date it has been possible to create β-like cells from related endodermal cell types by forcing the expression of developmental transcription factors, but not from more distant cell lineages like fibroblasts. One cell type of interest is the pancreatic insulin-producing β cell whose loss and/or dysfunction leads to diabetes. 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. ![]()
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