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Background  Pancreatic progenitors (PPs) co-expressing the two transcription factors (TFs) PDX1 and NKX6.1 are recognized as the indispensable precursors of functional pancreatic β cells. Here, we aimed to establish an efficient protocol for maximizing generation of PDX1 + /NKX6.1 +  PPs from human pluripotent stem cells (hPSCs).    Methods  In order to enhance the PDX1 + /NKX6.1 +  population, we manipulated in vitro culture conditions during differentiation by dissociating densely formed endodermal cells and re-plating them at different densities. These dissociated cells were subjected to an augmented duration of retinoid and fibroblast growth factor (FGF)10 signaling to induce higher PDX1 and NKX6.1 expression.    Results  Our optimized protocol dramatically increased the expression of NKX6.1, leading to an increase in the proportion of PDX1 + /NKX6.1 +  progenitors (~90%) in monolayer, higher than the previously published protocols, as well as upregulated key TFs controlling pancreatic development. The improved efficiency of pancreatic differentiation was complemented by an inhibited hepatic specification and an increased proliferation of NKX6.1 +  cells. Interestingly, we were able to enrich a novel PDX1 – /NKX6.1 +  population by manipulating the re-plating density; these oriented themselves in three-dimensional clusters. Further differentiation validated the ability of our PDX1 + /NKX6.1 +  progenitors to generate NGN3 +  endocrine progenitors.    Conclusions  We provide a novel technique that facilitates appropriate cellular rearrangement in monolayer culture to yield a high proportion of PDX1 + /NKX6.1 +  PPs with an elevated self-replicating capacity, thereby aiding scalable production of functional β cells from hPSCs in vitro. Our innovative method also enriches a novel NKX6.1 + /PDX1 –  population, with characteristics of proposed endocrine precursors, allowing further studies on deciphering routes to β-cell development.    Electronic supplementary material  The online version of this article (doi:10.1186/s13287-017-0759-z) contains supplementary material, which is available to authorized users.

Enhanced differentiation of human pluripotent stem cells into pancreatic progenitors co-expressing PDX1 and NKX6.1