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

Abstract

BackgroundPancreatic 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).MethodsIn 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.ResultsOur 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.ConclusionsWe 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 materialThe online version of this article (doi:10.1186/s13287-017-0759-z) contains supplementary material, which is available to authorized users.

Scalable generation of mesenchymal stem cells and adipocytes from human pluripotent stem cells

“…BrdU incorporation assay was performed as previously described [22] with some modifications. Briefly, the cells were incubated without or with BrdU (1:100; ThermoFisher Scientific, USA) for 19 h in the differentiation media.…”

Scalable Generation of Mesenchymal Stem Cells and Adipocytes from Human Pluripotent Stem Cells

“…BrdU incorporation assay was performed as previously described [23] with some modifications. Briefly, the cells were incubated without or with BrdU (1:100; ThermoFisher Scientific, Waltham, MA, USA) for 19 h in the differentiation media.…”

Stem Cell Therapy for Diabetes: Beta Cells versus Pancreatic Progenitors

“…Therefore, in vitro differentiation strategies employ a combination of small molecules or recombinant proteins to turn on or off these pathways at specific stages of the protocol [8,22]. Multiple groups have differentiated hPSCs to PDX1 and NKX6.1 co-expressing pancreatic progenitors in monolayer cultures [16,23], yielding even up to 90% of PDX1+/NKX6.1+ co-positive pancreatic progenitors [24,25] (Figure 3). Prior to the most recent improvements in protocols employing monolayer method, studies showed that cellular aggregation during differentiation process could improve the efficiency of pancreatic progenitors as compared to monolayer cultures [19,26].…”