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

Bushra Memon,Essam M Abdelalim,Manale Karam,Sara Al-Khawaga

doi:10.1186/s13287-017-0759-z

Bushra Memon, Essam M Abdelalim + Show 2 more

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https://doi.org/10.1186/s13287-017-0759-z

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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.

Highlights

Pancreatic progenitors (PPs) co-expressing the two transcription factors (TFs) pancreatic and duodenal homeobox 1 (PDX1) and NKX6.1 are recognized as the indispensable precursors of functional pancreatic β cells
Shortening stage 3 to 2 days led to generation of pancreatic progenitors expressing PDX1 and NKX6.1 (PDX1+/NKX6.1+) (Fig. 2a)
In conclusion, we demonstrated that manipulating the cell seeding density, cell-cell contact, and cues from the extracellular matrix are essential elements in improving pancreatic differentiation efficiency and proliferation, thereby providing a simplified differentiation method for generating pancreatic progenitors in vitro under adherent culture conditions

Summary

Introduction

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. Step-wise protocols have been designed to differentiate hPSCs into β cells by directing them along the stages of definitive endoderm, pancreatic foregut, pancreatic progenitors, and endocrine precursor cells that mature into insulin-secreting cells [3,4,5,6,7,8,9]. These protocols involve the use of specific growth factors or pharmacological molecules that regulate specific signaling pathways. This is marked by the reconstruction of crucial human developmental cues that include activation or inhibition of appropriate transcription factors (TFs) and alternative signaling pathways [3,4,5,6,7,8,9]

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