Rho-Associated Kinases and Non-muscle Myosin IIs Inhibit the Differentiation of Human iPSCs to Pancreatic Endoderm

Toyoda, Tarō; Kimura, Azuma; Tanaka, Hiromi; Ameku, Tomonaga; Mima, Atsushi; Hirose, Yurie; Nakamura, Masahiro; Watanabe, Akira; Osafune, Kenji

doi:10.1016/j.stemcr.2017.07.005

Cited by 24 publications

(33 citation statements)

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“…On the other hand, we found that re-plating of the dissociated endodermal cells at a lower density induced NKX6.1 expression in both PDX1 + /NKX6.1 + and PDX1 – /NKX6.1 + populations. Our results for NKX6.1 induction at very low (a quarter that of the endoderm) density within the novel PDX1 – /NKX6.1 + population is in accord with a recent study reporting that NKX6.1 + cells can be produced at low-density culture in the presence of ROCK-NM II inhibitor [ 28 ].…”

Section: Discussionsupporting

confidence: 92%

“…More recently, Nostro et al provided the optimum temporal window and cytokine cocktail for higher induction of NKX6.1 in pancreatic progenitors in adherent culture [ 19 ]. Another group showed the upregulating effect of high-density aggregate cultures on PDX1 + /NKX6.1 + pancreatic progenitors [ 27 , 28 ]. Herein, we present an efficient method for producing a high proportion of PDX1 + /NKX6.1 + pancreatic progenitors from hPSCs and maximizing their proliferative capacity.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2018

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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.

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Section: Discussionsupporting

confidence: 92%

Section: Introductionmentioning

confidence: 99%

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

et al. 2018

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“…This effect was independent of cell proliferation. At similar cell densities, inhibition of ROCK-NM II pathways improved pancreatic differentiation over controls, suggesting that this effect is due to aggregation ( Toyoda et al, 2017 ). High cell density cultures may therefore better recapitulate the local cell density in the developing pancreas, thereby increasing bias toward pancreatic endocrine fate.…”

Section: Cell–cell Interactionsmentioning

confidence: 99%

“…Forced aggregation of PSC-derived pancreatic progenitor cells increases the proportion of NKX6.1 + cells over standard 2D cultures ( Toyoda et al, 2015 ). In their later work, Toyoda et al (2017) demonstrated that inhibition of Rho-associated kinases (ROCK) and non-muscle myosin II (NM II) increased the fraction of NKX6.1 + cells by emulating an aggregate phenotype even in low density cultures, suggesting that the physical act of aggregation may not be necessary. In addition to this, recent studies have shown that insulin-secreting cells can be produced in planar, non-aggregate protocols by the timed depolymerization of the actin cytoskeleton ( Hogrebe et al, 2020 ).…”

Section: Cell–cell Interactionsmentioning

confidence: 99%

Developmentally-Inspired Biomimetic Culture Models to Produce Functional Islet-Like Cells From Pluripotent Precursors

Tran

Moraes

Hoesli

2020

Front. Bioeng. Biotechnol.

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Insulin-producing beta cells sourced from pluripotent stem cells hold great potential as a virtually unlimited cell source to treat diabetes. Directed pancreatic differentiation protocols aim to mimic various stimuli present during embryonic development through sequential changes of in vitro culture conditions. This is commonly accomplished by the timed addition of soluble signaling factors, in conjunction with cell-handling steps such as the formation of 3D cell aggregates. Interestingly, when stem cells at the pancreatic progenitor stage are transplanted, they form functional insulinproducing cells, suggesting that in vivo microenvironmental cues promote beta cell specification. Among these cues, biophysical stimuli have only recently emerged in the context of optimizing pancreatic differentiation protocols. This review focuses on studies of cell-microenvironment interactions and their impact on differentiating pancreatic cells when considering cell signaling, cell-cell and cell-ECM interactions. We highlight the development of in vitro cell culture models that allow systematic studies of pancreatic cell mechanobiology in response to extracellular matrix proteins, biomechanical effects, soluble factor modulation of biomechanics, substrate stiffness, fluid flow and topography. Finally, we explore how these new mechanical insights could lead to novel pancreatic differentiation protocols that improve efficiency, maturity, and throughput.

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“…Therefore, stem cell differentiation protocols have focused on inducing the expression of these two key markers. Aside from media components, the induction of PDX1 + /NKX6.1 + cells is affected by cell density in adherent cultures, and markedly improved with cell aggregation cultures and inhibition of Rho‐associated kinase or non‐muscle myosin II. Protocols have been developed to efficiently mass produce cells marked by PDX1 and NKX6.1, including in three‐dimensional suspension bioreactor systems, and these cells are able to mature into islet cells and effectively lower blood glucose in immunodeficient mice.…”

Section: Insulin‐producing Cells From Human Stem Cellsmentioning

confidence: 99%

Beta‐cell replacement strategies for diabetes

Kieffer

Woltjen

Osafune

et al. 2017

J of Diabetes Invest

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Diabetes is characterized by elevated levels of blood glucose as a result of insufficient production of insulin from loss or dysfunction of pancreatic islet β‐cells. Here, we review several approaches to replacing β‐cells that were recently discussed at a symposium held in Kyoto, Japan. Transplant of donor human islets can effectively treat diabetes and eliminate the need for insulin injections, supporting research aimed at identifying abundant supplies of cells. Studies showing the feasibility of producing mouse islets in rats support the concept of generating pigs with human pancreas that can serve as donors of human islets, although scientific and ethical challenges remain. Alternatively, in vitro differentiation of both human embryonic stem cells and induced pluripotent stem cells is being actively pursued as an islet cell source, and embryonic stem cell‐derived pancreatic progenitor cells are now in clinical trials in North America in patients with diabetes. Macro‐encapsulation devices are being used to contain and protect the cells from immune attack, and alternate strategies of immune‐isolation are being pursued, such as islets contained within long microfibers. Recent advancements in genetic engineering tools offer exciting opportunities to broaden therapeutic strategies and to probe the genetic involvement in β‐cell failure that contributes to diabetes. Personalized medicine might eventually become a possibility with genetically edited patient‐induced pluripotent stem cells, and the development of simplified robust differentiation protocols that ideally become standardized and automated. Additional efforts to develop a safe and effective β‐cell replacement strategy to treat diabetes are warranted.

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Rho-Associated Kinases and Non-muscle Myosin IIs Inhibit the Differentiation of Human iPSCs to Pancreatic Endoderm

Cited by 24 publications

References 24 publications

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

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

Developmentally-Inspired Biomimetic Culture Models to Produce Functional Islet-Like Cells From Pluripotent Precursors

Beta‐cell replacement strategies for diabetes

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