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Neural cells play an inhibitory role in pancreatic differentiation of pluripotent stem cells.

ABSTRACT: Pancreatic endocrine ?-cells derived from embryonic stem (ES) cells and induced pluripotent stem (iPS) cells have received attention as screening systems for therapeutic drugs and as the basis for cell-based therapies. Here, we used a 12-day ?-cell differentiation protocol for mouse ES cells and obtained several hit compounds that promoted ?-cell differentiation. One of these compounds, mycophenolic acid (MPA), effectively promoted ES cell differentiation with a concomitant reduction of neuronal cells. The existence of neural cell-derived inhibitory humoral factors for ?-cell differentiation was suggested using a co-culture system. Based on gene array analysis, we focused on the Wnt/?-catenin pathway and showed that the Wnt pathway inhibitor reversed MPA-induced ?-cell differentiation. Wnt pathway activation promoted ?-cell differentiation also in human iPS cells. Our results showed that Wnt signaling activation positively regulates ?-cell differentiation, and represent a downstream target of the neural inhibitory factor.

SUBMITTER: Nakashima R

PROVIDER: S-EPMC4738370 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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Neural cells play an inhibitory role in pancreatic differentiation of pluripotent stem cells.

Nakashima Ryutaro R Morooka Mayu M Shiraki Nobuaki N Sakano Daisuke D Ogaki Soichiro S Kume Kazuhiko K Kume Shoen S

Genes to cells : devoted to molecular & cellular mechanisms 20151030 12

Pancreatic endocrine β-cells derived from embryonic stem (ES) cells and induced pluripotent stem (iPS) cells have received attention as screening systems for therapeutic drugs and as the basis for cell-based therapies. Here, we used a 12-day β-cell differentiation protocol for mouse ES cells and obtained several hit compounds that promoted β-cell differentiation. One of these compounds, mycophenolic acid (MPA), effectively promoted ES cell differentiation with a concomitant reduction of neuronal ...[more]

PMID: 26514269

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Project description:Efficient and effective methods for converting human induced pluripotent stem cells into differentiated derivatives are critical for performing robust, large-scale studies of development and disease modelling, and for providing a source of cells for regenerative medicine. Here, we describe a 14-day neural differentiation protocol which allows for the scalable, simultaneous differentiation of multiple iPSC lines into cortical neural stem cells We currently employ this protocol to differentiate and compare sets of engineered iPSC lines carrying loss of function alleles in developmental disorder associated genes, alongside isogenic wildtype controls. Using RNA sequencing (RNA-Seq), we can examine the changes in gene expression brought about by each disease gene knockout, to determine its impact on neural development and explore mechanisms of disease. The 10-day Neural Induction period uses the well established dual-SMAD inhibition approach combined with Wnt/β-Catenin inhibition to selectively induce formation of cortical NSCs. This is followed by a 4-day Neural Maintenance period facilitating NSC expansion and rosette formation, and NSC cryopreservation. We also describe methods for thawing and passaging the cryopreserved NSCs, which are useful in confirming their viability for further culture. Routine implementation of immunocytochemistry Quality Control confirms the presence of PAX6-positive and/or FOXG1-positive NSCs and the absence of OCT4-positive iPSCs after differentiation. RNA-Seq, flow cytometry, immunocytochemistry (ICC) and RT-qPCR provide additional confirmation of robust presence of NSC markers in the differentiated cells. The broader utility and application of our protocol is demonstrated by the successful differentiation of wildtype iPSC lines from five additional independent donors. This paper thereby describes an efficient method for the production of large numbers of high purity cortical NSCs, which are widely applicable for downstream research into developmental mechanisms, further differentiation into postmitotic cortical neurons, or other applications such as large-scale drug screening experiments.

Dataset Information

Neural cells play an inhibitory role in pancreatic differentiation of pluripotent stem cells.

Publications

Neural cells play an inhibitory role in pancreatic differentiation of pluripotent stem cells.

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