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Tributyltin Inhibits Neural Induction of Human Induced Pluripotent Stem Cells.

ABSTRACT: Tributyltin (TBT), one of the organotin compounds, is a well-known environmental pollutant. In our recent study, we reported that TBT induces mitochondrial dysfunction, in human-induced pluripotent stem cells (iPSCs) through the degradation of mitofusin1 (Mfn1), which is a mitochondrial fusion factor. However, the effect of TBT toxicity on the developmental process of iPSCs was not clear. The present study examined the effect of TBT on the differentiation of iPSCs into the ectodermal, mesodermal, and endodermal germ layers. We found that exposure to nanomolar concentration of TBT (50?nM) selectively inhibited the induction of iPSCs into the ectoderm, which is the first step in neurogenesis. We further assessed the effect of TBT on neural differentiation and found that it reduced the expression of several neural differentiation marker genes, which were also downregulated by Mfn1 knockdown in iPSCs. Taken together, these results indicate that TBT induces developmental neurotoxicity via Mfn1-mediated mitochondrial dysfunction in iPSCs.

SUBMITTER: Yamada S

PROVIDER: S-EPMC6092327 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Tributyltin Inhibits Neural Induction of Human Induced Pluripotent Stem Cells.

Yamada Shigeru S Kubo Yusuke Y Yamazaki Daiju D Sekino Yuko Y Nomura Yoko Y Yoshida Sachiko S Kanda Yasunari Y

Scientific reports 20180814 1

Tributyltin (TBT), one of the organotin compounds, is a well-known environmental pollutant. In our recent study, we reported that TBT induces mitochondrial dysfunction, in human-induced pluripotent stem cells (iPSCs) through the degradation of mitofusin1 (Mfn1), which is a mitochondrial fusion factor. However, the effect of TBT toxicity on the developmental process of iPSCs was not clear. The present study examined the effect of TBT on the differentiation of iPSCs into the ectodermal, mesodermal ...[more]

PMID: 30108368

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Project description:IntroductionNeural induction of human induced pluripotent stem cells represents a critical switch in cell state during which pluripotency is lost and commitment to a neural lineage is initiated. Although many of the key transcription factors involved in neural induction are known, we know little of the temporal and causal relationships that are required for this state transition.MethodsHere, we have carried out a longitudinal analysis of the transcriptome of human iPSCs undergoing neural induction. Using the temporal relationships between the changing profile of key transcription factors and subsequent changes in their target gene expression profiles, we have identified distinct functional modules operative throughout neural induction.ResultsIn addition to modules that govern loss of pluripotency and gain of neural ectoderm identity, we discover other modules governing cell cycle and metabolism. Strikingly, some of these functional modules are retained throughout neural induction, even though the gene membership of the module changes. Systems analysis identifies other modules associated with cell fate commitment, genome integrity, stress response and lineage specification. We then focussed on OTX2, one of the most precociously activated transcription factors during neural induction. Our temporal analysis of OTX2 target gene expression identified several OTX2 regulated gene modules representing protein remodelling, RNA splicing and RNA processing. Further CRISPRi inhibition of OTX2 prior to neural induction promotes an accelerated loss of pluripotency and a precocious and aberrant neural induction disrupting some of the previously identified modules.DiscussionWe infer that OTX2 has a diverse role during neural induction and regulates many of the biological processes that are required for loss of pluripotency and gain of neural identity. This dynamical analysis of transcriptional changes provides a unique perspective of the widespread remodelling of the cell machinery that occurs during neural induction of human iPSCs.

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.

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Tributyltin Inhibits Neural Induction of Human Induced Pluripotent Stem Cells.

Publications

Tributyltin Inhibits Neural Induction of Human Induced Pluripotent Stem Cells.

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