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MicroRNA expression profiles of human iPSCs differentiation into insulin-producing cells

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Abstract

Aims

MicroRNAs are a class of small noncoding RNAs, which control gene expression by inhibition of mRNA translation. MicroRNAs are involved in the control of biological processes including cell differentiation. Here, we aim at characterizing microRNA expression profiles during differentiation of human induced pluripotent stem cells (hiPSCs) into insulin-producing cells.

Methods

We differentiated hiPSCs toward endocrine pancreatic lineage following a 18-day protocol. We analyzed genes and microRNA expression levels using RT real-time PCR and TaqMan microRNA arrays followed by bioinformatic functional analysis.

Results

MicroRNA expression profiles analysis of undifferentiated hiPSCs during pancreatic differentiation revealed that 347/768 microRNAs were expressed at least in one time point of all samples. We observed 18 microRNAs differentially expressed: 11 were upregulated (miR-9-5p, miR-9-3p, miR-10a, miR-99a-3p, miR-124a, miR-135a, miR-138, miR-149, miR-211, miR-342-3p and miR-375) and 7 downregulated (miR-31, miR-127, miR-143, miR-302c-3p, miR-373, miR-518b and miR-520c-3p) during differentiation into insulin-producing cells. Selected microRNAs were further evaluated during differentiation of Sendai-virus-reprogrammed hiPSCs using an improved endocrine pancreatic beta cell derivation protocol and, moreover, in differentiated NKX6.1+ sorted cells. Following Targetscan7.0 analysis of target genes of differentially expressed microRNAs and gene ontology classification, we found that such target genes belong to categories of major significance in pancreas organogenesis and development or exocytosis.

Conclusions

We detected a specific hiPSCs microRNAs signature during differentiation into insulin-producing cells and demonstrated that differentially expressed microRNAs target several genes involved in pancreas organogenesis.

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Acknowledgements

This work was supported by grants from the European Union [Project PEVNET (Project Number: 261441) in the Framework Program 7 (FP7)], from the Italian Ministry of Research (n. 2010JS3PMZ_008) and from Fondazione Roma. The editorial help of Maddalena Prencipe has been greatly appreciated. Silvia Pellegrini was supported by a fellowship from the Fondazione Diabete Ricerca (Società Italiana di Diabetologia—SID).

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Authors and Affiliations

  1. Diabetes Unit, Department of Medicine, Surgery and Neuroscience, University of Siena, Siena, Italy

    Guido Sebastiani, Marco Valentini, Giuseppina Emanuela Grieco, Giuliana Ventriglia, Laura Nigi, Francesca Mancarella & Francesco Dotta

  2. Fondazione Umberto di Mario ONLUS, Toscana Life Sciences, Siena, Italy

    Guido Sebastiani, Marco Valentini, Giuseppina Emanuela Grieco, Giuliana Ventriglia, Laura Nigi, Francesca Mancarella & Francesco Dotta

  3. Diabetes Research Institute, IRCCS San Raffaele Scientific Institute, Milan, Italy

    Silvia Pellegrini, Valeria Sordi & Lorenzo Piemonti

  4. Division of Neuroscience, Institute of Experimental Neurology (INSpe), IRCCS San Raffaele Hospital, Milan, Italy

    Gianvito Martino

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  1. Guido Sebastiani
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  2. Marco Valentini
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  3. Giuseppina Emanuela Grieco
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Correspondence to Francesco Dotta.

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Conflict of interest

Guido Sebastiani, Marco Valentini, Giuseppina Emanuela Grieco, Giuliana Ventriglia, Laura Nigi, Francesca Mancarella, Silvia Pellegrini, Gianvito Martino,Valeria Sordi, Lorenzo Piemonti and Francesco Dotta declare that they have no conflict of interest.

Ethical standard

All procedures performed were in accordance with the ethical standards and with the 1964 Declaration of Helsinki and its later amendments.

Human and animal rights

All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008 (5).

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Informed consent was obtained from all patients for being included in the study.

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Sebastiani, G., Valentini, M., Grieco, G.E. et al. MicroRNA expression profiles of human iPSCs differentiation into insulin-producing cells. Acta Diabetol 54, 265–281 (2017). https://doi.org/10.1007/s00592-016-0955-9

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