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Differentiation of Human Induced Pluripotent Stem Cells into Insulin-Like Cell Clusters with miR-186 and miR-375 by using chemical transfection

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Abstract

Diabetes mellitus is characterized by either the inability to produce insulin or insensitivity to insulin secreted by the body. Islet cell replacement is an effective approach for diabetes treatment; however, it is not sufficient for all the diabetic patients. MicroRNAs (miRNAs) are a class of small noncoding RNAs that play an important role in mediating a broad and expanding range of biological activities, such as pancreas development. The present study aimed to develop a protocol to efficiently differentiate human induced pluripotent stem (iPS) cells into islet-like cell clusters (ILCs) in vitro by using miR-186 and miR-375. The human iPS colonies were transfected with hsa-miR-186 and hsa-miR-375 by using siPORT™ NeoFX™ Transfection Agent, and the differentiation was compared to controls. Total RNA was extracted 24 and 48 h after transfection. The gene expressions of insulin, NGN3, GLUT2, PAX4, PAX6, KIR6.2, NKX6.1, PDX1, Glucagon, and OCT4 were then evaluated through real-time qPCR. On the third day, the potency of the clusters was assessed in response to high glucose levels. Dithizone (DTZ) was used to identify the existence of the β-cells. Besides, the presence of insulin and NGN3 proteins was investigated by immunocytochemistry. Morphological changes were observed on the first day after the chemical transfection, and cell clusters were formed on the third day. The expression of pancreatic specific transcription factors was increased on the first day and significantly increased on the second day. The ILCs were positive for insulin and NGN3 proteins in the immunocytochemistry. Besides, the clusters were stained with DTZ and secreted insulin in glucose challenge test. Overexpression of miR-186 and miR-375 can be an alternative strategy for producing ILCs from the iPS cells in a short time. This work provides a new approach by using patient-specific iPSCs for β-cell replacement therapy in diabetic patients.

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Acknowledgments

Research Improvement Center of Shiraz University of Medical Sciences and Ms. A. Keivanshekouh are appreciated for improving the use of English in the manuscript. The study was financially supported by a grant from Iran National Science Foundation (INSF).

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Authors and Affiliations

  1. Department of Biology, Science and Research Branch, Islamic Azad University, Fars, Iran

    Anahita Shaer

  2. Transplant Research Center, Shiraz University of Medical Sciences, Zand Street, Nemazi Hospital, 7193711351, Shiraz, Iran

    Anahita Shaer, Negar Azarpira & Mohammad Hosein Karimi

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  1. Anahita Shaer
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  2. Negar Azarpira
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  3. Mohammad Hosein Karimi
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Correspondence to Negar Azarpira.

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Shaer, A., Azarpira, N. & Karimi, M.H. Differentiation of Human Induced Pluripotent Stem Cells into Insulin-Like Cell Clusters with miR-186 and miR-375 by using chemical transfection. Appl Biochem Biotechnol 174, 242–258 (2014). https://doi.org/10.1007/s12010-014-1045-5

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