Lahmy Reyhaneh, Soleimani Masoud, Sanati Mohammad H, Behmanesh Mehrdad, Kouhkan Fatemeh, Mobarra Naser
Department of Genetics, Faculty of Biology Sciences, Tarbiat Modares University, Tehran, Iran.
Mol Biol Rep. 2014;41(4):2055-66. doi: 10.1007/s11033-014-3054-4. Epub 2014 Jan 29.
Islet transplantation is considered as an ultimate option for the treatment of type I diabetes. Human induced pluripotent stem cells (hiPSCs) have raised the possibility that patient-specific insulin-secreting cells might be derived from somatic cells through cell fate reprogramming. However, current protocols mostly rely on the use of several cytokines and inhibitors for directing differentiation towards pancreatic fate. Given the high manufacturing cost of these recombinant proteins, this approach is prohibitive for clinical applications. Knowing that microRNAs (miRNAs) are key players in various stages of pancreatic development, we present a novel and cost-effective strategy in which over-expression of miR-375 promotes pancreatic differentiation in hiPSCs in the absence of any other stimulator. We used a polycistronic viral vector expressing Sox2, Klf4, c-Myc, and Oct4 to drive hiPSCs from human foreskin fibroblasts. The established hiPSCs are similar to human embryonic stem cells in many aspects including morphology, passaging, surface and pluripotency markers, and gene expression. For differentiation induction, miR-375 was lentivirally overexpressed in these hiPSCs. Morphological assessment, immunocytochemistry, and expression analysis of islet marker genes confirmed that islet like cells were obtained in miR-375 transduced cells compared to controls. Our differentiated clusters secreted insulin in a glucose-dependant manner, showing in vitro functionality. We demonstrated for the first time that miRNAs might be ideal substitutes to induce pancreatic differentiation in hiPSCs. This work provides a new approach to study the role of miRNAs in pancreatic specification and increase the feasibility of using patient-specific iPSCs for beta cell replacement therapy for type I diabetes.
胰岛移植被认为是治疗I型糖尿病的最终选择。人类诱导多能干细胞（hiPSCs）增加了通过细胞命运重编程从体细胞中获得患者特异性胰岛素分泌细胞的可能性。然而，目前的方案大多依赖于使用几种细胞因子和抑制剂来引导细胞向胰腺命运分化。鉴于这些重组蛋白的高生产成本，这种方法在临床应用中令人望而却步。由于已知微小RNA（miRNAs）是胰腺发育各个阶段的关键参与者，我们提出了一种新颖且具有成本效益的策略，即miR-375的过表达在没有任何其他刺激物的情况下促进hiPSCs向胰腺分化。我们使用了一种表达Sox2、Klf4、c-Myc和Oct4的多顺反子病毒载体从人包皮成纤维细胞驱动生成hiPSCs。所建立的hiPSCs在许多方面与人类胚胎干细胞相似，包括形态、传代、表面和多能性标记以及基因表达。为了诱导分化，在这些hiPSCs中通过慢病毒使miR-375过表达。形态学评估、免疫细胞化学和胰岛标记基因的表达分析证实，与对照相比，在转导了miR-375的细胞中获得了胰岛样细胞。我们分化的细胞簇以葡萄糖依赖的方式分泌胰岛素，显示出体外功能。我们首次证明，miRNAs可能是诱导hiPSCs向胰腺分化的理想替代物。这项工作提供了一种新方法来研究miRNAs在胰腺特化中的作用，并提高了使用患者特异性iPSCs进行I型糖尿病β细胞替代治疗的可行性。
