Department of Medical Nanotechnology, Zanjan University of Medical Sciences, Zanjan, Iran.
Cell-Based Therapies Research Center, Digestive Disease Research Institute, Tehran University of Medical Sciences, Tehran, Iran.
Biochem Biophys Res Commun. 2020 Aug 27;529(3):526-532. doi: 10.1016/j.bbrc.2020.06.006. Epub 2020 Jul 14.
MiRNAs are small non-coding RNAs that are ordinarily involved in modulating mRNAs and stem cell differentiation. 3D nanofibrous scaffolds have an important role in the differentiation of stem cells due to their similarity to the extracellular matrix (ECM). In the present study, we tried to introduce a new approach to guiding the differentiation of conjunctiva mesenchymal stem cells (CJMSCs) into photoreceptor-like cells by hsa-miR-9-1 delivery on both 2D and 3D substrates. First, the CJMSCs were transduced by a lentiviral vector carrying miR-9 (pCDH + hsa-miR-9-1) and then cell transduction efficacy verified by using fluorescent microscopy, flow cytometry, and qPCR analyses. Silk Fibroin-poly-L-lactic acid (SF-PLLA) scaffold was fabricated by the electrospinning technique while the scaffold characteristics including morphology, chemical properties, and biocompatibility were evaluated by SEM, FTIR, and MTT assays, respectively. Then, the miR-9-CJMSCs were seeded on both TCPS and the scaffold; photoreceptor gene and protein expressions were evaluated by RT-qPCR and immunostaining after 14 and 21 days of transduction. More than 80% of CJMSCs were transduced and miR-9 expression was significantly higher in miR-9-CJMSCs compared with empty vector (EV)-CJMSCs. SEM and FTIR confirmed the fabrication of the SF/PLLA hybrid structure. RT-qPCR and immunostaining analyses showed that the specific photoreceptor genes and proteins were expressed in miR-9 transduced CJMSCs. Mir-9 induced CJMSCs into photoreceptor-like cells in a time-dependent manneron on both TCPS and nanofibrous scaffold.We have proved that hsa-miR-9-1 has the potency to guide the photoreceptor differentiation of mesenchymal stem cells and promote retinal regeneration.
miRNAs 是通常参与调节 mRNA 和干细胞分化的小非编码 RNA。3D 纳米纤维支架由于其与细胞外基质（ECM）的相似性，在干细胞分化中起着重要作用。在本研究中，我们试图通过在 2D 和 3D 基质上递呈 hsa-miR-9-1 来引入一种新方法，以指导结膜间充质干细胞（CJMSCs）向光感受器样细胞分化。首先，通过携带 miR-9 的慢病毒载体（pCDH+hsa-miR-9-1）转导 CJMSCs，然后通过荧光显微镜、流式细胞术和 qPCR 分析验证细胞转导效率。丝素蛋白-聚 L-乳酸（SF-PLLA）支架通过静电纺丝技术制备，通过 SEM、FTIR 和 MTT 分析分别评估支架的形态、化学性质和生物相容性。然后，将 miR-9-CJMSCs 接种在 TCPS 和支架上；在转导后 14 和 21 天，通过 RT-qPCR 和免疫染色评估光感受器基因和蛋白的表达。超过 80%的 CJMSCs 被转导，miR-9-CJMSCs 中的 miR-9 表达明显高于空载体（EV）-CJMSCs。SEM 和 FTIR 证实了 SF/PLLA 混合结构的制备。RT-qPCR 和免疫染色分析表明，在 miR-9 转导的 CJMSCs 中表达了特定的光感受器基因和蛋白。miR-9 以时间依赖的方式在 TCPS 和纳米纤维支架上诱导 CJMSCs 向光感受器样细胞分化。我们已经证明 hsa-miR-9-1 具有指导间充质干细胞向光感受器分化并促进视网膜再生的潜力。
