Koh Avin Ee-Hwan, Alsaeedi Hiba Amer, Rashid Munirah Binti Abd, Lam Chenshen, Harun Mohd Hairul Nizam, Ng Min Hwei, Mohd Isa Hazlita, Then Kong Yong, Bastion Mae-Lynn Catherine, Farhana Aisha, Khursheed Alam Mohammad, Subbiah Suresh Kumar, Mok Pooi Ling
Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Jouf University, Sakaka, Saudi Arabia.
Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Selangor, Malaysia.
Front Cell Dev Biol. 2021 Apr 27;9:652017. doi: 10.3389/fcell.2021.652017. eCollection 2021.
Mesenchymal stem cells (MSC) are highly regarded as a potential treatment for retinal degenerative disorders like retinitis pigmentosa and age-related macular degeneration. However, donor cell heterogeneity and inconsistent protocols for transplantation have led to varied outcomes in clinical trials. We previously showed that genetically-modifying MSCs to express erythropoietin (MSC) improved its regenerative capabilities . Hence, in this study, we sought to prove its potential by transplanting MSCs in a rat retinal degeneration model and analyzing its retinal transcriptome using RNA-Seq. Firstly, MSCs were cultured and expanded before being intravitreally transplanted into the sodium iodate-induced model. After the procedure, electroretinography (ERG) was performed bi-weekly for 30 days. Histological analyses were performed after the ERG assessment. The retina was then harvested for RNA extraction. After mRNA-enrichment and library preparation, paired-end RNA-Seq was performed. Salmon and DESeq2 were used to process the output files. The generated dataset was then analyzed using over-representation (ORA), functional enrichment (GSEA), and pathway topology analysis tools (SPIA) to identify enrichment of key pathways in the experimental groups. The results showed that the MSC-treated group had detectable ERG waves ( <0.05), which were indicative of successful phototransduction. The stem cells were also successfully detected by immunohistochemistry 30 days after intravitreal transplantation. An initial over-representation analysis revealed a snapshot of immune-related pathways in all the groups but was mainly overexpressed in the MSC group. A subsequent GSEA and SPIA analysis later revealed enrichment in a large number of biological processes including phototransduction, regeneration, and cell death ( <0.05). Based on these pathways, a set of pro-survival gene expressions were extracted and tabulated. This study provided an in-depth transcriptomic analysis on the MSC-treated retinal degeneration model as well as a profile of pro-survival genes that can be used as candidates for further genetic enhancement studies on stem cells.
间充质干细胞（MSC）被高度视为治疗视网膜色素变性和年龄相关性黄斑变性等视网膜退行性疾病的潜在疗法。然而，供体细胞的异质性以及移植方案的不一致导致临床试验结果各异。我们之前表明，对MSC进行基因改造使其表达促红细胞生成素（eMSC）可提高其再生能力。因此，在本研究中，我们试图通过将eMSC移植到大鼠视网膜变性模型中并使用RNA测序分析其视网膜转录组来证明其潜力。首先，培养并扩增MSC，然后将其玻璃体内注射到碘酸钠诱导的模型中。手术后，每两周进行一次视网膜电图（ERG）检查，持续30天。在ERG评估后进行组织学分析。然后收集视网膜进行RNA提取。在mRNA富集和文库制备后，进行双末端RNA测序。使用Salmon和DESeq2处理输出文件。然后使用过表达分析（ORA）、功能富集分析（GSEA）和通路拓扑分析工具（SPIA）对生成的数据集进行分析，以确定实验组中关键通路的富集情况。结果表明，eMSC治疗组可检测到ERG波（P<0.05），这表明光转导成功。玻璃体内注射后30天，通过免疫组织化学也成功检测到了干细胞。初步的过表达分析揭示了所有组中免疫相关通路的概况，但主要在eMSC组中过度表达。随后的GSEA和SPIA分析后来揭示了大量生物过程的富集，包括光转导、再生和细胞死亡（P<0.05）。基于这些通路，提取并列出了一组促生存基因表达。本研究对eMSC治疗的视网膜变性模型进行了深入的转录组分析，并提供了促生存基因概况，可作为干细胞进一步基因增强研究的候选基因。
