Gene expression profiling of tissue and hMSC xenografts in a rat postpartum urinary injury model

Periurethral human mesenchymal stem cell (hMSC) injections are associated with functional improvement in animal models of post-partum stress urinary incontinence (SUI). However, limited data exists on the role of hMSCs in modulating gene expression in tissue repair after urethral injury. We quantified temporal gene expression modulation in hMSCs, and injured rat urethral tissue, using RNA seq in an animal model of SUI, over a 3-day time period following urethral injury, and local hMSC therapy. We injected PKH fluorescent-labeled human hMSC into the periurethral space of rats, following a 4h vaginal distention (3 rats per time point). Control rats underwent vaginal distention injury only, and were sacrificed at 12h, 24h, 36h, 72h post injury. Rat urethral and vaginal tissues were frozen and sectioned. Fluorescent labeled hMSCs were distinguished from adjacent, unlabeled rat urethral tissue. RNA was prepared from urethral tissue obtained by laser dissection of frozen tissue sections and sequenced on an Illumina HiSeq 2500. Differentially expressed genes (DEGs) over 72h were evaluated using a 2-group t-test (p<0.05). Our transcriptional analyses identified candidate genes involved in tissue injury, that were broadly sorted by injury and exposure to hMSC, throughout the first 72h of acute phase of injury. DEGs in treated urethra, compared with untreated urethra, were functionally associated with tissue repair, angiogenesis, neurogenesis, and oxidative stress suppression. DEGs included a variety of cytokines, extracellular matrix stabilization and regeneration genes, cytokine signaling modification, cell cycle regulation, muscle differentiation and stabilization. Moreover, our results revealed DEGs changes in the hMSCs (PKH-labelled), which were harvested from injured urethra. The expressions are related to DNA damage repair, transcription activation, stem cell regulation, cell survival, apoptosis, self-renewal, cell proliferation, migration and injury response. Overall design: Laser captured rat urethral tissue or PKH labeled hMSC were obtained from 12 hr, 24 hr, 36 hr, or 72 hr post injury. Our submission contains the following samples: Urethral tissues from 3 control rats for every timepoint; urethral tissue and hMSC from 2 rats at 12 hr; urethral tissue and hMSC from 3 rats per timepoint at 24 hr, 36 hr, and 72 hr post-injury. In total we submitted 31 tissue samples for RNA seq analysis.

经尿道周围注射人源间充质干细胞（human mesenchymal stem cell, hMSC），可在产后压力性尿失禁（post-partum stress urinary incontinence, SUI）动物模型中实现功能改善。然而，目前关于hMSC在尿道损伤后组织修复过程中调控基因表达的相关数据仍较为匮乏。本研究基于SUI动物模型，在尿道损伤及局部hMSC治疗后的3天周期内，通过RNA测序（RNA sequencing, RNA-seq）量化分析hMSC与受损大鼠尿道组织的时序基因表达调控模式。

本研究先对大鼠进行4小时阴道扩张造模，随后经尿道周围间隙注射PKH荧光标记的hMSC，每个时间点设置3只大鼠。对照组大鼠仅接受阴道扩张损伤，分别于损伤后12h、24h、36h、72h处死取材。获取大鼠尿道与阴道组织后进行冷冻制片，通过荧光标记区分hMSC与未标记的宿主大鼠尿道组织。利用激光捕获显微切割技术从冷冻组织切片中分离尿道组织，提取RNA并在Illumina HiSeq 2500平台上完成测序。采用两独立样本t检验（p<0.05）对72小时内的差异表达基因（differentially expressed genes, DEGs）进行筛选分析。

转录组分析结果显示，在损伤急性期的前72小时内，与组织损伤相关的候选基因可根据损伤程度及hMSC暴露情况进行大致聚类。与未处理组尿道组织相比，hMSC处理组尿道组织中的DEGs功能富集于组织修复、血管生成、神经发生以及氧化应激抑制等通路。这些DEGs涵盖多种细胞因子、细胞外基质稳定与再生相关基因、细胞因子信号修饰因子、细胞周期调控因子、肌肉分化与稳定相关因子。此外，本研究还分析了从损伤尿道中分离获取的PKH标记hMSC的DEGs变化，其表达谱涉及DNA损伤修复、转录激活、干细胞调控、细胞存活、凋亡、自我更新、细胞增殖、迁移以及损伤应答等过程。

研究整体设计：分别于损伤后12h、24h、36h、72h四个时间点获取激光捕获的大鼠尿道组织或PKH标记的hMSC。本次提交的测序样本包括：各时间点对照组大鼠尿道组织各3例；损伤后12h时的大鼠尿道组织及hMSC样本各2例；损伤后24h、36h、72h三个时间点的大鼠尿道组织及hMSC样本各3例。本次研究共计提交31份组织样本用于RNA测序分析。