背景:骨髓间充质干细胞(BMSCs)在周围神经损伤过程中可以分化为雪旺细胞(SCs);在我们先前的研究中,我们发现SC来源的外泌体(SC-exos)具有直接诱导作用,而成纤维细胞来源的外泌体(Fb-exos)没有明显的诱导作用。神经干细胞(NSC)来源的外泌体(NSC-exos)的诱导作用也已得到广泛证实。然而,没有研究同时比较这三种细胞的诱导效果。因此,通过研究这三种细胞来源的外泌体对诱导BMSCs分化为SCs的影响,本研究探讨了不同外泌体在促进干细胞向SCs细胞分化中的作用,并通过RNA测序在两组之间进行比较,以进一步缩小目标基因和相关基因通路的范围,以研究其相关机制。
方法:我们从干细胞中提取外泌体,成纤维细胞(Fb)和神经干细胞(NSC),然后研究这些外泌体在不同培养条件下诱导分化为BMSCs的能力。荧光免疫法检测诱导细胞中关键蛋白和基因标记物的表达水平,蛋白质印迹和聚合酶链反应(PCR);然后,我们统计比较了不同条件下的相对诱导效应。最后,我们通过RNA-seq分析了三种类型的外泌体,以预测靶基因和相关基因通路。
结果:BMSCs通过三种培养基培养:常规(无诱导),在不同的培养条件下具有相同细胞来源的外来体的预诱导或预诱导+原始诱导培养基(ODM)。当分别添加三种不同类型的外泌体时,诱导BMSCs向SCs分化的整体显著增加(P<0.05)。诱导能力排序如下:诱导前+ODM+外泌体组>诱导前+外泌体组>非诱导+外泌体组。在相同的培养条件下使用来自不同细胞来源的外泌体,在三种培养条件下,我们观察到以下趋势:RSC96-exos组≥NSC-exos组>Fb-exos组。在RSC96-exos组和NSC-exos组中,将BMSCs诱导为SCs的总体能力显著更高。虽然两组的诱导效率比较无显著性差异,RSC96-exos组的总体诱导能力略高于NSC-exos组。通过将分化诱导结果与RNA-seq数据相结合,三种类型的外泌体分为三个比较组:RSC与NSC,RSCvs.Fb和NSCvs.Fb.我们在这三组中鉴定了203个差异表达的mRNA靶基因。两个差异表达基因同时上调,即核黄素激酶(RFK,ENSRNOG00000022273)和核糖体RNA加工36(Rrp36,ENSRNOG00000017836)。我们没有发现任何共同上调的miRNA靶基因,但确实确定了lncRNAs的一个靶基因,即ENSRNOG00000065005.分析确定了90个与mRNAs中的神经和轴突相关的GO术语;此外,KEGG富集和GASA分析确定了三组中的13种常见差异表达途径。
结论:我们的分析发现,诱导前+ODM+RSC96/NSC-exos培养条件最有利于诱导和分化。RSC96-exos和NSC-exos表现出BMSCs向SCs的显著更大的分化效率。虽然没有统计学差异,数据表明RSC96-exos具有优势的趋势我们确定了三组之间的203个差异表达的mRNA,并且两个差异表达的靶mRNA被上调,即核黄素激酶(RFK,ENSRNOG00000022273)和核糖体RNA加工36(Rrp36,ENSRNOG00000017836)。90个GO术语与神经和轴突有关。最后,我们在三种外泌体中鉴定出13条常见的差异表达途径.希望能够提高BMSCs诱导分化为SCs的效率,给患者带来希望,为临床治疗带来更多选择。
BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) can differentiate into Schwann cells (SCs) during peripheral nerve injury; in our previous research, we showed that SC-derived exosomes (SC-exos) played a direct induction role while fibroblast-derived exosomes (Fb-exos) had no obvious induction role. The induction role of neural stem cell (NSC)-derived exosomes (NSC-exos) has also been widely confirmed. However, no studies have compared the induction effects of these three types of cells at the same time. Therefore, by investigating the effect of these three cell-derived exosomes upon the induction of BMSCs to differentiate into SCs, this study explored the role of different exosomes in promoting the differentiation of stem cells into SCs cells, and conducted a comparison between the two groups by RNA sequencing to further narrow the range of target genes and related gene pathways in order to study their related mechanisms.
METHODS: We extracted exosomes from SCs, fibroblasts (Fb) and neural stem cells (NSC) and then investigated the ability of these exosomes to induce differentiation into BMSCs under different culture conditions. The expression levels of key proteins and gene markers were detected in induced cells by fluorescence immunoassays, western blotting and polymerase chain reaction (PCR); then, we statistically compared the relative induction effects under different conditions. Finally, we analyzed the three types of exosomes by RNA-seq to predict target genes and related gene pathways.
RESULTS: BMSCs were cultured by three media: conventional (no
induction), pre-
induction or pre-
induction + original
induction medium (ODM) with exosomes of the same cell origin under different culture conditions. When adding the three different types of exosomes separately, the overall induction of BMSCs to differentiate into SCs was significantly increased (P < 0.05). The
induction ability was ranked as follows: pre-
induction + ODM + exosome group > pre-
induction + exosome group > non-
induction + exosome group. Using exosomes from different cell sources under the same culture conditions, we observed the following trends under the three culture conditions: RSC96-exos group ≥ NSC-exos group > Fb-exos group. The overall ability to induce BMSCs into SCs was significantly greater in the RSC96-exos group and the NSC-exos group. Although there was no significant difference in induction efficiency when comparing these two groups, the overall induction ability of the RSC96-exos group was slightly higher than that of the NSC-exos group. By combining the differentiation induction results with the RNA-seq data, the three types of exosomes were divided into three comparative groups: RSC vs. NSC, RSC vs. Fb and NSC vs. Fb. We identified 203 differentially expressed mRNA target genes in these three groups. Two differentially expressed genes were upregulated simultaneously, namely riboflavin kinase (RFK, ENSRNOG00000022273) and ribosomal RNA processing 36 (Rrp36, ENSRNOG00000017836). We did not identify any co-upregulated target genes for the miRNAs, but did identify one target gene of the lncRNAs, namely ENSRNOG00000065005. Analysis identified 90 GO terms related to nerves and axons in the mRNAs; in addition, KEGG enrichment and GASA analysis identified 13 common differential expression pathways in the three groups.
CONCLUSIONS: Our analysis found that pre-induction + ODM + RSC96/NSC-exos culture conditions were most conducive with regards to
induction and differentiation. RSC96-exos and NSC-exos exhibited significantly greater differentiation efficiency of BMSCs into SCs. Although there was no statistical difference, the data indicated a trend for RSC96-exos to be advantageous We identified 203 differentially expressed mRNAs between the three groups and two differentially expressed target mRNAs were upregulated, namely riboflavin kinase (RFK, ENSRNOG00000022273) and ribosomal RNA processing 36 (Rrp36, ENSRNOG00000017836). 90 GO terms were related to nerves and axons. Finally, we identified 13 common differentially expressed pathways across our three types of exosomes. It is hoped that the efficiency of BMSCs induction differentiation into SCs can be improved, bringing hope to patients and more options for clinical treatment.