Khan Amir Ali, Huat Tee Jong, Al Mutery Abdullah, El-Serafi Ahmed Taher, Kacem Hassen Hadj, Abdallah Sallam Hasan, Reza Muhammed Faruque, Abdullah Jafri Malin, Jaafar Hasnan
Department of Applied Biology, College of Sciences, University of Sharjah, P.O. Box 27272, Emirate of Sharjah, United Arab Emirates.
Research Institute of Science and Engineering, University of Sharjah, P.O. Box 27272, Emirate of Sharjah, United Arab Emirates.
Cell Biosci. 2020 Oct 28;10:126. doi: 10.1186/s13578-020-00487-z. eCollection 2020.
Mesenchymal stem cells (MSCs) isolated from bone marrow have different developmental origins, including neural crest. MSCs can differentiate into neural progenitor-like cells (NPCs) under the influence of bFGF and EGF. NPCs can terminally differentiate into neurons that express beta-III-tubulin and elicit action potential. The main aim of the study was to identify key genetic markers involved in differentiation of MSCs into NPCs through transcriptomic analysis.
Total RNA was isolated from MSCs and MSCs-derived NPCs followed by cDNA library construction for transcriptomic analysis. Sample libraries that passed the quality and quantity assessments were subjected to high throughput mRNA sequencing using NextSeq®500. Differential gene expression analysis was performed using the DESeq2 R package with MSC samples being a reference group. The expression of eight differentially regulated genes was counter validated using real-time PCR.
In total, of the 3,252 differentially regulated genes between MSCs and NPCs with two or more folds, 1,771 were upregulated genes, whereas 1,481 were downregulated in NPCs. Amongst these differential genes, 104 transcription factors were upregulated, and 45 were downregulated in NPCs. Neurogenesis related genes were upregulated in NPCs and the main non-redundant gene ontology (GO) terms enriched in NPCs were the autonomic nervous system, cell surface receptor signalling pathways), extracellular structure organisation, and programmed cell death. The main non-redundant GO terms enriched in MSCs included cytoskeleton organisation cytoskeleton structural constituent, mitotic cell cycle), and the mitotic cell cycle process Gene set enrichment analysis also confirmed cell cycle regulated pathways as well as Biocarta integrin pathway were upregulated in MSCs. Transcription factors enrichment analysis by ChEA3 revealed Foxs1 and HEYL, amongst the top five transcription factors, inhibits and enhances, respectively, the NPCs differentiation of MSCs.
The vast differences in the transcriptomic profiles between NPCs and MSCs revealed a set of markers that can identify the differentiation stage of NPCs as well as provide new targets to enhance MSCs differentiation into NPCs.
从骨髓中分离出的间充质干细胞（MSC）具有不同的发育起源，包括神经嵴。在碱性成纤维细胞生长因子（bFGF）和表皮生长因子（EGF）的影响下，MSC可分化为神经祖细胞样细胞（NPC）。NPC可终末分化为表达β-III-微管蛋白并引发动作电位的神经元。本研究的主要目的是通过转录组分析确定参与MSC向NPC分化的关键遗传标记。
从MSC和MSC来源的NPC中分离总RNA，随后构建cDNA文库用于转录组分析。通过质量和数量评估的样本文库使用NextSeq®500进行高通量mRNA测序。使用DESeq2 R软件包进行差异基因表达分析，以MSC样本作为参考组。使用实时PCR对八个差异调节基因的表达进行反向验证。
在MSC和NPC之间总共3252个差异调节两倍或更多倍的基因中，1771个是上调基因，而在NPC中有1481个是下调基因。在这些差异基因中，104个转录因子在NPC中上调，45个下调。神经发生相关基因在NPC中上调，在NPC中富集的主要非冗余基因本体（GO）术语是自主神经系统、细胞表面受体信号通路、细胞外结构组织和程序性细胞死亡。在MSC中富集的主要非冗余GO术语包括细胞骨架组织、细胞骨架结构成分、有丝分裂细胞周期和有丝分裂细胞周期过程。基因集富集分析还证实细胞周期调节途径以及Biocarta整合素途径在MSC中上调。通过ChEA3进行的转录因子富集分析显示，在前五个转录因子中，Foxs1和HEYL分别抑制和增强MSC向NPC的分化。
NPC和MSC转录组图谱的巨大差异揭示了一组可识别NPC分化阶段的标记物，并为增强MSC向NPC的分化提供了新的靶点。
