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Next Generation Sequencing (RNA-Seq) of stem cell-treated rats for quantitative analyses of retinal transcriptomes

ABSTRACT: Mesenchymal stem cell (MSC) heterogeneity can lead to variable treatment outcomes. Genetically modifying MSCs to express erythropoietin could confer additional therapeutic benefits that may enhance standalone MSC treatments. The goals of this study are to compare NGS-derived retinal transcriptome profiling (RNA-seq) between healthy, retinal degenerated, and stem cell-treated rat retinal degeneration models for high-throughput data analysis. MSC, Wharton's jelly Mesecnhymal stem cells; MSCEPO, EPO-expressing MSC; DPSC, dental pulp MSC stem cells Methods: Retinal mRNA profiles of 31-day-old healthy, sodium iodate-administered (retinotoxic inducer), DPSC-treated, MSC-treated, and MSCEPO-treated Sprague Dawley rats were generated by deep sequencing, in triplicate, using Illumina Nextseq 500. The sequence reads that passed quality filters were analyzed at the transcript isoform level with Salmon, and then converted to the gene level count matrices for quantification with DESeq2. The quant assay kit was used for qPCR amplification. Results: Using the Salmon-DESeq2 pipeline, we mapped about 25-35 million sequence reads per sample to the rat genome (build Rnor 6.0) and identified 20,376 transcripts in the retinas of the control and experimental groups. Approximately 500-1000 genes were found to be differentially expressed using the arbitrary cutoff of Padj<.05 and fold change>±2.0. Hierarchical clustering of differentially expressed genes revealed profiles involved in cellular regeneration,cell death, and phototransduction, which indicated the therapeutic effectiveness of the stem cells on retinal survival. Gene set enrichment analysis and pathway topology analysis showed enrichment in the aforementioned processes, Padj<.05. Conclusions: Our study represents the first detailed analysis of MSCEPO-treated retinal transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for further investigations in MSC enhancement methods. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within the retina. We conclude that RNA-seq based retinal transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions, especially in regards to MSC-mediated tissue repair and phototransduction.

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE164152 | GEO | 2021/04/27

REPOSITORIES: GEO

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Project description:Primary cortical neurons were isolated from E15 mice and after 5 days in vitro were untreated or treated for 24 h with mesenchymal stem cell conditioned medium and then untreated or treated for a further 24 h with NMDA. Neuron gene expression was profiled and compared between the four different conditions (neurons, neurons+MSC cm, neurons+NMDA, neurons+MSC cm+NMDA) to investigate the molecular mechanisms of MSC neuroprotection. Mesenchymal stem cells (MSC) promote functional recovery in experimental models of central nervous system (CNS) pathology and are currently being tested in clinical trials for stroke, multiple sclerosis and CNS injury. Their beneficial effects are attributed to activation of endogenous CNS repair processes and immune regulation but their mechanisms of action are poorly understood. Here we investigated the neuroprotective effects of MSC in simplified MSC-neuron co-culture systems and in mice using models of glutamate excitotoxicity. MSC protected primary cortical neurons against glutamate (NMDA) receptor-induced death and conditioned medium from MSC (MSC cm), but not control NIH3T3 cells, was sufficient for this effect. MSC cm neuroprotection in mouse cortical neurons was reduced by neutralizing antibodies to bFGF and associated with altered gene expression in neurons towards an immature phenotype as well as reduced neuronal Grin1, Grin2a and Grin2b mRNA levels in response to NMDA stimulation. Further, MSC cm neuroprotection in rat retinal ganglion cells was associated with absence of glutamate-induced calcium influx. Adoptive transfer of EGFP+MSC in a mouse kainic acid seizure model reduced CA3 neuron damage and hippocampal astrocytosis and resulted in the increased expression of neuronal genes that are upregulated by MSC cm, Bmi1, Ddx4, Ezh1, in the hippocampus. These results show that MSC mediate direct neuroprotection against glutamate excitotoxicity by secreting bFGF, reducing glutamate receptor expression and function and altering neuron gene expression towards an immature pattern, and provide evidence for a link between the therapeutic effects of MSC and the activation of endogenous repair processes following CNS injury. In vitro cultures primary cortical neurons from mice were protected from glutamate excitotoxicity when pre-treated with MSC cm. Global gene expression changes induced in neurons before and after treatment with MSC cm and/or NMDA were investigated using a cDNA spotted macroarray filter. Four samples were analysed in duplicate: neurons alone (untreated), neurons+MSC cm, neurons+NMDA, neurons+MSC cm+NMDA.

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Next Generation Sequencing (RNA-Seq) of stem cell-treated rats for quantitative analyses of retinal transcriptomes

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