Unknown,Transcriptomics,Genomics,Proteomics

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MiRNA expression profiles from neurogenic differentiation of mesenchymal stem cells under conditioned microenvironments

ABSTRACT: Background: Studies suggested that mesenchymal stem cells (MSCs) have intrinsic neurogenic potential and can be differentiated into neural stem cell/ neural progenitor cells (NPCs) under specific microenvironment. Manipulation of growth factors is one of the popular method to achieve trans-lineage differentiation of MSCs. Synergistic effect of epidermal growth factor (EGF) and fibroblast growth factor 2 (bFGF) have been widely identified as basic requirement for neural differentiation to take place. Insulin-like growth factor 1 (IGF-1) also known as somatomedin C is an important growth promoting protein during embryonic development which control numerous cellular responses and biological systems. Our recent study has found that the combination of EGF, bFGF and IGF-1 could significantly improved the growth and survivability of MSCs-derived NPCs. Therefore, to understand the genomic mechanism underlying the differentiation in vitro, we have studied the miRNAs profile of MSCs-derived NPCs under IGF-1 influenced conditioned microenvironments. Objectives: To evaluate the effects of IGF-1 in trans-lineage differentiation of MSCs, we have induced MSCs into neural lineage in 3 groups; Group A (positive control) - EGF+bFGF, Group B (Treatment) - EGF+bFGF+IGF-1, and Group C (negative control/ untreated). To unravel the role of regulatory miRNAs involved in the early differentiation, we have performed detailed miRNA profiling for MSCs-derived NPCs at three time intervals (day 1, day 3 and day 5). The data has explored crucial miRNAs involved in early differentiation of MSCs into NPCs. Stage specific MSCs-derived NPCs at Passage 1 were collected for total RNA extraction at three time-points (D1, D3 and D5) and hybridization on Affymetrix miRNA geneChip 2.0 arrays. Each experiment were repeated three times independently (Exp 1, Exp 2 and Exp 3). Group A served as positive control (EGF+bFGF), Group B as treatment (EGF+bFGF+IGF-1) and Group C without growth factor as negative control.

ORGANISM(S): Rattus norvegicus

SUBMITTER: Tee Huat

PROVIDER: E-GEOD-60060 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Purpose: The goal of this study is to unravel the differential gene expression profiles (RNA-Seq) during the early differentiation of bone marrow mesenchymal stem cells (BMSCs) into neural progenitor-like stem cells (NPCs) under the influence of different growth factor combinations using high-throughput RNA sequencing method; Group A: EGF and bFGF, Group B: EGF + bFGF and IGF-1, and Control: untreated BMSCs. Methodology: mRNA profiles of BMSCs (Control) and BMSCs-derived NPCs under the influence of growth factor combinations (Group A and Group B) were generated by deep sequencing, in triplicate, using Illumina NextSeq500. Raw reads were evaluated for quality checks in terms of sequencing quality and contamination. Reads were trimmed and filtered using BBDuk. Reads were then aligned to the latest reference genome (rn6) with GTF from Ensembl (v99) using STAR aligner. Aligned reads were transformed into read counts per gene using the RSEM tool. Pairwise differential expression analysis was performed using the DESeq2 R package. qRT–PCR validation was performed using the RT² Profiler PCR Array Rat Custom array (Array ID: CAPR13765E). Results: Using an optimized data analysis workflow, we mapped about 50 million sequence reads per sample to the Rattus norvegicus genome (Rnor_6.0 Assembly) and identified an average of 27,146 transcripts in Group A, 25,850 transcripts in Group B, and 21,683 transcripts in the control group. All transcripts have a fragment count estimation of at least 10 counts per gene. Differential analysis was performed as pairwise grouping. Comparison of Group A vs MSC identified 3,252 differentially expressed genes. Thirteen significantly expressed genes with the potential roles during neural differentiation were randomly selected for validation with qRT–PCR. RNA-seq data had a linear relationship with qRT–PCR. Hierarchical clustering of differentially expressed genes uncovered several as yet uncharacterized genes that may contribute to neural differentiation of mesenchymal stem cells. Data from the present study analysis revealed a significant overlap yet provided complementary insights in transcriptome profiling of mesenchymal stem cells derived neural progenitor-like cells. Conclusion: Our study represents the first detailed analysis of mesenchymal stem cells derived neural progenitor-like cell transcriptomes, generated by RNA-seq technology. Our data has explored crucial and novel genes involved during the early differentiation of mesenchymal stem cells into neural progenitor-like cells under the influence of different growth factor combinations.

Project description:Adult mouse subventricular zone (SVZ) neural progenitor cells (NPCs) retain the capacity to generate multiple lineages in vitro and in vivo. Thus far, the mechanisms involved in the regulation of these cells have not been well elucidated. We have carried out RNA profiling of adult SVZ cell cultures undergoing differentiation, to identify pathways that regulate progenitor cell proliferation and to define a set of transcripts that can be used as molecular tools in the drug discovery process. We carried out a stepwise stratification of the results to identify transcripts specifically enriched in NPCs and validated some of these using comparative literature analysis, quantitative PCR and immunological techniques. The results show a set of transcription factors, secreted molecules, and plasma membrane markers which are differentially regulated during differentiation. Pathway analysis highlights alterations in IGF, Wnt and TGFbeta signalling cascades. Further characterization of these components could provide greater insight into the mechanisms involved in the regulation of neurogenesis in the adult brain. Mouse subventricular zone neural stem cells cultures were prepared from C57Bl6/J mice (Johansson et al.,1999) and cultured in suspension in DFEM/F12 1:1 supplemented with PSF, B27 supplement and EGF/ FGF-2 at 20ng/ml. RNA was isolated from either expanding neurospheres in the presence of FGF2 and EGF (Reynolds and Weiss, 1992; Gritti et al., 1999), or from cultures induced to differentiate upon growth factor withdrawal or serum exposure, either after 24 hours (in suspension, to avoid changes induced by substrate attachment) or 5 days (as adherent cultures in poly-lysine and laminin-coated plates). Experiments were conducted in triplicate from independent batches of SVZ preparations. As a control for potential effects of media changes, a set of identical cultures were included where cells were grown for a further 24 hours in fresh media containing FGF2 and EGF (20 ng/ml each, normal growth media, NGM). Fluorescently labelled aRNA from individual samples were competitively hybridised against a pool of labelled aRNA from undifferentiated reference (starting) material on custom Agilent microarrays representing ~22,500 mouse transcripts. Technical, fluor-reversed hybridisations were performed for every sample.

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MiRNA expression profiles from neurogenic differentiation of mesenchymal stem cells under conditioned microenvironments

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