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LC-MS/MS of Extracellular Vesicles from Pluripotent Stem Cells and Pluripotent-derived-Mesenchymal Stem Cell

ABSTRACT: Mesenchymal Stem/Stromal Cells (MSC) obtained from Pluripotent Stem Cells (PSC) constitute an interesting alternative to classical MSC in regenerative medicine. Amongst one of their many mechanism of action, MSC extracellular vesicles (EVs) raise as a suitable substitution of MSC in future cell-free based therapeutics approaches. EVs, unlike cells, do not elicit acute immune rejection and can be produced in large quantities and stored ready to use, until needed. Though therapeutic potential of MSC-EVs had already been proved, thorough characterization of them is still scarce. In this work we performed a label-free Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) proteomic approach to identify the most abundant proteins in EVs secreted from MSC derived from PSC (PD-MSC) and from their parental iPSC. Next, we compared both datasets finding that while iPSC EVs enclose proteins that modulates RNA and miRNA stability and protein sorting, PD-MSC EVs are rich in proteins that organize extracellular matrix, regulate locomotion and influence cell-substrate adhesion. Moreover, when compared to their respective cells, iPSC and iPSC EVs share a greater proportion of proteins while PD-MSC proteome seems to be more specific. Correlation and Principal Component Analysis consistently aggregate iPSC and iPSC EVs but segregate PD-MSC and their EVs. Altogether these findings suggest that during differentiation, PD-MSC EVs acquire a more specific set of proteins than their parental iPSC�s EVs have and that, arguably, this might confer them their therapeutic properties.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Carlos Luzzani

PROVIDER: MSV000081933 | MassIVE | Sat Jan 13 07:04:00 GMT 2018

SECONDARY ACCESSION(S): PXD008669

REPOSITORIES: MassIVE

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Project description:Induced pluripotent stem cells (iPSCs) hold great promise for in vitro disease modeling and cell replacement therapy for Parkinson’s disease (PD). Both applications crucially require an in-depth profiling of the disease-relevant, iPSC-derived cell type. Midbrain dopaminergic (mDA) neurons derived from pluripotent stem cells are of substantial interest because of their instrumental value for PD therapy. IPSC-derived mDA neuron-like cells have been generated, however, detailed genetic and epigenetic characterization of strictly purified in vitro generated DA neurons has so far lagged behind. We generated mouse Pitx3gfp/+ iPSC-derived DA neurons that, after fluorescent activated cell sorting (FACS) allowed comprehensive comparison to mesodiencephalic dopaminergic (mdDA) neurons from Fac-sorted Pitx3gfp/+ ventral midbrains. We performed detailed analysis of global gene expression and genome-scale DNA methylation of CpG islands (CGIs) by reduced representation bisulfite sequencing. The reprogramming pathway from fibroblasts to iPSCs left parental cell footprints for both gene expression and DNA methylation. However, most gene expression patterns of iPSC-derived DA neurons closely resembled that of primary mdDA neurons with the strongest correlations for mdDA specific genes. Also, for DNA methylation patterns, high similarities were found for the vast majority of CGIs when comparing primary mdDA neurons with iPSC-derived DA neurons. Additionally, we found de novo DNA methylation during in vitro differentiation for hundreds of genes specifically in lineage-committed neural precursors that persisted in iPSC-derived DA neurons. Our study provides novel detailed characteristics of iPSC-derived DA neurons in comparison to the primary cell type. These findings add important information to our knowledge about these biomedically highly valuable, in vitro generated neurons. Microarray expression study comparing 3 samples of facs-sorted, Pitx3-gfp positive cells from each experimental group to a common reference consisting of adult mouse midbrain RNA. Each sample was analysed in normal and opposite dye orientation.

Project description:Background: Extracellular vesicles (EVs) isolated from mesenchymal stem/stromal cells (MSCs) contribute to recovery of damaged tissue in animals models of human disease. We have previously shown that EVs isolated from porcine MSCs transport mRNA and miRNA capable of modulating several cellular pathways in recipient cells, yet their proteome remained to be profiled. Using a quantitative proteomic strategy, we sought to study the protein cargo of porcine MSC-derived EVs to identify candidate molecules for mediating their therapeutic effect. Methods: Autologous MSCs were collected from abdominal fat of 3 female domestic pigs, and MSC-derived EVs were subsequently isolated, cultured, and characterized by the expression of typical MSC and EV markers. LC-MS/MS proteomic analysis was performed and proteins classified using the Panther Classification System. Functional pathway analysis was performed with DAVID 6.7. Three candidate proteins were selected for validation and their expression in EVs and MSCs confirmed by Western blot. Results: Proteomics analysis identified 5,469 protein groups in MSCs and 4,937 in EVs. Average protein intensity was higher in MSCs compared to EVs (p<0.0001). Differential expression analysis revealed 128 proteins upregulated in EVs vs. MSCs (log2 fold change>10, p<0.05), whereas 563 proteins were excluded from EVs (log2 fold change<-10, p<0.05). Biological functional analysis of proteins enriched in EVs indicated a broad distribution, with the most frequently represented categories being proteins involved in angiogenesis, blood coagulation, apoptosis, extracellular matrix remodeling, and regulation of inflammatory responses. Proteins excluded from EVs were mostly nuclear proteins and proteins involved in nucleotide binding and RNA splicing. Conclusions: The present study provides novel proteomic characterization of the biological signatures of porcine adipose MSC-derived EVs. The selective cargo that EVs shuttle may define the spectrum of their roles in mediating MSC intercellular communication.

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LC-MS/MS of Extracellular Vesicles from Pluripotent Stem Cells and Pluripotent-derived-Mesenchymal Stem Cell

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