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Cell surface proteomics analysis indicates a neural lineage bias of rat bone marrow mesenchymal stromal cells.

ABSTRACT: Mesenchymal stromal cells (MSCs) are one of the most intensively studied stem cell types with application aims. However, the molecular characterisation and the relationship between the molecular characterisation and functional properties of MSCs are largely unknown. In this study, we purified the surface proteins from rat bone marrow MSCs (rBMMSCs) and characterised their surface proteome by LC-MS/MS. Moreover, we comparatively analysed the data from this study with the surface proteomics data of mouse and human embryonic stem (ES) cells and human mesenchymal stromal cells (hMSCs). The data showed that, in contrast to ES cells and human mesenchymal stromal cells, rBMMSCs possessed a surface proteomics pattern biased to neural and neural-endocrine lineages, indicating a neural/neural crest bias, and suggested a neural differentiation tendency of these cells. The different surface proteomics pattern between rBMMSCs and hMSCs also suggested that MSCs of different origin might possess a different lineage bias.

SUBMITTER: Zhao G

PROVIDER: S-EPMC3914342 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Cell surface proteomics analysis indicates a neural lineage bias of rat bone marrow mesenchymal stromal cells.

Zhao Guiyun G Ji Huijiao H Wang Shihao S Gu Bin B Gu Bin B Song Xiuli X Zhang Jiarong J Liu Yukan Y Chen Liangbiao L Zhang Ming M

BioMed research international 20140116

Mesenchymal stromal cells (MSCs) are one of the most intensively studied stem cell types with application aims. However, the molecular characterisation and the relationship between the molecular characterisation and functional properties of MSCs are largely unknown. In this study, we purified the surface proteins from rat bone marrow MSCs (rBMMSCs) and characterised their surface proteome by LC-MS/MS. Moreover, we comparatively analysed the data from this study with the surface proteomics data o ...[more]

PMID: 24551847

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Project description:Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are dynamic cells that can sense the environment, adapting their regulatory functions to different conditions. Accordingly, the therapeutic potential of BM-MSCs can be modulated by preconditioning strategies aimed at modifying their paracrine action. Although rat BM-MSCs (rBM-MSCs) have been widely tested in preclinical research, most preconditioning studies have employed human and mouse BM-MSCs. Herein, we investigated whether rBM-MSCs modify their phenotype and paracrine functions in response to Toll-like receptor (TLR) agonists. The data showed that rBM-MSCs expressed TLR3, TLR4, and MDA5 mRNA and were able to internalize polyinosinic-polycytidylic acid (Poly(I:C)), a TLR3/MDA5 agonist. rBM-MSCs were then stimulated with Poly(I:C) or with lipopolysaccharide (LPS, a TLR4 agonist) for 1?h and were grown under normal culture conditions. LPS or Poly(I:C) stimulation did not affect the viability or the morphology of rBM-MSCs and did not modify the expression pattern of key cell surface markers. Poly(I:C) did not induce statistically significant changes in the release of several inflammatory mediators and VEGF by rBM-MSCs, although it tended to increase IL-6 and MCP-1 secretion, whereas LPS increased the release of IL-6, MCP-1, and VEGF, three factors that were constitutively secreted by unstimulated cells. The neurotrophic activity of the conditioned medium from unstimulated and LPS-preconditioned rBM-MSCs was investigated using dorsal root ganglion explants, showing that soluble factors produced by unstimulated and LPS-preconditioned rBM-MSCs can stimulate neurite outgrowth similarly, in a VEGF-dependent manner. LPS-preconditioned cells, however, were slightly more efficient in increasing the number of regrowing axons in a model of sciatic nerve transection in rats. In conclusion, LPS preconditioning boosted the production of constitutively secreted factors by rBM-MSCs, without changing their mesenchymal identity, an effect that requires further investigation in exploratory preclinical studies.

Project description:BackgroundClinical applications have shown extracellular vesicles (EVs) to be a major paracrine effector in therapeutic responses produced by human mesenchymal stromal/stem cells (hMSCs). As the regenerative capacity of EVs is mainly ascribed to the transfer of proteins and RNA composing its cargo, and to the activity attributed by the protein surface markers, we sought to profile the protein composition of small EVs released from hMSCs to identify hMSC-EV biomarkers with potential clinical relevance.MethodsSmall EVs were produced and qualified from five human bone marrow MSC donors at low passage following a 48-h culture in exosome-depleted medium further processed by steps of centrifugation, filtration, and precipitation. Quantitative proteomic analysis comparing the protein profile of the EVs released from hMSCs and their parental cell was conducted using tandem mass tag labeling combined to mass spectrometry (LC-MS/MS) to identify enriched EV protein markers.ResultsNanoparticle tracking analysis showed no differences in the EV concentration and size among the five hMSC donors (1.83 × 1010 ± 3.23 × 109/mL), with the mode particle size measuring at 109.3 ± 5.7 nm. Transmission electron microscopy confirmed the presence of nanovesicles with bilayer membranes. Flow cytometric analysis identified commonly found exosomal (CD63/CD81) and hMSC (CD105/CD44/CD146) markers from released EVs in addition to surface mediators of migration (CD29 and MCSP). Quantitative proteomic identified 270 proteins significantly enriched by at least twofold in EVs released from hMSCs as compared to parental hMSCs, where neuropilin 1 (NRP1) was identified among 21 membrane-bound proteins regulating the migration and invasion of cells, as well as chemotaxis and vasculogenesis. Validation by western blot of multiple batches of EVs confirmed consistent enrichment of NRP1 in the nanovesicles released from all five hMSC donors.ConclusionThe identification and verification of NRP1 as a novel enriched surface marker from multiple batches of EVs derived from multiple hMSC donors may serve as a biomarker for the assessment and measurement of EVs for therapeutic uses.

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Cell surface proteomics analysis indicates a neural lineage bias of rat bone marrow mesenchymal stromal cells.

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Cell surface proteomics analysis indicates a neural lineage bias of rat bone marrow mesenchymal stromal cells.

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