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Quantitative Analysis of Differential Proteomes in Mouse Mesenchymal Stem Cells Converted to Salivary Epithelial Precursors
Severe hyposalivation often results from Sjögren’s syndrome or radiation therapy for head and neck cancer. Devastating consequences of hyposalivation, such as rampant dental caries and persistent oral candidiasis, compromise the quality of life in those patients. Clinical management for dry mouth typically involves simple palliative methods or secretagogues, which are designed to stimulate saliva secretion from residual salivary acinar cells present in the glands. However, direct interventions in chronic dryness have yet to be employed in the clinical setting. Despite numerous studies on salivary gland regeneration, the molecular basis governing stem cell transdifferentiation into salivary epithelial precursors (SEP) is largely unknown. Our previously published study clearly indicated mouse bone marrow-derived mesenchymal stem cells (BM-MSCs) can be differentiated into SEP in vitro when co-cultured with isolated primary salivary gland cells without cell-to-cell contact. Our current study utilized iTRAQ-LS-MS/MS-based quantitative proteomics to profile key regulatory factors involved in mouse MSC-to-SEP conversion. We identified 280 differentially expressed proteins in BM-MSCs over the course of 7 days of co-culture. Interestingly, protein expression of salivary transcription factors (STFs), such as transcription factor E2a (TCF3), high mobility group protein 20B (HMG20b), and ankyrin repeat domain-containing protein 56 (ANKRD56) were increased in a time-dependent manner. Notably, pancreas specific transcription factor 1a (PTF1α), muscle, intestine and stomach expression-1 (MIST-1) and achaete-scute complex homolog 3(ASCL3) were newly induced over time in differentiated MSCs. We also identified the expression of Ptf1α in the mouse salivary glands for the first time and verified its expression in independent batches of co-cultured MSCs by western blotting. Furthermore, simulation of the molecular network involving the identified STFs has demonstrated evidence for their perspective roles in salivary gland development during glandular maturation. Thus, our study provides the first extensive proteomic profile of MSC-to-SEP transdifferentiation and identifies novel STFs that may be critical for this process.
Sample Processing Protocol
All co-culture experiments were conducted in 6- or 24-well plates containing a 0.4 µm pore size polycarbonate membrane-based transwell insert (Millipore Millicell cell culture inserts, EMD Millipore). mMSCs (1.0×104 cells/cm2) were seeded on the collagen-coated lower chamber of the cell culture plate and incubated in Hepato-STIM media without serum for 12hr prior to co-culture experiments. Once mMSCs were well attached to the bottom of the plate, isolated pSGCs (6×104 cells/cm2) were seeded onto the membrane of the upper transwell insert. Cells in the co-culture system were maintained at 37°C and 5% CO2 in a humidified atmosphere without replacing the media and harvested at 1, 3, 5, or 7 days. Control mMSCs were cultured for each time point without pSGCs. Three biological replicates of control mMSCs and co-cultured MSCs that are identified expression of marker proteins and morphological changed were prepared for proteomics experiments. Protein lysates were extracted and quantified following a previous method (13). Protein assays were performed to quantify purified proteins by the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Inc., Rockford, IL, USA) with the SoftMax Pro Software v5.3 (Molecular Devices, LLC. Sunnyvale, CA, USA) under the SpectraMax M5 (Molecular Devices, LLC). For each sample, 100μg protein was dissolved in a dissolution buffer (AB Sciex,CIEX Inc., Foster City, CA, USA).
Data Processing Protocol
The samples were reduced, alkylated, trypsin-digested, and labeled following the manufacturer’s instructions for the iTRAQ Reagents 8-plex kit (AB Sciex, Inc.CIEX). Control mMSCs and each co-cultured mMSCs from three biological replicates were labeled with iTRAQ tags including tag swap. The iTRAQ-labeled peptide mixtures were desalted with C18-solid phase extraction (The Next Group, Inc., Southborough, MA, USA) and fractionated with a strong cation exchange (SCX) chromatography using a polysulfoethyl A column (2.1 × 100 mm, 5 µm, 300 Å; PolyLC, Inc., Columbia, MD, USA) including solvent A (25% (v/v) acetonitrile, 10mM ammonium formate, and 0.1% (v/v) formic acid (pH 2.8). Peptides were eluted with a flow rate of 200 µl/min at a linear gradient of 0–20% solvent B (25% (v/v) acetonitrile and 500 mM ammonium formate (pH 6.8) over 50 min followed by ramping up to 100% solvent B in 5 min. Sixteen fractions were collected by monitoring the absorbance at 280nm and lyophilized. A quadrupole time-of-flight (LTQ Orbitrap XL) MS system (Thermo Fisher Scientific, Bremen, GermanyInc.) was applied as described previously (14). It was interfaced with an Eksigent nano-LC AS2 system (Eksigent Technologies, LLC, ) using high energy collision dissociation (HCD). Each fraction was loaded onto an Agilent Zorbax 300SB-C18 trap column (0.3 mm id × 5 mm length, 5 µm particle size) with a flow rate of 5 µl/min for 10 min. Reversed-phase C18 chromatographic separation of peptides was carried out on a pre-packed BetaBasic C18 PicoFrit column (75 µm id × 10 cm length, New Objective, Inc.) at 300 nl/min using the following gradient: 5% B for 1 min as an equilibration status; 60% B for 99 min as a gradient; 90% B for 5 min as a washing status; 5% B for 10 min as an equilibration status (solvent A: 0.1% formic acid in 97% water, 3% ACN; solvent B: 0.1% formic acid in 97% ACN, 3% water).
Park YJ, Koh J, Kwon JT, Park YS, Yang L, Cha S. Uncovering stem cell differentiation factors for salivary gland regeneration by quantitative analysis of differential proteomes. PLoS One. 2017 Feb 3;12(2):e0169677 PubMed: 28158262
|#||Accession||Title||Proteins||Peptides||Unique Peptides||Spectra||Identified Spectra||View in Reactome|
|1||56840||no assay title provided (mzIdentML)||2||13||3||2055||10||
|2||56868||no assay title provided (mzIdentML)||480||5539||632||8365||2241||
|3||56841||no assay title provided (mzIdentML)||464||5907||610||8436||2252||
|4||56867||no assay title provided (mzIdentML)||209||2939||292||6903||1294||
|5||56842||no assay title provided (mzIdentML)||368||4247||514||7807||1912||
|6||56843||no assay title provided (mzIdentML)||77||1168||118||5279||610||
|7||56844||no assay title provided (mzIdentML)||73||473||83||3806||270||
|8||56864||no assay title provided (mzIdentML)||15||73||19||0||0||
|9||56845||no assay title provided (mzIdentML)||529||3722||801||7849||1911||
|10||56863||no assay title provided (mzIdentML)||692||5731||1064||8791||2796||