IntroductionBone Stro-1 and negative biomarkers like CD14, CD34, and
IntroductionBone defects are an important concern in fields such as orthopedics, dentistry, maxilla-facial and plastic surgery. Mesenchymal stem cells (MSCs) can differentiate into osteocytes and are used for bone formation to treat these diseases. MSCs are the type of cells used for differentiation into myocytes, adipocytes, chondrocytes and osteocytes. MSCs are mainly extracted from bone marrow, umbilical cords, adipocytes and they are found in other tissues. Most of the cells express metabolites, proteins or activate transcription factors if they are under condition of differentiation and these elements can be tracked and recorded using different techniques to assess cell differentiation and can be regarded as biomarkers. Because most of these biomarkers are protein, proteomics can help us find or track these biomarkers in different cellular stages.Mass Spectrophotometry (MS) is a technique to detect analytes based on their mass to charge ratio with high precision. Detection of almost every biological molecule that can be ionized by MS makes this technique useful in differentiation assessment. Several methods are used for differentiation assessment of MSCs for osteogenic differentiation these methods are divided into protein, phosphorylation and metabolomic detection which is shown in Figure 1. Each method is briefly described in this article. Protein detectionProteins are suitable biomarkers for detection of the MSCs sources and for verification of osteogenic proteins expression in stem cells. Stem cells isolated from several parts of body show variation in differentiation potential and phenotype. Surface biomarkers like CD166, CD90, CD105 and Stro-1 and negative biomarkers like CD14, CD34, and CD45 can help us determine the source of MSCs (1, 2). Also for verification of osteogenic proteins expression, some factors like Runt-related transcription factor 2 (RUNX2) that sometimes referred to as the master of osteogenic differentiation are useful. RUNX2 is a crucial transcription factor for osteogenic mineralization and it’s expressed during the first stages of osteogenic differentiation. Alkaline phosphatase (ALP) which is responsible for extracellular matrix mineralization (both the mRNA or protein expression can be used), Osteopontin (OPN) as early differentiation marker and Osteocalcin (OCN) as late differentiation marker are also used in experiments (3).In an experiment by Graneli et al. 2014 MSCs were isolated from bone marrow and the proteins of the cells labeled with C14 during the differentiation by growing in suitable culture environment. (Figure 1) The surface proteins were isolated and sorted using Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). Protein’s relative expression was measured using mass-spectrophotometer. The method used in this experiment was Liquid Chromatography-Tandem Mass Spectrophotometry (LC-MS/MS). This method is a suitable way for detection of proteins. The solution of proteins is treated with proteases for making the protein fragments shorter. some enzymes like trypsin are usually used for this purpose. the protein solution is injected into the instrument and proteins are sorted in the first chamber of instrument and are separated and transferred to vacuum chamber between the first and second chamber one after other and they are broken in a way that the friction occurs just in one place in average so that the proteins can be sequenced or their relative expression can be measured easily in the second chamber which is based on M/Z ratio of the molecules (5).Figure 1. Methods for differentiation assessment using MASS spectrometry. 1- Cells are extracted from human 2- cells are cultured under effect of differentiation factors. depending on method sometimes the cells are labeled 3- dominant phenotypes are used as biomarkers 4- depending on method metabolites are extracted and detected using Mass spectrometry technics 5- further experiments like electrophoresis and sequencing or Flow cytometry are applied for more information or more assurance of differentiation. (CID) Collision Induced Dissociation, (MS/MS) Tandem Mass Spectrometry, (EI) Electrospray Phosphorylation DetectionA majority of cellular events are based on protein phosphorylation and phosphrylation is known as one of the classes of biomarkers (6). Studies show that some pathways like Sma and Mothers against Decapentaplegic (SMAD) have an impact on osteogenic differentiation trigger (7). some other pathways may also affect the osteogenic differentiation, for example, activation of signaling pathways like Hedgehog, Wnt, PTHr-P and BMPs causes the activation of osteogenic transcription factors and as result, the osteogenic differentiation proteins are expressed and the differentiation begins to start (7). One of the important group of proteins in bone differentiation is Bone Morphogenetic Proteins (BMPs) that are originally discovered by their potential to induce bone formation signals. The phosphorylation and dephosphorylation of SMADs affect the BMP signaling pathway by activation or deactivation of this pathway and as result, it controls the osteogenic differentiation. One of the methods for determination of new phosphorylated sites in a protein is phosphospecific antibodies and the utilization of radioisotopes which is a laborious task; but mass spectroscopy by detection of inorganic phosphate neutral loss through Collision Induced Dissociation (CID), originated from phosphoserine, phosphotyrosine and phosphothreonine, can help us determine new phosphorylation sites (8)(Figure 1). In CID technic the molecules injected to the instrument are accelerated to high kinetic energy and are broken down by collision with neutral molecules and are identified by a detector or by other MS method like MS/MS.(9)databases like MOSCOT can be used for Mass spectrometry. One of the methods for identification of enhanced express in a protein is Mascot Delta Score (nMD Score) and that’s subtraction of top two scores for proteins in the spectrum and dividing it by top score again. to prevent from errors, scores less than 0.4 are neglected (8). Other mass spectrometry databases like NIST, VOS and FFNSC are used for this purpose too. Proteins can be sequenced by technics like MS/MS and as result, with this approach, the transcription factors involved in osteogenic differentiation can be quantified and identified.Cell metabolomics detection This is based on fact that each differentiated cell has different metabolic products that can be used as biomarker to identify the type of differentiation (Figure 1). In terms of Osteogenic differentiation, it is found that in vitro differentiated cells showed some metabolic phenotypes like the metabolic shift to OXPHOS or production of dexamethasone during differentiation. Studies show that stem cells can differentiate in absence of osteogenic signals but for effective differentiation, stem cells require osteoinductive agents like dexamethasone (10). ConclusionOf the methods used for stem cell differentiation assessment with MS, metabolomics is the newest way compared to the others and more specific and accurate in some circumstances. Phosphorylation is a good way of tracking the cell stages but because many cellular signaling pathways are based on phosphorylation it may cause some errors. However, using membrane surface biomarker is an efficient way to identify or verify the cellular stages of stem cells and it is the most commonly used method for differentiation assessment.