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      Visualization of Src and FAK Activity during the Differentiation Process from HMSCs to Osteoblasts

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          Abstract

          Non-receptor protein kinases FAK and Src play crucial roles in regulating cellular adhesions, growth, migration and differentiation. However, it remains unclear how the activity of FAK and Src is regulated during the differentiation process from mesenchymal stem cells (MSCs) to bone cells. In this study, we used genetically encoded FAK and Src biosensors based on fluorescence resonance energy transfer (FRET) to monitor the FAK and Src activity in live cells during the differentiation process. The results revealed that the FAK activity increased after the induction of differentiation, which peaked around 20–27 days after induction. Meanwhile, the Src activity decreased continuously for 27 days after induction. Therefore, the results showed significant and differential changes of FAK and Src activity upon induction. This opposite trend between FAK and Src activation suggests novel and un-coupled Src/FAK functions during the osteoblastic differentiation process. These results should provide important information for the biochemical signals during the differentiation process of stem cells toward bone cells, which will advance our understanding of bone repair and tissue engineering.

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          Most cited references 46

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          Multilineage potential of adult human mesenchymal stem cells.

          Human mesenchymal stem cells are thought to be multipotent cells, which are present in adult marrow, that can replicate as undifferentiated cells and that have the potential to differentiate to lineages of mesenchymal tissues, including bone, cartilage, fat, tendon, muscle, and marrow stroma. Cells that have the characteristics of human mesenchymal stem cells were isolated from marrow aspirates of volunteer donors. These cells displayed a stable phenotype and remained as a monolayer in vitro. These adult stem cells could be induced to differentiate exclusively into the adipocytic, chondrocytic, or osteocytic lineages. Individual stem cells were identified that, when expanded to colonies, retained their multilineage potential.
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            Matrix elasticity directs stem cell lineage specification.

            Microenvironments appear important in stem cell lineage specification but can be difficult to adequately characterize or control with soft tissues. Naive mesenchymal stem cells (MSCs) are shown here to specify lineage and commit to phenotypes with extreme sensitivity to tissue-level elasticity. Soft matrices that mimic brain are neurogenic, stiffer matrices that mimic muscle are myogenic, and comparatively rigid matrices that mimic collagenous bone prove osteogenic. During the initial week in culture, reprogramming of these lineages is possible with addition of soluble induction factors, but after several weeks in culture, the cells commit to the lineage specified by matrix elasticity, consistent with the elasticity-insensitive commitment of differentiated cell types. Inhibition of nonmuscle myosin II blocks all elasticity-directed lineage specification-without strongly perturbing many other aspects of cell function and shape. The results have significant implications for understanding physical effects of the in vivo microenvironment and also for therapeutic uses of stem cells.
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              Human adipose tissue is a source of multipotent stem cells.

              Much of the work conducted on adult stem cells has focused on mesenchymal stem cells (MSCs) found within the bone marrow stroma. Adipose tissue, like bone marrow, is derived from the embryonic mesenchyme and contains a stroma that is easily isolated. Preliminary studies have recently identified a putative stem cell population within the adipose stromal compartment. This cell population, termed processed lipoaspirate (PLA) cells, can be isolated from human lipoaspirates and, like MSCs, differentiate toward the osteogenic, adipogenic, myogenic, and chondrogenic lineages. To confirm whether adipose tissue contains stem cells, the PLA population and multiple clonal isolates were analyzed using several molecular and biochemical approaches. PLA cells expressed multiple CD marker antigens similar to those observed on MSCs. Mesodermal lineage induction of PLA cells and clones resulted in the expression of multiple lineage-specific genes and proteins. Furthermore, biochemical analysis also confirmed lineage-specific activity. In addition to mesodermal capacity, PLA cells and clones differentiated into putative neurogenic cells, exhibiting a neuronal-like morphology and expressing several proteins consistent with the neuronal phenotype. Finally, PLA cells exhibited unique characteristics distinct from those seen in MSCs, including differences in CD marker profile and gene expression.
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                Author and article information

                Affiliations
                [1 ]Biomaterials and Live Cell Imaging Institute, School of Metallurgy and Materials Engineering, Chongqing University of Science and technology, Chongqing, People’s Republic of China
                [2 ]Department of Bioengineering, University of Illinois, Urbana-Champaign, Urbana, Illinois, United States of America
                [3 ]Beckman Institute for Advanced Science and Technology, Center for Biophysics and Computational Biology, Institute for Genomic Biology, Department of Integrative and Molecular Physiology, University of Illinois, Urbana-Champaign, Urbana, Illinois, United States of America
                National Centre for Scientific Research, ‘Demokritos’, Greece
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: XL SL YW. Performed the experiments: XL. Analyzed the data: XL SL YZ CW WX YW. Contributed reagents/materials/analysis tools: YW. Wrote the paper: XL SL YZ CW WX YW.

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2012
                10 August 2012
                : 7
                : 8
                3416797
                22900044
                PONE-D-12-06619
                10.1371/journal.pone.0042709
                (Editor)

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Counts
                Pages: 7
                Funding
                This work was supported in part by grants from NIH HL098472, NS063405, NSF CBET0846429 (Y.W., S.L. and C.W.), Research Foundation of University of Illinois (S.L.) and the Natural Science Key Foundation Project of CQ in China (CSTC2012JJB0097), and Research Foundation of Chongqing University of Science & Technology (CK2010B18, CK2011Z03) (X.L., W.X.). Y.Z. was supported by the Beckman Graduate Fellowship.
                Categories
                Research Article
                Biology
                Anatomy and Physiology
                Musculoskeletal System
                Bone
                Biotechnology
                Tissue Engineering
                Developmental Biology
                Stem Cells
                Mesenchymal Stem Cells
                Cell Differentiation
                Molecular Cell Biology
                Signal Transduction
                Signaling Cascades
                Protein Kinase Signaling Cascade

                Uncategorized

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