Proteomic Profiling of Bone Marrow Mesenchymal Stem Cells upon Transforming Growth Factor β1 Stimulation

Wang, Daojing; Park, Jennifer S.; Chu, Julia; Krakowski, Ari; Luo, Kunxin; Chen, David J.; Li, Song

doi:10.1074/jbc.m407368200

Cited by 224 publications

(211 citation statements)

References 43 publications

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“…5 Because proteins presented in bone tissue are essential for all life processes of bone and are the most important final products of the information pathways, profiling those proteins is vital to understand the bone biology thoroughly. However, proteome research on bone is mainly focused on in vitro systems using bone cells, [6][7][8][9] such as osteoblasts and osteoclasts, to determine which proteins are expressed by a cell type under given experimental conditions, but they cannot establish what the actual protein profile of bone is. Recently, the extraction of proteins directly from bone for proteome analysis had been reported.…”

Section: Introductionmentioning

confidence: 99%

Method Development of Efficient Protein Extraction in Bone Tissue for Proteome Analysis

et al. 2007

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Exploring bone proteome is an important and challenging task for understanding the mechanisms of physiological/pathological process of bone tissue. However, classical methods of protein extraction for soft tissues and cells are not applicable for bone tissue. Therefore, method development of efficient protein extraction is critical for bone proteome analysis. We found in this study that the protein extraction efficiency was improved significantly when bone tissue was demineralized by hydrochloric acid (HCl). A sequential protein extraction method was developed for large-scale proteome analysis of bone tissue. The bone tissue was first demineralized by HCl solution and then extracted using three different lysis buffers. As large amounts of acid soluble proteins also presented in the HCl solution, besides collection of proteins in the extracted lysis buffers, the proteins in the demineralized HCl solution were also collected for proteome analysis. Automated 2D-LC-MS/MS analysis of the collected protein fractions resulted in the identification of 6202 unique peptides which matched 2479 unique proteins. The identified proteins revealed a broad diversity in the protein identity and function. More than 40 bone-specific proteins and 15 potential protein biomarkers previously reported were observed in this study. It was demonstrated that the developed extraction method of proteins in bone tissue, which was also the first large-scale proteomic study of bone, was very efficient for comprehensive analysis of bone proteome and might be helpful for clarifying the mechanisms of bone diseases.

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Section: Introductionmentioning

confidence: 99%

Method Development of Efficient Protein Extraction in Bone Tissue for Proteome Analysis

et al. 2007

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“…Human MSCs were acquired from Cambrex (East Rutherford, NJ) and cultured in MSC growth medium (Cambrex) for expansion without differentiation. After cell expansion, MSCs were subjected to flow cytometry analysis of surface marker expression (positive for CD105, CD166, CD29, and CD44 and negative for CD34, CD14, and CD45) to confirm the undifferentiated state of MSCs (28). Cells were kept in a humidified incubator at 37°C and supplemented with 5% CO 2 .…”

Section: Methodsmentioning

confidence: 99%

Anisotropic mechanosensing by mesenchymal stem cells

Kurpinski

Chu

Hashi

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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358

316

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Mesenchymal stem cells (MSCs) are a potential source for the construction of tissue-engineered vascular grafts. However, how vascular mechanical forces regulate the genetic reprogramming in MSCs is not well understood. Mechanical strain in the vascular wall is anisotropic and mainly in the circumferential direction. We have shown that cyclic uniaxial strain on elastic substrates causes the cells to align perpendicularly to the strain axis, which is different from that in the vascular wall. To simulate the vascular cell alignment and investigate the anisotropic mechanical sensing by MSCs, we used soft lithography to create elastomeric membranes with parallel microgrooves. This topographic pattern kept MSCs aligned parallel to the strain axis, and the cells were subjected to 5% cyclic uniaxial strain (1 Hz) for 2-4 days. DNA microarray analysis revealed global gene expression changes, including an increase in the smooth muscle marker calponin 1, decreases in cartilage matrix markers, and alterations in cell signaling (e.g., down-regulation of the Jagged1 signaling pathway). In addition, uniaxial strain increased MSC proliferation. However, when micropatterning was used to align cells perpendicularly to the axis of mechanical strain, the changes of some genes were diminished, and MSC proliferation was not affected. This study suggests that mechanical strain plays an important role in MSC differentiation and proliferation, and that the effects of mechanotransduction depend on the orientation of cells with respect to the strain axis. The differential cellular responses to the anisotropic mechanical environment have important implications in cardiovascular development, tissue remodeling, and tissue engineering. stem cell engineering ͉ differentiation ͉ genetic reprogramming ͉ uniaxial mechanical strain ͉ micropatterning

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“…TGF-b1 has been shown to upregulate the expression of alpha-smooth muscle actin (aSMA) in SMC 23 and MSC, 24,25 and RhoA is known to affect actin reorganization and contractile activity. 26 Myogenic differentiation was monitored by the aSMA promoter activity and confirmed by western blot and immunostaining.…”

Section: Introductionmentioning

confidence: 99%

A novel lentivirus for quantitative assessment of gene knockdown in stem cell differentiation

et al. 2012

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Loss of gene function is a valuable tool for screening genes in cellular processes including stem cell differentiation differentiation. However, the criteria for evaluating gene knockdown are usually based on end-point analysis and real-time, dynamic information is lacking. To overcome these limitations, we engineered a shRNA encoding LentiViral Dual Promoter vector (shLVDP) that enabled real-time monitoring of mesenchymal stem (MSC) differentiation and simultaneous gene knockdown. In this vector, the activity of the alpha-smooth muscle actin (aSMA) promoter was measured by the expression of a destabilized green fluorescent protein, and was used as an indicator of myogenic differentiation; constitutive expression of discosoma red fluorescent protein was used to measure transduction efficiency and to normalize aSMA promoter activity; and shRNA was encoded by a doxycycline (Dox)-regulatable H1 promoter. Importantly, the normalized promoter activity was independent of lentivirus titer allowing quantitative assessment of gene knockdown. Using this vector, we evaluated 11 genes in the TGF-b1 or Rho signaling pathway on SMC maturation and on MSC differentiation along the myogenic lineage. As expected, knockdown of genes such as Smad2/3 or RhoA inhibited myogenic differentiation, while knocking down the myogenic differentiation inhibitor, Klf4, increased aSMA promoter activity significantly. Notably, some genes for example, Smad7 or KLF4 showed differential regulation of myogenic differentiation in MSC from different anatomic locations such as bone marrow and hair follicles. Finally, Dox-regulatable shRNA expression enabled temporal control of gene knockdown and provided dynamic information on the effect of different genes on myogenic phenotype. Our data suggests that shLVDP may be ideal for development of lentiviral microarrays to decipher gene regulatory networks of complex biological processes such as stem cell differentiation or reprogramming.

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Proteomic Profiling of Bone Marrow Mesenchymal Stem Cells upon Transforming Growth Factor β1 Stimulation

Cited by 224 publications

References 43 publications

Method Development of Efficient Protein Extraction in Bone Tissue for Proteome Analysis

Method Development of Efficient Protein Extraction in Bone Tissue for Proteome Analysis

Anisotropic mechanosensing by mesenchymal stem cells

A novel lentivirus for quantitative assessment of gene knockdown in stem cell differentiation

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