Guanbin Song

RhoA/ROCK, cytoskeletal dynamics, and focal adhesion kinase are required for mechanical stretch-induced tenogenic differentiation of human mesenchymal stem cells.

Human bone marrow mesenchymal stem cells (hMSCs) have the potential to differentiate into tendon/ligament‐like lineages when they are subjected to mechanical stretching. However, the means through which mechanical stretch regulates the tenogenic differentiation of hMSCs remains unclear. This study examined the role of RhoA/ROCK, cytoskeletal organization, and focal adhesion kinase (FAK) in mechanical stretch‐induced tenogenic differentiation characterized by the up‐regulation of tendon‐related marker gene expression. Our findings showed that RhoA/ROCK and FAK regulated mechanical stretch‐induced realignment of hMSCs by regulating cytoskeletal organization and that RhoA/ROCK and cytoskeletal organization were essential to mechanical stretch‐activated FAK phosphorylation at Tyr397. We also demonstrated that this process can be blocked by Y‐27632 (a specific inhibitor of RhoA/ROCK), cytochalasin D (an inhibitor of cytoskeletal organization) or PF 573228 (a specific inhibitor of FAK). The results of this study suggest that RhoA/ROCK, cytoskeletal organization, and FAK compose a “signaling network” that senses mechanical stretching and drives mechanical stretch‐induced tenogenic differentiation of hMSCs. This work provides novel insights regarding the mechanisms of tenogenesis in a stretch‐induced environment and supports the therapeutic potential of hMSCs. J. Cell. Physiol. 227: 2722–2729, 2012.

Low-Level Shear Stress Induces Human Mesenchymal Stem Cell Migration Through the SDF-1/CXCR4 Axis Via MAPK Signaling Pathways

Mesenchymal stem cells (MSCs) are able to home and migrate into damaged tissues and are thus, considered an optimal therapeutic strategy for clinical use. We previously demonstrated that higher shear stress (>2 Pa) hindered human MSC (hMSC) migration, whereas lower shear stress (0.2 Pa) induced cell migration through mitogen-activated protein kinase (MAPK) pathways. Here the mechanisms underlying shear stress-induced hMSC migration have been studied further. An MSC monolayer was mechanically wounded and subsequently exposed to low-level shear stress of 0.2 Pa. Image analysis was performed to quantify cell migration speeds under both flow and static conditions. hMSCs along both upstream- and downstream edges of the wound migrated at a similar speed to cover the wounded area under static conditions, whereas shear stress induced cells along the downstream edge of the wound to migrate significantly faster than those along the upstream edge. We also found that shear stress upregulated the secretion of stromal-derived factor-1 (SDF-1), which stimulated its receptor CXCR4 expression in hMSCs until the cells covered the wounded area. A CXCR4 antagonist repressed both cell migration and activation of c-Jun N-terminal kinase (JNK) and p38 MAPK but did not affect extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. When MAPK activation in upstream- and downstream hMSCs was evaluated separately, ERK1/2 was activated earlier in downstream than in upstream cells. These results indicate that the SDF-1/CXCR4 axis mediates shear stress-induced hMSC migration through JNK and p38 MAPK pathways and that the difference in migration speeds between upstream- and downstream cells may be due to ERK1/2 activation.

Osteopontin Promotes Mesenchymal Stem Cell Migration and Lessens Cell Stiffness via Integrin β1, FAK, and ERK Pathways

The use of mesenchymal stem cells (MSCs) for therapeutic applications has attracted great attention because MSCs home to and engraft to injured tissues after in vivo administration. The expression of osteopontin (OPN) is elevated in response to injury and inflammation, and its role on rat bone marrow-derived mesenchymal stem cells (rMSCs)-directed migration has been elucidated. However, the signaling pathways through the activation of which OPN promotes rMSCs migration and the involvement of cell mechanics during OPN-mediating rMSCs migration have not been well studied. In this study, we found that OPN activated focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK) signaling pathways by the ligation of integrin β1 in rMSCs. Inhibitors of FAK and ERK pathways inhibited OPN-induced rMSCs migration, indicating the possible involvement of FAK and ERK activation in OPN-induced migration in rMSCs. In addition, atomic force microscopy analysis showed that OPN reduced cell stiffness in rMSCs via integrin β1, FAK, and ERK pathways, suggesting that the promotion of rMSCs migration might partially be contributing to the decrease in cell stiffness stimulated by OPN. To further examine the role of OPN on cell motility and stiffness, actin cytoskeleton of rMSCs was observed. The reduced well-defined F-actin filaments and the promoted formation of pseudopodia in rMSCs induced by OPN explained the reduction in cell stiffness and the increase in cell migration. The current study data have shown for the first time that OPN binding to integrin β1 promotes rMSCs migration through the activation of FAK and ERK pathways, which may be attributed to the change in cell stiffness caused by the reduction in the amount of organized actin cytoskeleton.

TLyP-1-conjugated Au-nanorod@SiO2 core-shell nanoparticles for tumor-targeted drug delivery and photothermal therapy

Mesoporous silica-coated Au nanorod (AuNR@SiO2) is one of the most important appealing nanomaterials for cancer therapy. The multifunctions of chemotherapy, photothermal therapy, and imaging of AuNR@SiO2 make it very useful for cancer therapy. In this study, AuNR@SiO2 was functionalized to deliver hydrophobic antitumor drug and to heat the targeted tumor with the energy of near-infrared (NIR). To carry out the function of targeting the tumor, tLyP-1, a kind of tumor homing and penetrating peptide, was engrafted to AuNR@SiO2. The fabricated AuNR@SiO2-tLyP-1 which was loaded with camptothecin (CPT) showed a robust, selective targeting and penetrating efficiency to Hela and MCF-7 cells and induced the death of these cells. When the micromasses of these AuNR@SiO2-tLyP-1 internalized cells were irradiated by NIR illumination, all the cells were killed instantaneously owing to the increased temperature caused by the surface plasma resonance (SPR) of the internalized AuNR@SiO2-tLyP-1. Moreover, the systematic toxicity of CPT-loaded AuNR@SiO2-tLyP-1 on human mesenchymal stem cells (hMSCs) was minimized, because the AuNR@SiO2-tLyP-1 selectively targeted and penetrated into the tumor cells, and little hydrophobic CPT was released into the culture medium or blood. This study indicates that the AuNR@SiO2-tLyP-1 drug delivery system (DDS) has great potential application for the chemo-photothermal cancer therapy.

Effect of Focal Adhesion Kinase on the Regulation of Realignment and Tenogenic Differentiation of Human Mesenchymal Stem Cells by Mechanical Stretch

Focal adhesion kinase (FAK) is a focal adhesion-associated protein kinase involved in cell adhesion and spreading. It is recruited as a participant in focal adhesion dynamics between cells and has a role in cell motility, differentiation, and survival. The role of FAK in the differentiation of human mesenchymal stem cells (hMSCs), however, is not well understood, particularly in terms of tenogenic differentiation. In this study, we reported that FAK regulates the mechanical stretch-induced realignment of hMSCs. We showed that FAK can be activated by mechanical stretch and, with a 10 μM PF 573228 (a novel small molecule inhibitor of FAK) treatment, FAK autophosphorylation at Tyr397 is significantly decreased. Moreover, our findings demonstrated that this decrease in FAK autophosphorylation at Tyr397 leads to the attenuation of upregulation of mechanical stretch-induced mRNA expression of tendon-related genes, including type I collagen, type III collagen, tenascin-C, and scleraxis. These results indicate that the FAK signaling molecule plays an important role in regulating cell realignment and tenogenic differentiation of hMSCs when induced by mechanical stretch. Collectively, our findings provide novel insight into the role of FAK in the realignment and mechanotransduction of hMSCs during the process of tenogenic differentiation induced by mechanical stretch.

Liver cancer stem cell markers: Progression and therapeutic implications

Cancer stem cells (CSCs) are a small subpopulation in cancer, have been proposed to be cancer-initiating cells, and have been shown to be responsible for chemotherapy resistance and cancer recurrence. The identification of CSC subpopulations inside a tumor presents a new understanding of cancer development because it implies that tumors can only be eradicated by targeting CSCs. Although advances in liver cancer detection and treatment have increased the possibility of curing the disease at early stages, unfortunately, most patients will relapse and succumb to their disease. Strategies aimed at efficiently targeting liver CSCs are becoming important for monitoring the progress of liver cancer therapy and for evaluating new therapeutic approaches. Herein, we provide a critical discussion of biological markers described in the literature regarding liver cancer stem cells and the potential of these markers to serve as therapeutic targets.

Role of p38, ERK1/2, focal adhesion kinase, RhoA/ROCK and cytoskeleton in the adipogenesis of human mesenchymal stem cells.

Adipogenesis is important to health and is thought occurring in the two stages of mesenchymal stem cell commitment to a preadipocyte fate and terminal differentiation of the preadipocyte. However, the mechanism of adipogenesis is still not clear. In this study, the roles of p38, extracellular regulated protein kinases 1/2 (ERK1/2), focal adhesion kinase (FAK), RhoA/ROCK, and cytoskeleton in both of the two stages of adipogenesis were assayed. Our results showed that the treatments of SB203580 (the inhibitor of p38) and U0126 (the inhibitor of ERK1/2) suppressed the adipogenesis induced by differentiation medium, and the treatments of PF573228 (a specific inhibitor of FAK), Y27632 (a specific inhibitor of RhoA/ROCK) and cytochalasin D (an inhibitor of cytoskeletal organization) promoted the adipogenesis. The treatments of SB203580 and U0126 significantly inhibited the adipogenic differentiation of hMSCs cultured in differentiation medium in the presence of PF573228, Y27632 or cytochalasin D. Moreover, the treatments of PF573228, Y27632 and cytochalasin D promoted p38 and ERK1/2 phosphorylations, and the treatments of U0126 and SB203580 decreased p38 and ERK1/2 phosphorylations, respectively. These results demonstrated that p38 and ERK1/2 played crucial positive roles in adipogenesis, and FAK, RhoA/ROCK and cytoskeleton played negative roles. Furthermore, FAK, RhoA/ROCK and cytoskeleton affected adipogenesis by regulating the activities of p38 and ERK1/2 which interacted with each other in the process of adipogenesis.

In vitro proliferation and osteogenic differentiation of mesenchymal stem cells on nanoporous alumina

Cell adhesion, migration, and proliferation are significantly affected by the surface topography of the substrates on which the cells are cultured. Alumina is one of the most popular implant materials used in orthopedics, but few data are available concerning the cellular responses of mesenchymal stem cells (MSCs) grown on nanoporous structures. MSCs were cultured on smooth alumina substrates and nanoporous alumina substrates to investigate the interaction between surface topographies of nanoporous alumina and cellular behavior. Nanoporous alumina substrates with pore sizes of 20 nm and 100 nm were used to evaluate the effect of pore size on MSCs as measured by proliferation, morphology, expression of integrin β1, and osteogenic differentiation. An MTT assay was used to measure cell viability of MSCs on different substrates, and determined that cell viability decreased with increasing pore size. Scanning electron microscopy was used to investigate the effect of pore size on cell morphology. Extremely elongated cells and prominent cell membrane protrusions were observed in cells cultured on alumina with the larger pore size. The expression of integrin β1 was enhanced in MSCs cultured on porous alumina, revealing that porous alumina substrates were more favorable for cell growth than smooth alumina substrates. Higher levels of osteoblastic differentiation markers such as alkaline phosphatase, osteocalcin, and mineralization were detected in cells cultured on alumina with 100 nm pores compared with cells cultured on alumina with either 20 nm pores or smooth alumina. This work demonstrates that cellular behavior is affected by variation in pore size, providing new insight into the potential application of this novel biocompatible material for the developing field of tissue engineering.

A synthetic mechano-growth factor E peptide promotes rat tenocyte migration by lessening cell stiffness and increasing F-actin formation via the FAK-ERK1/2 signaling pathway.

Tendon injuries are common in sports and are frequent reasons for orthopedic consultations. The management of damaged tendons is one of the most challenging problems in orthopedics. Mechano-growth factor (MGF), a recently discovered growth repair factor, plays positive roles in tissue repair through the improvement of cell proliferation and migration and the protection of cells against injury-induced apoptosis. However, it remains unclear whether MGF has the potential to accelerate tendon repair. We used a scratch wound assay in this study to demonstrate that MGF-C25E (a synthetic mechano-growth factor E peptide) promotes the migration of rat tenocytes and that this promotion is accompanied by an elevation in the expression of the following signaling molecules: focal adhesion kinase (FAK) and extracellular signal regulated kinase1/2 (ERK1/2). Inhibitors of the FAK and ERK1/2 pathways inhibited the MGF-C25E-induced tenocyte migration, indicating that MGF-C25E promotes tenocyte migration through the FAK-ERK1/2 signaling pathway. The analysis of the mechanical properties showed that the Youngs modulus of tenocytes was decreased through treatment of MGF-C25E, and an obvious formation of pseudopodia and F-actin was observed in MGF-C25E-treated tenocytes. The inhibition of the FAK or ERK1/2 signals restored the decrease in Youngs modulus and inhibited the formation of pseudopodia and F-actin. Overall, our study demonstrated that MGF-C25E promotes rat tenocyte migration by lessening cell stiffness and increasing pseudopodia formation via the FAK-ERK1/2 signaling pathway.

Mechano-growth factor induces migration of rat mesenchymal stem cells by altering its mechanical properties and activating ERK pathway

Mechano-growth factor (MGF) generated by cells in response to mechanical stimulation has been identified as a mechano effector molecule, playing a key role in regulating mesenchymal stem cell (MSC) function, including proliferation and migration. However, the mechanism(s) underlying how MGF-induced MSC migration occurs is still unclear. In the present study, MGF motivated migration of rat MSCs (rMSCs) in a concentration-dependent manner and optimal concentration of MGF at 50 ng/mL (defined as MGF treatment in this paper) was demonstrated. Notably, enhancement of mechanical properties that is pertinent to cell migration, such as cell traction force and cell stiffness were found to respond to MGF treatment. Furthermore, MGF increased phosphorylation of extracellular signal-regulated kinase (ERK), ERK inhibitor (i.e., PD98059) suppressed ERK phosphorylation, and abolished MGF-induced rMSC migration were found, demonstrating that ERK is involved molecule for MGF-induced rMSC migration. These in vitro evidences of MGF-induced rMSC migration and its direct link to altering rMSC mechanics and activating the ERK pathway, uncover the underlying biomechanical and biological mechanisms of MGF-induced rMSC migration, which may help find MGF-based application of MSC in clinical therapeutics.