Stiffness gradients mimicking in vivo tissue variation regulate mesenchymal stem cell fate

Mesenchymal stem cell (MSC) differentiation is regulated in part by tissue stiffness, yet MSCs can often encounter stiffness gradients within tissues caused by pathological, e.g., myocardial infarction ～8.7±1.5 kPa/mm, or normal tissue variation, e.g., myocardium ～0.6±0.9 kPa/mm; since migration predominantly occurs through physiological rather than pathological gradients, it is not clear whether MSC differentiate or migrate first. MSCs cultured up to 21 days on a hydrogel containing a physiological gradient of 1.0±0.1 kPa/mm undergo directed migration, or durotaxis, up stiffness gradients rather than remain stationary. Temporal assessment of morphology and differentiation markers indicates that MSCs migrate to stiffer matrix and then differentiate into a more contractile myogenic phenotype. In those cells migrating from soft to stiff regions however, phenotype is not completely determined by the stiff hydrogel as some cells retain expression of a neural marker. These data may indicate that stiffness variation, not just stiffness alone, can be an important regulator of MSC behavior.     

Phorbaketal A stimulates osteoblast differentiation through TAZ mediated Runx2 activation

Osteoporosis arises from an imbalance between osteoblastic bone formation and osteoclastic bone resorption. In this study, we screened molecules from marine natural products that stimulate osteoblast differentiation. We found that phorbaketal A significantly stimulates osteoblast differentiation in mesenchymal cells. Increased interaction of TAZ and Runx2 stimulated phorbaketal A-induced expression of osteoblastic marker genes. The activation of ERK was important for the stimulation of differentiation because an inhibitor of ERK blocked phorbaketal A-induced osteogenic differentiation. Taken together, the results showed that phorbaketal A stimulates TAZ-mediated osteoblast differentiation through the activation of ERK.     

Adult human mesenchymal stem cell differentiation to the osteogenic or adipogenic lineage is regulated by mitogen-activated protein kinase

Adult human mesenchymal stem cells are primary, multipotent cells capable of differentiating to osteocytic, chondrocytic, and adipocytic lineages when stimulated under appropriate conditions. To characterize the molecular mechanisms that regulate osteogenic differentiation, we examined the contribution of mitogen-activated protein kinase family members, ERK, JNK, and p38. Treatment of these stem cells with osteogenic supplements resulted in a sustained phase of ERK activation from day 7 to day 11 that coincided with differentiation, before decreasing to basal levels. Activation of JNK occurred much later (day 13 to day 17) in the osteogenic differentiation process. This JNK activation was associated with extracellular matrix synthesis and increased calcium deposition, the two hallmarks of bone formation. Inhibition of ERK activation by PD98059, a specific inhibitor of the ERK signaling pathway, blocked the osteogenic differentiation in a dose-dependent manner, as did transfection with a dominant negative form of MAP kinase kinase (MEK-1). Significantly, the blockage of osteogenic differentiation resulted in the adipogenic differentiation of the stem cells and the expression of adipose-specific mRNAs peroxisome proliferator-activated receptor gamma2, aP2, and lipoprotein lipase. These observations provide a potential mechanism involving MAP kinase activation in osteogenic differentiation of adult stem cells and suggest that commitment of hMSCs into osteogenic or adipogenic lineages is governed by activation or inhibition of ERK, respectively.     

Vitronectin and collagen I differentially regulate osteogenesis in mesenchymal stem cells

The roles of various soluble factors in promoting the osteogenic differentiation of adult mesenchymal stem cells (MSCs) have been widely studied, but little is known about how the extracellular matrix (ECM) instructs the phenotypic transition between growth and differentiation. To investigate this question, we cultured MSCs on purified vitronectin or type-I collagen, motivated by our earlier tissue engineering work demonstrating that MSC adhesion to polymer scaffolds is primarily mediated by the passive adsorption of these two ECM ligands from serum. Using alkaline phosphatase activity and matrix mineralization as indicators of the early and late stages of osteogenesis, respectively, we report here that both substrates supported differentiation, but the mechanism was substrate dependent. Specifically, osteogenesis on vitronectin correlated with enhanced focal adhesion formation, the activation of focal adhesion kinase (FAK) and paxillin, and the diminished activation of extracellular signal-regulated kinase (ERK) and phosphatidylinositol-3 kinase (PI3K) pathways. By contrast, MSCs on type-I collagen exhibited reduced focal adhesion formation, reduced activation of FAK and paxillin, and increased activation of ERK and PI3K. Inhibition of ERK and FAK blocked mineral deposition on both substrates, suggesting that the observed differences in signaling pathways ultimately converge to the same cell fate. Understanding these mechanistic differences is essential to predictably control the osteogenic differentiation of MSCs and widen their use in regenerative medicine.     

Laminin regulates the osteogenic differentiation of dental follicle cells via integrin-α2/-β1 and the activation of the FAK/ERK signaling pathway

Dental follicle cells (DFCs) are ideal for studies concerning the differentiation of dental precursor cells into alveolar osteoblasts and cementoblasts. Previous investigations have suggested that the extracellular matrix (ECM) protein laminin and the ECM receptor integrin-α2/-β1 play regulatory roles during the osteogenic differentiation of DFCs. Our present data indicate that laminin impairs alkaline phosphatase (ALP) activity following osteogenic induction while inducing integrin-α2/-β1 expression, osteogenic differentiation marker elevation, and DFC biomineralization. Integrin-α2/-β1 facilitates the laminin-dependent expression of osteogenic differentiation markers and the laminin-dependent inhibition of ALP activity. Moreover, these laminin-dependent effects on the osteogenic differentiation of DFCs can be reversed by the inhibition of the FAK/ERK signaling pathway. Thus, laminin regulates the inhibition of early osteogenic differentiation markers and the induction of late osteogenic differentiation markers via integrin-α2/-β1 and the activation of the FAK/ERK signaling pathway.     

Predicting YAP/TAZ nuclear translocation in response to ECM mechanosensing

Cells translate mechanical cues from the extracellular matrix (ECM) into signaling that can affect the nucleus. One pathway by which such nuclear mechanotransduction occurs is a signaling axis that begins with integrin-ECM bonds and continues through a cascade of chemical reactions and structural changes that lead to nuclear translocation of YAP/TAZ. This signaling axis is self-reinforcing, with stiff ECM promoting integrin binding and thus facilitating polymerization and tension in the cytoskeletal contractile apparatus, which can compress nuclei, open nuclear pore channels, and enhance nuclear accumulation of YAP/TAZ. We previously developed a computational model of this mechanosensing axis for the linear elastic ECM by assuming that there is a linear relationship between the nucleocytoplasmic ratio of YAP/TAZ and nuclear flattening. Here, we extended our previous model to more general ECM behaviors (e.g., viscosity, viscoelasticity, and viscoplasticity) and included detailed YAP/TAZ translocation dynamics based on nuclear deformation. This model was predictive of diverse mechanosensing responses in a broad range of cells. Results support the hypothesis that diverse mechanosensing phenomena across many cell types arise from a simple, unified set of mechanosensing pathways.     

MG63 osteoblast-like cells exhibit different behavior when grown on electrospun collagen matrix versus electrospun gelatin matrix

Electrospinning is a simple and efficient method of fabricating a non-woven polymeric nanofiber matrix. However, using fluorinated alcohols as a solvent for the electrospinning of proteins often results in protein denaturation. TEM and circular dichroism analysis indicated a massive loss of triple-helical collagen from an electrospun collagen (EC) matrix, and the random coils were similar to those found in gelatin. Nevertheless, from mechanical testing we found the Young's modulus and ultimate tensile stresses of EC matrices were significantly higher than electrospun gelatin (EG) matrices because matrix stiffness can affect many cell behaviors such as cell adhesion, proliferation and differentiation. We hypothesize that the difference of matrix stiffness between EC and EG will affect intracellular signaling through the mechano-transducers Rho kinase (ROCK) and focal adhesion kinase (FAK) and subsequently regulates the osteogenic phenotype of MG63 osteoblast-like cells. From the results, we found there was no significant difference between the EC and EG matrices with respect to either cell attachment or proliferation rate. However, the gene expression levels of OPN, type I collagen, ALP, and OCN were significantly higher in MG63 osteoblast-like cells grown on the EC than in those grown on the EG. In addition, the phosphorylation levels of Y397-FAK, ERK1/2, BSP, and OPN proteins, as well as ALP activity, were also higher on the EC than on the EG. We further inhibited ROCK activation with Y27632 during differentiation to investigate its effects on matrix-mediated osteogenic differentiation. Results showed the extent of mineralization was decreased with inhibition after induction. Moreover, there is no significant difference between EC and EG. From the results of the protein levels of phosphorylated Y397-FAK, ERK1/2, BSP and OPN, ALP activity and mineral deposition, we speculate that the mechanism that influences the osteogenic differentiation of MG63 osteoblast-like cells on EC and EG is matrix stiffness and via ROCK-FAK-ERK1/2.     

Comparative proteomic profiling of human osteoblast‐derived extracellular matrices identifies proteins involved in mesenchymal stromal cell osteogenic differentiation and mineralization

The extracellular matrix (ECM) is a dynamic component of tissue architecture that physically supports cells and actively influences their behavior. In the context of bone regeneration, cell‐secreted ECMs have become of interest as they reproduce tissue‐architecture and modulate the promising properties of mesenchymal stem cells (MSCs). We have previously created an in vitro model of human osteoblast‐derived devitalized ECM that was osteopromotive for MSCs. The aim of this study was to identify ECM regulatory proteins able to modulate MSC differentiation to broaden the spectrum of MSC clinical applications. To this end, we created two additional models of devitalized ECMs with different mineralization phenotypes. Our results showed that the ECM derived from osteoblast‐differentiated MSCs had increased osteogenic potential compared to ECM derived from undifferentiated MSCs and non‐ECM cultures. Proteomic analysis revealed that structural ECM proteins and ribosomal proteins were upregulated in the ECM from undifferentiated MSCs. A similar response profile was obtained by treating osteoblast‐differentiating MSCs with Activin‐A. Extracellular proteins were upregulated in Activin‐A ECM, whereas mitochondrial and membrane proteins were downregulated. In summary, this study illustrates that the composition of different MSC‐secreted ECMs is important to regulate the osteogenic differentiation of MSCs. These models of devitalized ECMs could be used to modulate MSC properties to regulate bone quality.     

Matrix stiffness controls cardiac fibroblast activation through regulating YAP via AT1R

Cardiac fibrosis is a common pathway leading to heart failure and involves continued activation of cardiac fibroblasts (CFs) into myofibroblasts during myocardium damage, causing excessive deposition of the extracellular matrix (ECM) and thus increases matrix stiffness. Increasing evidence has shown that stiffened matrix plays an important role in promoting CF activation and cardiac fibrosis, and several signaling factors mediating CF mechanotransduction have been identified. However, the key molecules that perceive matrix stiffness to regulate CF activation remain to be further explored. Here, we detected significantly increased expression and nuclear localization of Yes-associated protein (YAP) in native fibrotic cardiac tissues. By using mechanically regulated in vitro cell culture models, we found that a stiff matrix-induced high expression and nuclear localization of YAP in CFs, accompanied by enhanced cell activation. We also demonstrated that YAP knockdown decreased fibrogenic response of CFs and that YAP overexpression promoted CF activation, indicating that YAP plays an important role in mediating matrix stiffness-induced CF activation. Further mechanistic studies revealed that the YAP pathway is an important signaling branch downstream of angiotensin II type 1 receptor in CF mechanotransduction. The findings help elucidate the mechanism of fibrotic mechanotransduction and may contribute to the development of new approaches for treating fibrotic diseases.     

Human mesenchymal stem cells stimulated by TNF-alpha, LPS, or hypoxia produce growth factors by an NF kappa B- but not JNK-dependent mechanism

Understanding the mechanisms by which adult stem cells produce growth factors may represent an important way to optimize their beneficial paracrine and autocrine effects. Components of the wound milieu may stimulate growth factor production to promote stem cell-mediated repair. We hypothesized that tumor necrosis factor-alpha (TNF-alpha), endotoxin (LPS), or hypoxia may activate human mesenchymal stem cells (MSCs) to increase release of vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF2), insulin-like growth factor 1 (IGF-1), or hepatocyte growth factor (HGF) and that nuclear factor-kappa B (NF kappa B), c-Jun NH2-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) mediates growth factor production from human MSCs. To study this, human MSCs were harvested, passaged, divided into four groups (100,000 cells, triplicates) and treated as follows: 1) with vehicle; 2) with stimulant alone [24 h LPS (200 ng/ml), 24 h TNF-alpha (50 ng/ml), or 24 h hypoxia (1% O2)]; 3) with inhibitor alone [NF kappa B (PDTC, 1 mM), JNK (TI-JIP, 10 microM), or ERK (ERK Inhibitor II, 25 microM)]; and 4) with stimulant and the various inhibitors. After 24 h incubation, MSC activation was determined by measuring supernatants for VEGF, FGF2, IGF-1, or HGF (ELISA). TNF-alpha, LPS, and hypoxia significantly increased human MSC VEGF, FGF2, HGF, and IGF-1 production versus controls. Stem cells exposed to injury demonstrated increased activation of NF kappa B, ERK, and JNK. VEGF, FGF2, and HGF expression was significantly reduced by NF kappa B inhibition (50% decrease) but not ERK or JNK inhibition. Moreover, ERK, JNK, and NF kappa B inhibitor alone did not activate MSC VEGF expression over controls. Various stressors activate human MSCs to increase VEGF, FGF2, HGF, and IGF-1 expression, which depends on an NFkB mechanism.     

Rigid matrix supports osteogenic differentiation of stem cells from human exfoliated deciduous teeth (SHED)

Stem cell fate can be induced by the grade of stiffness of the extracellular matrix, depending on the developed tissue or complex tissues. For example, a rigid extracellular matrix induces the osteogenic differentiation in bone marrow derived mesenchymal stem cells (MSCs), while a softer surface induces the osteogenic differentiation in dental follicle cells (DFCs). To determine whether differentiation of ectomesenchymal dental precursor cells is supported by similar grades of extracellular matrices (ECMs) stiffness, we examined the influence of the surface stiffness on the proliferation and osteogenic differentiation of stem cells from human exfoliated deciduous teeth (SHED). Cell proliferation of SHED was significantly decreased on cell culture surfaces with a muscle-like stiffness. A dexamethasone-based differentiation medium induced the osteogenic differentiation of SHED on substrates of varying mechanical stiffness. Here, the hardest surface improved the induction of osteogenic differentiation in comparison to that with the softest stiffness. In conclusion, our study showed that the osteogenic differentiation of ectomesenchymal dental precursor cells SHED and DFCs are not supported by similar grades of ECM stiffness.     

Mechanically tuned 3 dimensional hydrogels support human mammary fibroblast growth and viability

Background

Carcinoma associated fibroblasts (CAFs or myofibroblasts) are activated fibroblasts which participate in breast tumor growth, angiogenesis, invasion, metastasis and therapy resistance. As such, recent efforts have been directed toward understanding the factors responsible for activation of the phenotype. In this study, we have investigated how changes in the mechanical stiffness of a 3D hydrogel alter the behavior and myofibroblast-like properties of human mammary fibroblasts (HMFs).

Results

Here, we utilized microbial transglutaminase (mTG) to mechanically tune the stiffness of gelatin hydrogels and used rheology to show that increasing concentrations mTG resulted in hydrogels with greater elastic moduli (G’). Upon encapsulation of HMFs in 200 (compliant), 300 (moderate) and 1100 Pa (stiff) mTG hydrogels, it was found that the HMFs remained viable and proliferated over the 7 day culture period. Specifically, rates of proliferation were greatest for HMFs in moderate hydrogels. Regarding morphology, HMFs in compliant and moderate hydrogels exhibited a spindle-like morphology while HMFs in stiff hydrogels exhibited a rounded morphology with several large cellular protrusions. Quantification of cell morphology revealed that HMFs cultured in all mTG hydrogels overall assumed a more elongated phenotype over time in culture; however, few significant differences in morphology were observed between HMFs in each of the hydrogel conditions. To determine whether matrix stiffness upregulated expression of ECM and myofibroblast markers, western blot was performed on HMFs in compliant, moderate and stiff hydrogels. It was found that ECM and myofibroblast proteins varied in expression during both the culture period and according to matrix stiffness with no clear correlation between matrix stiffness and a myofibroblast phenotype. Finally, TGF-β levels were quantified in the conditioned media from HMFs in compliant, moderate and stiff hydrogels. TGF-β was significantly greater for HMFs encapsulated in stiff hydrogels.

Conclusions

Overall, these results show that while HMFs are viable and proliferate in mTG hydrogels, increasing matrix stiffness of mTG gelatin hydrogels doesn’t support a robust myofibroblast phenotype from HMFs. These results have important implications for further understanding how modulating 3D matrix stiffness affects fibroblast morphology and activation into a myofibroblast phenotype.