Activation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) is needed for the TGFβ-induced chondrogenic and osteogenic differentiation of mesenchymal stem cells

The role of the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway on the osteogenesis of progenitor and stem cells has received a lot of attention due to conflicting results in the literature. ERK1/2 has been reported to be both activating and inhibitory to the osteogenesis of different cell types under varying culture conditions. This study focused specifically on the role of ERK1/2 on the chondrogenesis and osteogenesis of mesenchymal stem cells (MSC) induced by cytokine exposure. Bone marrow-derived MSC were cultured in three-dimensional fibrin gel scaffolds and stimulated down the chondrogenic and osteogenic programs by addition of TGF-β3 to and osteogenic buffer media. Cells were cultured under control conditions (no cytokine supplementation), treated with TGF-β3 or treated with PD98059 + TGF-β3 for 7 days. RT-PCR results show that addition of TGF-β3 significantly upregulates the phosphorylation of ERK1/2 and induces the cells down the chondrogenic and osteogenic pathways (as demonstrated by the significant upregulation of aggrecan, sox9, collagen types 1 & 2 gene expressions). Inhibition of ERK1/2 phosphorylation with PD98059 led to the abolishment of the upregulation of chondrogenic and osteogenic-specific gene expressions. These results demonstrate that ERK1/2 is needed for the chondrogenic and osteogenic differentiation of MSC as induced by TGF-β3 supplementation.     

NSAIDS inhibit in vitro MSC chondrogenesis but not osteogenesis: implications for mechanism of bone formation inhibition in man

The non-steroidal anti-inflammatory drugs (NSAIDs) are widely used for analgesia but may inhibit bone formation. We investigated whether the reported NSAID effect on bone is related to inhibition of bone marrow mesenchymal stem cell (MSC) proliferation and osteogenic and chondrogenic differentiation and evaluated both cyclooxygenase (COX)-1 and COX-2 specific drugs. The effects of seven COX-1 and COX-2 inhibitors on MSC proliferation and osteogenic and chondrogenic differentiation were tested using Vybrant, sodium 3′-[1-(phenylaminocarbonyl)- 3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate (XTT), functional and quantitative assays of MSC differentiation. The MSC expression of COX-1 and COX-2 and prostaglandin E2 (PGE-2) levels were evaluated serially during lineage differentiation by quantitative PCR and ELISA. None of the NSAIDs at broad range of concentration (range 10⁻³ to 100 μg/ml) significantly affected MSC proliferation. Surprisingly, MSC osteogenic differentiation inhibition was not evident. However, NSAIDs affected chondrogenic potential with a reduction in sulphated glycosaminoglycans (sGAG) content by 45% and 55% with diclofenac and ketorolac, respectively (P < 0.05 compared to controls). Parecoxib and meloxicam, more COX-2 specific reagents inhibited sGAG to a lesser degree, 22% and 27% respectively (P < 0.05 compared to controls). Cartilage pellet immunohistochemistry confirmed the above results. Pellet chondrogenesis was associated with increased COX-1 expression levels but not COX-2, and COX-1 specific drugs suppressed MSC PGE-2 more than COX-2 specific inhibitors. These findings suggest that NSAIDs may inhibit bone formation via blockage of MSC chondrogenic differentiation which is an important intermediate phase in normal endochondral bone formation.     

Effect of parathyroid hormone on prechondroblast differentiation

Parathyroid hormone (PTH) exerts an anabolic effect that is manifested upon periodic exposure to the hormone and a catabolic effect that is observed in case of a prolonged uninterrupted exposure to high doses of the hormone. The latter effect is characteristic of a range of pathologies, such as the hyperfunction of the parathyroid glands. The analysis of the effects of periodic and continuous administration of PTH fragment 1–34 on the differentiation of bovine prechondroblasts maintained in chondrogenic and osteogenic media was the aim of the present study. Alcian blue staining of monolayers of cultured cells maintained in a chondrogenic medium revealed the stimulation of chondrogenesis (manifested as higher intensity of matrix staining) in the case of continuous PTH administration. Alizarin red staining of monolayers of cultured cells maintained in an osteogenic medium revealed more intensive mineralization of the matrix in plates with cells that were exposed to PTH in a periodical manner. The results of the histochemical study were confirmed using electrophoretic detection of osteocrin and type II collagen.     

阻断ERK1/2激酶可增强骨形态发生蛋白9诱导的间充质干细胞成骨分化

骨形态发生蛋白9(bone morphogenetic protein 9,BMP9)具有很强的诱导间充质干细胞定向成骨分化的能力.但对于其所涉及的相关分子机理了解并不深入.利用BMP9重组腺病毒感染间充质干细胞,Western blot检测ERK1/2激酶的磷酸化,ERK1/2的特异性抑制剂PD98059阻断ERK1/2活性,或以RNA干扰抑制ERK1/2表达,通过体外细胞实验和体内动物实验,初步分析和揭示ERK1/2对于BMP9诱导的间充质干细胞成骨分化的调控作用及其可能机制.结果发现:BMP9可以促进ERK1/2激酶的磷酸化,ERK1/2抑制剂PD98059可增强由BMP9诱导的碱性磷酸酶(alkaline phosphatase,ALP)活性、骨桥蛋白(osteopontin,OPN)表达和钙盐沉积,并促进由BMP9诱导的Runx2基因的表达和转录活性,以及Smad经典途径的活化;而RNA干扰导致ERK1/2基因沉默同样也可进一步促进BMP9诱导的ALP活性和钙盐沉积,并促进BMP9诱导的间充质干细胞在裸鼠皮下异位成骨.因此,BMP9可以促进ERK1/2蛋白激酶的活化,而阻断ERK1/2蛋白激酶可进一步增强BMP9诱导的成骨分化,ERK1/2极可能对于BMP9诱导的间充质干细胞成骨分化起着负向调控作用.     

BMP9经JNK激酶途径调控间充质干细胞C3H10T1/2成骨分化

为了证实JNK激酶在骨形态发生蛋白9(bone morphogenetic proteins 9,BMP9) 诱导间充质干细胞C3H10T1/2成骨分化中的作用,利用重组腺病毒将BMP9导入间充质干细胞C3H10T1/2. 通过碱性磷酸酶(ALP)活性测定、钙盐沉积实验、荧光素酶报告基因检测、Western印迹和组织化学染色等方法,检测BMP9是否可经JNK激酶途径调控间充质干细胞C3H10T1/2向成骨分化.动物实验验证在RNA沉默JNK蛋白激酶后,对BMP9诱导间充质干细胞C3H10T1/2向成骨分化的影响．结果发现,BMP9可以增强JNK 激酶的磷酸化;利用JNK抑制剂SP600125抑制JNK激酶活性后,BMP9诱导的间充质干细胞C3H10T1/2的早期成骨指标ALP活性和晚期指标钙盐沉积均受到抑制,而且经典SMAD信号的活化也相应受到抑制;RNA干扰沉默JNK基因表达后,同样也可抑制BMP9 诱导的C3H10T1/2细胞的ALP活性和裸鼠皮下异位成骨．因此表明,BMP9可活化JNK激酶途径从而诱导间充质干细胞C3H10T1/2向成骨分化．     

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.     

Effect of expansion media containing fibroblast growth factor-2 and dexamethasone on the chondrogenic potential of human adipose-derived stromal cells

hASCs [human ASCs (adipose derived stromal cells)] proliferate more rapidly in the presence of basic FGF-2 (fibroblast growth factor-2) and Dex (dexamethasone). We have examined the effects of expanding hASCs in media containing these two factors on their chondrogenic differentiation potential. Results show that the addition of FGF-2 and Dex to the expansion medium does not remarkably alter the chondrogenic potential of the cells induced by BMP-6 (bone morphogenetic protein-6), based on chondrogenic gene expression, sGAG (sulfated glycosaminoglycan) accumulation and immunohistochemical observation. This is in direct contrast to previously reported promotion of the osteogenic and adipogenic potential of hASCs by these two factors. Therefore, an expansion medium containing FGF-2, with or without Dex, is appropriate for the fast expansion of hASCs without compromising chondrogenic potential.     

Chip-Based Comparison of the Osteogenesis of Human Bone Marrow- and Adipose Tissue-Derived Mesenchymal Stem Cells under Mechanical Stimulation

Adipose tissue-derived stem cells (ASCs) are considered as an attractive stem cell source for tissue engineering and regenerative medicine. We compared human bone marrow-derived mesenchymal stem cells (hMSCs) and hASCs under dynamic hydraulic compression to evaluate and compare osteogenic abilities. A novel micro cell chip integrated with microvalves and microscale cell culture chambers separated from an air-pressure chamber was developed using microfabrication technology. The microscale chip enables the culture of two types of stem cells concurrently, where each is loaded into cell culture chambers and dynamic compressive stimulation is applied to the cells uniformly. Dynamic hydraulic compression (1 Hz, 1 psi) increased the production of osteogenic matrix components (bone sialoprotein, oateopontin, type I collagen) and integrin (CD11b and CD31) expression from both stem cell sources. Alkaline phosphatase and Alrizarin red staining were evident in the stimulated hMSCs, while the stimulated hASCs did not show significant increases in staining under the same stimulation conditions. Upon application of mechanical stimulus to the two types of stem cells, integrin (β1) and osteogenic gene markers were upregulated from both cell types. In conclusion, stimulated hMSCs and hASCs showed increased osteogenic gene expression compared to non-stimulated groups. The hMSCs were more sensitive to mechanical stimulation and more effective towards osteogenic differentiation than the hASCs under these modes of mechanical stimulation.     

Differential expression of CCN-family members in primary human bone marrow-derived mesenchymal stem cells during osteogenic, chondrogenic and adipogenic differentiation

BACKGROUND: The human cysteine rich protein 61 (CYR61, CCN1) as well as the other members of the CCN family of genes play important roles in cellular processes such as proliferation, adhesion, migration and survival. These cellular events are of special importance within the complex cellular interactions ongoing in bone remodeling. Previously, we analyzed the role of CYR61/CCN1 as an extracellular signaling molecule in human osteoblasts. Since mesenchymal stem cells of bone marrow are important progenitors for various differentiation pathways in bone and possess increasing potential for regenerative medicine, here we aimed to analyze the expression of CCN family members in bone marrow-derived human mesenchymal stem cells and along the osteogenic, the adipogenic and the chondrogenic differentiation. RESULTS: Primary cultures of human mesenchymal stem cells were obtained from the femoral head of patients undergoing total hip arthroplasty. Differentiation into adipocytes and osteoblasts was done in monolayer culture, differentiation into chondrocytes was induced in high density cell pellet cultures. For either pathway, established differentiation markers and CCN-members were analyzed at the mRNA level by RT-PCR and the CYR61/CCN1 protein was analyzed by immunocytochemistry.RT-PCR and histochemical analysis revealed the appropriate phenotype of differentiated cells (Alizarin-red S, Oil Red O, Alcian blue, alkaline phosphatase; osteocalcin, collagen types I, II, IX, X, cbfa1, PPARgamma, aggrecan). Mesenchymal stem cells expressed CYR61/CCN1, CTGF/CCN2, CTGF-L/WISP2/CCN5 and WISP3/CCN6. The CYR61/CCN1 expression decreased markedly during osteogenic differentiation, adipogenic differentiation and chondrogenic differentiation. These results were confirmed by immuncytochemical analyses. WISP2/CCN5 RNA expression declined during adipogenic differentiation and WISP3/CCN6 RNA expression was markedly reduced in chondrogenic differentiation. CONCLUSION: The decrease in CYR61/CCN1 expression during the differentiation pathways of mesenchymal stem cells into osteoblasts, adipocytes and chondrocytes suggests a specific role of CYR61/CCN1 for maintenance of the stem cell phenotype. The differential expression of CTGF/CCN2, WISP2/CCN5, WISP3/CCN6 and mainly CYR61/CCN1 indicates, that these members of the CCN-family might be important regulators for bone marrow-derived mesenchymal stem cells in the regulation of proliferation and initiation of specific differentiation pathways.     

Cyclic strain enhances matrix mineralization by adult human mesenchymal stem cells via the extracellular signal-regulated kinase (ERK1/2) signaling pathway

Physical stimuli play critical roles in the development, regeneration, and pathology of many mesenchymal tissues, most notably bone. While mature bone cells, such as osteoblasts and osteocytes, are clearly involved in these processes, the role of their progenitors in mechanically mediated tissue responses is unknown. In this study, we investigated the effect of cyclic substrate deformation on the proliferation and osteogenic differentiation of human mesenchymal stem cells (hMSCs). Application of equibiaxial cyclic strain (3%, 0.25Hz) to hMSCs cultured in osteogenic media inhibited proliferation and stimulated a 2.3-fold increase in matrix mineralization over unstrained cells. The strain stimulus activated the extracellular signal-regulated kinase (ERK1/2) and p38 mitogen-activated protein kinase pathways, but had no effect on c-Jun N-terminal kinase phosphorylation or activity. Strain-induced mineralization was largely mediated by ERK1/2 signaling, as inhibition of ERK1/2 attenuated calcium deposition by 55%. Inhibition of the p38 pathway resulted in a more mature osteogenic phenotype, suggesting an inhibitory role for p38 signaling in the modulation of strain-induced osteogenic differentiation. These results demonstrate that mechanical signals regulate hMSC function, suggesting a critical role for physical stimulation of this specific cell population in mesenchymal tissue formation.     

Cyclic hydrostatic compression stimulates chondroinduction of C3H/10T1/2 cells

While the potential for intermittent hydrostatic pressure to promote cartilaginous matrix synthesis is well established, its potential to influence chondroinduction remains poorly understood. This study examined the effects of relatively short- and long-duration cyclic hydrostatic compression on the chondroinduction of C3H/10T1/2 murine embryonic fibroblasts by recombinant human bone morphogenetic protein-2 (rhBMP-2). Cells were seeded at high density into round bottom wells of a 96-well plate and supplemented with 25 ng/ml rhBMP-2. Experimental cultures were subjected to either 1,800 cycles/day or 7,200 cycles/day of 1 Hz sinusoidal hydrostatic compression to 5 MPa (applied 10 min on/10 min off) for 3 days. Non-pressurized control and experimental cultures were maintained in static culture for an additional 5 days. Cultures were then analyzed for alcian blue staining intensity, DNA and sulfated glycosaminoglycan (sGAG) content, and for the rate of collagen synthesis. Whereas cultures subjected to 1,800 pressure cycles exhibited no significant differences (statistical or qualitative) compared to controls, those subjected to 7,200 cycles stained more intensely with alcian blue, contained nearly twice as much sGAG, and displayed twice the rate of collagen synthesis as non-pressurized controls. This study demonstrates the potential for cyclic hydrostatic compression to stimulate chondrogenic differentiation of the C3H/10T1/2 cell line in a duration-dependent manner.     

Regulation of osteogenic and chondrogenic differentiation of mesenchymal stem cells in PEG-ECM hydrogels

Bone-marrow-derived mesenchymal stem cells (MSCs) are candidates for regeneration applications in musculoskeletal tissue such as cartilage and bone. Various soluble factors in the form of growth factors and cytokines have been widely studied for directing the chondrogenic and osteogenic differentiation of MSCs, but little is known about the way that the composition of extracellular matrix (ECM) components in three-dimensional microenvironments plays a role in regulating the differentiation of MSCs. To define whether ECM components influence the regulation of osteogenic and chondrogenic differentiation by MSCs, we encapsulated MSCs in poly-(ethylene glycol)-based (PEG-based) hydrogels containing exogenous type I collagen, type II collagen, or hyaluronic acids (HA) and cultured them for up to 6 weeks in chondrogenic medium containing transforming growth factor-β1 (10 ng/ml) or osteogenic medium. Actin cytoskeleton organization and cellular morphology were strongly dependent on which ECM components were added to the PEG-based hydrogels. Additionally, chondrogenic differentiation of MSCs was marginally enhanced in collagen-matrix-based hydrogels, whereas osteogenic differentiation, as measured by calcium accumulation, was induced in HA-containing hydrogels. Thus, the microenvironments created by exogenous ECM components seem to modulate the fate of MSC differentiation.     

Dynamic compression and co-culture with nucleus pulposus cells promotes proliferation and differentiation of adipose-derived mesenchymal stem cells

Adipose-derived stem cells (ASCs) are a set of multi potent stem cells potentially used in cartilage tissue engineering. We hypothesized that the effect of dynamic compression and co-culture with nucleus pulposus cells (NPCs) promotes ASC proliferation and chondrogenic differentiation. A controlled dynamic compression loading device was utilized to stimulate ASCs obtained from Sprague Dawley (SD) rats and identified by flow cytometry. The proliferation index was measured by carboxyfluorescein succinimidyl ester (CFSE) staining. Dynamic compression, as well as co-culture enhanced chondrogenic differentiation of ASCs as indicated by the expression of SOX-9, type-II collagen and aggrecan, which were measured by real-time PCR and Western blot. In our study, we found dynamic compression promoted the proliferation of ASCs and induced its differentiation into NP-like cells. Combination of dynamic compression and co-culture showed an additive effect on NP-like cell differentiation.     

The role of BMP‐7 in chondrogenic and osteogenic differentiation of human bone marrow multipotent mesenchymal stromal cells in vitro

This study addresses the role of bone morphogenetic protein‐7 (BMP‐7) in chondrogenic and osteogenic differentiation of human bone marrow multipotent mesenchymal stromal cells (BM MSCs) in vitro. BM MSCs were expanded and differentiated in the presence or absence of BMP‐7 in monolayer and three‐dimensional cultures. After 3 days of stimulation, BMP‐7 significantly inhibited MSC growth in expansion cultures. When supplemented in commonly used induction media for 7–21 days, BMP‐7 facilitated both chondrogenic and osteogenic differentiation of MSCs. This was evident by specific gene and protein expression analyses using real‐time PCR, Western blot, histological, and immunohistochemical staining. BMP‐7 supplementation appeared to enhance upregulation of lineage‐specific markers, such as type II and type IX collagens (COL2A1, COL9A1) in chondrogenic and secreted phosphoprotein 1 (SPP1), osteocalcin (BGLAP), and osterix (SP7) in osteogenic differentiation. BMP‐7 in the presence of TGF‐β3 induced superior chondrocytic proteoglycan accumulation, type II collagen, and SOX9 protein expression in alginate and pellet cultures compared to either factor alone. BMP‐7 increased alkaline phosphatase activity and dose‐dependently accelerated calcium mineralization of osteogenic differentiated MSCs. The potential of BMP‐7 to promote adipogenesis of MSCs was restricted under osteogenic conditions, despite upregulation of adipocyte gene expression. These data suggest that BMP‐7 is not a singular lineage determinant, rather it promotes both chondrogenic and osteogenic differentiation of MSCs by co‐ordinating with initial lineage‐specific signals to accelerate cell fate determination. BMP‐7 may be a useful enhancer of in vitro differentiation of BM MSCs for cell‐based tissue repair. J. Cell. Biochem. 109: 406–416, 2010. © 2009 Wiley‐Liss, Inc.     

Osteogenic and chondrogenic differentiation by adipose-derived stem cells harvested from GFP transgenic mice

Recent studies suggest that human adipose tissue contains pluripotent stem cells similar to bone marrow-derived stem cells. Taking advantage of homogeneously marked cells from green fluorescent protein (GFP) transgenic mice, we have previously demonstrated that bone marrow-derived stromal cells (BSCs) differentiate into a variety of cell lineages both in vitro and in vivo. In the present study, we extend this approach to characterize adipose tissue-derived stromal cells, sometimes called processed lipoaspirate (PLA) cells. Adipose-derived stromal cells (ASCs) were isolated from inguinal fat pads of GFP transgenic mice after extensive washing with phosphate-buffered saline and treatment with collagenase. After primary culture in a control medium (Dulbecco's modified Eagle's medium+10% fetal bovine serum) and expansion to two passages, the cells were incubated in either an osteogenic medium (Dulbecco's modified Eagle's medium+10% fetal bovine serum+dexamethasone+ascorbate-2-phosphate+beta-glycerophosphate) or a chondrogenic medium (Dulbecco's modified Eagle's medium+1% fetal bovine serum+insulin+ascorbate-2-phosphate+transforming growth factor-beta1) for 2-4 weeks to induce osteogenesis and chondrogenesis, respectively. Osteogenic differentiation was assessed by von Kossa and alkaline phosphatase staining, while chondrogenic differentiation was assessed by Alcian blue staining. Expression of osteocyte specific osteopontin, osteocalcin, and alkaline phosphatase, and chondrocyte specific aggrecan and type II/X collagen was confirmed by RT-PCR. ASCs incubated in the osteogenic medium were stained positively for von Kossa and alkaline phosphatase staining. Expression of osteocyte specific genes, except osteocalcin, was also detected. Incubation with chondrogenic medium induced Alcian blue positive cells and expression of aggrecan and type II/X collagen genes. No osteochondrogenic differentiation was observed in cells incubated in the control medium. ASCs from GFP transgenic mice have both osteogenic and chondrogenic potential in vitro. Since this cell population can be easily identified through fluorescence microscopy, it may be an ideal source of ASCs for further experiments on stem cell biology and tissue engineering.     

Focal adhesion kinase signaling controls cyclic tensile strain enhanced collagen I-induced osteogenic differentiation of human mesenchymal stem cells

Focal adhesion kinase (FAK) is a key integrator of integrin-mediated signals from the extracellular matrix to the cytoskeleton and downstream signaling molecules. FAK is activated by phosphorylation at specific tyrosine residues, which then stimulate downstream signaling including the ERK1/2 pathway, leading to a variety of cellular responses. In this study, we examined the effects of FAK point mutations at tyrosine residues (Y397, Y925, Y861, and Y576/7) on osteogenic differentiation of human mesenchymal stem cells exposed to collagen I and cyclic tensile strain. Our results demonstrate that FAK signaling emanating from Y397, Y925, and to a lesser extent Y576/7, but not from Y861, controls osteogenic differentiation through an ERK1/2 pathway, as measured by expression levels of key osteogenesis marker genes and subsequent matrix mineralization. These data indicate that FAK is a critical decision maker in extracellular matrix/strain-enhanced osteogenic differentiation.