Cytoskeletal organization of human mesenchymal stem cells (MSC) changes during their osteogenic differentiation

Human MSCs have been studied to define the mechanisms involved in normal bone remodeling and the regulation of osteogenesis. During osteogenic differentiation, MSCs change from their characteristic fibroblast-like phenotype to near spherical shape. In this study, we analyzed the correlation between the organization of cytoskeleton of MSCs, changes in cell morphology, and the expression of specific markers (alkaline phosphatase activity and calcium deposition) of osteogenic differentiation. For osteoblastic differentiation, cells were cultured in a culture medium supplemented with 100 nM dexamethasone, 10 mM beta- glycerophosphate, and 50 microg/ml ascorbic acid. The organization of microfilaments and microtubules was examined by inmunofluorescence using Alexa fluor 594 phalloidin and anti alpha-tubulin monoclonal antibody. Cytochalasin D and nocodazole were used to alter reversibly the cytoskeleton dynamic. A remarkable change in cytoskeleton organization was observed in human MSCs during osteogenic differentiation. Actin cytoskeleton changed from a large number of thin, parallel microfilament bundles extending across the entire cytoplasm in undifferentiated MSCs to a few thick actin filament bundles located at the outermost periphery in differentiated cells. Under osteogenic culture conditions, a reversible reorganization of microfilaments induced by an initial treatment with cytochalasin D but not with nocodazole reduced the expression of differentiation markers, without affecting the final morphology of the cells. The results indicate that changes in the assembly and disassembly kinetics of microfilaments dynamic of actin network formation may be critical in supporting the osteogenic differentiation of human MSCs; also indicated that the organization of microtubules appears to have a regulatory role on the kinetic of this process.     

Adipose-derived stem cells: An appropriate selection for osteogenic differentiation

Mesenchymal stem cells (MSCs) are a major component of various forms of tissue engineering. MSCs have self-renewal and multidifferential potential. Osteogenic differentiation of MSCs is an area of attention in bone regeneration. One form of MSCs are adipose-derived stem cells (ASCs), which can be simply harvested and differentiated into several cell lineages, such as chondrocytes, adipocytes, or osteoblasts. Due to special properties, ASCs are frequently used in vitro and in vivo bone regeneration. Identifying factors involved in osteogenic differentiation of ASCs is important for better understanding the mechanism of osteogenic differentiation. Different methods are used to stimulate osteogenesis of ASCs in literature, including common osteogenic media, growth factors, hormones, hypoxia, mechanical and chemical stimuli, genetic modification, and nanotechnology. This review article provides an overview describing the isolation procedure, characterization, properties, current methods for osteogenic differentiation of ASCs, and their basic biological mechanism.     

Donepezil hydrochloride as a novel inducer for osteogenic differentiation of mesenchymal stem cells on PLLA scaffolds in vitro

Over the past decades, bone defects caused by illness or trauma have been the most common traumatic injuries in humans and treatment of orthopedic infections has always been a serious challenge to experts in the world. In this project, poly L-lactic acid (PLLA) nanofibrous scaffolds were synthesized as a nontoxic, eco-friendly, and cost-effective scaffold by the electrospinning technique. Then, the impact of PLLA on the cell proliferation and osteogenic differentiation of human mesenchymal stem cells (hMSCs) was assayed in the presence and absence of donepezil hydrochloride (DH) which was prescribed in patients with Alzheimer's disease. Also, hMSCs were seeded on PLLA scaffold in the presence (PLLA-DH) and absence of 1 μg mL^-1 of DH under osteogenic induction media. Osteogenic differentiation of hMSCs was assessed by specific bone-related tests including alkaline phosphatase (ALP) activity, Alizarin red and von Kossa staining, calcium content assay. Also, Osteocalcin and osteopontin were evaluated as osteogenic proteins as well as ALP, osteonectin, osteocalcin, collagen type I (Col-I) and Runx2 as osteogenic genes via immunocytochemistry (ICC) and Real-time PCR analyses. The obtained data showed the higher ALP enzyme activity and biomineralization, more intensity during von Kossa staining as well as the increase in the expression rate of osteogenic related gene and protein markers in differentiated hMSCs on PLLA-DH. In conclusion, the present study revealed that the combination of PLLA scaffold with DH provides a scope to develop a suitable matrix in bone tissue engineering applications.     

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.     

Comparative osteogenic transcription profiling of various fetal and adult mesenchymal stem cell sources

Human mesenchymal stem cells (MSC) from adult and fetal tissues are promising candidates for cell therapy but there is a need to identify the optimal source for bone regeneration. We have previously characterized MSC populations in first trimester fetal blood, liver, and bone marrow and we now evaluate their osteogenic differentiation potential in comparison to adult bone marrow MSC. Using quantitative real-time RT-PCR, we demonstrated that 16 osteogenic-specific genes (OC, ON, BSP, OP, Col1, PCE, Met2A, OPG, PHOS1, SORT, ALP, BMP2, CBFA1, OSX, NOG, IGFII) were expressed in both fetal and adult MSC under basal conditions and were up-regulated under osteogenic conditions both in vivo and during an in vitro 21-day time-course. However, under basal conditions, fetal MSC had higher levels of osteogenic gene expression than adult MSC. Upon osteogenic differentiation, fetal MSC produced more calcium in vitro and reached higher levels of osteogenic gene up-regulation in vivo and in vitro. Second, we observed a hierarchy within fetal samples, with fetal bone marrow MSC having greater osteogenic potential than fetal blood MSC, which in turn had greater osteogenic potential than fetal liver MSC. Finally, we found that the level of gene expression under basal conditions was positively correlated with both calcium secretion and gene expression after 21 days in osteogenic conditions. Our findings suggest that stem cell therapy for bone dysplasias such as osteogenesis imperfecta may benefit from preferentially using first trimester fetal blood or bone marrow MSC over fetal liver or adult bone marrow MSC.     

GNAS knockdown suppresses osteogenic differentiation of mesenchymal stem cells via activation of Hippo signaling pathway

Bone marrow-derived mesenchymal stem cells (BMSCs) are a suitable option for cell-based tissue engineering therapies due to their ability to renew and differentiate into multiple different tissue types, such as bone. Over the last decade, the effect of GNAS on the regulation of osteoblast differentiation has attracted great attention. Herein, this study aimed to explore the role of GNAS in osteogenic differentiation of MSCs. A total of 85 GNAS^f/f male mice were selected for animal experiments and 10 GNAS^f/f male mice for BMSC isolation to conduct cell experiments. The mice and BMSCs were treated with Verteporfin (a Hippo signaling pathway inhibitor) to inhibit the Hippo signaling pathway or recombinant adenovirus-expressing Cre to knockout the GNAS expression. Next, computed tomography scan, Von Kossa staining, and alizarin red staining were performed to detect osteogenic differentiation ability. Moreover, immunohistochemistry and alkaline phosphatase (ALP) staining were used to assess the expression of Oc and Osx in femur tissues and ALP activity. At last, the expression of GNAS, osteogenic markers, and factors related to the Hippo signaling pathway was evaluated. Initially, the results displayed successful knockout of the GNAS gene from mice and BMSCs. Moreover, the data indicated that GNAS knockout inhibits expression of Oc, Osx, ALP, BMP-2, and Runx2, and ALP activity. Additionally, GNAS knockout promotes activation of the Hippo signaling pathway, so as to repress osteogenic differentiation. Collectively, depleted GNAS exerts an inhibitory role in osteogenic differentiation of MSCs by activating Hippo signaling pathway, providing a candidate mediator for osteoporosis.     

Cross‐talk between EGF and BMP9 signalling pathways regulates the osteogenic differentiation of mesenchymal stem cells

Mesenchymal stem cells (MSCs) are multipotent progenitors, which give rise to several lineages, including bone, cartilage and fat. Epidermal growth factor (EGF) stimulates cell growth, proliferation and differentiation. EGF acts by binding with high affinity to epidermal growth factor receptor (EGFR) on the cell surface and stimulating the intrinsic protein tyrosine kinase activity of its receptor, which initiates a signal transduction cascade causing a variety of biochemical changes within the cell and regulating cell proliferation and differentiation. We have identified BMP9 as one of the most osteogenic BMPs in MSCs. In this study, we investigate if EGF signalling cross‐talks with BMP9 and regulates BMP9‐induced osteogenic differentiation. We find that EGF potentiates BMP9‐induced early and late osteogenic markers of MSCs in vitro, which can be effectively blunted by EGFR inhibitors Gefitinib and Erlotinib or receptor tyrosine kinase inhibitors AG‐1478 and AG‐494 in a dose‐ and time‐dependent manner. Furthermore, EGF significantly augments BMP9‐induced bone formation in the cultured mouse foetal limb explants. In vivo stem cell implantation experiment reveals that exogenous expression of EGF in MSCs can effectively potentiate BMP9‐induced ectopic bone formation, yielding larger and more mature bone masses. Interestingly, we find that, while EGF can induce BMP9 expression in MSCs, EGFR expression is directly up‐regulated by BMP9 through Smad1/5/8 signalling pathway. Thus, the cross‐talk between EGF and BMP9 signalling pathways in MSCs may underline their important roles in regulating osteogenic differentiation. Harnessing the synergy between BMP9 and EGF should be beneficial for enhancing osteogenesis in regenerative medicine.     

Bioinformatics analysis of the biological changes involved in the osteogenic differentiation of human mesenchymal stem cells

The mechanisms underlying the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) remain unclear. In the present study, we aimed to identify the key biological processes during osteogenic differentiation. To this end, we downloaded three microarray data sets from the Gene Expression Omnibus (GEO) database: GSE12266, GSE18043 and GSE37558. Differentially expressed genes (DEGs) were screened using the limma package, and enrichment analysis was performed. Protein‐protein interaction network (PPI) analysis and visualization analysis were performed with STRING and Cytoscape. A total of 240 DEGs were identified, including 147 up‐regulated genes and 93 down‐regulated genes. Functional enrichment and pathways of the present DEGs include extracellular matrix organization, ossification, cell division, spindle and microtubule. Functional enrichment analysis of 10 hub genes showed that these genes are mainly enriched in microtubule‐related biological changes, that is sister chromatid segregation, microtubule cytoskeleton organization involved in mitosis, and spindle microtubule. Moreover, immunofluorescence and Western blotting revealed dramatic quantitative and morphological changes in the microtubules during the osteogenic differentiation of human adipose‐derived stem cells. In summary, the present results provide novel insights into the microtubule‐ and cytoskeleton‐related biological process changes, identifying candidates for the further study of osteogenic differentiation of the mesenchymal stem cells.     

Secreted microvesicular miR‐31 inhibits osteogenic differentiation of mesenchymal stem cells

Damage to cells and tissues is one of the driving forces of aging and age‐related diseases. Various repair systems are in place to counteract this functional decline. In particular, the property of adult stem cells to self‐renew and differentiate is essential for tissue homeostasis and regeneration. However, their functionality declines with age (Rando, 2006). One organ that is notably affected by the reduced differentiation capacity of stem cells with age is the skeleton. Here, we found that circulating microvesicles impact on the osteogenic differentiation capacity of mesenchymal stem cells in a donor‐age‐dependent way. While searching for factors mediating the inhibitory effect of elderly derived microvesicles on osteogenesis, we identified miR‐31 as a crucial component. We demonstrated that miR‐31 is present at elevated levels in the plasma of elderly and of osteoporosis patients. As a potential source of its secretion, we identified senescent endothelial cells, which are known to increase during aging in vivo (Erusalimsky, 2009). Endothelial miR‐31 is secreted within senescent cell‐derived microvesicles and taken up by mesenchymal stem cells where it inhibits osteogenic differentiation by knocking down its target Frizzled‐3. Therefore, we suggest that microvesicular miR‐31 in the plasma of elderly might play a role in the pathogenesis of age‐related impaired bone formation and that miR‐31 might be a valuable plasma‐based biomarker for aging and for a systemic environment that does not favor cell‐based therapies whenever osteogenesis is a limiting factor.     

Coexpression of osteogenic and adipogenic differentiation markers in selected subpopulations of primary human mesenchymal progenitor cells

Knowledge of the basic mechanisms controlling osteogenesis and adipogenesis might provide new insights into the prevention of osteoporosis and age-related osteopenia. With the help of magnetic cell sorting and fluorescence activated cell sorting (FACS), osteoblastic subpopulations of mesenchymal progenitor cells were characterized. Alkaline phosphatase (AP) negative cells expressed low levels of osteoblastic and adipocytic markers. AP positive cells expressed adipocytic markers more strongly than the AP negative cell populations, thus suggesting that committed osteoblasts exhibit a greater adipogenic potential. AP negative cells differentiated to the mature osteoblastic phenotype, as demonstrated by increased AP-activity and osteocalcin secretion under standard osteogenic culture conditions. Surprisingly, this was accompanied by increased expression of adipocytic gene markers such as peroxisome proliferator-activated receptor-gamma2, lipoprotein lipase and fatty acid binding protein. The induction of adipogenic markers was suppressed by transforming growth factor-beta1 (TGF-beta1) and promoted by bone morphogenetic protein 2 (BMP-2). Osteogenic culture conditions including BMP-2 induced both the formation of mineralized nodules and cytoplasmic lipid vacuoles. Upon immunogold electron microscopic analysis, osteoblastic and adipogenic marker proteins were detectable in the same cell. Our results suggest that osteogenic and adipogenic differentiation in human mesenchymal progenitor cells might not be exclusively reciprocal, but rather, a parallel event until late during osteoblast development.     

Depletion of HOXA5 inhibits the osteogenic differentiation and proliferation potential of stem cells from the apical papilla

Mesenchymal stem cells (MSCs) are a prospective cell source for tissue regeneration due to their self‐renewal abilities and potential to differentiate into different cell lineages, but the molecular mechanisms of the directed differentiation and proliferation are still unknown. Recently, multiple studies have indicated the crucial role of HOX genes in MSC differentiation and proliferation. However, the role of HOXA5 in MSCs remains unknown. Here, we investigated HOXA5 function in stem cells from the apical papilla (SCAPs). After HOXA5 depletion, the results showed a significant decrease in ALP activity and a weakened mineralization ability of SCAPs. The real‐time RT‐PCR results showed prominently lessened expression of OPN and BSP. The CCK8 and CFSE results displayed inhibited proliferation of SCAPs, and flow cytometry assays revealed arrested cell cycle progression at the S phase. Furthermore, we found that depletion of HOXA5 upregulated p16^INK4A and p18^INK4C and downregulated the Cyclin A. Our research demonstrated that depletion of HOXA5 inhibited osteogenic differentiation and repressed cell proliferation by arresting cell cycle progression at the S phase via p16^INK4A, p18^INK4C, and Cyclin A in SCAPs, indicating that HOXA5 has a significant role in maintaining the proliferation and differentiation potential of dental‐tissue‐derived MSCs.