Resveratrol mediated modulation of Sirt-1/Runx2 promotes osteogenic differentiation of mesenchymal stem cells: potential role of Runx2 deacetylation.

Osteogenic repair in response to bone injury is characterized by activation and differentiation of mesenchymal stem cells (MSCs) to osteoblasts. This study determined whether activation of Sirt-1 (a NAD(+)-dependent histone deacetylase) by the phytoestrogen resveratrol affects osteogenic differentiation. Monolayer and high-density cultures of MSCs and pre-osteoblastic cells were treated with an osteogenic induction medium with/without the Sirt-1 inhibitor nicotinamide or/and resveratrol in a concentration dependent manner. MSCs and pre-osteoblastic cells differentiated to osteoblasts when exposed to osteogenic-induction medium. The osteogenic response was blocked by nicotinamide, resulting in adipogenic differentiation and expression of the adipose transcription regulator PPAR-γ (peroxisome proliferator-activated receptor). However, in nicotinamide-treated cultures, pre-treatment with resveratrol significantly enhanced osteogenesis by increasing expression of Runx2 (bone specific transcription factor) and decreasing expression of PPAR-γ. Activation of Sirt-1 by resveratrol in MSCs increased its binding to PPAR-γ and repressed PPAR-γ activity by involving its cofactor NCoR (nuclear receptor co-repressor). The modulatory effects of resveratrol on nicotinamide-induced expression of PPAR-γ and its cofactor NCoR were found to be mediated, at least in part, by Sirt-1/Runx2 association and deacetylation of Runx2. Finally, knockdown of Sirt-1 by using antisense oligonucleotides downregulated the expression of Sirt-1 protein and abolished the inhibitory effects of resveratrol, namely nicotinamide-induced Sirt-1 suppression and Runx2 acetylation, suggesting that the acetylated content of Runx2 is related to downregulated Sirt-1 expression. These data support a critical role for Runx2 acetylation/deacetylation during osteogenic differentiation in MSCs in vitro. (242 words in abstract)

Differentiated Stem Cells Derived from Rabbit Adipose Tissue Exhibited in ‎Vitro Adipogenesis and Osteogenesis

The multipotent characteristic of rabbit adipose-derived stem cells makes them available and ‎convenient sources for isolating mesenchymal stem cells. The aim of this study was to assess ‎the differentiation in rabbit adipose-derived stem cells pre-committed to produce several ‎mesenchymal lineages in response to inductive extracellular cues to multipotent stromal cells. ‎Three grams of adipose tissue was taken from a subcutaneous region of the nape of the neck ‎and was carefully isolated to obtain mesenchymal stem cells for expanded by fourth passage. In ‎the 4th passage, active growth of mesenchymal stem cells was observed. Furthermore, the ‎research demonstrated the inherent ability of rabbit MSCs to induce differentiation in ‎osteogenic and adipogenic lineages. These mesenchymal stem cells were successfully isolated ‎from adipose tissue which differentiated into either osteocytes or adipocyte-like cells after 21 ‎and 14 days of culturing in specific osteogenic and adipogenic media, respectively. The ‎remarkable differentiation potential of rabbit mesenchymal stem cells is indicated by ‎mineralized deposition to the osteocytes and lipid droplets accumulated in the cytoplasm lipid ‎vacuoles in the adipocytes‎

Characterization of Mandible and Femur Canine Mesenchymal Stem Cells: A Pilot Study

Mesenchymal stem cells (MSCs) are emerging donor grafts for bone regeneration in dentistry. MSCs are phenotypically and functionally skeletal site- specific based on extensive studies using human and rodent MSCs but there is paucity of information on canine MSCs (cMSCs) and their regenerative applications in veterinary dentistry. We hypothesized that cMSCs are functionally skeletal-site specific and that mandible cMSCs (M-cMSCs) are highly osteogenic relative to femur cMSCs (F-cMSCs). Trabecular bone samples were obtained from mandible and femur of 2 healthy beagle dogs (ages: 3 weeks, females). Primary M-cMSCs and F-cMSCs were established in culture. Using early passage cells, colony-forming units (CFU), cell proliferation and population doubling capacity were assessed. Using established induction culture conditions, in vitro osteogenesis, chondrogenesis, adipogenesis, and neurogenesis were also assessed. Western blotting and real time PCR were used to assess the following osteogenic markers: alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin (OCN) and osteopontin (OPN). Chondrogenesis was assessed using pellet culture method and histologic sections were stained with Alcian blue; adipogenically induced-cultures were stained with Oil Red O. Neural differentiation was evaluated using morphological analysis and immunostaining to nestin and βIII-tubulin antibodies. Furthermore, in vivo osteogenesis was assessed using the mouse model of in vivo bone regeneration. Transplants were harvested at 6, 8 and 12 weeks for histological analysis.The M-cMSCs demonstrated 1.5 to 2 fold increases in cell proliferation (p =0.006) and life span (five more passages of survival) relative to F-cMSCs. Similar pattern was displayed by M-cMSCs based on expression levels of BSP (14 days p=0.05), ALP (14 days p= 0.004) and OCN (14 days p= 0.03) but OPN levels were not significantly different. Adipogenesis based on number of stained lipid droplets per unit area in M-cMSCs was significant higher than F-cMSCs (p=0.007) and chondrogenic response was also significant higher in M-cMSCs compared with F-cMSCs (4 weeks p= 0.009). Canine MSCs induced substantial in vivo bone formation. The canine MSCs phenotypic and functional properties are site-dependent as the M-cMSCs were apparently more responsive to multi-lineage differentiation relative to F-cMSCs. While the sample size in this study is limited, our findings are still consistent with previous studies using human, mouse and rat MSCs for site-to-site comparative characterizations (Akintoye et al, 2006; Yoshimura et al, 2007; Aghaloo et al, 2010; Lee et al, 2011). Additionally, it is imperative to further confirm these in a larger sample size and in other dog breeds since dogs exhibit an extremely wide range of body physique. New information will advance our understanding of pre-clinical applications of orofacial MSCs as donor graft materials for oral bone regeneration

Differential properties of human ACL and MCL stem cells may be responsible for their differential healing capacity

<p>Abstract</p> <p>Background</p> <p>The human anterior cruciate ligament (hACL) and medial collateral ligament (hMCL) of the knee joint are frequently injured, especially in athletic settings. It has been known that, while injuries to the MCL typically heal with conservative treatment, ACL injuries usually do not heal. As adult stem cells repair injured tissues through proliferation and differentiation, we hypothesized that the hACL and hMCL contain stem cells exhibiting unique properties that could be responsible for the differential healing capacity of the two ligaments.</p> <p>Methods</p> <p>To test the above hypothesis, we derived ligament stem cells from normal hACL and hMCL samples from the same adult donors using tissue culture techniques and characterized their properties using immunocytochemistry, RT-PCR, and flow cytometry.</p> <p>Results</p> <p>We found that both hACL stem cells (hACL-SCs) and hMCL stem cells (hMCL-SCs) formed colonies in culture and expressed stem cell markers nucleostemin and stage-specific embryonic antigen-4 (SSEA-4). Moreover, both hACL-SCs and hMCL-SCs expressed CD surface markers for mesenchymal stem cells, including CD44 and CD90, but not those markers for vascular cells, CD31, CD34, CD45, and CD146. However, hACL-SCs differed from hMCL-SCs in that the size and number of hACL-SC colonies in culture were much smaller and grew more slowly than hMCL-SC colonies. Moreover, fewer hACL-SCs in cell colonies expressed stem cell markers STRO-1 and octamer-binding transcription factor-4 (Oct-4) than hMCL-SCs. Finally, hACL-SCs had less multi-differentiation potential than hMCL-SCs, evidenced by differing extents of adipogenesis, chondrogenesis, and osteogenesis in the respective induction media.</p> <p>Conclusions</p> <p>This study shows for the first time that hACL-SCs are intrinsically different from hMCL-SCs. We suggest that the differences in their properties contribute to the known disparity in healing capabilities between the two ligaments.</p

Characterization and comparison of cell frequency, growth, and multipotential differentiation of adult mesenchymal stromal cells derived from equine bone marrow and adipose tissue

Equine bone marrow-derived mesenchymal stromal cells (MSCs) and adipose tissue-derived stromal cells (ASCs) were compared for frequency within their respective tissues, cell doubling characteristics and differentiation multipotential in culture based on histochemical staining and compositional analysis. Equine MSCs and ASCs from young adult horses were harvested and isolated from sternal bone marrow and supragluteal subcutaneous adipose tissue, respectively, and grown up to passage 10 (P10) to determine cell doubling characteristics. Limit dilution assays were performed on primary and passaged (P2, P4) MSCs and ASCs to determine the frequency of colony forming units with a fibroblastic phenotype (CFU-F), and the frequency of MSC differentiation into adipocytes (CFU-Ad) and osteoblasts (CFU-Ob). Pellet cultures of MSCs and ASCs at P2 were performed in chondrogenic media with or without transforming growth factor (TGF-â3) and bone morphogenic protein (BMP-6). Collagen type II expression, glycosaminoglycan and DNA content and pellet size were measured. Primary MSCs doubled more slowly than subsequent MSC passages (DT = 4.9 ± 1.6 days compared to 1.4 ± 0.22 days). Doubling time of ASCs (2.1 ± 0.9 days) was significantly slower than that of MSCs. Primary MSC frequency was 1 in 4,224 ± 3,265 nucleated BM cells while the frequency of ASC was 1 in 2.3 ± 0.4 nucleated stromal vascular fraction cells. Primary and subcultured MSCs showed robust adipogenic and osteogenic differentiation potential. MSC pellet cultures developed collagen type II expression by Day 7 and hyaline matrix by Day 14. ASC pellets only exhibited mild matrix or collagen formation under electron microscopic examination but showed no immunohistochemical expression of collagen type II. MSC pellets supplemented with growth factors were larger (p \u3c0.0033) and showed significant increases in GAG concentration by Day 14 compared with all other pellets of both cell types (P\u3c.0001). The frequency, in vitro growth rate, and adipogenic and osteogenic differentiation potential of young adult horses are similar to those documented for MSCs of other species, whereas in vitro growth rate of ASCs differs from human ASCs. MSCs show earlier osteogenesis compared with ASCs and more robust chondrogenesis in the presence or absence of human recombinant TGFâ3 and BMP6

Effects of nutrients, mainly from mediterranean dietary foods, on mesenchymal stem derived cells: growth or differentiation

During the last decade the interest for the mesenchymal cells is growing due to their possible uses in therapies to treat certain degenerative pathologies. Mesenchymal stem cells have been found in the bone marrow and they have been shown to be responsible for bone repair and fat cells production. Mesenchymal stromal cells can be obtained from a wide variety of tissues in addition to bone marrow and can differentiate into many other cell types. The study of cell differentiation and programming provides new models for drug discovery and cell therapy that now overcomes gene therapy. Senescence, cancer development and degenerative diseases depend on mesenchymal cells contribution to tissue homeostasis. On the other hand, diet and life style are included among risk factors, which can contribute to the success of pharmacological treatments. This review focuses on nutrients from Mediterranean diet and supplements, which have been shown to influence mesenchymal stem cells and cells derived from them. Dietary intake of nutrients impairs both in vitro and in vivo observations, this review aims to gather the results about the effects of food compounds on mesenchymal cells from which adipocytes and osteoblasts derive. Amino acids and proteins, carbohydrates, lipids, fatty acids and vegetable secondary metabolites, differently act on mesenchymal cells bearing on modulation of gene expression and controlling the fate of cell lineages. Remarkable, the analysis of literature shows that the main effect of nutrients on mesenchymal cells is the stimulation of transcription factors which address the cells toward proliferation or differentiation. For instance, carbohydrates, simple or complex, and lipids appear to stimulate the PPAR receptors, whereas proteins and amino acids result to act on the mTOR system and they can also stimulate the MyoD-1 transcription factor and cooperating proteins. In conclusion, nutrients can promote cell growth and differentiation of mesenchymal cells

Characterization and differentiation potential of mesenchymal stem cells isolated from multiple canine adipose tissue sources

Background Mesenchymal stem cells (MSCs) are undifferentiated cells that can give rise to a mesoderm lineage. Adipose-derived MSCs are an easy and accessible source for MSCs isolation, although each source of MSC has its own advantages and disadvantages. Our study identifies a promising source for the isolation and differentiation of canines MSCs. For this purpose, adipose tissue from inguinal subcutaneous (SC), perirenal (PR), omental (OM), and infrapatellar fat pad (IPFP) was isolated and processed for MSCs isolation. In the third passage, MSCs proliferation/metabolism, surface markers expression, in vitro differentiation potential and quantitative reverse transcription PCR (CD73, CD90, CD105, PPARγ, FabP4, FAS, SP7, Osteopontin, and Osteocalcin) were evaluated. Results Our results showed that MSCs derived from IPFP have a higher proliferation rate, while OM-derived MSCs have higher cell metabolism. In addition, MSCs from all adipose tissue sources showed positive expression of CD73 (NT5E), CD90 (THY1), CD105 (ENDOGLIN), and very low expression of CD45. The isolated canine MSCs were successfully differentiated into adipogenic and osteogenic lineages. The oil-red-O quantification and adipogenic gene expression (FAS, FabP4, and PPARγ) were higher in OM-derived cells, followed by IPFP-MSCs. Similarly, in osteogenic differentiation, alkaline phosphatase activity and osteogenic gene (SP7 and Osteocalcin) expression were higher in OM-derived MSCs, while osteopontin expression was higher in PR-derived MSCs. Conclusion In summary, among all four adipose tissue sources, OM-derived MSCs have better differentiation potential toward adipo- and osteogenic lineages, followed by IPFP-MSCs. Interestingly, among all adipose tissue sources, MSCs derived from IPFP have the maximum proliferation potential. The characterization and differentiation potential of canine MSCs isolated from four different adipose tissue sources are useful to assess their potential for application in regenerative medicine

Differentiation Potential of the Human Adipose Stem Cells in Response to Regulation of ROCK, FAK and MEK-ERK Signaling Pathways

Background and aims: Adipose stem cells (ASCs), obtained from adipose tissue, are a promising cell source for bone tissue engineering applications. ASCs exhibit stable growth and proliferation in vitro and they possess multi-lineage differentiation capacity into various cell lineages including adipocytes, osteoblasts and chondrocytes. The differentiation process of mesenchymal stem cells (MSC) is known to be regulated through cell attachment and morphology, however, the intracellular details of the regulation remain unidentified. The aim of this study was to enlighten the signaling events in cell attachment or morphology -mediated differentiation in hASCs. The significance of Rho-kinase ROCK), Focal adhesion kinase (FAK) and Mitogen-activated protein kinase/Extracellular signal-regulated kinase (MEK-ERK) signaling to hASC differentiation potential towards osteoblasts and adipocytes were analyzed. Methods: To assess the significance of ROCK, FAK and MEK-ERK signaling pathways to hASC differentiation, specific inhibitor molecules targeted to these cascades were used: ROCK1 inhibitor Y-27632 2HCl, FAK inhibitor PF-562271 and MEK inhibitor PD98059. The inhibitor effect on cell viability and proliferation were assessed with LIVE/DEAD® assay and CyQUANT® assay, respectively. The hASC adipogenic differentiation was determined by Oil Red O staining, and the osteogenic differentiation was assessed by quantitative alkaline phosphatase assay (qALP), and qualitative and quantitative Alizarin Red staining. The intracellular protein activation was evaluated with Western Blot analysis method. Results: The results indicated that cell attachment to the culture platform and to the extracellular matrix (ECM) is critical for cell viability, proliferation and induction of the osteogenic differentiation of hASCs. Disruption of the cell adhesion with FAK or ROCK inhibition suppressed the osteogenic differentiation. Cell morphology guided hASC lineage commitment: spread cytoskeleton induced osteogenesis while rounded shape favoured commitment to adipogenesis. ROCK was found to be a positive regulator of osteogenesis and the inhibition of ROCK enhanced adipogenesis in the hASC lines studied. Also functional MEK-ERK pathway was required for various intracellular processes regulating cell proliferation and differentiation in hASCs. Conclusions: Taken together, the cell adhesion is an essential part and a prerequisite of many intracellular functions in hASC differentiation. Based on this study, ROCK, FAK and MEK-ERK signaling cascades were involved in the osteogenic differentiation of hASCs. An active ROCK protein worked as a negative regulator of adipogenesis, representing the influence of cell morphology to the differentiation potential of hASCs. The results indicate, that the differentiation mechanisms of hASCs could be supported by enhancing the cell adhesion and regulating the morphology of the cells