Endothelial and cancer cells interact with mesenchymal stem cells via both microparticles and secreted factors

Tightly associated with blood vessels in their perivascular niche, human mesenchymal stem cells (MSCs) closely interact with endothelial cells (ECs). MSCs also home to tumours and interact with cancer cells (CCs). Microparticles (MPs) are cell-derived vesicles released into the extracellular environment along with secreted factors. MPs are capable of intercellular signalling and, as biomolecular shuttles, transfer proteins and RNA from one cell to another. Here, we characterize interactions among ECs, CCs and MSCs via MPs and secreted factors in vitro. MPs and non-MP secreted factors (Sup) were isolated from serum-free medium conditioned by human microvascular ECs (HMEC-1) or by the CC line HT1080. Fluorescently labelled MPs were prepared from cells treated with membrane dyes, and cytosolic GFP-containing MPs were isolated from cells transduced with CMV-GFP lentivirus. MSCs were treated with MPs, Sup, or vehicle controls, and analysed for MP uptake, proliferation, migration, activation of intracellular signalling pathways and cytokine release. Fluorescently labelled MPs fused with MSCs, transferring the fluorescent dyes to the MSC surface. GFP was transferred to and retained in MSCs incubated with GFP-MPs, but not free GFP. Thus, only MP-associated cellular proteins were taken up and retained by MSCs, suggesting that MP biomolecules, but not secreted factors, are shuttled to MSCs. MP and Sup treatment significantly increased MSC proliferation, migration, and MMP-1, MMP-3, CCL-2/MCP-1 and IL-6 secretion compared with vehicle controls. MSCs treated with Sup and MPs also exhibited activated NF-κB signalling. Taken together, these results suggest that MPs act to regulate MSC functions through several mechanisms

Promotion of human mesenchymal stem cell osteogenesis by PI3-kinase/Akt signaling, and the influence of caveolin-1/cholesterol homeostasis

Introduction: Stem cells are considered an important resource for tissue repair and regeneration. Their utilization in regenerative medicine will be aided by mechanistic insight into their responsiveness to external stimuli. It is likely that, similar to all other cells, an initial determinant of stem cell responsiveness to external stimuli is the organization of signaling molecules in cell membrane rafts. The clustering of signaling molecules in these cholesterol-rich membrane microdomains can affect the activity, specificity, cross-talk and amplification of cell signaling. Membrane rafts fall into two broad categories, non-caveolar and caveolar, based on the absence or presence, respectively, of caveolin scaffolding proteins. We have recently demonstrated that caveolin-1 (Cav-1) expression increases during, and knockdown of Cav-1 expression enhances, osteogenic differentiation of human bone marrow derived mesenchymal stem cells (MSCs). The increase in Cav-1 expression observed during osteogenesis is likely a negative feedback mechanism. We hypothesize that focal adhesion signaling pathways such as PI3K/Akt signaling may be negatively regulated by Cav-1 during human MSC osteogenesis. Methods: Human bone marrow MSCs were isolated from femoral heads obtained after total hip arthroplasty. MSCs were incubated in standard growth medium alone or induced to osteogenically differentiate by the addition of supplements (β-glycerophosphate, ascorbic acid, dexamethasone, and 1,25-dihydroxyvitamin D3). The activation of and requirement for PI3K/Akt signaling in MSC osteogenesis were assessed by immunoblotting for phosphorylated Akt, and treatment with the PI3K inhibitor LY294002 and Akt siRNA, respectively. The influences of Cav-1 and cholesterol membrane rafts on PI3K/Akt signaling were investigated by treatment with Cav-1 siRNA, methyl-β-cyclodextrin, or cholesterol oxidase, followed by cellular sub-fractionation and/or immunoblotting for phosphorylated Akt. Results: LY294002 and Akt siRNA inhibited MSC osteogenesis. Methyl-β-cyclodextrin, which disrupts all membrane rafts, inhibited osteogenesis. Conversely, Cav-1 siRNA and cholesterol oxidase, which displaces Cav-1 from caveolae, enhanced Akt signaling induced by osteogenic supplements. In control cells, phosphorylated Akt began to accumulate in caveolae after 10 days of osteogenic differentiation. Conclusions: PI3K/Akt signaling is a key pathway required for human MSC osteogenesis, and it is likely that localization of active Akt in non-caveolar and caveolar membrane rafts positively and negatively contributes to osteogenesis, respectively

The less-often-traveled surface of stem cells: Caveolin-1 and caveolae in stem cells, tissue repair and regeneration

Stem cells are an important resource for tissue repair and regeneration. While a great deal of attention has focused on derivation and molecular regulation of stem cells, relatively little research has focused on how the subcellular structure and composition of the cell membrane influences stem cell activities such as proliferation, differentiation and homing. Caveolae are specialized membrane lipid rafts coated with caveolin scaffolding proteins, which can regulate cholesterol transport and the activity of cell signaling receptors and their downstream effectors. Caveolin-1 is involved in the regulation of many cellular processes, including growth, control of mitochondrial antioxidant levels, migration and senescence. These activities are of relevance to stem cell biology, and in this review evidence for caveolin-1 involvement in stem cell biology is summarized. Altered stem and progenitor cell populations in caveolin-1 null mice suggest that caveolin-1 can regulate stem cell proliferation, and in vitro studies with isolated stem cells suggest that caveolin-1 regulates stem cell differentiation. The available evidence leads us to hypothesize that caveolin-1 expression may stabilize the differentiated and undifferentiated stem cell phenotype, and transient downregulation of caveolin-1 expression may be required for transition between the two. Such regulation would probably be critical in regenerative applications of adult stem cells and during tissue regeneration. We also review here the temporal changes in caveolin-1 expression reported during tissue repair. Delayed muscle regeneration in transgenic mice overexpressing caveolin-1 as well as compromised cardiac, brain and liver tissue repair and delayed wound healing in caveolin-1 null mice suggest that caveolin-1 plays an important role in tissue repair, but that this role may be negative or positive depending on the tissue type and the nature of the repair process. Finally, we also discuss how caveolin-1 quiescence-inducing activities and effects on mitochondrial antioxidant levels may influence stem cell aging. © 2013 BioMed Central Ltd

Biology of platelet-rich plasma and its clinical application in cartilage repair

Platelet-rich plasma (PRP) is an autologous concentrated cocktail of growth factors and inflammatory mediators, and has been considered to be potentially effective for cartilage repair. In addition, the fibrinogen in PRP may be activated to form a fibrin matrix to fill cartilage lesions, fulfilling the initial requirements of physiological wound healing. The anabolic, anti-inflammatory and scaffolding effects of PRP based on laboratory investigations, animal studies, and clinical trials are reviewed here. In vitro, PRP is found to stimulate cell proliferation and cartilaginous matrix production by chondrocytes and adult mesenchymal stem cells (MSCs), enhance matrix secretion by synoviocytes, mitigate IL-1β-induced inflammation, and provide a favorable substrate for MSCs. In preclinical studies, PRP has been used either as a gel to fill cartilage defects with variable results, or to slow the progression of arthritis in animal models with positive outcomes. Findings from current clinical trials suggest that PRP may have the potential to fill cartilage defects to enhance cartilage repair, attenuate symptoms of osteoarthritis and improve joint function, with an acceptable safety profile. Although current evidence appears to favor PRP over hyaluronan for the treatment of osteoarthritis, the efficacy of PRP therapy remains unpredictable owing to the highly heterogeneous nature of reported studies and the variable composition of the PRP preparations. Future studies are critical to elucidate the functional activity of individual PRP components in modulating specific pathogenic mechanisms. © 2014 Xie et al.; licensee BioMed Central Ltd

Three years of stem cell research & therapy

Editorial\ud \ud This year we celebrate three years of publication of Stem Cell Research & Therapy. In this short space of time, as the number of journals in the stem cell and regenerative medicine field has grown and continues to grow dramatically, Stem Cell Research & Therapy has become established as the destination for high quality open access research into stem cell therapeutics

Cartilage stem/progenitor cells are activated in osteoarthritis via interleukin-1β/nerve growth factor signaling

Introduction: Interleukin-1β (IL-1β) and nerve growth factor (NGF) are key regulators in the pathogenesis of inflammatory arthritis; specifically, IL-1β is involved in tissue degeneration and NGF is involved in joint pain. However, the cellular and molecular interactions between IL-1β and NGF in articular cartilage are not known. Cartilage stem/progenitor cells (CSPCs) have recently been identified in osteoarthritic (OA) cartilage on the basis of their migratory properties. Here we hypothesize that IL-1β/NGF signaling is involved in OA cartilage degeneration by targeting CSPCs. Method: NGF and NGF receptor (NGFR: TrkA and p75NTR) expression in healthy and OA human articular cartilage and isolated chondrocytes was determined by immunostaining, qRT-PCR, flow cytometry and western blot. Articular cartilage derived stem/progenitor cells were collected and identified by stem/progenitor cell characteristics. 3D-cultured CSPC pellets and cartilage explants were treated with NGF and NGF neutralizing antibody, and extracellular matrix changes were examined by sulfated glycosaminoglycan (GAG) release and MMP expression and activity. Results: Expression of NGF, TrkA and p75NTR was found to be elevated in human OA cartilage. Cellular changes upon IL-1β and/or NGF treatment were then examined. NGF mRNA and NGFR proteins levels were upregulated in cultured chondrocytes exposed to IL-1β. NGF was chemotactic for cells isolated from OA cartilage. Cells isolated on the basis of their chemotactic migration towards NGF demonstrated stem/progenitor cell characteristics, including colony-forming ability, multi-lineage differentiation potential, and stem cell surface markers. The effects of NGF perturbation in cartilage explants and 3D-cultured CSPCs were next analyzed. NGF treatment resulted in extracellular matrix catabolism indicated by increased sGAG release and MMP expression and activity; conversely, treatment with NGF neutralizing antibody inhibited increased MMP levels, and enhanced tissue inhibitor of matrix metalloprotease-1 (TIMP1) expression in OA cartilage explants. NGF blockade with neutralizing antibody also affected cartilage matrix remodeling in 3D-CSPC pellet cultures. Conclusion: Our results strongly suggest that NGF signaling is a contributing factor in articular cartilage degeneration in OA, which likely targets a specific subpopulation of progenitor cells, the CSPCs, affecting their migratory and matrix remodeling activities. These findings provide novel cellular/signaling therapeutic targets in osteoarthritic cartilage

Stem cell- and growth factor-based regenerative therapies for avascular necrosis of the femoral head

Avascular necrosis (AVN) of the femoral head is a debilitating disease of multifactorial genesis, predominately affects young patients, and often leads to the development of secondary osteoarthritis. The evolving field of regenerative medicine offers promising treatment strategies using cells, biomaterial scaffolds, and bioactive factors, which might improve clinical outcome. Early stages of AVN with preserved structural integrity of the subchondral plate are accessible to retrograde surgical procedures, such as core decompression to reduce the intraosseous pressure and to induce bone remodeling. The additive application of concentrated bone marrow aspirates, ex vivo expanded mesenchymal stem cells, and osteogenic or angiogenic growth factors (or both) holds great potential to improve bone regeneration. In contrast, advanced stages of AVN with collapsed subchondral bone require an osteochondral reconstruction to preserve the physiological joint function. Analogously to strategies for osteochondral reconstruction in the knee, anterograde surgical techniques, such as osteochondral transplantation (mosaicplasty), matrix-based autologous chondrocyte implantation, or the use of acellular scaffolds alone, might preserve joint function and reduce the need for hip replacement. This review summarizes recent experimental accomplishments and initial clinical findings in the field of regenerative medicine which apply cells, growth factors, and matrices to address the clinical problem of AVN. © 2012 BioMed Central Ltd

Effect of urbanization on bone mineral density: A Thai epidemiological study

BACKGROUND: The incidence of fractures in rural populations is lower than in urban populations, although the reason for this difference is unclear. This cross-sectional study was designed to examine the difference in bone mineral density (BMD), a primary predictor of fracture risk, between urban and rural Thai populations. METHODS: Femoral neck and lumbar spine BMD was measured by dual-energy X-ray absorptiometry (GE Lunar, Madison, WI) in 411 urban and 436 rural subjects (340 men and 507 women), aged between 20 and 84 years. Body mass index (BMI) was calculated from weight and height. RESULTS: After adjusting for age and body weight in an analysis of covariance model, femoral neck BMD in rural men and women was significantly higher than those in urban men and women (P < 0.001), but the difference was not observed at the lumbar spine. After stratifying by sex, age group, and BMI category, the urban-rural difference in femoral neck BMD became more pronounced in men and women aged <50 years and with BMI ≥ 25 kg/m(2). CONCLUSIONS: These data suggest that femoral neck BMD in rural men and women was higher than their counterparts in urban areas. This difference could potentially explain part of the urban-rural difference in fracture incidence