Adult tissue-derived mesenchymal stem cells (MSCs) were identified almost five decades ago. Their self-renewal ability, differentiation multipotency, and anti-inflammatory and immunomodulatory properties suggest potential roles in tissue responses in diseases, and tissue remodeling and repair. However, how MSCs participate in pathophysiological processes and to what extent MSCs can be applied as immunomodulatory therapeutic agents remain incompletely understood. This article provides an overview of current research activities on the biology of MSCs, their regulation, and their therapeutic potential in autoimmune diseases and allotransplantation.

Adult mesenchymal stem cells are a resource for autologous and allogeneic cell therapies for immune-modulation and regenerative medicine. However, patients most in need of such therapies are often of advanced age. Therefore, the effects of the aged milieu on these cells and their intrinsic aging in vivo are important considerations. Furthermore, these cells may require expansion in vitro before use as well as for future research. Their aging in vitro is thus also an important consideration. Here, we focus on bone marrow mesenchymal stem cells (BMSCs), which are unique compared to other stem cells due to their support of hematopoietic cells in addition to contributing to bone formation. BMSCs may be sensitive to age-related diseases and could perpetuate degenerative diseases in which bone remodeling is a contributory factor. Here, we review (1) the characterization of BMSCs, (2) the characterization of in vivo-aged BMSCs, (3) the characterization of in vitro-aged BMSCs, and (4) potential approaches to optimize the performance of aged BMSCs. This article is part of a Special Issue entitled “Stem Cells and Bone”.

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.

Purpose: Stem cells are an important resource for tissue repair and regeneration. The organization of signaling molecules in the stem cell membrane likely plays an important, but incompletely understood, role in governing stem cell phenotype and responsiveness to external stimuli. We have recently demonstrated that caveolin-1 (Cav-1) acts to inhibit osteogenesis of human bone marrow derived mesenchymal stem cells (MSCs). Cav-1 is a scaffolding protein of caveolae lipid rafts of the plasma membrane with an affinity for many signaling molecules. Others have shown that caveolar endocytosis of activated integrins contributes to the repression of rat MSC osteogenesis on soft matrices, at least partially by the co-internalization of BMP receptors. Given that caveolae are capable of integrin internalization, integrin focal adhesion signaling activates Akt, and Cav-1 is known to bind to Akt, we hypothesize that Cav-1 inhibition of osteogenesis may be mediated via repressing Akt signaling. The purpose of this study was to test the relationship between Akt signaling, Cav-1 and lipid rafts during MSC osteogenesis. Methods: MSCs were isolated from the bone marrow of femoral heads obtained following total hip replacement surgery with IRB approval. MSCs with or without disruption to Cav-1 expression (using siRNA), lipid rafts (using methyl-β-cyclodextrin, MβCD) or PI3K/Akt signaling (using the inhibitor LY294002) were cultured in growth medium with 10% FBS (GM) or GM supplemented with 5 mM β-glycerophosphate, 50 μg/ml ascorbate, 10 nM 1,25-dihydroxyvitamin D3, and 10 nM dexamethasone (OM) to induce osteogenesis. Total cell lysates were collected in RIPA buffer at short time intervals after initial addition of GM or OM to determine Akt activity. Osteogenesis was assessed on the basis of alkaline phosphatase (ALP) activity using p-nitrophenylphosphate substrate on day 4, and by alizarin red staining for matrix mineralization on day 21. Caveolae and non-caveolar lipid rafts were isolated from cells grown for 10 days in GM or OM using a sucrose density centrifugation method. The gradient fractions were harvested and probed for Cav-1 and Akt by western blotting. Results: As previously described, upon culturing in OM, MSCs with siRNA-mediated Cav-1 knockdown showed enhanced osteogenesis, i.e., increased ALP activity and alizarin red staining, compared to control MSCs. Akt phosphorylation was seen 30 min after the addition of OM, but it was unclear if this signaling was enhanced in MSCs with reduced Cav-1 expression due to MSC donor variation. LY294002 completely abolished Akt phosphorylation, and significantly reduced ALP activity and alizarin red staining induced by OM in a dose dependent manner. Pre-treatment of MSCs with MβCD also dose dependently inhibited ALP activity induced by OM. Akt was detected in lipid rafts, more to non-caveolar lipid rafts than to caveolae, and its abundance in lipid rafts seemed to be elevated in osteogenically differentiating MSCs. Conclusion: These results show: (1) the involvement of PI3K/Akt signaling in MSC osteogenesis, because OM induced Akt phosphorylation and osteogenesis were both inhibited by the PI3K inhibitor LY294002; (2) the requirement of membrane cholesterol in MSC osteogenic differentiation, as MβCD, which binds to and removes cholesterol from cell membranes, inhibited osteogenesis; and (3) the concentration of Akt in non-caveolar cholesterol-rich lipid rafts during MSC osteogenesis, because Akt was immunoblot-detected in lipid rafts and appeared more abundant in non-caveolar lipid rafts in response to OM. We are currently investigating the dependence of pro-osteogenic PI3K/Akt signaling on membrane cholesterol, and whether Akt localized to caveolae is inhibited by Cav-1, a known suppressor of osteogenesis. Supported in part by a grant from the Commonwealth of PA Dept of Health

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.

Caveolin-1 is a scaffolding protein of cholesterol-rich caveolae lipid rafts in the plasma membrane. In addition to regulating cholesterol transport, caveolin-1 has the ability to bind a diverse array of cell signaling molecules and regulate cell signal transduction in caveolae. Currently, there is little known about the role of caveolin-1 in stem cells. It has been reported that the caveolin-1 null mouse has an expanded population of cells expressing stem cell markers in the gut, mammary gland, and brain, suggestive of a role for caveolin-1 in stem cell regulation. The caveolin-1 null mouse also has increased bone mass and an increased bone formation rate, and its bone marrow-derived mesenchymal stem cells (MSCs) have enhanced osteogenic potential. However, the role of caveolin-1 in human MSC osteogenic differentiation remains unexplored. In this study, we have characterized the expression of caveolin-1 in human bone marrow derived MSCs. We show that caveolin-1 protein is enriched in density gradient-fractionated MSC plasma membrane, consisting of ~100 nm diameter membrane-bound vesicles, and is distributed in a punctate pattern by immunofluoresence localization. Expression of caveolin-1 increases in MSCs induced to undergo osteogenic differentiation, and siRNA-mediated knockdown of caveolin-1 expression enhances MSC proliferation and osteogenic differentiation. Taken together, these findings suggest that caveolin-1 normally acts to regulate the differentiation and renewal of MSCs, and increased caveolin-1 expression during MSC osteogenesis likely acts as a negative feedback to stabilize the cell phenotype.