Suspension cultures of bone-marrow-derived mesenchymal stem cells: effects of donor age and glucose level.

Both ageing and diabetes are associated with reduced numbers and functional viability of mesenchymal stem cells (MSCs) in vivo which in turn lead to degenerative pathologies of the musculoskeletal system. The overall aim of this study was to elucidate the effects of age and raised glucose levels on the proliferation and self-renewal of rat nonadherent bone marrow MSCs (Na-BM-MSCs) in suspension cultures. MSC cultures isolated from 3- and 12-month-old rats were maintained using the "pour-off" method for up to 14 days in media containing different glucose levels and the phenotype, growth characteristics, colony forming unit-fibroblastic (CFU-f) numbers, and pluripotency characteristics of these cells were determined. This study indicates that rat adult bone marrow harbors pluripotent Na-BM-MSCs that seem to be unaffected by ageing during in vitro expansion. The Na-BM-MSCs express the pluripotency markers Oct4, Sox2, and Nanog. It was found that culture in high-glucose-containing medium had a negative effect on colony formation and differentiation. In contrast to classical MSC cultures, the generation of colonies by Na-BM-MSCs in suspension culture was not reduced in the older animals. The Na-BM-MSCs were found to express the pluripotency markers Oct4, Sox2, and Nanog, suggesting a more primitive stage of differentiation as compared with adherent MSCs. These data indicate that rat adult bone marrow harbors a population of pluripotent Na-BM-MSCs that appear to be relatively unaffected by ageing during in vitro expansion in suspension.

Cytocentrifugation: a convenient and efficient method for seeding tendon-derived cells into monolayer cultures or 3-D tissue engineering scaffolds.

Tendon and ligament injuries are very common, requiring some 200,000 reconstructions per year in the USA. Autografting can be used to repair these but donor tissue is limited and harvesting leads to morbidity at the graft sites. Tissue engineering has been used to grow simple tissues such as skin, cartilage and bone and due to their low vascularity and simple structure, tendons should be ideal candidates for such an approach. Scaffolds are essential for tissue engineering as they provide structure and signals that regulate growth. However, they present a physical barrier to cell seeding with the majority of the cells congregating at the scaffold surface. To address this we used centrifugation to enhance penetration of tendon-derived cells to the centres of 3-D scaffolds. The process had no apparent deleterious effects on the cells and both plating efficiency and cell distribution improved. After attachment the cells continued to proliferate and deposit a collagenous matrix. Scaffold penetration was investigated using layers of Azowipes allowing the separation and examination of individual leaves. At relatively low g-forces, cells penetrated a stack of 6 Azowipes leaving cells attached to each leaf. These data suggest that cytocentrifugation improves the penetration and homogeneity of tendon derived cells in 3-D and monolayer cultures.

Use of rapidly mineralising osteoblasts and short periods of mechanical loading to accelerate matrix maturation in 3D scaffolds.

MLO-A5 cells are a fully differentiated osteoblastic cell line with the ability to rapidly synthesise mineralised extracellular matrix (ECM). We used MLO-A5 cells to develop a system for studying the mechanical modulation of bone matrix formation in 3D using a cyclic compressive loading stimulus. Polyurethane (PU) open cell foam scaffolds were seeded with MLO-A5 cells under static conditions and loaded in compression at 1 Hz, 5% strain in a sterile fluid-filled chamber. Loading was applied for only 2 h per day on days 5, 10 and 15 of culture and cell-seeded scaffolds were assayed on days 10, 15 and 20 of culture. Collagen content as assayed by Sirius red was significantly (2 fold) higher at days 15 and 20 in loaded samples compared with static controls. Calcium content as assayed by alizarin red was significantly (4 fold) higher by day 20. The number of viable cells as assayed by MTS was higher in loaded samples at day 10 but there was no difference by days 15 and 20. Loaded samples also had higher stiffness in compression by the end of the experiment. The mRNA expression of type I collagen, osteopontin and osteocalcin was higher, after a single bout of loading, in loaded than in non-loaded samples as assayed by RT-PCR. In conclusion, mineralisation by fully differentiated osteoblasts, MLO-A5s, was shown to be highly sensitive to mechanical loading, with short bouts of mechanical loading having a strong effect on mineralised matrix production. The 3D system developed will be useful for systematic investigation of the modulators of in vitro matrix mineralisation by osteoblasts in mechanobiology and tissue engineering studies.

Stressed stem cells: Temperature response in aged mesenchymal stem cells.

Mesenchymal stem cells (MSCs) derived from young (6 week) and aged (56 week) Wistar rats were cultured at standard (37 degrees C) and reduced (32 degrees C) temperature and compared for age markers and stress levels. (ROS, NO, TBARS, carbonyls, lipofuscin, SOD, GPx, apoptosis, proteasome activity) and heat shock proteins (HSP27, -60, -70, -90). Aged MSCs display many of the stress markers associated with aging in other cell types, but results vary across marker categories and are temperature dependant. In young MSCs, culturing at reduced temperature had a generally beneficial effect: the anti-apoptotic heat shock proteins HSP 27, HSP70, and HSP90 were up-regulated; pro-apoptotic HSP60 was downregulated; SOD, GPx increased; and levels in ROS, NO, TBARS, carbonyl, and lipofuscin were diminished. Apoptosis was reduced, but also proteasome activity. In contrast, in aged MSCs, culturing at reduced temperature generally produced no 'beneficial' changes in these parameters, and can even have detrimental effects. Implications for tissue engineering and for stem cell gerontology are discussed. The results suggest that a 'hormesis' theory of stress response can be extended to MSCs, but that cooling cultivation temperature stress produces positive effects in young cells only.

Effect of reduced culture temperature on antioxidant defences of mesenchymal stem cells.

Mesenchymal stem cells (MSC) promise to be valuable therapeutic tools but, due to their low numbers, require considerable in vitro expansion before use. This leads to in vitro aging, the accumulation of intracellular oxidative damage, and subsequently a decreased potential for proliferation and differentiation. Optimised culture conditions might help to reduce oxidative damage in MSC in vitro, and therefore, as reduced temperature is known to reduce oxidative stress in other somatic cells, we have investigated the effect of reduced temperature on rat MSC viability, differentiation, and oxidative damage. Temperature reduction did not affect MSC viability but increased differentiation and reduced apoptosis. Oxidative-damage-related indices were improved; reactive oxide species, nitric oxide, thiobarbituric acid reactive substances, carbonyl, and lipofuscin levels were reduced and glutathione peroxidase and superoxide dimutase levels increased. Levels of antiapoptotic heat shock proteins (HSP-27, -70, and -90) were raised and levels of the proapoptotic HSP-60 reduced. These data demonstrate that culturing MSC at reduced temperature decreases the accumulation of oxidative damage and therefore would probably improve long-term viability and successful engraftment of MSC used for tissue engineering or cell therapeutic purposes.

Age-related impairment of mesenchymal progenitor cell function.

In most mesenchymal tissues a subcompartment of multipotent progenitor cells is responsible for the maintenance and repair of the tissue following trauma. With increasing age, the ability of tissues to repair themselves is diminished, which may be due to reduced functional capacity of the progenitor cells. The purpose of this study was to investigate the effect of aging on rat mesenchymal progenitor cells. Mesenchymal progenitor cells were isolated from Wistar rats aged 3, 7, 12 and 56 weeks. Viability, capacity for differentiation and cellular aging were examined. Cells from the oldest group accumulated raised levels of oxidized proteins and lipids and showed decreased levels of antioxidative enzyme activity. This was reflected in decreased fibroblast colony-forming unit (CFU-f) numbers, increased levels of apoptosis and reduced proliferation and potential for differentiation. These data suggest that the reduced ability to maintain mesenchymal tissue homeostasis in aged mammals is not purely due to a decline in progenitor cells numbers but also to a loss of progenitor functionality due to the accumulation of oxidative damage, which may in turn be a causative factor in a number of age-related pathologies such as arthritis, tendinosis and osteoporosis.

Glucose-induced replicative senescence in mesenchymal stem cells.

Mesenchymal stem cells (MSCs) show great promise for use in a variety of cell-based therapies. Because isolated primary mesenchymal stem cells are low in numbers, in vitro expansion is necessary. However, the expansion potential is limited and in vitro aging leads to loss of multipotency and replicative senescence. Stress induced by culture conditions is likely to be a major cause of replicative senescence and reduced multipotency of MSC and optimization of culture conditions might be able to reduce this. Caloric restriction (CR) is the only established method to delay aging and extend lifespan. In vitro caloric restriction experiments are rare, but have demonstrated beneficial effects. Therefore, we investigated the effect of culture medium glucose concentration on the proliferative and differentiation potential of mesenchymal stem cells. Reduction in glucose concentrations led to decreased apoptosis and an increased rate of MSC proliferation and increased the number and size of fibroblastic colonies in the colony-forming unit assay.

hPTH-fragments (53-84) and (28-48) antagonize the stimulation of calcium release and repression of alkaline phosphatase activity by hPTH-(1-34) in vitro.

Different C-terminal fragments of parathyroid hormone (PTH)-(1-84) in blood participate in the regulation of calcium homeostasis by PTH-(1-84), and an antagonizing effect for the large carboxyl-terminal parathyroid hormone (C-PTH)-fragment (7-84) on calcium release has been described in vivo and in vitro. In this study the smaller C-PTH-fragment (53-84) and mid-regional PTH fragment (28-48), which represent discrete areas of activity in the PTH-(7-84) molecule, were assayed for their effects on calcium release and alkaline phosphatase (ALP) activity in a chick bone organ culture system. Neither PTH-(28-48) nor PTH-(53-84) had any effect on calcium release into the medium and both fragments stimulated ALP activity in the bone tissue, suggesting that the cAMP/PKA signalling pathway was not affected by these fragments. However they suppressed the calcium release induced by PTH-(1-34) and attenuated the down regulation of ALP activity caused by PTH-(1-34), suggesting that the effect on the cAMP/PKA signalling pathway may be indirectly. In conclusion, the study shows that the PTH-fragments (53-84) and (28-48) antagonize the PTH-(1-34) induced effects on calcium release and inhibition of ALP activity in a chick bone organ culture system.

Glucocorticoids inhibit tenocyte proliferation and Tendon progenitor cell recruitment.

Corticosteroid injection is commonly used to treat tendon injuries but is often associated with tendon rupture and impaired tendon healing. The effects of dexamethasone on tenocytes have been studied in vitro but only using high concentrations of dexamethasone in monolayer cultures of tenocytes over short periods of time. We have therefore investigated the effects of physiological and pharmacological concentrations of dexamethasone on monolayer cultures of tenocytes over extended time periods. We have also used fibroblastic-colony forming unit cultures to examine the effects of dexamethasone on a progenitor cell population located in tendons. Culturing tenocytes in the presence of dexamethasone for a period of 24 days resulted in a concentration-related decrease in cell number and collagen synthesis as compared to control cultures. This effect was time dependent with cell number in both dexamethasone-treated and control cultures leveling off after 14 days with the control cultures reaching higher cell densities. In contrast in control cultures, collagen accumulation continued to increase until week 4, whereas in the presence of dexamethasone, this tended to level off after 14 days. To study the role of progenitor cell recruitment, the effects of dexamethasone were investigated using the fibroblastic-colony forming unit assay. Treatment with dexamethasone at concentrations of 0.1 nM to 10 microM leads to a progressive reduction in mean colony size as compared to control cultures. Colony number remained constant at concentrations below 10 nM but fell progressively at concentrations above this. In conclusion, dexamethasone reduces both cell number and collagen synthesis in tenocyte cultures in a concentration-dependent manner by both direct effects on tenocyte proliferation and collagen accumulation, and also by modulating the recruitment of tendon progenitor cells.

Aging of mesenchymal stem cells.

The role of adult mesenchymal stem cells (MSC) in tissue maintenance and regeneration has received significant attention of late. Questions arise to what extent these cells are either subject to, or causes of aging; whether age-related changes in these cells are due to intrinsic factors or induced by the somatic environment. This review collates and examines recent data in support of these different theories. By means of introduction, a brief overview is given of current MSC definitions and their basic role in tissue regeneration followed by a comparative analysis of gerontological studies involving MSC. Evidence for extrinsic aging and various aging markers relating to morphology, proliferation, signalling, senescence markers, telomeres and telomerase, and other indicators is discussed. We observe that while the literature might often appear to conflict, many apparent discrepancies are attributable to inconsistent methods of extracting and isolating MSC which in fact contains various subsets of adult stem cells, varying not only in their differentiation potential but also in their vulnerability to senescence--ranging from quasi-somatic lifespan to perennial vigour. Thus, mesenchymal stem cells emerge as both subject to and key mediators of organismal aging.

Megakaryocyte-bone marrow stromal cell aggregates demonstrate increased colony formation and alkaline phosphatase expression in vitro.

Bone marrow stromal cells (BMSCs) possess certain stem celllike properties and can differentiate to adopt a number of mesenchymal phenotypes. BMSCs are usually investigated in vitro as homogeneous single-cell suspensions; however, these preparations lose much of their osteogenic capacity. Using the fibroblastic colony-forming unit assay, we have compared the proliferation and capacity to express alkaline phosphatase of BMSC-containing aggregates of bone marrow cells with single-cell suspensions of bone marrow cells from the same source. Aggregates were separated from single cells by density gradient centrifugation or cell sieving. The aggregate and single-cell preparations gave rise to similar numbers of colonies; however, the colonies produced by the aggregates were larger and expressed higher levels of alkaline phosphatase. When the aggregates were dissociated, colonies still formed; however, they expressed negligible levels of alkaline phosphatase. Immunomagnetic selection and immunofluorescent staining for CD61, N-methyl-D-aspartate (NMDA) receptor subunit 1, and acetylcholinesterase showed that the majority of the aggregates giving rise to osteoblastic colonies contained megakaryocytes. These data demonstrate that removing BMSCs from their normal environment reduces their osteoblastic capacity and that to achieve their maximal differentiation, BMSCs require direct physical contact with accessory cells such as megakaryocytes. These findings may be of direct relevance to the use of BMSCs for tissue-engineering purposes.

EMD273316 & EMD95833, type 4 phosphodiesterase inhibitors, stimulate fibroblastic-colony formation by bone marrow cells via direct inhibition of PDE4 and the induction of endogenous prostaglandin synthesis.

BACKGROUND: Type 4 phosphodiesterase (PDE4) inhibitors have been shown to stimulate bone formation in vivo and to stimulate osteoblastic differentiation in vitro. As one possible mechanism for the stimulation of bone formation is the recruitment of osteoprogenitor cells from the bone marrow, we have investigated the effect of the PDE4 inhibitors EMD273316, EMD95833, EMD249615 and EMD 219906 on fibroblastic colony formation by whole bone marrow cells and on the ability of these colonies to adopt an osteoblastic phenotype. RESULTS: All four agents stimulated colony formation in a concentration dependent manner, however, in the case of EMD273316 & EMD95833, the effect was evident at lower concentrations and the addition of prostaglandin E2 (PGE2) was not necessary for maximal stimulation. It was subsequently found that co-incubation with indomethacin reduced the stimulatory effects of EMD273316 & EMD95833 but had no effect on the actions of EMD249615 and EMD 219906 and that EMD273316 & EMD95833 stimulated the synthesis of endogenous PGE2 by whole bone marrow cells whereas EMD249615 and EMD 219906 had no significant effect. CONCLUSIONS: These data suggest that EMD249615, EMD 219906, EMD273316 & EMD95833 can promote the recruitment of bone marrow osteoprogenitor cells leading to a stimulation of bone formation via their direct inhibitory effects on PDE4. The actions of EMD273316 & EMD95833 however, are augmented by their ability to stimulate endogenous prostanoids synthesis which acts synergistically with their direct effects on PDE4.

Mouse embryo-derived NIH3T3 fibroblasts adopt an osteoblast-like phenotype when treated with 1alpha,25-dihydroxyvitamin D(3) and dexamethasone in vitro.

This study examines the capability of NIH3T3 fibroblasts to express osteoblastic markers following stimulation with a number of hormones and growth factors in vitro. Of the agents tested, 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) dose-dependently induced alkaline phosphatase (ALP) activity in NIH3T3 cells, and this effect was enhanced by the addition of dexamethasone (Dex), which when administered alone caused no detectable ALP expression. The combined use of 1,25(OH)(2)D(3) and Dex also stimulated the synthesis of osteocalcin, and osteopontin. Furthermore, cells treated with the both hormones, in the presence of beta-glycerophosphate and l-ascorbic acid, formed mineralized plaques, indicating an osteoblast (OB) phenotype. By contrast, the differentiation induced by 1,25(OH)(2)D(3) or 1,25(OH)(2)D(3) plus Dex was significantly antagonized by transforming growth factor-beta1 and all trans-retinoic acid. These data indicate that NIH3T3 cells have the potential to adopt an OB-like phenotype and may prove to be a convenient model for studying the early events of osteogenic differentiation and the specific interactions of 1,25(OH)(2)D(3) with glucocorticoids in controlling this process in vitro.

Recruitment, augmentation and apoptosis of rat osteoclasts in 1,25-(OH)2D3 response to short-term treatment with 1,25-dihydroxyvitamin D3 in vivo.

BACKGROUND: Although much is known about the regulation of osteoclast (OC) formation and activity, little is known about OC senescence. In particular, the fate of of OC seen after 1,25-(OH)2D3 administration in vivo is unclear. There is evidence that the normal fate of OC is to undergo apoptosis (programmed cell death). We have investigated the effect of short-term application of high dose 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on OC apoptosis in an experimental rat model. METHODS: OC recruitment, augmentation and apoptosis was visualised and quantitated by staining histochemically for tartrate resistant acid phosphatase (TRAP), double staining for TRAP/ED1 or TRAP/DAPI, in situ DNA fragmentation end labelling and histomorphometric analysis. RESULTS: Short-term treatment with high-dose 1,25-(OH)2D3 increased the recruitment of OC precursors in the bone marrow resulting in a short-lived increase in OC numbers. This was rapidly followed by an increase in the number of apoptotic OC and their subsequent removal. The response of OC to 1,25-(OH)2D3 treatment was dose and site dependent; higher doses producing stronger, more rapid responses and the response in the tibiae being consistently stronger and more rapid than in the vertebrae. CONCLUSIONS: This study demonstrates that (1) after recruitment, OC are removed from the resorption site by apoptosis (2) the combined use of TRAP and ED1 can be used to identify OC and their precursors in vivo (3) double staining for TRAP and DAPI or in situ DNA fragmentation end labelling can be used to identify apoptotic OC in vivo.

Histochemical localization of alkaline phosphatase activity in decalcified bone and cartilage.

We have developed methodology that enables alkaline phosphatase (ALP) to be histochemically stained reproducibly in decalcified paraffin-embedded bone and cartilage of rodents. Proximal tibiae and fourth lumbar vertebrae were fixed in periodate-lysine-paraformaldehyde (PLP) fixative, decalcified in an EDTA-G solution, and embedded in paraffin. In the articular cartilage of the proximal tibia, ALP activity was localized to the hypertrophic chondrocytes and cartilage matrix of the deep zone and the maturing chondrocytes of the intermediate zone. The cells and matrix in the superficial zone did not exhibit any enzyme activity. In tibial and vertebral growth plates, a progressive increase in ALP expression was seen in chondrocytes and cartilage matrix, with activity being weakest in the proliferative zone, higher in the maturing zone, and highest in the hypertrophic zone. In bone tissue, ALP activity was detected widely in pre-osteoblasts, osteoblasts, lining cells on the surface of trabeculae, some newly embedded osteocytes, endosteal cells, and subperiosteal cells. In areas of new bone formation, ALP activity was detected in osteoid. In the bone marrow, about 20% of bone marrow cells expressed ALP activity. In adult rats, the thickness of the growth plates was less and ALP activity was enhanced in maturing and hypertrophic chondrocytes, cartilage matrix in the hypertrophic zone, and primary spongiosa. This is the first time that ALP activity has been successfully visualized histochemically in decalcified, paraffin-embedded mineralized tissues. This technique should prove to be a very convenient adjunct for studying the behavior of osteoblasts during osteogenesis.