CD200 positive human mesenchymal stem cells suppress TNF-alpha secretion from CD200 receptor positive macrophage-like cells

Human mesenchymal stem cells (hMSCs) display immunosuppressive properties in vitro and the potential has also been transferred successfully to clinical trials for treatment of autoimmune diseases. OX-2 (CD200), a member of the immunoglobulin superfamily, is widely expressed in several tissues and has recently been found from hMSCs. The CD200 receptor (CD200R) occurs only in myeloid-lineage cells. The CD200-CD200R is involved in down-regulation of several immune cells, especially macrophages. The present study on 20 hMSC lines shows that the CD200 expression pattern varied from high (CD200Hi) to medium (CD200Me) and low (CD200Lo) in bone marrow-derived mesenchymal stem cell (BMMSC) lines, whereas umbilical cord blood derived mesenchymal stem cells (UCBMSCs) were constantly negative for CD200. The role of the CD200-CD200R axis in BMMSCs mediated immunosuppression was studied using THP-1 human macrophages. Interestingly, hMSCs showed greater inhibition of TNF-α secretion in co-cultures with IFN-γ primed THP-1 macrophages when compared to LPS activated cells. The ability of CD200Hi BMMSCs to suppress TNF-α secretion from IFN-γ stimulated THP-1 macrophages was significantly greater when compared to CD200Lo whereas UCBMSCs did not significantly reduce TNF-α secretion. The interference of CD200 binding to the CD200R by anti-CD200 antibody weakened the capability of BMMSCs to inhibit TNF-α secretion from IFN-γ activated THP-1 macrophages. This study clearly demonstrated that the efficiency of BMMSCs to suppress TNF-α secretion of THP-1 macrophages was dependent on the type of stimulus. Moreover, the CD200-CD200r axis could have a previously unidentified role in the BMMSC mediated immunosuppression.     

Cell proliferation of human bone marrow mesenchymal stem cells on biodegradable microcarriers enhances in vitro differentiation potential

Objectives: For reasons of provision of highly‐specific surface area and three‐dimensional culture, microcarrier culture (MC) has garnered great interest for its potential to expand anchorage‐dependent stem cells. This study utilizes MC for in vitro expansion of human bone marrow mesenchymal stem cells (BMMSCs) and analyses its effects on BMMSC proliferation and differentiation. Materials and methods: Effects of semi‐continuous MC compared to control plate culture (PC) and serial bead‐to‐bead transfer MC (MC bead‐T) on human BMMSCs were investigated. Cell population growth kinetics, cell phenotypes and differentiation potential of cells were assayed. Results: Maximum cell density and overall fold increase in cell population growth were similar between PCs and MCs with similar starting conditions, but lag period of BMMSC growth differed substantially between the two; moreover, MC cells exhibited reduced granularity and higher CXCR4 expression. Differentiation of BMMSCs into osteogenic and adipogenic lineages was enhanced after 3 days in MC. However, MC bead‐T resulted in changes in cell granularity and lower osteogenic and adipogenic differentiation potential. Conclusions: In comparison to PC, MC supported expansion of BMMSCs in an up‐scalable three‐dimensional culture system using a semi‐continuous process, increasing potential for stem cell homing ability and osteogenic and adipogenic differentiation.     

Bone morphogenetic protein-2 enhances bone regeneration mediated by transplantation of osteogenically undifferentiated bone marrow-derived mesenchymal stem cells

We have hypothesized that human bone marrow-derived mesenchymal stem cells (BMMSCs), that are not osteogenically differentiated prior to implantation, would regenerate bone extensively in vivo once exogenous bone morphogenetic protein-2 (BMP-2) was delivered to the implantation site. BMP-2 released from heparin-conjugated poly(lactic-co-glycolic acid) (HCPLGA) scaffolds stimulates osteogenic differentiation of cultured BMMSCs. Upon implantation, undifferentiated BMMSCs on BMP-2-loaded HCPLGA scaffolds induce far more extensive bone formation than either undifferentiated BMMSCs or osteogenically differentiated BMMSCs on HCPLGA scaffolds. These BMP-2-loaded HCPLGA scaffolds could prove invaluable for in vivo regeneration of bone from undifferentiated human BMMSCs.     

Autophagy controls mesenchymal stem cell properties and senescence during bone aging

Bone marrow‐derived mesenchymal stem cells (BMMSCs) exhibit degenerative changes, including imbalanced differentiation and reduced proliferation during aging, that contribute to age‐related bone loss. We demonstrate here that autophagy is significantly reduced in aged BMMSCs compared with young BMMSCs. The autophagy inhibitor 3‐methyladenine (3‐MA) could turn young BMMSCs into a relatively aged state by reducing their osteogenic differentiation and proliferation capacity and enhancing their adipogenic differentiation capacity. Accordingly, the autophagy activator rapamycin could restore the biological properties of aged BMMSCs by increasing osteogenic differentiation and proliferation capacity and decreasing adipogenic differentiation capacity. Possible underlying mechanisms were explored, and the analysis revealed that autophagy could affect reactive oxygen species and p53 levels, thus regulating biological properties of BMMSCs. In an in vivo study, we found that activation of autophagy restored bone loss in aged mice. In conclusion, our results suggest that autophagy plays a pivotal role in the aging of BMMSCs, and activation of autophagy could partially reverse this aging and may represent a potential therapeutic avenue to clinically treat age‐related bone loss.     

Canonical Wnt signaling differently modulates osteogenic differentiation of mesenchymal stem cells derived from bone marrow and from periodontal ligament under inflammatory conditions

Background

Cellular plasticity and complex functional requirements of the periodontal ligament (PDL) assume a local stem cell (SC) niche to maintain tissue homeostasis and repair. Here, pathological alterations caused by inflammatory insults might impact the regenerative capacities of these cells. As bone homeostasis is fundamentally controlled by Wnt-mediated signals, it was the aim of this study to characterize the SC-like capacities of cells derived from PDL and to investigate their involvement in bone pathophysiology especially regarding the canonical Wnt pathway.

Methods

PDLSCs were investigated for their SC characteristics via analysis of cell surface marker expression, colony forming unit efficiency, proliferation, osteogenic differentiation and adipogenic differentiation, and compared to bone marrow derived mesenchymal SCs (BMMSCs). To determine the impact of both inflammation and the canonical Wnt pathway on osteogenic differentiation, cells were challenged with TNF-α, maintained with or without Wnt3a or DKK-1 under osteogenic induction conditions and investigated for p-IκBα, p-NF-κB, p-Akt, β-catenin, p-GSK-3β, ALP and Runx2.

Results

PDLSCs exhibit weaker adipogenic and osteogenic differentiation capacities compared to BMMSCs. TNF-α inhibited osteogenic differentiation of PDLSCs more than BMMSCs mainly through regulating canonical Wnt pathway. Blocking the canonical Wnt pathway by DKK-1 reconstituted osteogenic differentiation of PDLSCs under inflammatory conditions, whereas activation by Wnt3a increased osteogenic differentiation of BMMSCs.

Conclusions

Our results suggest a diverse regulation of the inhibitory effect of TNF-α in BMMSCs and PDLSCs via canonical Wnt pathway modulation.

General significance

These findings provide novel insights on PDLSC SC-like capacities and their involvement in bone pathophysiology under the impact of the canonical Wnt pathway.     

Let-7a promotes periodontal bone regeneration of bone marrow mesenchymal stem cell aggregates via the Fas/FasL-autophagy pathway

Periodontal bone regeneration using bone marrow mesenchymal stem cell (BMMSC) transplantation is a promising method; however, the method for osteogenic differentiation of BMMSCs needs to be improved. In this research, we sought to identify the roles of let-7a in the osteogenesis of BMMSCs and to provide a potential method for periodontal bone regeneration. Our previous study revealed that Fas/FasL is a target of let-7a. In this study, we demonstrated that let-7a overexpression significantly enhanced BMMSC-CAs osteogenesis both in vitro and in vivo. Mechanistically, upregulation of Fas/FasL using the rfas/rfaslg plasmid obstructed the osteogenesis of BMMSCs by inhibiting autophagy. Furthermore, we confirmed that overexpression of let-7a activated autophagy and alleviated the inhibited osteogenesis by the autophagy inhibitor 3-MA and the rfas/rfaslg plasmid of BMMSCs. In general, our findings showed that let-7a promoted the osteogenesis of BMMSCs through the Fas/FasL-autophagy pathway, suggesting that the application of let-7a in BMMSC-CAs based periodontal bone regeneration could be a promising strategy.     

Pulsed electromagnetic fields accelerate proliferation and osteogenic gene expression in human bone marrow mesenchymal stem cells during osteogenic differentiation

Osteogenesis is a complex series of events involving the differentiation of mesenchymal stem cells to generate new bone. In this study, we examined the effect of pulsed electromagnetic fields (PEMFs) on cell proliferation, alkaline phosphatase (ALP) activity, mineralization of the extracellular matrix, and gene expression in bone marrow mesenchymal stem cells (BMMSCs) during osteogenic differentiation. Exposure of BMMSCs to PEMFs increased cell proliferation by 29.6% compared to untreated cells at day 1 of differentiation. Semi‐quantitative RT‐PCR indicated that PEMFs significantly altered temporal expression of osteogenesis‐related genes, including a 2.7‐fold increase in expression of the key osteogenesis regulatory gene cbfa1, compared to untreated controls. In addition, exposure to PEMFs significantly increased ALP expression during the early stages of osteogenesis and substantially enhanced mineralization near the midpoint of osteogenesis. These results suggest that PEMFs enhance early cell proliferation in BMMSC‐mediated osteogenesis, and accelerate the osteogenesis. Bioelectromagnetics 31:209–219, 2010. © 2009 Wiley‐Liss, Inc.     

乙酰水杨酸预处理骨髓间充质干细胞对实验性牙周炎大鼠的治疗作用

本研究旨在探讨应用乙酰水杨酸(ASA)预处理的骨髓间充质干细胞(BMMSCs)治疗对大鼠牙周炎模型中的牙周骨修复的影响。通过建立大鼠牙周炎动物模型并使用ASA和BMMSCs联和治疗大鼠,本研究检测了体外BMMSCs的成骨分化、成脂分化、碱性磷酸酶(ALP)活性及成骨相关基因(ALP和OCN)的表达,并检测大鼠相关炎症因子(TNF-α,IL-17和IL-10)水平。结果显示,使用成骨培养基诱导BMMSCs后,可清晰地观察到BMMSCs的成骨分化和成脂分化。体外研究显示,60μg/mL的ASA显著促进了体外BMMSCs的增殖,提高了碱性磷酸酶(ALP)活性,促进了钙沉积和上调了成骨相关基因(ALP和OCN)的表达。此外,与未治疗的牙周炎大鼠比较,经ASA-BMMSCs治疗的牙周炎大鼠的TNF-α和IL-17水平显著下降,而IL-10显著升高。本研究表明,60μg/mL的ASA显著促进了体外BMMSCs的增殖和成骨分化。ASA和BMMSCs联用能够调节大鼠体内相关细胞因子的表达,并减轻炎症反应,可能是牙周炎治疗和牙周骨再生的有效方法。     

Galanin is highly expressed in bone marrow mesenchymal stem cells and facilitates migration of cells both in vitro and in vivo

Galanin peptide has recently been found to be highly abundant in early embryonic mouse mesenchyme, while galanin and its receptors are expressed in embryonic mouse stem cells. Bone marrow mesenchymal stem cells (BMMSCs) represent the primary source for adult stem cell therapy. In this study we examined the abundance of galanin and its receptors in BMMSCs and evaluated its possible function. Galanin mRNA and protein were highly expressed in BMMSCs cultures up to four passages, while among the three galanin receptor subtypes (GalR1, GalR2, and GalR3) only GalR2 and to a lesser extent GalR3 were expressed. Using chemotaxis and wound assays we found that galanin protein increased the migration of BMMSCs. Furthermore, increased serum galanin levels in a galanin transgenic model enhanced the mobilization (homing) of injected BMMSCs in vivo. These data suggest a role for galanin in BMMSC migration, probably through activation of the GalR2 receptor.     

Type 2 diabetes alters mesenchymal stem cell secretome composition and angiogenic properties

This study aimed at characterizing the impact of type 2 diabetes mellitus (T2DM) on the bone marrow mesenchymal stem cell (BMMSC) secretome and angiogenic properties. BMMSCs from Zucker diabetic fatty rats (ZDF) (a T2DM model) and Zucker LEAN littermates (control) were cultured. The supernatant conditioned media (CM) from BMMSCs of diabetic and control rats were collected and analysed. Compared to results obtained using CM from LEAN‐BMMSCs, the bioactive content of ZDF‐BMMSC CM (i) differently affects endothelial cell (HUVEC) functions in vitro by inducing increased (3.5‐fold; P < 0.01) formation of tubule‐like structures and migration of these cells (3‐fold; P < 0.001), as well as promotes improved vascular formation in vivo, and (ii) contains different levels of angiogenic factors (e.g. IGF1) and mediators, such as OSTP, CATD, FMOD LTBP1 and LTBP2, which are involved in angiogenesis and/or extracellular matrix composition. Addition of neutralizing antibodies against IGF‐1, LTBP1 or LTBP2 in the CM of BMMSCs from diabetic rats decreased its stimulatory effect on HUVEC migration by approximately 60%, 40% or 40%, respectively. These results demonstrate that BMMSCs from T2DM rats have a unique secretome with distinct angiogenic properties and provide new insights into the role of BMMSCs in aberrant angiogenesis in the diabetic milieu.     

骨髓间充质干细胞向角膜上皮细胞分化及其免疫调节作用

目的：探讨大鼠骨髓间充质干细胞（rBMMSCs）转分化为角膜上皮的潜能,并在体外共培养体系中研究rBMMSCs对促炎细胞因子干扰素-γ（IFN-γ）和肿瘤坏死因子-α（TNF-α）刺激下的人角膜上皮细胞（hCECs）的免疫调节作用。方法采用聚蔗糖梯密度离心法获得rBMMSCs,并通过上皮细胞培养微环境来诱导rBMMSCs分化为上皮样细胞。通过免疫组织化学方法鉴定CD29、CD34、CK5＆8和ZO-1等标记物在rBMMSCs及诱导的上皮样细胞中的表达。流式细胞术用来分析CD29/CD34的表达及细胞分化过程中表达量的变化。hCECs单独培养或与rBMMSCs共培养,并采用IFN-γ/TNF-α刺激24或48 h。通过流式细胞术来分析细胞间黏附分子-1（ICAM-1）于IFN-γ/TNF-α刺激前后在hCECs上的表达,并通过黏附分析实验验证rBMMSC条件培养基对单核细胞黏附于IFN-γ/TNF-α刺激后的hCECs的作用。多组间比较采用单因素方差分析（ANOVA）,两组间比较采用双侧t检验。结果成功分离rBMMSCs,细胞表达CD29,但不表达CD34。在上皮细胞培养条件中培养5 d,大约4﹪的rBMMSCs可分化为上皮样细胞。此类细胞失去了CD29的标志,转为表达CK5＆8和ZO-1。IFN-γ/TNF-α能显著上调hCECs中ICAM-1的表达,在IFN-γ/TNF-α处理24 h和48 h后,ICAM-1分别呈现10倍和8倍的升高,分别达到4524±554.2和3107±329.6（P=0.0025,0.0014）。但与MSC共同培养时,上调作用被显著抑制,ICAM-1平均值为1356±325.6（24 h）与1323±106.6（48 h）（P=0.0079,0.0024）。MSC条件培养基可显著抑制单核细胞对hCECs的黏附作用,黏附细胞数从（10.01±3.01）×10^3/ml细胞降至（2.21±0.19）×10^3/ml细胞（P=0.0271）。结论rBMMSCs可转分化为角膜上皮样细胞,并抑制由促炎细胞因子诱导的ICAM-1在hCECs上的表达,同时对促炎细胞因子诱导的单核细胞的黏附性具有抑制作用,提示BMMSCs具有在角膜炎症疾病和损伤修复中的治疗潜能。     

雌激素缺乏导致的骨质疏松发病过程中T淋巴细胞对骨髓间充质干细胞增殖和成骨分化的影响及与TNF-α的关系

探讨骨质疏松发病过程中T淋巴细胞对骨髓间充质干细胞（bonemarrow-derived mesenchymalstem cells,BMMSC）增殖分化的影响。选用健康雌性小鼠行双侧卵巢切除术（ovariectomy,OVX）,建立绝经后骨质疏松模型。选用同一批次健康小鼠行双侧卵巢脂肪组织部分切除,建立假手术组（sham）,Micro-CT确立模型成功建立。将sham组、OVX组、sham＋anti—TNFα组、OVX＋anti—TNFα组中T淋巴细胞与BMMSC共培养．ELISA检测sham组与OVX组T＇N-巴细胞上清液中TNF-α表达的差异,MTT法检测四组共培养体系中BMMSC生长曲线：成骨诱导后碱性磷酸酶和钙化结节茜素红染色法检测BMMsc成骨能力差异：ImPcR检测小鼠BMMSC成骨相关基因Runx2、碱性磷酸酶（alkaline phosphatase,ALP）的表达。结果显示,与sham组相比,OVX组中BMMsc的增殖受到了抑制,成骨分化减弱（P〈O．05）,OVXanti—TNF-α刺激组较OVX组增殖显著升高沪〈0．05）,成骨分化能力显著增强（P〈0．05）。以上结果证明,在雌激素缺乏下的T淋巴细胞能影响BMMSC增殖及成骨分化能力,这可能与T淋巴细胞表达TNF-α增强相关。     

Effect of low‐level laser therapy and oxytocin on osteoporotic bone marrow‐derived mesenchymal stem cells

Postmenopausal osteoporosis (OP) is a major concern for public health. Low‐level laser therapy (LLLT) has a positive effect on the health of bone marrow mesenchymal stem cells (BMMSCs). The purpose of this study is to evaluate the influence of LLLT and oxytocin (OT) incubation—individually and in combination—on osteoporotic BMMSCs in ovariectomized rats. Twelve female rats were randomized into two groups to undergo either a sham surgery (sham group) or ovariectomy‐induced osteoporosis (OVX group). MSCs harvested from the BM of healthy and OVX rats underwent culture expansion. There were five groups. In Groups one (sham‐BMMSC) and two (OVX‐BMMSC) the cells were held in osteogenic condition medium without any intervention. In the group three (OT), OT incubation with optimum dose was performed for 48 h (two times, 10^?12 molar). In Group four, laser‐treated‐OVX‐BMMSCs were treated with optimum protocol of LLLT (one time, 1.2 J/cm²). In Group five (laser + OT group), the OT incubation plus the laser irradiation was performed. The biostimulatory effect of LLLT is demonstrated by a significant increase in the viability of OVX‐BMMSCs, cell cycle, and extracellular levels of Transforming growth factor beta (TGF‐β), insulin‐like growth factor‐I (IGF‐I), and Alkaline phosphatase (ALP) compared to control OVX‐BMMSCs and/or the sham group. OT incubation and laser + OT incubation have a positive effect on OVX‐BMMSCs. However, LLLT is more effective statistically. We conclude that LLLT significantly improved cell viability, enhanced the osteogenic potential of the OVX‐BMMSCs, and increased the extracellular levels of the TGF‐β, IGF‐I, and ALP.     

The effects of conditioned media generated by polarized macrophages on the cellular behaviours of bone marrow mesenchymal stem cells

Macrophages (Mφs) are involved in a variety of physiological and pathological events including wound healing and tissue regeneration, in which they play both positive and negative roles depending on their polarization state. In this study, we investigated the cellular behaviours of bone marrow mesenchymal stem cells (BMMSCs) after incubation in different conditioned media (CMs) generated by unpolarized Mφs (M0) or polarized Mφs (M1 and M2). Mφ polarization was induced by stimulation with various cytokines, and CMs were obtained from in vitro Mφ cultures termed CM0, CM1 and CM2 based on each Mφ phenotype. We found that CM1 supported the proliferation and adipogenic differentiation of BMMSCs, whereas CM0 had a remarkable effect on cell osteogenic differentiation. To a certain degree, CM2 also facilitated BMMSC osteogenesis; in particular, cells incubated with CM2 exhibited an enhanced capacity to form robust stem cell sheets. Although incubation with CM1 also increased production of extracellular matrix components, such as fibronectin, COL‐1 and integrin β1during sheet induction, the sheets generated by CM2‐incubated cells were thicker than those generated by CM1‐incubated cells (P < 0.001). Our data suggest that each Mφ phenotype has a unique effect on BMMSCs. Fine‐tuning Mφ polarization following transplantation may serve as an effective method to modulate the therapeutic potential of BMMSCs.     

Effect of Fatty Acids on Human Bone Marrow Mesenchymal Stem Cell Energy Metabolism and Survival

Successful stem cell therapy requires the optimal proliferation, engraftment, and differentiation of stem cells into the desired cell lineage of tissues. However, stem cell therapy clinical trials to date have had limited success, suggesting that a better understanding of stem cell biology is needed. This includes a better understanding of stem cell energy metabolism because of the importance of energy metabolism in stem cell proliferation and differentiation. We report here the first direct evidence that human bone marrow mesenchymal stem cell (BMMSC) energy metabolism is highly glycolytic with low rates of mitochondrial oxidative metabolism. The contribution of glycolysis to ATP production is greater than 97% in undifferentiated BMMSCs, while glucose and fatty acid oxidation combined only contribute 3% of ATP production. We also assessed the effect of physiological levels of fatty acids on human BMMSC survival and energy metabolism. We found that the saturated fatty acid palmitate induces BMMSC apoptosis and decreases proliferation, an effect prevented by the unsaturated fatty acid oleate. Interestingly, chronic exposure of human BMMSCs to physiological levels of palmitate (for 24 hr) reduces palmitate oxidation rates. This decrease in palmitate oxidation is prevented by chronic exposure of the BMMSCs to oleate. These results suggest that reducing saturated fatty acid oxidation can decrease human BMMSC proliferation and cause cell death. These results also suggest that saturated fatty acids may be involved in the long-term impairment of BMMSC survival in vivo.     

Effects of strontium on proliferation and differentiation of rat bone marrow mesenchymal stem cells

Strontium ranelate (SrR) was an effective anti-osteoporotic drug to increase bone formation and decrease bone resorption. However, reports about the effect of SR on osteoblastic and adipocytic differentiation from bone marrow mesenchymal stem cells (BMMSCs) are limited. The purpose of this study is to evaluate whether SrR affects the ability of BMMSCs to differentiate into osteoblasts or adipocytes. Rat BMMSCs were identified by flow cytometry and exposed to SR (0.1 and 1.0mMSr(2+)) under osteogenic or adipogenic medium for 1 and 2weeks. The proliferation and differentiation of BMMSCs were analyzed by MTT, alkaline phosphatase (ALP), Oil red O staining, quantitative real-time RT-PCR and Western blot assays. SrR significantly inhibited the proliferation, increased osteoblastic but decreased adipocytic differentiation of rat BMMSCs dose-dependently. In osteogenic medium, SrR increased the expression of ALP, the mRNA levels of Cbfa1/Runx2, bone sialoprotein, and osteocalcin by RT-PCR, and the protein levels of Cbfa1/Runx2 by Western blot. In adipogenic medium, SrR decreased the mRNA levels of PPARγ2, adipocyte lipid-binding protein 2 (aP2/ALBP), and lipoprotein lipase (LPL) by RT-PCR, and the protein expression of PPARγ in Western blot analysis. These results indicated that the effects of SrR to promote osteoblastic but inhibit adipocytic differentiation of BMMSCs might contribute to its effect on osteoporosis treatment.     

Enhancement of ectopic bone formation by bone morphogenetic protein-2 delivery using heparin-conjugated PLGA nanoparticles with transplantation of bone marrow-derived mesenchymal stem cells

This study was performed to determine if a combination of previously undifferentiated bone marrow-derived mesenchymal stem cells (BMMSCs) and exogenous bone morphogenetic protein-2 (BMP-2) delivered via heparin-conjugated PLGA nanoparticles (HCPNs) would extensively regenerate bone in vivo. In vitro testing found that the HCPNs were able to release BMP-2 over a 2-week period. Human BMMSCs cultured in medium containing BMP-2-loaded HCPNs for 2 weeks differentiated toward osteogenic cells expressing alkaline phosphatase (ALP), osteopontin (OPN) and osteocalcin (OCN) mRNA, while cells without BMP-2 expressed only ALP. In vivo testing found that undifferentiated BMMSCs with BMP-2-loaded HCPNs induce far more extensive bone formation than either implantation of BMP-2-loaded HCPNs or osteogenically differentiated BMMSCs. This study demonstrates the feasibility of extensive in vivo bone regeneration by transplantation of undifferentiated BMMSCs and BMP-2 delivery via HCPNs. Sung Eun Kim and Oju Jeon equally contributed to this work     

Aromatic Amino Acid Activation of Signaling Pathways in Bone Marrow Mesenchymal Stem Cells Depends on Oxygen Tension

The physiologic oxygen pressures inside the bone marrow environment are much lower than what is present in the peripheral circulation, ranging from 1–7%, compared to values as high as 10–13% in the arteries, lungs and liver. Thus, experiments done with bone marrow mesenchymal stem cells (BMMSCs) using standard culture conditions may not accurately reflect the true hypoxic bone marrow microenvironment. However, since aging is associated with an increased generation of reactive oxygen species, experiments done under 21%O₂ conditions may actually more closely resemble that of the aging bone marrow environment. Aromatic amino acids are known to be natural anti-oxidants. We have previously reported that aromatic amino acids are potent agonists for stimulating increases in intracellular calcium and phospho-c-Raf and in promoting BMMSC differentiation down the osteogenic pathway. Our previous experiments were performed under normoxic conditions. Thus, we next decided to compare a normoxic (21% O₂) vs. a hypoxic environment (3% O₂) alone or after treatment with aromatic amino acids. Reverse-phase protein arrays showed that 3% O₂ itself up-regulated proliferative pathways. Aromatic amino acids had no additional effect on signaling pathways under these conditions. However, under 21%O₂ conditions, aromatic amino acids could now significantly increase these proliferative pathways over this “normoxic” baseline. Pharmacologic studies are consistent with the aromatic amino acids activating the extracellular calcium-sensing receptor. The effects of aromatic amino acids on BMMSC function in the 21% O2 environment is consistent with a potential role for these amino acids in an aging environment as functional anti oxidants.     

Effect of pulsed electromagnetic field on the proliferation and differentiation potential of human bone marrow mesenchymal stem cells

Pulsed electromagnetic fields (PEMFs) have been used clinically to slow down osteoporosis and accelerate the healing of bone fractures for many years. The aim of this study is to investigate the effect of PEMFs on the proliferation and differentiation potential of human bone marrow mesenchymal stem cells (BMMSC). PEMF stimulus was administered to BMMSCs for 8 h per day during culture period. The PEMF applied consisted of 4.5 ms bursts repeating at 15 Hz, and each burst contained 20 pulses. Results showed that about 59% and 40% more viable BMMSC cells were obtained in the PEMF‐exposed cultures at 24 h after plating for the seeding density of 1000 and 3000 cells/cm², respectively. Although, based on the kinetic analysis, the growth rates of BMMSC during the exponential growth phase were not significantly affected, 20–60% higher cell densities were achieved during the exponentially expanding stage. Many newly divided cells appeared from 12 to 16 h after the PEMF treatment as revealed by the cell cycle analysis. These results suggest that PEMF exposure could enhance the BMMSC cell proliferation during the exponential phase and it possibly resulted from the shortening of the lag phase. In addition, according to the cytochemical and immunofluorescence analysis performed, the PEMF‐exposed BMMSC showed multi‐lineage differentiation potential similar to the control group. Bioelectromagnetics 30:251–260, 2009. © 2009 Wiley‐Liss, Inc.