Proliferation and differentiation of bone marrow stromal cells under hypoxic conditions

Low oxygen tension is a potent differentiation inducer of numerous cell types and an effective stimulus of many gene expressions. Here, we described that under 8% O(2), bone marrow stromal cells (MSCs) exhibited proliferative and morphologic changes. The level of differentiated antigen H-2Dd and the number of G(2)/S/M phase cells increased evidently under 8% O(2) condition. Also, the proportion of wide, flattened, and epithelial-like cells (which were alkaline phosphatase staining positive) in MSCs increased significantly. When cultured in adipogenic medium, there was a 5- to 6-fold increase in the number of lipid droplets under hypoxic conditions compared with that in normoxic culture. We also demonstrated the existence of MSC differentiation under hypoxic conditions by electron microscopy. Expression of Oct4 was inhibited under 8% O(2) condition, but after adipocyte differentiation in normoxic culture and hypoxia-mimicking agents cobalt chloride (CoCl(2)) and deferoxamine mesylate (DFX) treatments, Oct4 was still expressed in MSCs. These results indicate hypoxia accelerates MSC differentiation and hypoxia and hypoxia-mimicking agents exert different effects on MSC differentiation.     

RANKL from bone marrow adipose lineage cells promotes osteoclast formation and bone loss

Receptor activator of NF‐κB ligand (RANKL) is essential for osteoclast formation and bone remodeling. Nevertheless, the cellular source of RANKL for osteoclastogenesis has not been fully uncovered. Different from peripheral adipose tissue, bone marrow (BM) adipose lineage cells originate from bone marrow mesenchymal stromal cells (BMSCs). Here, we demonstrate that adiponectin promoter‐driven Cre expression (Adipoq^Cre ) can target bone marrow adipose lineage cells. We cross the Adipoq^Cre mice with rankl^fl/fl mice to conditionally delete RANKL from BM adipose lineage cells. Conditional deletion of RANKL increases cancellous bone mass of long bones in mice by reducing the formation of trabecular osteoclasts and inhibiting bone resorption but does not affect cortical bone thickness or resorption of calcified cartilage. Adipoq^Cre; rankl^fl/fl mice exhibit resistance to estrogen deficiency and rosiglitazone (ROS)‐induced trabecular bone loss but show bone loss induced by unloading. BM adipose lineage cells therefore represent an essential source of RANKL for the formation of trabecula osteoclasts and resorption of cancellous bone during remodeling under physiological and pathological conditions. Targeting bone marrow adiposity is a promising way of preventing pathological bone loss.     

Chromosomal variability of human mesenchymal stem cells cultured under hypoxic conditions

Bone marrow derived human mesenchymal stem cells (hMSCs) have attracted great interest from both bench and clinical researchers because of their pluripotency and ease of expansion ex vivo. However, these cells do finally reach a senescent stage and lose their multipotent potential. Proliferation of these cells is limited up to the time of their senescence, which limits their supply, and they may accumulate chromosomal changes through ex vivo culturing. The safe, rapid expansion of hMSCs is critical for their clinical application. Chromosomal aberration is known as one of the hallmarks of human cancer, and therefore it is important to understand the chromosomal stability and variability of ex vivo expanded hMSCs before they are used widely in clinical applications. In this study, we examined the effects of culturing under ambient (20%) or physiologic (5%) O(2) concentrations on the rate of cell proliferation and on the spontaneous transformation of hMSCs in primary culture and after expansion, because it has been reported that culturing under hypoxic conditions accelerates the propagation of hMSCs. Bone marrow samples were collected from 40 patients involved in clinical research. We found that hypoxic conditions promote cell proliferation more favourably than normoxic conditions. Chromosomal aberrations, including structural instability or aneuploidy, were detected in significantly earlier passages under hypoxic conditions than under normoxic culture conditions, suggesting that amplification of hMSCs in a low-oxygen environment facilitated chromosomal instability. Furthermore, smoothed hazard-function modelling of chromosomal aberrations showed increased hazard after the fourth passage under both sets of culture conditions, and showed a tendency to increase the detection rate of primary karyotypic abnormalities among donors aged 60 years and over. In conclusion, we propose that the continuous monitoring of hMSCs will be required before they are used in therapeutic applications in the clinic, especially when cells are cultured under hypoxic conditions.     

Characterization and hepatogenic differentiation of mesenchymal stem cells from human amniotic fluid and human bone marrow: a comparative study

Since stem cells can differentiate into hepatocyte, stem cell-based therapy becomes a potential alternative treatment for terminal liver diseases. However, an appropriate source of human mesenchymal stem cells (hMSCs) for hepatocytes has not yet been clearly elucidated. The aim of the present study was to investigate the in vitro biological characterization and hepatic differentiation potential of human amniotic fluid-derived mesenchymal stem cells (AF-hMSCs) and human bone marrow-derived mesenchymal stem cells (BM-hMSCs). Our results show that AF-hMSCs possess higher proliferation and self-renewal capacity than BM-hMSCs. Cytogenetic studies indicate that AF-hMSCs are as genetically stabile as BM-hMSCs. Following incubation with specific hepatogenic agents, AF-hMSCs showed a higher hepatic differentiation potential than BM-hMSCs. Expression of several liver-specific markers was significantly greater in AF-hMSCs than in BM-hMSCs, as shown by real time RT-PCR and immunofluorescence (IF). In conclusion, AF-hMSCs possess superior potential for hepatic differentiation, making them more suitable for diverse terminal liver diseases.     

NK cells in allogeneic bone marrow transplantation

NK cells, until recently an ignored subset of lymphocytes, have begun to emerge as important cytotoxic effectors. It is now accepted that NK cells together with T cells constitute major actors in graft-versus-leukemia reaction after allogeneic bone marrow transplantation (BMT). Over the last several years the mechanisms regulating the activation of NK cells have been the subject of intense investigations encouraged by the clinical implications that these studies will have. This article provides a general overview of NK-cells biology and regulation pertinent to their function in allogeneic BMT, followed by a review of the in vivo preclinical and clinical evidence for the beneficial effect of NK cells in the adoptive immunotherapy of leukemia.     

Bone marrow mesenchymal stem cells enhance autophagy and help protect cells under hypoxic and retinal detachment conditions

Our study aimed to evaluate the protective role and mechanisms of bone marrow mesenchymal stem cells (BMSCs) in hypoxic photoreceptors and experimental retinal detachment. The cellular morphology, viability, apoptosis and autophagy of hypoxic 661w cells and cells cocultured with BMSCs were analysed. In retinal detachment model, BMSCs were intraocularly transplanted, and then, the retinal morphology, outer nuclear layer (ONL) thickness and rhodopsin expression were studied as well as apoptosis and autophagy of the retinal cells. The hypoxia-induced apoptosis of 661w cells obviously increased together with autophagy levels increasing and peaking at 8 hours after hypoxia. Upon coculturing with BMSCs, hypoxic 661w cells had a better morphology and fewer apoptosis. After autophagy was inhibited, the apoptotic 661w cells under the hypoxia increased, and the cell viability was reduced, even in the presence of transplanted BMSCs. In retina-detached eyes transplanted with BMSCs, the retinal ONL thickness was closer to that of the normal retina. After transplantation, apoptosis decreased significantly and retinal autophagy was activated in the BMSC-treated retinas. Increased autophagy in the early stage could facilitate the survival of 661w cells under hypoxic stress. Coculturing with BMSCs protects 661w cells from hypoxic damage, possibly due to autophagy activation. In retinal detachment models, BMSC transplantation can significantly reduce photoreceptor cell death and preserve retinal structure. The capacity of BMSCs to reduce retinal cell apoptosis and to initiate autophagy shortly after transplantation may facilitate the survival of retinal cells in the low-oxygen and nutrition-restricted milieu after retinal detachment.     

Adenovirus-mediated BMP2 expression in human bone marrow stromal cells

Recombinant adenoviral vectors have been shown to be potential new tools for a variety of musculoskeletal defects. Much emphasis in the field of orthopedic research has been placed on developing systems for the production of bone. This study aims to determine the necessary conditions for sustained production of high levels of active bone morphogenetic protein 2 (BMP2) using a recombinant adenovirus type 5 (Ad5BMP2) capable of eliciting BMP2 synthesis upon infection and to evaluate the consequences for osteoprogenitor cells. The results indicate that high levels (144 ng/ml) of BMP2 can be produced in non-osteoprogenitor cells (A549 cell line) by this method and the resultant protein appears to be three times more biologically active than the recombinant protein. Surprisingly, similar levels of BMP2 expression could not be achieved after transduction with Ad5BMP2 of either human bone marrow stromal cells or the mouse bone marrow stromal cell line W20-17. However, human bone marrow stromal cells cultured with 1 microM dexamethasone for four days, or further stimulated to become osteoblast-like cells with 50 microg/ml ascorbic acid, produced high levels of BMP2 upon Ad5BMP2 infection as compared to the undifferentiated cells. The increased production of BMP2 in adenovirus transduced cells following exposure to 1 microM dexamethasone was reduced if the cells were not given 50 microg/ml ascorbic acid. When bone marrow stromal cells were allowed to become confluent in culture prior to differentiation, BMP2 production in response to Ad5BMP2 infection was lost entirely. Furthermore, the increase in BMP2 synthesis seen during differentiation was greatly decreased when Ad5BMP2 was administered prior to dexamethasone treatment. In short, the efficiency of adenovirus mediated expression of BMP2 in bone marrow stromal cells appears to be dependent on the differentiation state of these cells.     

Effect of hypoxic treatment on bone marrow cells that are able to migrate to the injured liver

Restricted numbers and poor regenerative properties limit the use of adult stem cells. We tested the effect of hypoxic treatment as a method by which to increase cell migration. Bone marrow cells (BMCs) were cultured under oxygen saturations of 0.1, 3, and 20% for 24 h. After hypoxic treatment, BMCs of apoptotic fraction were decreased. The expression of CXCR4 was noticeably increased in the hypoxia-treated BMCs and their migration in response to SDF-1α was enhanced compared with cells cultured under normoxic condition. Hypoxic BMCs had a higher degree of engraftment to the CCl₄-injured liver than the normoxic cells. Hypoxic treatment of BMCs may have merits in decreasing apoptosis of those cells as well as in enhancing cellular migration to SDF-1α, the chemokine which binds to BMCs expressed CXCR4 and to the injured tissue, such as CCl₄ damaged liver.     

Fluorescence correlation spectroscopy reveals topological segregation of the two tumor necrosis factor membrane receptors

The proinflammatory cytokine tumor necrosis factor (TNF) binds two distinct plasma membrane receptors, TNFR1 and TNFR2. We have produced different receptor mutants fused with enhanced green fluorescent protein to study their membrane dynamics by fluorescence correlation spectroscopy (FCS). TNFR1 mutants show diffusion constants of approximately 1.2 × 10^− 9 cm²/s and a broad distribution of diffusion times, which is hardly affected by ligand binding. However, cholesterol depletion enhances their diffusion, suggesting a constitutive affinity to cholesterol rich membrane microdomains. In contrast, TNFR2 and mutants thereof diffuse rather fast (D? = 3.1 × 10^− 9 cm²/s) with a marked reduction after 30 min of TNF treatment (D? = 0.9 × 10^− 9 cm²/s). This reduction cannot be explained by the formation of higher ordered receptor clusters, since the fluorescence intensity of TNF treated receptors indicate the presence of a few receptor molecules per complex only. Together, these data point to a topological segregation of the two TNF receptors in different microcompartments of the plasma membrane independent of the cytoplasmic signaling domains of the receptors.     

Feasibility and efficacy of bone tissue engineering using human bone marrow stromal cells cultivated in serum-free conditions

Current standard techniques for bone tissue engineering utilize ex vivo expanded osteogenic cells. However, ex vivo expansion requires serum, which may hinder clinical applications. Here, we report the feasibility and efficacy of bone tissue engineering with human bone marrow stromal cells (BMSCs) expanded in serum-free conditions. Bone marrow was aspirated from 4 healthy donors and adherent cells were cultured in either serum-free medium (STEMPRO^® MSC SFM) or conventional serum-containing medium (α-MEM supplemented with 10% serum). Efficacy of expansion was greater in serum-free medium. Phenotypically, serum-free expanded BMSCs were smaller in cell-size and showed expression of CD105⁺⁺ and CD146^dim. After osteogenic induction, serum-free expanded BMSCs showed lower alkaline phosphatase activity. However, they showed higher responsiveness to induction. In vivo bone-forming ability was also confirmed. In conclusion, bone tissue engineering with serum-free expanded BMSCs is feasible and as efficient as that obtained with BMSCs expanded in conventional serum-containing medium.     

Sildenafil attenuates hypoxic pulmonary remodelling by inhibiting bone marrow progenitor cells

The recruitment of bone marrow (BM)‐derived progenitor cells to the lung is related to pulmonary remodelling and the pathogenesis of pulmonary hypertension (PH). Although sildenafil is a known target in PH treatment, the underlying molecular mechanism is still elusive. To test the hypothesis that the therapeutic effect of sildenafil is linked to the reduced recruitment of BM‐derived progenitor cells, we induced pulmonary remodelling in rats by two‐week exposure to chronic hypoxia (CH, 10% oxygen), a trigger of BM‐derived progenitor cells. Rats were treated with either placebo (saline) or sildenafil (1.4 mg/kg/day ip) during CH. Control rats were kept in room air (21% oxygen) with no treatment. As expected, sildenafil attenuated the CH‐induced increase in right ventricular systolic pressure and right ventricular hypertrophy. However, sildenafil suppressed the CH‐induced increase in c‐kit⁺ cells in the adventitia of pulmonary arteries. Moreover, sildenafil reduced the number of c‐kit⁺ cells that colocalize with tyrosine kinase receptor 2 (VEGF‐R2) and CD68 (a marker for macrophages), indicating a positive effect on moderating hypoxia‐induced smooth muscle cell proliferation and inflammation without affecting the pulmonary levels of hypoxia‐inducible factor (HIF)‐1α. Furthermore, sildenafil depressed the number of CXCR4⁺ cells. Collectively, these findings indicate that the improvement in pulmonary haemodynamic by sildenafil is linked to decreased recruitment of BM‐derived c‐kit⁺ cells in the pulmonary tissue. The attenuation of the recruitment of BM‐derived c‐kit⁺ cells by sildenafil may provide novel therapeutic insights into the control of pulmonary remodelling.     

Isolation of mesenchymal stem cells from human placenta: comparison with human bone marrow mesenchymal stem cells

The presence within bone marrow of a population of mesenchymal stem cells (MSCs) able to differentiate into a number of different mesenchymal tissues, including bone and cartilage, was first suggested by Friedenstein nearly 40 years ago. Since then MSCs have been demonstrated in a variety of fetal and adult tissues, including bone marrow, fetal blood and liver, cord blood, amniotic fluid and, in some circumstances, in adult peripheral blood. MSCs from all of these sources can be extensively expanded in vitro and when cultured under specific permissive conditions retain their ability to differentiate into multiple lineages including bone, cartilage, fat, muscle, nerve, glial and stromal cells. There has been great interest in these cells both because of their value as a model for studying the molecular basis of differentiation and because of their therapeutic potential for tissue repair and immune modulation. However, MSCs are a rare population in these tissues. Here we tried to identify cells with MSC-like potency in human placenta. We isolated adherent cells from trypsin-digested term placentas and examined these cells for morphology, surface markers, and differentiation potential and found that they expressed several stem cell markers. They also showed endothelial and neurogenic differentiation potentials under appropriate conditions. We suggest that placenta-derived cells have multilineage differentiation potential similar to MSCs in terms of morphology and cell-surface antigen expression. The placenta may prove to be a useful source of MSCs.     

Fibroblast growth factor 2 facilitates the differentiation of transplanted bone marrow cells into hepatocytes

We have developed an in vivo mouse model, the green fluorescent protein (GFP)/carbon tetrachloride (CCl₄) model, and have previously reported that transplanted GFP-positive bone marrow cells (BMCs) differentiate into hepatocytes via hepatoblast intermediates. Here, we have investigated the growth factors that are closely related to the differentiation of transplanted BMCs into hepatocytes, and the way that a specific growth factor affects the differentiation process in the GFP/CCl₄ model. We performed immunohistochemical analysis to identify an important growth factor in our model, viz., fibroblast growth factor (FGF). In liver samples, the expression of FGF1 and FGF2 and of FGF receptors (FGFRs; FGFR1, FGFR2) was significantly elevated with time after bone marrow transplantation (BMT) compared with other factors, and co-expression of GFP and FGFs or FGFRs could be detected. We then analyzed the effect and molecular mechanism of FGF signaling on the enhancement of BMC differentiation into hepatocytes by immunohistochemistry, immunoblotting, and microarray analysis. Treatment with recombinant FGF (rFGF), especially rFGF2, elevated the repopulation rate of GFP-positive cells in the liver and significantly increased the expression of both Liv2 (hepatoblast marker) and albumin (hepatocyte marker). Administration of rFGF2 at BMT also raised serum albumin levels and improved the survival rate. Transplantation of BMCs with rFGF2 specifically activated tumor necrosis factor-alpha (TNF-α) signaling. Thus, FGF2 facilitates the differentiation of transplanted BMCs into albumin-producing hepatocytes via Liv2-positive hepatoblast intermediates through the activation of TNF-α signaling. Administration of FGF2 in combination with BMT improves the liver function and prognosis of mice with CCl₄-induced liver damage. This study was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (nos. 13470121, 13770262, 15790348, 16390211, and 16590597) and for translational research from the Ministry of Health, Labor and Welfare (H-trans-5).     

Purmorphamine enhances osteogenic activity of human osteoblasts derived from bone marrow mesenchymal cells

Purmorphamine is a novel small molecule with osteogenesis-inducing activity in multipotent mesenchymal progenitor cells, but there has been no evaluation of its effect on human cells to date. The aim of this study was to investigate the induction of osteogenic activity by purmorphamine in human osteoblasts differentiated from bone marrow mesenchymal cells. Cells were cultured in 24-well plates at a density of 2x10(4)/well in medium containing 1, 2 or 3 microM purmorphamine, or vehicle. At 7, 14 and 21 days, cell proliferation, viability, and alkaline phosphatase (ALP) activity were evaluated. Bone-like nodule formation was evaluated at 21 days. Purmorphamine did not affect cell proliferation or viability, but increased ALP activity and bone-like nodule formation. These results indicate that events related to osteoblast differentiation, including increased ALP activity and bone-like nodule formation, are enhanced by purmorphamine.     

Tumor necrosis factor alpha gene variants do not display allelic imbalance in circulating myeloid cells

Carriage of the TNF −308 A allele (rs1800629 A) has been associated with increased serum TNF-α levels, the development of sepsis syndrome, and fatal outcome, in severely traumatized patients (Menges et al., 2008 [1]). Herein, we analysed the putative allelic imbalance of TNF-α release from myeloid cells. Circulating peripheral blood cells from healthy human blood donors (n = 104) and monocyte-derived macrophages (n = 158) were analysed for their ex vivo capacity of TNF-α expression. Our findings indicate that carriage of the TNF −308 A allele is not associated with high TNF-α expression in circulating human leucocytes and monocyte-derived macrophages. Other cellular sources, e.g. tissue-resident cells like mast cells and/or tissue specific macrophages might be the cellular source of high TNF-α serum levels shortly after trauma.     

Ex vivo differentiation of human adult bone marrow stem cells into cardiomyocyte-like cells

Bone marrow mesenchymal stem cells have been shown to transdifferentiate into cardiomyocytes after 5-azacytidine treatment or co-culturing with rodent cardiomyocytes. We investigate if adult human bone marrow stem cells can be differentiated ex vivo into cardiomyocyte-like cells (CLCs) independent of cytotoxic agents or co-culturing technique. Sternal bone marrow was collected from 16 patients undergoing coronary artery bypass surgery. Mesenchymal stem cells were differentiated in a cardiomyogenic differentiation medium containing insulin, dexamethasone, and ascorbic acid. Differentiation towards CLCs was determined by induced expression of cardiomyocyte-specific proteins. Differentiated CLCs expressed multiple structural and contractile proteins that are associated with cardiomyocytes. Thin filament associated myofibrillar proteins were detected early in the cells, with cardiac troponin I, sarcomeric tropomyosin, and cardiac titin among the first expressed. Some CLCs were found to develop into a nascent cardiomyocyte phenotype with cross-striated myofibrils characterized by alpha-actinin-positive Z bands after 4-5 passages in differentiated culture. These lineage-defined CLCs may be potentially useful for repairing damaged myocardium.