Modification of the bone marrow MSC population in a xenograft model of early multiple myeloma

Multiple myeloma (MM) is a hematological malignancy characterized by clonal proliferation of abnormal plasma cells. MM dysregulates the homeostasis of the bone niche cells like osteoclasts and osteoblasts, responsible for the bone maintenance leading to bone loss and hypercalcemia, as well as the normal immune cells leading to immunodeficiency and anemia. Osteoblasts are part of the cell population differentiating from mesenchymal stem cells (MSC). MSC also gives rise to other cell types such as adipocytes and chondrocytes. It has been observed that adipocytes support MM growth by increasing its survival and chemo-resistance. As adipocytes originate from MSC, the understanding of early modifications in the MSC population during the disease progression is of paramount importance and may help for early diagnosis of MM. Herein, we have evaluated the modification of the MSC population in the bone niche in an in vivo model of MM. Our results showed that before an observable engraftment of MM in the bone niche, the proportion of MSC population is significantly decreased, while a significant increase in adipocyte related genes such as PPARγ and CEBPα expression appears, with no difference in osteogenic differentiation. These results suggest that the bone niche is switching to a “fatty” marrow which would create an adequate microenvironment for MM. This led us to screen for and identify modulated adipokines in the sera of this in vivo MM-mice model. Such changes could reflect early signs of MM and potentially be exploited as detection biomarkers of the disease.     

Impaired differentiation potential of human trabecular bone mesenchymal stromal cells from elderly patients

Background aimsAdvances in bone tissue engineering with mesenchymal stromal cells (MSC) as an alternative to conventional orthopedic procedures has opened new horizons for the treatment of large bone defects. Bone marrow (BM) and trabecular bone are both sources of MSC. Regarding clinical use, we tested the potency of MSC from different sources.MethodsWe obtained MSC from 17 donors (mean age 64.6 years) by extensive washing of trabecular bone from the femoral head and trochanter, as well as BM aspirates of the iliac crest and trochanter. The starting material was evaluated by histologic analysis and assessment of colony-forming unit–fibroblasts (CFU-F). The MSC populations were compared for proliferation and differentiation potential, at RNA and morphologic levels.ResultsMSC proliferation potential and immunophenotype (expression of CD49a, CD73, CD90, CD105, CD146 and Stro-1) were similar whatever the starting material. However, the differentiation potential of MSC obtained by bone washing was impaired compared with aspiration; culture-amplified cells showed few Oil Red O-positive adipocytes and few mineralized areas and formed inconsistent Alcian blue-positive high-density micropellets after growth under adipogenic, osteogenic and chondrogenic conditions, respectively. MSC cultured with 1 ng/mL fibroblast growth factor 2 (FGF-2) showed better differentiation potential.ConclusionsTrabecular bone MSC from elderly patients is not good starting material for use in cell therapy for bone repair and regeneration, unless cultured in the presence of FGF-2.     

Transplanted mesenchymal stromal cells are unable to migrate to the bone surface and subsequently improve osteogenesis in glucocorticoid-induced osteoporosis

Long-term or high-dose use of glucocorticoids causes bone loss and low bone formation. We previously demonstrated that dexamethasone (Dex) administration caused the shifted differentiation balance of mesenchymal stromal cells (MSCs) to favor adipogenic lineage over osteoblastic lineage, which is one of the key mechanisms for Dex-induced osteoporosis (DIO). These findings indicate that supplementing functional allogeneic MSCs could be a therapeutic strategy for DIO. Here, we found that transplanting MSCs by intramedullary injection had little effect in promoting new bone formation. Fluorescent-labeled lineage tracing revealed that 1 week after transplantation, green fluorescent protein (GFP)-MSCs were found to migrate to the bone surface (BS) in control mice but not in DIO mice. As expected, GFP-MSCs on the BS were mostly Runx2-positive; however, GFP-MSCs located away from the BS failed to differentiate into osteoblasts. We further discovered that the levels of transforming growth factor beta 1 (TGF-β1), one of the main chemokines for MSC migration, is significantly decreased in the bone marrow fluid of DIO mice, which is insufficient to direct MSC migration. Mechanistically, Dex inhibits TGF-β1 expression by down-regulating its promoter activity, which decreases bone matrix–deposited TGF-β1 as well as active TGF-β1 released during osteoclast-mediated bone resorption. This study indicates that blocking MSC migration in osteoporotic BM contributes to bone loss and suggests that MSC mobilization to the BS may be a promising target for treating osteoporosis.     

人参皂苷Rg1对MDA诱导小鼠间充质干细胞凋亡的保护作用

目的：观察人参皂苷Rg1 （Ginsenoside Rg1,GS-Rg1）对丙二醛（Malondialdehyde,MDA）诱导的小鼠骨髓间充质干细胞（Mesenchymal stem cells,MSCs）凋亡的保护作用,并探讨其作用的可能机制.方法：以不同剂量（10、50、100 mg/L）人参皂苷Rg1预处理24 h,在小鼠骨髓MSC体外培养体系中加入MDA,TUNEL法,流式细胞术检测MSC凋亡率,Q-RT-PCR和Westen印迹分析检测Bcl-2、Bax和Caspase-3表达.结果：GS-Rg1可以减少TUNEL阳性细胞百分率及亚G1峰凋亡细胞百分率,增加Bcl-2mRNA及蛋白的表达水平,降低Bax和Caspase-3mRNA及蛋白表达水平.结论：GS-Rg1对MDA诱导小鼠间充质干细胞凋亡具有保护作用,其作用机制可能与增加Bc1-2表达,降低Bax和Caspase-3表达有关.     

The influence of mesenchymal stem cells on bone tissue regeneration upon implantation of demineralized bone matrix

Mesenchymal stem cells (MSC) are resident pluripotent cells of bone marrow stroma. MSC are able to differentiate into chondroblasts, adipocytes, neurons, glia, cardiomyocytes, or osteoblasts. The problem of MSC usage in cell therapy of bone defects is widely discussed at present. The experiments were carried out using rats of inbred line Wistar-Kyoto. MSC were isolated from bone marrow and cultivated in vitro. Demineralized bone matrices (DBM) were obtained from parietal bones of rats and hens. Part of DBM was loaded with MSC. Bone defects were made in cranium parietal regions. DBM with or without MSC or metal plates were transplanted in these regions. It was shown that the application of MSC increased angiogenesis and osteogenesis in the damaged bone. The implantation of rat's DBM with MSC led to the formation of a full value bone. MSC suppressed inflammation, when transplantation of hen's DBM was carried out. The application of MSC always improved bone tissue regeneration.     

Orthogonal potency analysis of mesenchymal stromal cell function during ex vivo expansion

Adult bone marrow mesenchymal stromal cells (MSCs) have cross-functional, intrinsic potency that is of therapeutic interest. Their ability to regenerate bone, fat, and cartilage, modulate the immune system, and nurture the growth and function of other bone marrow hematopoietic stem/progenitor cells have all been evaluated by transplant applications of MSCs. These applications require the isolation and expansion scaled cell production. To investigate biophysical properties of MSCs that can be feasibly utilized as predictors of bioactivity during biomanufacturing, we used a low-density seeding model to drive MSCs into proliferative stress and exhibit the hallmark characteristics of in vitro aging. A low-density seeding method was used to generate MSCs from passages 1–7 to simulate serial expansion of these cells to maximize yield from a single donor. MSCs were subjected to three bioactivity assays in parallel to ascertain whether patterns in MSC age, size, and shape were associated with the outcomes of the potency assays. MSC age was found to be a predictor of adipogenesis, while cell and nuclear shape was strongly associated to hematopoietic-supportive potency. Together, these data evaluate morphological changes associated with cell potency and highlight new strategies for purification or alternatives to assessing MSC quality.     

Strontium ranelate rebalances bone marrow adipogenesis and osteoblastogenesis in senescent osteopenic mice through NFATc/Maf and Wnt signaling

With aging, bone marrow mesenchymal stromal cell (MSC) osteoblast differentiation decreases whereas MSC differentiation into adipocytes increases, resulting in increased adipogenesis and bone loss. Here, we investigated whether activation of cell signaling by strontium ranelate (SrRan) can reverse the excessive adipogenic differentiation associated with aging. In murine MSC cultures, SrRan increased Runx2 expression and matrix mineralization and decreased PPARγ2 expression and adipogenesis. This effect was associated with increased expression of the Wnt noncanonical representative Wnt5a and adipogenic modulator Maf and was abrogated by Wnt- and nuclear factor of activated T-cells (NFAT)c antagonists, implying a role for Wnt and NFATc/Maf signaling in the switch in osteoblastogenesis to adipogenesis induced by SrRan. To confirm this finding, we investigated the effect of SrRan in SAMP6 senescent mice, which exhibit decreased osteoblastogenesis, increased adipogenesis, and osteopenia. SrRan administration at a clinically relevant dose level increased bone mineral density, bone volume, trabecular thickness and number, as shown by densitometric, microscanning, and histomorphometric analyses in long bones and vertebrae. This attenuation of bone loss was related to increased osteoblast surface and bone formation rate and decreased bone marrow adipocyte volume and size. The restoration of osteoblast and adipocyte balance induced by SrRan was linked to increased Wnt5a and Maf expression in the bone marrow. The results indicate that SrRan acts on lineage allocation of MSCs by antagonizing the age-related switch in osteoblast to adipocyte differentiation via mechanisms involving NFATc/Maf and Wnt signaling, resulting in increased bone formation and attenuation of bone loss in senescent osteopenic mice.     

Osteoblastic cells: differentiation and trans-differentiation

The osteoblast is the bone forming cell and is derived from mesenchymal stem cells (MSC) present among the bone marrow stroma. MSC are capable of multi-lineage differentiation into mesoderm-type cells such as osteoblasts and adipocytes. Understanding the mechanisms underlying osteoblast differentiation from MSC is a central topic in bone biology that can provide insight into mechanisms of bone maintenance and also novel pharmacological targets to increase osteoblast differentiation and consequently bone formation.     

脐带间充质干细胞与再生障碍性贫血

再生障碍性贫血(aplastic anemia,AA)是由于物理、化学、生物或不明因素作用使骨髓造血干细胞和骨髓微环境严重受损,造成骨髓造血功能降低或衰竭,以全血细胞减少为主要表现的一组综合征。间充质干细胞(MSC)是属于中胚层的一类多能干细胞,主要存在于结缔组织和器官间质中。不仅具有多向分化潜能,还有多种免疫调节作用。许多研究表明MSCs抑制同种异体效应T淋巴细胞增殖,还能下调T淋巴细胞表面的活化分子CD25、CD38、CD69的表达。MSCs对DCs的分化、成熟和活化具有抑制作用,改变DCs的细胞因子分泌,使成熟的DCl分泌TNF-a减少,DC2分泌IL-10增加,从而抑制T淋巴细胞增殖。MSCs能够抑制IL-2和IL-15介导的NK细胞增殖,使IL-2刺激NK细胞分泌的IFN-y减少。通过这些机制来干预再生障碍性贫血,同时研究发现MSCs免疫原性较低。尤其脐带MSC,因为其独特优势,目前已经成为干细胞干预治疗AA的研究热点。     

Reduced reactivation from dormancy but maintained lineage choice of human mesenchymal stem cells with donor age

Mesenchymal stem cells (MSC) are promising for cell-based regeneration therapies but up to date it is still controversial whether their function is maintained throughout ageing. Aim of this study was to address whether frequency, activation in vitro, replicative function, and in vitro lineage choice of MSC is maintained throughout ageing to answer the question whether MSC-based regeneration strategies should be restricted to younger individuals. MSC from bone marrow aspirates of 28 donors (5-80 years) were characterized regarding colony-forming unit-fibroblast (CFU-F) numbers, single cell cloning efficiency (SSCE), osteogenic, adipogenic and chondrogenic differentiation capacity in vitro. Alkaline phosphatase (ALP) activity, mineralization, Oil Red O content, proteoglycan- and collagen type II deposition were quantified. While CFU-F frequency was maintained, SSCE and early proliferation rate decreased significantly with advanced donor age. MSC with higher proliferation rate before start of induction showed stronger osteogenic, adipogenic and chondrogenic differentiation. MSC with high osteogenic capacity underwent better chondrogenesis and showed a trend to better adipogenesis. Lineage choice was, however, unaltered with age. CONCLUSION: Ageing influenced activation from dormancy and replicative function of MSC in a way that it may be more demanding to mobilize MSC to fast cell growth at advanced age. Since fast proliferation came along with high multilineage capacity, the proliferation status of expanded MSC rather than donor age may provide an argument to restrict MSC-based therapies to certain individuals.     

Mesenchymal stem cell transplantation increases expression of vascular endothelial growth factor in papain-induced emphysematous lungs and inhibits apoptosis of lung cells

BackgroundPulmonary emphysema is characterized by loss of alveolar structures. We have found that bone marrow (BM) mesenchymal stem cell (MSC) transplantation ameliorates papain-induced pulmonary emphysema. However, the underlying mechanism is not completely understood. It has been shown that blocking the vascular endothelial growth factor (VEGF) signaling pathway leads to apoptosis of lung cells and pulmonary emphysema, and MSC are capable of secreting VEGF. We hypothesized that MSC transplantation may have a protective effect on pulmonary emphysema by increasing VEGF-A expression and inhibiting apoptosis of lung cells.MethodsWe examined the morphology and expression of VEGF-A in rat lung after papain treatment and MSC transplantation. We also used a co-culture system in which MSC and cells prepared from papain-treated lungs or control lungs were cultured together. The levels of VEGF-A in cells and culture medium were determined, and apoptosis of cultured lung cells was evaluated.ResultsVEGF-A expression in rat lungs was decreased after papain treatment, which was partly rescued by MSC transplantation. MSC production of VEGF-A was increased when MSC were co-cultured with cells prepared from papain-treated lungs. Furthermore, the apoptosis of papain-treated lung cells was inhibited when co-cultured with MSC. The induction of MSC production of VEGF-A by papain-treated lung cells was inhibited by adding anti-tumor necrosis factor (TNF)-α antibody to the medium.ConclusionsThe protective effect of MSC transplantation on pulmonary emphysema may be partly mediated by increasing VEGF-A expression and inhibiting the apoptosis of lung cells. TNF-α released from papain-treated lung cells induces MSC to secret VEGF-A.     

Recent highlights on bone stem cells: a report from Bone Stem Cells 2009, and not only…

The use of stem cells has opened new prospects for the treatment of orthopaedic conditions characterized by large bone defects. However, many issues still exist to which answers are needed before routine, large-scale application becomes possible. Bone marrow stromal cells (MSC), which are clonogenic, multipotential precursors present in the bone marrow stroma, are generally employed for bone regeneration. Stem cells with multilineage differentiation similar to MSC have also been demonstrated in adipose tissue, peripheral blood, umbilical cord and amniotic fluid. Each source presents its own advantages and drawbacks. Unfortunately, no unique surface antigen is expressed by MSC, and this hampers simple MSC enrichment from heterogeneous populations. MSC are identified through a combination of physical, morphological and functional assays. Different in vitro and in vivo models have been described for the research on bone stem cells. These models should predict the in vivo bone healing capacity of MSC and if the induced osteogenesis is similar to the physiological one. Although stem cells offer an exciting possibility of a renewable source of cells and tissues for replacement, orthopaedic applications often represent case reports whereas controlled randomized trials are still lacking. Further biological aspects of bone stem cells should be elucidated and a general consensus on the best models, protocols and proper use of scaffolds and growth factors should be achieved.     

Mesenchymal stromal cells augment CD4+ and CD8+ T-cell proliferation through a CCL2 pathway

Background aimsMesenchymal stromal cells (MSC) derived from bone marrow are immunosuppressive in vitro and in vivo. Recent evidence, however, has shown that in certain settings, MSC can also be immunostimulatory. The mechanisms involved in this process are largely unknown.MethodsMouse spleen T cells were stimulated with allogeneic mixed lymphocyte reaction (MLR) or anti-CD3/CD28 beads and treated with autologous bone marrow MSC or MSC-conditioned medium. CD4+ and CD8+ T-cell proliferation was analyzed after treatment.ResultsWe show that MSC have both suppressive and stimulatory functions toward T cells after stimulation with anti-CD3/CD28 beads or in an MLR. This depended on the ratio of MSC to responder T cells, with low numbers of MSC increasing and higher numbers inhibiting T-cell proliferation. Immunostimulatory function was mediated, in part, by soluble factors. MSC immunosuppression of the MLR was indirect and related to inhibition of antigen-presenting cell maturation. Direct effects of MSC-conditioned medium during anti-CD3/CD28 stimulated proliferation were entirely stimulatory and required the presence of the T-cell receptor. MSC supernatant contained both CCL2 and CCL5 at high levels, but only CCL2 level correlated with the ability to augment proliferation. An anti-CCL2 antibody blocked this proliferative activity.ConclusionsCCL2 plays an important role in the immunostimulatory function of MSC, and we further hypothesize that the immunomodulatory role of MSC is determined by a balance between inhibitory and stimulatory factors, suggesting the need for caution when these cells are investigated in clinical protocols.     

Plasticity and banking potential of cultured adipose tissue derived mesenchymal stem cells

The present day research on stem cells is yet not filled to the gunwales. The correlation of stem cell technology with tissue repair still has a long way to go. Since Embryonic stem cells are a kind of thorn inside when it comes to therapeutics, there emerged few potent contemporary sources of stem cells. Though bone marrow proves to be the pioneer among these, they lose themselves to adipose tissue in various aspects. The major shortcoming of bone marrow lies in lieu of its loss in potency with age. Adipose tissue puts up a tough competition among leading edge stem cell sources like cord blood and cord matrix. Adipose tissue wins over its counterparts in that it possesses astounding proliferation potency in vitro and holds a prominent stand in showcasing in vivo tissue repair efficacy. In spite of its precedence, the whole enchilada of adipose derived stem cells is still in its salad days. In our work we aim at excogitating the Mesenchymal stem cell population present in cultured adipose derived stem cells, in a wide perspective. Furthermore, the coalition of cell adhesion molecules with the proliferation potency of MSC and analysis of growth curve of ADSC was also paid accolade. The presence of robust MSC with immense differentiation and transdifferentiation potency was endorsed by lucrative differentiation of P3 cells into mesodermal and neuronal lineages. Additionally, mesenchymal stem cells exhibiting coherent expression of surface markers at P3 in all samples can be cryopreserved for therapeutic applications.     

Mesenchymal stem cells in arthritis: role of bone marrow microenvironment

Based on their capacity to suppress immune responses, multipotent mesenchymal stromal cells (MSCs) are intensively studied for regenerative medicine. Moreover, MSCs are potent immunomodulatory cells that occur through the secretion of soluble mediators including nitric oxide, transforming growth factor beta, and HLAG5. The MSCs, however, are also able to express inflammatory mediators such as prostaglandin E₂ or IL-6. MSCs in the bone marrow are in close contact with T cells and B cells, and they regulate immunological memory by organizing defined numbers of dedicated survival niches for plasma cells and memory T cells in the bone marrow. The role of MSCs in arthritis remains controversial - in some studies, murine allogeneic MSCs are able to decrease arthritis; in other studies, MSCs worsen the local inflammation. A recent paper in Arthritis Research and Therapy shows that bone marrow MSCs have decreased osteoblastic potential in rheumatoid arthritis, which may be related to chronic inflammation or to loss of expression of IL-1 receptor agonist. That article raises the importance of the bone marrow microenvironment for MSC biology.     

Mesenchymal stem cell aging: Mechanisms and influences on skeletal and non-skeletal tissues

The aging population and the incidence of aging-related diseases such as osteoporosis are on the rise. Aging at the tissue and organ levels usually involves tissue stem cells. Human and animal model studies indicate that aging affects two aspects of mesenchymal stem cell (MSC): a decrease in the bone marrow MSC pool and biased differentiation into adipocyte at the cost of osteoblast, which underlie the etiology of osteoporosis. Aging of MSC cells is also detrimental to some non-skeletal tissues, in particular the hematopoietic system, where MSCs serve as a niche component. In addition, aging compromises the therapeutic potentials of MSC cells, including cells isolated from aged individuals or cells cultured for many passages. Here we discuss the recent progress on our understanding of MSC aging, with a focus on the effects of MSC aging on bone remodeling and hematopoiesis and the mechanisms of MSC aging.     

Homing and migration of mesenchymal stromal cells: How to improve the efficacy of cell therapy?

Mesenchymal stromal cells(MSCs) are currently being investigated for use in a wide variety of clinical applications. For most of these applications, systemic delivery of the cells is preferred. However, this requires the homing and migration of MSCs to a target tissue. Although MSC hominghas been described, this process does not appear to be highly efficacious because only a few cells reach the target tissue and remain there after systemic administration. This has been ascribed to low expression levels of homing molecules, the loss of expression of such molecules during expansion, and the heterogeneity of MSCs in cultures and MSC culture protocols. To overcome these limitations, different methods to improve the homing capacity of MSCs have been examined. Here, we review the current understanding of MSC homing, with a particular focus on homing to bone marrow. In addition, we summarize the strategies that have been developed to improve this process. A better understanding of MSC biology, MSC migration and homing mechanisms will allow us to prepare MSCs with optimal homing capacities. The efficacy of therapeutic applications is dependent on efficient delivery of the cells and can, therefore, only benefit from better insights into the homing mechanisms.