Culture Of Human Mesenchymal Stem Cells Research Articles

Human mesenchymal stem cell proliferation and osteogenic differentiation during long-term ex vivo cultivation is not age dependent

Mesenchymal stem cells (MSCs) are of major clinical interest for the development of cell-based strategies to treat musculoskeletal diseases including critical-size bone defects caused by trauma, degenerative disorders, or infections. Elderly people mainly suffer from critical-size bone defects from the rising incidence of trauma, osteoporosis, and arthroplasties. In this study we investigated the influence of donor age on proliferation and osteogenic differentiation in long-term ex vivo cultures of primary human MSCs from patients in different age groups. Fifteen patients (8 men/7 women) comprised three age groups: (I) <50 years, (II) 50-65 years, and (III) >65 years. MSCs harvested from bone marrow derived from routine surgical procedures were isolated and cultured in standard medium over eight passages. Osteogenic differentiation was induced by dexamethasone (10 nM), ascorbic acid (300 μM), and β-glycerophosphate (3.5 mM). Osteogenic differentiation capacity of MSCs was quantified by alkaline phosphatase (ALP) activity, fluorescence-activated cell sorting (FACS) analysis of the surface markers CD9, CD90, CD54, CD166, CD105, CD44, and CD73, and RT-PCR for Coll I and II, Cbfa 1, ALP, OC, BSP1, and GAPDH genes characterized the phenotypic changes during monolayer expansion. In vitro chondrogenic differentiation was analyzed by immunohistochemistry and RT-PCR. Progenitor cells could be expanded in the long term from all bone marrow donations. FACS single staining analysis from MSCs showed no significant difference between the age groups. The surface antigen CD166 was predominantly found in all cell cultures independently of differentiation stage. Comparison of expanded and differentiated MSCs within a single age group showed that undifferentiated MSCs had higher CD44 levels. Osteogenic stimulation of MSCs was confirmed by measuring ALP activity. The highest ALP activity was found in probands of the age group >65 years. Additionally, we observed a tendency toward male-specific ALP increase during differentiation. Osteogenic marker gene expression in MSCs was detected by RT-PCR. No significant expression differences were detected between the three donor age groups. Micromass culture of MSCs resulted histologically and immunohistologically in a chondrogenic phenotype. Elderly osteoprogenitor cell donors are a highly clinically relevant patient population. In summary, cultivation leads to a reduced osteogenic differentiation capacity regardless of age. Because donor age does not affect osteogenic differentiation potential, it should not be used as an exclusion criterion for autologous transplantation of human adult MSCs.

Characterization and biocompatibility of nanohybrid scaffold prepared via in situ crystallization of hydroxyapatite in chitosan matrix

Hydroxyapatite (HAP) precursor solution was first mixed with an acetic acid chitosan (CS) solution. The mixture was then lyophilized to form the original scaffold, which stored the HAP precursors. The nano HAP crystallized homogeneously from the CS matrix during the alkaline treatment to form a nanohybrid scaffold. Scanning electron microscopy (SEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR) were used to investigate the fabrication process of the nanohybrid scaffold. XRD results showed that the in situ deposited mineral (HAP) in the scaffold has phase structure similar to natural bone. FTIR and XPS results indicated that CS's hydroxyl group, amino and amide regulated the nano HAP crystallization process, which resulted in the nano homogeneous distribution of nano HAP and provided nano topographical features for the nanohybrid scaffold. MTT testing and SEM images of human bone mesenchymal stem cells (hBMSCs) cultures revealed the attachment and growth of hBMSCs in the biocomposite scaffold. Cell morphology and viability data showed that the nanohybrid composite scaffold is suitable for use in bioapplications.

Defined high protein content surfaces for stem cell culture

Unlocking the clinical potential of stem cell based therapies requires firstly elucidation of the biological mechanisms which direct stem cell fate decisions and thereafter, technical advances which allow these processes to be driven in a fully defined culture environment. Strategies for the generation of defined surfaces for human embryonic stem cell (hESC) and mesenchymal stem cell (MSC) culture remain in their infancy. In this paper we outline a simple, effective and efficient method for presenting proteins or peptides on an otherwise non-fouling Layer-by-Layer (LbL) self-assembled surface of hyaluronic acid (HA) and chitosan (CHI). We are able to generate a surface that has both good temporal stability and the ability to direct biological outcomes based on its defined surface composition. Surface functionalization is achieved through suspending the selected extracellular matrix (ECM) protein domain or extracted full-length protein in buffer containing a cross-linking agent ( N-hydroxysulfosuccinimide/ N-(3-Dimethylaminopropyl)- N′-ethylcarbodiimide hydrochloride) over the LbL HA-CHI surface and then allowing the solvent to evaporate overnight. This simple, but important step results in remarkable protein deposition efficiencies often exceeding 50%, whereas traditional cross-linking methods result in such poor deposition of non-collagenous proteins that a.) quantification of bound amounts of protein is outside the resolution of commonly utilized protein assays, and b.) these surfaces are both unable to support cell attachment and growth. The utility of the protein-modified HA-CHI surfaces is demonstrated through the identification of specific hESC attachment efficiencies and through directing MSC osteogenic outcomes on these fully defined surfaces. This simple and scalable method is shown to enable the development of defined stem cell culture conditions, as well as the elucidation of the fundamental biological processes necessary for the realization of stem cell based therapies.

TRANSPLANTATION AND CELLULAR ENGINEERING: Adipose tissue mesenchymal stem cell expansion in animal serum‐free medium supplemented with autologous human platelet lysate

BACKGROUND: Mesenchymal stem cells (MSCs) have been considered for human regenerative therapy applications, and safe culture and expansion protocols are needed especially in the context of interspecies contamination. Human platelet lysate (PL) has been proposed as animal serum substitute during in vitro MSC expansion. In this work, a simplified and efficient method to obtain autologous PL to replace animal serum in cell culture applications is described.STUDY DESIGN AND METHODS: PL obtained by freezing and centrifugation procedures was tested as medium supplement for human adipose mesenchymal stem cell (hASC) culture. Differential proliferation, immunophenotypic changes, and differentiation under PL or fetal bovine serum (FBS) were assessed.RESULTS: In contrast to 10% FBS supplementation, cell population doubling time was significantly lower when hASCs were cultured with the same concentration of PL (PL 22.9 ± 1.5 hr vs. FBS 106.7 ± 6.5 hr, t test, p &lt; 0.05). Furthermore, hASCs maintained with 2.5% PL supplementation also showed satisfactory results. Immunophenotypic analysis revealed no differences between hASCs cultivated with PL or FBS supplementation and both cultures retained the potential to differentiate into adipose cells. These results demonstrate that autologous PL obtained from the same donor can be used as animal serum substitute in hASC culture.CONCLUSIONS: Taken together, evidence is provided that platelets provided by a single donor are sufficient to obtain PL for hASC propagation for clinical‐scale applications mitigating the potential untoward side effects associated with the use of animal‐derived reagents.

Silk fibroin/hyaluronan scaffolds for human mesenchymal stem cell culture in tissue engineering

The design of new bioactive scaffolds mimicking the physiologic environment present during tissue formation is an important frontier in biomaterials research. Herein, we evaluated scaffolds prepared from blends of two biopolymers: silk fibroin and hyaluronan. Our rationale was that such blends would allow the combination of silk fibroin's superior mechanical properties with the biological characteristics of hyaluronan. We prepared scaffolds with porous microstructures by freeze-drying aqueous solutions of silk fibroin and hyaluronan and subsequent incubation in methanol to induce water insolubility of silk fibroin. Hyaluronan acted as an efficient porogenic excipient for the silk fibroin scaffolding process, allowing the formation of microporous structures within the scaffolds under mild processing conditions. Mesenchymal stem cells were seeded on silk fibroin/hyaluronan scaffolds and cultured for three weeks. Histology of the constructs after cell culture showed enhanced cellular ingrowth into silk fibroin/hyaluronan scaffolds as compared to plain silk fibroin scaffolds. In the presence of tissue-inductive stimuli, in vitro stem cell culture on silk fibroin/hyaluronan scaffolds resulted in more efficient tissue formation when measured by glycosaminoglycan and type-I and type-III collagen gene expression, as compared to plain silk fibroin scaffolds. In conclusion, our data encourages further exploration of silk fibroin/hyaluronan scaffolds as biomimetic platform for mesenchymal stem cells in tissue engineering.

Culture of human synovium-derived mesenchymal stem cells from human knee joint and its multipotent differentiation in vitro.

：Objective To study themethod of isolating and culturing human synovium-derived mes-enchymal stem cels (SMSCs)from osteoarthritic knee joints and to investigate whether SMSCs could differ-entiate intoosteoblasts, adipocytes and chondrocytes in vitro. Methods Cell populations wereenzymatieally released from the synovial membrane obtained from human knee joints withend-stage osteoarthritis. Mono-clone was obtained by seleeting as primary SMSCs. SMSCswere characterized by FACS-analysis. SMSCs at passage 3 were used to investigate theirmulti-lineage potential in vitro. Upon treatments, phenotypes of cell cultures wereanalyzed by histo- and immunohistochemistry and by semi-quantitative reversetranscription-polymerase ehain reaction for the expression of lineage-related markers.Results Primary SMSCs, showed polygon and star shape. After passaged, it exhibited roundeels and spindle cells. Consequently, homoge-neous populations of fibroblast-like cellswere observed. SMSCs were positive for CD90 and CD44, negative for CD34, CD45 and CD71,respectively. Under appropriate euhure conditions, SMSCs were induced to dif-ferentiate tothe adipocyte, osteocyte, and chondrocyte lineages, which were stained positively by OilRed O staining, ALP staining, Alizarin red staining and Toluidine blue staining,respectively. Expression of adi-pogenic markers (Lipoprotein lipase, Adiponectin and PPARγ2)and osteogenic markers (ALP, Osteopontin and Osteocalein) were all detected. Type Ⅱ collagen was stained positivelyby immunohistochemistry stain-ing. Conclusion Human SMSCs can be isolated from knee jointsof end-stage OA. These cells have the a-bility to proliferate extensively in culture, andthey have the potential to differentiate into adipogenic, os-teogenic and chondrogeniclineage in vitro. Key words: Synovialmembrane; Mesenchymal stem cell; Cell differentiation; Adipocytes; Os-teoblasts; Chondrocytes

Sustained release of BMP-2 in a lipid-based microtube vehicle

Sustained release systems have been developed for the use of growth factors in tissue engineering applications. However, many of these systems continue to have limitations associated with low loading efficiencies and reduced biological activity after release. In this paper, we utilized a lipid-based microtube system for the sustained release of BMP-2. The lipid microtubes were fabricated using a self-assembly method, in order to avoid the use of harsh organic solvents that may damage the protein. BMP-2 was loaded into the microtubes by rehydrating dried microtubes in the protein solution. The loading efficiency and release kinetics of BMP-2 in the microtubes were measured using in vitro immunoassays. Loading efficiency was found to be dependent on microtube concentration. The potential for this system to deliver biologically active BMP-2 was assessed using the alkaline phosphatase assay and von Kossa staining on human mesenchymal stem cell cultures. The results demonstrate that the lipid microtube system is able to provide sustained delivery of biologically active BMP-2 and thereby induce osteogenic differentiation.

Fabrication and properties of injectable β-tricalcium phosphate particles/fibrin gel composite scaffolds for bone tissue engineering

β-Tricalcium phosphate (β-TCP) particles with a size of ~ 200 nm were blended with fibrinogen and thrombin to obtain an injectable composite scaffold. Clotting time of the composite systems was compared with the fibrin gel control. Results show that incorporation of the β-TCP particles could pronouncedly prolong the clotting time, and endow the resulted scaffold with a denser microstructure. The β-TCP particles were evenly distributed in the fibrin gel, with an increased particle size in a scaffold of a larger amount of the particles. Elastic modulus of the scaffold was increased with the increase of β-TCP particles' content too. In vitro human mesenchymal stem cell culture showed that both cytoviability and cell number were increased along with increase of the β-TCP particle amount. At a 1.5% β-TCP particle, the highest activity of alkaline phosphatase was measured. Since the fibrin gel itself is already very biocompatible, compounding with the β-TCP particles may enable the resulted scaffold more suitable for bone regeneration, with the preserved merit of potential injection in situ.

Mesenchymal stem cells: biology and clinical potential in type 1 diabetes therapy

Mesenchymal stem cells (MSCs) can be derived from adult bone marrow, fat and several foetal tissues. In vitro, MSCs have the capacity to differentiate into multiple mesodermal and non-mesodermal cell lineages. Besides, MSCs possess immunosuppressive effects by modulating the immune function of the major cell populations involved in alloantigen recognition and elimination. The intriguing biology of MSCs makes them strong candidates for cell-based therapy against various human diseases. Type 1 diabetes is caused by a cell-mediated autoimmune destruction of pancreatic β-cells. While insulin replacement remains the cornerstone treatment for type 1 diabetes, the transplantation of pancreatic islets of Langerhans provides a cure for this disorder. And yet, islet transplantation is limited by the lack of donor pancreas. Generation of insulin-producing cells (IPCs) from MSCs represents an attractive alternative. On the one hand, MSCs from pancreas, bone marrow, adipose tissue, umbilical cord blood and cord tissue have the potential to differentiate into IPCs by genetic modification and/or defined culture conditions In vitro. On the other hand, MSCs are able to serve as a cellular vehicle for the expression of human insulin gene. Moreover, protein transduction technology could offer a novel approach for generating IPCs from stem cells including MSCs. In this review, we first summarize the current knowledge on the biological characterization of MSCs. Next, we consider MSCs as surrogate β-cell source for islet transplantation, and present some basic requirements for these replacement cells. Finally, MSCs-mediated therapeutic neovascularization in type 1 diabetes is discussed.

Enhancement of Osteogenesis by Concanavalin a in Human Bone Marrow Mesenchymal Stem Cell Cultures

This study investigates concanavalin A (ConA) as a novel factor that may enhance osteogenesis of mesenchymal stem cells (MSCs) in vitro. Various factors, such as cytokine bone morphogenetic protein-2 (BMP-2), have been studied for their possible promotion of MSC osteogenesis in vivo and in vitro. However, the factor that might be safer, more effective, and less expensive than these has not been determined. We therefore cultured human MSCs in osteogenic medium in the presence or absence of ConA, and used calcium assays to compare the effects of ConA and BMP-2 on MSC calcification. We also used enzyme-linked immunosorbent assay (ELISA) and quantitative polymerase chain reaction (PCR) to evaluate the expression levels of bone-specific markers. ConA and BMP-2 enhanced calcification with comparable effectiveness. The combination of ConA and BMP-2 further enhanced calcification slightly but significantly. ConA also increased osteocalcin and BMP-2 protein levels in MSC culture medium. Furthermore, ConA increased osteocalcin, RUNX2, BMP-2, BMP-4, and BMP-6 mRNA expression levels. However, the gene expression pattern of ConA-stimulated MSCs was different from that of MSCs stimulated by BMP-2. Together, these results suggest that ConA and BMP-2 enhance MSC osteogenesis via different pathways. ConA-induced bone formation in MSC cultures may be useful in regenerative medicine or tissue engineering in clinical studies, as well as in basic research on bone formation.

Skeletal Muscle Regeneration: MSC versus Satellite Cells

Problem Differentiating stem cells into the myogenic linage in order to create functional muscle tissue is a challenging endeavour. In this work, adipose-derived mesenchymal stem cells (MSC) and satellite cells derived from muscle biopsies were compared regarding proliferation and myogenic differentiation potential under standardized cell culture conditions. This data was obtained in order to discover the most promising type of stem cell for regeneration of muscle tissue and to determine the optimal culture conditions for later clinical use. Methods Human MSC were isolated from adipose tissue, and primary human skeletal myoblasts were extracted from muscle biopsies by enzymatic digestion. Proliferation was analysed using the AlamarBlue® assay. Gene expression of marker genes – such as Myogenin, Myo D, Myf 5 and MHC – were analysed by RT-PCR. Immunostainings against desmin and sarcomeric-actin were performed as differentiation markers. Results MSC cell cultures showed a greater proliferation rate compared with satellite cell cultures. In both stem cell cultures, myogenic differentiation/heritage could be verified by immunostainings against the muscle-specific marker desmin. Gene expression and protein analysis revealed a more stable differentiation of human satellite cell cultures. Conclusion Characterization of both human MSC cultures and satellite cell cultures – and thereby an understanding of myogenesis – might lead to their clinical usage in skeletal muscle tissue engineering. The results in this study appear to indicate that human satellite cell cultures have a more stable differentiation under in vitro conditions and that they might offer a greater potential for skeletal muscle tissue engineering purposes. Significance Our study contributes to the understanding of myogenic differentiation of MSC and satellite cells and helps to improve culture systems for later clinical utilization.

Comparison of Human Serum with Fetal Bovine Serum for Expansion and Differentiation of Human Synovial MSC: Potential Feasibility for Clinical Applications

The aim of this study was to evaluate the effect of human serum (HS) on growth and differentiation capacity of human synovium-derived mesenchymal stem cells (MSC) in comparison to cells grown in fetal bovine serum (FBS). Human MSCs were isolated from the synovium of knee joints of three donors and the cells were cultured individually in varying concentrations of allogenic HS or FBS. Bovine MSCs were isolated from synovium and cultured in the same manner. Cell proliferation was assessed by the tetrazolium assay after passage 3. The capacity for chondrogenic and osteogenic differentiation was investigated in specific media followed by 1,9-dimethylmethylene blue assay and alcian blue staining, or by alizarin red staining, respectively. Human MSCs proliferated significantly more rapidly in the presence of HS than with equivalent levels of FBS. Chondrogenic or osteogenic differentiation occurred to nearly identical levels in HS or FBS. The results of this study indicate that HS is superior for the culture of human MSCs compared with FBS in terms of cellular expandability, without losing chondrogenic or osteogenic differentiation capacity. Coupled with the advantage in eliminating the potential risk accompanied with the use of xeno-derived materials, pooled, well-characterized HS could be a useful reagent to promote cellular expansion for clinical synovial stem cell-based therapy.

Effect of dynamic 3‐D culture on proliferation, distribution, and osteogenic differentiation of human mesenchymal stem cells

Ex vivo engineering of autologous bone tissue as an alternative to bone grafting is a major clinical need. In the present study, we evaluated the effect of 3-D dynamic spinner flask culture on the proliferation, distribution, and differentiation of human mesenchymal stem cells (MSCs). Immortalized human MSCs were cultured on porous 75:25 PLGA scaffolds for up to 3 weeks. Dynamically cultured cell/scaffold constructs demonstrated a 20% increase in DNA content (21 days), enhanced ALP specific activity (7 days and 21 days), a more than tenfold higher Ca2+ content (21 days), and significantly increased transcript levels of early osteogenesis markers (e.g., COL1A1, BMP2, RUNX-2) as compared with static culture. Despite the formation of a dense superficial cell layer, markedly increased cell ingrowth was observed by fluorescence microscopy on day 21. Furthermore, increased extracellular matrix deposition was visualized by scanning electron microscopy after 1 and 3 weeks of dynamic culture. The observed increased ingrowth and osteogenic differentiation of 3-D dynamically cultured human MSCs can be explained by generation of fluid shear stress and enhanced mass transport to the interior of the scaffold mimicking the native microenvironment of bone cells. This study provides evidence for the effectiveness of dynamic culture of human MSCs during the initial phase of ex vivo osteogenesis.

Neuropotency of Human Mesenchymal Stem Cell Cultures: Clonal Studies Reveal the Contribution of Cell Plasticity and Cell Contamination

Various studies have shown neuropotency of bone marrow-derived human mesenchymal stem cells (hMSC) based on the appearance of cells with neural phenotype before or after neural induction protocols. However, to date, it is unclear which mechanisms account for this observation. We hypothesized that neural phenotypes observed in hMSC cultures can be because of both intrinsic cell plasticity and contamination by cells of neural origin. Therefore, we characterized 38 clones from hMSC cultures by assessing their adipogenic/osteogenic potential with specific mesenchymal differentiation protocols, and their molecular neural phenotype by RT-PCR analysis before and after exposure to a defined neural stem cell (NSC) medium for 8 days (neural protocol). We found 33 clones with mesenchymal potential and 15 of them also showed a neural phenotype. As neural phenotypes were maintained during the neural protocol, this suggested neural cell plasticity in 39% of all clones through pluripotency. Importantly, we were able to induce neural phenotypes in 11 of mesenchymal clones applying the neural protocol, demonstrating neural cell plasticity in 29% of all clones through the mechanism of transdifferentiation. Finally, 2 of 5 nonmesenchymal clones (5% of all clones) displayed a neural phenotype indicating neural cell contamination of hMSC cultures. In conclusion, we found 2 different ways of neuropotency of hMSC cultures: cell plasticity and cell contamination.

Pulsed electromagnetic fields enhance BMP‐2 dependent osteoblastic differentiation of human mesenchymal stem cells

Mesenchymal stem cells (MSCs) express an osteoblastic phenotype when treated with BMP-2, and BMP-2 is used clinically to induce bone formation although high doses are required. Pulsed electromagnetic fields (PEMF) also promote osteogenesis in vivo, in part through direct action on osteoblasts. We tested the hypothesis that PEMF enhances osteogenesis of MSCs in the presence of an inductive stimulus like BMP-2. Confluent cultures of human MSCs were grown on calcium phosphate disks and were treated with osteogenic media (OM), OM containing 40 ng/mL rhBMP-2, OM + PEMF (8 h/day), or OM + BMP-2 + PEMF. MSCs demonstrated minor increases in alkaline phosphatase (ALP) during 24 days in culture and no change in osteocalcin. OM increased ALP and osteocalcin by day 6, but PEMF had no additional effect at any time. BMP-2 was stimulatory over OM, and PEMF + BMP-2 synergistically increased ALP and osteocalcin. PEMF also enhanced the effects of BMP-2 on PGE2, latent and active TGF-beta1, and osteoprotegerin. Effects of PEMF on BMP-2-treated cells were greatest at days 12 to 20. These results demonstrate that PEMF enhances osteogenic effects of BMP-2 on MSCs cultured on calcium phosphate substrates, suggesting that PEMF will improve MSC response to BMP-2 in vivo in a bone environment.

Multilineage Potential of Stable Human Mesenchymal Stem Cell Line Derived from Fetal Marrow

Human bone marrow contains two major cell types, hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). MSCs possess self-renewal capacity and pluripotency defined by their ability to differentiate into osteoblasts, chondrocytes, adipocytes and muscle cells. MSCs are also known to differentiate into neurons and glial cells in vitro, and in vivo following transplantation into the brain of animal models of neurological disorders including ischemia and intracerebral hemorrhage (ICH) stroke. In order to obtain sufficient number and homogeneous population of human MSCs, we have clonally isolated permanent and stable human MSC lines by transfecting primary cell cultures of fetal human bone marrow MSCs with a retroviral vector encoding v-myc gene. One of the cell lines, HM3.B10 (B10), was found to differentiate into neural cell types including neural stem cells, neurons, astrocytes and oligodendrocytes in vitro as shown by expression of genetic markers for neural stem cells (nestin and Musashi1), neurons (neurofilament protein, synapsin and MAP2), astrocytes (glial fibrillary acidic protein, GFAP) and oligodendrocytes (myelin basic protein, MBP) as determined by RT-PCR assay. In addition, B10 cells were found to differentiate into neural cell types as shown by immunocytochical demonstration of nestin (for neural stem cells), neurofilament protein and β-tubulin III (neurons) GFAP (astrocytes), and galactocerebroside (oligodendrocytes). Following brain transplantation in mouse ICH stroke model, B10 human MSCs integrate into host brain, survive, differentiate into neurons and astrocytes and induce behavioral improvement in the ICH animals. B10 human MSC cell line is not only a useful tool for the studies of organogenesis and specifically for the neurogenesis, but also provides a valuable source of cells for cell therapy studies in animal models of stroke and other neurological disorders.

Immunomodulatory Thalidomide Analogs Do Not Affect Osteoprogenitor Differentiation and Low Concentration of Bortezomib Promotes It, While Both Agents Suppress Osteoclast Differentiation.

Bone disease (BD) is a frequent characteristic of multiple myeloma (MM) and leads to severe clinical complications. BD in MM is due to not only the activation of osteoclast but also the impairment of osteoblast differentiation. Treatment for MM using high-dose melphalan followed by tandem auto-stem cell transplantation (tandem ASCT) and new drugs such as bortezomib (PS-341), thalidomide, and lenalidomide have improved OS. Interestingly, some reports have shown high serum bone-specific alkaline phosphatase levels after bortezomib-combined therapy in MM patients, and administration of proteasome inhibitor induced bone formation accompanied by the expression of bone morphogenetic protein-2 (BMP-2) in mice. However, the effects of immunomodulatory compounds on osteoblast or osteoclast differentiation remain unknown. We investigated the effects of bortezomib (PS-341) and immunomodulatory thalidomide analogs (immunomodulatory compounds; CC-4047, CC-6032, and CC-5013, or lenalidomide) on osteoblast and osteoclast differentiation in vitro. We used a human mesenchymal stem cell (MSCs) culture system for osteoblast differentiation and normal peripheral blood mononuclear cells for osteoclast differentiation. First, we tried to determine the working concentration of bortezomib and immunomodulatory compounds in our culture system of osteoblast and osteoclast differentiation by treatment of MM cell lines (KMS21BM, U266, and RPMI8226) with bortezomib (0.5–5.0nM) and immunomodulatory compounds (10μM). Low bortezomib concentrations of not more than 2.5nM did not show any apoptotic effect on these MM cell lines, and the apoptotic effects of immunomodulatory compounds on MM cells differed among MM cells and these compounds themselves. Subsequently, these agents (bortezomib; 0.5–5.0nM, immunomodulatoy compounds; 10μM) were added into the osteoprogenitor cell culture media or the media for osteoclast differentiation with M-CSF and receptor activator of NF-κB ligand (RANKL). We analyzed ALP activity and mineralized nodular formation by von Kossa staining as markers of osteoblast differentiation, and analyzed TRAP-positive multinucleated cells as mature osteoclasts. Furthermore, we analyzed the expression of BMP2 on osteoprogenitor cells in the treatment with bortezomib. The low bortezomib concentrations (0.5 and 1.0nM) increased ALP activity compared with that of the control, and mineralized nodular formation at less than 2.5nM was equal to that in the control. BMP2 mRNA content was found to increase in a time-dependent manner in osteoprogenitor cell culture medium that was independent of the treatment with bortezomib. However, these low bortezomib concentrations did not show either anti-myeloma or fixed anti-osteoclast effects despite the possibility of suppression of osteoclast differentiation even at doses of 1.0 and 2.5nM bortezomib according to cases. In contrast, immunomodulatory compounds at 10μM showed an anti-osteoclast effect without cytotoxicity to osteoblast differentiation, at which dose myeloma cell lines underwent apoptosis. These findings might improve the treatment strategy for MM patients without damage to BM stromal cells by combining of bortezomib with immunomodulatory compounds such as lenalidomide.

Resveratrol enhances proliferation and osteoblastic differentiation in human mesenchymal stem cells via ER-dependent ERK1/2 activation.

In the present study, we investigated the in vitro effect of resveratrol (RSVL), a polyphenolic phytoestrogen, on cell proliferation and osteoblastic maturation in human bone marrow-derived mesenchymal stem cell (HBMSC) cultures. RSVL (10(-8)-10(-5) M) increased cell growth dose-dependently, as measured by [(3)H]-thymidine incorporation, and stimulated osteoblastic maturation as assessed by alkaline phosphatase (ALP) activity, calcium deposition into the extracellular matrix, and the expression of osteoblastic markers such as RUNX2/CBFA1, Osterix and Osteocalcin in HBMSCs cell cultures. Further studies found that RSVL (10(-6)M) resulted in a rapid activation of both extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) signaling in HBMSCs cultures. The effects of RSVL were mimicked by 17beta-estrodial (10(-8) M) and were abolished by estrogen receptor (ER) antagonist ICI182780. An ERK1/2 pathway inhibitor, PD98059, significantly attenuated RSVL-induced ERK1/2 phosphorylation, consistent with the reduction of cell proliferation and osteoblastic differentiation as well as expression of osteoblastic markers. In contrast, SB203580, a p38 MAPK pathway blocker, blocked RSVL-induced p38 phosphorylation, but resulted in an increase of cell proliferation and a more osteoblastic maturation. These data suggest that RSVL stimulates HBMSCs proliferation and osteoblastic differentiation through an ER-dependent mechanism and coupling to ERK1/2 activation.

Human marrow mesenchymal stem cell culture: serum‐free medium allows better expansion than classical alpha‐minimal essential medium (MEM)

Human marrow mesenchymal stem cell culture: serum‐free medium allows better expansion than classical alpha‐minimal essential medium (MEM)

Human autologous serum obtained using a completely closed bag system as a substitute for foetal calf serum in human mesenchymal stem cell cultures

The major problem in cell therapy is the possibility of viral or bacterial infection and immune reactions. Therefore, it is expected of culture cells which are intended to be re-implanted with autologous serum rather than conventional bovine serum. Cell therapy with human mesenchymal stem cells (hMSC), differentiating to various cells, is thought to be curative. To culture hMSC with human autologous serum (HAS) and re-implant them for cell therapy, we developed a completely closed bag system separating serum, comparing proliferation and multipotency of hMSC cultured in HAS with those in foetal calf serum (FCS). HAS was simply, safely and efficiently obtained with the developed closed bag system. Cell proliferation of hMSC cultured in HAS was greater than that in FCS. hMSC, exposed to the defined induction medium containing HAS as well as FCS, differentiated into osteoblasts and adipocytes. These findings suggest that HAS obtained with the developed closed bag system is advantageous in a point of decrease in risk of virus or bacterial infection and foreign protein contamination and enhancement of proliferation of hMSC.