Mesenchymal Stem Cell

Bioreactors provide a dynamic culture system for efficient exchange of nutrients and mechanical stimulus necessary for the generation of effective tissue engineered bone grafts (TEBG). We have shown that biaxial rotating (BXR) bioreactor-matured human fetal mesenchymal stem cell (hfMSC) mediated-TEBG can heal a rat critical sized femoral defect. However, it is not known whether optimal bioreactors exist for bone TE (BTE) applications. We systematically compared this BXR bioreactor with three most commonly used systems: Spinner Flask (SF), Perfusion and Rotating Wall Vessel (RWV) bioreactors, for their application in BTE. The BXR bioreactor achieved higher levels of cellularity and confluence (1.4e2.5x, p < 0.05) in large 785 mm 3 macroporous scaffolds not achieved in the other bioreactors operating in optimal settings. BXR bioreactor-treated scaffolds experienced earlier and more robust osteogenic differentiation on von Kossa staining, ALP induction (1.2e1.6Â, p < 0.01) and calcium deposition (1.3 e2.3Â, p < 0.01). We developed a Micro CT quantification method which demonstrated homogenous distribution of hfMSC in BXR bioreactor-treated grafts, but not with the other three. BXR bioreactor enabled superior cellular proliferation, spatial distribution and osteogenic induction of hfMSC over other commonly used bioreactors. In addition, we developed and validated a non-invasive quantitative micro CT-based technique for analyzing neo-tissue formation and its spatial distribution within scaffolds.

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Over the past few years, substantial progress has been made in the field of stem cell regeneration of the intervertebral disc. Autogenic mesenchymal stem cells in animal models can arrest intervertebral disc degeneration or even partially regenerate it and the effect is suggested to be dependent on the severity of degeneration. Mesenchymal stem cells (MSCs) are able to escape alloantigen recognition which is an advantage for allogenic transplantation. A number of injectable scaffolds have been described and various methods to pre-modulate MSCs’ activity have been tested. In future, work will need to address the use of mesenchymal stem cells in large animal models and the fate of the implanted mesenchymal stem cells, particularly in the long term, in animals. This review examines the state-of-the-art in the field of stem cell regeneration of the intervertebral disc, and critically discusses, with scientific support, the issues involved, before stem cells could be used in human subjects.

Umbilical cord blood (UCB) contains a high number of primitive progenitor cells, allowing UCB to be used as a source of hematopoietic progenitors for clinical transplantation. However the rate of UCB CD34 z stem cells graft is low. Mesenchymal stem cells (MSCs) have been implicated in playing an important role in hematopoietic stem cell engraftment.

The ability to repair tissue via percutaneous means may allow interventional pain physicians to manage a wide variety of diseases including peripheral joint injuries and osteoarthritis. This review will highlight the developments in cellular medicine that may soon permit interventional pain management physicians to treat a much wider variety of clinical conditions and highlight an interventional case study using these technologies To determine if isolated and expanded human autologous mesenchymal stem cells could effectively regenerate cartilage and meniscal tissue when percutaneously injected into knees. Case Study Private Interventional Pain Management practice. An IRB approved study with a consenting volunteer in which mesenchymal stem cells were isolated and cultured ex-vivo from bone marrow aspiration of the iliac crest. The mesenchymal stem cells were then percutaneously injected into the subject&#39;s knee with MRI proven degenerative joint disease. Pre- and post-treatment su...

The clinically known importance of patient sex as a major risk factor for compromised bone healing is poorly reflected in animal models. Consequently, the underlying cellular mechanisms remain elusive. Because mesenchymal stem cells (MSCs) are postulated to regulate tissue regeneration and give rise to essential differentiated cell types, they may contribute to sex-specific differences in bone healing outcomes. Methods: We investigated sex-specific variations in bone healing and associated differences in MSC populations. A 1.5 mm osteotomy gap in the femora of 8 male and 8 female 12-month-old Sprague-Dawley rats was stabilized by an external fixator. Healing was analyzed in terms of biomechanical testing, bridging and callus size over time (radiography at 2, 4, and 6 weeks after surgery), and callus volume and geometry by μCT at final follow-up. MSCs were obtained from bone marrow samples of an age-matched group of 12 animals (6 per gender) and analyzed for numbers of colony-forming units (CFUs) and their capacity to differentiate and proliferate. The proportion of senescent cells was determined by β-galactosidase staining. Results: Sex-specific differences were indicated by a compromised mechanical competence of the callus in females compared with males (maximum torque at failure, p = 0.028). Throughout the follow-up, the crosssectional area of callus relative to bone was reduced in females (p ≤ 0.01), and the bridging of callus was delayed (p 2weeks = 0.041). μCT revealed a reduced callus size (p = 0.003), mineralization (p = 0.003) and polar moment of inertia (p = 0.003) in female animals. The female bone marrow contained significantly fewer MSCs, represented by low CFU numbers in both femora and tibiae (p femur = 0.017, p tibia = 0.010). Functional characteristics of male and female MSCs were similar. Conclusion: Biomechanically compromised and radiographically delayed bone formation were distinctive in female rats. These differences were concomitant with a reduced number of MSCs, which may be causative for the suboptimal bone healing.

An ideal scaffold that provides a combination of suitable mechanical properties along with biological signals is required for successful ligament/tendon regeneration in mesenchymal stem cell-based tissue engineering strategies. Among the various fibre-based scaffolds that have been used, hybrid fibrous scaffolds comprising both microfibres and nanofibres have been recently shown to be particularly promising. This study developed a biohybrid fibrous scaffold system by coating bioactive bFGF-releasing ultrafine PLGA fibres over mechanically robust slowly-degrading degummed knitted microfibrous silk scaffolds. On the ECM-like biomimetic architecture of ultrafine fibres, sustained release of bFGF mimicked the ECM in function, initially stimulating mesenchymal progenitor cell (MPC) proliferation, and subsequently, their tenogeneic differentiation. The biohybrid scaffold system not only facilitated MPC attachment and promoted cell proliferation, with cells growing both on ultrafine PLGA fibres and silk microfibres, but also stimulated tenogeneic differentiation of seeded MPCs. Upregulated gene expression of ligament/tendon-specific ECM proteins and increased collagen production likely contributed to enhancing mechanical properties of the constructs, generating a ligament/tendon analogue that has the potential to be used to repair injured ligaments/tendons.

Mesenchymal stem cells (MSC) are multipotent cells that differentiate into osteoblasts, myocytes, chondrocytes and adipocytes as well as insulin-producing cells. The mechanism underlying their in vivo differentiation is not clear and is thought to be caused by spontaneous cell fusion or factors present in the microenvironment. However, their ease of isolation, high 'ex-vivo' expansion potential and ability to differentiate into multiple lineages make them attractive tools for potential use in cell therapy. MSC have been isolated from several tissues, including bone/bone marrow, fat, Wharton's jelly, umbilical cord blood, placenta and pancreas. The 'immunosuppressive' property of human MSC makes them an important candidate for cellular therapy in allogeneic settings. Use of allogeneic MSC for repair of large defects may be an alternative to autologous and allogeneic tissue-grafting procedures. An allogeneic approach would enable MSC to be isolated from any donor, expanded and cryopreserved, providing a readily available source of progenitors for cell replacement therapy.

Putative stem cells have been isolated from various tissue fluids such as synovial fluid, amniotic fluid, menstrual blood, etc. Recently the presence of nestin positive putative mammary stem cells has been reported in human breast milk. However, it is not clear whether they demonstrate multipotent nature. Since human breast milk is a non-invasive source of mammary stem cells, we were interested in examining the nature of these stem cells. In this pursuit, we could succeed in isolating and expanding a mesenchymal stem cell-like population from human breast milk. These cultured cells were examined by immunofluorescent labeling and found positive for mesenchymal stem cell surface markers CD44, CD29, SCA-1 and negative for CD33, CD34, CD45, CD73 confirming their identity as mesenchymal stem cells. Cytoskeletal protein marker analysis revealed that these cells expressed mesenchymal stem cells markers, namely, nestin, vimentin, smooth muscle actin and also manifests presence of E-Cadherin, an epithelial to mesenchymal transition marker in their early passages. Further we tested the multipotent differentiation potential of these cells and found that they can differentiate into adipogenic, chondrogenic and oesteogenic lineage under the influence of specific differentiation cocktails. This means that these mesenchymal stem cells isolated from human breast milk could potentially be "reprogrammed" to form many types of human tissues. The presence of multipotent stem cells in human milk suggests that breast milk could be an alternative source of stem cells for autologous stem cell therapy although the significance of these cells needs to be determined.

Bone formation in the embryo, and during adult fracture repair and remodeling, involves the progeny of a small number of cells called mesenchymal stem cells (MSCs). These cells continuously replicate themselves, while a portion become committed to mesenchymal cell lineages such as bone, cartilage, tendon, ligament, and muscle. The differentiation of these cells, within each lineage, is a complex multistep pathway involving discrete cellular transitions much like that which occurs during hematopoiesis. Progression from one stage to the next depends on the presence of specific bioactive factors, nutrients, and other environmental cues whose exquisitely controlled contributions orchestrate the entire differentiation phenomenon. An understanding of the cellular and molecular events of osteogenic differentiation of MSCs provides the foundation for the emergence of a new therapeutic technology for cell therapy. The isolation and in vitro mitotic expansion of autologous human MSCs will support the development of novel protocols for the treatment of many clinically challenging conditions. For example, local bone defects can be repaired through site-directed delivery of MSCs in an appropriate carrier vehicle. Generalized conditions, such as osteoporosis, may be treatable by systemic administration of culture-expanded autologous MSCs or through biopharmaceutical regimens based on the discovery of critical regulatory molecules in the differentiation process. With this in mind, we can begin to explore therapeutic options that have never before been available.

Poor angiogenesis within tissue-engineered grafts has been identified as a main challenge limiting the clinical introduction of bone tissue-engineering (BTE) approaches for the repair of large bone defects. Thick BTE grafts often exhibit poor cellular viability particularly at the core, leading to graft failure and lack of integration with host tissues. Various BTE approaches have been explored for improving vascularisation in tissue-engineered constructs and are briefly discussed in this review. Recent investigations relating to co-culture systems of endothelial and osteoblast-like cells have shown evidence of BTE efficacy in increasing vascularization in thick constructs. This review provides an overview of key concepts related to bone formation and then focuses on the current state of engineered vascularized co-culture systems using bone repair as a model. It will also address key questions regarding the generation of clinically relevant vascularized bone constructs as well as potential directions and considerations for research with the objective of pursuing engineered co-culture systems in other disciplines of vascularized regenerative medicine. The final objective is to generate serious and functional long-lasting vessels for sustainable angiogenesis that will enable enhanced cellular survival within thick voluminous bone grafts, thereby aiding in bone formation and remodelling in the long term. However, more evidence about the quality of blood vessels formed and its associated functional improvement in bone formation as well as a mechanistic understanding of their interactions are necessary for designing better therapeutic strategies for translation to clinical settings. Figure 1. (A) Hierarchical structure of bone from a sub-nanostructure of collagen molecules to a nanostructure of cylindrically arranged microfibrils to lamellar layers forming the macrostructure of cortical bone (adapted from Rho et al. (1998). (B) Vascularised bone anatomy and microenvironmental influences such as biomechanical, microarchitectural and low oxygen tension factors Y. Liu et al.

On June 2008, the first human tissue-engineered trachea replacement was performed using decellularized (de-antigenised) cadaveric donor trachea, seeded with recipient epithelial cells on the internal surface of the graft and mesenchymal stem-cell-derived chondrocytes on the external. During the followup, cytological analysis at 4 postoperative days showed a migration of the stem-cells derived chondrocytes from the outer to the inner surface of the first 2 cm of the graft length. With the aim to rationalize these clinical findings, and under the hypothesis that cellular migration is driven by the oxygen gradients developing from the external part of the construct (exposed to O 2 deficiency) towards the better oxygenated epithelial region, an accurate computational model of oxygen transport in the trachea engineered construct was developed and solved using finite element method (FEM). Results confirm that critical limitation to oxygen transport prevalently occurs from proximal to middle section, within the first 2.8 cm of longitudinal length, in good agreement with experimental observation. In the proximal section, recognized as the most critical part of the engineered construct, the severe O 2 mass transfer limitation causes a drastic reduction of the diffusive flux within a distance of 650 mm. At cell density of 1 Â 10 7 cells/cm 3 , the 30% c.a of the total section area is under oxygen deficiency (O 2 partial pressure below the critical threshold of 38 mmHg). Along the whole tracheal construct, the Thiele modulus ranges within 2.3 and 3.7 in the external chondrocyte compartment, confirming thus the importance of the mass transfer limitation to oxygen diffusion rate. In general, the efficiency of the O 2 transport reduces considerably in the region close to proximal section.

Nanostructured materials are believed to play a fundamental role in orthopedic research because bone itself has a structural hierarchy at the first level in the nanometer regime. Here, we report on titanium oxide (TiO 2 ) surface nanostructures utilized for orthopedic implant considerations. Specifically, the effects of TiO 2 nanotube surfaces for bone regeneration will be discussed. This unique 3D tube shaped nanostructure created by electrochemical anodization has profound effects on osteogenic cells and is stimulating new avenues for orthopedic material surface designs. There is a growing body of data elucidating the benefits of using TiO 2 nanotubes for enhanced orthopedic implant surfaces. The current trends discussed within foreshadow the great potential of TiO 2 nanotubes for clinical use.

Osterix (Osx) is a transcription factor required for the differentiation of preosteoblasts into fully functioning osteoblasts. However, the pattern of Osx activation during preosteoblast differentiation and maturation has not been clearly defined. Our aim was to study Osx activation during these processes in osteoblasts differentiating from murine and human embryonic stem cells (ESC). To do this, we constructed an Osx-GFP fusion protein reporter system to track Osx translocation within the cells. The distribution of Osx-GFP at representative stages of differentiation was also investigated by screening primary osteoblasts, mesenchymal stem cells, synoviocytes, and pre-adipocytes. Our experiments revealed that Osx-GFP protein was detectable in the cytoplasm of cultured, differentiated ESC 4 days after plating of enzymatically dispersed embryoid bodies. Osterix-GFP protein became translocated into the nucleus on day 7 following transfer of differentiated ESC to osteogenic medium. After 14 days of differentiation, cells showing nuclear translocation of Osx-GFP formed rudimentary bone nodules that continued to increase in number over the following weeks (through day 21). We also found that Osx translocated into the nuclei of mesenchymal stem cells (C3H10T1/2) and pre-osteoblasts (MC3T3-E1) and showed partial activation in pre-adipocytes (MC3T3-L1). These data suggest that Osx activation occurs at a very early point in the differentiation of the mesenchymal-osteoblastic lineage.

Hematopoietic stem and progenitor cells (HSPC) reside in the bone marrow (BM) niche and serve as a reservoir for mature blood cells throughout life. Aging in the BM is characterized by low-grade chronic inflammation that could contribute to the reduced functionality of aged HSPC. Mesenchymal stromal cells (MSC) in the BM support HSPC self-renewal. However, changes in MSC function with age and the crosstalk between MSC and HSPC remain understudied. Here, we conducted an extensive characterization of senescence features in BM-derived MSC from young and aged healthy donors. Aged MSC displayed an enlarged senescent-like morphology, a delayed clonogenic potential and reduced proliferation ability when compared to younger counterparts. Of note, the observed proliferation delay was associated with increased levels of SA-β-galactosidase (SA-β-Gal) and lipofuscin in aged MSC at early passages and a modest but consistent accumulation of physical DNA damage and DNA damage response (DDR) activation. Consistent with the establishment of a senescence-like state in aged MSC, we detected an increase in pro-inflammatory senescence-associated secretory phenotype (SASP) factors, both at the transcript and protein levels. Conversely, the immunomodulatory properties of aged MSC were significantly reduced. Importantly, exposure of young HSPC to factors secreted by aged MSC induced pro-inflammatory genes in HSPC and impaired HSPC clonogenic potential in a SASP-dependent manner. Altogether, our results reveal that BMderived MSC from aged healthy donors display features of senescence and that, during aging, MSC-associated secretomes contribute to activate an inflammatory transcriptional program in HSPC that may ultimately impair their functionality.

The last decade has witnessed the development of cell-based therapy as a major biomedical research area, including the treatment of musculoskeletal diseases. Both differentiated and undifferentiated stem cells have been used as starting cell sources. In particular, the use of multipotent adult mesenchymal stem cells holds great promise for future therapeutic strategies. In addition to the cell type used, the cell delivery system is also of critical importance in cell-based therapy. Cell delivery may be achieved by direct cell injection or by grafting engineered constructs derived by cell seeding into natural or synthetic biomaterial scaffolds. While direct injection is the most direct and convenient means of cell delivery, the latter approach is capable of producing three-dimensional engineered tissues with mechanical properties compatible with those of various musculoskeletal tissues. This review will focus on the functional approach of using biomaterial scaffold materials as cell carriers for musculoskeletal applications, as well as the use of cell-based gene therapy for tissue engineering and regeneration.

Background: Articular cartilage has very limited intrinsic regenerative capacity, making cell-based therapy a tempting approach for cartilage repair. Cell tracking can be a major step towards unraveling and improving the repair process of these therapies. We studied superparamagnetic iron oxides (SPIO) for labeling human bone marrow-derived mesenchymal stem cells (hBMSCs) regarding effectivity, cell viability, long term metabolic cell activity, chondrogenic differentiation and hBMSC secretion profile. We additionally examined the capacity of synovial cells to endocytose SPIO from dead, labeled cells, together with the use of magnetic resonance imaging (MRI) for intra-articular visualization and quantification of SPIO labeled cells.

Since synthetic nerve conduits do not exhibit the characteristics of regeneration, they are generally inadequate substitutes for autologous nerve graft in the repair of long peripheral nerve defects. To resolve this problem, in this study, we constructed a nerve regeneration characteristics-containing nerve graft through integrating xenogeneic acellular nerve matrix (ANM) with autologous neural differentiated adipose-derived mesenchymal stem cells (ADSCs). Xenogeneic ANM was processed by a protocol removing cells and myelin sheath completely, meanwhile preserving growth factors and extracellular matrix (ECM) microstructure of natural nerve, such as porous and basal lamina tube. Cytocompatibility and immunocompatibility evaluation revealed that ANM could support cell attachment and proliferation, and did not stimulate vigorous host reject response. After inoculation of neural differentiated ADSCs onto ANM, differentiated cells were observed to align along longitudinal axis of ANM, resembling band of büngner, and persistently express NGF, GDNF, and BDNF. In vivo, neural differentiated ADSCs also presented glial cell characteristics and promote nerve regeneration 7 days post transplantation. We repaired 1 cm Sprague Dawley rat sciatic nerve defects using this nerve graft construction and nerve gap regeneration was indicated by electrophysiology, retrograde labeling and histology analysis. Therefore, we conclude that constructed nerve graft, offering nerve regeneration characteristics, hold great promise to replace autologous in repair peripheral nerve defect.

Successful cell therapy will depend on the ability to monitor transplanted cells. With cell labeling, it is important to demonstrate efficient long term labeling without deleterious effects on cell phenotype and differentiation capacity. We demonstrate long term (7 weeks) retention of superparamagnetic iron oxide particles (SPIO) by mesenchymal stem cells (MSCs) in vivo, detectable by MRI. In vitro, multilineage differentiation (osteogenic, chondrogenic and adipogenic) was demonstrated by histological evaluation and molecular analysis in SPIO labeled and unlabeled cells. Gene expression levels were comaparable to unlabeled controls in adipogenic and chondrogenic conditions however not in the osteogenic condition. MSCs seeded into a scaffold for 21 days and implanted subcutaneously into nude mice for 4 weeks, showed profoundly altered phenotypes in SPIO labeled samples compared to implanted unlabeled control scaffolds, indicating chondrogenic differentiation. This study demonstrates long term MSC traceability using SPIO and MRI, uninhibited multilineage MSC differentiation following SPIO labeling, though with subtle but significant phenotypical alterations.

a r c h i v e s o f o r a l b i o l o g y 5 5 ( 2 0 1 0 ) 1 8 5 -1 9 4 Leukocyte Platelet concentrate Mesenchymal stem cell Platelet-rich fibrin (PRF) Platelet-rich plasma (PRP) a b s t r a c t Background: Choukroun's platelet-rich fibrin (PRF) is an autologous leukocyte-and plateletrich fibrin biomaterial. The purpose of this study was to analyse the in vitro effects of PRF on human bone mesenchymal stem cells (BMSC), harvested in the oral cavity after preimplant endosteal stimulation.

Craniofacial tissue engineering promises the regeneration or de novo formation of dental, oral, and craniofacial structures lost to congenital anomalies, trauma, and diseases. Virtually all craniofacial structures are derivatives of mesenchymal cells. Mesenchymal stem cells are the offspring of mesenchymal cells following asymmetrical division, and reside in various craniofacial structures in the adult. Cells with characteristics of adult stem cells have been isolated from the dental pulp, the deciduous tooth, and the periodontium. Several craniofacial structures-such as the mandibular condyle, calvarial bone, cranial suture, and subcutaneous adipose tissue-have been engineered from mesenchymal stem cells, growth factor, and/or gene therapy approaches. As a departure from the reliance of current clinical practice on durable materials such as amalgam, composites, and metallic alloys, biological therapies utilize mesenchymal stem cells, delivered or internally recruited, to generate craniofacial structures in temporary scaffolding biomaterials. Craniofacial tissue engineering is likely to be realized in the foreseeable future, and represents an opportunity that dentistry cannot afford to miss.

Objective: To track superparamagnetic iron oxide nanoparticle (SPIO)-labeled, bone-derived mesenchymal stem cells (MSCs) by in vivo magnetic resonance imaging (MRI) with a 1.5 T-system following injection of engineered autologous MSCs into the knee joint cavity in rabbit articular cartilage defect models. Methods: Rabbit MSCs were labeled with SPIO and a transfection agent. Cell viability, proliferation and differentiation capacity were assessed in vitro using appropriate functional assays. Cells underwent GRE T2*-weighted MRI in vitro. The autologous MSCs seeded in chitosan and glycerophosphate (C-GP) gel were injected into the knee joint cavity of rabbit models for cartilage defects. All rabbits underwent GRE T2*weighted MRI 1, 4, 8 and 12 weeks post-injection. MR imaging findings were compared histologically. Results: Nanoparticles were stained with Prussian blue and observed by transmission electron microscopy inside the cells. Cell viability, proliferation, and differentiation were comparable between labeled and non-labeled cells. After intra-articular injection of labeled autologous MSCs, marked hypointense signal void areas representing the injected MSCs can be observed for at least 12 weeks on GRE T2*-weighted images. At 12 weeks post-injection, labeled MSCs migrated into the synovial fluid at the suprapatellar bursa, the popliteal space site and subchondral bone of the femur but no MSCs were detected in the defect. Histochemical staining confirmed the presence of Prussian blue-positive cells and BrdU-positive cells. Conclusions: MRI would be an efficient noninvasive technique to visually track SPIO-labeled seed cells in vivo; the engineered autologous MSCs do not actively participate in the repair of articular cartilage defects following intra-articular injection.

Background IL-21 is secreted by activated T cells and modulates immune cell functions with both proinflammatory and anti-inflammatory effects. IL-21 receptor (IL-21R), homologous to IL-2Rβ and IL-4Rα, associates with the gamma common chain upon ligand binding. It was recently described that IL-21R is overexpressed in the inflamed synovial membrane and on leucocytes of rheumatoid arthritis patients. Objective Previously we have shown that blockade of the IL-21 pathway with soluble IL-21R-Fc resulted in a reduction of clinical signs of arthritis in rodent models. To understand potential mechanisms of IL-21 regulation in arthritis, we analyzed serum immunoglobulin levels, and cytokine expression in the paws, serum, and collagen-restimulated splenocytes, in response to IL-21 pathway blockade. Methods Arthritis was induced in DBA/1 male mice with bovine type II collagen. Animals were treated with either soluble mIL-21R-Fc, which neutralizes murine IL-21 bioactivity, with TNFRII-Fc or with control IgG. Spleens from each group of treated mice were cultured in vitro with collagen and assayed for cytokine secretion. Cytokines and anticollagen-specific IgG levels were also measured in the serum by ELISA. Cytokine mRNA levels in the paws were evaluated by quantitative PCR analysis. Results Treatment of mice with IL-21R-Fc or TNFRII-Fc reduced clinical and histological signs of collagen-induced arthritis. IL-6 mRNA in paws and serum IL-6 levels were decreased after IL-21R-Fc treatment. IFNγ mRNA was increased in paws of IL-21R-Fctreated mice. Collagen-specific spleen cell responses from IL-21R-Fc-treated mice exhibited increased IFNγ and IL-2, and reduced IL-6 and IL-17 levels. Serum levels of total IgG 1 were also reduced in response to IL-21R-Fc treatment. Conclusion These data demonstrate a role for IL-21 in the modulation of collagen-specific T-cell responses and the pathology of arthritis, supporting a rationale for blockade of the IL-21 pathway in rheumatoid arthritis.

There is great interest in genetic modification of bone marrow-derived mesenchymal stem cells (MSC), not only for research purposes but also for use in (autologous) patient-derived-patientused transplantations. A major drawback of bulk methods for genetic modifications of (stem) cells, like bulk-electroporation, is its limited yield of DNA transfection (typically then 10%). This is even more limited when cells are present at very low numbers, as is the case for stem cells. Here we present an alternative technology to transfect cells with high efficiency (.75%), based on single cell electroporation in a microfluidic device. In a first experiment we show that we can successfully transport propidium iodide (PI) into single mouse myoblastic C2C12 cells. Subsequently, we show the use of this microfluidic device to perform successful electroporation of single mouse myoblastic C2C12 cells and single human MSC with vector DNA encoding a green fluorescent-erk1 fusion protein (EGFP-ERK1 (MAPK3)). Finally, we performed electroporation in combination with live imaging of protein expression and dynamics in response to extracellular stimuli, by fibroblast growth factor (FGF-2). We observed nuclear translocation of EGFP-ERK1 in both cell types within 15 min after FGF-2 stimulation. Due to the successful and promising results, we predict that microfluidic devices can be used for highly efficient small-scale 'genetic modification' of cells, and biological experimentation, offering possibilities to study cellular processes at the single cell level. Future applications might be small-scale production of cells for therapeutic application under controlled conditions.

Owing to their resistance to protein adsorption, poly(ethylene glycol) (PEG) hydrogels are unable to support the adhesion of human mesenchymal stem cells (hMSCs). Human MSCs attach to various substrates through cell adhesion proteins, and osteopontin is an important cell adhesion protein for both MSCs and osteoblasts found in bone. As such, we hypothesized that cell adhesion to a PEG hydrogel could be dramatically improved by actively promoting the formation of a mineral phase throughout the hydrogel. This was accomplished using the photoreactive, phosphate-containing molecule ethylene glycol methacrylate phosphate (EGMP). When EGMP was incorporated into the PEG hydrogel, it resulted in the formation of a bone-like mineral phase, as verified using compositional analysis and X-ray diffraction. Furthermore, cell viability of gel-encapsulated hMSCs was increased in the presence of EGMP from 15% in the absence of EGMP to 97% in the presence of 50 mM EGMP. This improvement in cell viability was thought to be due to the ability of mineralized hydrogels to sequester cell-secreted osteopontin. It was found that EGMP-containing PEGDA hydrogels are able to sequester the important cell adhesion protein osteopontin, and these hydrogels promoted hMSC adhesion and spreading. r

Introduction Several different synthetic and allograft bone graft substitutes are used clinically to treat large bone defects. In contrast to the ''gold standard'' of autologous bone grafts, these do not contain bone-forming (MSC) or vessel-forming (EPC) cells. In order to achieve the same level of success enjoyed by autologous bone grafts, they must be compatible with mesenchymal stem cells (MSC) and endothelial progenitor cells (EPC). In a previous study, we seeded MSC onto six different bone graft substitutes and then measured the cell adhesion, viability, differentiation, and morphology. In the present study, we seeded both MSC and EPC onto the same six bone graft substitutes and measured the same parameters. Methods In vitro, 125,000 MSC and 125,000 EPC were seeded onto Chronos Ò , Vitoss Ò , Actifuse Ò , Biobase Ò , Cerabone Ò , and Tutoplast Ò . Cell adhesion (fluorescence microscopy) and viability (MTT assay) were measured on days 2, 6, and 10. Osteogenic (cbfa-1, alkaline phosphatase [ALP], osteocalcin, collagen-1 alpha [Col1A]) and endothelial (von Willebrand factor [vWF], vascular endothelial growth factor [VEGF], kinase domain receptor [KDR]) gene expression were analyzed by reverse transcriptase polymerase chain reaction (RT-PCR). Morphology was described by scanning electron microscopy (SEM) at day 2. Results MSC adhered significantly better to Tutoplast Ò , Chronos Ò , Actifuse Ò , and Biobase Ò . EPC adhered better to Actifuse Ò , Chronos Ò , Biobase Ò , and Tutoplast Ò . Viability increased over time when seeded on Tutoplast Ò and Chronos Ò . Osteogenic and endothelial gene expression were detectable at day 10 in cells seeded on Chronos Ò , Actifuse Ò , and Tutoplast Ò . The best morphology of MSC and EPC was found on Tutoplast Ò , Chronos Ò , Actifuse Ò , and Biobase Ò . Conclusion When bone graft substitutes are used to help fill large defects, it is important that their interaction with these cells be supportive of bone healing.

The epithelium-specific Ets transcription factor, PDEF, plays a role in prostate and breast cancer, although its precise function has not been established. In prostate cancer, PDEF is involved in regulating prostate-specific antigen expression via interaction with the androgen receptor and NKX3.1, and down-regulation of PDEF by antiproliferative agents has been associated with reduced PDEF expression. We now report that reduced expression of PDEF leads to a morphologic change, increased migration and invasiveness in prostate cancer cells, reminiscent of transforming growth factor B (TGFB) function and epithelial-to-mesenchymal transition. Indeed, inhibition of PDEF expression triggers a transcriptional program of genes involved in the TGFB pathway, migration, invasion, adhesion, and epithelial dedifferentiation. Our results establish PDEF as a critical regulator of genes involved in cell motility, invasion, and adhesion of prostate cancer cells.

Previous studies have shown the relevance of bone marrow-derived MSCs (BM-MSCs) in controlling graft-versus-host disease (GVHD) after allogeneic transplantation. Since adipose tissue-derived MSCs (Ad-MSCs) may constitute a good alternative to BM-MSCs, we have expanded MSCs derived from human adipose tissue (hAd-MSCs) and mouse adipose tissue (mAd-MSCs), investigated the immunoregulatory properties of these cells, and evaluated their capacity to control GVHD in mice. The phenotype and immunoregulatory properties of expanded hAd-MSCs were similar to those of human BM-MSCs. Moreover, hAd-MSCs inhibited the proliferation and cytokine secretion of human primary T cells in response to mitogens and allogeneic T cells. Similarly, ex vivo expanded mAd-MSCs had an equivalent immunophenotype and exerted immunoregulatory properties similar to those of hAd-MSCs. Moreover, the infusion of mAd-MSCs in mice transplanted with haploidentical hematopoietic grafts controlled the lethal GVHD that occurred in control recipient mice. These findings constitute the first experimental proof that Ad-MSCs can efficiently control the GVHD associated with allogeneic hematopoietic transplantation, opening new perspectives for the clinical use of Ad-MSCs.

Once articular cartilage is injured, it has a very limited capacity for self-repair. Although current surgical therapeutic procedures to cartilage repair are clinically useful, they cannot restore a normal articular surface. Current research offers a growing number of bioactive reagents, including proteins and nucleic acids, that may be used to augment different aspects of the repair process. As these agents are difficult to administer effectively, gene transfer approaches are being developed to provide their sustained synthesis at sites of repair.

The architecture of an engineered tissue substitute plays an important role in modulating tissue growth. A novel poly(D,L-lactide-co-glycolide) (PLGA) structure with a unique architecture produced by an electrospinning process has been developed for tissue-engineering applications. Electrospinning is a process whereby ultra-fine fibers are formed in a high-voltage electrostatic field. The electrospun structure, composed of PLGA fibers ranging from 500 to 800 nm in diameter, features a morphologic similarity to the extracellular matrix (ECM) of natural tissue, which is characterized by a wide range of pore diameter distribution, high porosity, and effective mechanical properties. Such a structure meets the essential design criteria of an ideal en-gineered scaffold. The favorable cell-matrix interaction within the cellular construct supports the active biocompatibility of the structure. The electrospun nanofibrous structure is capable of supporting cell attachment and proliferation. Cells seeded on this structure tend to maintain phenotypic shape and guided growth according to nanofiber orientation. This novel biodegradable scaffold has potential applications for tissue engineering based upon its unique architecture, which acts to support and guide cell growth.

Cord blood (CB) is an important alternative source of hematopoietic stem cells (HSCs) for transplantation today. The principal drawbacks of cord blood transplantation are the limited number of hematopoietic stem cells and a long time to engraftment. Several promising approaches for engraftment enhancement are under intensive investigation. Such are transplantation with two cord blood units, co transplantation of cord blood and haploidentical HSCs and different methods for expansion of cord blood hematopoietic stem cells. In addition there are several ways for improving of homing of HSCs such as coinfusion of CB hematopoietic stem cells and mesenchymal stem cells, administration of parathyroid hormone (PTH), intra-bone transplantation and targeting the CXCR4/SDF1 system. These strategies are expected to increase the availability of transplantation to adults, for whom the chance to find a cord blood suitable for a single unit transplant is small. Recent advances in elucidation of the molecular mechanisms responsible for the proliferation and self-renewal of hematopoietic stem cells may bring further improvement of the outcomes of cord blood transplantation.

Low intensity pulsed ultrasound (LIPUS) has been used extensively for fracture healing. Many LIPUS parameters have been studied in depth except for pulse repetition frequency (PRF). However, to examine this parameter, a custom LIPUS system needed to be designed since commercial systems have very little control over parameter modification. Specifically, the PRF of commercial systems is typically set to either 100 Hz or 1 kHz and cannot be changed. A custom system was used to explore the effects of PRF on human adipose derived adult stem cells (hASCs) and bone marrow derived mesenchymal stem cells (hMSCs). The PRF was found to significantly affect cellular response to LIPUS stimulation for both cell types. Additionally, LIPUS was shown to strongly accelerate osteogenic differentiation of hASCs based on amount of calcium accretion normalized by total DNA.

Bone marrow is a viscous tissue that resides in the confines of bones and houses the vitally important pluripotent stem cells. Due to its confinement by bones, the marrow has a unique mechanical environment which has been shown to be affected from external factors, such as physiological activity and disuse. The mechanical environment of bone marrow can be defined by determining hydrostatic pressure, fluid flow induced shear stress, and viscosity. The hydrostatic pressure values of bone marrow reported in the literature vary in the range of 10.7-120 mmHg for mammals, which is generally accepted to be around one fourth of the systemic blood pressure. Viscosity values of bone marrow have been reported to be between 37.5 and 400 cP for mammals, which is dependent on the marrow composition and temperature. Marrow's mechanical and compositional properties have been implicated to be changing during common bone diseases, aging or disuse. In vitro experiments have demonstrated that the resident mesenchymal stem and progenitor cells in adult marrow are responsive to hydrostatic pressure, fluid shear or to local compositional factors such as medium viscosity. Therefore, the changes in the mechanical and compositional microenvironment of marrow may affect the fate of resident stem cells in vivo as well, which in turn may alter the homeostasis of bone. The aim of this review is to highlight the marrow tissue within the context of its mechanical environment during normal physiology and underline perturbations during disease.