Effects of FGF2 and FGF9 on osteogenic differentiation of bone marrow-derived progenitors

Bone repair is a major concern in reconstructive surgery. Transplants containing osteogenically committed mesenchymal stem cells (MSCs) provide an alternative source to the currently used autologous bone transplants which have limited supply and require additional surgery to the patient. A major drawback, however is the lack of a critical mass of cells needed for successful transplantation. The purpose of the present study was to test the effects of FGF2 and FGF9 on expansion and differentiation of MSCs in order to establish an optimal culture protocol resulting in sufficient committed osteogenic cells required for successful in vivo transplantation. Bone marrow-derived MSCs cultured in αMEM medium supplemented with osteogenic supplements for up to three passages (control medium), were additionally treated with FGF2 and FGF9 in various combinations. Cultures were evaluated for viability, calcium deposition and in vivo osteogenic capacity by testing subcutaneous transplants in nude mice. FGF2 had a positive effect on the proliferative capacity of cultured MSCs compared to FGF9 and control medium treated cultures. Cultures treated with FGF2 followed by FGF9 showed an increased amount of extracted Alizarin red indicating greater osteogenic differentiation. Moreover, the osteogenic capacity of cultured cells transplanted in immunodeficient mice revealed that cells that were subjected to treatment with FGF2 in the first two passages and subsequently to FGF9 in the last passage only, were more successful in forming new bone. It is concluded that the protocol using FGF2 prior to FGF9 is beneficial to cell expansion and commitment, resulting in higher in vivo bone formation for successful bone tissue engineering.     

Therapeutic potential of in utero mesenchymal stem cell (MSCs) transplantation in rat foetuses with spina bifida aperta

Neural tube defects (NTDs) are complex congenital malformations resulting from incomplete neurulation in embryo. Despite surgical repair of the defect, most of the patients who survive with NTDs have a multiple system handicap due to neuron deficiency of the defective spinal cord. In this study, we successfully devised a prenatal surgical approach and transplanted mesenchymal stem cells (MSCs) to foetal rat spinal column to treat retinoic acid induced NTDs in rat. Transplanted MSCs survived, grew and expressed markers of neurons, glia and myoblasts in the defective spinal cord. MSCs expressed and perhaps induced the surrounding spinal tissue to express neurotrophic factors. In addition, MSC reduced spinal tissue apoptosis in NTD. Our results suggested that prenatal MSC transplantation could treat spinal neuron deficiency in NTDs by the regeneration of neurons and reduced spinal neuron death in the defective spinal cord.     

骨碎补结合组织工程软骨促进软骨再生的实验研究

目的:评估骨碎补结合组织工程软骨治疗对实验兔软骨缺陷模型软骨再生的疗效。方法:将h IGF-1基因转染MSCs,并与脱细胞真皮基质(ADM)构建组织工程软骨。24只新西兰白兔随机分为A、B、C、D四组,A、C组进行自体软骨移植,B、D组进行改建的细胞-ADM移植。C、D组用40%骨碎补汤喂养4周,150 m L/d。第12周处死实验动物,分离缺损关节软骨部位,蜡块包埋染色,通过总体形态评价软骨再生组织。采用组织学评分评估再生软骨质量。采用甲苯胺蓝染色评价缺损部位产生软骨糖胺聚糖的情况。结果:与B组比较,C组和D组的新生软骨覆盖度、新骨髓的颜色、缺损边缘和表面粗糙度均显著提高(P0.05);再生软骨的组织学评分软骨表面评分显著改善(P0.05)。C组与D组具有比其他组更好的基质、细胞分布和表面指数。并且有较厚的透明样软骨组织,具有正常的糖胺聚糖产生。表明该治疗方法可以通过再生透明样软骨且没有不良事件来减少软骨缺陷。结论:工程软骨结合骨碎补治疗可显著改善兔膝关节软骨缺损修复的质量,为临床治疗软骨病变提供重要理论依据。     

Comparison of mesenchymal tissues-derived stem cells for in vivo chondrogenesis: suitable conditions for cell therapy of cartilage defects in rabbit

We previously compared mesenchymal stem cells (MSCs) from a variety of mesenchymal tissues and demonstrated that synovium-MSCs had the best expansion and chondrogenic ability in vitro in humans and rats. In this study, we compared the in vivo chondrogenic potential of rabbit MSCs. We also examined other parameters to clarify suitable conditions for in vitro and in vivo cartilage formation. MSCs were isolated from bone marrow, synovium, adipose tissue, and muscle of adult rabbits. Proliferation potential and in vitro chondrogenic potential were compared. Toxicity of the tracer DiI for in vitro chondrogenesis was also examined. MSCs from each tissue were embedded in collagen gel and transplanted into full thickness cartilage defects of rabbits. Cartilage matrix production was compared histologically. The effects of cell density and periosteal patch on the in vivo chondrogenic potential of synovium-MSCs were also examined. Synovium- and muscle-MSCs had a higher proliferation potential than other cells. Pellets from synovium- and bone-marrow-MSCs showed abundant cartilage matrix. DiI had no significant influence on in vitro cartilage formation. After transplantation into cartilage defects, synovium- and bone-marrow-MSCs produced much more cartilage matrix than other cells. When synovium-MSCs were transplanted at a higher cell density and with a periosteal patch, more abundant cartilage matrix was observed. Thus, synovium- and bone-marrow-MSCs had greater in vivo chondrogenic potential than adipose- and muscle-MSCs, but synovium-MSCs had the advantage of a greater proliferation potential. Higher cell density and a periosteum patch were needed to obtain a high production of cartilage matrix by synovium-MSCs.     

Phenotypic characterization of craniofacial bone marrow stromal cells: unique properties of enhanced osteogenesis,cell recruitment,autophagy, and apoptosis resistance

Previous studies have shown that craniofacial bone marrow stromal cells (MSCs) have greater osteogenic potential than appendicular bone MSCs. However, detailed phenotypic characterization of MSCs from bone marrow in the different sites remains unclear. To investigate bone repair and regeneration of craniofacial MSCs and the regulatory mechanisms underlying their unique properties, we compared osteogenesis, cell recruitment, autophagy, and apoptosis resistance of MSCs from the mandible (M-MSCs) to those from tibia (T-MSCs) in vitro and in vivo. Compared with T-MSCs, M-MSCs formed more colonies, possessed stronger proliferation activity, exhibited higher expression of pluripotency genes such as Oct4 and Nanog, and held stronger osteogenic differentiation in osteogenic medium. Moreover, M-MSCs had greater autophagy and anti-apoptotic capacities than T-MSCs under hypoxia and serum deprivation conditions. M-MSCs were found to be more capable of recruiting more MSCs than T-MSCs. When these MSCs were transplanted into mandible critical-sized defects, more bone formed in the M-MSC-treated animals than in their T-MSC counterparts. Collectively, these findings reveal that MSCs have unique characteristics and bone-repairing properties from the mandible as compared with those from tibia, presumably by enhanced osteogenic potential, cell recruitment, autophagy and apoptosis resistance.     

Survival of rat functional dental pulp cells in vascularized tissue engineering chambers

Regenerative endodontics aims to preserve, repair or regenerate the dental pulp tissue. Dental pulp stem cells, have a potential use in dental tissue generation. However, specific requirements to drive the dental tissue generation are still obscured. We established an in vivo model for studying the survival of dental pulp cells (DPC) and their potential to generate dental pulp tissue. DPC were mixed with collagen scaffold with or without slow release bone morphogenic protein 4 (BMP-4) and fibroblast growth factor 2 (FGF2). The cell suspension was transplanted into a vascularized tissue engineering chamber in the rat groin. Tissue constructs were harvested after 2, 4, 6, and 8 weeks and processed for histomorphological and immunohistochemical analysis. After 2 weeks newly formed tissue with new blood vessel formation were observed inside the chamber. DPC were found around dentin, particularly around the vascular pedicle and also close to the gelatin microspheres. Cell survival, was confirmed up to 8 weeks after transplantation. Dentin Sialophosphoprotein (DSPP) positive matrix production was detected in the chamber, indicating functionality of dental pulp progenitor cells. This study demonstrates the potential of our tissue engineering model to study rat dental pulp cells and their behavior in dental pulp regeneration, for future development of an alternative treatment using these techniques.     

Human peripheral blood-derived mesenchymal stem cells with NTRK1 over-expression enhance repairing capability in a rat model of Parkinson’s disease

The potency of mesenchymal stem cells (MSCs) for tissue repair and regeneration is mainly based on their ability to secret beneficial molecules. Administration of MSCs has been proposed as an innovative approach and is proved by a number of clinical trials to a certain degree for the therapy of many diseases including Parkinson’s disease (PD). However, the efficacy of MSCs alone is not significant. We investigated the effect of neurotrophic tyrosine receptor kinase 1 (NTRK1) overexpressed peripheral blood MSCs (PB-MSCs) on PD rat model. NTRK1 was overexpressed in PB-MSCs, which were then injected into PD rat model, Dopaminergic (DA) neuron regeneration and rotational performance was assessed. We found that DA neuron repair was increased in lesion site, rotational performance was also improved in MSC transplanted PD rat, with most potent effect in NTRK1 overexpressed PB-MSC transplanted PD rat. Our results indicate that overexpression of NTRK1 in MSCs could be an optimized therapeutic way via MSCs for PD treatment.     

Repair of injured articular and growth plate cartilage using mesenchymal stem cells and chondrogenic gene therapy

Injuries to the articular cartilage and growth plate are significant clinical problems due to their limited ability to regenerate themselves. Despite progress in orthopedic surgery and some success in development of chondrocyte transplantation treatment and in early tissue-engineering work, cartilage regeneration using a biological approach still remains a great challenge. In the last 15 years, researchers have made significant advances and tremendous progress in exploring the potentials of mesenchymal stem cells (MSCs) in cartilage repair. These include (a) identifying readily available sources of and devising appropriate techniques for isolation and culture expansion of MSCs that have good chondrogenic differentiation capability, (b) discovering appropriate growth factors (such as TGF-beta, IGF-I, BMPs, and FGF-2) that promote MSC chondrogenic differentiation, (c) identifying or engineering biological or artificial matrix scaffolds as carriers for MSCs and growth factors for their transplantation and defect filling. In addition, representing another new perspective for cartilage repair is the successful demonstration of gene therapy with chondrogenic growth factors or inflammatory inhibitors (either individually or in combination), either directly to the cartilage tissue or mediated through transducing and transplanting cultured chondrocytes, MSCs or other mesenchymal cells. However, despite these rapid pre-clinical advances and some success in engineering cartilage-like tissue and in repairing articular and growth plate cartilage, challenges of their clinical translation remain. To achieve clinical effectiveness, safety, and practicality of using MSCs for cartilage repair, one critical investigation will be to examine the optimal combination of MSC sources, growth factor cocktails, and supporting carrier matrixes. As more insights are acquired into the critical factors regulating MSC migration, proliferation and chondrogenic differentiation both ex vivo and in vivo, it will be possible clinically to orchestrate desirable repair of injured articular and growth plate cartilage, either by transplanting ex vivo expanded MSCs or MSCs with genetic modifications, or by mobilising endogenous MSCs from adjacent source tissues such as synovium, bone marrow, or trabecular bone.     

Growth and differentiation of fetal rat limb buds transplanted in athymic (nude) mice

Limb buds of day 14 rat fetuses were cut into pieces and transplanted into the subcutaneous tissue of athymic (nude) mice. In day 14 fetal limbs, mesenchymal cells have begun to condense to form cartilaginous anlage, but no cartilage has been formed. Within 7 days after grafting, masses of hyaline cartilage developed. Numerous osteoblasts appeared, and new bone formation began by 14 days. By 20 days, osteoclasts appeared, and the formation of bone trabeculae and marrow cavities progressed. The cytological characteristics of chondrocytes, osteoblasts and osteoclasts were essentially the same as those seen in vivo. Many grafts developed into long bones, having the diaphysis and epiphysis. The mode of chondrogenesis and osteogenesis in the grafts was histologically similar to the corresponding process in vivo, although the differentiation was slower in the grafted limbs. Since the grafted limb buds showed remarkable growth and tissue differentiation for at least several weeks, this heterotransplantation system would be of potential use for the study of bone formation and resorption as well as for developmental toxicological studies.     

Bone tissue engineering using marrow stromal cells

Bone tissue defects cause a significant socioeconomic problem, and bone is the most frequently transplanted tissue beside blood. Autografting is considered the gold standard treatment for bone defects, but its utility is limited due to donor site morbidity. Hence much research has focused on bone tissue engineering as a promising alternative method for repair of bone defects. Marrow stromal cells (MSCs) are considered to be potential cell sources for bone tissue engineering. In bone tissue engineering using MSCs, bone is formed through intramembranous and endochondral ossification in response to osteogenic inducers. Angiogenesis is a complex process mediated by multiple growth factors and is crucial for bone regeneration. Vascular endothelial growth factor plays important roles in bone tissue regeneration by promoting the migration and differentiation of osteoblasts, and by inducing angiogenesis. Scaffold materials used for bone tissue engineering include natural components of bone, such as calcium phosphate and collagen I, and biodegradable polymers such as poly(lactide-coglycolide) However, ideal scaffolds for bone tissue engineering have yet to be found. Bone tissue engineering has been successfully used to treat bone defects in several human clinical trials to regenerate bone defects. Through investigation of MSC biology and the development of novel scaffolds, we will be able to develop advanced bone tissue engineering techniques in the future.     

Differentiation of Human Umbilical Cord Mesenchymal Stem Cells into Prostate-Like Epithelial Cells In Vivo

Although human umbilical cord mesenchymal stem cells (hUC-MSCs) have been identified as a new source of MSCs for potential application in regenerative medicine, their full potential of differentiation has not been determined. In particular, whether they have the capability to differentiate into epithelial cells of endodermal origin such as the prostate epithelial cells is unknown. Here we report that when hUC-MSCs were combined with rat urogenital sinus stromal cells (rUGSSs) and transplanted into the renal capsule in vivo, they could differentiate into prostate epithelial-like cells that could be verified by prostate epithelial cell-specific markers including the prostate specific antigen. The prostatic glandular structures formed in vivo displayed similar cellular architecture with lumens and branching features as seen for a normal prostate. In addition, the human origin of the hUC-MSCs was confirmed by immunocytochemistry for human nuclear antigen. These findings together indicate that hUC-MSCs have the capability to differentiate into epithelial-like cells that are normally derived from the endoderm, implicating their potential applications in tissue repair and regeneration of many endoderm-derived internal organs.     

Bone‐forming capacity of adult human nasal chondrocytes

Nasal chondrocytes (NC) derive from the same multipotent embryological segment that gives rise to the majority of the maxillofacial bone and have been reported to differentiate into osteoblast‐like cells in vitro. In this study, we assessed the capacity of adult human NC, appropriately primed towards hypertrophic or osteoblastic differentiation, to form bone tissue in vivo. Hypertrophic induction of NC‐based micromass pellets formed mineralized cartilaginous tissues rich in type X collagen, but upon implantation into subcutaneous pockets of nude mice remained avascular and reverted to stable hyaline‐cartilage. In the same ectopic environment, NC embedded into ceramic scaffolds and primed with osteogenic medium only sporadically formed intramembranous bone tissue. A clonal study could not demonstrate that the low bone formation efficiency was related to a possibly small proportion of cells competent to become fully functional osteoblasts. We next tested whether the cues present in an orthotopic environment could induce a more efficient direct osteoblastic transformation of NC. Using a nude rat calvarial defect model, we demonstrated that (i) NC directly participated in frank bone formation and (ii) the efficiency of survival and bone formation by NC was significantly higher than that of reference osteogenic cells, namely bone marrow‐derived mesenchymal stromal cells. This study provides a proof‐of‐principle that NC have the plasticity to convert into bone cells and thereby represent an easily available cell source to be further investigated for craniofacial bone regeneration.     

Use of platelet lysate for bone regeneration - are we ready for clinical translation?

Current techniques to improve bone regeneration following trauma or tumour resection involve the use of autograft bone or its substitutes supplemented with osteoinductive growth factors and/or osteogenic cells such as mesenchymal stem cells(MSCs).Although MSCs are most commonly grown in media containing fetal calf serum,human platelet lysate(PL) offers an effective alternative.Bone marrow- derived MSCs grown in PLcontaining media display faster proliferation whilst maintaining good osteogenic differentiation capacity.Limited pre-clinical investigations using PL-expanded MSCs seeded onto osteoconductive scaffolds indicate good potential of such constructs to repair bone in vivo.In an alternative approach,nude PL-coated scaffolds without seeded MSCs have been proposed as novel regenerative medicine devices.Even though methods to coat scaffolds with PL vary,in vitro studies suggest that PL allows for MSC adhesion,migration and differentiation inside these scaffolds.Increased new bone formation and vascularisation in comparison to uncoated scaffolds have also been observed in vivo.This review outlines the state-of-the-art research in the field of PL for ex vivo MSC expansion and in vivo bone regeneration.To minimise inconsistency between the studies,further work is required towards standardisation of PL preparation in terms of the starting material,platelet concentration,leukocyte depletion,and the method of platelet lysis.PL quality control procedures and its "potency" assessment are urgently needed,which could include measurements of key growth and attachment factors important for MSC maintenance and differentiation.Furthermore,different PL formulations could be tailor-made for specific bone repair indications.Such measures would undoubtedly speed up clinical translation of PL-based treatments for bone regeneration.     

菲立磁标记食蟹猴骨髓间充质干细胞的脑内移植示踪研究

中枢神经系统损伤后的再生修复问题一直是神经科学领域关注的重点之一,骨髓间充质干细胞移植治疗拓宽了人类中枢神经系统损伤的治疗前景,而非侵入性的磁共振成像能活体追踪移植细胞,评价移植效果。应用菲立磁标记食蟹猴骨髓来源的间充质干细胞,在脑立体定位仪引导下,自体脑内移植。结果显示,菲立磁标记间充质干细胞的有效率高达90%以上,移植区磁共振影像呈明显的低信号改变。标记的间充质干细胞移植后在脑内存活,并向周围的脑实质内迁移。移植8周后,发现移植细胞通过血管向对侧脑部迁移,但并未发现移植细胞向神经细胞分化。这些结果提示,菲立磁可用于标记、追踪脑内移植的食蟹猴骨髓间充质干细胞,标记的移植细胞可在脑内存活、迁移。     

CXCR4 Receptor Overexpression in Mesenchymal Stem Cells Facilitates Treatment of Acute Lung Injury in Rats

Novel therapeutic regimens for tissue renewal incorporate mesenchymal stem cells (MSCs) as they differentiate into a variety of cell types and are a stem cell type that is easy to harvest and to expand in vitro. However, surface chemokine receptors, such as CXCR4, which are involved in the mobilization of MSCs, are expressed only on the surface of a small proportion of MSCs, and the lack of CXCR4 expression may underlie the low efficiency of homing of MSCs toward tissue damage, which results in a poor curative effect. Here, a rat CXCR4 expressing lentiviral vector was constructed and introduced into MSCs freshly prepared from rat bone marrow. The influence of CXCR4 expression on migration, proliferation, differentiation, and paracrine effects of MSCs was examined in vitro. The in vivo properties of CXCR4-MSCs were also investigated in a model of acute lung injury in rats induced by lipopolysaccharide. Expression of CXCR4 in MSCs significantly enhanced the chemotactic and paracrine characteristics of the cells in vitro but did not affect self-renewal or differentiation into alveolar and vascular endothelial cells. In vivo, CXCR4 improved MSC homing and colonization of damaged lung tissue, and furthermore, the transplanted CXCR4-MSCs suppressed the development of acute lung injury in part by modulating levels of inflammatory molecules and the neutrophil count. These results indicated that efficient mobilization of MSCs to sites of tissue injury may be due to CXCR4, and therefore, increased expression of CXCR4 may improve their therapeutic potential in the treatment of diseases where tissue damage develops.     

Noncanonical Wnt-4 signaling enhances bone regeneration of mesenchymal stem cells in craniofacial defects through activation of p38 MAPK

Mesenchymal stem cells (MSCs) are multipotent cells that can be differentiated into osteoblasts and provide an excellent cell source for bone regeneration and repair. Recently, the canonical Wnt/beta-catenin signaling pathway has been found to play a critical role in skeletal development and osteogenesis, implying that Wnts can be utilized to improve de novo bone formation mediated by MSCs. However, it is unknown whether noncanonical Wnt signaling regulates osteogenic differentiation. Here, we find that Wnt-4 enhanced in vitro osteogenic differentiation of MSCs isolated from human adult craniofacial tissues and promoted bone formation in vivo. Whereas Wnt-4 did not stabilize beta-catenin, it activated p38 MAPK in a novel noncanonical signaling pathway. The activation of p38 was dependent on Axin and was required for the enhancement of MSC differentiation by Wnt-4. Moreover, using two different models of craniofacial bone injury, we found that MSCs genetically engineered to express Wnt-4 enhanced osteogenesis and improved the repair of craniofacial defects in vivo. Taken together, our results reveal that noncanonical Wnt signaling could also play a role in osteogenic differentiation. Wnt-4 may have a potential use in improving bone regeneration and repair of craniofacial defects.     

Bone tissue formation from human embryonic stem cells in vivo

Although the use of embryonic stem cells in the assisted repair of musculoskeletal tissues holds promise, a direct comparison of this cell source with adult marrow-derived stem cells has not been undertaken. Here we have compared the osteogenic differentiation potential of human embryonic stem cells (hESC) with human adult-derived stem cells in vivo. hESC lines H7, H9, the HEF-1 mesenchymal-like, telomerized H1 derivative, the human embryonic kidney epithelial cell line HEK293 (negative control), and adult human mesenchymal stem cells (hMSC) were either used untreated or treated with osteogenic factors for 4 days prior to injection into diffusion chambers and implantation into nude mice. After 11 weeks in vivo chambers were removed, frozen, and analyzed for evidence of bone, cartilage, and adipose tissue formation. All hESCs, when pretreated with osteogenic (OS) factors gave rise exclusively to bone in the chambers. In contrast, untreated hESCs (H9) formed both bone and cartilage in vivo. Untreated hMSCs did not give rise to bone, cartilage, or adipose tissue in vivo, while pretreatment with OS factors engendered both bone and adipose tissue. These data demonstrate that hESCs exposed to OS factors in vitro undergo directed differentiation toward the osteogenic lineage in vivo in a similar fashion to that produced by hMSCs. These findings support the potential future use of hESC-derived cells in regenerative medicine applications.     

Nanoindentation measurements of biomechanical properties in mature and newly formed bone tissue surrounding an implant

The characterization of the biomechanical properties of newly formed bone tissue around implants is important to understand the osseointegration process. The objective of this study is to investigate the evolution of the hardness and indentation modulus of newly formed bone tissue as a function of healing time. To do so, a nanoindentation device is employed following a multimodality approach using histological analysis. Coin-shaped implants were placed in vivo at a distance of 200 μm from the cortical bone surface, leading to an initially empty cavity of 200 μm * 4.4 mm. Three New Zealand White rabbits were sacrificed after 4, 7, and 13 weeks of healing time. The bone samples were embedded and analyzed using histological analyses, allowing to distinguish mature and newly formed bone tissue. The bone mechanical properties were then measured in mature and newly formed bone tissue. The results are within the range of hardness and apparent Young's modulus values reported in previous literature. One-way ANOVA test revealed a significant effect of healing time on the indentation modulus (p < 0.001, F = 111.24) and hardness (p < 0.02, F = 3.47) of bone tissue. A Tukey-Kramer analysis revealed that the biomechanical properties of newly formed bone tissue (4 weeks) were significantly different from those of mature bone tissue. The comparison with the results obtained in Mathieu et al. (2011, "Micro-Brillouin Scattering Measurements in Mature and Newly Formed Bone Tissue Surrounding an Implant," J. Biomech. Eng., 133, 021006). shows that bone mass density increases by approximately 13.5% between newly formed bone (7 weeks) and mature bone tissue.     

Murine mesenchymal stem cells exhibit a restricted repertoire of functional chemokine receptors: comparison with human

Mesenchymal stem cells (MSCs) are non-haematopoeitic, stromal cells that are capable of differentiating into mesenchymal tissues such as bone and cartilage. They are rare in bone marrow, but have the ability to expand many-fold in culture, and retain their growth and multi-lineage potential. The properties of MSCs make them ideal candidates for tissue engineering. It has been shown that MSCs, when transplanted systemically, can home to sites of injury, suggesting that MSCs possess migratory capacity; however, mechanisms underlying migration of these cells remain unclear. Chemokine receptors and their ligands play an important role in tissue-specific homing of leukocytes. Here we define the cell surface chemokine receptor repertoire of murine MSCs from bone marrow, with a view to determining their migratory activity. We also define the chemokine receptor repertoire of human MSCs from bone marrow as a comparison. We isolated murine MSCs from the long bones of Balb/c mice by density gradient centrifugation and adherent cell culture. Human MSCs were isolated from the bone marrow of patients undergoing hip replacement by density gradient centrifugation and adherent cell culture. The expression of chemokine receptors on the surface of MSCs was studied using flow cytometry. Primary murine MSCs expressed CCR6, CCR9, CXCR3 and CXCR6 on a large proportion of cells (73+/-11%, 44+/-25%, 55+/-18% and 96+/-2% respectively). Chemotaxis assays were used to verify functionality of these chemokine receptors. We have also demonstrated expression of these receptors on human MSCs, revealing some similarity in chemokine receptor expression between the two species. Consequently, these murine MSCs would be a useful model to further study the role of chemokine receptors in in vivo models of disease and injury, for example in recruitment of MSCs to inflamed tissues for repair or immunosuppression.