Recombinant Stromal Cell-derived Factor-1 Research Articles

KLF9 inhibits the proliferation, invasion, and migration of renal cell carcinoma through the SDF-1/CXCR4 axis.

Altered Krüppel-like factor 9 (KLF9) expression can regulate the progression of several cancers, including renal cell carcinoma (RCC). This study was conducted to investigate the role of KLF9 in the proliferation, invasion, and migration of RCC cells via regulation of stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4). The expression patterns of KLF9, SDF-1, and CXCR4 in the experimental cell lines were determined by real-time quantitative polymerase chain reaction and Western blotting. After transfection of the KLF9 siRNA and KLF9 pcDNA, cell proliferation, invasion, and migration were evaluated by experiments including cell counting kit-8, colony formation, and Transwell assays. The binding of KLF9 to the SDF-1 promoter was analyzed by chromatin immunoprecipitation and dual-luciferase assay. The rescue experiment was performed using the recombinant SDF-1 protein and KLF9 pcDNA. KLF9 was downregulated in the RCC cells. KLF9 knockdown induced the proliferation, invasion, and migration of RCC cells, whereas KLF9 overexpression elicited the opposite roles. Mechanically, KLF9 bound to the SDF-1 promoter, repressed SDF-1 transcription, and reduced the SDF-1/CXCR4 expression levels. Activation of the SDF-1/CXCR4 axis attenuated the inhibitory role of KLF9 overexpression in RCC cell growth. Ordinarily, KLF9 suppressed the proliferation, invasion, and migration of RCC cells by repressing the SDF-1/CXCR4 signaling.

Mesenchymal stromal cell-associated migrasomes: a new source of chemoattractant for cells of hematopoietic origin

BackgroundMultipotent mesenchymal stromal cells (MSCs) are precursors of various cell types. Through soluble factors, direct cell–cell interactions and other intercellular communication mechanisms such as extracellular vesicles and tunneling nanotubes, MSCs support tissue homeostasis. In the bone marrow microenvironment, they promote hematopoiesis. The interaction between MSCs and cancer cells enhances the cancer and metastatic potential. Here, we have demonstrated that plastic-adherent MSCs isolated from human bone marrow generate migrasomes, a newly discovered organelle playing a role in intercellular communication.ResultsMigrasomes are forming a network with retraction fibers behind the migrating MSCs or surrounding them after membrane retraction. The MSC markers, CD44, CD73, CD90, CD105 and CD166 are present on the migrasome network, the latter being specific to migrasomes. Some migrasomes harbor the late endosomal GTPase Rab7 and exosomal marker CD63 indicating the presence of multivesicular bodies. Stromal cell-derived factor 1 (SDF-1) was detected in migrasomes, suggesting that they play a chemoattractant role. Co-cultures with KG-1a leukemic cells or primary CD34+ hematopoietic progenitors revealed that MSC-associated migrasomes attracted them, a process intercepted by the addition of AMD3100, a specific CXCR4 receptor inhibitor, or recombinant SDF-1. An antibody directed against CD166 reduced the association of hematopoietic cells and MSC-associated migrasomes. In contrast to primary CD34+ progenitors, leukemic cells can take up migrasomes.ConclusionOverall, we described a novel mechanism used by MSCs to communicate with cells of hematopoietic origin and further studies are needed to decipher all biological aspects of migrasomes in the healthy and transformed bone marrow microenvironment.1ydn92XG6FRTxRi1UCUyVwVideo

A Hydrogel as a Bespoke Delivery Platform for Stromal Cell-Derived Factor-1.

The defined self-assembly of peptides (SAPs) into nanostructured bioactive hydrogels has great potential for repairing traumatic brain injuries, as they maintain a stable, homeostatic environment at an injury site, preventing further degeneration. They also present a bespoke platform to restore function via the naturalistic presentation of therapeutic proteins, such as stromal-cell-derived factor 1 (SDF-1), expressed by meningeal cells. A key challenge to the use of the SDF protein, however, is its rapid diffusion and degradation. Here, we engineered a homeostatic hydrogel produced by incorporating recombinant SDF-1 protein within a self-assembled peptide hydrogel to create a supportive milieu for transplanted cells. Our hydrogel can concomitantly deliver viable primary neural progenitor cells and sustained active SDF-1 to support the nascent graft, resulting in increased neuronal differentiation. Moreover, this homeostatic hydrogel can ensure a healthy and larger graft core without impeding neuronal fiber growth and innervation. These findings demonstrate the regenerative potential of these hydrogels to improve the integration of grafted cells to treat neural injuries and diseases.

A Study of Avascular Necrosis of the Femoral Head Using Danshen Combined with Marrow Stromal Cells In Vitro

As avascular necrosis of the femoral head (ANFH) has been one of the major problem plaguing the medical profession and Marrow Stromal Cells (MSCs) in orthopedic exploration for recent years, the aim of this study was to investigate the possible chemotactic effect in vitro treated by Danshen combined with transplantation of MSCs in ANFH. The MSCs were extracted from rabbit tibia and femur. The inhibition rate of Danshen in MSCs proliferation was assayed by MTT. Chemokine receptor expression was determined by RT-PCR. Chemotaxis migration was assayed by Boyden chamber. Results showed that high concentration of Danshen injection on the proliferation of MSCs have a certain inhibition rate. Less than 50 μl/ml did not substantially affect the proliferation of MSCs. After treatment with Danshen injection, it can promote chemokine receptors CCR2, CX3CR1 and CXCR4 expression in MSCs. Danshen could significantly promote cell migration after mixed with 50 ng/ml recombinant monocyte chemoattratant protein-1 (rMCP-1) and recombinant stromal cellderived factor-1 (rSDF-1). In conclusion, culture of MSCs can express chemokine receptors CCR2, CX3CR1 and CXCR4 in vitro. Danshen injection can increase the expression of chemokine receptor CCR2, CX3CR1, and CXCR4, which is beneficial to the migration of MSCs. In conclusion, the chemotactic effects of rMCP-1 and rSDF-1 on the chemotaxis of MSCs were positively correlated with the concentrations of rMCP-1 and rSDF-1 and significantly increased combined with Danshen.

Enhanced critical-size calvarial bone healing by ASCs engineered with Cre/loxP-based hybrid baculovirus.

Calvarial bone repair remains challenging for adults. Although adipose-derived stem cells (ASCs) hold promise to heal bone defects, use of ASCs for critical-size calvarial bone repair is ineffective. Stromal cell-derived factor 1 (SDF-1) is a chemokine capable of triggering stem cell migration. Although recombinant SDF-1 protein is co-delivered with other molecules including BMP-2 to facilitate calvarial bone repair, these approaches did not yield satisfactory healing. This study aimed to exploit a newly developed Cre/loxP-based hybrid baculovirus for efficient gene delivery and prolonged transgene expression in ASCs. We demonstrated that transduction of rat ASCs with the hybrid Cre/loxP-based baculovirus enabled robust and sustained expression of functional BMP-2 and SDF-1. Expression of BMP-2 or SDF-1 alone failed to effectively induce rat ASCs osteogenesis and healing of critical-size calvarial bone defects. Nonetheless, prolonged BMP-2/SDF-1 co-expression in ASCs synergistically activated both Smad and ERK1/2 pathways and hence potentiated the osteogenesis. Consequently, transplantation of the hybrid baculovirus-engineered, BMP-2/SDF-1-expressing ASCs/scaffold constructs potently healed the critical-size (6mm) calvarial bone defects (filling ≈70% of defect volume), which considerably outperformed the calvarial bone repair using BMP-2/SDF-1 delivered with biomaterial-based scaffolds. These data implicated the potential of Cre/loxP-based hybrid baculovirus vector for ASCs engineering and calvarial bone healing.

Effect of stromal cell-derived factor-1 on myocardial apoptosis and cardiac function recovery in rats with acute myocardial infarction

The aim of the study was to investigate the effect of stromal cell-derived factor-1 (SDF-1) on myocardial apoptosis and cardiac function recovery in rats with acute myocardial infarction (AMI) and the mechanism of the Toll-like receptor (TLR)-4/nuclear factor-κB (NF-κB) signaling pathway. A total of 64 healthy male F344 rats were randomly divided into the sham operation, model, SDF-1 intervention and SDF-1 antibody groups, with 16 rats in each group. The method of Olivette was used to establish the AMI model by ligation of the left anterior descending artery. Day 1 after establishing the animal model, the rats in the SDF-1 intervention group were injected with 10 µl recombinant SDF-1 (400 ng/ml) in five regions including the myocardial infarction area and the four surrounding areas. The rats in the model group were injected with 10 µl normal saline including the myocardial infarction area and the four surrounding areas, and those in the SDF-1 antibody group were injected with 1 ml SDF-1 antibody (2 µg/ml). Four rats were sacrificed after 1, 3, 7 and 14 days after the intervention, and the analysis was carried out. TUNEL in situ labeled apoptotic cells were used for cell counting, and immunohistochemical staining was performed to measure vascular density. The animal echocardiographic measurement was for the left ventricular end-diastolic diameter (LVEDd), left ventricular end-systolic diameter (LVESd), left ventricular fractional shortening (FS) and ejection fraction (EF) values. The results showed that the number of apoptotic cells in the SDF-1 treatment group was significantly lower than those in the other groups at each time-point. The vessel densities in the 3–14 days were significantly greater than those in other groups. At each time-point, the LVEDd and LVESd values were smaller compared with the model group, but greater than the sham operation group and decreased over time. FS and EF values were higher than those in the model group at each time-point, but less than those of the sham operation group and increased over time. The expression levels of TLR-4 and NF-κB at each time-point were significantly higher than those of the remaining groups (p<0.05). In conclusion, SDF-1 is capable of decreasing the apoptosis of cardiac muscle cells in AMI, promoting angiogenesis and improving cardiac function, which may be associated with the activation of the TLR-4/NF-κB signaling pathway.

The role of SDF‐1 and CXCR4 on odontoblastic differentiation in human dental pulp cells

To examine the role of stromal cell-derived factor 1 (SDF-1) signalling during odontogenic differentiation in human dental pulp cells (HDPCs). Human dental pulp cells were treated with differentiation medium, recombinant human SDF-1, neutralizing antibody for SDF-1 or CXCR4, pertussis toxin (PTX) and AMD3100. The expression of SDF-1 and its receptor chemokine receptor type 4 (CXCR4) was measured by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. Odontoblastic differentiation was determined using alkaline phosphatase (ALP) activity assay, mineralized nodule formation and marker mRNAs by RT-PCR. Marked upregulation of SDF-1 and CXCR4 mRNA and protein was observed in cells grown 7days in osteogenic induction medium. The addition of recombinant human SDF-1 to HDPCs significantly (P<0.05) increased ALP activity, mineralized nodule formation and odontoblast marker mRNAs in a dose-dependent manner. Blocking SDF-1 signalling using antibodies against SDF-1 or CXCR4, or the G-protein-coupled receptor inhibitor PTX, and CXCR4 inhibitor AMD3100, strongly suppressed induction of odontogenic differentiation in HDPCs. Odontoblastic differentiation was stimulated by SDF-1 activation and repressed by SDF-1/CXCR4 inhibition. Thus, SDF-1/CXCR4 signalling may be a new therapeutic target and strategy to promote repair and regeneration in endodontics.

The Effect of Recombinant Stromal Cell-Derived Factor-1 Treatment on Hypoxic-Ischemic Brain Injury in Neonatal Mice

Stromal cell-derived factor-1 (SDF-1) plays a critical role in adult brain injury repair including neurogenesis and vasculogenesis. However, the effects of recombinant SDF-1(rSDF-1) treatment on hypoxic-ischemic (HI) brain injury in neonatal mice are not clear. Seven-day-old (P7) C57BL/6J mice were divided into sham group, control group, and rSDF-1 group. Mice brains were collected to determine histopathological damage and the expression level of SDF-1, caspase-3, and Ki67 on P8, P10, P14, and P21. Passive avoidance and elevated plus-maze tests were also assessed on P20 and P21. Compared with control group, rSDF-1 treatment increased the weight ratio of left and right brain hemisphere (p < 0.05), reduced the number of electric foot shocks (p < 0.05) and the percentage of time spent in the open arms (p < 0.05), meanwhile, increased the retention latency (p < 0.05) and the percentage of time spent in the enclosed arms (p < 0.05) on P20 and P21. High expression of Ki67 and low expression of caspase-3 were also observed. This study showed that rSDF-1 treatment effectively alleviated brain injury shown by reducing in caspase-3 expression and increasing in Ki67 expression. Moreover, rSDF-1 treatment significantly improved behavioral performances in juvenile mice after HI.

OV6+ tumor-initiating cells contribute to tumor progression and invasion in human hepatocellular carcinoma

Accumulating evidence suggests the involvement of tumor-initiating cells (T-ICs) in cancer genesis, but whether liver T-ICs contribute to HCC invasion and metastasis remains unclear. OV6(+) T-ICs were isolated from SMMC7721 and HuH7 cell lines by magnetic sorting. Characteristics of T-ICs were assessed by in vitro and mouse xenograft assays. Expression of OV6 was determined by immunostaining in specimens from 218 HCC patients, and Kaplan-Meier survival analysis was used to determine the correlation of OV6 expression with prognosis. OV6(+) T-ICs isolated from HCC cell lines not only possess a higher capacity to form tumor spheroids in vitro, but also had a greater potential to form tumors when implanted in non-obese diabetic/severe combined immunodeficient mice, suggesting their elevated self-renewal capacity and tumorigenicity. Moreover, OV6(+) T-ICs exhibited more invasive and metastatic potentials both in vitro and in vivo. Patients with more OV6(+) tumor cells were associated with aggressive clinicopathologic features and poor prognosis. CXCR4 is expressed at higher levels in OV6(+) cells. Recombinant stromal cell-derived factor-1 (SDF-1) treatment expanded the OV6(+) HCC T-ICs population, by sustaining the stem cell property of OV6(+) cells. The SDF-1 effect was blocked by a specific CXCR4 inhibitor, AMD3100, or transfection of siRNA targeting CXCR4. OV6(+) HCC cells may represent a subpopulation of T-ICs with augmented invasion and metastasis potential, which contribute to progression and metastasis of HCC. The SDF-1/CXCR4 axis also provides therapeutic targets for elimination of liver T-ICs.

Stromal cell‐derived factor 1 (SDF‐1) and its receptor CXCR4 in the formation of postburn hypertrophic scar (HTS)

Recent data support the involvement of stromal cell-derived factor 1 (SDF-1) in the homing of bone marrow-derived stem cells to wound sites during skeletal, myocardial, vascular, lung, and skin wound repair as well as some fibrotic disorders via its receptor CXCR4. In this study, the role of SDF-1/CXCR4 signaling in the formation of hypertrophic scar (HTS) following burn injury and after treatment with systemic interferon α2b (IFNα2b) is investigated. Studies show SDF-1/CXCR4 signaling was up-regulated in burn patients, including SDF-1 level in HTS tissue and serum as well as CD14+ CXCR4+ cells in the peripheral blood mononuclear cells. In vitro, dermal fibroblasts constitutively expressed SDF-1 and deep dermal fibroblasts expressed more SDF-1 than superficial fibroblasts. Lipopolysaccharide increased SDF-1 gene expression in fibroblasts. Also, recombinant SDF-1 and lipopolysaccharide stimulated fibroblast-conditioned medium up-regulated peripheral blood mononuclear cell mobility. In the burn patients with HTS who received subcutaneous IFNα2b treatment, increased SDF-1/CXCR4 signaling was found prior to treatment which was down-regulated after IFNα2b administration, coincident with enhanced remodeling of their HTS. Our results suggest that SDF-1/CXCR4 signaling is involved in the development of HTS by promoting migration of activated CD14+ CXCR4+ cells from the bloodstream to wound sites, where they may differentiate into fibrocyte and myofibroblasts and contribute to the development of HTS.

Protease-Resistant Stromal Cell–Derived Factor-1 for the Treatment of Experimental Peripheral Artery Disease

Peripheral artery disease is a potentially incapacitating disease for which pharmacological options are limited. Stromal cell-derived factor-1 (SDF-1) is a chemokine that attracts endothelial progenitor cells and promotes angiogenesis. Therapeutic use of SDF-1 in hindlimb ischemia may be challenged by proteolytic degradation. We hypothesized that protease-resistant variants of SDF-1 can increase blood flow in an experimental model of hindlimb ischemia. We screened a peptide library for mutations in SDF-1 that provide resistance to matrix metalloproteinase cleavage. Recombinant SDF-1 proteins carrying the mutations were designed, expressed, and purified, and activity of mutant proteins was tested with receptor activation assays and in vivo Matrigel plug assays. SSDF-1(S4V), which is resistant to both dipeptidylpeptidase IV/CD26 and matrix metalloproteinase-2 cleavage, was active in vitro and induced angiogenesis in vivo. We then designed and purified fusion proteins of SSDF-1 and SSDF-1(S4V) with the sequence of self-assembling peptide nanofibers for incorporation into nanofibers. In a blinded and randomized hindlimb ischemia mouse study, SSDF-1(S4V) delivery by nanofibers improved blood flow as measured by laser Doppler from 23.1±1.9% (untreated control) to 55.1±5.7% 6 weeks after surgery (P<0.001). Nanofibers alone or SSDF-1 delivered by nanofibers did not improve blood flow. Furthermore, SSDF-1(S4V) delivered by nanofibers increased formation of new arterioles. In vitro, SSDF-1(S4V) attracts smooth muscle cells but does not induce mitosis. SDF-1 engineered to be resistant to dipeptidylpeptidase IV/CD26 and matrix metalloproteinase-2 cleavage and delivered by nanofibers improves blood flow in a model of peripheral artery disease.

Continuous Delivery of Stromal Cell-Derived Factor-1 from Alginate Scaffolds Accelerates Wound Healing

Proper wound diagnosis and management is an increasingly important clinical challenge and is a large and growing unmet need. Pressure ulcers, hard-to-heal wounds, and problematic surgical incisions are emerging at increasing frequencies. At present, the wound-healing industry is experiencing a paradigm shift towards innovative treatments that exploit nanotechnology, biomaterials, and biologics. Our study utilized an alginate hydrogel patch to deliver stromal cell-derived factor-1 (SDF-1), a naturally occurring chemokine that is rapidly overexpressed in response to tissue injury, to assess the potential effects SDF-1 therapy on wound closure rates and scar formation. Alginate patches were loaded with either purified recombinant human SDF-1 protein or plasmid expressing SDF-1 and the kinetics of SDF-1 release were measured both in vitro and in vivo in mice. Our studies demonstrate that although SDF-1 plasmid- and protein-loaded patches were able to release therapeutic product over hours to days, SDF-1 protein was released faster (in vivo K(d) 0.55 days) than SDF-1 plasmid (in vivo K(d) 3.67 days). We hypothesized that chronic SDF-1 delivery would be more effective in accelerating the rate of dermal wound closure in Yorkshire pigs with acute surgical wounds, a model that closely mimics human wound healing. Wounds treated with SDF-1 protein (n = 10) and plasmid (n = 6) loaded patches healed faster than sham (n = 4) or control (n = 4). At day 9, SDF-1-treated wounds significantly accelerated wound closure (55.0 +/- 14.3% healed) compared to nontreated controls (8.2 +/- 6.0%, p < 0.05). Furthermore, 38% of SDF-1-treated wounds were fully healed at day 9 (vs. none in controls) with very little evidence of scarring. These data suggest that patch-mediated SDF-1 delivery may ultimately provide a novel therapy for accelerating healing and reducing scarring in clinical wounds.

Activation of Signal Transducers and Activators of Transcription 3 and Focal Adhesion Kinase by Stromal Cell-Derived Factor 1 Is Required for Migration of Human Mesenchymal Stem Cells in Response to Tumor Cell-Conditioned Medium

Mesenchymal stem cells (MSCs) migrate to tumors both in vitro and in vivo. Gene expression profiling analysis reveals that stromal cell-derived factor 1 (SDF-1) is significantly upregulated in MSCs exposed to tumor cell-conditioned medium, when compared with cells treated with control medium, suggesting that SDF-1 signaling is important in mediating MSC migration. This study investigates downstream signaling during MSC migration in response to tumor cell-conditioned medium and recombinant SDF-1 protein treatments. We observed that both recombinant SDF-1 and tumor cell-conditioned medium were able to activate downstream signaling via signal transducer and activator of transcription 3 (STAT3) and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) as revealed by increased phosphorylation of STAT3 and ERK1/2 in human MSCs (hMSCs). Significant impairment of in vitro migration was observed in the presence of MAPK/ERK kinase (MEK) inhibitor PD98059, whereas two Janus kinase 2 (Jak2) inhibitors completely abolished migration induced by tumor cell-conditioned medium. Impaired MSC migration correlated with decreased levels of phosphorylated STAT3 and ERK1/2, suggesting that SDF-1 stimulation activates Jak2/STAT3 as well as MEK/ERK1/2 signaling, which in turn promotes migration of MSCs toward tumor cells. Furthermore, stimulation of hMSCs with recombinant SDF-1 and tumor cell-conditioned medium also significantly activated the focal adhesion kinases (FAKs) and paxillin, which correlated with reorganization of F-actin filaments in hMSCs. Decreased phosphorylation of FAK and paxillin as well as disruption of cytoskeleton organization was observed following Jak2 and MEK inhibitor treatment. Taken together, our results provide insight into the molecular pathways responsible for MSC migration toward the tumor microenvironment and may provide the molecular basis for modifying MSCs for therapeutic purposes.

Engineering Human Tumor-specific Cytotoxic T Cells to Function in a Hypoxic Environment

Hypoxia occurs in many tumors and reduces the effectiveness of radio- and chemotherapy. Hypoxia also impedes immune responses to tumors, reducing T lymphocyte production of cytokines such as interleukin-2 (IL-2) and interferon gamma, as well as the survival and proliferation of these cells. We constructed a lentiviral vector encoding a bidirectional hypoxia-inducible responsive element (HRE) derived from human vascular endothelial growth factor, which drives the hIL-2 gene and a marker gene. We used a model of human B cell lymphoma to show that tumor-specific T cells modified with this vector upregulate hIL-2 expression when oxygen tension is low in vitro and in vivo. The consequence of this effect is to increase T-cell survival and proliferation whilst sustaining effector function, even in O(2) concentrations as low as 1%. The phenotype of the transduced cells is unchanged, as is their ability to migrate to tumor. HRE-IL-2-modified cytotoxic T lymphocytes (CTLs) produce faster and more complete tumor regression than parental CTLs and increase overall survival. Hypoxia-resistant T cells may thus be of value in the treatment of human tumors in which areas of hypoxia may otherwise account for resistance to this therapeutic strategy.

Recruitment of osteoclast precursors by stromal cell derived factor-1 (SDF-1) in giant cell tumor of bone

Giant cell tumor (GCT) of bone is a unique bone lesion that is characterized by an excessive number of multinucleated osteoclasts. GCT consists of neoplastic stromal cells, multinucleated osteoclasts and their precursors, thus serving as a naturally occurring human disease model for the study of osteoclastogenesis. It still remains unclear how stromal cells of GCT recruit osteoclast precursors. In the present study, we characterized the cellular components of GCT and confirmed the presence of CD14 +-monocytes/CD68 +-macrophages and CD34 +-hematopoetic stem cells that express CXCR4, a specific receptor for SDF-1; SDF-1 gene expression and presence of SDF-1 protein were confirmed by real time RT-PCR, in situ hybridization, and immunohistochemistry in the GCT tissue and cultured cells. SDF-1 was present at 25–50 ng/ml in the conditioned media from the GCT cultures, which is in the range of physiological chemotactic concentration. Migration of osteoclast precursors was 2.5-fold higher in response to GCT conditioned media compared to the control media; and migration was inhibited by an average of 36% with anti-SDF-1 neutralizing antibody or competing recombinant SDF-1. These results suggest that SDF-1 is one of the significant chemoattractant factors involved in the recruitment of hematopoietic osteoclast precursor cells during tumor-induced osteoclastogenesis.

Tumor progression in osteosarcoma (OS): Role of the chemokine receptor CXCR4 and of its ligand stromal-cell derived factor 1 (SDF-1)

9021 Background. Despite intensive chemotherapy and surgery treatment, lung and bone metastases develop in about 30% of OS patients. Mechanisms for this preferential metastatic behaviour are largely unknown. The CXCR4/SDF-1 system was shown to play a role in the homing of neoplastic cells in breast cancer as well as in rabdhomyosarcoma. This hypothesis was tested in vitro and in vivo in a mouse model. Methods. Human OS cell lines MG-63, SJSA, U2-OS and HOS were studied in standard colture conditions. Cell lines were evaluated for: CXCR4 expression by FACS analysis (mAb anti-CXCR4, BD Pharmingen); SDF-1 levels in conditioned medium (antibody sandwich ELISA, RD modulation of CXCR4 expression on OS cells by exposure to recombinant human SDF-1 (rhSDF-1). The adhesion and migration of SJSA cells were tested on a monolayer of bone marrow stromal cells in response to rhSDF-1. We evaluated lung metastasis development in mouse model (Balb-c nu/nu) with and without monoclonal antibody anti-CXCR4 (T134) t...

Interactions of chemokines and chemokine receptors mediate the migration of mesenchymal stem cells to the impaired site in the brain after hypoglossal nerve injury.

Mesenchymal stem cells (MSCs), cultured ex vivo, recently were shown to be able to migrate into sites of brain injuries when transplanted systemically or locally, suggesting that MSCs possess migratory capacity. However, the mechanisms underlying the migration of these cells remain unclear. In this study, we examined the role of some chemokines and their receptors in the trafficking of rat MSCs (rMSCs) in a rat model of left hypoglossal nerve injury. rMSCs transplanted into the lateral ventricles of the rat brain migrated to the avulsed hypoglossal nucleus, where the expression of chemokines, stromal-cell-derived factor 1 (SDF-1), and fractalkine was observed to be increased. This increase temporally paralleled the migration of rMSCs into the avulsed nucleus at 1 and 2 weeks after operation. It has been found that rMSCs express CXCR4 and CX3CR1, the respective receptors for SDF-1 and fractalkine, and other chemokine receptors, CCR2 and CCR5. Furthermore, in vitro analysis revealed that recombinant human SDF-1 alpha (rhSDF-1alpha) and recombinant rat fractalkine (rrfractalkine) induced the migration of rMSCs in a G-protein-dependent manner. Intracerebral injection of rhSDF-1alpha has also been shown to stimulate the homing of transplanted rMSCs to the site of injection in the brain. These data suggest that the interactions of fractalkine-CX3CR1 and SDF-1-CXCR4 could partially mediate the trafficking of transplanted rMSCs. This study provides an important insight into the understanding of the mechanisms governing the trafficking of transplanted rMSCs and also significantly expands the potential role of MSCs in cell therapy for brain injuries and diseases.

Stromal cell-derived factor 1 is secreted by meningeal cells and acts as chemotactic factor on neuronal stem cells of the cerebellar external granular layer

The cerebellar external granular layer (EGL) is an unusually long-lasting neural proliferative zone positioned immediately beneath the pial surface. Its position and stability critically depend on meningeal cells, as their selective destruction leads to its rapid dispersal, creating massive cortical ectopia. Similar ectopias have recently been described as a side effect of deficiency for stromal cell-derived factor 1 (SDF-1), a chemoattractant for haematopoietic precursor cell migration. Here we show that SDF-1 is present in meningeal cells in vivo and in vitro, where it is secreted in functionally relevant concentrations into the medium. Correspondingly, the SDF-1 receptor (termed CXCR4) can be demonstrated on stem cells of the external granular layer, but is absent on postmitotic cells commencing their final inward migration. We show that SDF-1 is concentrated by heparan sulphate proteoglycans highly expressed in the EGL in a laminar fashion, which thus might act to locally restrict SDF-1 action to the EGL in a kind of step gradient. In vitro, SDF-1 chemotactically attracts neuronal cells isolated from the external, but not from the internal granular layer, in a Boyden chamber assay in concentrations found in meningeal cell-conditioned medium. Selective removal of SDF-1 from conditioned media by immunoprecipitation abolishes their chemoattractive action, which can be reconstituted again by the addition of recombinant SDF-1. Meningeal cells are thus an important source for the expression of SDF-1 during brain development, which – comparable to its role in haematopoiesis – appears to be a key factor attracting precursor cells to their proliferative compartment.

Active movement of T cells away from a chemokine.

Movement towards or away from a given stimulus guides the directional migration of prokaryotes, simple eukaryotes and neurons. As bi-directional cues may influence entry and exit of immune effector cells from tissue sites, we evaluated the migratory responses of T-cell subsets to varying concentrations of the chemokine stromal cell derived factor-1 (SDF-1). There was selective repulsion of subpopulations of T cells at high concentrations of recombinant SDF-1 or naturally occurring bone marrow-derived SDF-1, which could be inhibited by pertussis toxin and antibody against the chemokine receptor CXCR4. Distinct sensitivity profiles to genistein, herbimycin and 8-Br-cAMP biochemically distinguished movement of cells towards or away from an SDF-1 gradient. In vivo, antigen-induced T-cell recruitment into the peritoneal cavity was reversed by high but not low concentrations of SDF-1. The phenomenon of movement away from a chemokine represents a previously unknown mechanism regulating the localization of mature T cells. It adds to the functional repertoire of chemokines that may participate in immune physiology and may be applied therapeutically to alter the immune response.

Overexpression of the chemokine receptor CXCR4 in B cell chronic lymphocytic leukemia is associated with increased functional response to stromal cell-derived factor-1 (SDF-1)

The chemokine receptor CXCR4 and its ligand stromal cell-derived factor-1 (SDF-1) play an important role in trafficking of normal lymphocytes, monocytes, as well as hematopoietic stem- and progenitor cells. SDF-1 constitutively produced by bone marrow stromal cells acts as a chemoattractant supporting the homing of stem cells and may also contribute to the tropism of malignant cells for the bone marrow. Low-grade lymphoproliferative disorders, particularly B cell chronic lymphocytic leukemia (B-CLL), are characterized by the presence of bone marrow infiltration. Therefore, we analyzed expression of the chemokine receptor CXCR4 in B-CLL, and investigated the functional effect of SDF-1 on the malignant cells. By flow cytometry, CXCR4 was consistently expressed on circulating CLL cells at a fluorescence intensity four-fold greater than that of normal B cells, and three-fold greater than that of CD19+/CD5+ cells from the normal bone marrow. CXCR4 was functionally active as demonstrated by a rapid flux of intracellular free calcium in response to SDF-1, which was significantly reduced by the partially blocking CXCR4 antibody 12G5. Moreover, transendothelial migration of B-CLL cells in vitro was stimulated by conditioned medium from bone marrow stromal cells due to its content of SDF-1, as suggested by reduced migration after addition of the CXCR4 antibody 12G5. In accordance with the CXCR4 overexpression, migration of CLL cells was more efficiently stimulated by recombinant SDF-1 compared to migration of normal B cells. We conclude that CXCR4 is overexpressed and functionally active in B-CLL, and may therefore contribute to the tropism of B-CLL cells for the bone marrow stroma.