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1.  Atypical PKC-ζ regulates SDF-1–mediated migration and development of human CD34+ progenitor cells 
Journal of Clinical Investigation  2005;115(1):168-176.
The chemokine stromal cell–derived factor–1 (SDF-1) and its receptor, CXCR4, play a major role in migration, retention, and development of hematopoietic progenitors in the bone marrow. We report the direct involvement of atypical PKC-ζ in SDF-1 signaling in immature human CD34+-enriched cells and in leukemic pre-B acute lymphocytic leukemia (ALL) G2 cells. Chemotaxis, cell polarization, and adhesion of CD34+ cells to bone marrow stromal cells were found to be PKC-ζ dependent. Overexpression of PKC-ζ in G2 and U937 cells led to increased directional motility to SDF-1. Interestingly, impaired SDF-1–induced migration of the pre-B ALL cell line B1 correlated with reduced PKC-ζ expression. SDF-1 triggered PKC-ζ phosphorylation, translocation to the plasma membrane, and kinase activity. Furthermore we identified PI3K as an activator of PKC-ζ, and Pyk-2 and ERK1/2 as downstream targets of PKC-ζ. SDF-1–induced proliferation and MMP-9 secretion also required PKC-ζ activation. Finally, we showed that in vivo engraftment, but not homing, of human CD34+-enriched cells to the bone marrow of NOD/SCID mice was PKC-ζ dependent and that injection of mice with inhibitory PKC-ζ pseudosubstrate peptides resulted in mobilization of murine progenitors. Our results demonstrate a central role for PKC-ζ in SDF-1–dependent regulation of hematopoietic stem and progenitor cell motility and development.
PMCID: PMC539190  PMID: 15630457
2.  HGF, SDF-1, and MMP-9 are involved in stress-induced human CD34+ stem cell recruitment to the liver 
Journal of Clinical Investigation  2003;112(2):160-169.
Hematopoietic stem cells rarely contribute to hepatic regeneration, however, the mechanisms governing their homing to the liver, which is a crucial first step, are poorly understood. The chemokine stromal cell–derived factor-1 (SDF-1), which attracts human and murine progenitors, is expressed by liver bile duct epithelium. Neutralization of the SDF-1 receptor CXCR4 abolished homing and engraftment of the murine liver by human CD34+ hematopoietic progenitors, while local injection of human SDF-1 increased their homing. Engrafted human cells were localized in clusters surrounding the bile ducts, in close proximity to SDF-1–expressing epithelial cells, and differentiated into albumin-producing cells. Irradiation or inflammation increased SDF-1 levels and hepatic injury induced MMP-9 activity, leading to both increased CXCR4 expression and SDF-1–mediated recruitment of hematopoietic progenitors to the liver. Unexpectedly, HGF, which is increased following liver injury, promoted protrusion formation, CXCR4 upregulation, and SDF-1–mediated directional migration by human CD34+ progenitors, and synergized with stem cell factor. Thus, stress-induced signals, such as increased expression of SDF-1, MMP-9, and HGF, recruit human CD34+ progenitors with hematopoietic and/or hepatic-like potential to the liver of NOD/SCID mice. Our results suggest the potential of hematopoietic CD34+/CXCR4+cells to respond to stress signals from nonhematopoietic injured organs as an important mechanism for tissue targeting and repair.
PMCID: PMC164291  PMID: 12865405
3.  Stromal derived factor-1 regulates bone morphogenetic protein 2-induced osteogenic differentiation of primary mesenchymal stem cells 
Stromal derived factor-1 (SDF-1) is a chemokine signaling molecule that binds to its transmembrane receptor CXC chemokine receptor-4 (CXCR4). While we previously detected that SDF-1 was co-required with bone morphogenetic protein 2 (BMP2) for differentiating mesenchymal C2C12 cells into osteoblastic cells, it is unknown whether SDF-1 is similarly involved in the osteogenic differentiation of mesenchymal stem cells (MSCs). Therefore, here we examined the role of SDF-1 signaling during BMP2-induced osteogenic differentiation of primary MSCs that were derived from human and mouse bone marrow. Our data showed that blocking of the SDF-1/CXCR4 signal axis or adding SDF-1 protein to MSCs significantly affected BMP2-induced alkaline phosphatase (ALP) activity and osteocalcin (OCN) synthesis, markers of preosteoblasts and mature osteoblasts, respectively. Moreover, disrupting the SDF-1 signaling impaired bone nodule mineralization during terminal differentiation of MSCs. Furthermore, we detected that blocking of the SDF-1 signaling inhibited the BMP2-induced early expression of Runt-related factor-2 (Runx2) and osterix (Osx), two “master” regulators of osteogenesis, and the SDF-1 effect was mediated via intracellular Smad and Erk activation. In conclusion, our results demonstrated a regulatory role of SDF-1 in BMP2-induced osteogenic differentiation of MSCs, as perturbing the SDF-1 signaling affected the differentiation of MSCs towards osteoblastic cells in response to BMP2 stimulation. These data provide novel insights into molecular mechanisms underlying MSC osteogenesis, and will contribute to the development of MSC therapies for enhancing bone formation and regeneration in broad orthopaedic situations.
PMCID: PMC2992806  PMID: 20362069
Bone morphogenetic protein 2; CXC chemokine receptor-4; Mesenchymal stem cell; Osteogenic differentiation; Stromal derived factor-1
4.  SDF-1α/CXCR4 Axis Mediates The Migration of Mesenchymal Stem Cells to The Hypoxic-Ischemic Brain Lesion in A Rat Model 
Cell Journal (Yakhteh)  2015;16(4):440-447.
Transplantation of mesenchymal stem cells (MSCs) can promote functional recovery of the brain after hypoxic-ischemic brain damage (HIBD). However, the mechanism regulating MSC migration to a hypoxic-ischemic lesion is poorly understood. Interaction between stromal cell-derived factor-1α (SDF-1α) and its cognate receptor CXC chemokine receptor 4 (CXCR4) is crucial for homing and migration of multiple stem cell types. In this study, we investigate the potential role of SDF-1α/CXCR4 axis in mediating MSC migration in an HIBD model.
Materials and Methods
In this experimental study, we first established the animal model of HIBD using the neonatal rat. Bone marrow MSCs were cultured and labeled with 5-bromo-21-deoxyuridine (BrdU) after which 6×106 cells were intravenously injected into the rat. BrdU positive MSCs in the hippocampus were detected by immunohistochemical analyses. The expression of hypoxia-inducible factor-1α (HIF-1α) and SDF-1α in the hippocampus of hypoxic-ischemic rats was detected by Western blotting. To investigate the role of hypoxia and SDF-1α on migration of MSCs in vitro, MSCs isolated from normal rats were cultured in a hypoxic environment (PO2=1%). Migration of MSCs was detected by the transwell assay. The expression of CXCR4 was tested using Western blotting and flow cytometry.
BrdU-labeled MSCs were found in the rat brain, which suggested that transplanted MSCs migrated to the site of the hypoxic-ischemic brain tissue. HIF-1α and SDF-1α significantly increased in the hippocampal formations of HIBD rats in a time-dependent manner. They peaked on day 7 and were stably expressed until day 21. Migration of MSCs in vitro was promoted by SDF-1α under hypoxia and inhibited by the CXCR4 inhibitor AMD3100. The expression of CXCR4 on MSCs was elevated by hypoxia stimulation as well as microdosage treatment of SDF-1α.
This observation illustrates that SDF-1α/CXCR4 axis mediate the migration of MSCs to a hypoxic-ischemic brain lesion in a rat model.
PMCID: PMC4297482  PMID: 25685734
Mesenchymal Stem Cells; Migration; SDF-1α; CXCR 4
5.  Cationic Liposome-Mediated CXCR4 Gene Delivery into Hematopoietic Stem/Progenitor Cells: Implications for Clinical Transplantation and Gene Therapy 
Stem Cells and Development  2011;21(10):1587-1596.
The chemokine stromal cell-derived factor (SDF)-1α/CXCL12 and its receptor CXC chemokine receptor 4 (CXCR4) play a crucial role in the homing/engraftment and retention of hematopoietic stem/progenitor cells (HSPCs) in the bone marrow. It has been shown using the viral gene transfer technique that CXCR4 overexpression on human CD34+ HSPC significantly improves their engraftment in murine models. However, clinical trials with gene therapy have revealed safety concerns related to the immunogenicity of the viral carriers, due to the random integration of viral genes into the host genome. Therefore, a method for CXCR4 gene delivery into HSPC that is safe, nonviral, and highly efficient is needed to improve clinical transplantation and gene therapies. In this work, we investigated the nonviral CXCR4 gene delivery into HSPC using the cationic liposome agent IBAfect. We used CD34+ cells from cord blood and the models of immature hematopoietic cells expressing CD34 antigen, namely, leukemic cell lines KG-1a and KG-1. Transfection efficiency was determined by flow cytometric analysis 12, 24, 48, and 72 h after transfection, and the viability of cells analyzed by trypan blue exclusion and MTS assays. The functional response of CXCR4-transfected HSPC toward an SDF-1α gradient was determined by chemotaxis assay. We found that ∼25% transfection is achieved for KG-1a and KG-1 cells and 20% for HSPC, and that the viability of CXCR4-transfected HSPC is not significantly altered. More importantly, overexpression of CXCR4 using IBAfect significantly increased the chemotaxis of KG-1 cells and HSPC toward SDF-1α. However, we tested 2 other commercially available cationic liposomes (Lipofectamine 2000 and 1,2-dioleoyl-3-trimethylammonium-propane [DOTAP]) in parallel, and we found that they failed to deliver the CXCR4 gene into cells under the same conditions. These results suggest that IBAfect-mediated in vitro gene delivery to overexpress CXCR4 on HSPC is a safe and efficient technique with great potential for improving the efficacy of HSPC transplantation and gene therapy protocols.
PMCID: PMC3376461  PMID: 22047530
6.  Stromal cell derived factor-1: its influence on invasiveness and migration of breast cancer cells in vitro, and its association with prognosis and survival in human breast cancer 
Breast Cancer Research  2005;7(4):R402-R410.
Stromal cell-derived factor (SDF)-1 (CXC chemokine ligand-12) is a member of the CXC subfamily of chemokines, which, through its cognate receptor (CXC chemokine receptor [CXCR]4), plays an important role in chemotaxis of cancer cells and in tumour metastasis. We conducted the present study to evaluate the effect of SDF-1 on the invasiveness and migration of breast cancer cells, and we analyzed the expression of SDF-1 and its relation to clinicopathological features and clinical outcomes in human breast cancer.
Expression of SDF-1 mRNA in breast cancer, endothelial (HECV) and fibroblast (MRC5) cell lines and in human breast tissues were studied using RT-PCR. MDA-MB-231 cells were transfected with a SDF-1 expression vector, and their invasiveness and migration was tested in vitro. In addition, the expression of SDF-1 was investigated using immunohistochemistry and quantitative RT-PCR in samples of normal human mammary tissue (n = 32) and mammary tumour (n = 120).
SDF-1 expression was identified in MRC5, MDA-MB-435s and MDA-MB-436 cell lines, but CXCR4 expression was detected in all cell lines and breast tissues. An autocrine loop was created following transfection of MDA-MB-231 (which was CXCR4 positive and SDF-1 negative) with a mammalian expression cassette encoding SDF-1 (MDA-MB-231SDF1+/+) or with control plasmid pcDNA4/GFP (MDA-MB-231+/-). MDA-MB-231SDF1+/+ cells exhibited significantly greater invasion and migration potential (in transfected cells versus in wild type and empty MDA-MB-231+/-; P < 0.01). In mammary tissues SDF-1 staining was primarily seen in stromal cells and weakly in mammary epithelial cells. Significantly higher levels of SDF-1 were seen in node-positive than in node-negative tumours (P = 0.05), in tumours that metastasized (P = 0.05), and tumours from patients who died (P = 0.03) than in tumours from patients who were disease free. It was most notable that levels of SDF-1 correlated significantly with overall survival (P = 0.001) and incidence-free survival (P = 0.035).
SDF-1 can increase the invasiveness and migration of breast cancer cells. Its levels correlated with node involvement and long-term survival in patients with breast cancer. SDF-1 may therefore have potential value in assessing clinical outcomes of patients with breast cancer.
PMCID: PMC1175055  PMID: 15987445
7.  A Transduced Living Hyaline Cartilage Graft Releasing Transgenic Stromal Cell-Derived Factor-1 Inducing Endogenous Stem Cell Homing In Vivo 
Tissue Engineering. Part A  2013;19(9-10):1091-1099.
Stromal cell-derived factor-1 (SDF-1), also known as a homing factor, is a potent chemokine that activates and directs mobilization, migration, and retention of certain cell species via systemic circulation. The responding homing cells largely consist of activated stem cells, so that, in case of tissue lesions, such SDF-1-induced cell migration may execute recruitment of endogenous stem cells to perform autoreparation and compensatory regeneration in situ. In this study, a recombinant adenoviral vector carrying SDF-1 transgene was constructed and applied to transduce a novel scaffold-free living hyaline cartilage graft (SDF-t-LhCG). As an engineered transgenic living tissue, SDF-t-LhCG is capable of continuously producing and releasing SDF-1 in vitro and in vivo. The in vitro trials were examined with ELISA, while the in vivo trials were subsequently performed via a subcutaneous implantation of SDF-t-LhCG in a nude mouse model, followed by series of biochemical and biological analyses. The results indicate that transgenic SDF-1 enhanced the presence of this chemokine in mouse's circulation system; in consequence, SDF-1-induced activation and recruitment of endogenous stem cells were also augmented in both peripheral blood and SDF-t-LhCG implant per se. These results were obtained via flow cytometry analyses on mouse blood samples and implanted SDF-t-LhCG samples, indicating an upregulation of the CXCR4+(SDF-1 receptor) cell population, accompanied by upregulation of the CD34+, CD44+, and Sca-1+ cell populations as well as a downregulation of the CD11b+ cell population. With the supply of SDF-1-recruited endogenous stem cells, enhanced chondrogenesis was observed in SDF-t-LhCG implants in situ.
PMCID: PMC3609611  PMID: 23167782
8.  The role of CXCR7 on the adhesion, proliferation and angiogenesis of endothelial progenitor cells 
Previous studies confirmed that stromal cell-derived factor 1 (SDF-1) was a principal regulator of retention, migration and mobilization of haematopoietic stem cells and endothelial progenitor cells (EPCs) during steady-state homeostasis and injury. CXC chemokine receptor 4 (CXCR4) has been considered as the unique receptor of SDF-1 and as the only mediator of SDF-1-induced biological effects for many years. However, recent studies found that SDF-1 could bind to not only CXCR4 but also CXC chemokine receptor 7 (CXCR7). The evidence that SDF-1 binds to the CXCR7 raises a concern how to distinguish the potential contribution of the SDF-1/CXCR7 pathway from SDF-1/CXCR4 pathway in all the processes that were previously attributed to SDF-1/CXCR4. In this study, the role of CXCR7 in EPCs was investigated in vitro. RT-PCR, Western blot and flow cytometry assay demonstrate that both CXCR4 and CXCR7 were expressed highly in EPCs. The adhesion of EPCs induced by SDF-1 was inhibited by blocking either CXCR4 or CXCR7 with their antibodies or antagonists. SDF-1 regulated the migration of EPCs via CXCR4 but not CXCR7. However, the transendothelial migration of EPCs was inhibited by either blocking of CXCR4 or CXCR7. Both CXCR7 and CXCR4 are essential for the tube formation of EPCs induced by SDF-1. These results suggested that both CXCR7 and CXCR4 are important for EPCs in response to SDF-1, indicating that CXCR7 may be another potential target molecule for angiogenesis-dependent diseases.
PMCID: PMC4373330  PMID: 21418513
CXC chemokine receptor 7; stromal cell-derived factor 1; endothelial progenitor cells; CXC chemokine receptor 4; angiogenesis
9.  Conditioned medium from umbilical cord mesenchymal stem cells induces migration and angiogenesis 
Molecular Medicine Reports  2015;12(1):20-30.
Umbilical cord mesenchymal stem cells (UC-MSCs) have been suggested as a candidate for various clinical applications, however, major limitations include the lack of organ-specific accumulation and low survival rates of transplanted cells. In the present study, it was hypothesized that the paracrine effects of UC-MSCs may enhance stem cell-based tissue repair and regeneration by promoting the specific homing of stem/progenitor cells and the overall ability to drive them to the damaged area. UC-MSCs-derived conditioned medium (UC-CM) was analyzed using liquid chip and ELISA techniques. In vitro tube formation assays of human umbilical vein endothelial cells (HUVECs) and UC-MSCs were then performed to assess the angiogenic properties of UC-CM. Subsequently, UC-MSCs, HUVECs and fibroblasts were labeled with PKH26 for an in vivo cell migration assay. The expression levels of C-X-C chemokine receptor 4 (CXCR4), C-C chemokine receptor 2 (CCR2) and c-met were determined in the UC-MSCs, HUVECs and fibroblasts using reverse transcription-quantitative polymerase chain reaction and flow cytometry. UC-CM was incubated with or without antibodies, and the contribution of stromal cell-derived factor 1 (SDF-1), monocyte chemotactic protein 1 (MCP-1) and hepatocyte growth factor (HGF) on the migration of cells was investigated in vitro. The results demonstrated that UC-MSCs secreted different cytokines and chemokines, including increased quantities of SDF-1, MCP-1 and HGF, in addition to the angiogenic factors, vascular cell adhesion protein-1, interleukin-8, insulin-like growth factor-1 and vascular endothelial growth factor. The total lengths of the tubes were significantly increased in the UC-MSCs and HUVECs incubated in UC-CM compared with those incubated in Dulbecco’s modified Eagle’s medium. In vivo cell migration assays demonstrated that UC-CM was a chemotactic stimulus for the UC-MSCs and HUVECs. In vitro Matrigel migration and scratch healing assays demonstrated that UC-CM increased the migration of CXCR4-postive or/and CCR2-positive cells in a dose-dependent manner. In addition, different molecules were screened under antibody-based blocking migration conditions. The data revealed that the SDF-1/CXCR4 and MCP-1/CCR2 axes were involved in the chemoattractive activity of UC-CM and suggested that the effective paracrine factor of UC-CM is a large complex rather than a single factor. The results of the present study supported the hypothesis that UC-MSCs release soluble factors, which may extend the therapeutic applicability of stem cells.
PMCID: PMC4438972  PMID: 25739039
umbilical cord mesenchymal stem cells; paracrine; angiogenesis; migration
10.  Essential but differential role for CXCR4 and CXCR7 in the therapeutic homingof human renal progenitor cells 
Recently, we have identified a population of renal progenitor cells in human kidneys showing regenerative potential for injured renal tissue of SCID mice. We demonstrate here that among all known chemokine receptors, human renal progenitor cells exhibit high expression of both stromal-derived factor-1 (SDF-1) receptors, CXCR4 and CXCR7. In SCID mice with acute renal failure (ARF), SDF-1 was strongly up-regulated in resident cells surrounding necrotic areas. In the same mice, intravenously injected renal stem/progenitor cells engrafted into injured renal tissue decreased the severity of ARF and prevented renal fibrosis. These beneficial effects were abolished by blocking either CXCR4 or CXCR7, which dramatically reduced the number of engrafting renal progenitor cells. However, although SDF-1–induced migration of renal progenitor cells was only abolished by an anti-CXCR4 antibody, transendothelial migration required the activity of both CXCR4 and CXCR7, with CXCR7 being essential for renal progenitor cell adhesion to endothelial cells. Moreover, CXCR7 but not CXCR4 was responsible for the SDF-1–induced renal progenitor cell survival. Collectively, these findings suggest that CXCR4 and CXCR7 play an essential, but differential, role in the therapeutic homing of human renal progenitor cells in ARF, with important implications for the development of stem cell–based therapies.
PMCID: PMC2271008  PMID: 18268039
11.  Stromal Cell-Derived Factor-1 Receptor CXCR4-Overexpressing Bone Marrow Mesenchymal Stem Cells Accelerate Wound Healing by Migrating into Skin Injury Areas 
Cellular Reprogramming  2013;15(3):206-215.
Stromal cell-derived factor-1 (SDF-1) and its membrane receptor C-X-C chemokine receptor type 4 (CXCR4) are involved in the homing and migration of multiple stem cell types, neovascularization, and cell proliferation. This study investigated the hypothesis that bone marrow–derived mesenchymal stem cells (BMSCs) accelerate skin wound healing in the mouse model by overexpression of CXCR4 in BMSCs. We compared SDF-1 expression and skin wound healing times of BALB/c mice, severe combined immunodeficiency (SCID) mice, and immune system–deficient nude mice after 60Co radiation–induced injury of their bone marrow. The occurrence of transplanted adenovirus-transfected CXCR4-overexpressing male BMSCs in the wound area was compared with the occurrence of untransfected male BALB/c BMSCs in 60Co-irradiated female mice skin wound healing areas by Y chromosome marker analyses. The wound healing time of BALB/c mice was 14.00±1.41 days, whereas for the nude and SCID mice it was 17.16±1.17 days and 19.83±0.76 days, respectively. Male BMSCs could be detected in the surrounding areas of 60Co-irradiated female BALB/c mice wounds, and CXCR4-overexpressing BMSCs accelerated the wound healing time. CXCR4-overexpressing BMSCs migrate in an enhanced manner to skin wounds in a SDF-1–expression-dependent manner, thereby reducing the skin wound healing time.
PMCID: PMC3666362  PMID: 23713431
12.  Bone-derived SDF-1 stimulates IL-6 release via CXCR4, ERK and NF-κB pathways and promotes osteoclastogenesis in human oral cancer cells 
Carcinogenesis  2008;29(8):1483-1492.
Oral squamous cell carcinoma (SCC) has a striking tendency to invade to bone. The chemokine stromal cell-derived factor-1 (SDF-1) is constitutively secreted by osteoblasts and plays a key role in homing of hematopoietic cells to the bone marrow. Interleukin (IL)-6 plays an important role in osteoclastogenesis. Herein, we found that SDF-1α increased the secretion of IL-6 in cultured human SCC cells, as shown by reverse transcriptase–polymerase chain reaction and enzyme-linked immunosorbent assay. SDF-1α also increased the surface expression of chemokine receptor 4 (CXCR4) in SCC cells. CXCR4-neutralizing antibody, CXCR4-specific inhibitor (AMD3100) or small interfering RNA against CXCR4 inhibited SDF-1α-induced increase IL-6 production. The transcriptional regulation of IL-6 by SDF-1α was mediated by phosphorylation of extracellular signal-regulated kinases (ERKs) and activation of the nuclear factor-kappa B (NF-κB) components p65 and p50. The binding of p65 and p50 to the NF-κB element on the IL-6 promoter was enhanced by SDF-1α. In addition, IL-6 antibody antagonized the SCC-conditioned medium-increased osteoclastogenesis. These results suggested that SDF-1α from osteoblasts could induce release of IL-6 in human SCC cells via activation of CXCR4, ERK and NF-κB pathway and thereby promote osteoclastogenesis.
PMCID: PMC2516485  PMID: 18310089
13.  Proteolytic processing of SDF-1α by matrix metalloproteinase-2 impairs CXCR4 signaling and reduces neural progenitor cell migration 
Protein & cell  2012;3(11):875-882.
Neural stem cells and neural progenitor cells (NPCs) exist throughout life and are mobilized to replace neurons, astrocytes and oligodendrocytes after injury. Stromal cell-derived factor 1 (SDF-1, now named CXCL12) and its receptor CXCR4, an α-chemokine receptor, are critical for NPC migration into damaged areas of the brain. Our previous studies demonstrated that immune activated and/or HIV-1-infected human monocyte-derived- macrophages (MDMs) induced a substantial increase of SDF-1 production by human astrocytes. However, matrix metalloproteinase (MMP)-2, a protein up-regulated in HIV-1-infected macrophages, is able to cleave four amino acids from the N-terminus of SDF-1, resulting in a truncated SDF-1(5–67). In this study, we investigate the diverse signaling and function induced by SDF-1α and SDF-1(5–67) in human cortical NPCs. SDF-1(5–67) was generated by incubating human recombinant SDF-1α with MMP-2 followed by protein determination via mass spectrometry, Western blotting and ELISA. SDF-1α induced time-dependent phosphorylation of extracellular signal-regulated kinases (ERK) 1/2, Akt-1, and diminished cyclic adenosine monophosphate (cAMP). In contrast, SDF-1(5–67) failed to induce these signaling. SDF-1α activation of CXCR4 induced migration of NPCs, an effect that is dependent on ERK1/2 and Akt-1 pathways; whereas SDF-1(5–67) failed to induce NPC migration. This observation provides evidence that MMP-2 may affect NPC migration through post-translational processing of SDF-1α.
PMCID: PMC3973535  PMID: 23143873
proteolysis; chemokine; neurogenesis; migration
14.  The Expanding Family of Bone Marrow Homing Factors for Hematopoietic Stem Cells: Stromal Derived Factor 1 Is Not the Only Player in the Game 
The Scientific World Journal  2012;2012:758512.
The α-chemokine stromal derived factor 1 (SDF-1), which binds to the CXCR4 and CXCR7 receptors, directs migration and homing of CXCR4+ hematopoietic stem/progenitor cells (HSPCs) to bone marrow (BM) and plays a crucial role in retention of these cells in stem cell niches. However, this unique role of SDF-1 has been recently challenged by several observations supporting SDF-1-CXCR4-independent BM homing. Specifically, it has been demonstrated that HSPCs respond robustly to some bioactive lipids, such as sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P), and migrate in response to gradients of certain extracellular nucleotides, including uridine triphosphate (UTP) and adenosine triphosphate (ATP). Moreover, the responsiveness of HSPCs to an SDF-1 gradient is enhanced by some elements of innate immunity (e.g., C3 complement cascade cleavage fragments and antimicrobial cationic peptides, such as cathelicidin/LL-37 or β2-defensin) as well as prostaglandin E2 (PGE2). Since all these factors are upregulated in BM after myeloblative conditioning for transplantation, a more complex picture of homing emerges that involves several factors supporting, and in some situations even replacing, the SDF-1-CXCR4 axis.
PMCID: PMC3373139  PMID: 22701372
15.  Stromal-Derived Factor-1 and Its Receptor, CXCR4, Are Constitutively Expressed by Mouse Liver Sinusoidal Endothelial Cells: Implications for the Regulation of Hematopoietic Cell Migration to the Liver During Extramedullary Hematopoiesis 
Stem Cells and Development  2011;21(12):2142-2151.
Stromal-derived factor (SDF)-1 is the main regulating factor for trafficking/homing of hematopoietic stem cells (HSC) to the bone marrow (BM). It is possible that this chemokine may also play a fundamental role in regulating the migration of HSC to several organs during extramedullary hematopoiesis. Because liver sinusoidal endothelial cells (LSEC) constitute an extramedullary niche for HSC, it is possible that these cells represent one of the main cellular sources of SDF-1 at the liver. Here, we show that LSEC express SDF-1 at the mRNA and protein level. Biological assays showed that conditioned medium from LSEC (LSEC-CM) stimulated the migration of BM progenitor lineage-negative (BM/Lin−) cells. This effect was significantly reduced by AMD3100, indicating that the SDF-1/CXCR4 axis is involved in the stimulatory migrating effect induced by LSEC-CM. Early localization of HSC in SDF-1–expressing LSEC microenvironment together with increased levels of this chemokine in hepatic homogenates was found in an experimental model of liver extramedullary hematopoiesis. Flow cytometry studies showed that LSEC express the CXCR4 receptor. Functional assays showed that activation of this receptor by SDF-1 stimulated the migration of LSEC and increased the expression of PECAM-1. Our findings suggest that LSEC through the production of SDF-1 may constitute a fundamental niche for regulation of HSC migration to the liver. To our knowledge, this is the first report showing that LSEC not only express and secrete SDF-1, but also its receptor CXCR4.
PMCID: PMC3411357  PMID: 22121892
16.  Overexpression of SDF-1α Enhanced Migration and Engraftment of Cardiac Stem Cells and Reduced Infarcted Size via CXCR4/PI3K Pathway 
PLoS ONE  2012;7(9):e43922.
Cardiac stem cells (CSCs) can home to the infarcted area and regenerate myocardium. Stromal cell-derived factor-1α/C-X-C chemokine receptor type 4 (SDF-1α/CXCR4) axis is pivotal in inducing CSCs migration. However, the mechanisms remain unclear. This study set out to detect if SDF-1α promotes migration and engraftment of CSCs through the CXCR4/PI3K (phosphatidylinositol 3-kinase) pathway. In the in vitro experiment, c-kit+ cells were isolated from neonatal mouse heart fragment culture by magnetic cell sorting. Fluorescence-activated cell sorting results demonstrated that a few c-kit+ cells expressed CD45 (4.54%) and Sca-1 (2.58%), the hematopoietic stem cell marker. Conditioned culture could induce c-kit+ cells multipotent differentiation, which was confirmed by cardiac troponin I (cTn-I), α-smooth muscle actin (α-SMA), and von Willebrand factor (vWF) staining. In vitro chemotaxis assays were performed using Transwell cell chambers to detect CSCs migration. The results showed that the cardiomyocytes infected with rAAV1-SDF-1α-eGFP significantly increased SDF-1α concentration, 5-fold more in supernatant than that in the control group, and subsequently attracted more CSCs migration. This effect was diminished by administration of AMD3100 (10 µg/ml, CXCR4 antagonist) or LY294002 (20 µmol/L, PI3K inhibitor). In myocardial infarction mice, overexpression of SDF-1α in the infarcted area by rAAV1-SDF-1α-eGFP infection resulted in more CSCs retention to the infarcted myocardium, a higher percentage of proliferation, and reduced infarcted area which was attenuated by AMD3100 or ly294002 pretreatment. These results indicated that overexpression of SDF-1α enhanced CSCs migration in vitro and engraftment of transplanted CSCs and reduced infarcted size via CXCR4/PI3K pathway.
PMCID: PMC3439464  PMID: 22984452
17.  VEGF/SDF-1 promotes cardiac stem cell mobilization and myocardial repair in the infarcted heart 
Cardiovascular Research  2011;91(3):402-411.
The objective of this study was to investigate whether vascular endothelial growth factor (VEGF) secreted by mesenchymal stem cells (MSC) improves myocardial survival and the engraftment of implanted MSC in infarcted hearts and promotes recruitment of stem cells through paracrine release of myocardial stromal cell-derived factor-1α (SDF-1α).
Methods and results
VEGF-expressing MSC (VEGFMSC)-conditioned medium enhanced SDF-1α expression in heart slices and H9C2 cardiomyoblast cells via VEGF and the vascular endothelial growth factor receptor (VEGFR). The VEGFMSC-conditioned medium markedly promoted cardiac stem cell (CSC) migration at least in part via the SDF-1α/CXCR4 pathway and involved binding to VEGFR-1 and VEGFR-3. In vivo, VEGFMSC-stimulated SDF-1α expression in infarcted hearts resulted in massive mobilization and homing of bone marrow stem cells and CSC. Moreover, VEGF-induced SDF-1α guided the exogenously introduced CSC in the atrioventricular groove to migrate to the infarcted area, leading to a reduction in infarct size. Functional studies showed that VEGFMSC transplantation stimulated extensive angiomyogenesis in infarcted hearts as indicated by the expression of cardiac troponin T, CD31, and von Willebrand factor and improved the left ventricular performance, whereas blockade of SDF-1α or its receptor by RNAi or antagonist significantly diminished the beneficial effects of VEGFMSC.
Exogenously expressed VEGF promotes myocardial repair at least in part through SDF-1α/CXCR4-mediated recruitment of CSC.
PMCID: PMC3139446  PMID: 21345805
Myocardial infarction; VEGF; SDF-1α; Cardiac stem cell; Migration
18.  Identification of E-Selectin as a Novel Target for the Regulation of Post-Natal Neovascularization: Implications for Diabetic Wound Healing 
Annals of Surgery  2010;252(4):625-634.
We previously reported that stromal cell-derived factor-1α (SDF-1α, a homing signal for recruiting endothelial progenitor cells (EPC) to areas of neovascularization), is down-regulated in diabetic wounds 1. We now investigate signals whereby mature endothelial cells (EC) and circulating EPC achieve SDF-1α-mediated EPC homing.
SDF-1α in diabetic wounds were therapeutically increased by injection of SDF-1α–engineered bone marrow-derived fibroblasts versus control cells (N= 48 (20, NOD), (28, STZ-C57)). PCR-array gene expression differences were validated by Western blotting and immunohistochemistry. The role of adhesion molecule(s) in mediating SDF-1α-induced EPC homing and wound healing was furthered studied using antagonists in vitro and in vivo.
Increasing wound SDF-1α via cell-base therapy promotes healing in diabetic mice (~20% increase in healing rates by day 3, p=0.006). SDF-1α increased EC-EPC adhesion and specifically upregulated E-selectin expression in human microvascular EC (2.3-fold increase, p<0.01). This effect was also significant in blood vessels of the experimental mice and resulted in increased wound neovascularization. The regulatory effects of SDF-1α on EC-EPC adhesion and EPC homing were specifically mediated by E-selectin, as the application of E-selectin antagonists significantly inhibited SDF-1α-induced EC-EPC adhesion, EPC homing, wound neovascularization, and wound healing.
SDF-1α–engineered cell-based therapy promotes diabetic wound healing in mice by specifically upregulating E-selectin expression in mature EC leading to increase EC-EPC adhesion, EPC homing and increased wound neovascularization. These findings provide novel insight into the signals underlying the biological effect of SDF-1α on EPC homing and point to E-selectin as a new potential target for therapeutic manipulation of EPC trafficking in diabetic wound healing.
PMCID: PMC3391745  PMID: 20881769
19.  Bone Marrow Mesenchymal Stem Cells for Improving Hematopoietic Function: An In Vitro and In Vivo Model. Part 2: Effect on Bone Marrow Microenvironment 
PLoS ONE  2011;6(10):e26241.
The aim of the present study was to determine how mesenchymal stem cells (MSC) could improve bone marrow (BM) stroma function after damage, both in vitro and in vivo. Human MSC from 20 healthy donors were isolated and expanded. Mobilized selected CD34+ progenitor cells were obtained from 20 HSCT donors. For in vitro study, long-term bone marrow cultures (LTBMC) were performed using a etoposide damaged stromal model to test MSC effect in stromal confluence, capability of MSC to lodge in stromal layer as well as some molecules (SDF1, osteopontin,) involved in hematopoietic niche maintenance were analyzed. For the in vivo model, 64 NOD/SCID recipients were transplanted with CD34+ cells administered either by intravenous (IV) or intrabone (IB) route, with or without BM derived MSC. MSC lodgement within the BM niche was assessed by FISH analysis and the expression of SDF1 and osteopontin by immunohistochemistry. In vivo study showed that when the stromal damage was severe, TP-MSC could lodge in the etoposide-treated BM stroma, as shown by FISH analysis. Osteopontin and SDF1 were differently expressed in damaged stroma and their expression restored after TP-MSC addition. Human in vivo MSC lodgement was observed within BM niche by FISH, but MSC only were detected and not in the contralateral femurs. Human MSC were located around blood vessels in the subendoestal region of femurs and expressed SDF1 and osteopontin. In summary, our data show that MSC can restore BM stromal function and also engraft when a higher stromal damage was done. Interestingly, MSC were detected locally where they were administered but not in the contralateral femur.
PMCID: PMC3197625  PMID: 22028841
20.  Stem cell attraction via SDF-1α expressing fat tissue grafts 
Mesenchymal stromal cell (MSCs) are key cellular components for site-specific tissue regeneration. The chemokine stromal derived factor 1 alpha (SDF-1α) is known to attract stem cells via the C-X-C chemokine receptor-4 (CXCR4) receptor. The aim of the study was to develop a model for stem cell attraction using SDF-1α overexpressing fat tissue grafts. Murine MSCs were lentiviral transduced to express the genes for enhanced green fluorescent protein, firefly luciferace, and human CXCR4 (hCXCR4). Murine fat tissue was adenoviral transduced to express SDF-1α and red fluorescent protein transgenes. MSCs were cultured on transwells with SDF-1α containing supernatants from transduced fat tissue. The numbers of migrated MSCs in four groups (with hCXCR4 positive (+) or hCXCR4 negative (−) MSCs with or without SDF-1α containing supernatant) were investigated. After 36 h of culture, 9025 ± 925 cells migrated through the membrane of the transwells in group 1 (CXCR4+/SDF-1α+), 4817 ± 940 cells in group 2 (CXCR4-/SDF-1α+), 2050 ± 766 cells in group 3 (CXCR4+/SDF-1α−), and 2108 ± 426 cells in group 4 (CXCR4-/SDF-1α-). Both, the presence of SDF-1α and the expression of hCXCR4 significantly increased the migration rates (p < 0.0001). MSCs overexpressing the CXCR4 receptor by lentiviral transduction are highly attracted by medium from SDF-1α expressing fat tissue in vitro. Thus, SDF-1α activated tissue grafts may be a strategy to enhance site-specific musculoskeletal tissue regeneration.
PMCID: PMC4056675  PMID: 23281045
MSC migration; SDF-1; CXCR4; adenovirus; lentivirus
21.  Rapid mobilization of hematopoietic progenitors by AMD3100 and catecholamines is mediated by CXCR4-dependent SDF-1 release from bone marrow stromal cells 
Leukemia  2011;25(8):1286-1296.
Steady-state egress of hematopoietic progenitor cells can be rapidly amplified by mobilizing agents such as AMD3100, the mechanism, however, is poorly understood. We report that AMD3100 increased the homeostatic release of the chemokine SDF-1 to the circulation in mice and non-human primates. Neutralizing antibodies against CXCR4 or SDF-1 inhibited both steady-state and AMD3100-induced SDF-1 release and reduced egress of murine progenitor cells over mature leukocytes. Intra-bone injection of biotinylated SDF-1 also enhanced release of this chemokine and murine progenitor cell mobilization. AMD3100 directly induced SDF-1 release from CXCR4+ human bone marrow osteoblasts and endothelial cells and activated uPA in a CXCR4/JNK-dependent manner. Additionally, ROS inhibition reduced AMD3100-induced SDF-1 release, activation of circulating uPA and mobilization of progenitor cells. Norepinephrine treatment, mimicking acute stress, rapidly increased SDF-1 release and progenitor cell mobilization, while β2-adrenergic antagonist inhibited both steady-state and AMD3100-induced SDF-1 release and progenitor cell mobilization in mice. In conclusion, this study reveals that SDF-1 release from bone marrow stromal cells to the circulation emerges as a pivotal mechanism essential for steady state egress and rapid mobilization of hematopoietic progenitor cells, but not mature leukocytes.
PMCID: PMC4175714  PMID: 21494253
Rapid mobilization; AMD3100; catecholamines; uPA; SDF-1/CXCR4; hematopoietic progenitor cells
22.  Stromal Cell-Derived Factor-1β Potentiates Bone Morphogenetic Protein-2-Stimulated Osteoinduction of Genetically Engineered Bone Marrow-Derived Mesenchymal Stem Cells In Vitro 
Tissue Engineering. Part A  2012;19(1-2):1-13.
Skeletal injuries are among the most prevalent clinical problems and bone marrow-derived mesenchymal stem/stromal cells (BMSCs) have successfully been used for the treatment thereof. Stromal cell-derived factor-1 (SDF-1; CXCL12) is a member of the CXC chemokine family with multiple splice variants. The two most abundant variants, SDF-1α and SDF-1β, share identical amino acid sequences, except for four additional amino acids at the C-terminus of SDF-1β, which may mediate surface stabilization via glycosaminoglycans and protect SDF-1β from proteolytic cleavage, rendering it twice as potent as SDF-1α. Increasing evidence suggests that SDF-1 is involved in bone formation through regulation of recruitment, engraftment, proliferation, and differentiation of stem/progenitor cells. The underlying molecular mechanisms, however, have not yet been fully elucidated. In this study, we tested the hypothesis that SDF-1β can potentiate bone morphogenetic protein-2 (BMP-2)-stimulated osteogenic differentiation and chemotaxis of BMSCs in vitro. Utilizing retrovirus-mediated gene transfer to generate novel Tet-Off-SDF-1β BMSCs, we found that conditional SDF-1β expression is tightly regulated by doxycycline in a dose-dependent and temporal fashion, leading to significantly increased SDF-1β mRNA and protein levels. In addition, SDF-1β was found to enhance BMP-2-stimulated mineralization, mRNA and protein expression of key osteogenic markers, and regulate BMP-2 signal transduction via extracellular signal-regulated kinases 1/2 (Erk1/2) phosphorylation in genetically engineered BMSCs in vitro. We also showed that SDF-1β promotes the migratory response of CXC chemokine receptor 4 (CXCR4)-expressing BMSCs in vitro. Taken together, these data support that SDF-1β can play an important role in BMP-2-stimulated osteogenic differentiation of BMSCs and may exert its biological activity in both an autocrine and paracrine fashion.
PMCID: PMC3530941  PMID: 22779446
23.  Effect of Dual Treatment with SDF-1 and BMP-2 on Ectopic and Orthotopic Bone Formation 
PLoS ONE  2015;10(3):e0120051.
The potent stem cell homing factor stromal cell-derived factor-1 (SDF-1) actively recruits mesenchymal stem cells from circulation and from local bone marrow. It is well established that bone morphogenetic protein-2 (BMP-2) induces ectopic and orthotopic bone formation. However, the exact synergistic effects of BMP-2 and SDF-1 in ectopic and orthotopic bone regeneration models have not been fully investigated. The purpose of this study was to evaluate the potential effects of simultaneous SDF-1 and BMP-2 treatment on bone formation.
Materials and Methods
Various doses of SDF-1 were loaded onto collagen sponges with or without BMP-2.These sponges were implanted into subcutaneous pockets and critical-size calvarial defects in C57BL/6 mice. The specimens were harvested 4 weeks post-surgery and the degree of bone formation in specimens was evaluated by histomorphometric and radiographic density analyses. Osteogenic potential and migration capacity of mesenchymal cells and capillary tube formation of endothelial cells following dual treatment with SDF-1 and BMP-2 were evaluated with in vitro assays.
SDF-1-only-treated implants did not yield significant in vivo bone formation and SDF-1 treatment did not enhance BMP-2-induced ectopic and orthotopic bone regeneration. In vitro experiments showed that concomitant use of BMP-2 and SDF-1 had no additive effect on osteoblastic differentiation, cell migration or angiogenesis compared to BMP-2 or SDF-1 treatment alone.
These findings imply that sequence-controlled application of SDF-1 and BMP-2 must be further investigated for the enhancement of robust osteogenesis in bone defects.
PMCID: PMC4363323  PMID: 25781922
24.  Stromal Cell-Derived Factor-1β Mediates Cell Survival through Enhancing Autophagy in Bone Marrow-Derived Mesenchymal Stem Cells 
PLoS ONE  2013;8(3):e58207.
Bone marrow-derived mesenchymal stem/stromal cells (BMSCs) hold great potential for cell-based therapy, yet the therapeutic efficacy remains uncertain. Transplanted BMSCs often fail to engraft within the bone marrow (BM), in part due to the poor survival of donor cells in response to inflammatory reactions, hypoxia, oxidative stress, or nutrient starvation. Two basic cell processes, apoptosis and autophagy, could potentially be responsible for the impaired survival of transplanted BMSCs. However, the functional relationship between apoptosis and autophagy in BMSC homeostasis is complex and not well understood. The stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) signaling axis appears to be critical in maintaining proliferation and survival of BM stem cell populations through improving cell proliferation and survival in response to stress; however, the exact mechanisms remain unclear. We recently described novel genetically engineered Tet-Off-SDF-1β BMSCs, which over-express SDF-1β under tight doxycycline-control, thus providing an ideal model system to investigate the isolated effects of SDF-1β. In this study we tested the hypothesis that SDF-1β can mediate cell survival of BMSCs in vitro through increasing autophagy. We found that SDF-1β had no effect on BMSC proliferation; however, SDF-1β significantly protected genetically engineered BMSCs from H2O2-induced cell death through increasing autophagy and decreasing caspase-3-dependent apoptosis. Taken together, we provide novel evidence that the SDF-1/CXCR4 axis, specifically activated by the SDF-1β isoform, plays a critical role in regulating BMSC survival under oxidative stress through increasing autophagy.
PMCID: PMC3589360  PMID: 23472159
25.  Sdf-1 (CXCL12) induces CD9 expression in stem cells engaged in muscle regeneration 
Understanding the mechanism of stem cell mobilization into injured skeletal muscles is a prerequisite step for the development of muscle disease therapies. Many of the currently studied stem cell types present myogenic potential; however, when introduced either into the blood stream or directly into the tissue, they are not able to efficiently engraft injured muscle. For this reason their use in therapy is still limited. Previously, we have shown that stromal-derived factor-1 (Sdf-1) caused the mobilization of endogenous (not transplanted) stem cells into injured skeletal muscle improving regeneration. Here, we demonstrate that the beneficial effect of Sdf-1 relies on the upregulation of the tetraspanin CD9 expression in stem cells.
The expression pattern of adhesion proteins, including CD9, was analysed after Sdf-1 treatment during regeneration of rat skeletal muscles and mouse Pax7-/- skeletal muscles, that are characterized by the decreased number of satellite cells. Next, we examined the changes in CD9 level in satellite cells-derived myoblasts, bone marrow-derived mesenchymal stem cells, and embryonic stem cells after Sdf-1 treatment or silencing expression of CXCR4 and CXCR7. Finally, we examined the potential of stem cells to fuse with myoblasts after Sdf-1 treatment.
In vivo analyses of Pax7-/- mice strongly suggest that Sdf-1-mediates increase in CD9 levels also in mobilized stem cells. In the absence of CXCR4 receptor the effect of Sdf-1 on CD9 expression is blocked. Next, in vitro studies show that Sdf-1 increases the level of CD9 not only in satellite cell-derived myoblasts but also in bone marrow derived mesenchymal stem cells, as well as embryonic stem cells. Importantly, the Sdf-1 treated cells migrate and fuse with myoblasts more effectively.
We suggest that Sdf-1 binding CXCR4 receptor improves skeletal muscle regeneration by upregulating expression of CD9 and thus, impacting at stem cells mobilization to the injured muscles.
PMCID: PMC4445299  PMID: 25890097

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