Stromal Cell-derived Factor-1 Gradient Research Articles

Engraftment and SDF-1/CXCR4 Signaling in Human CD34+ Hematopoietic Stem/Progenitor Cells Are Regulated By R4 Rgs Subfamily Proteins

Regulator of G-protein Signaling (RGS) family proteins are known to negatively regulate G-protein-coupled receptor signaling through their GTPase-accelerating activity. In several types of mature hematopoietic cells (e.g., B lymphocytes and megakaryocytes), responses to chemokines are subjected to regulation by R4 subfamily RGS proteins. However, their expression patterns and functional roles in hematopoietic stem and progenitor cells (HSC) are poorly characterized. Here, we showed that human CD34+ HSC derived from cord blood (CB, n = 12) expressed RGS1 , RGS2 , RGS3 , RGS5 , RGS13 , RGS16 and RGS18 at mRNA level, whereas RGS4 , RGS8 and RGS21 were undetectable. Exposure of CB CD34+ cells to the critical homing-related chemokine stromal cell-derived factor-1 (SDF-1) significantly increased RGS1 , RGS2 , RGS13 and RGS16 , and decreased RGS3 and RGS18 expressions (P ≤ 0.040, n = 5). Expressions of RGS1 , RGS13 and RGS16 were significantly higher in bone marrow (BM, n = 14) CD34+ cells when compared to mobilized peripheral blood (MPB, n = 14) CD34+ cells (P ≤ 0.002), while RGS3 and RGS18 expressions were lower in BM CD34+ cells (P ≤ 0.036), suggesting a SDF-1 and niche-dependent regulation of RGS expressions. Lentivirus-mediated overexpression of RGS1 , RGS13 or RGS16 significantly inhibited migration of CD34+ cells to a SDF-1 gradient (P ≤ 0.039, n = 4-5) butdid not alter SDF-1-induced actin polymerization (n = 3). In addition, phosphorylation of signaling molecules downstream of the SDF-1/CXCR4 axis, including Akt, ERK and STAT3 was significantly suppressed in RGS1 - and RGS16 -overexpressing CD34+ cells. In the NOD/SCID mouse xenotransplantation model, overexpression of RGS1 , RGS13 or RGS16 significantly reduced BM engraftment of CD34+ cells by 98% (P = 0.023, n = 7), 89% (P = 0.014, n = 8) and 87% (P = 0.015, n = 8) respectively. Transcriptome profiling (n = 4) of CD34+ cells identified 312 genes with differential expressions upon overexpression of RGS1 , RGS13 or RGS16 , with 32 genes commonly regulated. Gene function enrichment analysis revealed significant downregulation of genes involved in the migration (PROS1 , CCL1 , THBS1 , F2RL2 ), complement activation (C3AR1 , C5AR1 , C5AR2 ), and proteolysis (MMP14 , TIMP3 ) pathways, which were validated by qPCR (n = 8). Taken together, we provided the first evidence that R4 RGS members are expressed and regulated by the SDF-1 in human CD34+ HSC. We also presented data showing that RGS1 , RGS13 or RGS16 negatively regulated in vitro SDF-1-mediated migration and signaling as well as in vivo engraftment of CD34+ cells, suggesting that these RGS members might serve as a feedback mechanism to regulate the SDF-1/CXCR4 axis. Strategies to inhibit specific R4 RGS proteins could potentially enhance efficiency of HSC homing and engraftment, which are particularly important in the setting of CB transplantation. DisclosuresNo relevant conflicts of interest to declare.

R4 Rgs Subfamily Proteins Negatively Regulates SDF-1/CXCR4 Signaling in CD34+ Hematopoietic Stem and Progenitor Cells

RGS family proteins are known to negatively regulate G-protein-coupled receptor signaling through their GTPase-accelerating activity. In several types of hematopoietic cells (e.g., B lymphocytes and megakaryocytes), responses to stromal cell-derived factor-1 (SDF-1) are subjected to regulation by R4 subfamily RGS proteins. However, their expression patterns and functional roles in hematopoietic stem and progenitor cells (HSC) are poorly characterized. Here, we showed that human CD34+ HSC derived from cord blood (CB, n = 10) expressed 7 out of 10 R4 RGS proteins at mRNA level (RGS1-3, 5, 13, 16 and 18), whereas expressions of RGS4, 8 and 21 were undetectable. Exposure of CB CD34+ cells to SDF-1 significantly increased RGS1, 2, 13 and 16 expressions and decreased RGS3 and 18 expressions (P ≤ 0.0402, n = 5). Expressions of RGS1, 13 and 16 were significantly higher in bone marrow (BM, n = 10) CD34+ cells when compared to mobilized peripheral blood (MPB, n = 5) CD34+ cells (P ≤ 0.0160), while RGS3 and 18 expressions were lower in BM CD34+ cells (P ≤ 0.0471), suggesting a SDF-1- and niche-dependent regulation of RGS expressions. To investigate the potential involvement of RGS proteins in SDF-1-mediated homing-related functions, we introduced RGS overexpression constructs into CB CD34+ cells by lentiviral transduction. With >80% transduction efficiency, we showed that overexpression of RGS1, 13 and 16 but not RGS2 significantly inhibited migration of CD34+ cells to a SDF-1 gradient (P ≤ 0.0391, n = 4-5). Similarly, RGS1, 13 and 16 overexpression suppressed SDF-1-induced Akt phosphorylation (n = 2), but none of them affected SDF-1-mediated actin polymerization (n = 3). In the NOD/SCID mouse xenotransplantation model, preliminary results showed that bone marrow homing was impaired in RGS1- (16.3% reduction), RGS13- (12.7% reduction) or RGS16-overexpressing CD34+ cells (33.7% reduction). Taken together, we provided the first evidence that expressions of R4 RGS proteins are regulated by the SDF-1/CXCR4 axis in human CD34+ HSC. We also presented evidence that specific R4 RGS proteins (RGS1, 13 and 16) negatively regulate in vitro SDF-1-mediated responses and in vivo homing of CD34+ cells, suggesting that RGS proteins may serve as a feedback mechanism to regulate SDF-1/CXCR4 signaling. Strategies to inhibit RGS signaling could thus be a potential method for enhancing efficiency of HSC homing and long-term engraftment, which is particularly important in the setting of CB transplantation. DisclosuresNo relevant conflicts of interest to declare.

Dimethyl Sulfoxide (DMSO) Increases Percentage of CXCR4+ Hematopoietic Stem/Progenitor Cells, Their Responsiveness to an SDF-1 Gradient, Homing Capacities, and Survival

Cryopreservation of bone marrow (BM), mobilized peripheral blood (mPB), and cord blood (CB) hematopoietic stem/progenitor cells (HSPCs) is a routine procedure before transplantation. The most commonly used cryoprotectant for HSPCs is dimethyl sulfoxide (DMSO). The objective of this study was to evaluate the influence of DMSO on surface receptor expression and chemotactic activities of HSPCs. We found that 10 min of incubation of human mononuclear cells (MNCs) with 10% DMSO significantly increases the percentage of CXCR4(+), CD38(+), and CD34(+) cells, resulting in an increase of CD34(+), CD34(+)CXCR4(+), and CD34(+)CXCR4(+)CD38(-) subpopulations. Furthermore, DMSO significantly increased chemotactic responsiveness of MNCs and CXCR4(+) human hematopoietic Jurkat cell line to a stromal cell-derived factor-1 (SDF-1) gradient. Furthermore, we demonstrated enhanced chemotaxis of human clonogenic progenitor cells to an SDF-1 gradient, which suggests that DMSO directly enhances the chemotactic responsiveness of early human progenitors. DMSO preincubation also caused lower internalization of the CXCR4 receptor. In parallel experiments, we found that approximately 30% more of DMSO-preincubated human CD45(+) and CD45(+)CD34(+) cells homed to the mouse BM 24 h after transplantation in comparison to control cells. Finally, we demonstrated considerably higher (25 days) survival of mice transplanted with DMSO-exposed MNCs than those transplanted with the control cells. We show in this study an unexpected beneficial influence of DMSO on HSPC homing and suggest that a short priming with DMSO before transplantation could be considered a new strategy to enhance cell homing and engraftment.

Mononuclear cells and vascular repair in HHT.

Hereditary hemorrhagic telangiectasia (HHT) or Rendu–Osler–Weber disease is a rare genetic vascular disorder known for its endothelial dysplasia causing arteriovenous malformations and severe bleedings. HHT-1 and HHT-2 are the most prevalent variants and are caused by heterozygous mutations in endoglin and activin receptor-like kinase 1, respectively. An undervalued aspect of the disease is that HHT patients experience persistent inflammation. Although endothelial and mural cells have been the main research focus trying to unravel the mechanism behind the disease, wound healing is a process with a delicate balance between inflammatory and vascular cells. Inflammatory cells are part of the mononuclear cells (MNCs) fraction, and can, next to eliciting an immune response, also have angiogenic potential. This biphasic effect of MNC can hold a promising mechanism to further elucidate treatment strategies for HHT patients. Before MNC are able to contribute to repair, they need to home to and retain in ischemic and damaged tissue. Directed migration (homing) of MNCs following tissue damage is regulated by the stromal cell derived factor 1 (SDF1). MNCs that express the C-X-C chemokine receptor 4 (CXCR4) migrate toward the tightly regulated gradient of SDF1. This directed migration of monocytes and lymphocytes can be inhibited by dipeptidyl peptidase 4 (DPP4). Interestingly, MNC of HHT patients express elevated levels of DPP4 and show impaired homing toward damaged tissue. Impaired homing capacity of the MNCs might therefore contribute to the impaired angiogenesis and tissue repair observed in HHT patients. This review summarizes recent studies regarding the role of MNCs in the etiology of HHT and vascular repair, and evaluates the efficacy of DPP4 inhibition in tissue integrity and repair.

A Possible Role Of The Tetraspanin CD9 In Bone Marrow Retention and Engraftment Of Human CD34+ Hematopoietic Stem/Progenitor Cells

The stromal cell-derived factor-1 (SDF-1)/chemokine C-X-C receptor 4 (CXCR4) axis plays a critical role in homing, engraftment and retention of hematopoietic stem/progenitor cells. We previously demonstrated that expression of CD9 is a downstream signal of the SDF-1/CXCR4 axis, and that CD9 regulates short-term (20 hours) homing of cord blood (CB) CD34+ cells in the NOD/SCID mouse xenotransplantation model (Leung et al, Blood, 2011). Here, we provided further evidence that pretreatment of CB CD34+ cells with a CD9-neutralizing antibody significantly reduced their long-term (6 weeks) engraftment, as indicated by the presence of human CD45+ cells, in the recipient bone marrow and spleen by 70.9% (P = .0089) and 87.8% (P = .0179), respectively (n = 6). However, CD9 blockade did not bias specific lineage commitment, including the CD14+ monocytic, CD33+ myeloid, CD19+ B-lymphoid and CD34+ stem/progenitor cells (n = 4). We also observed an increase of the CD34+CD9+ subsets in the bone marrow (9.6-fold; P < .0001) and spleens (9.8-fold; P = .0014) of engrafted animals (n = 3-4). These data indicate that CD9 possesses important functions in regulating stem cell engraftment and its expression level on CD34+ cells is up-regulated in the target hematopoietic organs. Analysis of paired bone marrow (BM) and peripheral blood (PB) samples from healthy donors revealed a higher CD9 expression in BM-resident CD34+ cells (57.3% ± 8.1% CD9+ cells in BM vs. 29.3% ± 5.8% in PB; n = 5, P = 0.0478). Consistently, CD34+ cells in granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood (MPB) expressed lower levels of CD9 (33.8% ± 3.0% CD9+ cells, n = 24), when compared with those in BM (56.4% ± 4.9% CD9+ cells, n = 8, P = 0.0025). In vitro exposure of MPB CD34+ cells to SDF-1 significantly enhanced CD9 expression (1.55-fold increase, n = 4, P = 0.0103), concomitant with a 75.2% reduction in the CD34+CXCR4+ subsets (P = 0.0118). Treatment of NOD/SCID chimeric mice with G-CSF increased the frequency of circulating CD45+ cells (3.4-fold) and CD34+ cells (3.3-fold), and substantially decreased the CD34+CD9+ subsets in the BM from 75.8% to 30.8%. Importantly, the decline in CD9 levels during G-CSF mobilization was also observed in the CD34+CD38-/low primitive stem cell subpopulation. Interestingly, in vitro treatment of BM CD34+ cells with G-CSF did not affect CD9 expression (n = 3), suggesting that a signaling intermediate is required for G-CSF-mediated CD9 down-regulation in vivo. Transwell migration assay revealed a significant enrichment of CD9- cells that were migrated towards a SDF-1 gradient (n = 4 for BM CD34+ cells, P = 0.0074; n = 7 for CB CD34+ cells, P = 0.0258), implicating that CD9 might negatively regulate stem cell motility. In contrast, pretreatment with the CD9-neutralizing antibody inhibited adhesion of CD34+ cells to the osteoblastic cell line Saos-2 by 33.5% (n = 2). Our results collectively suggest a previously unrecognized role of CD9 in stem cell retention by dual regulation of cell motility and adhesion, and reveal a dynamic regulation of CD9 expression in the BM microenvironment, which might represent an important event in controlling stem cell homing and mobilization. Disclosures:No relevant conflicts of interest to declare.

Recruitment of exogenous mesenchymal stem cells in mandibular distraction osteogenesis by the stromal cell-derived factor-1/chemokine receptor-4 pathway in rats

Distraction osteogenesis is widely used in orthopaedic and craniofacial surgery. However, its exact mechanism is still poorly understood. The purpose of this study was to find out whether there is systemic recruitment of mesenchymal stem cells (MSC) to the neocallus in the distraction gap by the stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) axis during osteogenesis. We examined the migration of MSC towards a gradient of SDF-1 in vitro. We also transplanted MSC labelled with green fluorescent protein (GFP) intravenously, with or without treatment with CXCR4-blocking antibody, into rats that had had unilateral mandibular distraction osteogenesis, and investigated the distribution of cells labelled with GFP in the soft callus after 24h. We found that SDF-1 facilitated the migration potency of MSC both in vitro and in vivo, and this migration could be inhibited by AMD3100, an antagonist of CXCR4, and promoted by local infusion of exogenous SDF-1 into the distraction gap. This study provides a new insight into the molecular basis of how new bone is regenerated during distraction osteogenesis.

How the human spermatozoa sense the oocyte: a new role of SDF1-CXCR4 signalling

For fertilization to occur in mammals, ejaculated spermatozoa must reach the egg which, following ovulation has moved from the ovary into the Fallopian tube. Two active mechanisms of spermatozoa guidance have been shown in mammals: thermotaxis and chemotaxis. The identity of most of human spermatozoa chemoattractants is unknown, and herein we tested if SDF1 (chemokine stromal cell-derived factor-1) and its pathway is involved in spermatozoa chemotaxis. We found that SDF1 is expressed in the oocytes, endometrium and follicular fluid, as well as its specific receptor CXCR4 (chemokine CXC motif receptor 4) is expressed in the head of spermatozoa. By SDF1 gradient experiments, we stated that SDF1 is able to induce hyperactivation in spermatozoa leading to accumulation, to give rise to an increase in intracellular calcium concentration, and to preserve the mitochondrial status and not to induce acrosome reaction. Our findings suggest these phenomena could reflect spermatozoa chemotaxis, and that SDF1 action could represent an important event leading to egg fertilization, even if further studies regarding the link between spermatozoa accumulation and chemotaxis are mandatory. These data suggest that the SDF-1/CXCR4 signalling could be used to manipulate the human fertilization, to improve both the outcome of physiological or assisted reproduction, and to develop new contraceptive methods, by development of SDF1 or CXCR4 antagonist.

Stromal cell-derived factor-1 (SDF-1): homing factor for engineered regenerative medicine

Introduction: Stromal cell-derived factor-1α (SDF-1) is a chemokine that plays a major role in cell trafficking and homing of CD34+ stem cells. Studies employing SDF-1/CXCR4 have demonstrated its therapeutic potential in tissue engineering. During injury, cells from the injured organ highly express SDF-1, which causes an elevation of localized SDF-1 levels. This leads to recruitment and retention of circulating CD34+ progenitor cells at the injury site via chemotactic attraction toward a gradient of SDF-1. The general approaches for SDF-1 introduction in tissue engineering are direct protein incorporation into scaffolds and transplantation of SDF-1-overexpressing cells and both methods are successful in improving the regeneration of the damaged tissue/organ.Areas covered: The mechanisms of SDF-1-mediated homing via CXCR4 receptor and the success of SDF-1-based medical applications in mesenchymal stem cell (MSC) homing as well as areas such as therapeutic angiogenesis, wound healing and neuronal and liver regeneration.Expert opinion: Current SDF-1 delivery designs and platforms hold much room for improvement. Regardless of the different techniques of SDF-1 introduction, they have proved to be effective in recruitment of various stem/progenitor cells. The pursuit of SDF-1-related regenerative medicine has already begun. It is thus conceivable that its usage in the clinical setting will be a reality in the near future.

E0203 Atherosclerosis impairs CXCR4 expression and function in bone marrow cells

ObjectivesThe chemokine stromal cell-derived factor-1 (SDF-1) plays a critical role in mobilising CXCR4-positive precursor cells in the bone marrow and may be essential for efficient vascular regeneration and repair. We...

Hypothesis: Human Umbilical Cord Blood-derived Stromal Cells Promote Megakaryocytopoiesis Through the Influence of SDF-1 and PECAM-1

The development and maturation of megakaryocytes (MKs) is a complex and multistage cellular and biological process. The hematopoietic microenvironment plays an important role in megakaryocytopoiesis regulation. Stromal cells, an important ingredient in the hematopoietic microenvironment, may regulate the development of MKs via the adhesion with MKs and via augmentation of cytokine secretion. Our laboratory has previously isolated a novel population of stromal cells from human umbilical cord blood, called hUCBDSCs. Compared with hBMSCs, the hUCBDSCs express higher levels of stromal cell-derived factor-1 (SDF-1) and increase the colony-forming-unit-megakaryocytes (CFU-MK). Meanwhile, there are reports identified a migration defect in PECAM-1-deficient MKs in response to a gradient of SDF-1. Based on literature searches and our experimental findings, we present a hypothesis that hUCBDSCs, secreting high level of SDF-1, modulated the expression of PECAM-1 of MKs, to regulate the megakaryocyte development.

MT1-MMP association with membrane lipid rafts facilitates G-CSF−induced hematopoietic stem/progenitor cell mobilization

Soluble matrix metalloproteinases (MMPs) facilitate the egress of hematopoietic stem/progenitor cells (HSPC) from the bone marrow (BM) during granulocyte colony-stimulating factor (G-CSF)-induced mobilization. Because membrane-type (MT)1-MMP, which is localized on the leading edge of migrating cells, activates the latent forms of soluble MMPs, we investigated its role in HSPC mobilization. We examined the effect of G-CSF on the expression of MT1-MMP and its activities (proMMP-2 activation and migration) in hematopoietic cells. We also investigated the subcellular localization of MT1-MMP and the signaling pathways that regulate its expression and function in hematopoietic cells after exposure to G-CSF. We found that G-CSF increases MT1-MMP transcription and protein synthesis in hematopoietic cells; proMMP-2 activation in cocultures of HSPC with BM fibroblasts; chemoinvasion across reconstituted basement membrane Matrigel toward a stromal cell-derived factor-1 gradient, which is reduced by small interfering RNA silencing of MT1-MMP; and localization of MT1-MMP to membrane lipid rafts through a mechanism that is regulated by the phosphatidylinositol 3-kinase signaling pathway. Disruption of raft formation (by the cholesterol-sequestering agent methyl-beta-cyclodextrin) abrogated phosphatidylinositol 3-kinase phosphorylation and MT1-MMP incorporation into lipid rafts resulting in reduced proMMP-2 activation and HSPC migration. G-CSF-induced upregulation of MT1-MMP in hematopoietic cells and its enhanced incorporation into membrane lipid rafts contributes to proMMP-2 activation, which facilitates mobilization of HSPC.

Hypercholesterolemia promotes bone marrow cell mobilization by perturbing the SDF-1:CXCR4 axis

AbstractHypercholesterolemia is associated with elevated peripheral blood leukocytes and increased platelet levels, generally attributed to cholesterol-induced proinflammatory cytokines. Bone marrow (BM) cell mobilization and platelet production is achieved by disrupting the SDF-1:CXCR4 axis, namely with granulocyte colony-stimulating factor and/or CXCR4 antagonists. Here we show that high cholesterol disrupts the BM SDF-1:CXCR4 axis; promotes the mobilization of B cells, neutrophils, and progenitor cells (HPCs); and creates thrombocytosis. Hypercholesterolemia was achieved after a 30-day high-cholesterol feeding trial, resulting in elevated low-density lipoprotein (LDL) cholesterol levels and inversion of the LDL to high-density lipoprotein cholesterol ratio. Hypercholesterolemic mice displayed lymphocytosis, increased neutrophils, HPCs, and thrombocytosis with a lineage-specific decrease in the BM. Histologic analysis revealed that megakaryocyte numbers remained unaltered but, in high-cholesterol mice, they formed large clusters in contact with BM vessels. In vitro, LDL induced stromal cell–derived factor-1 (SDF-1) production, suggesting that megakaryocyte delocalization resulted from an altered SDF-1 gradient. LDL also stimulated B cells and HPC migration toward SDF-1, which was blocked by scavenger receptor class B type I (cholesterol receptor) inhibition. Accordingly, hypercholesterolemic mice had increased peripheral blood SDF-1 levels, increased platelets, CXCR4-positive B lymphocytes, neutrophils, and HPCs. High cholesterol interferes with the BM SDF-1:CXCR4 axis, resulting in lymphocytosis, thrombocytosis, and HPC mobilization.

Fifth complement cascade protein (C5) cleavage fragments disrupt the SDF-1/CXCR4 axis: Further evidence that innate immunity orchestrates the mobilization of hematopoietic stem/progenitor cells

Having previously demonstrated that the complement system modulates mobilization of hematopoietic stem/progenitor cells (HSPC) in mice, we investigated the involvement of C5 cleavage fragments (C5a/(desArg)C5a) in human HSPC mobilization. C5 cleavage fragments in the plasma were evaluated by enzyme-linked immunosorbent assay using human anti-(desArg)C5a antibody, and expression of the C5a/(desArg)C5a receptor (CD88) in hematopoietic cells by flow cytometry. We also examined the chemotactic responses of hematopoietic cells to C5 cleavage fragments and expression of stromal cell-derived factor-1 (SDF-1)-degrading proteases that perturb retention of HSPC in bone marrow, namely matrix metalloproteinase (MMP)-9, membrane type (MT) 1-MMP, and carboxypeptidase M. We found that plasma levels of (desArg)C5a are significantly higher in patients who are good mobilizers and correlate with CD34(+) cell and white blood cell counts in mobilized peripheral blood. C5 cleavage fragments did not chemoattract myeloid progenitors (colony-forming unit granulocyte-macrophage), but (desArg)C5a did strongly chemoattract mature nucleated cells. Consistently, CD88 was not detected on CD34(+) cells, but appeared on more mature myeloid precursors, monocytes, and granulocytes. Moreover, granulocyte colony-stimulating factor-mobilized peripheral blood mononuclear cells and polymorphonuclear cells had a significantly higher percentage of cells expressing CD88 than nonmobilized peripheral blood. Furthermore, C5a stimulation of granulocytes and monocytes decreased CXCR4 expression and chemotaxis toward an SDF-1 gradient and increased secretion of MMP-9 and expression of MT1-MMP and carboxypeptidase M. C5 cleavage fragments not only induce a highly proteolytic microenvironment in human bone marrow, which perturbs retention through the CXCR4/SDF-1 axis, but also strongly chemoattracts granulocytes, promoting their egress into mobilized peripheral blood, which is crucial for subsequent mobilization of HSPC.

SDF-1 Is Elevated in Trauma Patients at the Site of Injury

Introduction: Bone marrow derived cells (BMDC) migrate to the site of injury following trauma, which animal models suggest plays a crucial role in the healing of injured tissue. Stromal cell-derived factor-1 (SDF-1) is a chemokine which is mutually exclusive with the CXCR4 receptor found on BMDC. Multiple studies have shown the involvement of an SDF-1 gradient and the SDF-1/CXCR4 axis in homing of BMDC to injured tissue. To our knowledge, this is the first study showing elevation of SDF-1 in areas of injury in trauma patients and the presence of an SDF-1 gradient from the blood towards those areas. Methods: Peripheral blood (PB) was collected from trauma patients and volunteers. Chest tube (CT) fluid samples were collected from trauma patients with chest injuries requiring tube thoracostomy. After centrifugation, the supernatant were stored at -20 °C until assayed by ELISA for SDF-1. ELISA results were reported as nanogram of SDF-1 per milligram of total protein. Samples were categorized by initial base deficit (BD), injury severity score (ISS), age, gender, time from injury, survival, and mechanism of injury. The Mann-Whitney Test was used to determine significance. Results: Trauma patients were found to have significantly elevated SDF-1 levels in their PB (n=16) as compared to normal volunteers (n=21) (4.1 vs 2.4, p=0.0014). Furthermore, SDF-1 levels were significantly higher in trauma patient CT fluid (n=30) than in trauma patient PB (9.8 vs 4.1, p=<0.001). Both initial BD and mortality were associated with higher SDF-1 levels in CT fluid (Table 1). Conclusion: As predicted by the animal models, SDF-1 was elevated in the PB of trauma patients, with even greater levels found at the area of injury, supporting the notion of an SDF-1 gradient mechanism to promote homing of BMDC to injured tissue. The degree of shock appears to correlate to SDF-1 levels, with more severe shock resulting in greater elevation of SDF-1 levels. This study suggests that SDF-1 plays a role in the homing of BMDC to sites of injury in trauma patients.

Marginal expression of CXCR4 on c-kit+Sca-1+Lineage− hematopoietic stem/progenitor cells

Stromal cell-derived factor 1 (SDF-1) and its receptor CXCR4 are the key regulatory molecules of hematopoietic stem cell (HSC) migration and engraftment to the bone marrow (BM) microenvironment. However, the significance of the ligand-receptor complex on HSC in steady-state BM is not clear. There is currently a lack of information as to how CXCR4 is expressed on HSCs. We herein demonstrate that c-kit(+)Sca-1(+)Lineage(-) (KSL) cells freshly isolated from BM expressed very low to undetectable levels of CXCR4. Two hours of incubation at 37 degrees C quickly up-modulated the receptor expression on KSL cells. Protein synthesis was not required for this early stage up-regulation, thus suggesting the emergence of intracellularly pooled receptors to the cell surface. However, protein synthesis was involved at the later stage of up-regulation. The up-regulated CXCR4 was functional, as evidenced by the fact that the incubated KSL cells more efficiently migrated to the SDF-1 gradient in vitro. Therefore, although KSL cells are able to express functional CXCR4, the receptors are only marginally expressed in the steady-state BM microenvironment. These observations therefore indicate the limited role of the SDF-1-CXCR4 axis on HSC functionality in a static BM environment.

G-CSF Enhanced SDF-1 Gradient Between Bone Marrow and Liver Associated with Mobilization of Peripheral Blood CD34+ Cells in Rats with Acute Liver Failure

The role of stromal cell-derived factor-1 (SDF-1) in modulating massive liver damage is not well known. In this study, expression of SDF-1 in bone marrow and liver was investigated in rats with acute liver failure (ALF) when mobilized using granulocyte colony-stimulating factor (G-CSF). ALF was induced in rats by D-galactosamine (D-GalN). Starting after 2 hours following D-GalN induction, the animals were injected with G-CSF 50 microg/kg daily or saline as placebo for 5 days. The percentages of CD34+ cells in peripheral blood and the expression of SDF-1 in bone marrow and liver were then determined. The percentages of peripheral CD34+ cells demonstrated a transient increase in placebo rats following D-GalN induction and a significant increase in rats after G-CSF administration. SDF-1 expression showed a transient decrease in bone marrow and a transient increase in liver tissue from placebo rats. However, a significant decrease of SDF-1 expression in bone marrow and a remarkable increase in liver tissue were observed in animals from the G-CSF group. It was concluded that G-CSF can enhance the reduced expression of SDF-1 in bone marrow and increased expression in liver in ALF rats, forming a greater SDF-1 gradient, and chemoattracting CD34+ cells' migration from bone marrow to an injured liver.

Direct contact with mesenchymal stromal cells affects migratory behavior and gene expression profile of CD133 + hematopoietic stem cells during ex vivo expansion

To investigate the impact of direct contact between mesenchymal stromal cells (MSCs) and CD133(+) hematopoietic stem cells in terms of expansion potential, differentiation, migratory capacity, and gene expression profile. CD133(+)-purified hematopoietic progenitor cells were cultured for 7 days on subconfluent MSCs supplemented with growth-factor-containing medium. After ex vivo expansion, nonadherent and adherent cells were collected and analyzed separately. The adherent cell population was less differentiated than the nonadherent fraction. CXCR4 was upregulated in the adherent fraction, which was associated with a higher migration capacity toward a stromal cell-derived factor-1 gradient. Colony-forming unit granulocyte-macrophage and long-term culture-initiation cell assays demonstrated a higher clonogenicity and repopulating capacity of the adherent fraction. Genes involved in adhesion, cell-cycle control, motility, and self-renewal were more highly expressed in the adherent fraction. Adhesion and direct cell-to-cell contact with an MSC feeder layer supports ex vivo expansion, migratory potential, and stemness of CD133(+) hematopoietic progenitor cells.

The role of CXCR4/SDF-1, CD117/SCF, and c-met/HGF chemokine signalling in the mobilization of progenitor cells and the parameters of the left ventricular function, remodelling, and myocardial perfusion following acute myocardial infarction

Chemokines and their receptors induce the qualitative and quantitative changes of the pool of bone marrow-derived and resident cardiac stem/progenitor cells, which in turn can modulate myocardial recovery after myocardial infarction (MI). Primary role of the chemokines, such as stromal cell-derived factor-1 (SDF-1), stem cell factor (SCF), hepatocyte growth factor (HGF) and leukaemia inhibitory factor (LIF) is the mobilization and homing of the bone marrow-derived stem/progenitor cells to the infarcted myocardium. In the setting of the acute MI there is an increased expression of SDF-1, HGF, LIF in the peri-infarct zone. The most important chemokine–chemokine receptor axis is SDF-1/CXCR4 which is significantly upregulated during acute MI. It corresponds with the significant mobilization of cells expressing cardiac and endothelial markers, which also express the chemokine receptors CXCR4, c-met, CD117 and leukemia inhibitory factor receptor. Specific population of small non-haematopoietic cells expressing the CXCR4 receptor was identified, which have the capability of cardiogenic differentiation. The mobilization of stem/progenitor cells expressing the receptors for chemoattractant cytokines is significantly correlated with the improvement of left ventricular ejection fraction (LVEF) and remodelling in 1-year follow-up after the acute MI. Patients with reduced LVEF after MI have also impaired mobilization of the stem/progenitor cells. The number of circulating cells is inversely correlated with the infarct area and positively with myocardial perfusion. Also the migratory response of the endothelial progenitor cells (EPCs) to the SDF-1 gradient is positively correlated with their capability to improve the perfusion and is one of the factors that seem to determine the outcome of the progenitor cell-based therapy after acute MI.

MRNA Transfection of CXCR4-GFP Fusion—Simply Generated by PCR—Results in Efficient Migration of Primary Human Mesenchymal Stem Cells

We present a general, entirely PCR-based strategy to construct mRNAs coding for green fluorescent protein (GFP) fusion proteins from a cDNA pool. We exemplify our approach for the chemokine receptor CXCR4. mRNA transfection of the PCR-generated fusion of CXCR4-GFP into K562 cells or primary mesenchymal stem cells (MSCs) resulted in excellent viability (> 90%) with more than 90% of target cells expressing easily detectable CXCR4-GFP for > 72 h. The fusion protein was localized in the plasma membrane and was rapidly internalized upon incubation with the CXCR4 ligand stromal cell-derived factor-1 (SDF-1). Transwell migration experiments showed significantly increased migration of CXCR4-GFP mRNA-transfected MSCs toward a gradient of SDF-1, demonstrating that mRNA-mediated chemokine receptor overexpression allows for transient initiation of chemotaxis. The presented strategy to construct a PCR-based fluorescent fusion protein can be generally applied to other genes of interest to study their function by simple overexpression and easy detection in primary cells.

New developments with umbilical cord blood

In this issue of Cytotherapy, three papers present, between them, the derivation of mesenchymal stromal cells (MSC) and colony-forming unit megakaryocytes (CFU-Meg) from human umbilical cord blood (UCB) cells and their immuno-equivalence to adult bone marrow (BM)-derived MSC. Briefly, Choi et al. [1] describe the derivation of viable MSC from human UCB and use both phenotypic/ enzymatic expression and reverse transcriptase-polymerase chain reaction (RT-PCR) to demonstrate their adipoand osteogenic lineages. They use atelocollagen matrix and type II collagen synthesis to demonstrate the chondrogenic potential of these UCB-derived MSC. In another paper, Shirvaikar et al. [2] describe the generation of CFU-Meg from UCB in the presence of thrombopoietin (TPO) and interleukin-3 (IL-3) and demonstrate synthesis of both matrix metalloproteinase-9 (MMP) and the membrane-type (MT) variety, MT1-MMP. They also demonstrate expression of CXCR4, the unique receptor for the chemokine stromal cell-derived factor-1 (SDF-1), a chemokine important for both progenitor cell homing and up-regulation of MMP-9 and MT1-MMP. Lastly, Ennis et al. [3] explore the immunogenicity of human UCB perivascular cells (HUCPVC) and demonstrate immuno-equivalence to BM-derived MSC using standard lymphoproliferative assays and T-cell CD25 and CD45 cell-surface receptor expression. What is novel about marrow vs. UCB-derived MSC and SDF-1? MSC found in human BM are multipotent progenitors of mesenchymal tissues and have novel regenerative and immune properties [4]. SDF-1 exerts its effect via its unique receptor, CXCR4. SDF-1 is a potent chemoattractant for all cells expressing CXCR4, including T cells and hematopoietic stem cells. Although low levels of CXCR4 expression are found in minor subpopulations of cultured MSC, the majority of marrow and UCBderived MSC lack CXCR4 and, hence, do not migrate toward an SDF-1 gradient. In addition, SDF-1 stimulates pre-B-lymphocyte cell growth, is important in hematopoetic stem cell survival, and increases survival of human CD34 cells [5]. SDF-1 is important in stem cell migration and CXCR4 mediates its effects. SDF-1-induced migration appears to be dependent on CXCR4 expression. SDF-1 activates adhesion molecules on progenitor cells, and monoclonal antibodies (MAb) against SDF-1 inhibit transendothelial migration of hematopoetic progenitor cells [6]. SDF-1 activates CXCR4 CD34 cells and leads to their adhesion and transendothelial migration. In rhabdomyosarcoma cells that are CXCR4 , SDF-1 increases MMP-2 and decreases tissue inhibitors of MMP (TIMP) secretion [7]. Hence, SDF-1 may facilitate, through its actions on CXCR4, stem cell migration to injured tissue and, through its actions on MMP-2 and TIMP, stem cell-mediated repair of injured tissues. Taken together, observations by Shirvaikar et al. on this issue of up-regulation of metalloproteinases by SDF-1 emphasizes the critical role SDF-1 plays in UCB-derived regenerative cellular medicine. We have demonstrated previously that transduction of human MSC with a retroviral vector expressing CXCR4 improved in vitro migration to SDF-1 [8]. However, given safety and ethical concerns over retroviral transduction of human progenitor cells to be administered clinically, identification of a UCB source of progenitor cells with naturally sufficient CXCR4 expression to facilitate SDF-1mediated homing remains an important discovery and has strong potential in cell therapy strategies, both from a safety as well as a feasibility standpoint.