Functional potentials of human hematopoietic progenitor cells are maintained by mesenchymal stromal cells and not impaired by plerixafor

Background aimsMesenchymal stromal cells (MSCs) resemble an essential component of the bone marrow niche for maintenance of stemness of hematopoietic progenitor cells (HPCs). Perturbation of the C-X-C chemokine receptor type 4 (CXCR4)/stromal cell-derived factor-1α (SDF-1α) axis by plerixafor (AMD3100) mobilizes HPCs from their niche; however, little is known about how plerixafor affects interaction of HPCs and MSCs in vitro.MethodsWe monitored cell division kinetics, surface expression of CD34 and CXCR4, migration behavior and colony-forming frequency of HPCs on co-culture with MSCs either with or without exposure to plerixafor.ResultsCo-culture with MSCs significantly accelerated cell division kinetics of HPCs. Despite this, the proportion of CD34⁺ cells was significantly increased on co-culture, whereas the expression of CXCR4 was reduced. In addition, co-culture with MSCs led to significantly higher colony-forming capacity and enhanced migration rate of HPCs compared with mono-culture conditions. The composition of MSC sub-populations—and conversely their hematopoiesis supportive functions—may be influenced by culture conditions. We compared the stromal function of MSCs isolated with three different culture media. Overall, the supporting potentials of these MSC preparations were quite similar. Perturbation by the CXCR4-antagonist plerixafor reduced the cell division kinetics of HPCs on co-culture with MSCs. However, the progenitor cell potential of the HPCs as reflected by colony-forming capacity was not affected by plerixafor.ConclusionsThese results support the notion that the CXCR4/SDF-1α axis is critical for HPC-MSC interaction with regard to migration, adhesion and regulation of proliferation but not for maintenance of primitive progenitor cells.     

TNF inhibits production of stromal cell-derived factor 1 by bone stromal cells and increases osteoclast precursor mobilization from bone marrow to peripheral blood

Introduction

The objective of the present study was to investigate the role of the stromal cell-derived factor 1 (SDF-1)/CXCR4 axis in TNF-induced mobilization of osteoclast precursors (OCPs) from bone marrow.

Methods

OCPs were generated from bone marrow cells of TNF-transgenic mice or wild-type mice treated with TNF or PBS. The percentage of CD11b⁺/Gr-1^-/lo OCPs was assessed by fluorescence-activated cell sorting. OCP migration to the SDF-1 gradient and the osteoclast forming potency were assessed in chemotaxis/osteoclastogenic assays. SDF-1 expression was assessed by real-time RT-PCR, ELISA and immunostaining in primary bone marrow stromal cells, in the ST2 bone marrow stromal cell line, and in bones from TNF-injected mice.

Results

OCPs generated in vitro from wild-type mice migrated to SDF-1 gradients and subsequently gave rise to osteoclasts in response to RANKL and macrophage colony-stimulating factor. TNF reduced SDF-1 expression by ST2 cells. Bone marrow stromal cells from TNF-transgenic mice produced low levels of SDF-1. TNF treatment of wild-type mice decreased the SDF-1 concentration in bone marrow extracts and decreased the SDF-1 immunostaining of bone marrow stromal cells, and it also increased the circulating OCP numbers. The percentage of bone marrow CXCR4⁺ OCPs was similar in TNF-transgenic mice and wild-type littermates and in TNF-treated and PBS-treated wild-type mice.

Conclusion

Systemically elevated TNF levels inhibit bone marrow stromal cell production of SDF-1 and increase the release of bone marrow OCPs to the peripheral blood. Disruption of the SDF-1/CXCR4 axis by TNF may play an important role in mediating OCP mobilization from the bone marrow cavity in chronic inflammatory arthritis.     

Influence of a dual-injection regimen,plerixafor and CXCR4 on in utero hematopoietic stem cell transplantation and engraftment with use of the sheep model

Background aimsInadequate engraftment of hematopoietic stem cells (HSCs) after in utero HSC transplantation (IUHSCT) remains a major obstacle for the prenatal correction of numerous hereditary disorders. HSCs express CXCR4 receptors that allow homing and engraftment in response to stromal-derived factor 1 (SDF-1) ligand present in the bone marrow stromal niche. Plerixafor, a mobilization drug, works through the interruption of the CXCR4-SDF-1 axis.MethodsWe used the fetal sheep large-animal model to test our hypotheses that (i) by administering plerixafor in utero before performing IUHSCT to release fetal HSCs and thus vacating recipient HSC niches, (ii) by using human mesenchymal stromal/stem cells (MSCs) to immunomodulate and humanize the fetal BM niches and (iii) by increasing the CXCR4⁺ fraction of CD34⁺ HSCs, we could improve engraftment. Human cord blood-derived CD34⁺ cells and human bone marrow-derived MSCs were used for these studies.ResultsWhen MSCs were transplanted 1 week before CD34⁺ cells with plerixafor treatment, we observed 2.80% donor hematopoietic engraftment. Combination of this regimen with additional CD34⁺ cells at the time of MSC infusion increased engraftment levels to 8.77%. Next, increasing the fraction of CXCR4⁺ cells in the CD34⁺ population albeit transplanting at a late gestation age was not beneficial. Our results show engraftment of both lymphoid and myeloid lineages.ConclusionsPrior MSC and HSC cotransplantation followed by manipulation of the CXCR4–SDF-1 axis in IUHSCT provides an innovative conceptual approach for conferring competitive advantage to donor HSCs. Our novel approach could provide a clinically relevant approach for enhancing engraftment early in the fetus.     

Rapid hematopoietic progenitor mobilization by sulfated colominic acid

Hematopoietic progenitor cells (HPCs) can be mobilized from bone marrow (BM) to the blood by G-CSF. In this process, CXCR4 and CD26 play critical roles. Sulfated colominic acid (SCA) inhibits HIV entry, the step which requires CXCR4 and CD26 as co-receptors. Thus, we hypothesized that SCA would modulate HPC trafficking. We first found that SCA mobilized HPCs rapidly via CD26-independent mechanism. In vitro progenitor migration toward chemokine SDF-1 was significantly enhanced by SCA, and it was completely abrogated by CXCR4 inhibition. This likely originated from the inhibition of CXCR4 down-regulation after interaction with SDF-1. Serum SDF-1 level increased after SCA injection, whereas no change was observed in BM and bone. These results suggest that SCA induces HPC mobilization by modulating CXCR4 function resulting in attraction toward increased SDF-1 in the circulation. Furthermore, we confirmed an additive effect with G-CSF in mobilization. SCA may provide an efficacy in clinical mobilization.     

SDF-1/CXCR4轴在缺氧缺血性脑损伤中的研究进展

干细胞在许多组织器官显示巨大的细胞分化潜能,其治疗缺血缺氧性疾病成为当前研究的热点。已知局部缺血可诱导干细胞的动员,并能感受组织损伤而定向迁移到损伤区并进行分化。具有趋化因子受体4（CXC chemokine receptor 4,CXCR4）的干细胞迁移到高表达间质细胞来源的因子-1（stromal cell-derived factor-1,SDF-1）的组织区域,这种细胞的迁移运动能被CXCR4拈抗剂所阻断或通过CXCR4的过表达增强迁移的运动。SDF-1-CXCR4轴是体内各种类型的干细胞迁移及细胞在骨髓的滞留和归巢中的重要调节物质。本文就缺氧缺血性脑损伤的骨髓间质干细胞（bone marrow stromal cell,BMSC）治疗,SDF- 1-CXCR4轴在MSCs动员和损伤、修复中的作用作一综述。     

Tanshinone IIA and astragaloside IV promote the migration of mesenchymal stem cells by up-regulation of CXCR4

Mesenchymal stem cells (MSCs) have a therapeutic potential to treat cardiovascular diseases. However, a significant barrier to MSC therapy is insufficient MSC engraftment in ischemic myocardium after systemic administration. Here, we investigated the modulatory effects of tanshinone IIA and astragaloside IV on the migration of MSCs and further defined the underlying mechanisms. CXCR4 expression in MSCs was determined by using flow cytometry, real-time PCR, and western blotting. The results showed that CXCR4 expression was significantly higher in tanshinone IIA- and astragaloside IV-stimulated MSCs than that of the control. MSC migration toward stromal cell-derived factor-1α (SDF-1α) was studied using a transwell system. MSCs treated with tanshinone IIA and astragaloside IV showed stronger migration than that of the control. Moreover, this enhanced migration ability was abrogated by a CXCR4 inhibitor. In a rat acute myocardial infarction model, MSCs stimulated with tanshinone IIA and astragaloside IV were stained with Dio and injected into model rats via the tail vein. Dio-labeled cells in myocardium sections were observed by fluorescence microscopy. Tanshinone IIA- and astragaloside IV-stimulated MSCs showed enhanced capacities to home to ischemic myocardium sites. In addition, there was no significant difference in the SDF-1α expression among groups. These data suggest that tanshinone IIA and astragaloside IV regulate MSC mobilization, at least partially via modulation of the CXCR4 expression.     

A possible role for CXCR4 and its ligand, the CXC chemokine stromal cell-derived factor-1, in the development of bone marrow metastases in neuroblastoma

The homing of hemopoietic stem cells to the bone marrow is mediated by specific interactions occurring between CXCR4, which is expressed on hemopoietic stem cells, and its ligand, stromal cell-derived factor-1 (SDF-1), a CXC chemokine secreted by bone marrow stromal cells. In the present study we evaluated the possibility that neuroblastoma cells use a mechanism similar to that used by hemopoietic stem cells to home to the bone marrow and adhere to bone marrow stromal cells. Our study suggests that CXCR4 expression may be a general characteristic of neuroblastoma cells. SH-SY5Y neuroblastoma cells express not only CXCR4, but also its ligand, SDF-1. CXCR4 expression on SH-SY5Y neuroblastoma cells is tightly regulated by tumor cell-derived SDF-1, as demonstrated by the ability of neutralizing Abs against human SDF-1alpha to up-regulate CXCR4 expression on the tumor cells. The reduction in CXCR4 expression following short term exposure to recombinant human SDF-1alpha can be recovered as a result of de novo receptor synthesis. Recombinant human SDF-1alpha induces the migration of CXCR4-expressing SH-SY5Y neuroblastoma cells in CXCR4- and heterotrimeric G protein-dependent manners. Furthermore, SH-SY5Y cells interact at multiple levels with bone marrow components, as evidenced by the fact that bone marrow-derived constituents promote SH-SY5Y cell migration, adhesion to bone marrow stromal cells, and proliferation. These results suggest that SH-SY5Y neuroblastoma cells are equipped with adequate machinery to support their homing to the bone marrow. Therefore, the ability of neuroblastoma tumors to preferentially form metastases in the bone marrow may be influenced by a set of complex CXCR4-SDF-1 interactions.     

Identification of hepatic niche harboring human acute lymphoblastic leukemic cells via the SDF-1/CXCR4 axis

In acute lymphoblastic leukemia (ALL) patients, the bone marrow niche is widely known to be an important element of treatment response and relapse. Furthermore, a characteristic liver pathology observed in ALL patients implies that the hepatic microenvironment provides an extramedullary niche for leukemic cells. However, it remains unclear whether the liver actually provides a specific niche. The mechanism underlying this pathology is also poorly understood. Here, to answer these questions, we reconstituted the histopathology of leukemic liver by using patients-derived primary ALL cells into NOD/SCID/Yc (null) mice. The liver pathology in this model was similar to that observed in the patients. By using this model, we clearly demonstrated that bile duct epithelial cells form a hepatic niche that supports infiltration and proliferation of ALL cells in the liver. Furthermore, we showed that functions of the niche are maintained by the SDF-1/CXCR4 axis, proposing a novel therapeutic approach targeting the extramedullary niche by inhibition of the SDF-1/CXCR4 axis. In conclusion, we demonstrated that the liver dissemination of leukemia is not due to nonselective infiltration, but rather systematic invasion and proliferation of leukemic cells in hepatic niche. Although the contribution of SDF-1/CXCR4 axis is reported in some cancer cells or leukemic niches such as bone marrow, we demonstrated that this axis works even in the extramedullary niche of leukemic cells. Our findings form the basis for therapeutic approaches that target the extramedullary niche by inhibiting the SDF-1/CXCR4 axis.     

Cyclic stretch upregulates SDF-1α/CXCR4 axis in human saphenous vein smooth muscle cells

Cyclic stretch (CS) mediates different cellular functions in vascular smooth muscle cells and involves in neointimal hyperplasia and subsequent atherosclerosis of vein grafts. Here, we investigated whether CS can modulate stromal cell-derived factor-1α (SDF-1α)/CXCR4 axis in human saphenous vein smooth muscle cells. We found CS induced the upregulation of SDF-1α and CXCR4 in human saphenous vein smooth muscle cells in vitro, which was dependent on PI3K/Akt/mTOR pathway. Furthermore, CS augmented human saphenous vein smooth muscle migration and focal adhesion kinase (FAK) activation by PI3K/Akt/mTOR pathway. Interestingly, the upregulation of SDF-1α/CXCR4 axis was instrumental in CS-induced saphenous vein smooth muscle cell migration and FAK activation, as showed by AMD3100, an inhibitor of SDF-1α/CXCR4 axis, partially but significantly blocked the CS-induced cellular effects. Thus, those data suggested SDF-1α/CXCR4 axis involves in CS-mediated cellular functions in human saphenous vein smooth muscle cells.     

Cutting Edge: Acute and chronic exposure of immature B cells to antigen leads to impaired homing and SHIP1-dependent reduction in stromal cell-derived factor-1 responsiveness

An encounter of B cells with cognate self Ags in the periphery can lead to anergy, a condition characterized by altered anatomical localization, shortened life span, and refractility to Ag stimulation. We recently reported that an immature B cell encounter with cognate self-Ag in the bone marrow can also lead to anergy. In this study we show that anergic as well as acutely Ag-stimulated immature B cells are defective in stromal cell-derived factor-1 (SDF-1)-induced calcium mobilization and migration and do not localize to bone marrow following adoptive transfer. This hyporesponsiveness does not involve CXCR4 modulation. However, BCR signal-mediated hyporesponsiveness to SDF-1 is associated with phosphorylation of the 5-inositol phosphatase SHIP1 and requires SHIP1 expression. Therefore, an encounter with cognate Ag may, by preventing SDF-1-induced phosphatidylinositol 3,4,5-triphosphate accumulation, trigger premature emigration of immature B cells from bone marrow.     

基质衍生因子(SDF-1)对骨髓间充质干细胞定向迁移的影响

目的:研究基质细胞衍生因子-1(SDF-1)/CXCR4轴在骨髓间充质干细胞迁徙到受损胰腺中的作用。方法:密度梯度离心、贴壁培养骨髓间充质干细胞,建立STZ诱导糖尿病模型并制备正常和受损胰腺组织提取液,利用Transwell小室体外迁移体系观察不同浓度SDF-1和不同组织提取液对骨髓间充质干细胞的趋化作用,及SDF-1/CXCR4特异抑制剂AMD3100对骨髓间充质干细胞迁移的影响。结果:成功培养了骨髓间充质干细胞并建立了糖尿病大鼠模型。SDF-l对骨髓间充质干细胞有剂量依赖性的趋化作用,造模1周的胰腺组织提取液对骨髓间充质干细胞有明显的趋化作用,而这种作用可部分被SDF-1受体CXCR4的抑制剂AMD3100抑制。结论:受损胰腺组织提取液对骨髓间充质干细胞有明显的趋化作用,SDF-1/CXCR4轴可能在组织提取液趋化骨髓间充质干细胞迁移中起主要的作用。     

Regulation of plasticity and biological features of endothelial progenitor cells by MSC-derived SDF-1

Bone marrow (BM) is a source of mesenchymal stromal cells (MSCs) and endothelial progenitor cells (EPCs). MSCs provide a specific niche in the BM and biological features of EPCs may be changed with this niche. Stromal cell-derived factor 1 (SDF-1) secreted from primary BM-MSCs and biological features of this niche on EPC development are still yet to be understood. The aim of this study was to evaluate the role of SDF-1 produced by MSCs on EPC development. We applied the CRISPR/Cas9 system for the knock-out of the SDF-1 gene in BM-derived MSCs. BM-derived EPCs were then cocultured with MSCs^SDF-1−/− or MSCs^SDF-1+/+ to identify the role of MSC-derived SDF-1α on proliferation, migration and angiogenic activity of EPCs. Next, pre-expanded EPCs were harvested and co-transplanted with MSCs^SDF-1−/− or MSCs^SDF-1+/+ into sublethally irradiated mice to analyze the potency of these cells for marrow reconstitution. Our results revealed that proliferation, colony formation, migration and angiogenic activity of EPCs was significantly increased after coculture with MSCs^SDF-1+/+. We also found that co-transplantation of EPCs with MSCs^SDF-1+/+, in contrast to MSCs^SDF-1−/−, into irradiated mice resulted in marrow repopulation and hematologic recovery, leading to improved survival of transplanted mice. In conclusions, MSC-derived SDF-1 niche plays an important role in the development of EPCs and this niche is essential for bone marrow repopulation by these cells and can enhance the efficiency of EPC therapy for ischemic diseases.     

Lithium down-regulates the expression of CXCR4 in human neutrophils

The CXC chemokine receptor CXCR4 and its unique ligand SDF-1 (stromal-derived factor-1) play critical roles for the retention of hematopoietic cells within the bone marrow (BM) and for their mobilization into the circulation. Lithium often produces neutrophilia in psychiatric patients, but the mechanism of mobilization related to neutrophilia has not been fully clarified. We showed here that lithium dose-dependently reduces the levels of surface CXCR4 protein and mRNA in neutrophils, but not in lymphocytes. The chemotactic migration of neutrophils in response to SDF-1 was reduced after a pre-incubation with lithium. We provide evidence that lithium down-regulates the CXCR4 expression of neutrophils and it attenuates their responsiveness to SDF-1. Our studies support the concept that down-regulation of CXCR4 is one of the mechanisms by which causes neutrophilia.     

Insulin-like growth factor 1 enhances the migratory capacity of mesenchymal stem cells

Mesenchymal stem cells (MSCs) are attractive candidates for cell based therapies. However, the mechanisms responsible for stem cell migration and homing after transplantation remain unknown. It has been shown that insulin-like growth factor-1 (IGF-1) induces proliferation and migration of some cell types, but its effects on stem cells have not been investigated. We isolated and cultured MSC from rat bone marrow, and found that IGF-1 increased the expression levels of the chemokine receptor CXCR4 (receptor for stromal cell-derived factor-1, SDF-1). Moreover, IGF-1 markedly increased the migratory response of MSC to SDF-1. The IGF-1-induced increase in MSC migration in response to SDF-1 was attenuated by PI3 kinase inhibitor (LY294002 and wortmannin) but not by mitogen-activated protein/ERK kinase inhibitor PD98059. Our data indicate that IGF-1 increases MSC migratory responses via CXCR4 chemokine receptor signaling which is PI3/Akt dependent. These findings provide a new paradigm for biological effects of IGF-1 on MSC and have implications for the development of novel stem cell therapeutic strategies.     

Migration of human umbilical cord blood mesenchymal stem cells mediated by stromal cell-derived factor-1/CXCR4 axis via Akt, ERK, and p38 signal transduction pathways

Human mesenchymal stem cells (hMSCs) have been used for cell-based therapies in degenerative disease and as vehicles for delivering therapeutic genes to sites of injury and tumors. Recently, umbilical cord blood (UCB) was identified as a source for MSCs, and human UCB-derived MSCs (hUCB-MSCs) can serve as an alternative source of bone marrow-derived mesenchymal stem cells (BM-MSCs). However, migration signaling pathways required for homing and recruitment of hUCB-MSCs are not fully understood. Stromal cell-derived factor-1 (SDF-1), a ligand for the CXCR4 chemokine receptor, plays a pivotal role in mobilization and homing of stem cells and modulates different biological responses in various stem cells. In this study, expression of CXCR4 in hUCB-MSCs was studied by western blot analysis and the functional role of SDF-1 was assessed. SDF-1 induced the migration of hUCB-MSCs in a dose-dependent manner. The induced migration was inhibited by the CXCR4-specific peptide antagonist (AMD3100) and by inhibitors of phosphoinositide 3-kinase (LY294002), mitogen-activated protein kinase/extracellular signal related kinase (PD98059) and p38MAPK inhibitor (SB203580). hUCB-MSCs treated with SDF-1 displayed increased phosphorylation of Akt, ERK and p38, which were inhibited by AMD3100. Small-interfering RNA-mediated knock-down of Akt, ERK and p38 blocked SDF-1 induced hUCB-MSC migration. In addition, SDF-1-induced actin polymerization was also blocked by these inhibitors. Taken together, these results demonstrate that Akt, ERK and p38 signal transduction pathways may be involved in SDF-1-mediated migration of hUCB-MSCs.     

The role of SDF-1-CXCR4/CXCR7 axis in biological behaviors of adipose tissue-derived mesenchymal stem cells in vitro

Numerous studies have reported that CXCR4 and CXCR7 play an essential, but differential role in stromal cell-derived factor-1 (SDF-1)-inducing cell chemotaxis, viability and paracrine actions of BMSCs. Adipose tissue-derived mesenchymal stem cells (ADSCs) have been suggested to be potential seed cells for clinical application instead of bone marrow derived stroma cell (BMSCs). However, the function of SDF-1/CXCR4 and SDF-1/CXCR7 in ADSCs is not well understood. This study was designed to analyze the effect of SDF-1/CXCR4 and SDF-1/CXCR7 axis on ADSCs biological behaviors in vitro. Using Flow cytometry and Western blot methods, we found for the first time that CXCR4/CXCR7 expression was increased after treatment with SDF-1 in ADSCs. SDF-1 promoted ADSCs paracrine, proliferation and migration abilities. CXCR4 or CXCR7 antibody suppressed ADSCs paracrine action induced by SDF-1. The migration of ADSCs can be abolished by CXCR4 antibody, while the proliferation of ADSCs was only downregulated by CXCR7 antibody. Our study indicated that the angiogenesis of ADSCs is, at least partly, mediated by SDF-1/CXCR4 and SDF-1/CXCR7 axis. However, only binding of SDF-1/CXCR7 was required for proliferation of ADSCs, and CXCR7 was required for migration of ADSCs induced by SDF-1. Our studies provide evidence that the activation of either axis may be helpful to improve the effectiveness of ADSCs-based stem cell therapy.     

Stimulation of chondrocyte hypertrophy by chemokine stromal cell-derived factor 1 in the chondro-osseous junction during endochondral bone formation

During endochondral bone formation, chondrocytes undergo differentiation toward hypertrophy before they are replaced by bone and bone marrow. In this study, we found that a G-protein coupled receptor CXCR4 is predominantly expressed in hypertrophic chondrocytes, while its ligand, chemokine stromal cell-derived factor 1 (SDF-1) is expressed in the bone marrow adjacent to hypertrophic chondrocytes. Thus, they are expressed in a complementary pattern in the chondro-osseous junction of the growth plate. Transfection of a CXCR4 cDNA into pre-hypertrophic chondrocytes results in a dose-dependent increase of hypertrophic markers including Runx2, Col X, and MMP-13 in response to SDF-1 treatment. In organ culture SDF-1 infiltrates cartilage and accelerates growth plate hypertrophy. Furthermore, a continuous infusion of SDF-1 into the rabbit proximal tibial physis results in early physeal closure, which is accompanied by a transient elevation of type X collagen expression. Blocking SDF-1/CXCR4 interaction suppresses the expression of Runx2. Thus, interaction of SDF-1 and CXCR4 is required for Runx2 expression. Interestingly, knocking down Runx2 gene expression results in a decrease of CXCR4 mRNA levels in hypertrophic chondrocytes. This suggests a positive feedback loop of stimulation of chondrocyte hypertrophy by SDF-1/CXCR4, which is mediated by Runx2.