The homing of human cord blood stem cells to sites of inflammation

Efficient homing of human umbilical cord blood mesenchymal stem cells (hUCBSC) to inflammation sites is crucial for therapeutic use. In glioblastoma multiforme, soluble factors released by the tumor facilitate the migratory capacity of mesenchymal stem cells toward glioma cells. These factors include chemokines and growth inducers. Nonetheless, the mechanistic details of these factors involved in hUCBSC homing have not been clearly delineated. The present study is aimed to deduce specific factors involved in hUCBSC homing by utilizing a glioma stem cell-induced inflammatory lesion model in the mouse brain. Our results show that hUCBSC do not form tumors in athymic nude mice brains and do not elicit immune responses in immunocompetent SKH1 mice. Further, hUCBSC spheroids migrate and invade glioma spheroids, while no effect was observed on rat fetal brain aggregates. Several cytokines, including GRO, MCP-1, IL-8, IL-3, IL-10, Osteopontin and TGF-β2, were constitutively secreted in the naive hUCBSC-conditioned medium, while significant increases of IL-8, GRO, GRO-α, MCP-1 and MCP-2 were observed in glioma stem cell-challenged hUCBSC culture filtrates. Furthermore, hUCBSC showed a stronger migration capacity toward glioma stem cells in vitro and exhibited enhanced migration to glioma stem cells in an intracranial human malignant glioma xenograft model. Our results indicate that multiple cytokines are involved in recruitment of hUCBSC toward glioma stem cells, and that hUCBSC are a potential candidate for glioma therapy.     

Overexpression of the Mesenchymal Stem Cell Cxcr4 Gene in Irradiated Mice Increases the Homing Capacity of These Cells

The efficiency of the intravascular delivery of mesenchymal stem cells (MSCs) homing to bone marrow has been largely limited. This study aimed to evaluate the homing efficacy in irradiated mice of MSCs that have been engineered to overexpress the murine Cxcr4 gene. Mouse MSCs were infected by a lentivirus vector carrying Cxcr4. MSC migration was detected by an in vitro transwell migration assay. EGFP-positive MSCs were systemically injected into BALB/c mice and detected in bone marrow samples by flow cytometry. The concentration of mouse stromal-derived factor 1 was detected by ELISA. The plasma concentration of the inflammatory cytokines, interleukin (IL)-6, IL-10, MCP-1, IFN-γ, TNF-α, and IL-12p70, were determined by cytometric bead array. MSCs that overexpressed Cxcr4 displayed enhanced migration toward a stromal-derived factor 1 gradient. The transplantation of Cxcr4-overexpressing MSCs into irradiated mice leads to increased homing to the bone marrow. Moreover, the frequency of the EGFP-positive cells in a bone marrow infusion 24 h after total body irradiation was 2.2-fold more than at 4 h after irradiation. The concentration of both plasma and bone marrow stromal-derived factor 1 increased after irradiation, and this was positively correlated with the number of Cxcr4-overexpressing MSCs homing to the bone marrow. Moreover, compared with the control groups, the plasma levels of IL-6, IFN-γ, TNF-α, and MCP-1 and IL-12p70 in recipients infused with Cxcr4-overexpressing MSCs was significantly decreased. The level of IL-10 was increased, which correlated with changes in the Th1 and Th2 subset balance. MSCs that overexpressed Cxcr4 and were injected into irradiated mice had an enhanced homing capacity which was related to the bone marrow level of stromal-derived factor 1.     

CXC chemokine receptor 1 enhances the ability of human umbilical cord blood-derived mesenchymal stem cells to migrate toward gliomas

In this study, we showed that knocking-down interleukin-8 (IL-8) in glioma cells, or its receptor, CXC chemokine receptor 1 (CXCR1) in hUCB-MSCs reduced hUCB-MSC migration toward glioma cells in a Transwell chamber. In contrast, CXCR1-transfected hUCB-MSCs (CXCR1-MSCs) showed a superior capacity to migrate toward glioma cells in a Transwell chamber compared to primary hUCB-MSCs. Furthermore, these transfected cells also demonstrated the same ability to migrate toward tumors in mice bearing intracranial human gliomas as shown by histological and in vivo imaging analysis. Our findings indicate that overexpression of CXCR1 could be a useful tool for MSC-based gene therapy to achieve a sufficient quantity of therapeutic MSCs that are localized within tumors.     

应用骨髓干细胞治疗肝硬化研究进展

随着干细胞研究的深入和技术的发展,再生医学的干细胞疗法治疗肝脏疾病已成为研究热点。骨髓来源造血干细胞和间充质干细胞等在肝脏疾病治疗方面有巨大潜力。骨髓干细胞参与肝纤维化与肝硬化修复主要包括迁移、归巢与转化等过程,并需要多种细胞因子和趋化因子的协同作用促进肝细胞再生与减轻肝纤维化。本文拟对骨髓干细胞治疗肝硬化的最新研究进展进行综述。     

Paracrine interleukin-8 affects mesenchymal stem cells through the Akt pathway and enhances human umbilical vein endothelial cell proliferation and migration

Interleukin-8 (IL-8) promotes cell homing and angiogenesis, but its effects on activating human bone marrow mesenchymal stem cells (BMSCs) and promoting angiogenesis are unclear. We used bioinformatics to predict these processes. In vitro, BMSCs were stimulated in a high-glucose (HG) environment with 50 or 100 μg/ml IL-8 was used as the IL-8 group. A total of 5 μmol/l Triciribine was added to the two IL-8 groups as the Akt inhibitor group. Cultured human umbilical vein endothelial cells (HUVECs) were cultured in BMSCs conditioned medium (CM). The changes in proliferation, apoptosis, migration ability and levels of VEGF and IL-6 in HUVECs were observed in each group. Seventy processes and 26 pathways were involved in vascular development, through which IL-8 affected BMSCs. Compared with the HG control group, HUVEC proliferation absorbance value (A value), Gap closure rate, and Transwell cell migration rate in the IL-8 50 and IL-8 100 CM groups were significantly increased (P<0.01, n=30). However, HUVEC apoptosis was significantly decreased (P<0.01, n=30). Akt and phospho-Akt (P-Akt) protein contents in lysates of BMSCs treated with IL-8, as well as VEGF and IL-6 protein contents in the supernatant of BMSCs treated with IL-8, were all highly expressed (P<0.01, n=15). These analyses confirmed that IL-8 promoted the expression of 41 core proteins in BMSCs through the PI3K Akt pathway, which could promote the proliferation and migration of vascular endothelial cells. Therefore, in an HG environment, IL-8 activated the Akt signaling pathway, promoted paracrine mechanisms of BMSCs, and improved the proliferation and migration of HUVECs.     

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.     

Transmigration of Neural Stem Cells across the Blood Brain Barrier Induced by Glioma Cells

Transit of human neural stem cells, ReNcell CX, through the blood brain barrier (BBB) was evaluated in an in vitro model of BBB and in nude mice. The BBB model was based on rat brain microvascular endothelial cells (RBMECs) cultured on Millicell inserts bathed from the basolateral side with conditioned media (CM) from astrocytes or glioma C6 cells. Glioma C6 CM induced a significant transendothelial migration of ReNcells CX in comparison to astrocyte CM. The presence in glioma C6 CM of high amounts of HGF, VEGF, zonulin and PGE₂, together with the low abundance of EGF, promoted ReNcells CX transmigration. In contrast cytokines IFN-α, TNF-α, IL-12p70, IL-1β, IL-6, IL-8 and IL-10, as well as metalloproteinases -2 and -9 were present in equal amounts in glioma C6 and astrocyte CMs. ReNcells expressed the tight junction proteins occludin and claudins 1, 3 and 4, and the cell adhesion molecule CRTAM, while RBMECs expressed occludin, claudins 1 and 5 and CRTAM. Competing CRTAM mediated adhesion with soluble CRTAM, inhibited ReNcells CX transmigration, and at the sites of transmigration, the expression of occludin and claudin-5 diminished in RBMECs. In nude mice we found that ReNcells CX injected into systemic circulation passed the BBB and reached intracranial gliomas, which overexpressed HGF, VEGF and zonulin/prehaptoglobin 2.     

间充质干细胞影响肿瘤细胞增殖及其分子机理

间充质干细胞MSCs(mesenchymal stem cells)与肿瘤细胞间的相互作用是近年来肿瘤领域的研究热点之一.MSCs是一种多能干细胞,具有分化为成骨细胞、软骨细胞、脂肪细胞、纤维母细胞或肌肉细胞等多种间充质细胞的能力.MSCs在肿瘤细胞中表现出的归巢和转移能力为其成为潜在的抗肿瘤工具奠定了基础,MSCs转移到肿瘤细胞后参与重塑肿瘤微环境,并对其增殖、侵袭和转移等生物学行为产生重要影响.MSCs重塑肿瘤微环境后对肿瘤细胞的增殖究竟是促进还是抑制,相关文献报道有很大的争议.基于相关研究近况,主要综述骨髓间充质干细胞BMSCs(bone marrow derived mesenchymal stem cells)参与重塑肿瘤微环境对肿瘤细胞增殖的影响,并就已知的分子机理做一简要介绍.     

Interleukin-1 beta and tumor necrosis factor alpha inhibit migration activity of chondrogenic progenitor cells from non-fibrillated osteoarthritic cartilage

Introduction

The repair capability of traumatized articular cartilage is highly limited so that joint injuries often lead to osteoarthritis. Migratory chondrogenic progenitor cells (CPC) might represent a target cell population for in situ regeneration. This study aims to clarify, whether 1) CPC are present in regions of macroscopically intact cartilage from human osteoarthritic joints, 2) CPC migration is stimulated by single growth factors and the cocktail of factors released from traumatized cartilage and 3) CPC migration is influenced by cytokines present in traumatized joints.

Methods

We characterized the cells growing out from macroscopically intact human osteoarthritic cartilage using a panel of positive and negative surface markers and analyzed their differentiation capacity. The migratory response to platelet-derived growth factor (PDGF)-BB, insulin-like growth factor 1 (IGF-1), supernatants obtained from in vitro traumatized cartilage and interleukin-1 beta (IL-1β) as well as tumor necrosis factor alpha (TNF-α) were tested with a modified Boyden chamber assay. The influence of IL-1β and TNF-α was additionally examined by scratch assays and outgrowth experiments.

Results

A comparison of 25 quadruplicate marker combinations in CPC and bone-marrow derived mesenchymal stromal cells showed a similar expression profile. CPC cultures had the potential for adipogenic, osteogenic and chondrogenic differentiation. PDGF-BB and IGF-1, such as the supernatant from traumatized cartilage, induced a significant site-directed migratory response. IL-1β and TNF-α significantly reduced basal cell migration and abrogated the stimulative effect of the growth factors and the trauma supernatant. Both cytokines also inhibited cell migration in the scratch assay and primary outgrowth of CPC from cartilage tissue. In contrast, the cytokine IL-6, which is present in trauma supernatant, did not affect growth factor induced migration of CPC.

Conclusion

These results indicate that traumatized cartilage releases chemoattractive factors for CPC but IL-1β and TNF-α inhibit their migratory activity which might contribute to the low regenerative potential of cartilage in vivo.     

Enhanced Healing of Rat Calvarial Bone Defects with Hypoxic Conditioned Medium from Mesenchymal Stem Cells through Increased Endogenous Stem Cell Migration via Regulation of ICAM-1 Targeted-microRNA-221

The use of conditioned medium from mesenchymal stem cells may be a feasible approach for regeneration of bone defects through secretion of various components of mesenchymal stem cells such as cytokines, chemokines, and growth factors. Mesenchymal stem cells secrete and accumulate multiple factors in conditioned medium under specific physiological conditions. In this study, we investigated whether the conditioned medium collected under hypoxic condition could effectively influence bone regeneration through enhanced migration and adhesion of endogenous mesenchymal stem cells. Cell migration and adhesion abilities were increased through overexpression of intercellular adhesion molecule-1 in hypoxic conditioned medium treated group. Intercellular adhesion molecule-1 was upregulated by microRNA-221 in mesenchymal stem cells because microRNAs are key regulators of various biological functions via gene expression. To investigate the effects in vivo, evaluation of bone regeneration by computed tomography and histological assays revealed that osteogenesis was enhanced in the hypoxic conditioned medium group relative to the other groups. These results suggest that behavioral changes of endogenous mesenchymal stem cells through microRNA-221 targeted-intercellular adhesion molecule-1 expression under hypoxic conditions may be a potential treatment for patients with bone defects.     

Cord Blood Stem Cell-Mediated Induction of Apoptosis in Glioma Downregulates X-Linked Inhibitor of Apoptosis Protein (XIAP)

Background

XIAP (X-linked inhibitor of apoptosis protein) is one of the most important members of the apoptosis inhibitor family. XIAP is upregulated in various malignancies, including human glioblastoma. It promotes invasion, metastasis, growth and survival of malignant cells. We hypothesized that downregulation of XIAP by human umbilical cord blood mesenchymal stem cells (hUCBSC) in glioma cells would cause them to undergo apoptotic death.

Methodology/Principal Findings

We observed the effect of hUCBSC on two malignant glioma cell lines (SNB19 and U251) and two glioma xenograft cell lines (4910 and 5310). In co-cultures of glioma cells with hUCBSC, proliferation of glioma cells was significantly inhibited. This is associated with increased cytotoxicity of glioma cells, which led to glioma cell death. Stem cells induced apoptosis in glioma cells, which was evaluated by TUNEL assay, FACS analyses and immunoblotting. The induction of apoptosis is associated with inhibition of XIAP in co-cultures of hUCBSC. Similar results were obtained by the treatment of glioma cells with shRNA to downregulate XIAP (siXIAP). Downregulation of XIAP resulted in activation of caspase-3 and caspase-9 to trigger apoptosis in glioma cells. Apoptosis is characterized by the loss of mitochondrial membrane potential and upregulation of mitochondrial apoptotic proteins Bax and Bad. Cell death of glioma cells was marked by downregulation of Akt and phospho-Akt molecules. We observed similar results under in vivo conditions in U251- and 5310-injected nude mice brains, which were treated with hUCBSC. Under in vivo conditions, Smac/DIABLO was found to be colocalized in the nucleus, showing that hUCBSC induced apoptosis is mediated by inhibition of XIAP and activation of Smac/DIABLO.

Conclusions/Significance

Our results indicate that downregulation of XIAP by hUCBSC treatment induces apoptosis, which led to the death of the glioma cells and xenograft cells. This study demonstrates the therapeutic potential of XIAP and hUCBSC to treat malignant gliomas.     

壳聚糖薄膜成球培养对脐带间充质干细胞迁徙趋化的影响

基于细胞实验研究壳聚糖（chitosan,CS）薄膜成球培养技术对间充质干细胞(mesenchymal stem cells, MSCs)迁徙趋化特性的影响。从脐带组织中分离原代MSCs采取CS成球法培养,以常规贴壁培养MSCs作为对照,72 h后收集两组细胞分别进行划痕实验、Tranthwell迁徙实验观察并拍照记录,RT-PCR方法检测两种培养方式中MSCs迁徙相关基因表达水平的差异。研究结果显示,相较常规贴壁培养方式,CS培养组MSCs体外迁徙趋化能力增强,差异具有显著统计学意义（P<0.01）;CS成球培养组MSCs 中CXCR4、CXCR7、MCP-1、MMP-1、MMP-2、MMP-9、TIMP-2等迁徙相关基因表达均明显上调（P<0.01）。实验表明CS成球培养可显著促进MSCs的迁移趋化特性。     

Cytokine interactions in mesenchymal stem cells from cord blood

We used cytokine protein array to analyze the expression of cytokines from human cord blood-derived mesenchymal stem cells (CB-MSCs). Several cytokines, interleukins (IL), and growth factors, including ENA-78, GM-CSF, GRO, IL-1β, IL-6, IL-8, MCP-1, OSM, VEGF, FGF-4, FGF-7, FGF-9, GCP-2, IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IP-10, LIF, MIF, MIP-3α, osteoprotegerin, PARC, PIGF, TGF-β2, TGF-β3, TIMP-1, as well as TIMP-2, were secreted by CB-MSCs, while IL-4, IL-5, IL-7, IL-13, TGF-β1, TNF-α, and TNF-β were not expressed under normal growth conditions. IL-6, IL-8, TIMP-1, and TIMP-2 were the most abundant interleukins expressed by CB-MSCs. A set of growth factors were selected to evaluate their stimulatory effects on the IL6 secretion for CB-MSCs. IL-1β was the most important factor inducing CB-MSC to secret IL-6. The mechanism by which IL-1β promoted IL-6 expression in CB-MSCs was studied. By using various inhibitors of signal transduction, we found that activation of p38 mitogen-activated protein kinases (MAPK) and MAPK kinase (MEK) is essential in the IL-1β stimulated signaling cascade which leads to the increase in IL-6 synthesis. Additionally, continuous supplement of IL-1β in the CB-MSCs culture will facilitate adipogenic maturation of CB-MSCs as evidenced by the presence of oil drops in the CB-MSCs and secretion of leptin, a molecule marker of adipocytes. These results strongly suggest that cytokine induction and signal transduction are important for the differentiation of CB-MSCs.     

Macrophage contributes to radiation-induced anti-tumor abscopal effect on transplanted breast cancer by HMGB1/TNF-α signaling factors

Objectives: The roles of innate immunity including macrophages in radiation-induced abscopal effect (RIAE) are ambiguous. In this study, we evaluated the role of macrophage in RIAE and the interaction of cytokines in tumor microenvironment after irradiation.Materials and Methods: Transplanted tumor of breast cancer cells in BalB/C mice, severe combined immunodeficiency (SCID) mice and non-obese diabetic (NOD)-SCID mice were irradiated with fractionation doses to observe anti-tumor abscopal effect. The underlying mechanism of RIAE was investigated by treating the mice with TNF-α inhibitor or macrophage depletion drug and analyzing the alteration of macrophage distribution in tumors. A co-culture system of breast cancer cells and macrophages was applied to disclose the signaling factors and related pathways involved in the RIAE.Results: The growth of nonirradiated tumor was effectively suppressed in mice with normal or infused macrophages but not in mice with insufficiency/depletion of macrophage or TNF-α inhibition, where M1-macrophage was mainly involved. Investigation of the bystander signaling factors in vitro demonstrated that HMGB1 released from irradiated breast cancer cells promoted bystander macrophages to secret TNF-α through TLR-4 pathway and further inhibited the proliferation and migration of non-irradiated cancer cells by PI3K-p110γ suppression.Conclusions: HMGB1 and TNF-α contributes to M1-macrophages facilitated systemic anti-tumor abscopal response triggered by radiotherapy in breast cancer, indicating that the combination of immunotherapy and radiotherapy may has important implication in enhancing the efficiency of tumor treatment.     

Lysophospholipids increase IL-8 and MCP-1 expressions in human umbilical cord vein endothelial cells through an IL-1-dependent mechanism

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are both low-molecular-weight lysophospholipid (LPL) ligands which are recognized by the Edg family of G protein-coupled receptors (GPCRs). In endothelial cells, these two ligands activate Edg receptors resulting in cell proliferation and cell migration. Interleukin-8 (IL-8) is a C-X-C chemokine and acts as a chemoattractant of neutrophils, whereas monocyte chemoattractant protein-1 (MCP-1) is a C-C chemokine and functions mainly as a chemoattractant of monocytes/macrophages. Both factors are secreted from endothelial cells and have been implicated in the processes leading to atherosclerosis. We examined the effects of LPLs on the expression of IL-8 and MCP-1, key regulators of leukocyte recruitment in human umbilical cord vein endothelial cells (HUVECs). Work illustrated in this article showed that LPA and S1P enhanced IL-8 and MCP-1 mRNA expressions, and protein secretions in dose- and time-dependent fashions. Maximal mRNA expression appeared at 16 hr post-ligand treatment. Using prior treatments with chemical inhibitors, LPLs enhanced IL-8 and MCP-1 expressions through a Gi-, Rho-, and NFkappaB-dependent mechanism. In a chemotaxis assay system, LPL treatments of endothelial cells enhanced monocyte recruitment through upregulating IL-8 and MCP-1 protein secretions. Pre-incubation with AF12198, an IL-1 receptor antagonist or IL-1 functional blocking antibody both suppressed the enhanced effects elicited by LPLs of IL-8 and MCP-1 mRNA expressions in HUVECs. These results suggest that LPLs released by activated platelets might enhance the IL-8- and MCP-1-dependent chemoattraction of monocytes toward the endothelium through an IL-1-dependent mechanism, which may play an important role in facilitating wound-healing and inflammation processes.     

Alpinate Oxyphyllae extracts enhance the longevity and homing of mesenchymal stem cells and augment their protection against senescence in H9c2 cells

Adipose-derived mesenchymal stem cells (ADMSCs) are easily accessible and are attractive mesenchymal stem cells for use in regenerative medicine; however their application is frequently restricted due to various challenges present in the environment they are administered. Therefore ADMSCs are preferably preconditioned with various stimulating factors to overcome the barriers developed in any pathological conditions. Here we used ADMSCs from rat adipose based on the abundance of positive markers and preconditioned the cells with extracts from Alpinate Oxyphyllae Fructus (AOF), a traditional Chinese herb used for antiaging, associated various health benefits. The preconditioned stem cells were tested for their potential to drive H9c2 from doxorubicin (Dox)-induced aging. The AOF-treated stem cells enriched stemness in ADMSCs with respect to their stem cells' positive marker, and enhanced their longevity mechanism and elevated the stem cell homing-associated C-X-C chemokine receptor type 7 (CXCR7). The AOF preconditioned stem cells, when cocultured with H9c2 cells, showed effective protection to Dox-induced senescence and stem cell homing to damaged H9c2 cells. The presence of AOF provided greater protective effects in the Dox environment. In addition, AOF-pretreated ADMSCs showed enhanced migration than those treated with AOF in Dox environment. Therefore, our results show that administration of AOF preconditioned stem cells is potentially an effective strategy in the management of aging-associated cardiac disorders.