Chemotaxis Of Mesenchymal Stem Cells Research Articles

A Self-Homing and Traceable Cardiac Patch Leveraging Ferumoxytol for Spatiotemporal Therapeutic Delivery.

Mesenchymal stem cell (MSC)-based cardiac patches are envisioned to be a promising treatment option for patients with myocardial infarction. However, their therapeutic efficacy and duration are hampered due to their limited retention on the epicardium. We engineered a scaffold-free MSC sheet with an inherent ability to migrate into the infarcted myocardium, a strategy enabled by actively establishing a sustained intracellular hypoxic environment through the endocytosis of our FDA-approved ferumoxytol. This iron oxide nanoparticle stabilized hypoxia-induced factor-1α, triggering upregulation of the CXC chemokine receptor and subsequent MSC chemotaxis. Thus, MSCs integrated into 2/3 depth of the left ventricular anterior wall in a rat model of acute myocardial infarction and persisted for at least 28 days. This led to spatiotemporal delivery of paracrine factors by MSCs, enhancing cardiac regeneration and function. Ferumoxytol also facilitated the noninvasive MRI tracking of implanted MSCs. Our approach introduces a strategy for mobilizing MSC migration, holding promise for rapid clinical translation in myocardial infarction treatment.

Stiffness-dependent MSC homing and differentiation into CAFs - implications for breast cancer invasion.

Cellular heterogeneity and extracellular matrix (ECM) stiffening have been shown to be drivers of breast cancer invasiveness. Here, we examine how stiffness-dependent crosstalk between cancer cells and mesenchymal stem cells (MSCs) within an evolving tumor microenvironment regulates cancer invasion. By analyzing previously published single-cell RNA sequencing datasets, we establish the existence of a subpopulation of cells in primary tumors, secondary sites and circulatory tumor cell clusters of highly aggressive triple-negative breast cancer (TNBC) that co-express MSC and cancer-associated fibroblast (CAF) markers. By using hydrogels with stiffnesses of 0.5, 2 and 5 kPa to mimic different stages of ECM stiffening, we show that conditioned medium from MDA-MB-231 TNBC cells cultured on 2 kPa gels, which mimic the pre-metastatic stroma, drives efficient MSC chemotaxis and induces stable differentiation of MSC-derived CAFs in a TGFβ (TGFB1)- and contractility-dependent manner. In addition to enhancing cancer cell proliferation, MSC-derived CAFs on 2 kPa gels maximally boost local invasion and confer resistance to flow-induced shear stresses. Collectively, our results suggest that homing of MSCs at the pre-metastatic stage and their differentiation into CAFs actively drives breast cancer invasion and metastasis in TNBC.

Identification of a Specific Phage as Growth Factor Alternative Promoting the Recruitment and Differentiation of MSCs in Bone Tissue Regeneration.

Inefficient use and loss of exogenously implanted mesenchymal stem cells (MSCs) are major concerns in MSCs-based bone tissue engineering. It is a promising approach to overcome the above issues by recruiting and regulation of endogenous MSCs. However, there are few substances that can recruit MSCs effectively and specifically to the site of bone injury. In this study, we identified a phage clone (termed P11) with specific affinity for MSCs through phage display biopanning, and further investigated the effects of P11 on the cytological behavior of MSCs and macrophages. The results showed that P11 could bind MSCs specifically and promote the proliferation and migration of MSCs. Meanwhile, P11 could polarize macrophages to the M1 phenotype and significantly changed their morphology, which further enhanced the chemotaxis of MSCs. Additionally, RNA-seq results revealed that P11 could promote the secretion of osteogenesis-related markers in MSCs through the TPL2-MEK-ERK signaling pathway. Altogether, P11 has great potential to be used as growth factor alternatives in bone tissue engineering, with the advantages of cheaper and stable activity. Our study also advances the understanding of the effects of phages on macrophages and MSCs, and provides a new idea for the development in the field of phage-based tissue engineering.

Influence of Local Chemokine Delivery and Postoperative Peripheral Blood Mobilization on Achilles Tendon Repair

Category:Basic Sciences/Biologics; Sports; TraumaIntroduction/Purpose:Mesenchymal stem cells (MSCs) have demonstrated promise in augmenting the surgical repair of full thickness tendon tears. Despite this promise, obtaining a therapeutic concentration of MSCs is time-, labor- and resource intensive, and is subject to ever-tightening governmental regulations. Our group recently demonstrated that postoperative mobilization of MSCs from the marrow compartment and into peripheral blood via subcutaneous granulocyte- colony stimulating factor administration enhanced tendon-bone healing in a rat model of surgical repair of a full-thickness supraspinatus tendon tear. We hypothesized that combining postoperative stem cell mobilization with local delivery of a chemokine that stimulates MSC chemotaxis would enhance the surgical repair of a mid-substance Achilles tendon transection.Methods:Following IACUC approval, Achilles tendons were unilaterally frayed via multiple longitudinal scalpel blade nicks in 24 Lewis rats. Tendons were then surgically transected, followed by repair with braided non-absorbable suture in a modified-Kessler pattern. Based on randomization (n=6/group), the tendon anastomosis was coated with a resorbable chitosan hydrogel containing 100 ng of the chemokine, CCL5 (RANTES), or hydrogel without RANTES. Skin closure and anesthesia recovery were then performed; rats were allowed ad libitum activity. Beginning 24 hours postoperatively, rats received a daily subcutaneous injection of either a peripheral blood mobilizing agent, AMD3100 (5 mg/kg), or saline; dosing was repeated at 48 and 72 hours. At the 4- week endpoint, Achilles tendons harvested en bloc. Decalcified histologic sections were stained with H&E and Alcian Blue as well as immunostained for Collagen III, CD163, and CD68. Slides were digitized (40x magnification) and graded by blinded reviewers via modified-Bonar score to assess tendon integrity.Results:Rats that received local application of RANTES-loaded chitosan hydrogel had worse (higher) modified-Bonar subscores with respect to tenocyte morphology, collagen orientation, and ground substance staining, though none of these comparisons were statistically significant (p=0.068, p=0.672, and p=0.407, respectively). Animals that received subcutaneous AMD3100 alone, or in conjunction with RANTES-loaded chitosan hydrogel displayed the best (lowest) total modified Bonar scores, though these comparisons were also not statistically significant (p = 0.498). No significant differences were found with respect to CD163 (p=0.132) and CD68 (p=0.104) staining. Despite a lack of statistical significance, animals that received AMD3100 displayed a higher number of both macrophage subtypes. No differences were found with respect to Collagen III immunostaining (p=0.808).Conclusion:Tendon repair and regeneration remains a significant clinical challenge, which may be aided by MSC transplantation. The mobilization of endogenous marrow-derived MSCs into peripheral blood and subsequent recruitment via local chemokine delivery may be an efficacious way to enhance tendon repair without the need for ex vivo processing. We failed to demonstrate significant effects of stem cell mobilization via AMD3100 injections, and chemokine-directed recruitment, though trends were present indicating that AMD3100 alone may be beneficial. Additional work is required to draw any translatable conclusions from this research.

A Novel Strategy to Enhance Microfracture Treatment With Stromal Cell-Derived Factor-1 in a Rat Model.

BackgroundMicrofracture is one of the most widely used techniques for the repair of articular cartilage. However, microfracture often results in filling of the chondral defect with fibrocartilage, which exhibits poor durability and sub-optimal mechanical properties. Stromal cell-derived factor-1 (SDF-1) is a potent chemoattractant for mesenchymal stem cells (MSCs) and is expressed at high levels in bone marrow adjacent to developing cartilage during endochondral bone formation. Integrating SDF-1 into an implantable collagen scaffold may provide a chondro-conductive and chondro-inductive milieu via chemotaxis of MSCs and promotion of chondrogenic differentiation, facilitating more robust hyaline cartilage formation following microfracture.ObjectiveThis work aimed to confirm the chemoattractive properties of SDF-1 in vitro and develop a one-step method for incorporating SDF-1 in vivo to enhance cartilage repair using a rat osteochondral defect model.MethodsBone marrow-derived MSCs (BMSCs) were harvested from the femurs of Sprague–Dawley rats and cultured in low-glucose Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, with the medium changed every 3 days. Passage 1 MSCs were analyzed by flow cytometry with an S3 Cell Sorter (Bio-Rad). In vitro cell migration assays were performed on MSCs by labeling cells with carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE; Bio-Rad). For the microfracture model, a 1.6-mm-diameter osteochondral defect was created in the femoral trochleae of 20 Sprague–Dawley rats bilaterally until bone marrow spillage was seen under saline irrigation. One knee was chosen at random to receive implantation of the scaffold, and the contralateral knee was left unfilled as an empty control. Type I collagen scaffolds (Kensey Nash) were coated with either gelatin only or gelatin and SDF-1 using a dip coating process. The rats received implantation of either a gelatin-only scaffold (N = 10) or gelatin-and-SDF-1 scaffold (N = 10) at the site of the microfracture. Femurs were collected for histological analyses at 4- and 8-week time points post-operatively, and sections were stained with Safranin O/Fast Green. The samples were graded blindly by two observers using the Modified O’Driscoll score, a validated scoring system for chondral repair. A minimum of 10 separate grading scores were made per sample and averaged. Quantitative comparisons of cell migration in vitro were performed with one-way ANOVA. Cartilage repair in vivo was also compared among groups with one-way ANOVA, and the results were presented as mean ± standard deviation, with P-values < 0.05 considered as statistically significant.ResultsMSC migration showed a dose–response relationship with SDF-1, with an optimal dosage for chemotaxis between 10 and 100 ng/ml. After scaffold implantation, the SDF-1-treated group demonstrated complete filling of the cartilage defect with mature cartilage tissue, exhibiting strong proteoglycan content, smooth borders, and good incorporation into marginal cartilage. Modified O’Driscoll scores after 8 weeks showed a significant improvement of cartilage repair in the SDF-1 group relative to the empty control group (P < 0.01), with a trend toward improvement when compared with the gelatin-only-scaffold group (P < 0.1). No significant differences in scores were found between the empty defect group and gelatin-only group.ConclusionIn this study, we demonstrated a simple method for improving the quality of cartilage defect repair in a rat model of microfracture. We confirmed the chemotactic properties of SDF-1 on rat MSCs and found an optimized dosage range for chemotaxis between 10 and 100 ng/ml. Furthermore, we demonstrated a strategy to incorporate SDF-1 into gelatin–collagen I scaffolds in vivo at the site of an osteochondral defect. SDF-1-treated defects displayed robust hyaline cartilage resurfacing of the defect with minimal fibrous tissue, in contrast to the empty control group. The results of the in vitro and in vivo studies together suggest that SDF-1-mediated signaling may significantly improve the quality of cartilage regeneration in an osteochondral defect.

Glucocorticoid guides mobilization of bone marrow stem/progenitor cells via FPR and CXCR4 coupling

BackgroundOur previous studies have proved the efficient exogenous repairing responses via bone marrow stem and progenitor cells (BMSPCs). However, the trafficking of endogenous bone marrow stem and progenitor cells to and from the bone marrow (BM) is a highly regulated process that remains to be elucidated. We aimed to study the relative importance of the hypothalamic-pituitary-adrenal (HPA) axis in the glucocorticoid-induced BMSPC mobilization.MethodsThe circulating mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) were examined in Crh (+/+, −/−) mice after running stress or glucocorticoid mini-infusion. The MSCs and EPCs were investigated ex vivo after treatment with glucocorticoid and glucocorticoid receptor (GR) antagonist, RU486. The expression of chemotaxis receptors, N-formyl peptide receptor (FPR), and Cys-X-Cys receptor 4 (CXCR4) of MSCs and EPCs as well as their colocalization were investigated after treatment with glucocorticoid, glucocorticoid receptor (GR) antagonist (RU486), and FPR antagonist (Cyclosporin H).ResultsForced running stress increased circulating MSCs and EPCs in mice, which was blunted when Crh was knocked out, and positively related to the levels of serum glucocorticoid. Prolonged glucocorticoid mini-infusion imitated the stress-induced increase in circulating MSCs and EPCs in Crh+/+ mice and rescued the impaired mobilization in circulating MSCs and EPCs in Crh−/− mice. Meanwhile, glucocorticoid promoted the chemotaxis of MSCs and EPCs ex vivo via GR, inhibited by RU486 (10 μM). Concurrently, glucocorticoid increased the expression of FPR of MSCs and EPCs, but inhibited their expression of CXCR4, followed by their changing colocalization in the cytoplasm. The GC-induced colocalization of FPR and CXCR4 was blunted by Cyclosporin H (1 μM).ConclusionGlucocorticoid-induced CXCR4-FPR responsiveness selectively guides the mobilization of BMSPCs, which is essential to functional tissue repair.Graphical abstractSchematic view of the role of glucocorticoid on the mobilization of bone marrow-derived stem/progenitor cells subsets in the present study. The HPA axis activation promotes the release of glucocorticoid, which regulates the directional migration of MSCs and EPCs mainly via GR. The possible mechanisms refer to the signal coupling of FPR and CXCR4. Their two-sided changes regulated by glucocorticoid are involved in the egress of MSCs and EPCs from BM, which is helpful for wound healing. MSCs, mesenchymal stem cells; EPCs, endothelial progenitor cells.

Improving Chronic Diabetic Wound Healing through an Injectable and Self-Healing Hydrogel with Platelet-Rich Plasma Release.

Diabetic skin ulcer is one of the severe complications of diabetes mellitus, which has a high incidence and may cause death or disability. Platelet-rich plasma (PRP) is widely used in the treatment of diabetic wounds due to the effect of growth factors (GFs) derived from it. However, the relatively short half-life of GFs limits their applications in clinics. In addition, the presence of a large amount of proteases in the diabetic wound microenvironment results in the degradation of GFs, which further impedes angiogenesis and diabetic wound healing. In our study, we fabricated a self-healing and injectable hydrogel with a composite of chitosan, silk fibroin, and PRP (CBPGCTS-SF@PRP) for promoting diabetic wound healing. CBPGCTS-SF@PRP could protect PRP from enzymatic hydrolysis, release PRP sustainably, and enhance the chemotaxis of mesenchymal stem cells. The results showed that it could promote the proliferation of repair cells in vitro. Moreover, it could enhance wound healing by expediting collagen deposition, angiogenesis, and nerve repair in a type 2 diabetic rat model and a rat skin defect model. We hope that this study will offer a new treatment for diabetic nonhealing wounds in clinics.

SUN-120 Regional Hyperthermia Enhances Selective Mesenchymal Stem Cell Migration Towards the Tumor Stroma

The tumor homing characteristics of mesenchymal stem cells (MSCs) make them attractive vehicles for the tumor-specific delivery of therapeutic agents, such as the sodium iodide symporter (NIS). NIS is a theranostic protein that allows non-invasive monitoring of the in vivo biodistribution of functional NIS expression by radioiodine imaging as well as the therapeutic application of 131I. To enhance the actively recruitment of MSCs to growing tumor stroma and thereby trigger targeted delivery of the NIS gene to the tumor, we examined the combination with regional hyperthermia, as heat induces the secretion of immunomodulatory chemokines, cytokines and growth factors, well-known attractants of MSCs.Human hepatocellular carcinoma cells (HuH7) were heat-treated in a water bath at 41 °C for 1h, followed by incubation at 37 °C for 0-48h. mRNA and protein levels of chemokines involved in MSC migration was analyzed by RT-PCR and ELISA. Chemotaxis of MSCs in relation to a gradient of supernatants was tested in a 3D live cell tracking migration assay. In a subcutaneous HuH7 mouse xenograft tumor model, a single systemic injection of CMV-NIS-MSCs was applied 6h, 24h, 48h after or 24h, 48h before hyperthermia treatment and tumoral 123I accumulation was assessed by 123I-scintigraphy. Ex vivo NIS analysis of tumor sections was performed by RT-PCR and immunohistochemistry. The optimal imaging regime was then used for a 131I therapy study.Chemokine mRNA and protein analysis indicated a substantial increase in expression levels of chemokines and growth factors, involved in MSC tumor homing, after heat exposure. In addition, MSCs showed directed migration towards the supernatant of thermo-stimulated cancer cells. In vivo, with the optimal regime, we observed a significantly increased uptake of 123I in tumors of heat-treated animals (41 °C) when thermostimulated 24h after CMV-NIS-MSC injection compared to control animals (37 °C). Immunohistochemical staining of tumor sections showed strong tumoral NIS-specific immunoreactivity and RT-PCR an increased NIS mRNA expression in heat-treated tumors, thereby confirming tumor-selective, temperature-dependent MSC migration. CMV-NIS-MSC-mediated 131I therapy combined with regional hyperthermia resulted in a reduced tumor growth that was associated with prolonged survival of regional heat-treated animals compared to normothermic mice and to the saline control group.In summary, we have demonstrated a significantly increased, selective MSC migration towards the tumor stroma after regional hyperthermia in the 123I imaging study. The combination of MSC-mediated NIS gene therapy with mild regional hyperthermia resulting in stimulated therapeutic efficacy of NIS-mediated 131I therapy.

HIV glycoprotein gp120 enhances mesenchymal stem cell migration by upregulating CXCR4 expression

BackgroundHIV infection and/or the direct pathogenic effects of circulating HIV proteins impairs the physiological function of mesenchymal stem cells (MSCs), and contribute to the pathogenesis of age-related clinical comorbidities in people living with HIV. The SDF-1/CXCR4 pathway is vital for modulating MSC proliferation, migration and differentiation. HIV glycoprotein gp120 inhibits SDF-1 induced chemotaxis by downregulating the expression and function of CXCR4 in monocytes, B and T cells. The influence of gp120 on CXCR4 expression and migration in MSCs is unknown. MethodsWe investigated CXCR4 expression and SDF-1/CXCR4-mediated MSC migration in response to gp120, and its effect on downstream signaling pathways: focal adhesion kinase (FAK)/Paxillin and extracellular signal-regulated kinase (ERK). ResultsGp120 upregulated MSC CXCR4 expression. This potentiated the effects of SDF-1 in inducing chemotaxis; FAK/Paxillin and ERK pathways were over-activated, thereby facilitating actin stress fiber reorganization. CXCR4 blockage or depletion abrogated the observed effects. ConclusionGp120 from both T- and M- tropic HIV strains upregulated CXCR4 expression in MSCs, resulting in enhanced MSC chemotaxis in response to SDF-1. General significanceHIV infection and its proteins are known to disrupt physiological differentiation of MSC; increased gp120-driven migration amplifies the total MSC population destined for ineffective and inappropriate differentiation, thus contributing to the pathogenesis of HIV-related comorbidities. Additionally, given that MSCs are permissive to HIV infection, initial cellular priming by gp120 results in increased expression of CXCR4 and could lead to co-receptor switching and cell tropism changes in chronic HIV infection and may have implications against CCR5-knockout based HIV cure strategies.

Local application of IGFBP5 protein enhanced periodontal tissue regeneration via increasing the migration, cell proliferation and osteo/dentinogenic differentiation of mesenchymal stem cells in an inflammatory niche

BackgroundPeriodontitis is a widespread infectious disease ultimately resulting in tooth loss. The number of mesenchymal stem cells (MSCs) in patients with periodontitis is decreased, and MSC functions are impaired. Rescuing the impaired function of MSCs in periodontitis is the key for treatment, especially in a manner independent of exogenous MSCs. Our previous study found that overexpressed insulin-like growth factor binding protein 5 (IGFBP5) could promote exogenous MSC-mediated periodontal tissue regeneration. Here, we investigate the role of IGFBP5 protein in MSCs and periodontal tissue regeneration independent of exogenous MSCs in an inflammatory niche.MethodsTNFα was used to mimic the inflammatory niche. Lentiviral IGFBP5 shRNA was used to silence IGFBP5 and recombinant human IGFBP5 protein (rhIGFBP5) was used to stimulate the periodontal ligament stem cells (PDLSCs) and bone marrow stem cells (BMSCs). The effects of IGFBP5 on PDLSCs were evaluated using the scratch-simulated wound migration, Transwell chemotaxis, alkaline phosphatase (ALP) activity, Alizarin red staining, Cell Counting Kit-8, Western blot, Real-time PCR, Co-IP and ChIP assays. The swine model of periodontitis was used to investigate the functions of IGFBP5 for periodontal regeneration and its anti-inflammation effect.ResultsWe discovered that 0.5 ng/ml rhIGFBP5 protein enhanced the migration, chemotaxis, osteo/dentinogenic differentiation and cell proliferation of MSCs under the inflammatory condition. Moreover, 0.5 ng/ml rhIGFBP5 application could rescue the impaired functions of IGFBP5-silenced-MSCs in the inflammatory niche. Furthermore, local injection of rhIGFBP5 could promote periodontal tissue regeneration and relieve the local inflammation in a minipig model of periodontitis. Mechanistically, we found that BCOR negatively regulated the expression of IGFBP5 in MSCs. BCOR formed a protein complex with histone demethylase KDM6B and raised histone K27 methylation in the IGFBP5 promoter.ConclusionsThis study revealed that rhIGFBP5 could activate the functions of MSCs in an inflammatory niche, provided insight into the mechanism underlying the activated capacities of MSCs, and identified IGFBP5 as a potential cytokine for improving tissue regeneration and periodontitis treatment independent of exogenous MSCs and its potential application in dental clinic.

Myocardial apoptosis and mesenchymal stem cells with acute exercise.

Aerobic exercise confers many health benefits. However, numerous reports have shown that acute aerobic exercise can injure the heart. We tested the general hypothesis that acute moderate‐intensity exercise in rodents induces cardiomyocyte damage and stimulates mesenchymal stem cells (MSCs) to increase paracrine‐mediated protective effects on cardiomyocytes. A single session of treadmill running (13 m/min, 0% grade, for 45 min) in untrained C57BL/6 male mice (n = 18) increased cleaved poly ADP‐ribose polymerase (PARP), a marker of apoptosis, in the myocardium 24 h postexercise. Microarray analysis of mouse myocardium identified 11 relevant apoptotic genes and several shifts in matrix remodeling transcripts over the postexercise window. Postexercise cardiomyocyte death was recapitulated in neonatal rat cardiomyocytes (NRCMs) by culturing cells in 2% plasma harvested from exercised rats. The increased cell death observed in exercise‐treated NRCMs was attenuated by β‐adrenergic blockade, but not antioxidant treatment. MSC survival, proliferation, and chemotaxis showed no significant differences between sedentary and exercise plasma conditions, despite increased IL‐6, TNF‐α, IL‐1β, and IFN‐γ secretions from MSCs treated with exercise plasma. NRCM survival was increased nearly 500% when cocultured with MSCs, but this effect was not altered under exercise plasma culture conditions. Our results suggest acute moderate‐intensity aerobic treadmill running in exercise‐naïve rodents induces temporal cardiomyocyte death due to plasma‐borne factors, namely, catecholaminergic stress. Even though exercise conditions prompt an inflammatory response in MSCs, the exercise milieu does not alter the MSC‐protective phenotype on cardiomyocytes.

Norepinephrine inhibits mesenchymal stem cell chemotaxis migration by increasing stromal cell-derived factor-1 secretion by vascular endothelial cells via NE/abrd3/JNK pathway

Mesenchymal stem cells (MSCs), which are physiologically maintained in vascular endothelial cell (VEC)-based niches, play a critical role in tissue regeneration. Our previous studies demonstrated that sympathetic denervation could promote MSC mobilization, thereby enhancing bone formation in distraction osteogenesis (DO), a self-tissue engineering for craniofacial and orthopeadic surgeries. However, the mechanisms on how sympathetic neurotransmitter norepinephrine (NE) regulates MSC migration are not well understood. Here we showed that deprivation of NE by transection of cervical sympathetic trunk (TCST) inhibited stromal cell-derived factor-1 (SDF-1) expression in the perivascular regions in rat mandibular DO. In vitro studies showed that NE treatment markedly upregulated p-JNK and therefore stimulated higher SDF-1 expression in VECs than control groups, and siRNA knockdown of the abrd3 gene abolished the NE-induced p-JNK activation. On the other hand, osteoblasts differentiated from MSCs showed an increase in SDF-1 secretion with lack of NE. Importantly, NE-treated VECs inhibited the MSC chemotaxis migration along the SDF-1 concentration gradient as demonstrated in a novel 3-chamber Transwell assay. Collectively, our study suggested that NE may increase the SDF-1 secretion by VECs via NE/abrd3/JNK pathway, thereby inhibiting the MSC chemotaxis migration from perivascular regions toward bone trabecular frontlines along the SDF-1 concentration gradient in bone regeneration.

Multimodal Delivery of Isogenic Mesenchymal Stem Cells Yields Synergistic Protection from Retinal Degeneration and Vision Loss.

We previously demonstrated that subretinal injection (SRI) of isogenic mesenchymal stem cells (MSCs) reduced the severity of retinal degeneration in Royal College of Surgeons rats in a focal manner. In contrast, intravenous MSC infusion (MSCIV) produced panoptic retinal rescue. By combining these treatments, we now show that MSCIV supplementation potentiates the MSCSRI‐mediated rescue of photoreceptors and visual function. Electrophysiological recording from superior colliculi revealed 3.9‐fold lower luminance threshold responses (LTRs) and 22% larger functional rescue area from combined treatment compared with MSCSRI alone. MSCIV supplementation of sham (saline) injection also improved LTRs 3.4‐fold and enlarged rescue areas by 27% compared with saline alone. We confirmed the involvement of MSC chemotaxis for vision rescue by modulating C‐X‐C chemokine receptor 4 activity before MSCIV but without increased retinal homing. Rather, circulating platelets and lymphocytes were reduced 3 and 7 days after MSCIV, respectively. We demonstrated MSCSRI‐mediated paracrine support of vision rescue by SRI of concentrated MSC‐conditioned medium and assessed function by electroretinography and optokinetic response. MSC‐secreted peptides increased retinal pigment epithelium (RPE) metabolic activity and clearance of photoreceptor outer segments ex vivo, which was partially abrogated by antibody blockade of trophic factors in concentrated MSC‐conditioned medium, or their cognate receptors on RPE. These data support multimodal mechanisms for MSC‐mediated retinal protection that differ by administration route and synergize when combined. Thus, using MSCIV as adjuvant therapy might improve cell therapies for retinal dystrophy and warrants further translational evaluation. Stem Cells Translational Medicine 2017;6:444–457

Effects of mesenchymal stem cells surface CXC chemokine receptor 4 over-expression on repair of ischemia reperfusion injured kidney

Objective To observe the effects of mesenchymal stem cells (MSCs) surface CXC chemokine receptor 4 over-expression on the repair of kidney after ischemia reperfusion (I/R) injury. Methods The MSCs were co-cultured with I/R injured renal cell homogenate supernatant. The MSCs surface CXCR4 and stromal cells derived factor-1 (SDF-1α) protein levels were detected by Western blot, chemotactic ability of MSCs to SDF-1 was investigated by transwell test. The I/R injured renal model was made and pathological changes were observed in control group, I/R group, MSCs injection group and CXCR4 neutralize antibody group. Renal CXCR4 protein expression was measured by immunofluorescence histochemistry, SDF-1α、CXCR4、hepatocyte growth factor (HGF) and epidermal growth factor (EGF) mRNA were detected by Real-time quantitative polymerase chain reaction (RT-PCR). Comparisons among multiple groups were performed using One-way analysis of variance, and comparisons between groups were carried out using independent-sample t-test. Results In vitro, the SDF-1α protein expression markedly increased in I/R injured renal tissue homogenate, but the difference was not significant between I/R group and CXCR4 antibody group (t=0.862, P=0.403). MSCs surface CXCR4 protein expression increased significantly after co-cultured with I/R injured renal tissue homogenate (F=95.957, t=10.166, P <0.01) , and the chemotactic ability of MSCs to SDF-1 increased at the same time (F=82.459, t=6.826, P <0.01) , the CXCR4 protein expression (t=13.657, P <0.01) and the chemotactic ability (t=12.662, P <0.01) could be decreased by CXCR4 neutralize antibody. In vivo, renal tubular structure was destroyed in I/R group. After MSCs injection, the renal pathological injury improved rapidly, but the improvement could be inhibited by CXCR4 antibody. The expression of SDF-1α mRNA and level of SDF-1a protein increased in I/R group, but there was no significant difference among different groups (F=1.909, P=0.173). MSCs injection markedly up-regulated the CXCR4 protein and mRNA expression (F=6.663, P=0.006). Following the increase in CXCR4 expression, the expressions of HGF mRNA (F=11.898, P <0.01) and EGF mRNA (F=5.309, P <0.05) increased gradually which could be restrained by CXCR4 antibody (t=5.312, t=4.310, P <0.01). Conclusions I/R injured renal microenvironment markedly increased the mesenchymal stem cells surface CXCR4 expression, and increased CXCR4 expression can induce MSCs chemotaxis and stimulate the secretion of renal protective growth factors paracrine promoting the repair of the kidney. Key words: Mesenchymal stem cells; CXC chemokine receptor 4; Ischemia reperfusion injury; Rats; Renal injury; Chemotaxis; Paracrine; Recovery

Investigational Study of Mesenchymal Stem Cells on Lung Cancer Cell Proliferation and Invasion

背景与目的间充质干细胞(mesenchymal stem cells, MSC)是来源于中胚层的成体干细胞。有文献报道MSC通过向肿瘤组织的归巢和向间质成分分化，改变肿瘤微环境，影响肿瘤的生长和转移。但MSC在非小细胞肺癌(non-small cell lung cancer, NSCLC)中的作用报道较少，且不一致。本研究旨在探讨MSC向NSCLC细胞的趋化能力，以及其对NSCLC细胞的增殖和侵袭能力的作用。方法Transwell法检测MSC向肺癌细胞迁移能力，Thymidine嵌入实验检测MSC条件培养液对肺癌细胞增殖能力的影响，Real-time PCR法检测肺癌/MSC共培养后MSC表达白介素(interleukin-6, IL-6)、胰岛素样生长因子(insulinlike growth factor, IGF-1)、血管内皮生长因子(vascular endothelial growth factor, VEGF)和Dickkopf相关蛋白1(dickkopf-related protein 1, DKK1)的变化。建立人肺癌A549细胞裸鼠皮下荷瘤模型，给予MSC细胞，定期测量肿瘤体积变化。结果MSC可以向肺癌细胞趋化运动，其条件培养液可以促进肺癌细胞的增殖能力。肺癌细胞反过来可以促进MSC过表达IL-6、IGF-1、VEGF和DKK1。体内试验显示MSC注射组的肿瘤体积明显大于对照组。结论MSC可以向肺癌细胞趋化并促进肺癌的生长。反过来，肺癌细胞可以刺激MSC过表达生长因子进一步促进肿瘤生长。

Biodegradable chitosan microparticles induce delayed STAT-1 activation and lead to distinct cytokine responses in differentially polarized human macrophages in vitro

Current data suggest that chitosan activates wound macrophages to release endogenous factors that guide mesenchymal stem cell (MSC) to bone fractures. We tested the hypothesis that chitosan, a polymer containing glucosamine and N-acetyl glucosamine, stimulates macrophages in different polarization states to release functional MSC chemokines and mainly anabolic factors. Low-serum conditioned medium was collected from M0, M1 and M2a U937 macrophages previously differentiated with phorbol myristate acetate (PMA) and exposed or not for 24h to chitosan microparticles (80% degree of deacetylation, DDA, 130kDa). Chitosan particles were highly phagocytosed. Chitosan enhanced anabolic factor release from M0 and M2a macrophages (MCP-1, IP-10, MIP-1beta, IL-1ra, IL-10, PDGF), and IL-1beta release, with 25- to 400-fold excess IL-1ra over IL-1beta. In M1 macrophages, chitosan enhanced IL-1beta without enhancing or suppressing inflammatory factor release (IL-6, IP-10, IL-8). M0 and M2a macrophages, with or without chitosan stimulation, produced conditioned medium that promoted 2-fold more MSC chemotaxis than low-serum control medium, while M1-conditioned medium failed to induce MSC chemotaxis. Acetylated chitosan induced U937 macrophages to release IL-1ra without STAT-6 activation, and also induced a delayed STAT-1 activation/IP-10 release response that was not observed using non-biodegradable chitosan (98% DDA, 130kDa). In primary human macrophages, acetylated chitosan enhanced IL-1ra release without inducing IL-1beta, and required PMA priming to elicit STAT-1 activation and IP-10 release. We conclude that biodegradable chitosan particles enhance M0 and M2a macrophage anabolic responses independent of the IL4/STAT-6 axis, by inducing excess IL-1ra over IL-1beta and more chemokine release, without altering their inherent capacity to attract MSCs.

Leptin signaling is required for augmented therapeutic properties of mesenchymal stem cells conferred by hypoxia preconditioning.

Hypoxia preconditioning enhances the therapeutic effect of mesenchymal stem cells (MSCs). However, the mechanism underlying hypoxia-induced augmentation of the protective effect of MSCs on myocardial infarction (MI) is poorly understood. We show that hypoxia-enhanced survival, mobility, and protection of cocultured cardiomyocytes were paralleled by increased expression of leptin and cell surface receptor CXCR4. The enhanced activities were abolished by either knockdown of leptin with a selective shRNA or by genetic deficiency of leptin or its receptor in MSCs derived, respectively, from ob/ob or db/db mice. To characterize the role of leptin in the regulation of MSC functions by hypoxia and its possible contribution to enhanced therapeutic efficacy, cell therapy using MSCs derived from wild-type, ob/ob, or db/db mice was implemented in mouse models of acute MI. Augmented protection by hypoxia pretreatment was only seen with MSCs from wild-type mice. Parameters that were differentially affected by hypoxia pretreatment included MSC engraftment, c-Kit(+) cell recruitment to the infarct, vascular density, infarct size, and long-term contractile function. These data show that leptin signaling is an early and essential step for the enhanced survival, chemotaxis, and therapeutic properties of MSCs conferred by preculture under hypoxia. Leptin may play a physiological role in priming MSCs resident in the bone marrow endosteum for optimal response to systemic signaling molecules and subsequent tissue repair.

Interaction of Mesenchymal Stem Cells with Fibroblast-like Synoviocytes via Cadherin-11 Promotes Angiogenesis by Enhanced Secretion of Placental Growth Factor

Bone marrow-derived mesenchymal stem cells (MSC) exist in the synovium of patients with rheumatoid arthritis (RA), yet the role of MSC in RA is elusive. Placental growth factor (PlGF) expression is increased in RA synovial fluids, and blocking of PlGF attenuates progression of arthritis in mice. In this study, we observed that PlGF induced chemotaxis of MSC in a dose-dependent manner, which was blocked by anti-vascular endothelial growth factor receptor-1 peptide. MSC exposed to PlGF elicited increased phosphorylation of Akt and p38 MAPK. PlGF-mediated chemotaxis was inhibited by PI3K inhibitor (LY294002) and p38 MAPK inhibitor (SB203580), but not by ERK1/2 inhibitor (PD98059). Fibroblast-like synoviocytes (FLS) constitutively produced PlGF, but MSC released negligible amounts of PlGF. Of note, when FLS of RA patients and MSC were cocultured, PlGF production by FLS was significantly increased; such an increase was dependent on the number of added MSC. Moreover, coculture conditioned medium promoted chemotaxis of MSC and increased angiogenesis in Matrigel plugs assay, and these were suppressed by preincubation of the medium with anti-PlGF Ab. Transwell experiments revealed that MSC to FLS contact was required for the increase in PlGF production by coculture. Cadherin-11 was expressed both in FLS and MSC, and small interfering RNA knockdown of cadherin-11 in FLS significantly abrogated the enhanced PlGF production under coculture conditions. These data indicate that increased levels of PlGF in RA joints could induce the migration of MSC to the synovium, and interaction of migrated MSC with FLS via cadherin-11 may contribute to angiogenesis and chronic synovitis by enhancing the secretion of PlGF.

Transforming growth factor-β1 in the microenvironment of ischemia reperfusion-injured kidney enhances the chemotaxis of mesenchymal stem cells to stromal cell-derived factor-1 through upregulation of surface chemokine (C-X-C motif) receptor 4

Acute renal failure is one of the most common complications observed in hospitals. Although extensive studies have been carried out to search for therapeutic treatments, no effective cure has been established. In recent years, stem cell therapy for tissue engineering and repair has become a key area of study. Bone marrow mesenchymal stem cells (MSCs) have been demonstrated to exhibit a reparative role in ischemia reperfusion-injured kidney tissue, and the stromal cell-derived factor-1 (SDF-1)/chemokine (C-X-C motif) receptor 4 (CXCR4) axis has been found to play an important role in the migration and homing of MSCs. In the present study, transforming growth factor-β1 (TGF-β1) in the homogenate of the acute ischemia reperfusion-injured renal tissue was found to markedly increase the CXCR4 surface expression of MSCs, which contributes to the migration of MSCs to SDF-1. Neutralization of TGF-β1 inhibited the migration in an antibody concentration-dependent manner, through downregulation of CXCR4 localized to the membrane. These observations suggest a potential mechanism for MSC migration to the kidney which may provide a possible therapeutic target for curing acute renal failure.

CCL5/RANTES is a key chemoattractant released by degenerative intervertebral discs in organ culture

Release of chemotactic factors in response to tissue damage has been described for different musculoskeletal tissues, including the intervertebral disc (IVD). This study investigated the chemoattractants that are released by induced degenerative IVDs and may be involved in recruiting mesenchymal stem cells (MSCs). Bovine caudal discs were cultured within a bioreactor and loaded under conditions that mimicked physiological or degenerative settings. Between days 4-6, medium was replaced by PBS, which was subsequently used for proteomic, ELISA and immunoprecipitation analyses of secreted chemokines and cytokines. A Boyden chamber assay was used to observe human MSC migration towards native and chemokine depleted media. Gene expression levels of chemokine receptors in human MSCs were analysed, and CCL5 was localised in bovine and human IVD by immunohistochemistry. Proteomic analysis revealed the presence of CCL5 and CXCL6 within conditioned media. Higher concentrations of CCL5 were found in the degenerative media, and a relationship was found between interleukin-1β and CCL5 concentration. Chemokine immunoprecipitation showed that MSCs had a significantly reduced chemotactic migration towards CCL5-immunoprecipitated and CCL5/CXCL6 co-immunoprecipitated media, whilst CXCL6 depletion did not change MSC chemotaxis. MSCs showed a significant increase in mRNA expression of the CCL5 receptors, CCR1 and CCR4, upon culture in degenerative media. Furthermore, CCL5 was identified in bovine and human disc tissue by immunohistochemistry. Hence, CCL5 may be a key chemoattractant that is produced and released by the intervertebral disc cells. Therefore, these factors could be used to enhance stem/progenitor cell mobilisation in regenerative therapies for early stages of disc degeneration.