Mobilization Of Mesenchymal Stem Cells Research Articles

Functioning of whole bone marrow cell population in a model of L-arginine-induced pancreatitis

Objective The present study aimed to investigate the metabolic activity and adherence capacity of the whole bone marrow cell (wBMC) population and changes in bone marrow (BM) architecture after acute pancreatitis (AP) induction. Methods The disease was induced by injecting L-arginine (350 mg/100 g; i.p.). The control animals, animals injected with L-arginine, and animals pre-treated with allopurinol were included in this study. Serum and routine pathohistological analysis were conducted to confirm the induction of AP. The metabolic activity and adherence capacity of wBMC were evaluated in the MTT assay and methylene-blue test, respectively, and the alterations in pancreatic tissue and BM were examined on histological sections. In addition, immunohistochemical expression of β-catenin was assessed in the pancreatic tissue. Results The wBMC adherence capacity and their total number significantly increased after AP induction. The adherence capability of wBMC was reduced in rats pre-treated with allopurinol. There were no statistically significant changes in the metabolic activity of wBMCs. In the BM isolated from AP and allopurinol pre-treated animals, a discrete left shift in granulocytopoiesis was found, with a slight increase in the myeloid-to-erythroid ratio. Conclusions After AP induction, it was shown that wBMCs expressed increased adherence capacity and unchanged metabolic activity, while the alterations in BM may reflect the general activation of myelopoiesis, which would agree with the increased mobilization of mesenchymal stem cells. Also, a significant decrease in β-catenin expression in the pancreatic tissue was noticed.

GRANULOCYTE-COLONY STIMULATING FACTOR (G-CSF) ENHANCES THE MOBILIZATION OF BONE MARROW-DERIVED MESENCHYMAL STEM CELLS IN THE PERIPHERAL BLOOD OF BALB/C MICE

Mesenchymal stem cells (MSCs) have emerged in recent years as beneficial tools for cell therapy and autologous/allogeneic transplantation and their clinical harnessing considers the bone marrow (BM) and peripheral blood (PB). However, this requires a process of endogenous mobilization that offers a non-invasive alternative. Thus, granulocyte colony-stimulating factor (G-CSF) and AMD3100 (Plerixafor ® ) are well known and clinically approved to stimulate MSCs mobilization, but data on their potential activity in MSCs mobilization are still scarce. Evaluate the influence of G-CSF and AMD3100 stimulation on MSCs mobilization from BM to PB in BALB/c mice. For this study, 45 male BALB/c mice aged 3-4 weeks were used. They were divided into 5 groups: PBS+PBS control group (CG, n = 9), PBS+GCSF group (n = 9), PBS+AMD3100 group (n = 9), G-CSF+PBS group (n = 9) and G-CSF+AMD3100 group (n = 9). Pre-treatment was performed with intraperitoneal (i.p.) injections of phosphate-buffered saline (PBS, 160 μl/i.p.) or G-CSF (200 μg/kg/i.p.) for 4 consecutive days. On day 5 (D5), 24 hours after the last injection of PBS or G-CSF, mice were also injected with PBS or AMD3100 (5 mg/kg/i.p.). After 1 hour, BM and PB samples were subsequently collected. The mobilized CD14+CD73+CD90+CD105+ MSCs were then evaluated ex vivo using Flow Cytometry. Statistical analyzes were performed through GraphPad Prism (v.5) using One-way ANOVA test with Tukey's post-test. Based on this study, it was found that there were no statistically significant differences for MSCs mobilization in the BM in any of the studied groups; however, the PBS+AMD3100 group had the highest rate of MSCs enrichment within the BM. Notably, mice of the G-CSF+PBS group exhibited the highest rate of MSCs mobilization in the PB when compared to the CG (p < 0.0001), while the G-CSF+AMD3100 group showed no statistically significant difference for MSCs mobilization in PB. Furthermore, the mice of the PBS+G-CSF group and the PBS+AMD3100 group did not exhibit a significant increase in MSCs mobilization in the PB. With these results, it is verified that the pre-treatment with G-CSF is the most suitable to induce the MSCs mobilization to PB in a dose-dependent manner. Our data demonstrated that AMD3100 proved not to be suitable for MSCs mobilization in both BM and PB. FAPEAM, CAPES and CNPq.

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.

Age-Related Features of the Response of the Liver and Stem Cells during Modeling of Liver Cirrhosis.

We studied the age-related characteristics of the response of stem cells and liver in male Wistar rats to administration of carbon tetrachloride (CCl4) and ethanol. It was shown that modeling of liver cirrhosis caused inflammation, fibrosis, damage to sinusoidal capillaries, necrosis, and disturbances in the functional activity of hepatocytes in young rats. These processes were accompanied by mobilization of profibrotic mesenchymal stem cells (MSC), proinflammatory hematopoietic stem cells (HSC) and lymphocytes (CD45hiCD133+) from the bone marrow into the blood and migration to the liver. On the other hand, the number of hepatocyte precursors expressing Sox9 (cells of Hering's canal), immature cholangiocytes, Ito cells, oval cells, and endothelial cells of the liver sinusoids) sharply increased in the liver. In young rats, mobilization and migration of MSC, HSC, and hepatocyte precursors against the background of liver cirrhosis were more intensive than in old animals. The higher resistance of old rats to exposure is associated with age-related changes in the niches as well as in mobilization and migration of cells.

Intra‐articular Injection of PDGF-BB Explored in a Novel in Vitro Model Mobilizes Mesenchymal Stem Cells From the Synovium Into Synovial Fluid in Rats

Drugs that can induce mesenchymal stem cell (MSC) mobilization from synovium into synovial fluid will enable regenerative medicine in joints without use of exogenous MSCs. An in vitro synovial MSC migration model had previously been developed for screening but had problems in practical application. We herein developed a novel in vitro model, explored cytokines for synovial MSC mobilization with this model, and verified whether MSCs in synovial fluid increase following intra-articular injection of the cytokine. Human synovial MSCs embedded in a mixture of Matrigel and type 1 collagen hydrogel were placed on a culture insert and then put in medium containing migration factor. Of the six cytokines, we identified the one that mobilizes the highest number of MSCs. PDGF-BB or PBS was injected into rat knees, and 48h later, synovial fluid was collected and cultured. The cells were examined for MSC properties. PDGF-BB was the most effective for synovial MSC mobilization among six cytokines. The effect of PDGF-BB was inhibited by a PRGFR inhibitor. Injection of PDGF-BB into rat knees increased colony-forming cells in the synovial fluid. These cells had surface epitopes and multipotency comparable to MSCs and a higher capacity for proliferation, colony formation, and chondrogenesis. This novel in vitro model recapitulated the migration of MSCs from synovium into synovial fluid. Our exploration of cytokines revealed that PDGF-BB strongly induced in vitro synovial MSC migration, while intra-articular injection of PDGF-BB increased in vivo MSC numbers in synovial fluid in rats.

Neovascularization: The Main Mechanism of MSCs in Ischemic Heart Disease Therapy.

Mesenchymal stem cell (MSC) transplantation after myocardial infarction (MI) has been shown to effectively limit the infarct area in numerous clinical and preclinical studies. However, the primary mechanism associated with this activity in MSC transplantation therapy remains unclear. Blood supply is fundamental for the survival of myocardial tissue, and the formation of an efficient vascular network is a prerequisite for blood flow. The paracrine function of MSCs, which is throughout the neovascularization process, including MSC mobilization, migration, homing, adhesion and retention, regulates angiogenesis and vasculogenesis through existing endothelial cells (ECs) and endothelial progenitor cells (EPCs). Additionally, MSCs have the ability to differentiate into multiple cell lineages and can be mobilized and migrate to ischemic tissue to differentiate into ECs, pericytes and smooth muscle cells in some degree, which are necessary components of blood vessels. These characteristics of MSCs support the view that these cells improve ischemic myocardium through angiogenesis and vasculogenesis. In this review, the results of recent clinical and preclinical studies are discussed to illustrate the processes and mechanisms of neovascularization in ischemic heart disease.

SDF-1 mediates mesenchymal stem cell recruitment and migration via the SDF-1/CXCR4 axis in bone defect.

Recent studies have indicated the potential of stem cell therapy in combination with cytokines to restore the bone repair via migration and homing of stem cells to the defected area. The present study aimed to investigate the mobilization and recruitment of mesenchymal stem cells (MSCs) in response to SDF-1. Herein, the knockout rat model of the bone defect (BD) was treated with the induced membrane technique. Then, wild type Wistar rats and SDF-1-knockout rats were selected for the establishment of BD-induced membrane (BD-IM) models and bone-graft (BG) models. The number of MSCs was evaluated by flow cytometry, along with the expression pattern of the SDF-1/CXCR4 axis as well as osteogenic factors was identified by RT-qPCR and Western blot analyses. Finally, the MSC migration ability was assessed by the Transwell assay. Our data illustrated that in the induced membrane tissues, the number of MSCs among the BD-IM modeled rats was increased, whereas, a lower number was documented among BG modeled rats. Besides, we found that lentivirus-mediated over-expression of SDF-1 in BG modeled rats could activate the SDF-1/CXCR4 axis, mobilize MSCs into the defect area, and up-regulate the osteogenic proteins. Collectively, our study speculated that up-regulation of SDF-1 promotes the mobilization and migration of MSCs through the activation of the SDF-1/CXCR4 signal pathway.

Dimethyloxalyl Glycine Regulates the HIF-1 Signaling Pathway in Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are pluripotent stem cells with self-renewal and multidirectional differentiation capabilities. Dimethyloxalyl glycine (DMOG) mobilizes MSCs, and the hypoxia inducible factor-1 (HIF-1) signaling pathway plays an important role in MSC mobilization. We aimed to investigate the effect of DMOG on the HIF-1 pathway in MSCs. Rats were treated with DMOG, and the numbers of peripheral blood MSCs (PB-MSCs) and bone marrow MSCs (BM-MSCs) were detected by the Colony-forming unit fibroblastic (CFU-F) method. The growth curve, cell cycle and migration ability of PB-MSCs and BM-MSCs were detected by CCK-8, Flow cytometry and Transwell assays. Western blotting and real-time qPCR were used to detect the expression of the HIF-1 pathway. The number of bone marrow microvessels was detected by immunohistochemistry. DMOG significantly increased the numbers of PB-MSCs and BM-MSCs (P < 0.05). Further, the MSCs in peripheral blood and bone marrow still had the ability to proliferate and migrate after mobilization by DMOG. The expression levels of HIF-1α, stromal cell-derived factor-1α (SDF-1α) and vascular endothelial growth factor (VEGF) in MSCs were significantly regulated by DMOG (P < 0.05). The number of bone marrow microvessels decreased after the VEGF/VEGFR signaling pathway was blocked by SU5416 (P < 0.05). Therefore, these findings demonstrated that DMOG regulates the HIF-1α signaling pathway and promotes biological effects in MSCs.

Increased mobilization of mesenchymal stem cells in patients with acute respiratory distress syndrome undergoing extracorporeal membrane oxygenation.

The acute respiratory distress syndrome (ARDS) is characterized by pulmonary epithelial and endothelial barrier dysfunction and injury. In severe forms of ARDS, extracorporeal membrane oxygenation (ECMO) is often the last option for life support. Endothelial progenitor (EPC) and mesenchymal stem cells (MSC) can regenerate damaged endothelium and thereby improve pulmonary endothelial dysfunction. However, we still lack sufficient knowledge about how ECMO might affect EPC- and MSC-mediated regenerative pathways in ARDS. Therefore, we investigated if ECMO impacts EPC and MSC numbers in ARDS patients. Peripheral blood mononuclear cells from ARDS patients undergoing ECMO (n = 16) and without ECMO support (n = 12) and from healthy volunteers (n = 16) were isolated. The number and presence of circulating EPC and MSC was detected by flow cytometry. Serum concentrations of vascular endothelial growth factor (VEGF) and angiopoietin 2 (Ang2) were determined. In the ECMO group, MSC subpopulations were higher by 71% compared to the non-ECMO group. Numbers of circulating EPC were not significantly altered. During ECMO, VEGF and Ang2 serum levels remained unchanged compared to the non-ECMO group (p = 0.16), but Ang2 serum levels in non-survivors of ARDS were significantly increased by 100% (p = 0.02) compared to survivors. ECMO support in ARDS is specifically associated with an increased number of circulating MSC, most likely due to enhanced mobilization, but not with a higher numbers of EPC or serum concentrations of VEGF and Ang2.

Human Bone Marrow-Derived Mesenchymal Stem Cells Home via the PI3K-Akt, MAPK, and Jak/Stat Signaling Pathways in Response to Platelet-Derived Growth Factor.

Mesenchymal stem cells (MSCs) have great potential to improve clinical outcomes for many inflammatory and degenerative diseases either through intravenously delivered MSCs or through mobilization and migration of endogenous MSCs to injury sites, termed "stem cell homing." Stem cell homing involves the processes of attachment to and transmigration through endothelial cells lining the vasculature and migration through the tissue stroma to a site of injury or inflammation. Although the process of leukocyte transendothelial migration (TEM) is well understood, far less is known about stem cell homing. In this study, a transwell-based model was developed to monitor adherence and TEM of human MSCs in response to chemokine exposure. Specifically, transwell membranes lined with human synovial microvascular endothelial cells were partitioned from the tissue injury-mimetic site containing chemokine stromal cell-derived factor-1 (SDF-1). Two population subsets of MSCs were studied: migratory cells that initiated transmigration on the endothelial lining and nonmigratory cells. We hypothesized that cells would adhere to and migrate through the endothelial lining in response to SDF-1 exposure and that gene and protein expression changes would be observed between migratory and nonmigratory cells. We validated a vasculature model for MSC transmigration that showed increased expression of several genes and activation of proteins of the PI3K-Akt, MAPK, and Jak/Stat signaling pathways. These findings showed that MSC homing may be driven by activation of PDGFRA/PI3K/Akt, PDGFRA/MAPK/Grb2, and PDGFRA/Jak2/Stat signaling, as a result of SDF-1-stimulated endothelial cell production of platelet-derived growth factor. This model can be used to further investigate these key regulatory molecules toward the development of targeted therapies.

Circulating mesenchymal stem cells in sulfur mustard-exposed patients with long-term pulmonary complications

Sulfur mustard (SM) is a toxic agent that causes acute and long-term pulmonary complications. Recent evidence has shown the impact of SM on mesenchymal stem cells (MSCs). These cells have a critical role in repairing the damaged tissues. In this study, we evaluated the mobilization of MSCs in SM-exposed patients with long-term pulmonary complications. Fifty-nine SM-injured patients with prolonged pulmonary complications and 20 healthy individuals were included. Patients were classified based on taking drugs, having comorbidities, and severity of respiratory consequence. MSCs with phenotype of CD45-CD44+CD29+CD105+ were evaluated in peripheral blood using flow cytometry. Circulating MSCs were lower in SM-exposed patients compared to the control group (0.93 vs. 2.72 respectively, P = 0.005). No significant difference was observed in the MSC count between patients taking corticosteroids or antibiotics and those patients not taking them. Comorbidities like liver and kidney diseases had changed the count of MSCs in SM-exposed subjects. In addition, the frequency of MSCs did not show any association with the severity of long-term pulmonary complications. In conclusion, SM-exposure causes a decline in the frequency of circulating MSCs in survivors. The lower number of the peripheral MSC population in SM-exposed patients was not affected by taking corticosteroids or antibiotics, but comorbidities are probably involved in MSC frequency. The decreases observed in the number of circulating MSCs was not associated with the severity of the pulmonary complications; however, further studies in mustard lung models are required to demonstrate the therapeutic or pathologic role of MSCs in SM injuries.

Beneficial effect of polaprezinc on cardiac function post-myocardial infarction: A prospective and randomized clinical trial.

Background:Polaprezinc is clinically used for the treatment of gastric ulcers. It induces the mobilization of mesenchymal stem cells and the mRNA expression of insulin-like growth factor-1 in vascular endothelial cells in order to protect injured gastric tissue or skin.Methods:The current study population included 50 patients with primary acute myocardial infarction (AMI). After percutaneous coronary intervention, the subjects were randomly divided into 2 groups, namely, the nonpolaprezinc and polaprezinc groups. Peripheral blood and urinary samples were collected in a specific time to analyze zinc concentration, cardiac enzymes, and the levels of the inflammation marker interleukin-6. To evaluate the cardiac function, echocardiography was performed upon admission to the hospital and at 9 months post-AMI.Results:The urine and blood zinc levels of the polaprezinc group were higher compared with those of the non-polaprezinc group at 8 days after percutaneous coronary intervention. The mean interleukin-6/maximal creatine phosphokinase level was significantly reduced in the polaprezinc group (0.024 [0.003–0.066] vs. 0.076 [0.015–0.212], respectively; P = .045). In addition, echocardiography revealed that the ejection fraction of the nonpolaprezinc group was not significantly increased between day 3 and 9 months post-AMI (53 [49–60.8] vs. 59.5 [52–69.3], respectively; P = .015). However, a significant increase was detected in the ejection fraction of the polaprezinc group at the 2 time points (54 [51–57] vs. 62 [55–71], respectively; P < .01).Conclusions:The results of the present study suggest that polaprezinc has an anti-inflammatory effect and improves cardiac function after AMI.

Anti-depressive effect of Shuangxinfang on rats with acute myocardial infarction: Promoting bone marrow mesenchymal stem cells mobilization and alleviating inflammatory response

Bone marrow mesenchymal stem cells (BM-MSCs) are recruited to injured site for cardiac self-repairing in acute myocardial infarction (AMI), but the spontaneous mobilization of BM-MSCs is insufficient for self-repairing. Inflammation initiated by necrosis cardiomyocytes induced cardiac remodeling and depression. Given the anti-inflammatory effects of BM-MSCs and the inextricably relationship among inflammation, ventricular remodeling and depression following AMI, methods focused on enhancing BM-MSCs mobilization are promising. Shuangxinfang (Psycho-cardiology Formula, PCF) is a classical traditional Chinese medicine prescription. In this study, we explored its psycho-cardiology effects in rats with AMI and explore its potential mechanism. Our results showed PCF inhibited inflammation caused by injured myocardium, improved heart function and depression developed from myocardial infarction, and these might partly attribute to the higher BM-MSCs mobilization efficiency promoted by PCF.

VEGF with AMD3100 endogenously mobilizes mesenchymal stem cells and improves fracture healing.

ABSTRACTA significant number of fractures develop non‐union. Mesenchymal stem cell (MSC) therapy may be beneficial, however, this requires cell acquisition, culture and delivery. Endogenous mobilization of stem cells offers a non‐invasive alternative. The hypothesis was administration of VEGF and the CXCR4 antagonist AMD3100 would increase the circulating pool of available MSCs and improve fracture healing. Ex‐breeder female wistar rats received VEGF followed by AMD3100, or sham PBS. Blood prepared for culture and colonies were counted. P3 cells were analyzed by flow cytometry, bi‐differentiation. The effect of mobilization on fracture healing was evaluated with 1.5 mm femoral osteotomy stabilized with an external fixator in 12–14 week old female Wistars. The mobilized group had significantly greater number of cfus/ml compared to controls, p = 0.029. The isolated cells expressed 1.8% CD34, 35% CD45, 61% CD29, 78% CD90, and differentiated into osteoblasts but not into adipocytes. The fracture gap in animals treated with VEGF and AMD3100 showed increased bone volume; 5.22 ± 1.7 µm3 and trabecular thickness 0.05 ± 0.01 µm compared with control animals (4.3 ± 3.1 µm3, 0.04 ± 0.01 µm, respectively). Radiographic scores quantifying fracture healing (RUST) showed that the animals in the mobilization group had a higher healing score compared to controls (9.6 vs. 7.7). Histologically, mobilization resulted in significantly lower group variability in bone formation (p = 0.032) and greater amounts of bone and less fibrous tissue than the control group. Clinical significance: This pre‐clinical study demonstrates a beneficial effect of endogenous MSC mobilization on fracture healing, which may have translation potential to prevent or treat clinical fractures at risk of delayed or non‐union fractures. © 2018 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 37:1294–1302, 2019.

GM-CSF Enhances Mobilization of Bone Marrow Mesenchymal Stem Cells via a CXCR4-Medicated Mechanism.

This study was conducted to investigate the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on the mobilization of mesenchymal stem cells (MSCs) from the bone marrow (BM) into the peripheral blood (PB) in rats. GM-CSF was administered subcutaneously to rats at 50μg/kg body weight for 5 consecutive days. The BM and PB of rats were collected at 1, 3, and 5days during the administration for analysis. Upon GM-CSF administration, the number of mononuclear cells increased rapidly at day 1 both in the BM and PB. This number decreased gradually over time in the BM to below the initial amount by day 5, but was maintained at a high level in the PB until day 5. The colony-forming unit-fibroblasts were increased in the PB by 10.3-fold at day 5 of GM-CSF administration, but decreased in the BM. Compared to GM-CSF, granulocyte-colony stimulating factor (G-CSF) stimulated lower levels of MSC mobilization from the BM to the PB. Immunohistochemical analysis revealed that GM-CSF induced a hypoxic and proteolytic microenvironment and increased C-X-C chemokine receptor type 4 (CXCR4) expression in the BM. GM-CSF added to BM MSCs in vitro dose-dependently increased CXCR4 expression and cell migration. G-CSF and stromal cell derived factor-1 (SDF-1) showed similar results in these in vitro assays. Know-down of CXCR4 expression with siRNA significantly abolished GM-CSF- and G-CSF-induced MSC migration in vitro, indicating the involvement of the SDF-1-CXCR4 interaction in the mechanism. These results suggest that GM-CSF is a useful tool for mobilizing BM MSCs into the PB.

Nanoparticle-modified chitosan-agarose-gelatin scaffold for sustained release of SDF-1 and BMP-2.

BackgroundStromal cell-derived factor 1 (SDF-1) is an important chemokine for stem cell mobilization, and plays a critical role in mobilization of mesenchymal stem cells (MSCs). Bone morphogenetic protein 2 (BMP-2) plays a critical role in osteogenesis of MSCs. However, the use of SDF-1 and BMP-2 in bone tissue engineering is limited by their short half-lives and rapid degradation in vitro and in vivo.MethodsThe chitosan oligosaccharide/heparin nanoparticles (CSO/H NPs) were first prepared via self-assembly. Chitosan-agarose-gelatin (CAG) Scaffolds were then synthesized via gelation technology using cross-linked chitosan, agarose, and gelatin, and were modified by CSO/H NPs. The encapsulation efficiency and release kinetics of SDF-1 and BMP-2 were quantified using an enzyme-linked immunosorbent assay. A CCK-8 assays were used to evaluate biocompatibility of NP-modified scaffolds. The biological activity of the loaded SDF-1 and BMP-2 was evaluated using the transwell migration assay and osteogenic induction assay. An animal MSC recruitment model was used to study the ability of SDF-1 released from NP-modified scaffolds to induce migration of MSCs.ResultsIn this study, we developed a novel nanoparticle-modified CAG scaffold for the delivery of SDF-1 and BMP-2. CCK-8 assays demonstrated excellent biocompatibility of NP-modified scaffolds. In addition, we investigated the release of SDF-1 and BMP-2 from NP-modified scaffolds, and evaluated the effect of released SDF-1 on MSC migration. The effect of released BMP-2 on MSC osteogenesis was also examined. In vitro cell migration assays showed that SDF-1 released from NP-modified scaffolds retained its migration activity; osteogenesis studies demonstrated that released BMP-2 exhibited a strong ability to induce differentiation towards osteoblasts. Our in vivo recruitment assays showed continuous chemotactic response of MSCs to SDF-1 released from the NP-modified scaffold.ConclusionThe simplicity of synthesizing CSO/H NP-modified CAG scaffolds, combined with its high cytokine loading capacity and sustained release effect, renders NP-modified CAG scaffold an attractive candidate for sustained release of SDF-1 and BMP-2 to promote bone repair and regeneration.

Molecular Targeting to Expand the Therapeutic Ratio in Women with Curable Cervical Cancer

Approximately 30-40% of cervical cancer patients treated with radio(chemo)therapy (RTCT) develop recurrence that can be difficult to treat. New approaches for overcoming treatment failures are needed. The CXCL12/CXCR4 chemokine pathway promotes tumor growth and metastasis in various tumor sites and we have observed the pathway is upregulated in RT treated cervical cancers. Plerixafor, an inhibitor of this pathway, is approved clinically and our initial studies showed the addition of plerixafor to RTCT enhanced primary tumor response. The current studies examined: 1. Different ways of sequencing RTCT and plerixafor to maximize efficacy 2. Biomarkers of response to RTCT and plerixafor 3. Effects of plerixafor with RTCT on intestinal toxicity, an important dose-limiting consequence of treatment in these patients. Orthotopic cervix xenografts were treated with RT (30 Gy; 2 Gy/day) and weekly cisplatin (4mg/kg) with or without plerixafor (5mg/kg/day). Plerixafor was administered concurrently with RTCT (3 wks), adjuvantly after RTCT (3 wks) or continuously (6 wks). Biomarker response was evaluated at the end of the concurrent and adjuvant treatments. Tumor growth delay was assessed at later times. Tumor control was assessed after 50 Gy (2Gy/day-5 wks) with cisplatin and plerixafor. Late intestinal toxicity was assessed by histologic examination of the colorectal junction 90 days after treatment. Plerixafor, whether administered concurrently or adjuvantly, prolonged tumor growth delay following RTCT (30 Gy). Adjuvant plerixafor was associated with longer growth delay. Tumor cure was achieved at a higher RT dose of 50 Gy with RTCT+plerixafor. RTCT alone increased CXCL12/CXCR4 signalling, PD-L1 (immune checkpoint marker) expression and the tumor accumulation of myeloid cells. The addition of plerixafor during or after RTCT abrogated these effects. This suggests: 1) RTCT upregulation of the CXCL12/CXCR4 pathway leads to recruitment of immune populations into the tumor that confer treatment resistance and 2) RTCT-induced increases in PD-L1 expression may promote an immunosuppressed tumor microenvironment that impairs treatment response. Plerixafor reduced RT-related intestinal injury, suggesting a protective effect that may relate to modifications of immune cell infiltrates involved with the mobilization of mesenchymal stem cells from the marrow. Further investigation is needed to assess mechanisms underlying these radio-protective effects. Adding plerixafor to RTCT blunts RTCT-induced upregulation of the CXCL12/CXCR4 pathway and reduces the increase in tumor-associated myeloid cells. These benefits may apply to other tumors where RT plays a curative role. Plerixafor protects normal tissue from RT injury. Few if any drugs have been identified previously that both improve the effectiveness of RT and prevent side effects. The combination of RT and plerixafor warrants testing in clinical trials to validate these findings.

Naringin promotes cellular chemokine synthesis and potentiates mesenchymal stromal cell migration via the Ras signaling pathway.

Directional migration of mesenchymal stem cells (MSCs) is known to serve roles in bone fracture healing. Naringin is a traditional medicine used in China to treat bone injury and has been confirmed to act as a chemoattractant to MSCs. In the present study, the secretion of chemokines and stimulation of relevant signaling pathways by naringin were detected to determine the molecular mechanism of naringin-induced MSC migration. In these experiments, Quantibody® arrays were used to detect chemokines secreted by MSCs with or without the addition of naringin. The results revealed differential naringin-induced chemokine secretion of C-X-C motif chemokine (CXCL)5, CXCL6 and C-C motif chemokine 20. Furthermore, the Ras signaling pathway was markedly activated in the naringin-treated groups, suggesting that naringin may enhance the migrational ability of MSCs via Ras activation. Furthermore, naringin was able to promote the secretion of various chemokines derived from MSCs, which would, in turn, increase the mobility of MSCs. The aim of the present study was to provide novel candidate agents for clinical orthopedics and theoretical basis for the future improvement of adjunctive medication for bone fracture healing.

Impact of Wharton Jelly Peptide Р199 on fibroblast and mesenchyme skin cells functioning

Objective — to determine the effect of the sh­oligopeptide­72 on the activity and mobility of mesenchymal stem cells of the skin (MSC) and fibroblasts in vitro.Materials and methods. The primary culture of dermal fibroblasts and MSCs was obtained from healthy donors of 40 years. The metabolic and proliferative activity of cells was investigated using the MTT test and the « wound healing» test. The concentration of the polypeptide from 1 ng/ml to 1000 ng/ml was investigated.Results and discussion. The concentration range of 1—1000 ng/ml does not show any toxic or stimulating effects on differentiated fibroblasts. At the same time, MSCs of the human derma were sensitive to the action of the P199 polypeptide. The concentration of 1—2 ng/ml stimulates the metabolic activity of MSCs. Also, we have detected the differential effect of P199 on migration activity. It is shown stimulating effect on the mobility of MSC of the dermis, but not of dermal fibroblasts.Conclusions. Wharton Jelly Peptide P199 stimulates the metabolic, proliferative and migration activity of dermal MSCs. Skin fibroblasts Have not been affected.

Novel Lipid Signaling Mediators for Mesenchymal Stem Cell Mobilization during Bone Repair.

Mesenchymal stem and progenitor cells (MSCs), which normally reside in the bone marrow, are critical to bone health and can be recruited to sites of traumatic bone injury, contributing to new bone formation. The ability to control the trafficking of MSCs provides therapeutic potential for improving traumatic bone healing and therapy for genetic bone diseases such as hypophosphatasia. In this study, we explored the sphingosine-1-phosphate (S1P) signaling axis as a means to control the mobilization of MSCs into blood and possibly to recruit MSCs enhancing bone growth. Loss of S1P receptor 3 (S1PR3) leads to an increase in circulating CD45-/CD29+/CD90+/Sca1 putative mesenchymal progenitor cells, suggesting that blocking S1PR3 may stimulate MSCs to leave the bone marrow. Antagonism of S1PR3 with the small molecule VPC01091 stimulated acute migration of CD45-/CD29+/CD90+/Sca1+ MSCs into the blood as early as 1.5 hours after treatment. VPC01091 administration also increased ectopic bone formation induced by BMP-2 and significantly increased new bone formation in critically sized rat cranial defects, suggesting that mobilized MSCs may home to injuries to contribute to healing. We also explored the possibility of combining S1P manipulation of endogenous host cell occupancy with exogenous MSC transplantation for potential use in combination therapies. Importantly, reducing niche occupancy of host MSCs with VPC01091 does not impede engraftment of exogenous MSCs. Our studies suggest that MSC mobilization through S1PR3 antagonism is a promising strategy for endogenous tissue engineering and improving MSC delivery to treat bone diseases.