Microvesicles derived from mesenchymal stem cells: Potent organelles for induction of tolerogenic signaling

The adult bone contains a number of distinct populations of stem cells, including haematopoietic stem cells, mesenchymal stem cells, endothelial progenitor cells and fibrocytes. While haematopoietic stem cells are required to provide a lifelong supply of blood cells it is thought that the other populations of stem cells play a role in tissue regeneration and potentially disease. The chemokine CXCL12 is produced constitutively in the bone marrow and, acting via CXCR4, is critical in maintaining HSPCs in a quiescent state and retaining all subsets of stem and progenitor cells in the bone marrow environment. The cytokine G-CSF, used clinically to mobilize haematopoietic stem cells for bone marrow transplants, activates the sympathetic nervous system and bone marrow macrophages to reduce the expression of CXCL12 by bone marrow stromal cells, thereby promoting the exit of haematopoietic stem cells from the bone marrow. Understanding the molecular mechanisms underlying G-CSF stimulated mobilization has led to development of CXCR4 antagonists as fast acting mobilizing agents for haematopoietic stem cells. Evidence now suggests that CXCR4 antagonists can similarly mobilize distinct subsets of progenitor cells, namely the endothelial progenitor cells and mesenchymal stem cells, but this requires conditioning of the bone marrow with VEGF rather than G-CSF.     

Functional recovery of sciatic nerve through inside-out vein graft in rats

Objective: Present study aimed at further comprehensive functional, histomorphometrical and immunohistochemical assessment of peripheral nerve regeneration using rat sciatic nerve transection model.Methods: The 10-mm rat sciatic nerve gap was created in rats. In control group nerve stumps were sutured to adjacent muscle and in treatment group the gap was bridged using an inside-out vein graft. In sham-operated group the nerve was manipulated and left intact. All animals underwent walking track analysis test 4, 8, and 12 weeks after surgery.Subsequently, muscle mass measurement was performed to assess reenervation, histological examination to observe the sciatic nerve regeneration morphologically and immunohistochemistry to detect Schwann cells using anti S-100. Results were analyzed using a factorial ANOVA with two between-subjects factors. Bonferroni test for pairwise comparisons was used to examine the effect of treatments.Results: Functional analysis ofmyelinated nerve fibers showed that nerve function improved significantly in the time course in treatment group. However, quantitative morphometrical analysis of myelinated nerve fibers showed that there was no significant difference between 8 and 12 weeks in treatment group. Muscle weight ratio was bigger and weight loss of the gastrocnemius muscle was ameliorated by inside-out vein grafting. The position of positive immunohistochemical reactions further implied that regenerated axons and Schwann cell-like cells existed after vein grafting was performed, and was accompanied by the process of myelination and structural recovery of regenerated nerves.Conclusion: Functional analysis of peripheral nerve repair is far more reliable than quantitative morphometrical analysis     

Poly Lactic-co-Glycolic Acid Nanoparticles Containing Human Gastric Tumor Lysates as Antigen Delivery Vehicles for Dendritic Cell-Based Antitumor Immunotherapy

ABSTRACT

Stimulating antitumor T cells using dendritic cells (DCs) is a novel and promising method in cancer therapy. Poly lactic-co-glycolic acid is one of the best-known polymers used for encapsulating antigen to protect them against proteolytic enzymes. In this study, poly lactic-co-glycolic acid nanoparticles (NPs) were used as DC antigen delivery vehicles in a preclinical model of immunotherapy of gastric cancer. The DCs were generated from peripheral blood monocytes by conventional in vitro differentiation and loaded with either soluble tumor lysate or lysate encapsulated in NPs using a double emulsion/solvent evaporation technique. Morphology of NPs was determined by scanning electron microscopy. Tumor lysate, either in the soluble form or encapsulated in NPs, was loaded into DC and stimulatory capacity was compared using patient-derived autologous CD3+ T cells as responders. The amount of relevant cytokines produced by Ag-loaded DC and in DC/T cell cocultures was evaluated as a measure of initial DC stimulation and T-cell responses, respectively. Significance increases in expression of DC surface molecules (i.e., CD80, CD83, CD86, and Human Leukocyte Antigen–DR (HLA-DR)) and cytokine production by both DC and DC/T cell cocultures (i.e., interleukin (IL)-12:IL-10 and interferon [IFN]-γ:IL-4 ratios) was observed following loading with lysate NP versus controls. The results suggest that NP-encapsulated antigen can shift antitumor T-cell responses toward a Th1 bias, which potentially increases DC vaccine potency in clinical settings.     

PPD extract induces the maturation of human monocyte-derived dendritic cells

Dendritic cells (DCs) are the most powerful antigen presenting cells, capable of inducing T-dependent immune responses even in naive T cells. DCs are of special interest as cellular adjuvants for immunity induction in clinical settings and several methods for their generation and maturation are recently under investigation. The present study was set out to define the effects of PPD (Purified Protein Derivative), a mycobacterial extract used in the tuberculin skin test, on in vitro differentiation and maturation of human monocyte derived dendritic cells. Immature DCs were prepared from the peripheral blood monocytes of healthy volunteers by culturing in a medium supplemented with granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). The resultant cells were then stimulated with PPD extract and their properties such as cell morphology and the expression of key surface molecules were compared with tumor necrosis factor-alpha (TNF-alpha) stimulated immature DCs. Our results suggest that mycobacterial purified extract is as potent as TNF-alpha, a well-established DC stimulator, in induction of maturation in human monocyte derived DCs. We also ruled out the contribution of lipopolysaccharide (LPS) and beta-glucan contamination in maturation effect of PPD preparations. So, PPD as an examined safe material for in vivo consumption could be used to stimulate DC maturation in DC based immunotherapy protocols.     

Rat hair follicle stem cells differentiate and promote recovery following spinal cord injury

Emerging studies of treating spinal cord injury （SCI） with adult stem cells led us to evaluate the effects of transplantation of hair follicle stem cells in rats with a compression-induced spinal cord lesion. Here, we proposed a hypothesis that rat hair follicle stem cell transplantation can promote the recovery of injured spinal cord. Compression-induced spinal cord injury was induced in Wistar rats in this study. The bulge area of the rat vibdssa follicles was isolated, cultivated and characterized with nestin as a stem cell marker. 5-Bromo-2＇-deoxyuridine （BrdU） labeled bulge stem cells were transplanted into rats with spinal cord injury. Immunohistochemical staining results showed that some of the grafted cells could survive and differentiate into oligodendrocytes （receptor-interacting protein positive cells） and neuronal-like cells （~lll-tubulin positive cells） at 3 weeks after transplantation. In addition, recovery of hind limb locomotor function in spinal cord injury rats at 8 weeks following cell transplantation was assessed using the Basso, Beattie and Bresnahan （BBB） locomotor rating scale. The results demon- strate that the grafted hair follicle stem cells can survive for a long time period in vivo and differentiate into neuronal- and glial-like cells. These results suggest that hair follicle stem cells can promote the recovery of spinal cord injury.     

Phenotypic and functional comparison of two distinct subsets of programmable cell of monocytic origin (PCMOs)-derived dendritic cells with conventional monocyte-derived dendritic cells

Dendritic cells (DCs) are professional antigen-presenting cells with the ability to induce primary T-cell responses. They are commonly produced by culturing monocytes in the presence of IL-4 and GM-CSF (cells produced in this manner are called conventional DCs). Here we report the generation of two functionally distinct subsets of DCs derived from programmable cells of monocytic origin (PCMOs) in the presence of IL-3 or tumor necrosis factor alpha (TNF-α). Monocytes were treated with macrophage colony-stimulating factor (M-CSF) and IL-3 for 6 days and then incubated with IL-4 and IL-3 (for IL-3 DCs) or with IL-4, GM-CSF and TNF-α (for TNF-α DCs) for 7 days. Monocytes were then loaded with tumor lysate (used as antigen), and poly (I∶C) was added. The maturation factors TNF-α and monocyte conditioned medium (MCM) were added on days 4 and 5, respectively. The phenotypes of the DCs generated were characterized by flow cytometry, and the cells'' phagocytic activities were measured using FITC-conjugated latex bead uptake. T-cell proliferation and cytokine release were assayed using MTT and commercially available ELISA kits, respectively. We found that either IL-3DCs or TNF-α DCs induce T-cell proliferation and cytokine secretion; the cytokine release pattern showed reduced IL-12/IL-10 and IFN-γ/IL-4 ratios in both types of DCs and in DC-primed T-cell supernatant, respectively, which confirmed that the primed T cells were polarized toward aTh2-type immune response. We concluded that PCMOs are a new cell source that can develop into two functionally distinct DCs that both induce a Th2-type response in vitro. This modality can be used as a DC-based immunotherapy for autoimmune diseases.     

The use of undifferentiated bone marrow stromal cells for sciatic nerve regeneration in rats

In recent years, cell transplantation has become a focus of attention and reliable outcomes have been achieved in regeneration of the sciatic nerve. The effect of undifferentiated bone marrow stromal cells (BMSCs) on peripheral nerve regeneration was studied using a rat sciatic nerve regeneration model. A 10-mm sciatic nerve defect was bridged using an inside-out vein graft (IOVG) filled with undifferentiated BMSCs (2 × 10(7)cells/ml). In the control group, the vein was filled with phosphate buffer saline alone. The regenerated fibres were studied 4, 8 and 12 weeks after surgery. Assessment of nerve regeneration was based on functional (walking track analysis), histomorphometric and immunohistochemical (Schwann cell detection by S100 expression) criteria. The functional study confirmed significant recovery of regenerated axons in the IOVG/BMSC group (P<0.05). Quantitative morphometric analyses of regenerated fibres showed the number and diameter of myelinated fibres in the IOVG/BMSC group were significantly higher than in the control group (P<0.05). This demonstrates the potential for using undifferentiated BMSCs in peripheral nerve regeneration without the limitations of donor-site morbidity associated with isolation of Schwann cells. It also reduces costs because the interval between tissue collection and cell injection is reduced and the laboratory procedures are simpler compared to undifferentiated BMSCs.