Alteration of immunoregulatory genes expression in mesenchymal stromal cells upon priming with B18R as an interferon binding protein.

Objectives: The B18R protein encoded by the Vaccinia virus decoys Type 1 interferons and inhibits the activity of several type I IFN members. In vitro transcription protocols benefit from this molecule's involvement in enhancing cell viability by inhibiting interferon signal transduction. As a result of their immunomodulatory properties and potential to regenerate, mesenchymal stromal cells (MSCs) are increasingly considered an alternative treatment for a wide range of immune disorders. In this study, we investigated the modification of expression of several genes involved in immune-related pathways after preconditioning MSCs with two immune stimuli, including poly(I:C) and LPS. Materials and Methods: ASCs were isolated and primed with B18R, and after exposure to poly(I:C) and LPS, the expression of the same sets of genes as in the previous experiment was evaluated. Following total RNA isolation from primed cells and cDNA preparation, real-time quantitative PCR was performed for several immunomodulatory and immune-related genes, including IDO1, TDO2, COX-2, TGF- ß 1, TNF- α, IL-1 ß , IL-6, TLR3, TLR4, and MCP-1. Results: Pretreatment of MSCs with poly(I:C) and LPS significantly increased the expression of all mentioned genes, while upon the B18R challenge followed by poly(I:C) and LPS treatment, they were down-regulated. Finally, it was observed that the relative expression level of IFN -ß has significantly decreased in MSCs+B18R+poly(I:C) and LPS in comparison with these groups without B18R. Conclusion: The data indicated that the presence of B18R prevents the overexpression of several immune-related genes, which are overexpressed in the in vitro inflammatory environment.

Allogeneic adipose-derived mesenchymal stromal cell transplantation for refractory lupus nephritis: Results of a phase I clinical trial.

BACKGROUND: Mesenchymal stromal/stem cells (MSCs) are known for their immunomodulatory properties. This study was performed to analyse the effects of MSC transplantation on treatment-resistant lupus nephritis (LN). METHODS: In this phase I trial, nine biopsy-proven LN patients refractory to standard treatments underwent systemic infusion of 2 × 106 allogeneic adipose-derived (AD) MSCs/kg and were followed for 12 months post-intervention. RESULTS: The treatment protocol resulted in no major adverse events. Urine protein levels significantly decreased during the first month post-intervention (baseline vs. month 1 (median): 1800 vs. 1020, P = 0.008), followed by a gradual increase but remained significantly lower than baseline only up to the 3rd month. During the first 3 months post-intervention, complete renal response (proteinuria < 0.5 g/24 h) and partial response (proteinuria > 0.5 g/24 h, but > 50% decrease in proteinuria) were observed in 33.3% and 44.4% of the patients, respectively, though these rates declined thereafter. Median score of Systemic Lupus Erythematosus Disease Activity Index decreased significantly from 16 at the baseline to 6 at sixth months post-treatment (P = 0.007), though it slightly increased at the 12th month follow-up. CONCLUSIONS: Allogenic AD-MSC transplantation was associated with favourable safety and efficient to reduce urine protein excretion and disease activity; however, the maximum effect (greatest improvement in outcomes) was observed at 1 month based on the proteinuria, and 6 months post-intervention based on disease activity scores. A single dose of AD-MSCs may not be adequate to maintain long-term remission of refractory LN, and so, additional doses may be required.

Use of cerium oxide nanoparticles: a good candidate to improve skin tissue engineering.

Today advancements in nanotechnology have made extensive progress in tissue engineering. Application of cerium oxide nanoparticles (CeO2) has improved regenerative medicine due to their antioxidant properties. In this study, nanoparticles were used to increase the efficacy of skin substitutes. Human skin samples were decellularized using four methods and studied via histological stainings and DNA content analyses. Then CeO2 dispersing and its stability were investigated. The prepared acellular dermal matrices (ADMs) were immersed in CeO2 suspension and their effects were evaluated on growth of cultured human adipose derived-mesenchymal stem cells (hAd-MSCs) using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and histological methods. Moreover, their antioxidant properties were assessed based on DPPH degradation. Changes in the collagen contents of the scaffolds containing cells and CeO2 were also determined by electron microscopy and their tensile strength was compared to ADM. Our results indicated that use of trypsin/NaOH protocol resulted in most efficient cell removal while maintaining extracellular matrix (ECM) architecture. Among different dispersal methods, the approach using Dulbecco's modified Eagle's medium (DMEM), wetting with fetal bovine serum (FBS) and ultrasonic bath resulted in the best stability. Furthermore, it was shown that CeO2 not only had no toxicity on the cells, but also increased the growth and survival of hAd-MSCs by about 27%, improved free radical scavenging, as well as the amount of collagen and tensile strength of the scaffolds containing nanoparticles compared to the ADM. It can be concluded that the combination of ADM/CeO2/hAd-MSCs could be a step forward in skin tissue engineering.

Using paracrine effects of Ad-MSCs on keratinocyte cultivation and fabrication of epidermal sheets for improving clinical applications.

Recent advances in wound healing have made cell therapy a potential approach for the treatment of various types of skin defects such as trauma, burns, scars and diabetic leg ulcers. Cultured keratinocytes have been applied to burn patients since 1981. Patients with acute and chronic wounds can be treated with autologous/allograft cultured keratinocytes. There are various methods for cultivation of epidermal keratinocytes used in cell therapy. One of the important properties of an efficient cell therapy is the preservation of epidermal stem cells. Mesenchymal Stem Cells (MSCs) are major regulatory cells involved in the acceleration of wound healing via induction of cell proliferation, angiogenesis and stimulating the release of paracrine signaling molecules. Considering the beneficial effects of MSCs on wound healing, the main aim of the present study is investigating paracrine effects of Adipose-derived Mesenchymal Stem Cell (Ad-MSCs) on cultivation of keratinocytes with focusing on preservation of stem cells and their differentiation process. We further introduced a new approach for culturing isolated keratinocytes in vitro in order to generate epidermal keratinocyte sheets without using a feeder layer. To do so, Ad-MSC conditioned medium was applied as an alternative to commercial media for keratinocyte cultivation. In this study, the expression of several stem/progenitor cell (P63, K19 and K14) and differentition (K10, IVL and FLG) markers was examined using real time PCR on days 7, 14 and 21 of culture in keratinocytes in Ad-MSC conditioned medium. P63 and α6 integrin expression was also evaluated via flow cytometry. The results were compared with control group including keratinocytes cultured in EpiLife medium and our data indicated that this Ad-MSC conditioned medium is a good alternative for keratinocyte cultivation and producing epidermal sheets for therapeutic and clinical purposes. The reasons are the expression of stem cell and differentiation markers and overcoming the requirement for feeder layer which leads to a xenograft-free transplantation. Besides, this approach has low cost and is easier to perform. However, more in vitro and in vivo experiments as well as safety evaluation required before clinical applications.

The effect of platelet-rich plasma on human mesenchymal stem cell-induced bone regeneration of canine alveolar defects with calcium phosphate-based scaffolds.

OBJECTIVES: Autologous bone transplantation known as the "gold standard" to reconstruction of osseous defects has known disadvantages. This study was designed to explore the effects of hydroxy-apatite/tricalcium-phosphate (HA/TCP) and platelet-rich plasma (PRP) on the osteogenesis ability of human adipose-derived mesenchymal stem cells (hAdMSCs) in vitro and in vivo. MATERIALS AND METHODS: hAdMSCs were incubated with HA/TCP granules and/or PRP in vitro and then, cell proliferation and differentiation was assessed by MTT assay, AZR S staining and SEM examination. In vivo, four cylindrical defects were drilled in the mandibular bones of 5 mongrel dogs and divided randomly into the following groups: I-autologous crushed bone, II- no filling material, III- HA/TCP and PRP, IV- PRP-enriched hAdMSCs seeded on HA/TCP granules. Inserted hAdMSCs were labeled to trace their contribution to bone tissue regeneration. Finally, cell tracing and tissue regeneration were evaluated by immunohistochemistry and histomorphometry methods, respectively. RESULTS: In vitro, co-incubation with HA/TCP granules significantly reduced proliferation and osteogenic differentiation ability of hAdMSCs; while PRP application promoted these capacities (P<0.05). In vivo, PRP-enriched hAdMSCs seeded on HA/TCP granules induced considerable bone formation in osseous defects (P<0.05). It was obviously shown that hAdMSCs were incorporated into the newly-formed bone. CONCLUSION: Based on this study, application of stem cells could offer a helpful therapeutic tool in bone tissue regeneration. Although inserted hAdMSCs were identifiable throughout the newly-formed bone tissue, their few number could be an indicator of indirect role of hAdMSCs in tissue regeneration.

Effects of Human Adipose-derived Stem Cells and Platelet-Rich Plasma on Healing Response of Canine Alveolar Surgical Bone Defects.

BACKGROUND: Due to the known disadvantages of autologous bone grafting, tissue engineering approaches have become an attractive method for ridge augmentation in dentistry. To the best of our knowledge, this is the first study conducted to evaluate the potential therapeutic capacity of PRP-assisted hADSCs seeded on HA/TCP granules on regenerative healing response of canine alveolar surgical bone defects. This could offer a great advantage to alternative approaches of bone tissue healing-induced therapies at clinically chair-side procedures. METHODS: Cylindrical through-and-through defects were drilled in the mandibular plate of 5 mongrel dogs and filled randomly as following: I- autologous crushed mandibular bone, II- no filling material, III- HA/TCP granules in combination with PRP, and IV- PRP-enriched hADSCs seeded on HA/TCP granules. After the completion of an 8-week period of healing, radiographic, histological and histomorphometrical analysis of osteocyte number, newly-formed vessels and marrow spaces were used for evaluation and comparison of the mentioned groups. Furthermore, the buccal side of mandibular alveolar bone of every individual animal was drilled as normal control samples (n=5). RESULTS: Our results revealed that hADSCs subcultured on HA/TCP granules in combination with PRP significantly promoted bone tissue regeneration as compared with those defects treated only with PRP and HA/TCP granules (P<0.05). CONCLUSION: In conclusion, our results indicated that application of PRP-assisted hADSCs could induce bone tissue regeneration in canine alveolar bone defects and thus, present a helpful alternative in bone tissue regeneration.