Saman Eghtesad

Rapamycin ameliorates dystrophic phenotype in mdx mouse skeletal muscle.

Duchenne muscular dystrophy (DMD) Is an X-linked, lethal, degenerative disease that results from mutations In the dystrophin gene, causing necrosis and inflammation in skeletal muscle tissue. Treatments that reduce muscle fiber destruction and immune cell infiltration can ameliorate DMD pathology. We treated the mdx mouse, a model for DMD, with the immunosuppressant drug rapamycin (RAPA) both locally and systemically to examine its effects on dystrophic mdx muscles. We observed a significant reduction of muscle fiber necrosis in treated mdx mouse tibialis anterior (TA) and diaphragm (Dia) muscles 6 wks post-treatment. This effect was associated with a significant reduction in infiltration of effector CD4+ and CD8+ T cells in skeletal muscle tissue, while Foxp3+ regulatory T cells were preserved. Because RAPA exerts its effects through the mammalian target of RAPA (mTOR), we studied the activation of mTOR in mdx TA and Dia with and without RAPA treatment. Surprisingly, mTOR activation levels in mdx TA were not different from control C57BL/10 (B10). However, mTOR activation was different in Dia between mdx and B10; mTOR activation levels did not rise between 6 and 12 wks of age in mdx Dia muscle, whereas a rise in mTOR activation level was observed in B10 Dia muscle. Furthermore, mdx Dia, but not TA, muscle mTOR activation was responsive to RAPA treatment.

Comparative Evaluation of Differentiation Potential of Menstrual Blood- Versus Bone Marrow- Derived Stem Cells into Hepatocyte-Like Cells

Menstrual blood has been introduced as an easily accessible and refreshing stem cell source with no ethical consideration. Although recent works have shown that menstrual blood stem cells (MenSCs) possess multi lineage differentiation capacity, their efficiency of hepatic differentiation in comparison to other stem cell resources has not been addressed so far. The aim of this study was to investigate hepatic differentiation capacity of MenSCs compared to bone marrow-derived stem cells (BMSCs) under protocols developed by different concentrations of hepatocyte growth factor (HGF) and oncostatin M (OSM) in combination with other components in serum supplemented or serum-free culture media. Such comparison was made after assessment of immunophenotye, trans-differentiation potential, immunogenicity and tumorigeicity of these cell types. The differential expression of mature hepatocyte markers such as albumin (ALB), cytokeratin 18 (CK-18), tyrosine aminotransferase and cholesterol 7 alpha-hydroxylase activities (CYP7A1) at both mRNA and protein levels in differentiating MenSCs was significantly higher in upper concentration of HGF and OSM (P1) compared to lower concentration of these factors (P2). Moreover, omission of serum during differentiation process (P3) caused typical improvement in functions assigned to hepatocytes in differentiated MenSCs. While up-regulation level of ALB and CYP7A1 was higher in differentiated MenSCs compared to driven BMSCs, expression level of CK-18, detected level of produced ALB and glycogen accumulation were lower or not significantly different. Therefore, based on the overall comparable hepatic differentiation ability of MenSCs with BMSCs, and also accessibility, refreshing nature and lack of ethical issues of MenSCs, these cells could be suggested as an apt and safe alternative to BMSCs for future stem cell therapy of chronic liver diseases.

Efficient generation of functional hepatocyte‐like cells from menstrual blood‐derived stem cells

In recent years, the advantages of menstrual blood‐derived stem cells (MenSCs), such as minimal ethical considerations, easy access and high proliferative ability, have inspired scientists to investigate the potential of MenSCs in cell therapy of different diseases. In order to characterize the potency of these cells for future cell therapy of liver diseases, we examined the potential of MenSCs to differentiate into hepatocytes, using different protocols. First, the immunophenotyping properties and potential of MenSCs to differentiate into osteoblasts, adipocytes and chondrocytes were evaluated. Thereafter, the differentiation protocols developed by two concentrations of hepatocyte growth factor (HGF) and oncostatin M (OSM), in combination with other components in serum‐supplemented or serum‐free culture media, were also investigated. The sequential differentiation was monitored by real‐time PCR, immunostaining and functional assays. Our primary data revealed that the isolated MenSCs exhibited mesenchymal stem cell markers in parallel to OCT‐4 as an embryonic marker. Regardless of differentiation procedures, the developed cells expressed mature hepatocyte markers, such as albumin, tyrosine aminotransferase and cytokeratin‐18 at the mRNA and protein levels. They also showed functional properties of hepatocytes, including albumin secretion, glycogen storage and cytochrome P450 7A1 expression. However, the degree of differentiation was dependent on the concentrations of HGF and OSM. Indeed, omission of serum during the differentiation process caused typical improvement in hepatocyte‐specific functions. This study is a novel report demonstrating the differentiation potential of MenSCs into hepatocyte‐like cells. We recommend a complementary serum‐free differentiation protocol for enrichment of in vitro production of functional MenSC‐derived hepatocyte‐like cells that could lead to a major step toward applied stem cell therapy of chronic liver diseases. Copyright

Quercetin attenuates warfarin-induced vascular calcification in vitro independently from matrix Gla protein.

Background: Molecular mechanism(s) of warfarin-induced vascular calcification are not well known. Results: Inhibition of β-catenin signaling with shRNA or quercetin prevents osteoblastic transformation and calcification in VSMCs and calcification in aortic rings treated with warfarin, independent from MGP and protein carboxylation. Conclusion: Quercetin inhibits vascular calcification via β-catenin signaling. Significance: Quercetin may be instrumental in treatment of warfarin-induced vascular calcification. Warfarin can stimulate vascular calcification in vitro via activation of β-catenin signaling and/or inhibition of matrix Gla protein (MGP) carboxylation. Calcification was induced in vascular smooth muscle cells (VSMCs) with therapeutic levels of warfarin in normal calcium and clinically acceptable phosphate levels. Although TGF/BMP and PKA pathways are activated in calcifying VSMCs, pharmacologic analysis reveals that their activation is not contributory. However, β-catenin activity is important because inhibition of β-catenin with shRNA or bioflavonoid quercetin prevents calcification in primary human VSMCs, rodent aortic rings, and rat A10 VSMC line. In the presence of quercetin, reactivation of β-catenin using the glycogen synthase kinase-3β (GSK-3β) inhibitor LiCl restores calcium accumulation, confirming that quercetin mechanism of action hinges on inhibition of the β-catenin pathway. Calcification in VSMCs induced by 10 μm warfarin does not associate with reduced levels of carboxylated MGP, and inhibitory effects of quercetin do not involve induction of MGP carboxylation. Further, down-regulation of MGP by shRNA does not alter the effect of quercetin. These results suggest a new β-catenin-targeting strategy to prevent vascular calcification induced by warfarin and identify quercetin as a potential therapeutic in this pathology.

Comparative Effect of Human Platelet Derivatives on Proliferation and Osteogenic Differentiation of Menstrual Blood-Derived Stem Cells

Menstrual blood has been recognized as an easily accessible and inexpensive source of stem cells, in recent years. To establish a safe and efficient protocol for development of menstrual blood-derived stem cells (MenSCs) into osteoblasts, the effect of substitution of fetal bovine serum (FBS) with human platelet derivatives (HPDs) was evaluated during proliferation and osteogenic differentiation of MenSCs. To this aim, parallel experiments were carried out on cultured MenSCs in the presence of platelet-rich plasma, platelet-poor plasma, platelet gel supernatant, or human platelet releasate (HPR), and compared with cells cultured in conventional growth medium containing FBS. There was no significant difference between growth curves of cultured MenSCs in presence of different fortified media. However, the MenSCs demonstrated variant differentiation patterns in response to FBS replacement with HPDs. Mineralization, as judged by Alizarin red staining, was significantly higher in cells differentiated in the presence of HPR compared to cells that were fortified with other medium supplements. A greater osteocalcin production level, alkaline phosphatase activity, and mRNA expression of osteogenic-specific genes in differentiated MenSCs under HPR condition further confirmed our previous findings. Based on our data, FBS substitution by HPDs not only allows for successful MenSCs proliferation, but also promotes MenSCs development into osteoblasts. The effectiveness of HPR on osteogenic differentiation of MenSCs represents an important novel step toward safe and applied stem cell therapy of bone diseases.

The companions: regulatory T cells and gene therapy

Undesired immunological responses to products of therapeutic gene replacement have been obstacles to successful gene therapy. Understanding such responses of the host immune system to achieve immunological tolerance to a transferred gene product is therefore crucial. In this article, we review relevant studies of immunological responses to gene replacement therapy, the role of immunological tolerance mediated by regulatory T cells in down‐regulating the unwanted immune responses, and the interrelationship of the two topics.

Effect of rapamycin on immunity induced by vector-mediated dystrophin expression in mdx skeletal muscle

Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene. Therapeutic gene replacement of a dystrophin cDNA into dystrophic muscle can provide functional dystrophin protein to the tissue. However, vector-mediated gene transfer is limited by anti-vector and anti-transgene host immunity that causes rejection of the therapeutic protein. We hypothesized that rapamycin (RAPA) would diminish immunity due to vector-delivered recombinant dystrophin in the adult mdx mouse model for DMD. To test this hypothesis, we injected limb muscle of mdx mice with RAPA-containing, poly-lactic-co-glycolic acid (PLGA) microparticles prior to dystrophin gene transfer and analyzed treated tissue after 6 weeks. RAPA decreased host immunity against vector-mediated dystrophin protein, as demonstrated by decreased cellular infiltrates and decreased anti-dystrophin antibody production. The interpretation of the effect of RAPA on recombinant dystrophin expression was complex because of an effect of PLGA microparticles.

Effects of irradiating adult mdx mice before full-length dystrophin cDNA transfer on host anti-dystrophin immunity

Duchenne muscular dystrophy is a fatal, genetic disorder in which dystrophin-deficient muscle progressively degenerates, for which dystrophin gene transfer could provide effective treatment. The host immune response to dystrophin, however, is an obstacle to therapeutic gene expression. Understanding the dystrophin-induced host immune response will facilitate the discovery of strategies to prolong expression of recombinant dystrophin in dystrophic muscle. Using whole-body irradiation of the dystrophic mdx mouse before gene transfer, we temporally removed the immune system; a 600u2009rad dose removed peripheral immune cells, which were restored by self-reconstitution, and a 900u2009rad dose removed central and peripheral immune cells, which were restored by adoptive transfer of bone marrow from a syngeneic, dystrophin-normal donor. The anti-dystrophin humoral response was delayed and dystrophin expression was partially preserved in irradiated, vector-treated mice. Nonirradiated, vector-treated control mice lost muscle dystrophin expression completely, had an earlier anti-dystrophin humoral response and demonstrated muscle fibers focally surrounded with T cells. We conclude that dystrophin gene transfer induced anti-dystrophin humoral immunity and cell-mediated responses that were significantly diminished and delayed by temporal removal of the host central or peripheral immune cells. Furthermore, manipulation of central immunity altered the pattern of regulatory T cells in muscle.Duchenne muscular dystrophy is a fatal, genetic disorder in which dystrophin-deficient muscle progressively degenerates, for which dystrophin gene transfer could provide effective treatment. The host immune response to dystrophin, however, is an obstacle to therapeutic gene expression. Understanding the dystrophin-induced host immune response will facilitate the discovery of strategies to prolong expression of recombinant dystrophin in dystrophic muscle. Using whole-body irradiation of the dystrophic mdx mouse before gene transfer, we temporally removed the immune system; a 600 rad dose removed peripheral immune cells, which were restored by self-reconstitution, and a 900 rad dose removed central and peripheral immune cells, which were restored by adoptive transfer of bone marrow from a syngeneic, dystrophin-normal donor. The anti-dystrophin humoral response was delayed and dystrophin expression was partially preserved in irradiated, vector-treated mice. Nonirradiated, vector-treated control mice lost muscle dystrophin expression completely, had an earlier anti-dystrophin humoral response and demonstrated muscle fibers focally surrounded with T cells. We conclude that dystrophin gene transfer induced anti-dystrophin humoral immunity and cell-mediated responses that were significantly diminished and delayed by temporal removal of the host central or peripheral immune cells. Furthermore, manipulation of central immunity altered the pattern of regulatory T cells in muscle.

Binding of pro-migratory serum factors to electrospun PLLA nano-fibers

Architecture of the poly(l-lactic acid) (PLLA) scaffolds is known to affect protein affinity and binding strength. Here, we demonstrate that nanofibrous electrospun PLLA scaffolds reversibly absorb the pro-migratory serum factors that stimulate migration of vascular smooth muscle via an NFkB-dependent mechanism. Further, we demonstrate that mesenchymal stem cells seeded on the PLLA scaffolds do not enhance muscle migration but may maintain the ability of induced cells to migrate in an NFkB-independent manner. These findings further support the promising application of PLLA scaffolds for therapeutic angiogenesis and vascular graft engineering.

Gene therapy for muscular dystrophy reaches human clinical trial

tified than at other times. Consideration of the percentage decrease in cognitive ability in a general population may be a powerful way of indentifying new causes of abnormal brain development. Indeed, with this method, it is possible to include large numbers of subjects systematically, improving confidence intervals. What mechanism is responsible of the observed effect? This point is carefully discussed by the authors. One possibility is mother-to-child transmission of the virus, as for rubella, CMV, varicella-zoster virus, or human immunodeficiency virus-1 infection, in a small percentage of infected women (influenced by the specific virus and the intensity of viremia, the socalled “viral load”). As for other viruses, following flu pandemics, increases in spontaneous abortions and brain malformations have been described. However, the relatively small decrease in IQ described here in otherwise apparently normal individuals seems less consistent with direct viral replication in the fetal brain as the underlying cause. Instead, it suggests a role for indirect factors linked to the maternal illness (including soluble factors of inflammation and the consequences of transient breathing or eating difficulties in the mother) early in gestation.