Liver Macrophage Depletion Ameliorates The Effect of Mesenchymal Stem Cell Transplantation in a Murine Model of Injured Liver.

Mesenchymal stem cells (MSCs) therapy show different levels of effectiveness in the context of different types of liver damage, suggesting that the microenvironment of the injured liver is a key determinant for effective stem cell therapy. The objective was to assess the modulatory effect of hepatic stem cell niche components on the transplanted MSCs during liver injury induced by carbon tetrachloride (CCl4). Superparamagnetic iron oxide (SPIO)-labeled human MSCs were injected intravenously into mice treated with CCl4 and subjected to hepatic macrophage-depletion. Liver tissues were collected at different intervals post transplantation for subsequent histopathological, morphometric, immunohistochemical, gene expression and ultrastructural studies. The homing of the transplanted MSCs was evidenced by tracing them within the niche by iron staining and immunohistochemical studies. MSCs differentiated into hepatocyte-like cells and intimal smooth muscle cells as evidenced by their expression of human albumin and α-smooth muscle actin with a concomitant increase in the level of mouse hepatocyte growth factor. A post transplantation reduction in the liver fibro-inflammatory reaction was found and was promoted by liver macrophages depletion. Thus, it could be concluded from the present study that prior manipulation of the microenvironment is required to improve the outcome of the transplanted cells.

Correction of aberrant pre-mRNA splicing by antisense oligonucleotides in beta-thalassemia Egyptian patients with IVSI-110 mutation.

The splicing mutation in intron 1 of beta-globin gene (IVS1-110) is the most common mutation in Egyptian thalassemics that causes aberrant splicing of pre-mRNA and deficient beta-globin chain synthesis. Antisense oligonucleotides (ASONs) are compounds that redirect pre-mRNA splicing and modify gene expression. Our aim was ex vivo correction of the aberrant splicing of beta-globin110 pre-mRNA by ASON against the 3' aberrant splice site. Peripheral blood mononuclear cells of 10 thalassemic patients with IVS1-110 mutation were duplicated and 1 was treated with 20 micromoL/mL morpholino ASON targeted against the 3' aberrant splice site. The level of total hemoglobin (Hb), fetal Hb, and mRNA were estimated in the duplicate samples. Five cases (50%) showed correction with ASON treatment, of which 2 cases showed the appearance of corrected mRNA band with absence of the aberrant band and 3 cases showed an increased ratio of the corrected to the aberrant mRNA band from 2:1 to 3:1, and 4:1. The total Hb showed significant increase in the 5 corrected cases. In conclusion, ASON can restore correct splicing of beta-globin pre-mRNA leading to correct gene product in cultured erythropoietic cells. These results suggest the applicability of ASON for the treatment of thalassemia.

Ex vivo expansion of stem cells: defining optimum conditions using various cytokines.

With the increasing information on the number, quality, and characteristics of hematopoietic stem cells (HSC) in umbilical cord and placental blood, this material has been found to be efficacious as an alternative source of HSC for transplantation in children. In this study, we sought to define the optimal conditions for ex vivo expansion of cord blood (CB) stem cells. These conditions include: the combinations and concentrations of hematopoietic growth factors (stem cell factor [SCF], granulocyte-macrophage colony-stimulating factor [GM-CSF], interleukin [IL]-3, thrombopoietin [Tpo], IL-6 and Fms-like tyrosine kinase 3 ligand [Flt-3L]), the duration of culture, and the effect of serum supplementation. In this study, 2 protocols were applied for ex vivo expansion of CB stem cells. In protocol I, 20 CB samples were expanded in a static, serum-added, liquid culture for 7 and 11 days using 5 cytokine cocktails. In protocol II, 10 CB samples were expanded for 7 days using cytokines of cocktail 1, with and without IL-6 and Flt-3L, in serum-added and serum-free culture media. This protocol was intended to verify the effect of IL-6, Flt-3L, and the role of serum supplementation in short-term liquid culture. From the present study, it can be concluded that cocktail 1 is the cocktail of choice for ex vivo expansion of CB stem cells in serum-free, liquid culture expanded for 7 days. We can also conclude that culture expanded for 7 days is better than 11 days, as the fold expansion of CD34+ cells was not significantly increased or even decreased in some of the cocktails used. Moreover, the percent of CD95+ cells (apoptotic cells) was significantly increased on day 11 compared to day 7 in the cocktails tested.