Characterization of human adipose tissue-derived stromal cells isolated from diabetic patient's distal limbs with critical ischemia.

Adipose tissue is an abundant source of autologous adult stem cells that may bring new therapeutic perspectives on the treatment of diabetes and its complications. It is unclear whether adipose tissue-derived stromal cells (ASCs) of diabetic patients, constantly influenced by hyperglycaemia, have the same properties as non-diabetic controls. As an alternative source of ASCs, adipose tissue from distal limbs of diabetic patients with critical ischemia was isolated. ASCs were characterized in terms of cell surface markers, multilineage differentiation and the expression of vascular endothelial growth factor (VEGFA), chemokine-related genes and compared with non-diabetic controls. Flow cytometry analysis confirmed mesenchymal phenotypes in both diabetic and non-diabetic ASCs. Nevertheless, 40% of diabetic and 20% of non-diabetic ASC samples displayed high expressions of fibroblast marker, which inversely correlated with the expression of CD105. In diabetic patients, significantly decreased expression of VEGFA and chemokine receptor CXCR4 was found in fibroblast-positive ASCs, compared with their fibroblast-negative counterparts. Reduced osteogenic differentiation and the downregulation of chemokine CXCL12 were found in fibroblast-negative diabetic ASCs. Both diabetic and non-diabetic ASCs were differentiated into adipocytes and chondrocytes and did not reveal islet-like cell differentiation. According to this study, adipose tissue from distal limbs of diabetic patients is not satisfactory as an autologous ASC source. Hyperglycaemic milieu as well as other metabolic disorders linked to diabetes may have an influence on endogenous stem cell properties. The present study investigated the feasibility of autologous stem cell therapy in diabetic patients. ASCs isolated from the ischemic limb of diabetic patients were found to be less potent when compared phenotypically and functionally to control non-diabetic counterparts with no signs of limb ischemia. High expression of fibroblast markers associated with reduced expression of VEGFA as well as reduced osteogenic differentiation may have an impact on the effectiveness of autologous cell therapies in diabetic patients.

Intrinsic properties of tumour cells have a key impact on the bystander effect mediated by genetically engineered mesenchymal stromal cells.

BACKGROUND: Engineered mesenchymal stromal cells (MSC) have been used in many preclinical studies of gene directed enzyme/prodrug therapy. We aimed to compare the efficacy of two most frequently used systems, as well as evaluate the extent of a bystander effect mediated by therapeutic MSC towards cell lines derived from different tumours. METHODS: Two approaches were compared: (i) herpes simplex virus thymidine kinase (TK)/ganciclovir (GCV) and (ii) yeast cytosine deaminase fused with uracil phosphoribosyltransferase (CD::UPRT)/5-fluorocytosine (5-FC). The cytotoxic effect mediated by therapeutic MSC was evaluated in direct co-culture by a fluorimetric assay. The expression profile of tumour cells was analyzed by a quantitative polymerase chain reaction, and the ability of gap-junctional intercellular communication (GJIC) was evaluated by a dye transfer assay. RESULTS: Both systems were effective only on glioblastoma cells (8-MG-BA). The CD::UPRT-MSC/5-FC system showed efficiency on melanoma A375 cells. We decreased the sensitivity of 8-MG-BA cells and A375 cells to the CD::UPRT-MSC/5-FC system by pharmacological inhibition of thymidylate synthase, and we achieved a similar result in A375 cells by inhibition of thymidine phosphorylase. Although we demonstrated functional GJIC in A375 cells, TK-MSC were ineffective in mediating the bystander effect similarly to HeLa cells, which were also relatively resistant to CD::UPRT-MSC/5-FC treatment. TK-MSC/GCV treatment had a strong cytotoxic effect on MDA-MB-231 cells (breast carcinoma), whereas CD::UPRT-MSC/5-FC treatment failed as a result of overexpression of the gene for ABCC11. Transfection of the MDA-MB-231 cell line with small interference RNA specific to ABCC11 led to a significantly increased sensitivity to the CD::UPRT-MSC/5-FC approach. CONCLUSIONS: GJIC, expression of enzymes involved in drug metabolism and ABC transporters correlate with the response of tumour cells to treatment by MSC-expressing prodrug-converting genes.