Cloning and expressing of interleukine 2 in amniotic membrane-derived mesenchymal stem cells, as a potent feeder layer.

Abstract

The application of mesenchymal stem cells (MSCs) is rapidly expanding due to their unique properties in cell therapy, especially as the feeder layer in the ex-vivo expansion of immune cells. Also, Interleukin 2 (IL-2) is an essential human cytokine in the expansion of hematopoietic precursors and progenitors, i.e., NK cells and T cells, while there is no endogenous expression of IL-2 in MSCs. This study aimed to examine the potency of amniotic membrane (AM)-MSCs as the IL-2 secretory cells. IL-2-containing pCMV3-C-GFPspark shuttle vector was transformed in E.coli DH5-alpha. After cloning, the plasmid DNA was extracted and transfected in isolated AM-MSCs, by lipofectamine-2000. Then, the RNA and protein expression levels of exogenous IL-2 were evaluated 3 to 15 days after transfection, using ELISA and qRT-PCR. Fluorescent microscopy and flowcytometry assays were used for evaluating the GFP-positivity of transfected AM-MSCs, as IL-2 expression control. There was a significant increase in RNA expression of exogenous IL-2 in transfected AM-MSCs in 3 to 15 days after transfection. (p<0.001) Also, IL-2 concentration released in the medium was increased in 3rd day after transfection (611 pg/ml). However, the RNA and protein expression of IL-2 was reduced through passing the time. The results show AM-MSC is a suitable host for the expression and secretion of IL-2 as a critical cytokine in the ex-vivo expansion of hematopoietic precursors and progenitors, i.e., NK cells and T cells. Also, the survival time of IL-2 expression in AM-MSCs was long enough for use as a feeder layer.