Leukemia Inhibitory Factor (LIF) overexpression increases angiogenic potential of mouse derived bone marrow mesenchymal stem/stromal cells

Abstract

Background & Aim Mesenchymal stem/stromal cells (MSCs) have the ability to secrete bioactive molecules, exerting multiple biological effects, such as induction of regeneration of injured tissues and neovascularization. The therapeutic potential of MSCs can be increased by genetic modification for overexpression of specific growth factors. Here we produced mouse bone marrow-derived MSCs lines overexpressing leukemia inhibitory factor (LIF), to assess their potential on immunomodulation and angiogenesis. Methods, Results & Conclusion Methods A second generation lentiviral system was used to modify MSCs obtained from the bone marrow of C57Bl/6 mice. The coding sequence of hLIF was amplified by PCR and subcloned into a lentiviral transfer vector containing a constitutive promoter and a selection marker. hLIF expression and secretion was quantified by qRT-PCR and by ELISA. A selected cell line was characterized by immunophenotyping, trilineage differentiation assay, and gene expression. The immunomodulatory effects of hLIF were evaluated on lymphoproliferation assay. The proangiogenic potential of conditioned medium (CM) and MSC_LIF was assessed by wound healing assay. Additionally, in vitro tube formation of endothelial cells and in vivo Matrigel plug assay were performed for evaluation of angiogenesis and tissue vascularization. Results Transduced MSCs effectively expressed hLIF after puromycin selection, as shown by qRT-PCR and ELISA. MSC_LIF cells presented immunophenotype characteristic of MSCs and were able to differentiate into osteocytes, chondrocytes and adipocytes, indicating the maintenance of stromal phenotype after genetic modification. Moreover, the immunomodulatory capacity of MSC_LIF to inhibit lymphocyte proliferation was maintained, compared to wild-type(WT) MSCs. LIF-transgenic MSCs had an increased expression of angiogenesis-related genes and had a potent angiogenic activity in vitro, as shown by the significant higher number of endothelial tubes formed in mouse aortic ring assays co-cultured with MSC_LIF or incubated with CM, compared with MSCs and negative controls. Moreover, in a wound healing assay, HUVECs showed increased migratory capacity in the presence of MSC_LIF CM when compared to WT_MSCs. Conclusions We generated a genetically-modified mouse cell line overexpressing LIF, with proangiogenic effects that can be tested in different experimental models, allowing a better understanding of its therapeutic potential and the effects of hLIF on MSCs' biology and tissue regeneration.