Leukemia Inhibitory Factor (LIF) Overexpression Increases the Angiogenic Potential of Bone Marrow Mesenchymal Stem/Stromal Cells.

Girlaine Café Santos,Bruno Diaz Paredes,Vitor Fortuna,Gabriela Louise Sampaio,Daniela Nascimento Silva,Thaís Alves De Santana,Brysa Mariana Silveira,Iasmim Diniz Orge,Ricardo Ribeiro-Dos-Santos,Milena Botelho Pereira Soares

doi:10.3389/fcell.2020.00778

Abstract

Mesenchymal stem/stromal cells (MSCs) have the ability to secrete bioactive molecules, exerting multiple biological effects, such as tissue regeneration, reduction of inflammation, and neovascularization. The therapeutic potential of MSCs can be increased by genetic modification to overexpress cytokines and growth factors. Here we produced mouse MSCs overexpressing human leukemia inhibitory factor (LIF) to assess their proangiogenic potential in vitro and in vivo. Mouse bone marrow-derived MSCs were transduced by using a second-generation lentiviral system to express human LIF. Leukemia inhibitory factor expression was confirmed by RT-qPCR and by ELISA, allowing the quantification of the transcript and secreted protein, respectively. Flow cytometry analysis and trilineage differentiation assay showed that the MSC_LIF cell line maintained the immunophenotype and a multipotency characteristic of MSCs. The immunosuppressive activity of MSC_LIF was confirmed using a lymphoproliferation assay. Moreover, gene expression analysis demonstrated upregulation of genes coding for strategic factors in the neovascularization process, such as angiogenin, IL-8, MCP-1, and VEGF, and for the perivascular cell markers αSMA, Col4a1, SM22, and NG2. To evaluate the pro-angiogenic potential of MSC_LIF, we first tested its effects on endothelial cells obtained from umbilical vein in a scratch wound healing assay. Conditioned medium (CM) from MSC_LIF promoted a significant increase in cell migration compared to CM from control MSC. Additionally, in vitro tube formation of endothelial cells was increased by the presence of MSC_LIF, as shown in microvessel sprouting in aortic ring cultures. Finally, an in vivo Matrigel plug assay was performed, showing that MSC_LIF were more potent in promoting in vivo angiogenesis and tissue vascularization than control MSCs. In conclusion, LIF overexpression is a promising strategy to increase the proangiogenic potential of MSCs and sets precedents for future investigations of their potential applications for the treatment of ischemic diseases and tissue repair.

Highlights

The potential of mesenchymal stem/stromal cells (MSCs), a cell population obtainable from different sources in the adult organism, has been intensely explored in the past decades for the development of cell and gene therapies
Despite maintaining a fibroblastlike morphology, MSCs overexpressing LIF (MSC_LIF) cells displayed a change in their distribution in culture, showing a tendency to spontaneously organize in circular structures (Figure 2D), compared to nontransduced control cultures (Figure 2E)
The production of the protein was confirmed by enzyme-linked immunosorbent assay (ELISA), in the supernatants of MSC_LIF cells collected after 24 h (p < 0.001), 48 h (p < 0.001), and 72 h of culture (p < 0.001) (Figure 2G)

Summary

Introduction

The potential of mesenchymal stem/stromal cells (MSCs), a cell population obtainable from different sources in the adult organism, has been intensely explored in the past decades for the development of cell and gene therapies. The main therapeutic properties of MSCs are attributed to their ability to secrete an array of soluble bioactive molecules, such as cytokines and growth factors, important in the regulation of several biological processes, including inflammation and fibrosis, cell growth, tissue repair and angiogenesis (Caplan and Dennis, 2006; Parekkadan et al, 2007; Kim et al, 2019). Several studies have investigated the therapeutic potential of MSCs for ischemic diseases, stimulating the development of MSCs-based therapies and the identification of factors and molecules responsible for their proangiogenic potential (Gearing et al, 1987). The direct effect of LIF on endothelial cells appears to inhibit their angiogenic capacity, while populations of stem cells are stimulated to secrete important proangiogenic growth factors in the presence of LIF, contributing to neovascularization. The levels of LIF secretion by MSCs may be relevant for the therapeutic effects of these cells (Chen et al, 2014; Nicola and Babon, 2015)

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Results

Conclusion