Evaluation of the proangiogenic potential of human mesenchymal stem cells genetically modified to overexpress the Leukemia inhibitor factor

• Grant number: 22/11585-9
• Support Opportunities: Scholarships in Brazil - Program to Stimulate Scientific Vocations
• Start date: January 20, 2023
• End date: March 08, 2023
• Field of knowledge: Biological Sciences - Immunology - Applied Immunology
• Principal Investigator: Milena Botelho Pereira Soares
• Grantee: Isabella Theodoro Silva
• Host Institution: Centro de Pesquisas Gonçalo Moniz. Fundação Oswaldo Cruz (Fiocruz). Ministério da Saúde (Brasil). Salvador , SP, Brazil

Abstract

Peripheral arterial disease (PAD) is a chronic vascular disease whose most extreme outcome, critical lower limb ischemia (MCI), is one of the main causes of morbidity and mortality worldwide. Despite its alarming prevalence, PAD remains without effective treatment to recover lost vasculature. Angiogenesis is a biological process that coordinates the formation of new blood vessels, being an essential event for tissue repair, especially in cases of ischemic insult. Mesenchymal stem cells (MSCs) are cells capable of self-renewal, differentiation and secretion of molecules that mediate multiple biological effects, such as tissue regeneration and angiogenesis induction, characterizing an important regenerative medicine tool against ICM. The therapeutic potential of MSCs can be increased through the overexpression of factors of interest, via genetic modifications. Leukemia inhibitory factor (LIF) was recently described as a cytokine capable of increasing the pro-angiogenic potential of MSCs when overexpressed in this cell type. The present project aims to generate a lineage of human mesenchymal stem cells genetically modified to overexpress LIF (MSC_LIF) and to evaluate its pro-angiogenic potential in in vitro and in vivo models. Human MSCs will be isolated from umbilical cord and transduced through a second-generation lentiviral system to express the human LIF gene. The transduction efficiency and LIF protein production will be confirmed by RT-qPCR and ELISA, respectively. The characterization of the generated cells will be performed by the differentiation assay in the three mesodermal lineages and the immunophenotype was evaluated by flow cytometry. Cell proliferation will be evaluated by Population Doubling and CFU-F assay. The analysis of gene expression of the main markers of angiogenesis and perivascular cells will be performed by RT-qPCR. The potential for microvessel formation in vitro will be investigated from the budding assay of aortic rings co-cultured with MSC, MSC_LIF or even the conditioned medium of these cells. (AU)