Human Bone Mesenchymal Stem Cell Lentiviral Transduction For The Production Of Extracellular Vesicles Targeted To The Heart Limits Cell Line Expansion

Objectives: Human bone mesenchymal stem cell derived-extracellular vesicles (HBMSC-EV) have been found to have regenerative and anti-inflammatory effects in heart disease, but one of the main obstacles of this treatment modality is the lack of practical delivery to the heart. To make intravenous delivery possible, this study sought to create a human bone mesenchymal stem cell (HBMSC) that secretes HBMSC-EV which will avoid macrophage uptake and home to the heart through lentiviral genetic modification. Methods: The following HBMSC extracellular genetic modifications were selected: CD-47, an anti-phagocytic marker; cardiac targeting peptide (CTP, sequence APWHLSSQYSRT) and ischemic myocardium targeting peptide (IMTP, sequence CSTSMLKAC), peptides both found to have selective cardiac and ischemic myocardium uptake, respectively, through previous biopanning research. Through a 3 rd generation lentiviral system and subsequent antibiotic selection process, wild-type HBMSC were altered to carry these modifications. Results: Three HMBSC lines were created: 1) CD-47 only, 2) CD-47 and CTP anchored to the outer cell membrane by Lamp-2b, and 3) CD-47 and IMTP anchored by Lamp-2b. Preliminary findings show hindered cell proliferation and adherence - cell doubling time was significantly slowed by passage 6 (versus passage 10 in wild-type HBMSC). Also, the genetically modified HBMSC required a 1:2 subculture ratio (versus 1:3-4 in wild-type HBMSC). Conclusions: These genetically-modified HBMSC, engineered with a goal to be better suited for HBMSC-EV production in the treatment of cardiac disease, have significant limitations in cell proliferation and expansion, which will be a significant obstacle to the production of a meaningful quantity of HBMSC-EV. Ongoing studies are being carried out to characterize changes in HBMSC differentiation and in the cell-matrix.