Abstract 17501: Enhancing Cardiac Repair by Allogeneic MSC via Downregulation of B2M by CRISPR/Cas9 Technology
Introduction: Allogeneic mesenchymal stem cells (allo-MSC) are convenient cell source for stem cell based therapy. However, immune rejection is a major obstacle for clinical application of allo-MSC to improve cardiac function after myocardial infarction. Immune rejection is due to the presence of MHC class I which is increased during myocardial infarction. We sought to determine whether knockdown of B2M, the light chain of MHC class I, would allow allo-MSC to enhance myocardial repair in rat myocardial infarction model.
Methods and Results: We constructed a lentivirus expressing CRISPR-Cas9 and a CRISPR guide RNA targeting B2M. The lentivirus particles were transfected into MSC which was prepared from 2-week old Lewis rat bone marrow. This CRISPR approach reduced expression of B2M in MSC as assessed by Western blot and immunostaining. The CRISPR/Cas9 based knockdown approach did not alter the expression of MSC markers such as CD90 and CD105, or the functions of exosomes which mediate the paracrine effects of MSC. Exosome was isolated from MSC conditioned media by ultracentrifugation, and the expression of exosome marker CD63 was analyzed by Western blot and flow cytometry. The exosomes isolated from both B2M knockout MSC and normal MSC prevented H2O2-induced mitochondrial dysfunction in cardiomyocytes H9C2 as assessed by Rh123, and prevented apoptosis as assessed by Death ELISA and Annexin V. In a Sprague-Dawley rat myocardial infarction model, the allo-MSCs from Lewis rat with B2M knockdown promoted survival (TUNEL assay and BrdU assay), angiogenesis, restored cardiac function (EF and FS), and inhibited cardiac fibrosis (Massion Tricrome), to a similar extent as auto-MSCs.
Conclusion: This study demonstrates that allo-MSC with B2M knockdown by CRISPR/Cas9 is a convenient and useful cell source to treat myocardial infarction.
Author Disclosures: Y. Li: None. L. Shao: None. Y. Zhang: None. Z. Zhang: None. J. Wang: None. B. Lan: None. L. Zhang: None. C. Liang: None. X. Pan: None. B. Liu: None. F. Li: None. Y. Geng: None. Z. Shen: None.
- © 2016 by American Heart Association, Inc.