P319Proliferative induced cardiomyocyte precursor cells obtained by direct reprogramming and transcriptional selection

• Published in: European heart journal Vol. 40; no. Supplement_1
• Main Authors: Bachamanda Somesh, D, Klose, K, Juerchott, K, Krueger, U, Kunkel, D, Kurtz, A, Falk, V, Gossen, M, Stamm, C
• Format: Journal Article
• Language: English
• Published: Oxford University Press 01.10.2019
• Subjects: Basic Science - Cardiovascular Development and Anatomy: Stem Cells, Cell Cycle, Cell Senescence, Cell Death
• ISSN: 0195-668X; 1522-9645
• DOI: 10.1093/eurheartj/ehz747.0154

Abstract Objective Enriched populations of proliferative induced cardiomyocyte precursors (iCMPs) generated from cardiac fibroblasts (CF) could represent a potential population for use in therapeutic application. We developed a protocol for generation of iCMPs from cardiac fibroblasts by genetic reprogramming followed by transcriptional selection and induced maturation. Methods Cardiac fibroblasts were reprogrammed into induced cardiomyocyte precursor cells (iCMPs) via transduction with cardiomyogenesis-related transcription factors Gata4, Mef2c, TBX5 and Myocd. Pure population of iCMPs were obtained by molecular beacons (MB) based selection of MYH6/7 expression. The transcriptome was profiled by RNA sequencing. For maturation iCMPs were treated with 5'-azacytidine and cultured in medium containing ascorbic acid and TGFβ1. The cells were monitored regularly for formation of sarcomere structures, cardiac marker expression and contractions. iCMPs were transplanted into MI mice and monitored for 6 weeks. At the end of 6 weeks, the mice were sacrificed and hearts were explanted and cryopreserved to examine the heart structure and scare size. Cardiac function was monitored by echocardiography. Results iCMPs expressed troponin T, α-actinin and myosin heavy chain (MHC) protein observed by immunocytology. These iCMPs could robustly proliferative and maintain a stable phenotype. Global transcriptome analysis revealed that the iCMP gene expression profile is unique from those of the parental CFs and adult CMs. With Gene Ontology (GO) analysis, we found that iCMPs show upregulation of genes associated with cardiac development, differentiation and morphogenesis while they showed downregulation of genes associated to cell-proliferation in comparison to their parental CFs. Evaluation of selected gene sets showed downregulation of non-myocyte genes, upregulation of transcription factors and upregulation of certain functional and structural genes like ion channel genes and contractile genes. Maturation studies showed that iCMPs gradually changed morphology after stimulation with 5-Aza, as seen in immunofluorescence staining. Cells expressing cTnT showed formation of sarcomeric striations. In vivo translational studies showed reduction of heart function in control groups treated with PBS or CF whereas the iCMPs group showed an improvement in function, reflected by left ventricular ejection fraction and fractional shortening as evaluated by echocardiography. Additionally we noticed an improvement in left ventricular wall thickness at diastole in mice transplanted with iCMPs. Conclusions Gene expression pattern suggests that these iCMPs represent an intermediate state of cardiogenic population that can be expanded to yield therapeutic cell doses. When transplanted to mouse hearts following myocardial infarction, they improved cardiac function.