Abstract 11472: Direct Induction of Human Cardiac Progenitor Cells to Functional Cardiomyocytes by Defined Factors
Background- The mortality of hypoplastic left heart syndrome (HLHS), characterized by hypoplasia of the left heart and aorta, remains the highest among congenital heart disease. Although recent work suggests that three transcription factors are sufficient to induce cardiac fibroblasts to undergo cardiomyocyte-like cells in mice, the possible effects of reprogramming by these defined factors in human remain to be investigated.
Methods- Cardiac tissue specimens were obtained during cardiac surgery in patients with congenital heart disease. Cardiac progenitor cells (CPCs) were isolated and transduced with lentiviral vectors expressing human Tbx5, GATA4, and Mef2C. Differentiated human cardiomyocytes were characterized by real time RT-PCR, global gene expression profile, and immunohistochemistry. For cell tracking, alpha-MHC promoter directing fluorescent proteins (eGFP) were transduced as well as a mitochondoria labeling fluorescent dye, tetramethylrhodamine methyl ester perchlorate (TMRM), was used. Reprogrammed CPCs were kept static or stimulated with cyclic mechanical stretch (1Hz, 10% elongation) to test cardiogenic induction.
Results- RT-PCR analysis showed that HLHS-derived CPC clones exhibit significant reduction of cardiac transcription factors, Nkx2.5 and Hand1, compared with biventricles; however, lentiviral infection of human Tbx5, GATA4, and Mef2C directly converted HLHS-derived CPCs into functional cardiomyocytes. Cardiomocyte induction was detected by eGFP positive cells 7 days after infection and that was increased in number up to 15% at day-14. These observations were verified by the expression and protein levels of cardiac troponin-T and sarcomeric actin. TMRM uptake was found 45% and 60% of induced human cardiomyocytes at day-7 and -14, respectively. In addition, cyclic mechanical stretch significantly enhanced cardiac conversion by myocyte organization and elongation.
Conclusions- Cyclic mechanical stretch is a crucial physiological and pathological factor in the process of direct cardiac conversion of human CPCs. Our study provides a novel strategy to generate functional human cardiomyocytes to explore disease mechanisms as well as for cell therapy in patients with congenital heart malformation.
- Stem cell biology
- Stem/progenitor cells
- Congenital heart disease
- Heart failure
- Regenerative medicine stem cells
- © 2011 by American Heart Association, Inc.