Abstract 15091: Clonogenic Human Cardiac Stem Cells With Long Telomeres Can Be Isolated From Minute Endomyocardial Biopsies
Autologous cardiac stem cell (CSC) therapy has been implemented in patients with chronic heart failure of ischemic origin. The right atrial appendage, weighing ~1 g, was used for the isolation and expansion of CSCs in the SCIPIO trial; this protocol was also successfully employed ex vivo with myocardial specimens weighing ~5 mg. However, the possibility of utilizing actual endomyocardial biopsies for the preparation of CSCs and their clinical application remained to be determined. A positive result would indicate that patients with advanced heart failure could be enrolled in clinical trials. IRB approval was obtained to test this procedure in consented patients having an endomyocardial biopsy performed for diagnostic purposes. From these subjects, 1-2 additional small samples, weighing ~1 mg, were collected. A predetermined number of 15 such cases was included. The clinical information was purposely kept blinded. Based on previous quantitative data, 1 mg of myocardium contained ~4-6 undifferentiated CSCs.
Samples were dissociated, and cardiac cells were grown. At passage 2, cells were sorted for c-kit and expanded to a target number of 5 x 106 CSCs. Sixty-days were required from tissue digestion to the generation of 5 x 106 CSCs. Our protocol yielded positive results in all 15 cases. CSCs had a population doubling time of 34±7 hours, and a cloning efficiency of 1.1±0.6%. CSCs were then characterized by FACS: the expression of c-kit in non-clonal, lineage negative CSCs varied from 68% to 97%, averaging 80±10%, while the fraction of committed cells was 6±3%. Telomere length measured by flow FISH ranged from 7.2 to 12.2 kbp averaging 8.6±0.8 kbp. Telomerase activity evaluated by qPCR varied from 0.7 to 1.6 x 105 template copy number, with a mean of 1.1±0.4 x 105. The senescence-associated protein p16INK4a comprised 2 to 11% of CSCs, averaging 5.2±2.9%. With the exception of a 2-fold increase in p16INK4a-positive CSCs, all other parameters were consistent with previous studies using the atrial appendage and larger myocardial samples. In conclusion, a significant number of functionally competent CSCs can be obtained from small endomyocardial biopsies, dramatically expanding the target population and the patient enrollment potential of future clinical trials.
- © 2013 by American Heart Association, Inc.