Abstract 11040: Reprogramming Transgene Offload Releases the Cardiogenic Potential of iPS Cells
Objective: Induced pluripotent stem (iPS) cells are bioengineered from somatic sources to acquire embryonic-like developmental potential. This study aims to evaluate the impact of imposed stemness load on the cardiogenic competency required for bona fide regenerative applications of iPS cells.
Rationale: Nuclear reprogramming inculcates pluripotent capacity by which de novo tissue differentiation is enabled. Yet, introduction of ectopic reprogramming factors imposes a stemness burden that may desynchronize natural developmental schedules and confound cardiac lineage specification.
Methods: Targeted inclusion and exclusion of reprogramming transgenes (c-MYC, KLF4, OCT4 and SOX2) was achieved using a drug-inducible and removable cassette according to the piggyBac transposon/transposase system.
Results: Pulsed transgene overexpression, prior to iPS cell differentiation, hindered cardiogenic outcomes. Transgene removal enabled proficient differentiation of iPS cells into cardiac tissue, not observed in counterparts that maintained integrated transgenes. Transgene-free iPS cells yielded reproducible beating activity and robust expression of cardiac α-actinin, connexin 43, MLC2a, α/β-MHC and troponin I. Cardiomyocytes derived from transgene-removed iPS cells exhibited structural and functional maturity with an operational excitation-contraction machinery. Validating the acquired cardiogenic identity, calcium and sodium channel currents orchestrated action potential activity responsive to adrenergic stimulation absent from transgene-containing reprogrammed clones.
Conclusions: : A disproportionate stemness load caused by integrated reprogramming transgenes impacts the cardiogenic competency of iPS cells. Offload of reprogramming transgenes ensured integrity of cardiac developmental programs, underscoring the value of non-integrative approaches for derivation of cardiogenic regenerative biologics.
- © 2013 by American Heart Association, Inc.