iPS Cell-Derived Cardiogenicity is Hindered by Sustained Integration of Reprogramming Transgenes
Background—Nuclear reprogramming inculcates pluripotent capacity by which de novo tissue differentiation is enabled. Yet, introduction of ectopic reprogramming factors may desynchronize natural developmental schedules. This study aims to evaluate the impact of imposed transgene load on the cardiogenic competency of iPS cells.
Methods and Results—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. Pulsed transgene overexpression, prior to iPS cell differentiation, hindered cardiogenic outcomes. Delayed in counterparts with maintained integrated transgenes, transgene removal enabled proficient differentiation of iPS cells into functional cardiac tissue. Transgene-free iPS cells generated reproducible beating activity with robust expression of cardiac α-actinin, connexin 43, MLC2a, α/β-MHC and troponin I. While operational excitation-contraction coupling was demonstrable in the presence or absence of transgenes, factor-free derivatives exhibited an expedited maturing phenotype with canonical responsiveness to adrenergic stimulation.
Conclusions—A disproportionate stemness load, caused by integrated transgenes, impacts the cardiogenic competency of iPS cells. Offload of transgenes in engineered iPS cells ensures integrity of cardiac developmental programs, underscoring the value of non-integrative nuclear reprogramming for derivation of competent cardiogenic regenerative biologics.
- regenerative medicine
- induced pluripotent stem cells
- nuclear reprogramming
- stem cell
- Received July 24, 2013.
- Revision received June 3, 2014.
- Accepted June 16, 2014.