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iPS cell-derived cardiogenicity is hindered by sustained integration of reprogramming transgenes.

Circulation. Cardiovascular genetics (2014-08-01)
Almudena Martinez-Fernandez, Timothy J Nelson, Santiago Reyes, Alexey E Alekseev, Frank Secreto, Carmen Perez-Terzic, Rosanna Beraldi, Hoon-Ki Sung, Andras Nagy, Andre Terzic
ABSTRACT

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 effect of imposed transgene load on the cardiogenic competency of induced pluripotent stem (iPS) cells. 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, before 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, myosin light chain 2a, α/β-myosin heavy chain, and troponin I. Although 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. A disproportionate stemness load, caused by integrated transgenes, affects the cardiogenic competency of iPS cells. Offload of transgenes in engineered iPS cells ensures integrity of cardiac developmental programs, underscoring the value of nonintegrative nuclear reprogramming for derivation of competent cardiogenic regenerative biologics.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Monoclonal Anti-α-Actinin (Sarcomeric) antibody produced in mouse, clone EA-53, ascites fluid
Sigma-Aldrich
Anti-MYL7 antibody produced in rabbit, Prestige Antibodies® Powered by Atlas Antibodies, affinity isolated antibody, buffered aqueous glycerol solution
Sigma-Aldrich
Anti-Connexin 43 Antibody, CT, cytosolic, Chemicon®, from rabbit

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