title: iPS cell-derived cardiogenicity is hindered by sustained integration of reprogramming transgenes.
abstract: 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 effect of imposed transgene load on the cardiogenic competency of induced pluripotent stem (iPS) cells.
  
  
  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, 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.
  
  
  CONCLUSIONS
  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.
authors: From the Center for Regenerative Medicine (A.M.-F., T.J.N., S.R., A.E.A., A.T.), Marriott Heart Disease Research Program, Division of Cardiovascular Diseases (A.M.-F., S.R., A.E.A., A.T.), and Department of Physical Medicine and Rehabilitation (C.P.-T.), General Internal Medicine and Transplant Center (T.J.N., F.S., R.B.), Mayo Clinic, Rochester, MN; and Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada (H.-K.S., A.N.).
authors: Almudena
authors: A
authors: Martinez-Fernandez
authors: From the Center for Regenerative Medicine (A.M.-F., T.J.N., S.R., A.E.A., A.T.), Marriott Heart Disease Research Program, Division of Cardiovascular Diseases (A.M.-F., S.R., A.E.A., A.T.), and Department of Physical Medicine and Rehabilitation (C.P.-T.), General Internal Medicine and Transplant Center (T.J.N., F.S., R.B.), Mayo Clinic, Rochester, MN; and Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada (H.-K.S., A.N.).
authors: Timothy J
authors: TJ
authors: Nelson
authors: From the Center for Regenerative Medicine (A.M.-F., T.J.N., S.R., A.E.A., A.T.), Marriott Heart Disease Research Program, Division of Cardiovascular Diseases (A.M.-F., S.R., A.E.A., A.T.), and Department of Physical Medicine and Rehabilitation (C.P.-T.), General Internal Medicine and Transplant Center (T.J.N., F.S., R.B.), Mayo Clinic, Rochester, MN; and Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada (H.-K.S., A.N.).
authors: Santiago
authors: S
authors: Reyes
authors: From the Center for Regenerative Medicine (A.M.-F., T.J.N., S.R., A.E.A., A.T.), Marriott Heart Disease Research Program, Division of Cardiovascular Diseases (A.M.-F., S.R., A.E.A., A.T.), and Department of Physical Medicine and Rehabilitation (C.P.-T.), General Internal Medicine and Transplant Center (T.J.N., F.S., R.B.), Mayo Clinic, Rochester, MN; and Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada (H.-K.S., A.N.).
authors: Alexey E
authors: AE
authors: Alekseev
authors: From the Center for Regenerative Medicine (A.M.-F., T.J.N., S.R., A.E.A., A.T.), Marriott Heart Disease Research Program, Division of Cardiovascular Diseases (A.M.-F., S.R., A.E.A., A.T.), and Department of Physical Medicine and Rehabilitation (C.P.-T.), General Internal Medicine and Transplant Center (T.J.N., F.S., R.B.), Mayo Clinic, Rochester, MN; and Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada (H.-K.S., A.N.).
authors: Frank
authors: F
authors: Secreto
authors: From the Center for Regenerative Medicine (A.M.-F., T.J.N., S.R., A.E.A., A.T.), Marriott Heart Disease Research Program, Division of Cardiovascular Diseases (A.M.-F., S.R., A.E.A., A.T.), and Department of Physical Medicine and Rehabilitation (C.P.-T.), General Internal Medicine and Transplant Center (T.J.N., F.S., R.B.), Mayo Clinic, Rochester, MN; and Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada (H.-K.S., A.N.).
authors: Carmen
authors: C
authors: Perez-Terzic
authors: From the Center for Regenerative Medicine (A.M.-F., T.J.N., S.R., A.E.A., A.T.), Marriott Heart Disease Research Program, Division of Cardiovascular Diseases (A.M.-F., S.R., A.E.A., A.T.), and Department of Physical Medicine and Rehabilitation (C.P.-T.), General Internal Medicine and Transplant Center (T.J.N., F.S., R.B.), Mayo Clinic, Rochester, MN; and Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada (H.-K.S., A.N.).
authors: Rosanna
authors: R
authors: Beraldi
authors: From the Center for Regenerative Medicine (A.M.-F., T.J.N., S.R., A.E.A., A.T.), Marriott Heart Disease Research Program, Division of Cardiovascular Diseases (A.M.-F., S.R., A.E.A., A.T.), and Department of Physical Medicine and Rehabilitation (C.P.-T.), General Internal Medicine and Transplant Center (T.J.N., F.S., R.B.), Mayo Clinic, Rochester, MN; and Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada (H.-K.S., A.N.).
authors: Hoon-Ki
authors: HK
authors: Sung
authors: From the Center for Regenerative Medicine (A.M.-F., T.J.N., S.R., A.E.A., A.T.), Marriott Heart Disease Research Program, Division of Cardiovascular Diseases (A.M.-F., S.R., A.E.A., A.T.), and Department of Physical Medicine and Rehabilitation (C.P.-T.), General Internal Medicine and Transplant Center (T.J.N., F.S., R.B.), Mayo Clinic, Rochester, MN; and Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada (H.-K.S., A.N.).
authors: Andras
authors: A
authors: Nagy
authors: From the Center for Regenerative Medicine (A.M.-F., T.J.N., S.R., A.E.A., A.T.), Marriott Heart Disease Research Program, Division of Cardiovascular Diseases (A.M.-F., S.R., A.E.A., A.T.), and Department of Physical Medicine and Rehabilitation (C.P.-T.), General Internal Medicine and Transplant Center (T.J.N., F.S., R.B.), Mayo Clinic, Rochester, MN; and Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada (H.-K.S., A.N.). terzic.andre@mayo.edu.
authors: Andre
authors: A
authors: Terzic
mesh_terms: D000185:Actinin / Q000096:biosynthesis; D000818:Animals; D024722:Cardiac Myosins / Q000096:biosynthesis; D002454:Cell Differentiation; D002469:Cell Separation; D065150:Cellular Reprogramming; D018031:Connexin 43 / Q000096:biosynthesis; D004594:Electrophysiology; D005347:Fibroblasts / Q000378:metabolism; D005434:Flow Cytometry; D005821:Genetic Techniques; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D000090062:Kruppel-Like Factor 4; D051379:Mice; D008854:Microscopy, Electron; D018995:Myosin Heavy Chains / Q000096:biosynthesis; D018994:Myosin Light Chains / Q000096:biosynthesis; D019076:Transgenes; D019210:Troponin I / Q000096:biosynthesis
chemical_list: D018031:Connexin 43; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D018994:Myosin Light Chains; D019210:Troponin I; C107184:myosin light chain 2; D000185:Actinin; D024722:Cardiac Myosins; D018995:Myosin Heavy Chains
keywords: induced pluripotent stem cells; nuclear reprogramming; regeneration; regenerative medicine; stem cells