Nagy, András

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.Methods and Results—Targeted inclusion and exclusion of reprogramming transgenes (c-MYC,KLF4,OCT4, andSOX2) 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.

Targeted disruption of the histidine decarboxylase gene (HDC −/− ), the only histamine-synthesizing enzyme, led to a histamine-deficient mice characterized by undetectable tissue histamine levels, impaired gastric acid secretion, impaired passive cutaneous anaphylaxis, and decreased mast cell degranulation. We used this model to study the role of histamine in bone physiology. Compared with WT mice, HDC −/− mice receiving a histamine-free diet had increased bone mineral density, increased cortical bone thickness, higher rate of bone formation, and a marked decrease in osteoclasts. After ovariectomy, cortical and trabecular bone loss was reduced by 50% in HDC −/− mice compared with WT. Histamine deficiency protected the skeleton from osteoporosis directly, by inhibiting osteoclastogenesis, and indirectly, by increasing calcitriol synthesis. Quantitative RT-PCR showed elevated 25-hydroxyvitamin D-1α-hydroxylase and markedly decreased 25-hydroxyvitamin D-24-hydroxylase mRNA levels. Serum parameters confirming this indirect effect included elevated calcitriol, phosphorus, alkaline phosphatase, and receptor activator of NF-κB ligand concentrations, and suppressed parathyroid hormone concentrations in HDC −/− mice compared with WT mice. After ovariectomy, histamine-deficient mice were protected from bone loss by the combination of increased bone formation and reduced bone resorption.