Animal embryonic stem cells (ESCs) provide powerful tool for studies of early embryonic development, gene targeting, cloning, and regenerative medicine. However, the majority of attempts to establish ESC lines from large animals, especially ungulate mammals have failed. Recently, another type of pluripotent stem cells, known as induced pluripotent stem cells (iPSCs), have been successfully generated from mouse, human, monkey, rat and pig. In this study we show sheep fibroblasts can be reprogrammed to pluripotency by defined factors using a drug-inducible system. Sheep iPSCs derived in this fashion have a normal karyotype, exhibit morphological features similar to those of human ESCs and express AP, Oct4, Sox2, Nanog and the cell surface marker SSEA-4. Pluripotency of these cells was further confirmed by embryoid body (EB) and teratoma formation assays which generated derivatives of all three germ layers. Our results also show that the substitution of knockout serum replacement (KSR) with fetal bovine serum in culture improves the reprogramming efficiency of sheep iPSCs. Generation of sheep iPSCs places sheep on the front lines of large animal preclinical trials and experiments involving modification of animal genomes.

Aims Doxorubicin is a widely used chemotherapy drug, but its application is associated with cardiotoxicity. Free radical generation and mitochondrial dysfunction are thought to contribute to doxorubicin-induced cardiac failure. Angiotensin-converting enzyme (ACE) inhibitors are commonly used as cardioprotective agents and have recently been shown in clinical studies to be efficacious in the prevention of anthracycline induced heart failure. Here we evaluated a mechanism for these protective effects by testing the ability of the ACE inhibitor enalapril to preserve mitochondrial function in a model of chronic doxorubicin treatment in rats. Methods Sprague Dawley rats were divided into three groups and followed for a total of 10 weeks: a) control-untreated, b) Doxorubicin treated (Dox), and c) Doxorubicin + Enalapril treated (DE). Doxorubicin was administered via intraperitoneal injection at weekly intervals from week 2 through week 7. Enalapril was administered in the drinking water of the DE group for the study duration. Results Doxorubicin treatment produced a significant loss in left ventricular contractility (P< 0.05), decrease in mitochondrial function via impairment of state-3 respiration, decrease in the cytosolic fraction of ATP, and up-regulation of free radical production. Enalapril significantly attenuated the decrease in percent fractional shortening (P< 0.05) and prevented the doxorubicin-associated reduction in respiratory efficiency and cytosolic ATP content (P< 0.05). Importantly, enalapril also abolished the robust doxorubicin-induced increase in free radical formation. Conclusions Administration of enalapril attenuates doxorubicin-induced cardiac dysfunction via preservation of mitochondrial respiratory efficiency and reduction in doxorubicin-associated free radical generation.

SUMMARY In recent years, human embryonic stem (hES) cells have become a promising cell source for regenerative medicine. Although hES cells have the ability for unlimited self-renewal, potential adverse effects of long-term cell culture upon hES cells must be investigated before therapeutic applications of hES cells can be realized. Here we investigated changes in molecular profiles associated with young (<60 passages) and old (>120 passages) cells of the H9 hES cell line as well as young (<85 passages) and old (>120 passages) cells of the PKU1 hES cell line. Our results show that morphology, stem cell markers, and telomerase activity do not differ significantly between young and old passage cells. Cells from both age groups were also shown to differentiate into derivatives of all three germ layers upon spontaneous differentiation in vitro. Interestingly, mitochondrial dysfunction was found to occur with prolonged culture. Old passage cells of both the H9 and PKU1 lines were characterized by higher mitochondrial membrane potential, larger mitochondrial morphology, and higher reactive oxygen species (ROS) content compared to their younger counterparts. Teratomas derived from higher passage cells were also found to have an uneven preference for differentiation as compared to tumors derived from younger cells. These findings suggest that prolonged culture of hES cells may negatively impact mitochondrial function and possibly affect long-term pluripotency.

No abstract