Induced Pluripotent Stem Cells Produced From Cryopreserved Pygmy Sperm Whale (Kogia Breviceps) Lung Tissue

Concern over marine mammal health has risen in the last decade due to increases in mortality, unusual mortality events and previously undetected pathologies. Developing environmental trends, such as global warming and acute environmental insults, such as large-scale oil spills have been implicated. Unfortunately, the limited availability of biological samples hinders basic biological/toxicological studies. To compensate for the limited availability of samples and to make the most of rare samples that do become available, we have pursued methods to establish and expand cultures of primary cell types and reconstituted tissues from marine mammals for long-term use as surrogates for freshly isolated samples. To this end, we first developed a method to cryopreserve tissues from deceased/stranded individuals and thereby initiate establishment of a tissue bank biorepository. We were able to establish conditions and perform this successfully on lung tissue from a Pygmy Sperm Whale (PSW; Kogia breviceps). Using these conditions, we were able to establish cultures of viable primary lung cell types from tissue fragments that had been cryopreserved several months earlier (immediately after the stranding event). We then applied genetic or chemical means for generating induced pluripotent stem (iPS) cells to one of these primary cultures (lung fibroblasts). We observed that the genetic means, involving the forced expression of Klf4, Oct3/4, Sox2 and Myc, did produce PSW cells with long-term expansion and differentiative capability, while the chemical means using valproic acid (driving histone deacetylase inhibition and chromatin decondensation) did not. Finally, we employed specialized culture conditions to differentiate in bulk PSW cells with vascular endothelial and airway epithelial-like properties. The cryopreservation of viable tissue samples and the generation of iPS-like cells with unlimited expansion & pluripotent differentiative capacities from marine mammals is anticipated to have far-reaching impacts on levels ranging from basic biology to environmental policy related to stressors of marine and land mammalian health