Derivation of pluripotent stem cells from blastocysts and somatic cells in the domestic cat (Felis Catus)
Keefer, Carol L
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Pluripotent stem cells in the domestic cat model represent a huge potential for disease modeling, drug screening and regenerative medical treatments for feline species as well as for humans. However, current knowledge on deriving and maintaining stem cells has been obtained primarily from studies in mouse, rat, and human. Difficulties in attaining similar results in cats indicate the necessity to better understand pluripotency in this species. The hypothesis was that inadequate cytokine supplementation results in pluripotency loss along with declining transcription factors expression. The main goal of this project was to assess the effects of selected growth factors and inhibitors, in maintaining pluripotency in embryonic cells, and to attain pluripotency from fibroblasts by controlling expression of exogenous transcription factors. In the first study, conventional cytokine cocktails, leukemia inhibitory factor coupling with glycogen synthase inhibitor 3, and mitogen-activated protein kinase inhibitor (LIF and 2i) could partially maintain pluripotency regulatory circuitry in the cat. In this condition, embryonic cells reached a state that was not fully defined (neither naive nor primed). Overall, cell characterizations revealed a trend of pluripotency loss over time. In the second study, pluripotency was attained by forced expression of inducible exogenous transcription factors (NANOG, POU5F1, CMYC, and SOX2) and cultured in medium supplemented with the same cytokine combination identified in the first study. Notably, unlike previous reports in the cat, colonies with partial pluripotent features could be maintained after the transgenes were silenced. In addition to the protein and transcript markers for pluripotency, lineage marker dynamics were examined in pluripotent cells and embryoid bodies. The outcome suggested the cells generated with LIF and 2i had developed beyond the undifferentiated stage of ICM in expanded blastocyst. Collective results not only challenged the efficacy of the cytokines combinations LIF and 2i in maintaining feline pluripotency, but also suggest direction of research towards the species-specific signaling requirement in embryonic progression and stem cell derivation.