Zhou, R., Comizzoli, P., Wildt, D.E., Keefer, C.L. (2016). Defining Conditions That Maintain Pluripotency of Embryonic Stem Cells In Domestic Cats. Abstract no. 332, 49th Annual Meeting for the Society for the Study of Reproduction, San Diego, CA.
Ran Zhou, Pierre Comizzoli, David E. Wildt, Carol L. Keefer (2017) Specific Combinations of Cytokines Enhance Cell Proliferation and Expression of Pluripotency-Related Proteins in Inner Cell Mass Outgrowths in the Domestic Cat Model. Flash Talk 4.20. and ABST. 50th Annual Meeting for the Society for the Study of Reproduction, Washington, DC. [abbreviated]
When propagating pluripotent cells from early-stage embryos, the level of dependence on LIF/STAT3 and FGF2/MEK/ERK signaling pathways are species-specific. However, culture conditions that maintain pluripotency in embryonic stem cells (ESC) using various combinations of growth factors (GF; targeting LIF or FGF2 pathways) and inhibitors (targeting WNT/GSK3 or FGF2 pathways) have yet to be determined in the cat model. We previously observed that, in absence of GFs or inhibitors, pluripotency-related proteins NANOG and POU5F1 were decreased in outgrowths generated from inner cell mass (ICM) of cat blastocysts at 144 hours of culture. Additionally, presence of SOX2 was decreased at 72 hours, while cell numbers were increased at 96 hours but then decreased. The objective of the present study was to evaluate LIF and FGF2 singly and in combination with GSK3 or MEK inhibitors on cell proliferation and pluripotency marker expression in ICM outgrowths. No single cytokine maintained all three pluripotency markers to levels comparable to ICMs in cat blastocysts. Seventeen combinations were tested using IVP blastocysts (n = 3-5 per treatment). ICM outgrowths were assessed for cell number and pluripotency marker expression at 144 hours. Four combinations sustained SOX2, POU5F1 and NANOG expression at similar (P>0.05, ChiSquare after K-Clustering) levels compared to ICMs in blastocysts. Furthermore, two of these combinations enhanced (P < 0 .05) cell proliferation compared to outgrowths without GFs. In summary, specific cytokine combinations can maintain pluripotency and support proliferation over a longer interval in vitro than single cytokine supplementation, likely through synergistic activation of multiple pathways.