279 INNER CELL MASS-DERIVED BOVINE CELL CULTURES MAINTAIN PLURIPOTENCY UNDER CHEMICALLY DEFINED CONDITIONS OF DUAL KINASE INHIBITION

Abstract

Authentic embryonic pluripotent stem cells (ePSC), capable of giving rise to all cell types of an adult animal, are only available in mouse and rat. Here, we report the generation of bovine ePSC-like cells under minimal conditions. Inner cell masses were immunosurgically isolated from IVF bovine blastocysts and explanted on laminin/gelatine-coated substrates. Explants were cultured feeder-free under low oxygen (7%) in a chemically defined medium containing inhibitors of mitogen-activated protein kinase kinase (MAPKK) and glycogen synthase kinase-3 signalling (GSK3). Dual kinase inhibition (2i) was necessary to sustain expression of epiblast-specific pluripotency markers SOX2 and NANOG in the central colony, which comprised a multi-layered clump of tightly packed cells that was clearly demarcated from the surrounding monolayer outgrowth. In 2i, explanted inner cell mass (ICM) expanded from 51 ± 4 to 1102 ± 55 cells per colony and surrounding outgrowth within 6 days of culture, equivalent to ~4 to 5 population doublings, before passaging. Moreover, 2i suppressed apoptosis after mechanical passaging and the cell number per colony and outgrowth remained constant for up to 8 passages every 4 to 5 days, after which cultures were discontinued. As a proxy for cell proliferation, we quantified DNA synthesis following different 5-ethynyl-2′-deoxyuridine (EdU)-incorporation protocols. EdU pulse-labelling for 30 min revealed that in steady state, 20 to 30% of cells were in S-phase in primary and passaged colonies, respectively. After cumulative labelling for 24 h, almost all primary and passaged cells were cycling. Throughout this passaging regime, ICM-derived cell lines expressed a repertoire of core pluripotency-related factors (CDH1, OCT4, SALL4, SOX2, TCF3), including markers enriched in naïve pluripotent cells (DPPA3, KLF4, LIN28, NANOG, SOCS3, STAT3) and primordial germ cells (IIFITM3). Genes that are downregulated in primed pluripotent cells were either undetectable (FGF5, T-BRACHYURY) or downregulated (LEFTY) after passaging. These mRNA results were confirmed on the protein level, where OCT4, KLF4, SOX2, and NANOG, as well as SSEA-3/4 and TRA-1-60/-81, but not SSEA-1, remained widely expressed. A diagnostic feature of murine ePSC is the simultaneous presence of 2 active X chromosomes (X_a X_a) and OCT4. We derived cultures from ICM of female blastocysts, produced through IVF with sexed semen, and stained primary cultures on Day 6 with an antibody against trimethylated histone (H) 3 lysine (K) 27 (H3K27me3). Nuclear foci of intense H3K27me3 immunoreactivity were absent in most OCT4-positive cells (660/724 = 92%), indicating presence of X_a X_a. In suspension culture, bovine ePSC-like cells formed cystic embryoid bodies expressing ectoderm (TUBB3, GFAP, NES), endoderm (AFP), and mesoderm (SPP1) markers. Bovine ePSC-like cells after 3 passages showed a normal chromosome number in the majority of spreads (17/18 = 94%). Our short-term culture system provides a chemically defined screening platform for factors that maintain long-term proliferation and pluripotency of ePSC in cattle.

Supported by MSI C10X1002.