(48) Establishment of Bovine Extraembryonic Endoderm Stem Cells

Hao Ming¹, Giovanna N. Scatolin¹, Alejandro Ojeda², Zongliang Jiang¹

1.Department of Animal Sciences, Genetics Institute, University of Florida, Gainesville, FL 32610, USA

2.Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610, USA

 

Abstract Text:

During the mammalian pre-implantation development, the first lineage differentiation specifies the inner cell mass (ICM) and trophectoderm (TE) in the blastocyst; the ICM further differentiates into epiblast and hypoblast (or primitive endoderm) in blastocyst. Subsequently, hypoblast or primitive endoderm gives rise to the yolk sac by implantation and is critical to support early conceptus development by producing a spectrum of serum proteins, generating early blood cells, and transporting nutrient from the uterus to the embryo. Despite the importance of early development, our knowledge of hypoblast development, particularly in ruminant species, is limited owing to in vivo experimental feasibility and the lack of in vitro models.

Here we report that a chemical cocktail (FGF4, BMP4, IL-6, XAV939, and A83-01) enables de novo derivation and long-term culture of bovine extraembryonic endoderm cells (bXENs). The characterization of bXENs revealed that 1) bXENs maintained stable colony morphology and a normal diploid number of chromosomes (60) after long-term in vitro culture ( >30 passages as of now); 2) bXENs highly expressed hypoblast transcriptional factors (GATA6, SOX17, GATA4, as well as novel hypoblast lineage markers identified from single cell transcriptomic dataset of day 12 bovine embryos (CTSV, FETUB, APOA1, APOE)), but not epiblast and trophoblast markers, which is verified by qRT-PCR and RNA-seq results; 3) RNA-seq analysis showing that bXENs were resemble to human and mouse XENs, while distinct from ESCs of bovine, human and mouse at the transcriptome level; and 4) bXENs enriched gene markers (e.g., CDH1, IHH, LAMB1, SPARC) and pathways (e.g., migration, TNF, and Hippo pathways) of extraembryonic visceral endoderm (VE) and parietal endoderm (PE) markers, indicating its developmental capacity towards to yolk sac.

Given that both epiblast and hypoblast specify from ICM and the plasticity of two lineages are largely unknown, here we implemented a 3D co-culture model with our established robust bXENs and bEPSCs. It was found that the presence of bXENs and associated communications support the growth and stemness of bEPSCs. To further confirm the role of bXENs in promoting epiblast development, we treated bovine in vitro cultured embryos with defined small molecular cocktails sustaining bXENs (BMP4, FGF4, A83-01, XAV939, IL-6). Consistently, these bXEN signaling cocktails could effectively protect epiblast from differentiation or degeneration in embryos.

We have previously reported the successful generation of bovine blastoids by self-assembly of bEPSCs and bTSCs in tFACL+PD culture condition, which providing an accessible in vitro cell model for studying embryogenesis. However, the blastoids assembled by the two-lineage approach has shown lower proportion of hypoblast lineage compared to IVF blastocysts, which may limit their developmental capacity. Here we generated bovine  blastocyst-like structures by self-organization of bXENs, bEPSCs, and bTSCs as the ratio of 8:8:16 in aggrewell dish. The 3 lineage formed blastoids had a similar proportion of hypoblast and a slightly higher ratio of epiblast population compared to day 8 blastocysts, with majority hypoblast cells surrounding epiblasts.

The bovine XENs established in this study will provide a model to study bovine hypoblast development and early pregnancy failure.