Technical note: improving the efficiency of generating bovine extraembryonic endoderm cells

Area of Science:

• Developmental Biology
• Stem Cell Research
• Reproductive Biology

Background:

• Extraembryonic endoderm (XEN) formation is critical in early embryonic development.
• Studying XEN cell types in ruminants is limited due to a lack of suitable cell culture models.
• Previous work identified fibroblast growth factor 2 (FGF2) as crucial for bovine XEN development.

Purpose of the Study:

• To establish an efficient protocol for producing XEN cell cultures from bovine blastocysts.
• To characterize the established XEN cell cultures for their morphology, gene expression, and differentiation potential.
• To assess the viability and behavior of cryopreserved XEN cell cultures.

Main Methods:

• Culturing bovine blastocysts under modified conditions, including 20% fetal bovine serum and FGF2 replenishment.
• Assessing endoderm outgrowth formation rates compared to control conditions (bovine serum albumin supplemented).
• Morphological and molecular characterization of cultured cells, including transcript analysis for XEN markers (GATA4, GATA6) and visceral/parietal XEN markers.

Main Results:

• The refined protocol significantly increased endoderm outgrowth incidence to 81.6% ± 5.5%, compared to 33.3% ± 5.5% in controls.
• Cultured cells exhibited XEN-like morphology and expressed key XEN transcripts (GATA4, GATA6), along with visceral and parietal XEN markers.
• Two XEN cell lines were maintained for up to 6 weeks, retaining XEN markers and showing increased visceral/parietal transcript abundance, suggesting differentiation potential. Cryopreserved cells maintained similar characteristics.

Conclusions:

• An efficient protocol for generating primary bovine XEN cell cultures from blastocysts has been successfully developed.
• These XEN cell cultures maintain their identity and exhibit differentiation potential during prolonged culture.
• The developed protocol and cell lines offer a valuable tool for investigating the roles of XEN cells in ruminant embryogenesis, particularly in nutrient supply.