Examining and optimizing the embryoid body model for early embryonic germ layer development in diverse hiPSC lineages

Nora Lippai

Document Type

Article

Publication Date

2025

Keywords

JGM

Abstract

Embryoid bodies are a useful tool in the fields of developmental biology and genetics due to their utility as an in vivo model for early embryonic processes and method by which specialized transient cell types may be acquired. Preliminary work in our lab seeking to develop embryoid bodies from the KOLF2.2J iPSC line has indicated a disproportionate propensity in this lineage for neuroectoderm differentiation under neutral (non-directed) conditions, presenting a challenge to comprehensively recapitulate the development of all three germ layers through the embryoid body model in this cell line. We sought to assess the effect of altered growth conditions on embryoid bodies derived from this cell line, as well as a set of genetically diverse iPSC lines, to assess the effect of germ layer development through embryoid body formation. Through the modulation of growth conditions and embryoid body development protocols, we observe a conserved high propensity for ectoderm differentiation, specifically through the expression of the OTX2, PAX6, and TUBB3 markers across cell lines. However, preliminary results gathered through this investigation also indicate a cell-line specific role of low oxygen in enhancing expression of endoderm and mesoderm markers, such as FOXA2 and MIXL1. This work lays the foundation for reliable and reproducible embryoid body development across newly developed iPSC lineages representing diverse human populations, which may be applied and further developed towards the modulation of differentiation propensity towards the acquisition of an expanded panel of cell types in these lineages and others.

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