SETD3 regulates endoderm differentiation of mouse embryonic stem cells through canonical Wnt signaling pathway.

With self-renewal and pluripotency features, embryonic stem cells (ESCs) provide an invaluable tool to investigate early cell fate decisions. Pluripotency exit and lineage commitment depend on precise regulation of gene expression that requires coordination between transcription (TF) and chromatin factors in response to various signaling pathways. SET domain-containing 3 (SETD3) is a methyltransferase that can modify histones in the nucleus and actin in the cytoplasm. Through an shRNA screen, we previously identified SETD3 as an important factor in the meso/endodermal lineage commitment of mouse ESCs (mESC). In this study, we identified SETD3-dependent transcriptomic changes during endoderm differentiation of mESCs using time-course RNA-seq analysis. We found that SETD3 is involved in the timely activation of the endoderm-related gene network. The canonical Wnt signaling pathway was one of the markedly altered signaling pathways in the absence of SETD3. The assessment of Wnt transcriptional activity revealed a significant reduction in Setd3-deleted (setd3∆) mESCs coincident with a decrease in the nuclear pool of the key TF ß-catenin level, though no change was observed in its mRNA or total protein level. Furthermore, a proximity ligation assay (PLA) found an interaction between SETD3 and ß-catenin. We were able to rescue the differentiation defect by stably re-expressing SETD3 or activating the canonical Wnt signaling pathway by changing mESC culture conditions. Our results suggest that alterations in the canonical Wnt pathway activity and subcellular localization of ß-catenin might contribute to the endoderm differentiation defect of setd3∆ mESCs.

Directed Differentiation of Mouse Embryonic Stem Cells to Mesoderm, Endoderm, and Neuroectoderm Lineages.

The self-renewal and pluripotency features of mouse embryonic stem cells (mESCs) make them a great tool to study early mammalian development. Various signaling pathways that shape early mammalian development can be mimicked for in vitro mESC differentiation toward primitive lineages first and more specialized cell types later. Since the precise nature of the molecular mechanisms and the crosstalk between these signaling pathways is yet to be fully understood, there is a high level of variability in the efficiency and synchronicity among available differentiation protocols. Commitment of mESCs toward mesoderm, endoderm, or neuroectoderm lineages happens over only a few days and is highly efficient. Here, we provide protocols for the directed differentiation of mESCs toward these lineages in vitro.

Mouse Embryonic Stem Cell Culture in Serum-Containing or 2i Conditions.

With their unique capabilities of self-renewal and differentiation into three germ layers, mouse embryonic stem cells (mESCs) are widely used as an in vitro cellular model for early mammalian developmental studies. mESCs are traditionally cultured in high-serum and LIF-containing medium on a growth-deficient mouse embryonic fibroblast layer. A more recent culturing system with two inhibitors (for GSK3ß (CHIR99021) and MEK1/2 (PD0325901)) and LIF enables the derivation of mESC lines from various mouse strains. Here we describe methods for the mESC growth and maintenance in each medium composition as well as their adaptation to either condition.