Differentiation Control – RNAi and Wnt/Notch
RNAi
RNA interference (RNAi) is a cellular response to cytoplasmic double stranded RNA (dsRNA) in which single stranded RNA complementary to one or other strand is degraded. The effect is optimally mediated by oligoribonucleotides of 19-21 bases in length, which serve as “guides” to target complementary sequences. The ability of introduced dsRNA to regulate the abundance of endogenous mRNA was first documented in a number of animal species in 1998 and has now become a widely used tool with which to manipulate gene expression for in vitro applications and for animal research. The procedure is both simple and rapid compared to conventional knockout techniques and more importantly, gene expression is manipulated without genetic modification.
In collaboration with the CSCB, Axordia has undertaken proof of concept work that demonstrates that RNAi can be used to modulate the differentiation of HESCs.
Lineage-specific differentiation of human embryonal carcinoma cells induced by RNAi
In the phase contrast images, note the distinct morphology of the control-treated cells compared with those subjected to Oct4 RNAi.
Oct4 RNAi causes trophectodermal differentiation in HESCs (Matin et al 2004). The fluorescent image shows multiple nuclei (in blue) following Oct4 RNAi, indicating the formation of syncytial trophoblast.
WNT AND NOTCH PATHWAY MODULATION
The Wnt and Notch pathways are highly conserved cell-cell signalling mechanisms found in divergent organisms and play key roles in regulating cellular proliferation and differentiation during development. Manipulation of signalling activation of the Wnt and Notch pathways has been demonstrated to alter cell behaviour in a number of in vitro systems, including HESCs. For example, Sato et al demonstrated that a pharmacological agent that promotes Wnt signalling facilitates the maintenance of pluripotency in mouse and human ESCs (Nature Medicine, 2004). Also, Castelo-Branco et al have shown that Wnt activation can be used to direct ventral midbrain precursors to form dopaminergic neurons (Journal of Cell Science, 2004). Similarly, Notch signalling is involved in cell fate decisions that are highly relevant to Axordia’s R&D agenda, with experimentally proven roles that include controlling the rate of cell cycle in HESCs and facilitating the expansion of neural precursor cells (Oishi et al; Developmental Biology, 2004).
In order to examine the effects of Wnt and Notch signalling activity upon cellular behaviour in stem cell systems, a number of expression constructs have been engineered by Axordia.
Cells transfected with Axordia Wnt and Notch modulating research tools
A: human embryonal carcinoma cells expressing a fusion protein that combines an antagonist of the Wnt ligand family, SFRP4, with a green fluorescent protein (GFP). It is anticipated that this molecule is secreted by the cells and inhibits signalling activity.
B: expression of another engineered protein comprising the intracellular (IC) portion of the transmembrane Notch receptor, fused with GFP. As would be anticipated with native Notch IC, the fusion protein is localised only in the nucleus, where it controls the expression of target genes.
Both hEC cells and HESCs show dynamic regulation of multiple components of the Wnt and Notch systems during differentiation.