The authors in this paper demonstrate the plasticity of mouse organoids simply by changing their culture conditions. Manipulation of key pathways resulted in induction of quiescence within stem cells and directed differentiation:
Inhibition of the Wnt or Notch pathway and activation of the BMP pathway resulted in decreased Lgr5 expression within organoids due to differentiation, with the remaining Lgr5+ cells being in cycle.
In comparison, inhibition of the EGF pathway (through the receptor and withdrawal of EGF) induced quiescence within the organoid stem cells, with increased Lgr5 expression and decreased proliferation observed. Inhibition of the downstream MAPK pathway produced similar results.
The quiescent state of Lgr5+ cells was reversible and these cells were able to create new organoids, demonstrating the retention of stem cell capacity. In addition, gene expression analysis showed proliferation and differentiation reached near normal levels one week after reactivation of the EGF pathway. However, absolute numbers of EECs and expression of the EEC marker Chromogranin A (Chga) increased above normal levels upon reactivation.
Gene ontology and gene set enrichment analysis of quiescent, in comparison to active Lgr5+ cells, confirmed a loss of proliferation, which may be driven by MYC and E2F1, and a downregulation of metabolic pathways. In comparison, Chga and a signature related to TNF-α and JAK-STAT3 was upregulated.
Combined inhibition of the Wnt, Notch and MEK pathways resulted in high level differentiation into the EEC lineage (~50% of cells within an organoid) without increased cell death. The subtype of EEC generated was found to be determined somewhat by the region from which the organoids were made.
EEC are very rare and little is known about the processes involved in fate specification of the EEC subtypes and the secretion of hormones. Although rare, EEC regulate a wide variety of physical responses and have been implicated in diseases such as diabetes, depression and obesity. Therefore, this method of enrichment of EEC numbers within organoids could help elucidate key processes that could impact a broad therapeutic area.
Epistem have presented developmental work within the organoid model in the areas of epithelial regeneration and GI toxicity at various conferences.
Readouts in this model include induced changes in proliferation/organoid branching, differentiation, apoptosis, protein and gene expression.
Basak, O., et al. (2016). “Induced Quiescence of Lgr5+ Stem Cells in Intestinal Organoids Enables Differentiation of Hormone-Producing Enteroendocrine Cells.” Cell Stem Cell