Epigenetic changes during long term intestinal organoid culture

Organoids retain the morphological and molecular patterns of their tissue of origin, are self-organizing, relatively simple to handle and accessible to genetic engineering. Thus, they represent an optimal tool for studying mechanisms of tissue maintenance and aging. Long-term expansion under standard growth conditions, however, is accompanied by changes in growth pattern and kinetics. As a potential explanation of these alterations, epigenetic drifts in organoid culture have been suggested. Here, we study histone tri-methylation at lysine 4 (H3K4me3) and 27 (H3K27me3) and transcriptome profiles of intestinal organoids derived from mismatch repair (MMR)-deficient and control mice and cultured for 3 and 20 weeks, and compare them with data on their tissue of origin. We find that, besides the expected changes in short-term culture, organoids show profound changes in their epigenome also during long-term culture. The most prominent are epigenetic gene activation by H3K4me3 recruitment to previously unmodified genes and by H3K27me3 loss from originally bivalent genes. We show that long-term culture is linked to broad transcriptional changes that indicate an ongoing maturation and metabolic adaptation process. This process is disturbed in MMR-deficient mice, resulting in endoplasmic reticulum (ER)-stress and Wnt-activation. Our results can be explained in terms of a mathematical model assuming that epigenetic changes during long-term culture involve DNA de-methylation that ceases if the metabolic adaptation is disturbed.