Development of intestinal organoids as tissue surrogates: Cell composition and the Epigenetic control of differentiation

Abstract

Intestinal organoids are multicellular crypt-like structures that can be derived from adult intestinal stem cells (ISCs), embryonic stem cells (ESCs) or induced pluripotent stem cells (IPSCs). Here we show that intestinal organoids generated from mouse ESCs were enriched in ISCs and early progenitors. Treatment of these organoids with a γ-secretase inhibitor increased Math1 and decreased Hes1 expression, indicating Notch signaling regulates ISC differentiation in these organoids. Lgr5 and Tert positive ISCs constituted approximately 10% and 20% of the organoids. As found in native tissue, Lgr5 and Tert expressing cells resolved into two discreet populations, which were stable over time. Intestinal organoids derived from cancer-prone Apc(Min/+) mice showed similar numbers of ISCs, but had reduced Math1 expression, indicating a suppressed secretory cell differentiation potential (as found in intestinal tissue). Apc(Min/+) organoids were used to screen epigenetically active compounds for those that increased Math1 expression and organoid differentiation (including HDAC inhibitors, Sirtuin (SIRT) modulators and methyltransferase inhibitors). Broad-spectrum HDAC inhibitors increased both Math1 and Muc2 expression, indicating an ability to promote the suppressed secretory cell differentiation pathway. Other epigenetic compounds had a diverse impact on cell differentiation, with a strong negative correlation between those that activated the secretory marker Muc2 and those that activated the absorptive cell marker Fabp2. These data show that ESC-derived intestinal organoids can be derived in large numbers, contain distinct ISC types and can be used to screen for agents that promote cell differentiation through different lineage pathways.

Non-animal models of epithelial barriers (skin, intestine and lung) in research, industrial applications and regulatory toxicology

“…In this respect, mouse and human intestinal crypts are embedded in a three dimensional matrix such as Matrigel or Collagen I covered with medium basically mimicking the intestinal stem cell niche by providing factors of the Wnt-, EGF-, BMP-, and Notch-signaling pathways (Cao et al, 2013;Farin et al, 2012). Over several days crypt cells form spherical structures with a crypt-like lumen, referred to as human enterospheres.…”

The Hippo pathway in colorectal cancer

“…Furthermore, Paneth cells constitutively secrete Wnt3 ligand, but the additional source of Wnt proteins also exists in the surrounding stromal cells. Notch signalling in the intestinal epithelium is involved in inhibition of the secretory phenotype of the differentiating ISC [76].…”

Development of an enhanced human gastrointestinal epithelial culture system to facilitate patient-based assays

“…Recently, there have been substantial advances in mouse and human intestinal epithelial cell culture methods[2–16]. These methods can be generally categorized as those that start cultures with embryonic stem cells or induced pluripotent stem (iPS) cells (induced organoid culture)[6, 13] or with dissociated crypts/stem cells from intestinal tissue (organoid or spheroid culture)[2–5, 7–9, 11, 12, 14–16].…”

“…These methods can be generally categorized as those that start cultures with embryonic stem cells or induced pluripotent stem (iPS) cells (induced organoid culture)[6, 13] or with dissociated crypts/stem cells from intestinal tissue (organoid or spheroid culture)[2–5, 7–9, 11, 12, 14–16]. While iPS-derived organoids are valuable for investigating embryonic development, the length of time that is required to generate mature, differentiated intestinal epithelial cells may limit their utility for patient-directed experimental approaches.…”