Cao Li, Kuratnik Anton, Xu Wanli, Gibson Jason D, Kolling Frederick, Falcone Eric R, Ammar Mohammed, Van Heyst Michael D, Wright Dennis L, Nelson Craig E, Giardina Charles
Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut, 06269-3125.
Mol Carcinog. 2015 Mar;54(3):189-202. doi: 10.1002/mc.22089. Epub 2013 Sep 21.
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.
肠道类器官是多细胞隐窝样结构，可从成体肠道干细胞（ISC）、胚胎干细胞（ESC）或诱导多能干细胞（iPSC）中获得。在此我们表明，从小鼠ESC生成的肠道类器官富含ISC和早期祖细胞。用γ-分泌酶抑制剂处理这些类器官会增加Math1表达并降低Hes1表达，表明Notch信号通路调节这些类器官中的ISC分化。Lgr5和Tert阳性ISC分别约占类器官的10%和20%。正如在天然组织中所发现的，表达Lgr5和Tert的细胞分化为两个离散的群体，且随时间保持稳定。源自易患癌症的Apc(Min/+)小鼠的肠道类器官显示出相似数量的ISC，但Math1表达降低，表明分泌细胞分化潜能受到抑制（如在肠道组织中所见）。Apc(Min/+)类器官被用于筛选表观遗传活性化合物，以寻找那些能增加Math1表达和类器官分化的化合物（包括组蛋白去乙酰化酶（HDAC）抑制剂、沉默调节蛋白（SIRT）调节剂和甲基转移酶抑制剂）。广谱HDAC抑制剂增加了Math1和Muc2的表达，表明其具有促进受抑制的分泌细胞分化途径的能力。其他表观遗传化合物对细胞分化有不同影响，激活分泌标记物Muc2的化合物与激活吸收细胞标记物Fabp2的化合物之间存在强烈的负相关。这些数据表明，ESC来源的肠道类器官可以大量获得，包含不同类型的ISC，并且可用于筛选通过不同谱系途径促进细胞分化的试剂。
