An Lgr5-independent developmental lineage is involved in mouse intestinal regeneration II. An Lgr5-independent developmental lineage is involved in mouse intestinal regeneration II

Collagenase/dispase (CD fraction) treatment of intestinal tissue from adult mice generates cells growing in matrigel as immortal cystic spheroids in addition to differentiated organoids. Contrary to classical EDTA-derived organoids, these spheroids display poor intestinal differentiation and are independent of Rspondin/Noggin/EGF for growth. Their transcriptome resembles strikingly that of fetal intestinal spheroids, with downregulation of crypt base columnar cell (CBC) markers (Lgr5, Ascl2, Smoc2, Olfm4). In addition, they display upregulation of inflammatory and mesenchymal genetic programs, together with robust expression of YAP target genes. Lineage tracing, cell-sorting and single cell RNA sequencing experiments demonstrate that adult spheroid-generating cells belong to a hitherto undescribed developmental lineage, independent of Lgr5+ve CBCs, and are involved in regeneration of the epithelium following CBC ablation Overall design: Examination of the transcriptome of adult intestinal spheroids cultivated in ENR medium upon cells seeding directly in Matrigel.These two sample were compared with small intestinal organoids and fetal small intestinal spheroids presented in an already existed project from our own lab available at GEO under accession number GSE65395. The isolation and the culture conditions remained the same in both studies.

对成年小鼠肠道组织采用胶原酶/分散酶（CD组分）处理后，除可获得分化型类器官外，还可得到可在基质胶（Matrigel）中增殖的永生性囊性球体。与经典EDTA来源类器官不同，此类球体肠道分化能力较弱，且生长不依赖Rspondin、Noggin与EGF。其转录组与胎儿肠道球体的转录组极为相似，且隐窝基底柱状细胞（crypt base columnar cell, CBC）标志物（Lgr5、Ascl2、Smoc2、Olfm4）表达下调。此外，此类球体还存在炎症与间充质基因程序的上调表达，同时YAP靶基因的表达水平显著升高。谱系示踪、细胞分选与单细胞RNA测序实验表明，成年小鼠肠道球体的生成细胞隶属于一个此前未被报道的发育谱系，且不依赖Lgr5阳性CBCs，同时参与CBC消融后上皮组织的再生过程。实验设计：对直接接种于基质胶（Matrigel）中、在ENR培养基内培养的成年小鼠肠道球体进行转录组分析。本研究将这两份样本与本实验室此前一项已发表研究中的小肠类器官及胎儿小肠球体进行对比，该研究数据已上传至GEO数据库，登录号为GSE65395。两项研究中的组织分离与培养条件完全一致。