PCR duplicates were removed using PICARD-1

PCR duplicates were removed using PICARD-1.94 (http://picard.sourceforge.net). cells likely enforces the functional specificity of the adult intestinal tract. Using clonally-derived colonic epithelia, we show that toxins A or B of the enteric pathogen recapitulate the salient features of pseudomembranous colitis. The stability of the epigenetic commitment programs of these stem cells, coupled with their unlimited replicative expansion and maintained clonogenicity, suggests certain advantages for their use in disease modeling and regenerative medicine. Introduction While dominating prospective strategies for regenerative medicine, embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) face formidable challenges including risk of teratoma, complex guiding protocols for lineage specificity, and limited regenerative capacity of the lineages ultimately produced3C8. The success and promise of iPSCs have largely overshadowed efforts to harness stem cells intrinsic to regenerative tissues. Green and colleagues developed methods for cloning epidermal stem cells9 that form a stratified epithelium upon engraftment, and these methods have been successfully applied to corneal, thymic, and airway epithelia10C12. However, stem cells of columnar epithelial tissues resist cloning in a manner that maintains their immaturity during proliferative expansion, and instead must be carried forward as regenerative, differentiating organoids13C18. Despite their obvious potential in regenerative medicine and constant improvement19, the very low percentage of clonogenic cells in organoids limits the kinetics of their propagation as well as their utility for exploring the elemental stem cell. The present study reports the cloning and propagation of ground state human intestinal stem cells (ISCSox9 expression in fetal intestine, scale bar, 25um; colonies from intestine (n=10 biological replicates; colonies of ISC pedigree (n=30 independent experiments). Scale bar, 75um. ISC colony growth. Scale bar, 75um. ISC and TBSC pedigrees and ALI differentiation (tubulin, green; Muc5AC, red). Scale bar, 50um left, 25um right top, 25um bottom right; n=7 biological replicates; n=3 technical replicates; 3 independent experiments ALI-differentiated ISC. Scale bar, 50um. n=7 biological replicates; n=3 technical replicates; 3 independent experiments. PCA using 2158 genes (>2-fold, p<0.05 by Student t-Test) of ISC and TBSC and corresponding ALI-differentiated epithelia. Markers in ISC and TBSC. n=3 technical replicates. The clonogenicity of cells in the colonies was determined by N-Methyl Metribuzin single cell transfer to be greater than 50% (Fig. 1b). This high clonogenicity permits the rapid generation of single cell pedigree lines for expansion and characterization of lineage fates upon differentiation12 (Fig. 1b). Pedigree lines of ISCand tracheobronchial stem cells (TBSCformed a highly uniform, 3-D serpentine pattern, whereas TBSCproduced a stratified epithelium with apically positioned ciliated and goblet cells. Histological sections of differentiated ICSrevealed a columnar epithelium of villus-like structures marked by goblet (Muc2+), endocrine (chromogranin A+), and Paneth cells and polarized villin expression (Fig. 1d; Extended Data Fig. 1d), indicating the progeny of a single ISCcan give rise to all epithelial lineages typically found in the small intestine. Importantly, differentiation of these ground state stem cells is accomplished by exposure to an air-liquid interface rather than a removal of factors such as Wnt that maintain immaturity. While principal component analysis (PCA) of differentially expressed genes of ground state stem cells and ALI differentiated tissue showed great divergence as expected for columnar and stratified epithelia, the gene expression profiles of undifferentiated ISCand TBSCdiffered by less than 4% (>2.0-fold, p<0.05) (Fig. 1e). ISCshowed high expression of intestinal stem cell markers such as OLFM4, CD13322, Lgr523, and Lrig124, whereas those from the airways had the typical stem cells markers of stratified epithelia (Krt14, Krt5, and Tp6311) (Fig. 1f). Intestinal stem cell variation Approximately one in 2,000 cells from GRF2 duodenum (IduSC), jejunum (IjeSC), and ileum (IilSC) of a 21-week old fetal intestine form a colony (Fig. 2a). N-Methyl Metribuzin Although these colonies were morphologically indistinguishable in culture, whole genome expression analysis of multiple pedigrees showed a consistent, region-specific signature of 24C178 genes (>1.5-fold, p<0.05; Fig. 2b; Extended Data Fig. 2). Open in a separate window Figure 2 Stem cells from fetal small intestineDepiction of small intestine and clones derived from each. Scale bar, 400um; N-Methyl Metribuzin n=3 biological replicates. Heatmap of.