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Cloning and variation of ground state intestinal stem cells

Nature volume 522, pages 173–178 (2015)Cite this article

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Abstract

Stem cells of the gastrointestinal tract, pancreas, liver and other columnar epithelia collectively resist cloning in their elemental states. Here we demonstrate the cloning and propagation of highly clonogenic, ‘ground state’ stem cells of the human intestine and colon. We show that derived stem-cell pedigrees sustain limited copy number and sequence variation despite extensive serial passaging and display exquisitely precise, cell-autonomous commitment to epithelial differentiation consistent with their origins along the intestinal tract. This developmentally patterned and epigenetically maintained commitment of stem cells is likely to enforce the functional specificity of the adult intestinal tract. Using clonally derived colonic epithelia, we show that toxins A or B of the enteric pathogen Clostridium difficile 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 modelling and regenerative medicine.

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Figure 1: Cloning stem cells from fetal intestine.
Figure 2: Stem cells from fetal small intestine.
Figure 3: Stem cells of fetal colon.
Figure 4: Differential gene expression in stem cells of stratified and columnar epithelia.
Figure 5: Genomic stability of ISC in culture.
Figure 6: C. difficile toxin B effects on in vitro-generated colonic epithelia.

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Primary accessions

European Nucleotide Archive

Gene Expression Omnibus

Data deposits

Data sets generated for this study have been submitted to the National Center for Biotechnology Information Gene Expression Omnibus (GEO) database and the European Nucleotide Archive under accession numbers GSE66749 and SRP056402.

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Acknowledgements

This work was supported by grants from Connecticut Innovations (W.X., F.M.), the Joint Council Office of the Agency for Science Technology Research Agency (A*STAR), Singapore (W.X., F.M.), the National Medical Research Council, Singapore (BNB101677A to H.K.Y., F.M., and W.X.; BnB11dec063 to N.N., F.M. and W.X.), the Department of Defense (W81XWH-10-1-0289 to C.P.C.) and the National Institute of Health (AI09575504 to D.B.L.). We thank M. LaLande, B. Lane and B. Seet for support, B. Tennent, B. Knowles and T. McLaughlin for comments on the manuscript, J. Hammer for artwork, L. Lapierre and J. Franklin for histology evaluation. We thank H. Green for advice and support.

Author information

Author notes

  1. Xia Wang and Yusuke Yamamoto: These authors contributed equally to this work.

Authors and Affiliations

  1. The Jackson Laboratory for Genomic Medicine, Farmington, 06032, Connecticut, USA

    Xia Wang, Yusuke Yamamoto, Lane H. Wilson, Gang Ning, Yue Hong, Benoit Chevalier, Frank McKeon & Wa Xian

  2. Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, 06032, Connecticut, USA

    Lane H. Wilson & Wa Xian

  3. Genome Institute of Singapore, Agency for Science, Technology and Research, 138672, Singapore

    Ting Zhang, Florian Kern, Chiea Chuen Khor, Denis Bertrand, Lingyan Wu, Niranjan Nagarajan & Frank McKeon

  4. Department of Pathology, Brigham and Women's Hospital, Boston, 02118, Massachusetts, USA

    Brooke E. Howitt, Christopher P. Crum & Wa Xian

  5. Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, 37232, Tennessee, USA

    Melissa A. Farrow & D. Borden Lacy

  6. Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, 119228, Singapore

    Chiea Chuen Khor

  7. Department of Pediatrics, Division of Gastroenterology, The University of North Carolina at Chapel Hill, Chapel Hill, 27599, North Carolina, USA

    Francisco A. Sylvester

  8. Division of Digestive Diseases, Hepatology, and Nutrition, Connecticut Children's Medical Center, Hartford, 06106, Connecticut, USA

    Jeffrey S. Hyams

  9. Department of Medicine, University of Connecticut Health Center, Farmington, 06032, Connecticut, USA

    Thomas Devers

  10. Department of Microbiology and Immunobiology, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Roderick Bronson

  11. Department of Medicine, National University of Singapore, 119228, Singapore

    Khek Yu Ho, Frank McKeon & Wa Xian

  12. Multiclonal Therapeutics, Inc., Farmington, 06032, Connecticut, USA

    Frank McKeon & Wa Xian

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  1. Xia Wang
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Contributions

Experimental design and conception were done by W.X., F.M., D.B.L., K.Y.H. and C.P.C.; X.W. cloned and differentiated the intestinal stem cells with help from L.H.W., F.K., G.N., B.E.H. and Y.H.; Y.Y., X.W. prepared the genomic and gene expression analyses together with F.K., G.N., C.C.K. and L.W.; T.Z., D.B. and N.N. performed all computational and bioinformatics work. B.H. and C.P.C. obtained fetal tissues and F.A.S., J.S.H. and T.D. provided endoscopic biopsies, and R.B. analysed the xenografts. The C. difficile experiments were designed and executed by B.C., L.H.W., M.A.F. and D.B.L.; W.X. and F.M. wrote the manuscript with input from all other authors.

Corresponding authors

Correspondence to Frank McKeon or Wa Xian.

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Extended data figures and tables

Extended Data Figure 1 Loss of clonogenicity in differentiated ISC.

a, Schematic of ISC differentiation using either the γ-secretase inhibitor dibenzazepine (DBZ) or withdrawal of the Wnt regulator R-spondin 1 (Rspo1). ISCs were plated on day 0, DBZ added or Rspo1 removed at day 2, and colonies passaged en masse at day 7. At day 14, after 7 days of continuous growth, colonies were counted. b, Micrographs show immunofluorescence at day 7 colonies grown without Rspo1 or in the presence of DBZ for 5 days using antibodies to Ki67, chromogranin A (CHGA), keratin 20 (Krt20), E-cadherin (E-cad), and mucin 2 (Muc2). Scale bar, 50 μm; n = 4 technical replicates. c, Histogram shows colony formation in each condition normalized to control ISCs. n = 4 biological replicates; error bars, s.d. d, Staining of ALI-differentiated intestinal stem cells with monoclonal antibody HD6 directed to Paneth cells. Scale bar, 50 μm; n = 4 technical replicates.

Extended Data Figure 2 Intestinal stem cell expression profiles.

a, List of genes differentially expressed in ISC derived from duodenum, jejunum and ileum. These data correspond to heat map of Fig. 2b. b, Immunofluorescence labelling of ALI-differentiated ISCs from duodenum with antibodies against Tff2, mucin 5AC, villin, E-cadherin, and mucin 2. c, Immunofluorescence labelling of ALI-differentiated epithelia from jejunum stem cells with antibodies to E-cadherin, mucin 2, villin, and mucin 5AC. Scale bar, 50 μm; n = 10 technical replicates.

Extended Data Figure 3 Differential gene expression in epithelia derived from colonic stem cells.

Heat map of differentially expressed (>1.5-fold, P < 0.05) genes in ALI cultures derived from stem cell pedigrees of ascending, transverse, and descending colon.

Extended Data Figure 4 Differential gene expression across columnar and stratified epithelial stem cells.

a, Histograms of expression microarray signal intensity of selected genes across averaged intestine and colon ISCs, stratified epithelial stem cells, and stem cells of the fallopian tube (FT). Biological replicas n = 2–6 (FT = 2, stratified epithelia = 3, colon, intestine = 6); error bars, s.d. b, Dot plot showing expression microarray data of indicated genes for stem cell pedigrees (ISC; Duo, duodenum; Jej, jejunum; Ile, ileum; AC, ascending colon; TC, transverse colon; DC, descending colon) derived from various regions of the intestinal tract before and after air–liquid interface (ALI) differentiation. Biological replicas n = 2 (total 12 data sets) for stem cells, technical replicas n = 2 for ALI. c, Chart of aggregate P values by Student's t-test for gene expression changes between ground state stem cells and their ALI-differentiated counterparts.

Extended Data Figure 5 Genes affected by CNV and SNV events in intestinal stem cell pedigrees during passaging.

a, Summary of CNV (events (genes affected)) and non-synonymous SNV in pedigrees 1 and 2 at P5 to P20. b, Summary of genes altered by interstitial CNV amplifications (top) or deletions (bottom) in ISC pedigrees 3 to 7 at P5 and P25. c, Summary of genes sustaining non-synonymous SNV in five ISC pedigrees at P5 and P25.

Extended Data Figure 6 Whole-genome CNV profiles for intestinal stem cell pedigrees 3–7 at P5 and P25.

Regions marked by ovals represent aneuploidy.

Extended Data Figure 7 Impact of ISCGS passaging on ALI differentiation.

ALI differentiation of intestinal pedigree 2 initiated from cells at the indicated passage number. As indicated, histological sections of differentiated epithelia were stained with antibodies to either E-cadherin (ECAD, green) and mucin 2 (Muc2, red), or Ki67 (green) and chromogranin A (CHGA, red). Scale bar, 75 μm; n = 4 technical replicates.

Extended Data Figure 8 ISCGS tumorigenicity assays in immunodeficient mice.

a, Quantification of tumour formation assessments at 4–16 weeks following subcutaneous inoculation of two million cells of the indicated ISC pedigrees at passage 6 or passage 25 at 4–16 weeks. ‘Pool’ indicates total set of clones derived from P0 ileum culture before pedigree generation. ‘Cancer cells’ refers to propagating cells from case of high-grade serous ovarian cancer. b, Left, histological section through site of injection of 1 million cells from pedigree 3. Right, section of injection site stained with antibody (STEM121) to human epithelial cells (brown) revealing benign cysts. Scale bar, 15 μm.

Extended Data Figure 9 Dose- and time-dependency of TcdB pathology in ALI-generated colonic epithelia.

a, Immunofluorescence localization of adherens junction marker E-cadherin and tight junction marker claudin 3 in ALI-differentiated epithelia derived from transverse colon stem cells following exposure to 100 pM TcdB for the indicated durations. n = 4 technical replicates. Scale bar, 100 μm. b, Representative H&E images of ALI cultures at indicated times and concentration of TcdB exposure. Scale bar, 250 μm; n = 4 technical replicates. c, Gene set enrichment analysis of whole-genome expression data from colonic epithelia treated with 500pM TcdB for 24 h and control samples showing enriched KEGG pathway sets. NES, normalized enrichment score; NOM P value, nominal P value. d, 3D plot of upregulated genes at the indicated time points and dosages > twofold, P < 0.05). n = 2 technical replicates. e, Heat map of upregulated genes in 500 pM TcdB samples. The genes (237 genes) were chosen by cutoff values (> twofold, P < 0.05). Three time points (8, 16 and 24 h) are shown. f, 3D plot of downregulated genes at the indicated time points and dosages > twofold, P < 0.05). n = 2 technical replicates.

Extended Data Figure 10 Dose- and time-dependency of TcdA pathology in ALI-generated colonic epithelia.

a, Left, representative H&E images of ALI cultures at indicated times and concentration of TcdA exposure; right, immunofluorescence localization of adherens junction marker E-cadherin (ECAD; green) and mucin 2 (MUC2; red) in ALI-differentiated epithelia derived from transverse colon stem cells following incubation with 10 nM TcdA for the indicated durations. Scale bar, 100 μm; n = 4 technical replicates. b, 3D plot of histological scoring of representative H&E time points and concentrations performed by a gastrointestinal pathologist according to a standard 0–3 rating for colonic epithelial integrity. c, Distribution of tight junction marker claudin 3 (Cldn3) and adherens junction marker (Cdh17) following treatment of ALI colonic epithelium with TcdA for the indicated times and doses. Scale bar, 50 μm; n = 4 technical replicates. d, Histogram of permeability of ALI colonic epithelium (Papp) to small molecules (FD4, molecular mass 4,400 Da) following exposure to the indicated doses of TcdA for the indicated times.

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This file contains Supplementary Tables 1-3 and a further table containing a summary of CNV and exome capture sequencing run analysis for intestinal stem cells. (PDF 334 kb)

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Wang, X., Yamamoto, Y., Wilson, L. et al. Cloning and variation of ground state intestinal stem cells. Nature 522, 173–178 (2015). https://doi.org/10.1038/nature14484

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