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      Cloning and Variation of Ground State Intestinal Stem Cells

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          Summary

          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 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 C. 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 modeling and regenerative medicine.

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          A reserve stem cell population in small intestine renders Lgr5-positive cells dispensable.

          Hua Tian, Brian Biehs, Søren Warming … (2011)
          The small intestine epithelium renews every 2 to 5 days, making it one of the most regenerative mammalian tissues. Genetic inducible fate mapping studies have identified two principal epithelial stem cell pools in this tissue. One pool consists of columnar Lgr5-expressing cells that cycle rapidly and are present predominantly at the crypt base. The other pool consists of Bmi1-expressing cells that largely reside above the crypt base. However, the relative functions of these two pools and their interrelationship are not understood. Here we specifically ablated Lgr5-expressing cells in mice using a human diphtheria toxin receptor (DTR) gene knocked into the Lgr5 locus. We found that complete loss of the Lgr5-expressing cells did not perturb homeostasis of the epithelium, indicating that other cell types can compensate for the elimination of this population. After ablation of Lgr5-expressing cells, progeny production by Bmi1-expressing cells increased, indicating that Bmi1-expressing stem cells compensate for the loss of Lgr5-expressing cells. Indeed, lineage tracing showed that Bmi1-expressing cells gave rise to Lgr5-expressing cells, pointing to a hierarchy of stem cells in the intestinal epithelium. Our results demonstrate that Lgr5-expressing cells are dispensable for normal intestinal homeostasis, and that in the absence of these cells, Bmi1-expressing cells can serve as an alternative stem cell pool. These data provide the first experimental evidence for the interrelationship between these populations. The Bmi1-expressing stem cells may represent both a reserve stem cell pool in case of injury to the small intestine epithelium and a source for replenishment of the Lgr5-expressing cells under non-pathological conditions.
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            Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells.

            J. Rheinwald, H. Green (1975)
            Human diploid epidermis epidermal cells have been successfully grown in serial culture. To initiate colony formation, they require the presence of fibroblasts, but proliferation of fibroblasts must be controlled so that the epidermal cell population is not overgrown. Both conditions can be achieved by the use of lethally irradiated 3T3 cells at the correct density. When trypsinized human skin cells are plated together with the 3T3 cells, the growth of the human fibroblasts is largely suppressed, but epidermal cells grow from single cells into colonies. Each colony consists of keratinocytes ultimately forming a stratified squamous epithelium in which the dividing cells are confined to the lowest layer(s). Hydrocortisone is added to the medium, since in secondary and subsequent subcultures it makes the colony morphology more oderly and distinctive, and maintains proliferation at a slightly greater rate. Under these culture conditions, it is possible to isolate keratinocyte clones free of viable fibroblasts. Like human diploid fibroblasts, human diploid keratinocytes appear to have a finite culture lifetime. For 7 strains studied, the culture lifetime ranged from 20-50 cell generations. The plating efficiency of the epidermal cells taken directly from skin was usually 0.1-1.0%. On subsequent transfer of the cultures initiated from newborns, the plating efficiency rose to 10% or higher, but was most often in the range of 1-5% and dropped sharply toward the end of their culture life. The plating efficiency and culture lifetime were lower for keratinocytes of older persons.
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              Bmi1 is expressed in vivo in intestinal stem cells.

              Eugenio Sangiorgi, Mario R. Capecchi (2008)
              Bmi1 plays an essential part in the self-renewal of hematopoietic and neural stem cells. To investigate its role in other adult stem cell populations, we generated a mouse expressing a tamoxifen-inducible Cre from the Bmi1 locus. We found that Bmi1 is expressed in discrete cells located near the bottom of crypts in the small intestine, predominantly four cells above the base of the crypt (+4 position). Over time, these cells proliferate, expand, self-renew and give rise to all the differentiated cell lineages of the small intestine epithelium. The induction of a stable form of beta-catenin in these cells was sufficient to rapidly generate adenomas. Moreover, ablation of Bmi1(+) cells using a Rosa26 conditional allele, expressing diphtheria toxin, led to crypt loss. These experiments identify Bmi1 as an intestinal stem cell marker in vivo. Unexpectedly, the distribution of Bmi1-expressing stem cells along the length of the small intestine suggested that mammals use more than one molecularly distinguishable adult stem cell subpopulation to maintain organ homeostasis.
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                Author and article information

                Journal
                Journal ID (nlm-journal-id): 0410462
                Journal ID (pubmed-jr-id): 6011
                Journal ID (nlm-ta): Nature
                Journal ID (iso-abbrev): Nature
                Title: Nature
                ISSN (Print): 0028-0836
                ISSN (Electronic): 1476-4687
                Publication date Nihms-submitted: 22 April 2016
                Publication date (Electronic): 03 June 2015
                Publication date (Print): 11 June 2015
                Publication date PMC-release: 03 May 2016
                Volume: 522
                Issue: 7555
                Pages: 173-178
                Affiliations
                [1 ]The Jackson Laboratory for Genomic Medicine, Farmington, CT, USA
                [2 ]Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT, USA
                [3 ]Genome Institute of Singapore, A-STAR, Singapore
                [4 ]Department of Pathology, Brigham and Women’s Hospital, Boston, MA USA
                [5 ]Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, TN, USA
                [6 ]Division of Gastroenterology, Connecticut Children’s Medical Center, Hartford, CT, USA
                [7 ]Department of Medicine, University of Connecticut Health Center, Farmington, CT, USA
                [8 ]Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA, USA
                [9 ]Department of Medicine, National University Health System, Singapore
                [10 ]Multiclonal Therapeutics, Inc., Farmington, CT, USA
                Author notes
                [* ]Correspondence to: Wa Xian, Xianmckeon2014@ 123456gmail.com , 1-860-480-1188. Frank McKeon, 1-860-480-1263
                [11]

                these authors contributed equally to this work.

                Article
                Manuscript ID: NIHMS768270
                DOI: 10.1038/nature14484
                PMC ID: 4853906
                PubMed ID: 26040716
                SO-VID: 23bc7046-b690-4280-a030-bcb5ab0b3f07
                License:

                Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms

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