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Cloning of ground-state intestinal stem cells from endoscopic biopsy samples

Nature Protocols volume 15, pages 1612–1627 (2020)Cite this article

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Abstract

‘Adult’ or ‘somatic’ stem cells harbor an intrinsic ability to regenerate tissues. Heterogeneity of such stem cells along the gastrointestinal tract yields the known segmental specificity of this organ and may contribute to the pathology of certain enteric conditions. Here we detail technology for the generation of ‘libraries’ of clonogenic cells from 1-mm-diamter endoscopic biopsy samples from the human gastrointestinal tract. Each of the 150–300 independent clones in a typical stem cell library can be clonally expanded to billions of cells in a few weeks while maintaining genomic stability and the ability to undergo multipotent differentiation to the specific epithelia from which the sample originated. The key to this methodology is the intrinsic immortality of normal intestinal stem cells (ISCs) and culture systems that maintain them as highly immature, ground-state ISCs marked by a single-cell clonogenicity of 70% and a corresponding 250-fold proliferative advantage over spheroid technologies. Clonal approaches such as this enhance the resolution of molecular genetics, make genome editing easier, and may be useful in regenerative medicine, unravelling heterogeneity in disease, and facilitating drug discovery.

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Fig. 1: Schematic of cloning of gISCs from endoscopic biopsy samples.
Fig. 2: High clonogenicity facilitates the expansion of single cell–derived clones.
Fig. 3: Conservation of high clonogenicity of gISCs over proliferative time.
Fig. 4: Representative images of 3T3-J2 cells.
Fig. 5: Adaptation of gISC clone to ALI differentiation or organoid culture format.
Fig. 6: FACS profiling of GFP-labeled gISCs for monitoring of purification.

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The corresponding authors will provide all data presented in the article and address technical questions upon reasonable request.

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Acknowledgements

This work was supported by grants from the Cancer Prevention Research Institute of Texas (CPRIT; RR15014 to W.X. and RR15088 to F.M.), the National Institutes of Health (1R01DK115445-01A1 to W.X.; U24CA228550 and 1R01CA241600-01 to F.M.), the US Dept. of Defense (W81XWH-17-1-0123 to W.X.; W81XWH-19-1-0127 to C.P.C.), the Singapore National Medical Research Council (to K.Y.H. and F.M.), a University of Texas Presidential Award (to W.X.) and an American Gastroenterology Association Research and Development Pilot Award in Technology (to W.X.). W.X. and F.M. are CPRIT Scholars for Cancer Research. We thank all the members of the Xian–McKeon laboratory for helpful discussions and support. We thank H. Green and J. Rosen for advice and support. We thank H. Green for the generous gift of the 3T3-J2 fibroblast cell line.

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Author notes

  1. These authors contributed equally: Marcin Duleba, Yusuke Yamamoto.

Authors and Affiliations

  1. Stem Cell Center, Department of Biology and Biochemistry, University of Houston, Houston, Texas, USA

    Marcin Duleba, Rahul Neupane, Wei Rao, Jingzhong Xie, Yutao Qi, Audrey-Ann Liew, Suchan Niroula, Yanting Zhang, Rajasekaran Mahalingam, Shan Wang, Kristina Goller, Frank McKeon & Wa Xian

  2. Division of Cellular Signaling, National Cancer Center Research Institute, Tokyo, Japan

    Yusuke Yamamoto

  3. Department of Gastrointestinal Medical Oncology, Division of Cancer Medicine, University of Texas MD Anderson Cancer Center, Houston, Texas, USA

    Jaffer A. Ajani

  4. Tract Pharmaceuticals, Inc., Marlborough, Massachusetts, USA

    Matthew Vincent

  5. Departments of Medicine and Pathology, National University of Singapore, Singapore, Singapore

    Khek Yu Ho

  6. Division of Gastroenterology, Baylor College of Medicine, Houston, Texas, USA

    Jason K. Hou

  7. Division of Digestive Diseases, Hepatology, and Nutrition, Connecticut Children’s Medical Center, Hartford, Connecticut, USA

    Jeffrey S. Hyams

  8. Division of Gastroenterology, Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

    Francisco A. Sylvester

  9. Department of Pathology, Harvard Medical School and Brigham and Women’s Hospital, Boston, Massachusetts, USA

    Christopher P. Crum

Authors
  1. Marcin Duleba
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Contributions

W.X., Y.Y., M.D. and F.M. wrote the manuscript with input from all other authors. J.A.A., K.Y.H., J.K.H., J.S.H., F.A.S. and C.P.C. provided biopsy material; M.D., Y.Y., Y.Z. and R.N. cloned gISCs; J.X., S.W. and R.M. performed informatics analyses; A-A.L., Y.Q. and K.G. performed xenografts and ALI cultures; W.R. and R.N. performed the FACS analyses; and all authors collaborated on the concepts of this work.

Corresponding authors

Correspondence to Frank McKeon or Wa Xian.

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Competing interests

W.X., F.M., M.D. and M.V. have filed patents related to the technology used in the present work. W.X., M.V. and F.M. have financial interests in Tract Pharmaceuticals, Inc.

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Related links

Key references using this protocol

Wang, X. et al. Nature 522, 173–178 (2015): https://doi.org/10.1038/nature14484

Duleba, M. et al. Gastroenterology 156, 20–23 (2019): https://doi.org/10.1053/j.gastro.2018.08.062

Yamamoto, Y. et al. Nat. Commun. 7, 10380 (2016): https://doi.org/10.1038/ncomms10380

Integrated supplementary information

Supplementary Fig. 1 FACS profiling of unlabeled gISCs.

Left, Debris removed by forward scatter (FSC) vs back scatter (BSC). Middle, Singlet selection using singlets are gated using FSC area (FSC-A) versus FSC width (FSC-W). Right, Selection of GFP-positive cells using FITC-A versus BSC-A.

Supplementary Fig. 2 FACS profiling of unlabeled 3T3-J2 cells.

Left, Debris removed by forward scatter (FSC) vs back scatter (BSC). Middle, Singlet selection using singlets are gated using FSC area (FSC-A) versus FSC width (FSC-W). Right, Selection of GFP-positive cells using FITC-A versus BSC-A.

Supplementary Fig. 3 FACS profiling of GFP-gISC/3T3-J2 co-cultures.

Left, Debris removed by forward scatter (FSC) vs back scatter (BSC). Middle, Singlet selection using singlets are gated using FSC area (FSC-A) versus FSC width (FSC-W). Right, Selection of GFP-positive cells using FITC-A versus BSC-A.

Supplementary Fig. 4 FACS profiling of GFP-gISC/3T3-J2 co-cultures following removal of 3T3-J2 cells.

Left, Debris removed by forward scatter (FSC) vs back scatter (BSC). Middle, Singlet selection using singlets are gated using FSC area (FSC-A) versus FSC width (FSC-W). Right, Selection of GFP-positive cells using FITC-A versus BSC-A.

Supplementary information

Supplementary Information

Supplementary Figs. 1–4, Supplementary Table 1 and Supplementary Methods.

Reporting Summary

Supplementary Video 1

Time lapse of colony growth from gISCs in vitro.

Supplementary Video 2

Clonal expansion of gISCs in vitro.

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Duleba, M., Yamamoto, Y., Neupane, R. et al. Cloning of ground-state intestinal stem cells from endoscopic biopsy samples. Nat Protoc 15, 1612–1627 (2020). https://doi.org/10.1038/s41596-020-0298-4

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