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Tissue underlying the intestinal epithelium elicits proliferation of intestinal stem cells following cytotoxic damage

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Abstract

The goals of this study were to document the proliferative response of intestinal stem cells (ISCs) during regeneration after damage from doxorubicin (DXR), and to characterize the signals responsible for ISC activation. To this end, jejuni from DXR-treated mice were harvested for histology, assessment of ISC numbers and proliferation by flow cytometry, crypt culture, and RNA analyses. Histology showed that crypt depth and width were increased 4 days after DXR. At this time point, flow cytometry on tissue collected 1 h after EdU administration revealed increased numbers of CD24loUEA− ISCs and increased percentage of ISCs cycling. In culture, crypts harvested from DXR-treated mice were equally proliferative as those of control mice. Addition of subepithelial intestinal tissue (SET) collected 4 days after DXR elicited increased budding (1.4 ± 0.3 vs. 5.1 ± 1.0 buds per enteroid). Microarray analysis of SET collected 4 days after DXR revealed 1030 differentially expressed transcripts. Cross-comparison of Gene Ontology terms considered relevant to ISC activation pointed to 10 candidate genes. Of these, the epidermal growth factor (EGF) family member amphiregulin and the BMP antagonist chordin-like 2 were chosen for further study. In crypt culture, amphiregulin alone did not elicit significant budding, but amphiregulin in combination with BMP antagonism showed marked synergism (yielding 6.3 ± 0.5 buds per enteroid). These data suggest a critical role for underlying tissue in regulating ISC behavior after damage, and point to synergism between amphiregulin and chordin-like 2 as factors which may account for activation of ISCs in the regenerative phase.

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Acknowledgements

We wish to thank: Ajay Gulati, MD, and Richard Moffitt, PhD, for their input toward experimental design of this project; Allison Rogala, DVM, for histologic assessment of inflammation in DXR-treated intestinal tissue; and Carlton W. Anderson and Scott T. Magness, PhD, of the Center for Gastrointestinal Biology and Disease’s Advanced Analytics Core for their valuable technical assistance with the Fluidigm qRT-PCR. We additionally acknowledge Joe Galanko, PhD, for statistical advice, Marcus Muehlbauer, MD PhD, for constructive input on the revised manuscript, Carolyn Suitt (UNC CGIBD Histology Core), Mike Vernon (UNC Functional Genomics Core) and valuable assistance from the UNC Flow Cytometry Core. This work was supported by the National Institutes of Health (NIH) Grants P30 DK034987, and U01-DK085547. The latter grant is part of the Intestinal Stem Cell Consortium, a collaborative research project funded by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) and the National Institute of Allergy and Infectious Diseases. Kristen Seiler was supported by the Medical Student Research Training Program (MSRTP) from the NIDDK funded as a supplement to NIH grant T32-DK007737. The UNC Flow Cytometry Core Facility is supported in part by NCI Center Core Support Grant (P30 CA016086) to the UNC Lineberger Comprehensive Cancer Center.

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Authors and Affiliations

  1. Department of Medicine and Center for Gastrointestinal Biology and Disease, University of North Carolina at Chapel Hill, 4341 Medical Biomolecular Research Building (MBRB), CB# 7032, Chapel Hill, NC, 27599-7032, USA

    Kristen M Seiler, Erica L Schenhals, Richard J von Furstenberg, Bhavya K Allena, Brian J Smith, Denny Scaria, Michele N Bresler & Susan J Henning

  2. Department of Surgery and Center for Gastrointestinal Biology and Disease, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA

    Christopher M Dekaney

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  1. Kristen M Seiler
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Correspondence to Susan J Henning.

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Supplemental Fig. 1

Representative images (×40) of H&E-stained jejunal sections from: a control mice and b mice 4 days post-DXR treatment. Crypts are outlined in white. In addition to effects on crypt morphology, there were obvious differences in the lamina propria. Control mice (a) show normal intracrypt cellularity, whereas DXR-treated mice (b) show reduced numbers of crypts and clear evidence of inflammatory infiltration. (GIF 156 kb)

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Seiler, K.M., Schenhals, E.L., von Furstenberg, R.J. et al. Tissue underlying the intestinal epithelium elicits proliferation of intestinal stem cells following cytotoxic damage. Cell Tissue Res 361, 427–438 (2015). https://doi.org/10.1007/s00441-015-2111-1

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