Intestinal subepithelial myofibroblasts support the growth of intestinal epithelial stem cells

• Published in: PloS one 2014-01, Vol.9 (1), p.e84651-e84651
• Main Authors: Lei, Nan Ye, Jabaji, Ziyad, Wang, Jiafang, Joshi, Vaidehi S, Brinkley, Garrett J, Khalil, Hassan, Wang, Fengchao, Jaroszewicz, Artur, Pellegrini, Matteo, Li, Linheng, Lewis, Michael, Stelzner, Matthias, Dunn, James C Y, Martín, Martín G
• Format: Article
• Language: English
• Subjects: Analysis; Animal models; Animals; Bioengineering; Biology; Cell culture; Cell Culture Techniques; Cell Proliferation; Cell self-renewal; Cells, Cultured; Cluster Analysis; Crypts; Developmental biology; Differentiation; Epithelium; Flow cytometry; Gastroenterology; Gene expression; Gene Expression Profiling; Growth; Growth factors; Homeostasis; House mouse; Implantation; Interrogation; Intestinal Mucosa - cytology; Intestinal Mucosa - metabolism; Intestine; Laboratories; Medical research; Medicine; Mice; Mice, Transgenic; Molecular biology; Monoculture; Myofibroblasts - metabolism; Nutrition; Pediatrics; Physiology; Rodents; Stem cell transplantation; Stem cells; Stem Cells - cytology; Stem Cells - metabolism; Surgery; Surgical implants; Syngeneic grafts; Transcriptome; Wnt protein
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0084651

Intestinal epithelial stem cells (ISCs) are the focus of recent intense study. Current in vitro models rely on supplementation with the Wnt agonist R-spondin1 to support robust growth, ISC self-renewal, and differentiation. Intestinal subepithelial myofibroblasts (ISEMFs) are important supportive cells within the ISC niche. We hypothesized that co-culture with ISEMF enhances the growth of ISCs in vitro and allows for their successful in vivo implantation and engraftment. ISC-containing small intestinal crypts, FACS-sorted single ISCs, and ISEMFs were procured from C57BL/6 mice. Crypts and single ISCs were grown in vitro into enteroids, in the presence or absence of ISEMFs. ISEMFs enhanced the growth of intestinal epithelium in vitro in a proximity-dependent fashion, with co-cultures giving rise to larger enteroids than monocultures. Co-culture of ISCs with supportive ISEMFs relinquished the requirement of exogenous R-spondin1 to sustain long-term growth and differentiation of ISCs. Mono- and co-cultures were implanted subcutaneously in syngeneic mice. Co-culture with ISEMFs proved necessary for successful in vivo engraftment and proliferation of enteroids; implants without ISEMFs did not survive. ISEMF whole transcriptome sequencing and qPCR demonstrated high expression of specific R-spondins, well-described Wnt agonists that supports ISC growth. Specific non-supportive ISEMF populations had reduced expression of R-spondins. The addition of ISEMFs in intestinal epithelial culture therefore recapitulates a critical element of the intestinal stem cell niche and allows for its experimental interrogation and biodesign-driven manipulation.