title: Intestinal subepithelial myofibroblasts support the growth of intestinal epithelial stem cells.
abstract: 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.
authors.affiliation: Department of Bioengineering, University of California Los Angeles, Los Angeles, California, United States of America ; Department of Surgery, David Geffen School of Medicine at UCLA, University of California Los Angeles, Los Angeles, California, United States of America.
authors.forename: Nan Ye
authors.initials: NY
authors.lastname: Lei
authors.affiliation: Department of Surgery, David Geffen School of Medicine at UCLA, University of California Los Angeles, Los Angeles, California, United States of America.
authors.forename: Ziyad
authors.initials: Z
authors.lastname: Jabaji
authors.affiliation: Department of Pediatrics, Division of Gastroenterology and Nutrition, Mattel Children's Hospital and the David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America.
authors.forename: Jiafang
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Bioengineering, University of California Los Angeles, Los Angeles, California, United States of America.
authors.forename: Vaidehi S
authors.initials: VS
authors.lastname: Joshi
authors.affiliation: Department of Pediatrics, Division of Gastroenterology and Nutrition, Mattel Children's Hospital and the David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America.
authors.forename: Garrett J
authors.initials: GJ
authors.lastname: Brinkley
authors.affiliation: Department of Surgery, David Geffen School of Medicine at UCLA, University of California Los Angeles, Los Angeles, California, United States of America.
authors.forename: Hassan
authors.initials: H
authors.lastname: Khalil
authors.affiliation: Stowers Institute for Medical Research, Kansas City, Missouri, United States of America.
authors.forename: Fengchao
authors.initials: F
authors.lastname: Wang
authors.affiliation: Department of Molecular Cell and Developmental Biology, University of California Los Angeles, Los Angeles, California, United States of America.
authors.forename: Artur
authors.initials: A
authors.lastname: Jaroszewicz
authors.affiliation: Department of Molecular Cell and Developmental Biology, University of California Los Angeles, Los Angeles, California, United States of America.
authors.forename: Matteo
authors.initials: M
authors.lastname: Pellegrini
authors.affiliation: Stowers Institute for Medical Research, Kansas City, Missouri, United States of America ; Department of Pathology & Laboratory Medicine, University of Kansas Medical Center, Kansas City, Kansas, United States of America.
authors.forename: Linheng
authors.initials: L
authors.lastname: Li
authors.affiliation: Department of Pathology, Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California, United States of America.
authors.forename: Michael
authors.initials: M
authors.lastname: Lewis
authors.affiliation: Department of Surgery, David Geffen School of Medicine at UCLA, University of California Los Angeles, Los Angeles, California, United States of America ; Department of Surgery, Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California, United States of America.
authors.forename: Matthias
authors.initials: M
authors.lastname: Stelzner
authors.affiliation: Department of Bioengineering, University of California Los Angeles, Los Angeles, California, United States of America ; Department of Surgery, David Geffen School of Medicine at UCLA, University of California Los Angeles, Los Angeles, California, United States of America.
authors.forename: James C Y
authors.initials: JC
authors.lastname: Dunn
authors.affiliation: Department of Pediatrics, Division of Gastroenterology and Nutrition, Mattel Children's Hospital and the David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America.
authors.forename: Martín G
authors.initials: MG
authors.lastname: Martín
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D002478:Cells, Cultured; D016000:Cluster Analysis; D020869:Gene Expression Profiling; D007413:Intestinal Mucosa / Q000166:cytology* / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D058628:Myofibroblasts / Q000378:metabolism*; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism*; D059467:Transcriptome