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.

CD34 + mesenchymal cells are a major component of the intestinal stem cells niche at homeostasis and after injury

“…These data are consistent with the previously reported expression of Wnt2b and Grem1 by intestinal mesenchyme (7,11), and of an ill-defined intestinal mesenchyme supporting IESC growth in vitro (34). We further show that inhibition of Grem1 significantly reduces the generation of CD34 + CSC-induced spheroids, indicating that secretion of Grem1 by CD34 + CSCs plays a predominant role in maintenance of IESCs.…”

Non-animal models of epithelial barriers (skin, intestine and lung) in research, industrial applications and regulatory toxicology

“…The alpha-smooth muscle actin expressing intestinal subepithelial myofibroblasts (ISEMFs) are known to support long-term epithelial growth in vitro and wound healing, for instance (Lahar et al, 2011;Seltana et al, 2010). This effect may be caused by factors such as R-Spondin-2, a Wnt-agonist, but it is still unclear if cell-cell contacts are necessary (Lei et al, 2014). More research is needed to understand the complex interplay between the different cell types to maintain the intestinal stem cell niche and gut homeostasis.…”

Tissue Engineering Functional Gastrointestinal Regions: The Importance of Stem and Progenitor Cells

“…A currently available biopharmaceutical, Glut-2 (teduglutide), treats human SBS and stimulates in vitro cultured ISEMFs to produce IGF-1 (Leen et al 2011). ISEMFs appear to be a critical ingredient of engineered intestine because they grow to enclose enteroids implanted in vivo and without them enteroids cannot survive in vivo (Lei et al 2014). In culture media supplemented with insulin, transferrin, and EGF, enteroids died without R-spondin, but lived with ISEMFs and without exogenous R-spondin.…”