Intestinal Subepithelial Myofibroblasts Support in vitro and in vivo Growth of Human Small Intestinal Epithelium

Abstract

The intestinal crypt-niche interaction is thought to be essential to the function, maintenance, and proliferation of progenitor stem cells found at the bases of intestinal crypts. These stem cells are constantly renewing the intestinal epithelium by sending differentiated cells from the base of the crypts of Lieberkühn to the villus tips where they slough off into the intestinal lumen. The intestinal niche consists of various cell types, extracellular matrix, and growth factors and surrounds the intestinal progenitor cells. There have recently been advances in the understanding of the interactions that regulate the behavior of the intestinal epithelium and there is great interest in methods for isolating and expanding viable intestinal epithelium. However, there is no method to maintain primary human small intestinal epithelium in culture over a prolonged period of time. Similarly no method has been published that describes isolation and support of human intestinal epithelium in an in vivo model. We describe a technique to isolate and maintain human small intestinal epithelium in vitro from surgical specimens. We also describe a novel method to maintain human intestinal epithelium subcutaneously in a mouse model for a prolonged period of time. Our methods require various growth factors and the intimate interaction between intestinal sub-epithelial myofibroblasts (ISEMFs) and the intestinal epithelial cells to support the epithelial in vitro and in vivo growth. Absence of these myofibroblasts precluded successful maintenance of epithelial cell formation and proliferation beyond just a few days, even in the presence of supportive growth factors. We believe that the methods described here can be used to explore the molecular basis of human intestinal stem cell support, maintenance, and growth.

Use of Collagen Gel as an Alternative Extracellular Matrix for the In Vitro and In Vivo Growth of Murine Small Intestinal Epithelium

“…Our implantation scheme relies on co-culture with myofibroblasts. This limitation is not unique to the collagen system; we have observed that both murine (unpublished results) and human 7 crypts grown in Matrigel require myofibroblasts for successful subcutaneous in vivo engraftment.…”

“…7 The ISEMF were cultured using myofibroblast medium consisting of DMEM/Low Glucose/ GlutaMAX (Invitrogen), 10% FBS (Invitrogen), 1 · Antibiotic-Antimycotic (Invitrogen), 0.25 U/mL insulin (Sigma), 20 ng/mL recombinant murine EGF (PeproTech, Rocky Hill, NJ), and 10 mg/mL transferrin (Sigma).…”

“…3,4 Following the description of a novel method for their long-term in vitro culture, 5 there have been reports of successful culture of the various mucosal epithelia of murine stomach, murine colon, human small intestine, both benign and neoplastic human 1 colon, and human Barrett's esophagus. [6][7][8] Previous methods for in vivo implantation relied on initial isolation of stem cell clusters surrounded by neighboring mesenchyme, [9][10][11][12] whereas the current culture techniques appear to have obviated this requirement. 13 In vitro cultured small intestinal units have been variously termed enteroids, spheroids, and organoids by different groups.…”

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

“…Within the intestinal stem cell niche, signals to maintain the undifferentiated nature of these cells, and thereby the epithelial integrity, are provided by surrounding mesenchymal cells. 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).…”

Stroma provides an intestinal stem cell niche in the absence of epithelial Wnts

“…However, depletion of Paneth cells or knockout of Wnt3 in the epithelial cells of the intestine did not show an obvious in vivo phenotype (Durand et al, 2012;Farin et al, 2012), indicating that other functionally important Wnts are made by epithelial or stromal niche cells. Several groups have demonstrated that stromal cells can support the growth of intestinal epithelium in culture (Farin et al, 2012;Lahar et al, 2011). Intestinal stromal cells express multiple Wnts (Farin et al, 2012;Gregorieff et al, 2005 One approach to address the functionally relevant source of Wnts in the small intestine is to globally target their secretion.…”