This study describes a robust long-term methodology for either small or large intestinal culture, allowing sustained intestinal proliferation and multilineage differentiation for a range of 30 d to >350 d. The lack of long-term methodology for primary intestinal culture has been a substantial obstacle to exploration of intestinal stem and progenitor cell biology and more general questions of physiology, despite attempts to recapitulate normal intestinal epithelial growth and differentiation in vitro15–17
. Monolayer cultures have been complicated by rapid apoptosis18,19
, whereas organoid cultures have historically been unable to preserve viability lasting more than ~10 d21
. More recently developed 3D organoid cultures have been restricted to embryonic tissue and again have shown restricted viability, typically less than 14 d22
. Consequently, implantation of dissociated intestinal material into syngeneic hosts or immuno-compromised mice, either directly or with an intervening culture period, has been the only available tool with which to demonstrate the presence and potential of stem cells and progenitors in cultured intestine17
To overcome the difficulties described above, we have maintained minced intestinal fragments in a 3D matrix scaffold of type I collagen gel under an air-liquid interface. With growing evidence that the 3D matrix environment has a crucial role in facilitating the behavior of stem cells and tissue morphogenesis, organotypic 3D cultures have great advantages over both conventional two-dimensional cell approaches and animal models31–33
. The major features of 3D collagen gel models are derived from their ability to mimic normal tissue organization to induce appropriate polarity of epithelial cells, to induce behavior of fibroblasts including extracellular matrix remodeling and to induce signaling between cell-cell and cell-matrix interactions34,35
. Additionally, air-liquid interface methods allow long-term culture of various epithelial cell types via improved oxygenation in vitro36–38
. In the current method, substitution of conventional immersed conditions for an air-liquid interface markedly decreases viability, although sporadic short-term sphere formation and growth can be observed. Our results suggest that our 3D culture system enables mouse intestinal fragments to recapitulate intestinal epithelial growth and differentiation within the microenvironment of an in vitro
Adult or somatic stem cells generally have limited function without their niche8
. Our method allows primary intestinal epithelium to be cultured in close apposition to myofibroblasts, which have been proposed to be the candidate niche supporting ISCs and influencing intestinal epithelial growth1,9,10
. Indeed, the growth of individual intestinal spheres was highly correlated with the preceding growth of myofibroblasts, which lined the basal surface of the cultured intestinal epithelium. Thus, appropriate stromal cell growth seems to permit long-term culture and multilineage differentiation of intestinal epithelial cells without implantation into syngenic or immunocompromised host animals. Our results by no means exclude the possibility of culturing intestinal epithelium without a cellular niche, which could certainly be achieved with appropriate extracellular signals.
Cultures <30 d old were predominantly comprised of viable, robustly proliferating tissue, which was present but increasingly sporadic at extremely long culture durations of >200 d. Although we obtained the most prolonged culture with neonatal intestine, we observed limited culture for 7 to 10 d without RSpo1-Fc treatment and up to 4 weeks with RSpo1-Fc treatment using adult mouse intestine as starting material. These aforementioned limitations of long-term and adult culture may be secondary either to progressive deficits in either the stromal myofibroblasts or other niche components in vitro or to cell-intrinsic deficits in ISCs, transit amplifying cells or both.
Accurate recapitulation of the in vivo
Wnt and Notch dependence of the ISC niche represents a prominent feature of the intestinal sphere cultures. Indeed, Dkk1 and dibenzazepine treatment phenocopied the intestinal effects of in vivo
Wnt and Notch inhibition on proliferation and goblet cell differentiation, respectively11–13,25
. At the same time, the endogenous Wnt and Notch signaling within the intestinal cultures was sufficient to support vigorous expansion, and exogenously added RSpo1-Fc conferred a further induction of intestinal epithelial growth, even allowing limited expansion of adult intestinal cultures. In this regard, RSpo1-Fc induced expansion of transit amplifying cell populations, which express the Wnt target genes Cd44
in concert with PCNA+
proliferative activity. This preservation of the ISC niche probably underlies the successful support of long-term proliferation and differentiation observed in the current studies. These studies further illustrate the utility of extracellular Wnt agonists, particularly R-spondin-1, as a potent growth factor that markedly enhances the efficacy of intestinal culture.
The observed prolonged growth and differentiation (>350 d) suggest the presence of ISCs or extremely long-lived transit amplifying cells in the sphere cultures. Our detection of both Lgr5+ cells as well as cells derived from the Bmi1+ lineage within the intestinal cultures is consistent with the former possibility. The potential regulation of ISCs by extracellular Wnt signals has been a plausible but previously untested hypothesis. The observed increase in both Lgr5+ lineage– and Bmi1+ lineage–derived cells by RSpo1-Fc treatment in vitro is consistent with the direct regulation of ISCs by Wnt signals, although we can not exclude the alternative possibilities that transit amplifying cells expressing these markers may have arisen in culture, or that RSpo1-Fc–induced Lgr5+ cells merely reflect increased Wnt-dependent gene expression rather than increased ISC number. We observed similar increases in crypt Lgr5+ cells after in vivo adenovirus RSpo1-Fc treatment, demonstrating the predictive nature of the in vitro culture system, although in both cases our in situ hybridization analyses only allow conclusions regarding Lgr5 mRNA and not Lgr5 protein.
The availability of a robust intestinal culture system supporting the expression of populations expressing ISC markers, accurately recapitulating the ISC niche in both small and large intestine and applicable to both neonatal and adult tissues should greatly facilitate the study of intestinal stem cells and niche-ISC interactions. This model could also potentially be used to study intestinal epithelial interactions with other heterologous cell types, including neurons, smooth muscle, endothelial cells and immune cells. Furthermore, the enablement of primary intestinal culture should have widespread application to general studies of intestinal biology, including investigations of physiology, host-pathogen interactions, neoplasia, tissue engineering and regenerative medicine.