Functional engraftment of colon epithelium expanded in vitro from a single adult Lgr5+ stem cell

Abstract

Adult stem-cell therapy holds promise for the treatment of gastrointestinal diseases. Here we describe methods for long-term expansion of colonic stem cells positive for leucine-rich repeat containing G protein-coupled receptor 5 (Lgr5(+) cells) in culture. To test the transplantability of these cells, we reintroduced cultured GFP(+) colon organoids into superficially damaged mouse colon. The transplanted donor cells readily integrated into the mouse colon, covering the area that lacked epithelium as a result of the introduced damage in recipient mice. At 4 weeks after transplantation, the donor-derived cells constituted a single-layered epithelium, which formed self-renewing crypts that were functionally and histologically normal. Moreover, we observed long-term (>6 months) engraftment with transplantation of organoids derived from a single Lgr5(+) colon stem cell after extensive in vitro expansion. These data show the feasibility of colon stem-cell therapy based on the in vitro expansion of a single adult colonic stem cell.

The non-muscle-myosin-II heavy chain Myh9 mediates colitis-induced epithelium injury by restricting Lgr5+ stem cells

“…DSS-induced acute colitis is a widely employed model to study colitis, which is thought to be provoked by inflammation 13,38,39 . However, recent studies suggest that damage of colonic stem cells could be a major cause [13][14][15] . In addition, disruption of epithelial cell contacts could be important in the pathological process as the loss of tight junctions of colonic epithelial cells was observed in DSS-induced colitis [40][41][42] .…”

“…As colonic epithelium injuries are critical for colitis initiation [13][14][15] , we then accessed whether Myh9 monoallelic loss could alleviate DSS-induced acute colitis. The disease activity index (DAI) has been commonly used to evaluate the grade and extent of acute colitis 18 .…”

“…DSS causes damage to colonic epithelial cells of the basal crypts and affects the integrity of the mucosal barrier. Lgr5 þ colonic stem cells are sensitive to DSS treatment and play critical roles in colonic epithelium regeneration 14,15 . Therefore, the DSS-induced acute colitis is a good model for investigation of the injury and regeneration of colonic crypts and the functions of Lgr5 þ stem cells.…”

A Human Colonic Crypt Culture System to Study Regulation of Stem Cell-Driven Tissue Renewal and Physiological Function

“…The Clevers' Laboratory paved the way by capitalising on their seminal work on Wnt signalling, intestinal stem cells and cancer [8][9][10], and developed the mouse small intestinal organoid culture system [11,12]. This 'mini-gut' culture system formed the basis for the development of human organoid cultures from the intestinal epithelium [13,14,1] and from (inducible) pluripotent stem cells [15], both of which hold the potential for therapeutic transplantation [16,17]. For the study of native intact human colonic crypts, we adopted a similar approach whereby crypts are embedded in a basement membrane-derived matrix, Matrigel [18], instead of collagen I [7], and serum-free culture medium is supplemented with the intestinotrophic factors IGF-1 (activator of RAS signalling pathway) and R-spondin (activator of Wnt signals) in combination with inhibitors of BMP (Noggin or Gremlin proteins) and TGF (SB431542 or A83-01 small molecules) signalling pathways [18].…”

Modeling colorectal cancer as a 3-dimensional disease in a dish: the case for drug screening using organoids, zebrafish, and fruit flies

“…Additionally, recent work by the Watanabe laboratory (2012) [47] showed that organoids retain the functionality of the intestine when transplanted back into test animals. This was demonstrated by elegant transplantation experiments showing that GFP-labeled organoids can replace damaged colonic tissue when transplanted into colons of mice that had been treated with the colitis inducing chemical dextran sodium sulfate (DSS) [47]. Together these experiments demonstrate that stem cells cultured within organoids retain the capacity for stem cell self-renewal and differentiation.…”