Functional Engraftment of Colon Epithelium Expanded In Vitro from a Single Adult Lgr5+ Stem Cell EPITHELIAL REGENERATION IS one ered to hold promise for the treatment of Mamoru Watanabe of the critical steps necessary for the heal- various gastrointestinal disorders such as ing of wounds at the surface of small in- inflammatory bowel diseases, while it testine and colon. We have investigated was impossible to evaluate even its feasi- this process intensely and previously re- bility because of the technical difficulties MD, PhD Professor, Gastroenterology and Hepatology, Graduate School of Medical and Dental Sciences, TMDU ported that damaged epithelia of the hu- to expand intestinal epithelial stem cells man intestinal tract were partly rescued in vitro. cells.1) transplantation of organoids derived from Howev- Our group led by Drs. Shiro Yui and Tet- a single adult Lgr5+ colon stem cell after er, regeneration of wounded surfaces suya Nakamura recently succeeded to extensive in vitro expansion. In addition, mainly depends on the re-expansion of show the first positive evidence for these engrafted recipient mice had higher body epithelial cells after damage and adult ep- very challenging issues.2) In this paper, weights than ungrafted controls, implying ithelial stem-cell therapy has been consid- we firstly reported our original organoid a beneficial role of epithelial transplanta- Fig.1: Colonic Stem Cell Culture and Transplantation culture methodology, named TMDU pro- tion at least in DSS-induced acute colitis tocol for long-term expansion of colonic model. These observations clearly con- stem cells, in which cells that are positive firmed that Lgr5 marks genuine stem for leucine-rich repeat containing G pro- cells that retain their self-renewal and tein-coupled receptor 5 (Lgr5). In brief, multilineage-differentiation properties we used Type I collagen gel as an extra- even after prolonged culture, and also cellular matrix of this three-dimensional they exactly revealed the feasibility of co- culture protocol and the medium that we lon stem-cell therapy based on the in vitro used contained only five types of recom- expansion of a single adult colonic stem binant proteins (Wnt3a, R-spondin1, cell. by bone marrow-derived Lgr5+ stem cells In vitro expansion from a single stem cell Epithelial Transplantation by expanded stem cells Re-epithelialization of damaged epithelium by transplanted cultured epithelial cells Noggin, EGF and HGF) and BSA. Inter- Our study provided for the first time a estingly, Lgr5+ colonic stem cells ap- proof of principle that cultured Lgr5+ peared to expand unrestrictedly in terms cells can be used for stem-cell therapy of their proliferative capacity under this to repair damaged epithelium. The most defined and completely serum-free condi- out-standing message of this paper was tion, forming round cystic structures strongly highlighted in Nature as being called colon organoids which maintained “one-step closer to gut repair.”3) Although their original colonic identity. Secondly, further optimization is clearly needed, our we tested the transplantability of these study strongly implies that in vitro expan- cultured colonic epithelial cells by rein- sion and transplantation of gastrointesti- troducing GFP+ colon organoids into a nal stem cells may be a promising, sim- superficially damaged mouse colon, and ple and safe option of regenerative and found that transplanted donor cells readi- gene-therapy strategies for patients with ly integrated into the mouse colon, cover- severe gastrointestinal epithelial injuries. ing the area that lacked epithelium as a result of the introduced damage by DexDamaged epithelium tran Sodium Sulfate (DSS) in recipient mice. At four weeks after transplantation, Regeneration of normal colonic epithelium 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), histologically normal engraftment with 35 References 1. Ryuichi Okamoto, Tomoharu Yajima, Motomi Yamazaki, Takanori Kanai, Makio Mukai, Shinichiro Okamoto, Yasuo Ikeda, Toshifumi Hibi, Johji Inazawa, Mamoru Watanabe. Damaged epithelia regenerated by bone marrow-derived cells in the human gastrointestinal tract. Nature Medicine. 8(9): 1011-1017, 2002 2. Shiro Yui, Tetsuya Nakamura, Toshiro Sato, Yasuhiro Nemoto, Tomohiro Mizutani, Xiu Zheng, Shizuko Ichinose, Takashi Nagaishi, Ryuichi Okamoto, Kiichiro Tsuchiya, Hans Clevers, Mamoru Watanabe. Functional engraftment of colon epithelium expanded in vitro from a single adult Lgr5+ stem cell. Nature Medicine. 18(4): 618-623, 2012 3. Anisa Shaker, Deborah C. Rubin. One Step Closer to Gut Repair. Nature. 485: 181-182, 2012