In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration.
The Wnt target gene Lgr5 (leucine-rich-repeat-containing G-protein-coupled receptor 5) marks actively dividing stem cells in Wnt-driven, self-renewing tissues such as small intestine and colon, stomach and hair follicles. A three-dimensional culture system allows long-term clonal expansion of single Lgr5(+) stem cells into transplantable organoids (budding cysts) that retain many characteristics of the original epithelial architecture. A crucial component of the culture medium is the Wnt agonist RSPO1, the recently discovered ligand of LGR5. Here we show that Lgr5-lacZ is not expressed in healthy adult liver, however, small Lgr5-LacZ(+) cells appear near bile ducts upon damage, coinciding with robust activation of Wnt signalling. As shown by mouse lineage tracing using a new Lgr5-IRES-creERT2 knock-in allele, damage-induced Lgr5(+) cells generate hepatocytes and bile ducts in vivo. Single Lgr5(+) cells from damaged mouse liver can be clonally expanded as organoids in Rspo1-based culture medium over several months. Such clonal organoids can be induced to differentiate in vitro and to generate functional hepatocytes upon transplantation into Fah(-/-) mice. These findings indicate that previous observations concerning Lgr5(+) stem cells in actively self-renewing tissues can also be extended to damage-induced stem cells in a tissue with a low rate of spontaneous proliferation.
Hubrecht Institute for Developmental Biology and Stem Cell Research, University Medical Centre Utrecht, Netherlands.
Meritxell
M
Huch
Craig
C
Dorrell
Sylvia F
SF
Boj
Johan H
JH
van Es
Vivian S W
VS
Li
Marc
M
van de Wetering
Toshiro
T
Sato
Karien
K
Hamer
Nobuo
N
Sasaki
Milton J
MJ
Finegold
Annelise
A
Haft
Robert G
RG
Vries
Markus
M
Grompe
Hans
H
Clevers
D000483:Alleles; D000818:Animals; D001652:Bile Ducts / Q000166:cytology / Q000378:metabolism; D019070:Cell Lineage; D002999:Clone Cells / Q000166:cytology / Q000378:metabolism; D003470:Culture Media / Q000737:chemistry / Q000378:metabolism; D004195:Disease Models, Animal; D005260:Female; D055879:Gene Knock-In Techniques; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism* / Q000473:pathology; D006867:Hydrolases / Q000172:deficiency / Q000235:genetics; D008099:Liver / Q000166:cytology / Q000378:metabolism / Q000473:pathology; D008107:Liver Diseases / Q000378:metabolism / Q000473:pathology; D008297:Male; D051379:Mice; D039902:Multipotent Stem Cells / Q000166:cytology / Q000378:metabolism; D009940:Organoids / Q000166:cytology / Q000637:transplantation; D043562:Receptors, G-Protein-Coupled / Q000819:agonists / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D012038:Regeneration; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism*; D019699:Thrombospondins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D020176:Tyrosinemias / Q000378:metabolism / Q000473:pathology; D060449:Wnt Signaling Pathway
D003470:Culture Media; C493597:Lgr5 protein, mouse; C518903:RSPO1 protein, mouse; D043562:Receptors, G-Protein-Coupled; D019699:Thrombospondins; D006867:Hydrolases; C020156:fumarylacetoacetase