Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis

Abstract

Lgr5 marks adult stem cells in multiple adult organs and is a receptor for the Wnt-agonistic R-spondins (RSPOs). Intestinal, stomach and liver Lgr5+ stem cells grow in 3D cultures to form ever-expanding organoids, which resemble the tissues of origin. Wnt signalling is inactive and Lgr5 is not expressed under physiological conditions in the adult pancreas. However, we now report that the Wnt pathway is robustly activated upon injury by partial duct ligation (PDL), concomitant with the appearance of Lgr5 expression in regenerating pancreatic ducts. In vitro, duct fragments from mouse pancreas initiate Lgr5 expression in RSPO1-based cultures, and develop into budding cyst-like structures (organoids) that expand five-fold weekly for >40 weeks. Single isolated duct cells can also be cultured into pancreatic organoids, containing Lgr5 stem/progenitor cells that can be clonally expanded. Clonal pancreas organoids can be induced to differentiate into duct as well as endocrine cells upon transplantation, thus proving their bi-potentiality.

Spontaneous regeneration of cochlear supporting cells after neonatal ablation ensures hearing in the adult mouse

“…Lgr5 + cells in the GER do not contribute significantly to the regeneration of IBCs and IPhCs, a surprising result given that Lgr5 is a marker of stem cells in other tissues (59)(60)(61)(62) and that Lgr5 + cells proliferate and transdifferentiate into HCs after genetic manipulations in the neonatal organ of Corti (34,38,40). However, we found that other cells in the GER, targeted with GlastCreER T induced with tamoxifen at P0 and P1, are required for the replacement of at least a fraction of IBCs/IPhCs.…”

Single Lgr5- or Lgr6-expressing taste stem/progenitor cells generate taste bud cells ex vivo

“…However, in other cases, such as Lgr5 + cells isolated from pancreatic ducts, no insulin-producing cells are found in long-term organoid cultures, indicating the requirement for additional extrinsic factors or intrinsic reprogramming (11). To determine whether any mature taste receptor cells are produced in organoids derived from Lgr5 + cells, we performed double or triple immunostaining of cultured organoids, using antibodies against the taste signaling components gustducin (G protein for sweet, bitter, and umami signaling), T1R3 (taste receptor type 1 member 3, a common subunit of the sweet T1R2/T1R3 and the umami T1R1/T1R3 taste receptor) (23)(24)(25), and carbonic anhydrase 4 (CA4; a marker of sour taste cells) (26).…”

“…Lgr5 is also known to mark actively cycling stem cells in small intestine, colon, stomach, and hair follicle, as well as quiescent stem cells in liver, pancreas, and cochlea (8). Isolated Lgr5 + adult stem cells from multiple tissues are able to generate so-called organoid structures ex vivo (9)(10)(11). For instance, Sato and colleagues (10) developed a 3D culture system to grow crypt-villus organoids from single intestinal stem cells; all differentiated cell types were found in these structures, indicating the multipotent nature of these cells.…”

Drug discovery through stem cell-based organoid models

“…49 Drug discovery today is at a crossroads: while increasingly large and 50 varied compound libraries are synthesized and tested in primary 51 screens, the promise of the identified lead compounds remains largely 52 unrealized. Indeed, while tremendous investments in automation 53 have enabled the costs and turnaround time for large to medium-scale 54 primary screening to fall significantly [1], the gap between lead com- 55 pound validation and success in the clinic is still wide, suggesting that 56 a process still beset by significant limitations in efficiency.…”

“…Indeed, there have also been in the last year reports of such diverse 496 organoids as the pituitary gland [52], inner ear [49], pancreas [53,54] …”