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Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/Rspondin 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 signaling 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) which expand 5-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

ORGANISM(S): Mus musculus

PROVIDER: GSE50103 | GEO | 2013/09/17

SECONDARY ACCESSION(S): PRJNA216069

REPOSITORIES: GEO

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

Project description: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 signaling 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) which expand 5-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 We generated arrays from whole pancreas, islet, acinar and duct (Sox9+ sorted) cells and pancreas derived cultures maintained in our defined medium. For the clustering analysis we substracted the pancreas array to all arrays. Genes 2 fold differentially expressed in the ductal array were used for the analsyis. For the genes enriched in organoid cultures compared to pancreas we substracted the organoid culture arrays to the pancreas array. Genes >2-fold differentially expressed were used for the analysis.

2013-09-17 | E-GEOD-50103 | biostudies-arrayexpress

In vitro expansion of single adult Lgr5+ liver cells induced by Wnt-driven regeneration

Project description:The Wnt target gene Lgr5 marks actively dividing stem cells in Wnt-driven, self-renewing tissues such as small intestine and colon, stomach and hair follicles. A 3D culture system allows long-term clonal expansion of single Lgr5+ stem cells into transplantable organoids that retain many characteristics of the original epithelial architecture. A crucial component of the culture medium is the Wnt agonist Rspo, the recently discovered ligand of Lgr5. Here we show that Lgr5-LacZ is not expressed in healthy adult liver, yet that small Lgr5-LacZ+ cells appear near bile ducts upon damage, coinciding with robust activation of Wnt signaling. As shown by lineage tracing using a novel Lgr5-ires-CreERT2 knock-in allele, damage-induced Lgr5+ cells generate hepatocytes and bile ducts in vivo. Single Lgr5+ cells from damaged liver can be clonally expanded as organoids in Rspo1-based culture medium over multiple months. Such clonal organoids can be induced to differentiate in vitro and to generate functional hepatocytes upon transplantation into FAH-/- mice. These findings imply that previous findings on Lgr5+ stem cells in actively self-renewing tissues extend to damage-induced stem cells in a tissue with a low rate of spontaneous self-renewal. We first generated arrays from multiple wildtype tissues including muscle, white adipose tissues, brown adipose tissues, liver and pancreas. Then we generated arrays from liver derived cultures maintained in different conditions, and compared the expression profile with the corresponding parental tissues and other non-related tissues.

2013-01-28 | E-GEOD-32210 | biostudies-arrayexpress

Gene expression profiles of DLD1 cells in which GATA6 or LGR5 expression was suppressed.

Project description:Mutations in APC or β-catenin that cause aberrant activation of Wnt signaling are responsible for the initiation of colorectal tumor development. LGR5 is specifically expressed in stem cells of the intestine, stomach and hair follicle, and plays essential roles in maintaining tissue homeostasis. LGR5-positive stem cells have been shown to be responsible for the intestinal adenoma initiated by some mutations in APC . Furthermore, it has recently been reported that Lgr5, which is associated with the Frizzled/Lrp Wnt receptor complex, interacts with R-spondins and thereby activates Wnt signaling. However, the function of LGR5 in colorectal tumorigenesis has been unclear. Here we show that LGR5 is required for the tumorigenicity of colorectal cancer cells. We also show that the transcription factor GATA6 directly enhances the expression of LGR5. DLD1 cells were infected with a lentivirus expressing an shRNA targeting GATA6 or LGR5.

2011-10-17 | E-GEOD-32987 | biostudies-arrayexpress

Self-Renewing Tri-Potent Stem/Progenitor-like Cells from Adult Human Pancreas

Project description:Stem and progenitor cells in the adult human pancreas provide an under-explored resource for regenerative medicine. Using micro-manipulation and methylcellulose-containing colony/organoid assays, we identified cells within the human cadaveric exocrine pancreas that fulfill the definition of a stem cell: able to self-renew and differentiate. Exocrine tissues were collected after the isolation of endocrine cells, dissociated into single cells, and plated into a 3-dimensional semisolid medium. We found that some pancreatic ductal cells gave rise to cystic colonies/organoids containing pancreatic duct, acinar, and endocrine lineage cells. These cells self-renewed and expanded approximately 300-fold over 9 weeks. When transplanted into diabetic mice, colonies/organoids lowered blood glucose levels and gave rise to insulin-expressing endocrine cells. Thus, stem/progenitor-like cells capable of self-renewal and differentiation either preexist in the adult human pancreas or readily adapt in culture. These human cells have implications for therapy in diabetes patients.

2021-07-02 | GSE153834 | GEO

Characterization of human pancreatic tissue and organoids reveals subpopulation of intercalated duct cells with progenitor traits

Project description:Pancreatic duct organoids have emerged as a valuable tool for investigating pancreas biology and physiology through in vitro modeling. Despite their widespread use, a comprehensive understanding of the cellular heterogeneity of organoids and their underlying tissue remains elusive. Here we performed single-cell transcriptomic profiling of human adult pancreatic exocrine tissue and their derived organoids, revealing a gradient of keratin expression within the ductal network that clustered cells into two distinct low- and high-keratin subpopulations. Keratin-high cells display a higher capacity to form organoids while keratin-low cells express stemness associated markers. Finally, comparative gene expression analysis between ductal tissue and derived organoids revealed a partial recapitulation of the in vivo heterogeneity. These findings further our knowledge on pancreatic duct organoids and pave the way for future investigations into the heterogeneity of the pancreatic ductal syste

2024-04-09 | GSE263364 | GEO

In vitro expansion of single adult Lgr5+ liver cells induced by Wnt-driven regeneration

2013-01-28 | GSE32210 | GEO

Gene expression profiles of DLD1 cells in which GATA6 or LGR5 expression was suppressed.

2011-10-18 | GSE32987 | GEO

Single-cell-resolved differentiation of human induced pluripotent stem cells into pancreatic duct-like organoids on a microwell chip

Project description:Creating in vitro models of diseases of the pancreatic ductal compartment requires a comprehensive understanding of the developmental trajectories of pancreas-specific cell types. Here, we report the single-cell characterization of the differentiation of pancreatic duct-like organoids (PDLOs) from human induced pluripotent stem cells (hiPSCs) on a microwell chip that facili-tates the uniform aggregation and chemical induction of hiPSC-derived pancreatic progenitors. Using time-resolved single-cell transcriptional profiling and immunofluorescence imaging of the forming PDLOs, we identified differentiation routes from pan-creas progenitors through ductal intermediates to two types of mature duct-like cell and a few non-ductal cell types. PDLO subpopulations expressed either mucins or the cystic fibrosis transmembrane conductance regulator, and resembled human adult duct cells. We also used the chip to uncover ductal markers relevant to pancreatic carcinogenesis, and to establish PDLO co-cultures with stellate cells, which allowed for the study of epithelial–mesenchymal signalling. The PDLO microsystem could be used to establish patient-specific pancreatic duct models.

2021-07-08 | GSE162547 | GEO

Modelling Plasticity and Dysplasia of Pancreatic Ductal Organoids Derived from Human Pluripotent Stem Cells

Project description:Full protein measurements from in vitro differentiation of the human embryonic stem cell line HUES8 into pancreatic progenitors (PP) and pancreatic duct-like organoids (PDLOs). Protein intensities were quantified by mass spectrometry analysis from PPs at day 13 and from PDLOs at day 59. Please see related publication “Modelling Plasticity and Dysplasia of Pancreatic Ductal Organoids Derived from Human Pluripotent Stem Cells” for experimental details.

2021-03-19 | PXD018785 | Pride

Producing enteroendocrine cells from Lgr5+ intestinal stem cells by manipulating quiescence

Project description:Lgr5+ adult intestinal stem cells are highly proliferative throughout life. Single Lgr5+ stem cells can be cultured into 3D epithelial organoids containing all cell types at nearnormal ratios. Culture conditions to generate the main cell types have been established previously, but signals inducing the various types of enteroendocrine cells (EECs) have remained elusive. Here we generate quiescent Lgr5+ stem cells in vitro by inhibition of the EGF-receptor (EGFR) and mitogen-associated protein kinase (MAPK) signaling pathways in organoids, a state that can be readily reversed. Quiescent Lgr5+ stem cells gain a distinct molecular signature, biased towards EEC differentiation. Indeed, combined inhibition of Wnt, Notch and MAPK pathways efficiently generates a diversity of EEC subtypes in vitro. Our observations uncouple Wnt-dependent stem cell maintenance from EGF-dependent proliferation and cell fate choice, and provide an in vitro approach for the study of the elusive EECs.

2017-01-03 | GSE80636 | GEO

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