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Long term culture of genome-stable bipotent progenitor cells from adult human liver

ABSTRACT: Despite the enormous replication potential of the human liver, there are currently no culture systems available that sustain hepatocyte replication and/or function in vitro. We have shown previously that single mouse Lgr5+ liver stem cells can be expanded as epithelial organoids in vitro and can be differentiated into functional hepatocytes in vitro and in vivo. We now describe conditions allowing long-term expansion of adult bile duct-derived bi-potent progenitor cells from human liver. The expanded cells are highly stable at the chromosome and structural level, while single base changes occur at very low rates. The cells can readily be converted into functional hepatocytes in vitro and upon transplantation in vivo. Organoids from α1-antitrypsin deficiency and Alagille Syndrome patients mirror the in vivo pathology. Clonal long-term expansion of primary adult liver stem cells opens up experimental avenues for disease modeling, toxicology studies, regenerative medicine and gene therapy.

ORGANISM(S): Homo sapiens

PROVIDER: GSE63859 | GEO | 2015/01/15

SECONDARY ACCESSION(S): PRJNA269269

REPOSITORIES: GEO

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Long term culture of genome-stable bipotent progenitor cells from adult human liver

Project description:Despite the enormous replication potential of the human liver, there are currently no culture systems available that sustain hepatocyte replication and/or function in vitro. We have shown previously that single mouse Lgr5+ liver stem cells can be expanded as epithelial organoids in vitro and can be differentiated into functional hepatocytes in vitro and in vivo. We now describe conditions allowing long-term expansion of adult bile duct-derived bi-potent progenitor cells from human liver. The expanded cells are highly stable at the chromosome and structural level, while single base changes occur at very low rates. The cells can readily be converted into functional hepatocytes in vitro and upon transplantation in vivo. Organoids from Î±1-antitrypsin deficiency and Alagille Syndrome patients mirror the in vivo pathology. Clonal long-term expansion of primary adult liver stem cells opens up experimental avenues for disease modeling, toxicology studies, regenerative medicine and gene therapy. We generated arrays from whole human liver tissue, and human liver derived cultures maintained in our defined medium. Genes 4 fold differentially expressed were used for the clustering analsyis (see matrix 1). For the genes enriched in organoid cultures grown in ERFHNic+A compared to ERFHnic we normalized the arrays by substracting the liver tissue 1 array to the culture arrays. Then we calculated the fold difference between the ERFHNic+A and the ERFHNic samples (see matrix 2).

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Small molecule-mediated reprogramming of human hepatocytes into bipotent progenitor cells

Project description:Currently, much effort is directed to the development of new cell sources for clinical therapy using cell fate conversion approaches by small molecules. Direct lineage reprogramming to a progenitor state has been reported in terminally differentiated rodent hepatocytes, yet remains a challenge in human hepatocytes. Human hepatocytes were isolated from healthy and diseased donor livers and reprogrammed into progenitor cells by two small molecules, A83-01 and CHIR99021 (AC), in the presence of EGF and HGF. The stemness properties of human chemically derived hepatic progenitors (hCdHs) were tested by standard in vitro and in vivo assays and transcriptome profiling. We developed a robust culture system for generating hCdHs with therapeutic potential. The use of HGF proved to be an essential determinant of fate conversion process. Based on functional evidence, activation of HGF/MET signal transduction system collaborated with A83-01 and CHIR99021 to allow a rapid expansion of progenitor cells through activation of ERK pathway. hCdHs expressed hepatic progenitor marker genes and proteins, and could self-renew for at least 10 passages while retaining normal karyotype and potential to differentiate into functional hepatocytes and biliary epithelial cells in vitro. RNASeq gene expression profiling confirmed transcriptional reprogramming of hCdHs toward a progenitor state and suppression of mature hepatocyte transcripts. Upon intrasplenic transplantation into immunocompromised mice with acute liver injury, hCdHs effectively repopulated damaged parenchyma. Our study is a first report of successful reprogramming of human hepatocytes to a population of proliferating bipotent cells with regenerative potential. hCdHs may provide a nove tool that permits expansion and genetic manipulation of patient-specific progenitors to study regeneration and repair of diseased liver.

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

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Integrative gene regulatory analysis reveals transcriptional mechanisms required in mature human hepatocytes and during in vitro hepatocyte differentiation (RNA-Seq)

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Integrative gene regulatory analysis reveals transcriptional mechanisms required in mature human hepatocytes and during in vitro hepatocyte differentiation (ATAC-Seq)

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Conversion of terminally committed mouse hepatocytes to culturable bipotent progenitor cells

Project description:A challenge for advancing approaches to liver regeneration is loss of functional differentiation capacity when hepatocyte progenitors are maintained in culture. Recent lineage-tracing studies have shown that mature hepatocytes (MHs) convert to an immature state during chronic liver injury, and we investigated whether this conversion could be recapitulated in vitro and if such converted cells could represent a source of expandable hepatocytes. We report that a cocktail of small molecules, Y-27632, A-83-01 and CHIR99021, can convert rat and mouse MHs in vitro into proliferative bipotent cells, which we term chemically induced liver progenitors (CLiPs). CLiPs can differentiate into both MHs and biliary epithelial cells that can form functional ductal structures. CLiPs in long-term culture did not lose their proliferative capacity or their hepatic differentiation ability, and rat CLiPs were shown to extensively repopulate chronically injured liver tissue. Thus our study advances the goals of liver regenerative medicine. Transcriptomic analysis for mouse CLiPs at P1 which underwent hepatic induction (Matrix 1). Transcriptomic analysis for mouse CLiPs at P11-12 which underwent hepatic induction (Matrix 2).

2016-11-15 | GSE87579 | GEO

R-spondin 1 and noggin facilitate expansion of resident stem cells from non-damaged gallbladders.

Project description:Pioneering studies within the last few years have allowed the in vitro expansion of tissue-specific adult stem cells from a variety of endoderm-derived organs, including the stomach, small intestine and colon. Here we derived organoids from mouse gallbladder tissue (gallbladder organoids), from mouse liver (including the extrahepatic biliary ducts and gallbladder; liver organoids) and from mouse small intestine tissue (intestinal organoids). RNA was prepared from these organoids and used to assay expression of 21,258 genes using Affymetrix gene expression arrays. RNA was also prepared from mouse gallbladder, liver and small intestine tissues and used to assay gene expression in these tissues. Finally, gallbladder organoids were induced to differentiate by removing R-spondin 1 and noggin from the culture media and subjected to gene expression array analysis. RNA was extracted from mouse gallbladder organoids, differentiated gallbladder organoids, liver organoids, small intestine organoids, gallbladder tissue, liver tissue and small intestine tissue and then used for hybridization of Affymetrix gene expression microarrays.

2016-04-06 | E-GEOD-77461 | biostudies-arrayexpress

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Project description:Hepatocellular carcinoma (HCC) and cholangiocarcinoma (ICC) are two main forms liver cancers with poor prognosis. Models for studying HCC and ICC development using human liver cells are urgently needed. Organoids serve as in vitro models for cancer studies as it recapitulates in vivo structures and microenvironment of solid tumors. Herein, we established liver cancer organoid models by introducing specific mutations into human induced hepatocyte (hiHep)-derived organoids. c-MYC and hRASG12V overexpression in hiHep organoids with repressed p53 activation by large T led to distinct HCC and ICC signatures. With these oncogenic mutations, the neoplastic hiHep organoids formed cancerous structures and possessed cancer-specific hallmarks. Comprehensive transcriptional analysis of liver cancer organoids revealed genes and pathways with disease-stage-specific alterations. Notably, with RAS mutations, hiHep organoids acquired biliary trans-differentiation, and showed a process of conversion from hepatocytes to ICC. To sum up, we have established a useful and convenient in vitro human organoid systems modeling liver cancer development.

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