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An in vitro expansion system for generation of human iPS cell-derived hepatic progenitor-like cells exhibiting a bipotent differentiation potential.

ABSTRACT: Hepatoblasts, hepatic stem/progenitor cells in liver development, have a high proliferative potential and the ability to differentiate into both hepatocytes and cholangiocytes. In regenerative medicine and drug screening for the treatment of severe liver diseases, human induced pluripotent stem (iPS) cell-derived mature functional hepatocytes are considered to be a potentially good cell source. However, induction of proliferation of these cells is difficult ex vivo. To circumvent this problem, we generated hepatic progenitor-like cells from human iPS cells using serial cytokine treatments in vitro. Highly proliferative hepatic progenitor-like cells were purified by fluorescence-activated cell sorting using antibodies against CD13 and CD133 that are known cell surface markers of hepatic stem/progenitor cells in fetal and adult mouse livers. When the purified CD13(high)CD133(+) cells were cultured at a low density with feeder cells in the presence of suitable growth factors and signaling inhibitors (ALK inhibitor A-83-01 and ROCK inhibitor Y-27632), individual cells gave rise to relatively large colonies. These colonies consisted of two types of cells expressing hepatocytic marker genes (hepatocyte nuclear factor 4? and ?-fetoprotein) and a cholangiocytic marker gene (cytokeratin 7), and continued to proliferate over long periods of time. In a spheroid formation assay, these cells were found to express genes required for mature liver function, such as cytochrome P450 enzymes, and secrete albumin. When these cells were cultured in a suitable extracellular matrix gel, they eventually formed a cholangiocytic cyst-like structure with epithelial polarity, suggesting that human iPS cell-derived hepatic progenitor-like cells have a bipotent differentiation ability. Collectively these data indicate that this novel procedure using an in vitro expansion system is useful for not only liver regeneration but also for the determination of molecular mechanisms that regulate liver development.

SUBMITTER: Yanagida A

PROVIDER: S-EPMC3723819 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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An in vitro expansion system for generation of human iPS cell-derived hepatic progenitor-like cells exhibiting a bipotent differentiation potential.

Yanagida Ayaka A Ito Keiichi K Chikada Hiromi H Nakauchi Hiromitsu H Kamiya Akihide A

PloS one 20130725 7

Hepatoblasts, hepatic stem/progenitor cells in liver development, have a high proliferative potential and the ability to differentiate into both hepatocytes and cholangiocytes. In regenerative medicine and drug screening for the treatment of severe liver diseases, human induced pluripotent stem (iPS) cell-derived mature functional hepatocytes are considered to be a potentially good cell source. However, induction of proliferation of these cells is difficult ex vivo. To circumvent this problem, w ...[more]

PMID: 23935837

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Project description:Background and aimLiver fibrosis is prevalent among chronic diseases of the liver and represents a major health burden worldwide. Growth differentiation factor 7 (GDF7), a member of the TGFβ protein superfamily, has been recently investigated for its role in repair of injured organs, but its role in chronic liver diseases remains unclear. Here, we examined hepatic GDF7 expression and its association with development and progression of human liver fibrosis. Moreover, we determined the source and target cells of GDF7 in the human liver.MethodsGDF7 expression was analyzed in fibrotic and healthy human liver tissues by immunohistochemistry and qPCR. Cell-specific accumulation of GDF7 was examined by immunofluorescence through co-staining of cell type-specific markers on formalin-fixed paraffin-embedded human liver tissues. Public single cell RNA sequence databases were analyzed for cell type-specific expression of GDF7. In vitro, human liver organoids and LX-2 hepatic stellate cells (LX-2) were treated with recombinant human GDF7. Human liver organoids were co-cultured with activated LX-2 cells to induce an autocrine signaling circuit of GDF7 in liver organoids.ResultsGDF7 protein levels were elevated in fibrotic liver tissue, mainly detected in hepatocytes and cholangiocytes. In line, GDF7 mRNA was mainly detected in liver parenchymal cells. Expressions of BMPR1A and BMPR2, encoding GDF7 receptors, were readily detected in hepatocytes, cholangiocytes and stellate cells in vivo and in vitro. In vitro, recombinant GDF7 promoted liver organoid growth and enhanced expression of the progenitor cell markers (LGR5, AXIN2), but failed to activate LX-2 cells. Still, activated LX-2 cells induced GDF7 and LGR5 expression in co-cultured human liver organoids.ConclusionsCollectively, this study reveals a role of GDF7 in liver fibrosis and suggests a potential pro-regenerative function that can be utilized for amelioration of hepatic fibrosis caused by chronic liver disease.

Project description:Background & aimsUpon liver injury in which hepatocyte proliferation is compromised, liver progenitor cells (LPCs), derived from biliary epithelial cells (BECs), differentiate into hepatocytes. Little is known about the mechanisms of LPC differentiation. We used zebrafish and mouse models of liver injury to study the mechanisms.MethodsWe used transgenic zebrafish, Tg(fabp10a:CFP-NTR), to study the effects of compounds that alter epigenetic factors on BEC-mediated liver regeneration. We analyzed zebrafish with disruptions of the histone deacetylase 1 gene (hdac1) or exposed to MS-275 (an inhibitor of Hdac1, Hdac2, and Hdac3). We also analyzed zebrafish with mutations in sox9b, fbxw7, kdm1a, and notch3. Zebrafish larvae were collected and analyzed by whole-mount immunostaining and in situ hybridization; their liver tissues were collected for quantitative reverse transcription polymerase chain reaction. We studied mice in which hepatocyte-specific deletion of β-catenin (Ctnnb1flox/flox mice injected with Adeno-associated virus serotype 8 [AAV8]-TBG-Cre) induces differentiation of LPCs into hepatocytes after a choline-deficient, ethionine-supplemented (CDE) diet. Liver tissues were collected and analyzed by immunohistochemistry and immunoblots. We performed immunohistochemical analyses of liver tissues from patients with compensated or decompensated cirrhosis or acute on chronic liver failure (n = 15).ResultsLoss of Hdac1 activity in zebrafish blocked differentiation of LPCs into hepatocytes by increasing levels of sox9b mRNA and reduced differentiation of LPCs into BECs by increasing levels of cdk8 mRNA, which encodes a negative regulator gene of Notch signaling. We identified Notch3 as the receptor that regulates differentiation of LPCs into BECs. Loss of activity of Kdm1a, a lysine demethylase that forms repressive complexes with Hdac1, produced the same defects in differentiation of LPCs into hepatocytes and BECs as observed in zebrafish with loss of Hdac1 activity. Administration of MS-275 to mice with hepatocyte-specific loss of β-catenin impaired differentiation of LPCs into hepatocytes after the CDE diet. HDAC1 was expressed in reactive ducts and hepatocyte buds of liver tissues from patients with cirrhosis.ConclusionsHdac1 regulates differentiation of LPCs into hepatocytes via Sox9b and differentiation of LPCs into BECs via Cdk8, Fbxw7, and Notch3 in zebrafish with severe hepatocyte loss. HDAC1 activity was also required for differentiation of LPCs into hepatocytes in mice with liver injury after the CDE diet. These pathways might be manipulated to induce LPC differentiation for treatment of patients with advanced liver diseases.

Dataset Information

An in vitro expansion system for generation of human iPS cell-derived hepatic progenitor-like cells exhibiting a bipotent differentiation potential.

Publications

An in vitro expansion system for generation of human iPS cell-derived hepatic progenitor-like cells exhibiting a bipotent differentiation potential.

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