Reversible transition between hepatocytes and liver progenitors for in vitro hepatocyte expansion.
Ontology highlight
ABSTRACT:
SUBMITTER: Wu H
PROVIDER: S-EPMC5520858 | biostudies-literature | 2017 May
REPOSITORIES: biostudies-literature
Publications
Reversible transition between hepatocytes and liver progenitors for in vitro hepatocyte expansion.
Wu Han H Zhou Xu X Fu Gong-Bo GB He Zhi-Ying ZY He Zhi-Ying ZY Wu Hong-Ping HP You Pu P Ashton Charles C Wang Xin X Wang Hong-Yang HY Yan He-Xin HX
Cell research 20170404 5
Similar Datasets
Project description:Despite decades of investigation on the proliferation of adult human primary hepatocytes, their expansion in vitro still remains challenging. To later be able to consider hepatocytes as a cell therapy alternative or bridge to liver transplantation, dramatically impeded by a shortage in liver donors, the first step is having an almost unlimited source of these cells. The banking of transplantable hepatocytes also implies a protocol for their expansion that can be compatible with large-scale production. We show that adult human primary hepatocytes when grown in 3D organoids are easily amplified, providing a substantial source of functional hepatocytes ready for transplantation. Following their plating, differentiated human hepatocytes are amplified during a transient and reversible step as liver progenitors, and can subsequently be converted back to mature differentiated hepatocytes. The protocol we propose is not only compatible with automated and high-throughput cell culture systems, thanks to the expansion of hepatocytes in suspension, but also guarantees the generation of a high number of functional cells from the same patient sample, with a relatively easy set up.
| S-EPMC5974235 | biostudies-literature
Project description:The liver has great regenerative capacity after functional mass loss caused by injury or disease. Many studies have shown that primary hepatocyte-derived exosomes, which can deliver biological information between cells, promote the regenerative process of the liver. However, the yield of exosomes is very limited. Recent studies have demonstrated that exosome-mimetic nanovesicles (NVs) can be prepared from cells with almost 100 times the production yield compared with exosomes. Thus, this study investigated the therapeutic capacity of exosome-mimetic NVs from primary hepatocytes in liver regeneration. Exosome-mimetic NVs were prepared by serial extrusions of cells through polycarbonate membranes, and the yield of these NVs was more than 100 times that of exosomes. The data indicated that the NVs could promote hepatocyte proliferation and liver regeneration by significantly enhancing the content of sphingosine kinase 2 in recipient cells. To the best of our knowledge, this is the first time that exosome-mimetic NVs from primary hepatocytes have been prepared, and these NVs have components similar to exosomes from primary hepatocytes and, in some respects, biofunctions similar to exosomes. Strategies inspired by this study may lead to substitution of exosomes with exosome-mimetic NVs for biofunctional purposes, including utilization in tissue repair and regeneration.
| S-EPMC5802835 | biostudies-literature
Project description:ObjectiveThe death rate of mature hepatocytes is chronically increased in various liver diseases, triggering responses that prevent liver atrophy, but often cause fibrosis. Mice with targeted disruption of inhibitor kappa B kinase (Ikk) in hepatocytes (HEP mice) provide a model to investigate this process because inhibiting Ikk-nuclear factor-kappaB (NF-kappaB) signalling in hepatocytes increases their apoptosis.MethodsCell proliferation, apoptosis, progenitors, fibrosis and production of Hedgehog (Hh) ligands (progenitor and myofibroblast growth factors) were compared in HEP and control mice before and after feeding methionine choline-deficient ethionine-supplemented (MCDE) diets. Ikkbeta was deleted from primary hepatocytes to determine the effects on Hh ligand production; Hh signalling was inhibited directly in progenitors to determine the effects on viability. Liver sections from patients were examined to assess relationships between hepatocyte production of Hh ligands, accumulation of myofibroblastic cells and liver fibrosis.ResultsDisrupting the Ikk-NF-kappaB pathway in hepatocytes inhibited their proliferation but induced their production of Hh ligands. The latter provided viability signals for progenitors and myofibroblasts, enhancing accumulation of these cell types and causing fibrogenesis. Findings in the mouse models were recapitulated in diseased human livers.ConclusionDying mature hepatocytes produce Hh ligands which promote the compensatory outgrowth of progenitors and myofibroblasts. These results help to explain why diseases that chronically increase hepatocyte death promote cirrhosis.
| S-EPMC3632642 | biostudies-literature
Project description:Hepatocytes are very difficult to expand in vitro. A few studies have demonstrated that chemical cocktails with growth factors or Wnt ligands can support long-term expansion of hepatocytes via dedifferentiation. However, the culture conditions are complex, and clonal expansion of hepatic progenitors with full differentiation capacity are rarely reported. Here, we discover IL6, combined with EGF and HGF, promotes long-term expansion (>30 passages in ~150 days with theoretical expansion of ~1035 times) of primary mouse hepatocytes in vitro in simple 2D culture, by converting hepatocytes into induced hepatic progenitor cells (iHPCs), which maintain the capacity of differentiation into hepatocytes. IL6 also supports the establishment of single hepatocyte-derived iHPC clones. The summation of the downstream STAT3, ERK and AKT pathways induces a number of transcription factors which support rapid growth. This physiological and simple way may provide ideas for culturing previously difficult-to-culture cell types and support their future applications.
| S-EPMC9708838 | biostudies-literature
Project description:Adult liver progenitor cells are biliary-like epithelial cells that emerge only under injury conditions in the periportal region of the liver. They exhibit phenotypes of both hepatocytes and bile ducts. However, their origin and their significance to injury repair remain unclear. Here, we used a chimeric lineage tracing system to demonstrate that hepatocytes contribute to the progenitor pool. RNA-sequencing, ultrastructural analysis, and in vitro progenitor assays revealed that hepatocyte-derived progenitors were distinct from their biliary-derived counterparts. In vivo lineage tracing and serial transplantation assays showed that hepatocyte-derived proliferative ducts retained a memory of their origin and differentiated back into hepatocytes upon cessation of injury. Similarly, human hepatocytes in chimeric mice also gave rise to biliary progenitors in vivo. We conclude that human and mouse hepatocytes can undergo reversible ductal metaplasia in response to injury, expand as ducts, and subsequently contribute to restoration of the hepatocyte mass.
| S-EPMC4254170 | biostudies-literature
Project description:Congenital disorders of the biliary tract are the primary reason for pediatric liver failure and ultimately for pediatric liver transplant needs. Not all causes of these disorders are well understood, but it is known that liver fibrosis occurs in many of those afflicted. The goal of this study is to develop a simple yet robust model that recapitulates physico-mechanical and cellular aspects of fibrosis mediated via hepatic stellate cells (HSCs) and their effects on biliary progenitor cells. Liver organoids were fabricated by embedding various HSCs, with distinctive abilities to generate mild to severe fibrotic environments, together with undifferentiated liver progenitor cell line, HepaRG, within a collagen I hydrogel. The fibrotic state of each organoid was characterized by examination of extracellular matrix (ECM) remodeling through quantitative image analysis, rheometry, and qPCR. In tandem, the phenotype of the liver progenitor cell and cluster formation was assessed through histology. Activated HSCs (aHSCs) created a more severe fibrotic state, exemplified by a more highly contracted and rigid ECM, as well higher relative expression of TGF-β, TIMP-1, LOXL2, and COL1A2 as compared to immortalized HSCs (LX-2). Within the more severe fibrotic environment, generated by the aHSCs, higher Notch signaling was associated with an expansion of CK19+ cells as well as the formation of larger, more densely populated cell biliary like-clusters as compared to mild and non-fibrotic controls. The expansion of CK19+ cells, coupled with a severely fibrotic environment, are phenomena found within patients suffering from a variety of congenital liver disorders of the biliary tract. Thus, the model presented here can be utilized as a novel in vitro testing platform to test drugs and identify new targets that could benefit pediatric patients that suffer from the biliary dysgenesis associated with a multitude of congenital liver diseases.
| S-EPMC8459590 | biostudies-literature
Project description:Background & aimsCurrent research focuses on developing alternative strategies to restore decreased liver mass prior to the onset of end-stage liver disease. Cell engraftment/repopulation requires regeneration in normal liver, but we have shown that severe liver injury stimulates repopulation without partial hepatectomy (PH). We have now investigated whether a less severe injury, secondary biliary fibrosis, would drive engraftment/repopulation of ectopically transplanted mature hepatocytes.MethodsDuctular proliferation and progressive fibrosis in dipeptidyl-peptidase IV (DPPIV)(-) F344 rats was induced by common bile duct ligation (BDL). Purified DPPIV(+)/green fluorescent protein (GFP)(+) hepatocytes were infused without PH into the spleen of BDL rats and compared to rats without BDL.ResultsWithin one week, transplanted hepatocytes were detected in hepatic portal areas and at the periphery of expanding portal regions. DPPIV(+)/GFP(+) repopulating cell clusters of different sizes were observed in BDL rats but not untreated normal recipients. Surprisingly, some engrafted hepatocytes formed CK-19/claudin-7 expressing epithelial cells resembling cholangiocytes within repopulating clusters. In addition, substantial numbers of hepatocytes engrafted at the intrasplenic injection site assembled into multicellular groups. These also showed biliary "transdifferentiation" in the majority of intrasplenic injection sites of rats that received BDL but not in untreated recipients. PCR array analysis showed upregulation of osteopontin (SPP1). Cell culture studies demonstrated increased Itgβ4, HNF1β, HNF6, Sox-9, and CK-19 mRNA expression in hepatocytes incubated with osteopontin, suggesting that this secreted protein promotes dedifferentiation of hepatocytes.ConclusionsOur studies show that biliary fibrosis stimulates liver repopulation by ectopically transplanted hepatocytes and also stimulates hepatocyte transition towards a biliary epithelial phenotype.
| S-EPMC5137249 | biostudies-literature
Project description:Background & aimsChronic failure of mechanisms that promote effective regeneration of dead hepatocytes causes replacement of functional hepatic parenchyma with fibrous scar tissue, ultimately resulting in cirrhosis. Therefore, defining and optimizing mechanisms that orchestrate effective regeneration might prevent cirrhosis. We hypothesized that effective regeneration of injured livers requires hepatocytes to evade the growth-inhibitory actions of TGFβ, since TGFβ signaling inhibits mature hepatocyte growth but drives cirrhosis pathogenesis.MethodsWild-type mice underwent 70% partial hepatectomy (PH); TGFβ expression and signaling were evaluated in intact tissue and primary hepatocytes before, during, and after the period of maximal hepatocyte proliferation that occurs from 24-72 h after PH. To determine the role of Yap1 in regulating TGFβ signaling in hepatocytes, studies were repeated after selectively deleting Yap1 from hepatocytes of Yap1flox/flox mice.ResultsTGFβ expression and hepatocyte nuclear accumulation of pSmad2 and Yap1 increased in parallel with hepatocyte proliferative activity after PH. Proliferative hepatocytes also upregulated Snai1, a pSmad2 target gene that promotes epithelial-to-mesenchymal transition (EMT), suppressed epithelial genes, induced myofibroblast markers, and produced collagen 1α1. Deleting Yap1 from hepatocytes blocked their nuclear accumulation of pSmad2 and EMT-like response, as well as their proliferation.ConclusionInteractions between the TGFβ and Hippo-Yap signaling pathways stimulate hepatocytes to undergo an EMT-like response that is necessary for them to grow in a TGFβ-enriched microenvironment and regenerate injured livers.Lay summaryThe adult liver has an extraordinary ability to regenerate after injury despite the accumulation of scar-forming factors that normally block the proliferation and reduce the survival of residual liver cells. We discovered that liver cells manage to escape these growth-inhibitory influences by transiently becoming more like fibroblasts themselves. They do this by reactivating programs that are known to drive tissue growth during fetal development and in many cancers. Understanding how the liver can control programs that are involved in scarring and cancer may help in the development of new treatments for cirrhosis and liver cancer.
| S-EPMC6349217 | biostudies-literature
Project description:Liver regeneration after injury is normally mediated by proliferation of hepatocytes, although recent studies have suggested biliary epithelial cells (BECs) can differentiate into hepatocytes during severe liver injury when hepatocyte proliferation is impaired. We investigated the effect of hepatocyte-specific β-catenin deletion in recovery from severe liver injury and BEC-to-hepatocyte differentiation. To induce liver injury, we administered choline-deficient, ethionine-supplemented (CDE) diet to three different mouse models, the first being mice with deletion of β-catenin in both BECs and hepatocytes (Albumin-Cre; Ctnnb1flox/flox mice). In our second model, we performed hepatocyte lineage tracing by injecting Ctnnb1flox/flox ; Rosa-stopflox/flox -EYFP mice with the adeno-associated virus serotype 8 encoding Cre recombinase under the control of the thyroid binding globulin promoter, a virus that infects only hepatocytes. Finally, we performed BEC lineage tracing via Krt19-CreERT ; Rosa-stopflox/flox -tdTomato mice. To observe BEC-to-hepatocyte differentiation, mice were allowed to recover on normal diet following CDE diet-induced liver injury. Livers were collected from all mice and analyzed by quantitative real-time polymerase chain reaction, western blotting, immunohistochemistry, and immunofluorescence. We show that mice with lack of β-catenin in hepatocytes placed on the CDE diet develop severe liver injury with impaired hepatocyte proliferation, creating a stimulus for BECs to differentiate into hepatocytes. In particular, we use both hepatocyte and BEC lineage tracing to show that BECs differentiate into hepatocytes, which go on to repopulate the liver during long-term recovery. Conclusion: β-catenin is important for liver regeneration after CDE diet-induced liver injury, and BEC-derived hepatocytes can permanently incorporate into the liver parenchyma to mediate liver regeneration.
| S-EPMC6351199 | biostudies-literature
Project description:Current dogma asserts that the foetal liver (FL) is an expansion niche for recently specified haematopoietic stem cells (HSCs) during ontogeny. Indeed, between embryonic day of development (E)12.5 and E14.5, the number of transplantable HSCs in the murine FL expands from 50 to about 1,000. Here we used a non-invasive, multi-colour lineage tracing strategy to interrogate the embryonic expansion of murine haematopoietic progenitors destined to contribute to the adult HSC pool. Our data show that this pool of fated progenitors expands only two-fold during FL ontogeny. Although Histone2B-GFP retention in vivo experiments confirmed substantial proliferation of phenotypic FL-HSC between E12.5 and E14.5, paired-daughter cell assays revealed that many mid-gestation phenotypic FL-HSCs are biased to differentiate, rather than self-renew, relative to phenotypic neonatal and adult bone marrow HSCs. In total, these data support a model in which the FL-HSC pool fated to contribute to adult blood expands only modestly during ontogeny.
| S-EPMC10026622 | biostudies-literature