Background/Aims Oval cells (OCs), putative hepatic stem cells, may give rise to liver cancers. We developed a carcinogenesis regimen, based upon induction of OC proliferation prior to carcinogen ...exposure. In our model, rats subjected to 2-acetylaminofluorene/ partial-hepatectomy followed by aflatoxin injection (APA regimen) developed well-differentiated hepatocholangiocarcinomas. The aim of this study was to establish and characterize cancer cell lines from this animal model. Methods Cancer cells were cultured from animals sacrificed eight months after treatment, and single clones were selected. The established cell lines, named LCSCs, were characterized, and their tumorigenicity was assessed in vivo . The roles of granulocyte-colony stimulating factor (G-CSF) and hepatocyte growth factor (HGF) in LCSC growth, survival and motility were also investigated. Results From primary tumors, six cell lines were developed. LCSCs shared with the primary tumors the expression of various OC-associated markers, including cMet and G-CSF receptor. In vitro , HGF conferred protection from death by serum withdrawal. Stimulation with G-CSF increased LCSC growth and motility, while the blockage of its receptor inhibited LCSC proliferation and migration. Conclusions Six cancer cell lines were established from our model of hepatocholangiocarcinoma. HGF modulated LCSC resistance to apoptosis, while G-CSF acted on LCSCs as a proliferative and chemotactic agent.
Background & Aims:
Recruitment and proliferation of Thy-1
+ oval cells is a hallmark of liver regeneration after 2-acetylaminofluorene (2-AAF)/partial hepatectomy (PHx) in rats. To understand the ...molecular mechanism underlying this process, we investigated the role of connective tissue growth factor (CTGF), one of the candidate genes differentially expressed in Thy-1
+ oval cells, in this liver injury model.
Methods:
Northern and Western analyses were performed to examine the induction of CTGF in total liver homogenate. Quantitative real-time polymerase chain reaction (PCR), immunofluorescent staining, and in situ hybridization were performed to confirm the expression and localization of CTGF in Thy-1
+ oval cells. Finally, a known inhibitor of CTGF synthesis, Iloprost, was administered to 2-AAF/PHx treated rats to investigate the effect of Iloprost on oval cell response.
Results:
CTGF was found to be up-regulated at both the RNA and protein levels and occurred concurrently with an up-regulation of transforming growth factor β1 (TGF-β1). Sorted Thy-1
+ oval cells expressed a high level of CTGF gene in a quantitative PCR assay. Colocalization of Thy-1 antigen and
ctgf signals by in situ hybridization further confirmed that Thy-1
+ oval cells were a source of CTGF. Iloprost administration blocked CTGF induction in treated animals but did not affect TGF-β1 expression. The inhibition of CTGF induction by Iloprost was associated with a significant decrease in oval cell proliferation and a lower level of α-fetoprotein expression as compared with control animals.
Conclusions:
These results show that CTGF induction is important for robust oval cell response after 2-AAF/PHx treatment in rats.
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Advancement of bioprinting technology is limited by the availability of materials that both facilitate bioprinting logistics as well as support cell viability and function by ...providing tissue-specific cues. Herein we describe a modular hyaluronic acid (HA) and gelatin-based hydrogel toolbox comprised of a 2-crosslinker, 2-stage polymerization technique, and the capability to provide tissue specific biochemically and mechanically accurate signals to cells within biofabricated tissue constructs. First, we prepared and characterized several tissue-derived decellularized extracellular matrix-based solutions, which contain complex combinations of growth factors, collagens, glycosaminoglycans, and elastin. These solutions can be incorporated into bioinks to provide the important biochemical cues of different tissue types. Second, we employed combinations of PEG-based crosslinkers with varying molecular weights, geometries (linear, 4-arm, and 8-arm), and functional groups to yield hydrogel bioinks that supported extrusion bioprinting and the capability to achieve final construct shear stiffness values ranging from approximately 100Pa to 20kPa. Lastly, we integrated these hydrogel bioinks with a 3-D bioprinting platform, and validated their use by bioprinting primary liver spheroids in a liver-specific bioink to create in vitro liver constructs with high cell viability and measurable functional albumin and urea output. This hydrogel bioink system has the potential to be a versatile tool for biofabrication of a wide range of tissue construct types.
Biochemical and mechanical factors both have important implications in guiding the behavior of cells in vivo, yet both realms are rarely considered together in the context of biofabrication in vitro tissue construct models. We describe a modular hydrogel system that (1) facilitates extrusion bioprinting of cell-laden hydrogels, (2) incorporates tissue-specific factors derived from decellularized tissue extracellular matrix, thus mimicking biochemical tissue profile, and (3) allows control over mechanical properties to mimic the tissue stiffness. We believe that employing this technology to attend to both the biochemical and mechanical profiles of tissues, will allow us to more accurately recapitulate the in vivo environment of tissues while creating functional 3-D in vitro tissue constructs that can be used as disease models, personalized medicine, and in vitro drug and toxicology screening systems.
Glycogen storage disease type Ia (GSDIa) is caused by a genetic defect in the hepatic enzyme glucose-6-phosphatase (G6Pase-α), which manifests as life-threatening hypoglycemia with related metabolic ...complications. A G6Pase-α knockout (KO) mouse model was generated to study potential therapies for correcting this disorder. Since then, gene therapy studies have produced promising results, showing long-term improvement in liver histology and glycogen metabolism. Under existing protocols, however, untreated KO pups seldom survived weaning. Here, we present a thorough characterization of the G6Pase-α KO mouse, as well as the husbandry protocol for rearing this strain to adulthood. These mice were raised with only palliative care, and characterized from birth through 6 months of age. Once KO mice have survived the very frail weaning period, their size, agility, serum lipids and glycemic control improve dramatically, reaching levels approaching their wild-type littermates. In addition, our data reveal that adult mice lacking G6Pase-α are able to mate and produce viable offspring. However, liver histology and glycogen accumulation do not improve with age. Overall, the reliable production of mature KO mice could provide a critical tool for advancing the GSDIa field, as the availability of a robust enzyme-deficient adult offers a new spectrum of treatment avenues that would not be tolerated by the frail pups. Most importantly, our detailed characterization of the adult KO mouse provides a crucial baseline for accurately gauging the efficacy of experimental therapies in this important model.
Current practices in drug development have led to therapeutic compounds being approved for widespread use in humans, only to be later withdrawn due to unanticipated toxicity. These occurrences are ...largely the result of erroneous data generated by in vivo and in vitro preclinical models that do not accurately recapitulate human physiology. Herein, a human primary cell- and stem cell-derived 3D organoid technology is employed to screen a panel of drugs that were recalled from market by the FDA. The platform is comprised of multiple tissue organoid types that remain viable for at least 28 days, in vitro. For many of these compounds, the 3D organoid system was able to demonstrate toxicity. Furthermore, organoids exposed to non-toxic compounds remained viable at clinically relevant doses. Additional experiments were performed on integrated multi-organoid systems containing liver, cardiac, lung, vascular, testis, colon, and brain. These integrated systems proved to maintain viability and expressed functional biomarkers, long-term. Examples are provided that demonstrate how multi-organoid 'body-on-a-chip' systems may be used to model the interdependent metabolism and downstream effects of drugs across multiple tissues in a single platform. Such 3D in vitro systems represent a more physiologically relevant model for drug screening and will likely reduce the cost and failure rate associated with the approval of new drugs.
Somatostatin (SST) is a regulatory peptide that activates G protein-coupled receptors comprised of five members (somatostatin receptors (SSTRs) 1–5). Despite the broad use of SST and its analogs in ...clinical practice, the spectrum of SST activities has been incompletely defined. Recently, it has been demonstrated that SST can be a chemoattractant for hematopoietic precursor cells. Since hepatic oval cells (HOCs) share common characteristics with hematopoietic stem cells, we hypothesized that SST could act as a chemoattractant for HOCs by stimulating SSTRs. Reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot assay revealed an increased expression of SST in the 2-acetyl-aminofluorene (2AAF)/partial hepatectomy (PHx) HOC induction model. Immunohistochemical staining showed the expression of SST in 2AAF/PHx-treated rat liver, as compared to normal liver. Proliferation and migration assays demonstrated that the increase of SST was related to migration of HOCs, but not their proliferation. RT-PCR and quantitative real-time PCR showed that SSTR4 was preferentially expressed by HOCs. Western blot assay and immunohistochemical staining confirmed the expression of SSTR4 by HOCs. In addition, pretreatment with anti-SSTR4 antibody cultures resulted in a dramatic reduction of cell migration as compared to that of control. Lastly, SST stimulated the rearrangement of actin filaments in HOCs, while HOCs treated with anti-SSTR4 antibody failed to do so. These results suggest a positive role for SST in the migration of HOCs, and that this effect is mediated through SSTR4.
BACKGROUND AND AIMS: Activation of the oval cell compartment occurs in the liver when hepatocytes are functionally compromised and/or unable to divide. Our goal was to investigate the systemic ...signals responsible for determining the efficiency of oval cell-mediated liver regeneration, focusing on the Notch signaling cascade. METHODS: The established oval cell induction protocol of 2-acetylaminofluorine (2-AAF) implantation followed by 70% surgical resection of the liver (partial hepatectomy, PH) was employed in a rat model. This oval cell induction model was further combined with injections of a γ-secretase inhibitor (GSI XX) to examine the effects of Notch inhibition on oval cell-aided regeneration of the liver. RESULTS: Notch signaling was found to be upregulated at the peak of oval cell induction during 2AAF-PH alone. Treatment with GSI XX led to interruption of the Notch signal, as shown by a decrease in expression of Hes1. While there was a robust oval cell response seen at day 11 post-PH, there was a measurable delay in differentiation when Notch was inhibited. This was confirmed morphologically as well as by immunohistochemistry for the oval cell markers, α-fetoprotein, OV-6, and CK19. The hepatocytes seen at day 22 demonstrated an enhanced hepatocellular mitoinhibition index (p21(Waf1)/Ki67), suggestive of dysregulated proliferation and cell cycle progression. Moreover, these hepatocytes exhibited decreased expression of hepatocyte functional markers, such as cytochrome P450 and glucose-6-phosphatase-α. CONCLUSIONS: Taken together, these results identify the Notch signaling pathway as a potent regulator of differentiation and proliferation in oval cells, which is necessary for functional for repair of the liver by oval cells.
Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as organ replacements for implantation in patients, ...and also, when created on a smaller size scale as model "organoids" that can be used in in vitro systems for drug and toxicology screening. Despite development of a wide variety of bioprinting devices, application of bioprinting technology can be limited by the availability of materials that both expedite bioprinting procedures and support cell viability and function by providing tissue-specific cues. Here we describe a versatile hyaluronic acid (HA) and gelatin-based hydrogel system comprised of a multi-crosslinker, 2-stage crosslinking protocol, which can provide tissue specific biochemical signals and mimic the mechanical properties of in vivo tissues. Biochemical factors are provided by incorporating tissue-derived extracellular matrix materials, which include potent growth factors. Tissue mechanical properties are controlled combinations of PEG-based crosslinkers with varying molecular weights, geometries (linear or multi-arm), and functional groups to yield extrudable bioinks and final construct shear stiffness values over a wide range (100 Pa to 20 kPa). Using these parameters, hydrogel bioinks were used to bioprint primary liver spheroids in a liver-specific bioink to create in vitro liver constructs with high cell viability and measurable functional albumin and urea output. This methodology provides a general framework that can be adapted for future customization of hydrogels for biofabrication of a wide range of tissue construct types.
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We have reported a method for the decellularization of intact rat livers by perfusion with increasing concentrations of detergents. This procedure results in a decellularized organ that ...maintains an intact capsule and retains an appropriate distribution of extracellular matrix proteins and associated mucopolysaccharides. Immunoassay demonstrated that many growth factors were retained on the decellularized matrix. Several growth factors known to regulate hepatocyte phenotype, including PDGF, IGFBP‐3 and BMP‐7, were found to be increased in matrices from regenerating livers. Equilibration of the decellularized matrix in serum increased the levels of many of these factors. Decellularization by an alternative, enzyme based method resulted in the retention of a different repertoire of growth factors. The liver progenitor cell line, WB344, was able to colonize these matrices and differentiate into mature hepatocytes, but no differentiation was observed on plastic alone. These data indicate that matrices generated from livers in different growth states and by different methods may each be best suited for specific applications.
Increasing survival rates of children following cancer treatment have resulted in a significant population of adult survivors with the common side effect of infertility. Additionally, the ...availability of genetic testing has identified Klinefelter syndrome (classic 47,XXY) as the cause of future male infertility for a significant number of prepubertal patients. This study explores new spermatogonia stem cell (SSC)-based fertility therapies to meet the needs of these patients. Testicular cells were isolated from cryopreserved human testes tissue stored from XY and XXY prepubertal patients and propagated in a two-dimensional culture. Cells were then incorporated into a 3D human testicular organoid (HTO) system. During a 3-week culture period, HTOs maintained their structure, viability, and metabolic activity. Cell-specific PCR and flow cytometry markers identified undifferentiated spermatogonia, Sertoli, Leydig, and peritubular cells within the HTOs. Testosterone was produced by the HTOs both with and without hCG stimulation. Upregulation of postmeiotic germ cell markers was detected after 23 days in culture. Fluorescence in situ hybridization (FISH) of chromosomes X, Y, and 18 identified haploid cells in the in vitro differentiated HTOs. Thus, 3D HTOs were successfully generated from isolated immature human testicular cells from both euploid (XY) and Klinefelter (XXY) patients, supporting androgen production and germ cell differentiation in vitro.
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