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Hepatocyte Markers (hepatocyte + marker)
Selected AbstractsNeoHepatocytes From Alcoholics and Controls Express Hepatocyte Markers and Display Reduced Fibrogenic TGF-,/Smad3 Signaling: Advantage for Cell Transplantation?ALCOHOLISM, Issue 4 2010Sabrina Ehnert Background:, Liver transplantation is the only definitive treatment for end stage liver disease. Donor organ scarcity raises a growing interest in new therapeutic options. Recently, we have shown that injection of monocyte-derived NeoHepatocytes can increase survival in rats with extended liver resection. In order to apply this technology in humans with chronic liver diseases in an autologous setting, we generated NeoHepatocytes from patients with alcoholic liver disease and healthy controls and compared those to human hepatocytes. Methods:, We generated NeoHepatocytes from alcoholics with Child A and B cirrhosis and healthy controls. Hepatocytes marker expression and transforming growth factor (TGF)-, signaling was investigated by RT-PCR, Western blot, immunofluorescent staining, and adenoviral reporter assays. Glucose and urea was measured photometrically. Phase I and II enzyme activities were measured using fluorogenic substrates. Neutral lipids were visualized by Oil Red O staining. Results:, There was no significant difference in generation and yield of NeoHepatocytes from alcoholics and controls. Hepatocyte markers, e.g., cytokeratin18 and alcohol dehydrogenase 1, increased significantly throughout differentiation. Glucose and urea production did not differ between alcoholics and controls and was comparable to human hepatocytes. During differentiation, phase I and II enzyme activities increased, however remained significantly lower than in human hepatocytes. Fat accumulation was induced by treatment with insulin, TGF-, and ethanol only in differentiated cells and hepatocytes. TGF-, signaling, via Smad transcription factors, critically required for progression of chronic liver disease, was comparable among the investigated cell types, merely expression of Smad1 and -3 was reduced (,30 and ,60%) in monocytes, programmable cells of monocytic origin, and NeoHepatocytes. Subsequently, expression of TGF-, regulated pro-fibrogenic genes, e.g., connective tissue growth factor and fibronectin was reduced. Conclusions:, Generation of NeoHepatocytes from alcoholics, displaying several features of human hepatocytes, offers new perspectives for cell therapeutic approaches, as cells can be obtained repeatedly in a noninvasive manner. Furthermore, the autologous setting reduces the need for immunosuppressants, which may support recovery of patients which are declined for liver transplantation. [source] Culture and Characterization of Human Hepatocytes Obtained after Graft Reduction for Liver Transplantation: A Reliable Source of Cells for a Bioartificial LiverARTIFICIAL ORGANS, Issue 7 2004Mariana Barbich Abstract:, This article describes results obtained when human liver cells obtained from reduced grafts are cultured in a chemically defined medium. Remnants of livers after reduction for pediatric transplantation were processed by a multiple cannulation system through the existing vasculature, which allowed the homogeneous perfusion of collagenase. The graft weight ranged between 55 and 1000 g (median value: 145.6 g). The yield ranged between 0.13 × 106 and 38 × 106 cells/g of tissue (median value 14.73 × 106 cells/g), and the viability was 61.17 ± 27.43%. The total number of cells ranged between 57.6 × 106 and 12 150 × 106 cells (median value: 740 × 106 cells). Cells were cultured for 30 days. Albumin synthesis was observed during the first 2 weeks, with a peak value at day 6 (27.85 ± 1.77 µg/mL). Urea production was detected during the first week (peak value at day 6: 17.12 ± 2.11 mg/dL). Light microscopy showed the presence of cells in a monolayer. Biliary pigments were observed at day 20. By immunohistochemistry, positive cells for albumin, for hepatocyte marker, cytokeratin 19, CD 34, CD 68, and for alpha actin for smooth muscle, were observed. Our results showed that hepatocytes obtained from reduced liver grafts are easily cultured and are able to maintain viability and functionality in vitro. This alternative source of human cells maintained under controlled culture conditions may play an important role in the development of a bioartificial liver. [source] Human skin fibroblasts: From mesodermal to hepatocyte-like differentiation,HEPATOLOGY, Issue 5 2007Philippe A. Lysy The phenotypic homology of fibroblasts and mesenchymal stem cells (MSCs) has been recently described. Our study investigated the in vitro potential of human skin fibroblasts to differentiate into mesodermal (osteocyte and adipocyte) and endodermal (hepatocyte) cell lineages by comparison with human bone marrow (hBM) MSCs. The endodermal potential of fibroblasts was then explored in vivo in a mouse model of liver injury. Fibroblasts were able to acquire osteocyte and adipocyte phenotypes as assessed by cytochemistry and gene expression analyses. After exposure to a specific differentiation cocktail, these cells presented hepatocyte-like morphology and acquired liver-specific markers on protein and gene expression levels. Furthermore, these fibroblast-derived hepatocyte-like cells (FDHLCs) displayed the ability to store glycogen and synthesize small amounts of urea. By gene expression analysis, we observed that fibroblasts remained in a mesenchymal-epithelial transition state after hepatocyte differentiation. Moreover, FDHLCs lost their hepatocyte-like phenotype after dedifferentiation. In vivo, human fibroblasts infused directly into the liver of hepatectomized severe combined immunodeficient (SCID) mice engrafted in situ and expressed hepatocyte markers (albumin, alpha-fetoprotein, and cytokeratin 18) together with the mesodermal marker fibronectin. Despite lower liver-specific marker expression, the in vitro and in vivo differentiation profile of fibroblasts was comparable to that of mesenchymal-derived hepatocyte-like cells (MDHLCs). In conclusion, our work demonstrates that human skin fibroblasts are able to display mesodermal and endodermal differentiation capacities and provides arguments that these cells share MSCs features both on the phenotypic and functional levels. (HEPATOLOGY 2007;46:1574,1585.) [source] Timing and sequence of differentiation of embryonic rat hepatocytes along the biliary epithelial lineageHEPATOLOGY, Issue 3 2003Robbert G. E. Notenboom To study the differentiation of hepatocytes along the biliary epithelial lineage in vivo, embryonic day 14 (E14) rat hepatocytes were isolated by differential centrifugation and transplanted as single-cell suspensions into the spleen of adult syngeneic rats. Hepatocytes and cholangiocytes were identified and their maturation characterized by the level of expression of ,-fetoprotein (AFP), glutamate dehydrogenase (GDH), and carbamoyl phosphate synthetase I (CPS); annexin IV, annexin V, cytokeratin 19 (CK-19), and cystic fibrosis transmembrane conductance regulator (CFTR); and electron microscopy. By correlating morphologic changes with the timing in the expression of these markers, we show that the organization of the transplanted E14 hepatocytes into lobular structures is accompanied by the formation and maturation of bile ducts around these developing lobules. Morphologic differentiation of the emerging bile ducts was accompanied by a gradual loss of hepatocyte markers and a gradual acquisition of cholangiocyte markers, with markers identifying a large-cholangiocyte phenotype appearing latest. Once fully differentiated, the intrasplenic liver lobules developed cholestatic features. The accompanying proliferation of bile ducts was due to cholangiocyte proliferation, but ductular transformation of hepatocytes was also observed. In conclusion, (1) bile duct formation at the interface between hepatocytes and connective tissue is an inherent component of liver development and (2) the susceptibility of developing hepatocytes to bile duct-inducing signals is highest in the fetal liver but that (3) this capacity is not irreversibly lost in otherwise mature hepatocytes. [source] Derivation, characterization, and phenotypic variation of hepatic progenitor cell lines isolated from adult ratsHEPATOLOGY, Issue 2 2002Li Yin Liver progenitor cells (LPCs) cloned from adult rat livers following allyl alcohol injury express hematopoietic stem cell and early hepatic lineage markers when cultured on feeder layers; under these conditions, neither mature hepatocyte nor bile duct, Ito, stellate, Kupffer cell, or macrophage markers are detected. These phenotypes have remained stable without aneuploidy or morphological transformation after more than 100 population doublings. When cultured without feeder layers, the early lineage markers disappear, and mature hepatocyte markers are expressed; mature hepatocytic differentiation and cell size are also augmented by polypeptide and steroidal growth factors. In contrast to hepatocytic potential, duct-like structures and biliary epithelial markers are expressed on Matrigel. Because they were derived without carcinogens or mutagens, these bipotential LPC lines provide novel tools for models of cellular plasticity and hepatocarcinogenesis, as well as lines for use in cellular transplantation, gene therapy, and bioreactor construction. [source] Human embryonic stem cells and liver diseases: From basic research to future clinical applicationJOURNAL OF DIGESTIVE DISEASES, Issue 1 2008Zheng WANG Human embryonic stem cells (hESC) provide access to the earliest stages of human development and because of their high proliferation capability, pluripotency and low immunogenicity may serve as a potential source of specialized cells for regenerative medicine. hESC-derived hepatocyte-like cells exhibit characteristic hepatocyte morphology, express hepatocyte markers and are capable of executing a range of hepatocyte functions. However, there are many challenges and obstacles to be overcome before the use of hESC and hESC-derived hepatocyte-like cells in clinical practice can be realized. Here, we highlight some of the recent efforts in this area, in hope of providing insights toward this complex yet important area of therapeutical modality for treating patients with liver disease. [source] Retinoic acid signalling induces the differentiation of mouse fetal liver-derived hepatic progenitor cellsLIVER INTERNATIONAL, Issue 10 2009Jiayi Huang Abstract Background: Hepatic progenitor cells (HPCs) can be isolated from fetal liver and extrahepatic tissues. Retinoic acid (RA) signalling plays an important role in development, although the role of RA signalling in liver-specific progenitors is poorly understood. Aims: We sought to determine the role of RA in regulating hepatic differentiation. Methods: RNA was isolated from liver tissues of various developmental stages. Liver marker expression was assessed by reverse transcriptase-polymerase chain reaction and immunofluorescence staining. Reversibly immortalized HPCs derived from mouse embryonic day 14.5 (E14.5) liver (aka, HP14.5) were established. Albumin promoter-driven reporter (Alb-GLuc) was used to monitor hepatic differentiation. Glycogen synthesis was assayed as a marker for terminal hepatic differentiation. Results: Retinoic acid receptor (RAR)-,, retinoid X receptor (RXR)-, and RXR-, expressed in E12.5 to postnatal day 28 liver samples. Expression of RAR-, and RXR-, was low perinatally, whereas RAR-, was undetectable in prenatal tissues and increased postnatally. Retinal dehydrogenase 1 and 2 (Raldh1 and Raldh2) were expressed in all tissues, while Raldh3 was weakly expressed in prenatal samples but was readily detected postnatally. Nuclear receptor corepressors were highly expressed in all tissues, while expression of nuclear co-activators decreased in perinatal tissues and increased after birth. HP14.5 cells expressed high levels of early liver stem cell markers. Expression of RA signalling components and coregulators was readily detected in HP14.5. RA was shown to induce Alb-GLuc activity and late hepatocyte markers. RA was further shown to induce glycogen synthesis in HP14.5 cells, an important function of mature hepatocytes. Conclusions: Our results strongly suggest that RA signalling may play an important role in regulating hepatic differentiation. [source] | ||||||||||