Functional Characteristics Of Hepatocytes Research Articles

The fate of hepatocyte cell line derived from a liver injury model with long-term in vitro passage

Orthotopic Liver Transplantation (OLT) is the therapy of choice for the treatment of end-stage liver disease, but the severe shortage of organ donors, complex and expensive surgical procedure and increased mortality of prospective organ recipient limit the use of OLT. To overcome this problem the technique of hepatocyte transplantation has been considered as an alternative to OLT. Hepatocyte transplantation is less invasive, cost effective cryo-preservable and can be distributed from single donor to multiple recipients. In this study we have established and characterize the hepatocyte cell line possessing the morphological and functional characteristics of hepatocytes from chemically injured liver. Hence we hypothesized that the hepatocyte cell line derived from liver injury can be used for hepatocyte transplantation. To induce the priming of hepatocyte, mice were fed with 3,5-diethoxycarbonyl-1,4-dihydro-collidine (DDC) diet for 3 weeks and hepatocytes were obtained by two step collagenase perfusion method, hepatocytes hence obtained was expended by >300 passages and tested for various hepatocyte specific functions. The cell line derived from liver injury model retains morphological and functional characteristics of hepatocytes in long-term in vitro culture. These cells transplanted to mice showed significant survival rate. The Hepatocyte cell line derived from liver injury model were used for hepatocyte transplantation and showed the significantly higher survival rate.

Development of Porous Hydrogel Scaffolds with Multiple Cues for Liver Tissue Engineering

Development of tissue-engineered construct for hepatic regeneration remains challenging due to the lack of potential milieu to control the trans-differentiation of hepatocytes. In the present study, galactose containing poly(vinyl alcohol) (PVA) and gelatin (8:2 and 9:1) hydrogels impregnated with hepatocyte growth factor (HGF)-loaded nanoparticles (241 ± 56 nm) using freeze/thaw technique was fabricated. The scaffolds with the HGF encapsulation efficiency of 57 ± 2.9% exhibited two-stage release kinetics for 30 days. Hepatogenic potential of the fabricated scaffolds were evaluated by determining the adhesion, viability, and proliferation with functional activity of rat primary hepatocytes for 28 days. Scaffolds of 8:2 ratios of PVA and gelatin maintained spheroidal morphology of hepatocytes and exhibited significantly higher metabolic secretions for 28 days (p < 0.05). Hence, galactose-based PVA-gelatin (8:2) hydrogel scaffold could be a promising alternative substrate to retain the phenotype and functional characteristics of hepatocytes due to the co-existence of bioactive motif and trophic factors. The developed 3D scaffold with growth factors encapsulated nanospheres possesses characteristic features for hepatic regeneration and is proposed to be a candidate material for further invivo evaluation

In Vitro Differentiation of Rat Bone Marrow Mesenchymal Stem Cells into Hepatocytes

To investigate the mechanism and regulation of differentiation from bone marrow mesenchymal stem cells (BMSCs) into hepatocytes and to find a new source for therapies of hepatic diseases. We isolated BMSCs for subsequent differentiation in the presence of hepatocyte growth factor (HGF) or beta-nerve growth factor (beta-NGF). Cell morphology was observed and cell surface phenotypings were detected by flow cytometry. a1-antitrypsin (AAT) expression of the hepatocytes was confirmed by immunocytochemistry and albumin expression was validated by real time PCR and western blotting. The expression of high-affinity nerve growth factor receptor (TrkA) and the activation of Erk pathway were detected by western blotting. Hepatocyte functional activity was confirmed by uptake of indocyanine green (ICG) assay. Small round cells appeared in the presence of HGF on day 10 or beta-NGF on day 12. Differentiated cells expressed albumin and had functional characteristics of hepatocytes, such as uptake of ICG. BMSCs were positive for TrkA. HGF and beta-NGF significantly upregulated the protein levels of phospho-Erk. BMSCs could differentiate into hepatocytes in the differentiation media including HGF or beta-NGF. Combination of HGF and beta-NGF significantly increased the efficiency of hepatic differentiation.

Human IPS Cells Differentiate Into Hepatocyte-Like Cells With the Phenotypic and Functional Characteristics of Hepatocytes

Human IPS Cells Differentiate Into Hepatocyte-Like Cells With the Phenotypic and Functional Characteristics of Hepatocytes

The effect of recombinant E-cadherin substratum on the differentiation of endoderm-derived hepatocyte-like cells from embryonic stem cells

Generation of specific lineages of cells from embryonic stem (ES) cells is pre-requisite to use these cells in pre-clinical applications. Here, we developed a recombinant E-cadherin substratum for generation of hepatic progenitor populations at single cell level. This artificial acellular feeder layer supports the stepwise differentiation of ES cells to cells with characteristics of definitive endoderm, hepatic progenitor cells, and finally cells with phenotypic and functional characteristics of hepatocytes. The efficient differentiation of hepatic endoderm cells (approximately 55%) together with the absence of neuroectoderm and mesoderm markers suggests the selective induction of endoderm differentiation. The co-expression of E-cahderin and alpha-fetoprotein (approximately 98%) suggests the important role of E-cadherin as a surface marker for the enrichment of hepatic progenitor cells. With extensive expansion, approximately 92% albumin expressing cells can be achieved without any enzymatic stress and cell sorting. Furthermore, these mouse ES cell-derived hepatocyte-like cells showed higher morphological similarities to primary hepatocytes. In conclusion, we demonstrated that E-cadherin substratum can guide differentiation of ES cells into endoderm-derived hepatocyte-like cells. This recombinant extracellular matrix could be effectively used as an in vitro model for studying the mechanisms of early stages of liver development even at single cell level.

Human embryonic and rat adult stem cells with primitive endoderm-like phenotype can be fated to definitive endoderm, and finally hepatocyte-like cells.

Stem cell-derived hepatocytes may be an alternative cell source to treat liver diseases or to be used for pharmacological purposes. We developed a protocol that mimics mammalian liver development, to differentiate cells with pluripotent characteristics to hepatocyte-like cells. The protocol supports the stepwise differentiation of human embryonic stem cells (ESC) to cells with characteristics of primitive streak (PS)/mesendoderm (ME)/definitive endoderm (DE), hepatoblasts, and finally cells with phenotypic and functional characteristics of hepatocytes. Remarkably, the same protocol can also differentiate rat multipotent adult progenitor cells (rMAPCs) to hepatocyte-like cells, even though rMAPC are isolated clonally from cultured rat bone marrow (BM) and have characteristics of primitive endoderm cells. A fraction of rMAPCs can be fated to cells expressing genes consistent with a PS/ME/DE phenotype, preceding the acquisition of phenotypic and functional characteristics of hepatocytes. Although the hepatocyte-like progeny derived from both cell types is mixed, between 10–20% of cells are developmentally consistent with late fetal hepatocytes that have attained synthetic, storage and detoxifying functions near those of adult hepatocytes. This differentiation protocol will be useful for generating hepatocyte-like cells from rodent and human stem cells, and to gain insight into the early stages of liver development.

Deciduous and Permanent Dental Pulp Mesenchymal Cells Acquire Hepatic Morphologic and Functional Features In Vitro

Introduction Mesenchymal stem cells display extensive proliferative capacity of multilineage differentiation. The stromal compartment of mesenchymal tissues is considered to harbor stem cells. We assessed the endodermal differentiation of mesenchymal cells from deciduous and wisdom tooth pulp. Methods Dental mesenchymal cells were isolated and expanded in vitro. After cell cultures had been established, cells were characterized using known stem cell markers. For hepatic differentiation the media was supplemented with hepatic growth factor, dexamethasone, Insulin-Transferrin-Selenium-X, and oncostatin. Results Both cultures showed a number of cells positive for specific hepatic markers including α-fetoprotein, albumin, and hepatic nuclear factor 4α after differentiation. Also, small clusters of cells positive for insulin-like growth factor 1 were found. The concentration of urea increased significantly in the media. Moreover, a significant amount of glycogen was found in the cells. Conclusion Because the cells proved to produce specific hepatic proteins and to start functions specific for hepatocytes, such as storing glycogen and urea production, we may state that the mesenchymal cell cultures from wisdom and deciduous tooth pulp acquired morphologic and functional characteristics of hepatocytes.

Induction of hepatic specification of human adipose-derived stem cells (hADSCs) in vitro

To induce hepatic differentiation of human adipose-derived stem cells (hADSCs) in vitro. hADSCs were isolated from human adipose tissue and treated with improved hepatic medium containing HGF, bFGF and FGF4. After 7 days of culture, OSM was added to the culture media. Cell growth during hepatic differentiation was evaluated by CCK8 assay. Morphology of differentiation was examined under light microscope. Liver specific genes and proteins were detected by RT-PCR analysis and immunohistochemical staining, respectively. And functional characteristics of hepatocytes were also examined. The number of hADSCs cultured in the improved hepatic media was increased significantly in comparison to hADSCs cultured in control media from 5 days to 21 days (t=6.59, 8.69, 15.94 and 24.64, respectively, P<0.05). The hADSCs-derived hepatocyte-like cells exhibited hepatocyte morphology, expressed hepatocyte markers, possessed hepatocyte-specific activities, such as uptake and excretion of indocyanine green, glycogen storage and albumin production. hADSCs can be induced into hepatocyte-like cells in this differentiation system. And this differentiation system promoted the growth of hADSCs.

Trans-differentiation of hepatocytes from mouse bone marrow mesenchymal stem cells induced by combi-nation of cytokines

Objective To establish an effective method for inducing mouse bone marrow mesen-ehymal stem cells (MSCs) into hepatocytes. Methods Isolated MSCs were selected by plastic adher-ence, differentiated in Iscove's Modified Dulbecco's medium supplemented with 10% new bovine ser-um (NBS), 20 ng/mL hepatocyte growth factor (HGF), 10 ng/mL fibroblast growth factor-4 (FGF-4) and 10 ng/ml oncostatin m (OSM) for 20 days'induction. The medium was changed every 4 days.Results When MSCs were cultured with HGF, FGF-4 and OSM, cuboidal morphology, which was a characteristic of hepatocytes, was observed, and the differentiated ceils also expressed marker genes specific to liver cells in a time-dependent manner, a-fetoprotein (AFP) was expressed on day 10, and CK18, ALB and TAT were detected on day 20, which was in consistent with the immunofluoresence results. Differentiated cells further demonstrated these cells also acquired functional characteristics of hepatocytes of storing glycogen. Conclusion Mouse MSCs can differentiate into hepatocytes when in-duced by HGF, FGF-4 and OSM. Key words: Mesenchymal; stem; cell; Hepatocyte; Differentiation; HGF; FGF-4; OSM

Identification and Characterization of a Novel Prespheroid 3-Dimensional Hepatocyte Monolayer on Galactosylated Substratum

Three-dimensional (3D) hepatocyte spheroids mimicking the structural and functional characteristics of hepatocytes in vivo were self-assembled onto a galactosylated polyethylene terephthalate (PET) substratum, and the dynamic process of spheroid formation was investigated using time-lapse confocal microscopy. Hepatocytes cultured on this galactosylated substratum formed small cell-aggregates within 12 h, which gradually merged into "island-like" clusters at approximately 1 day and spread to form prespheroid monolayer within 2 days; the prespheroid monolayer was stretched to fold into compact and larger 3D spheroids after 3 days. We compared the expressions of F-actin (cytoskeleton), phosphorylated focal adhesion kinase (p-FAK, cell-substratum interactions) and E-cadherin (cell-cell interactions) during the dynamic process of 3D hepatocyte spheroid formation with the dynamic process of 2D hepatocyte monolayer formation on collagen substratum. Hepatocytes in the prespheroid monolayer stage exhibited the strongest cell-substratum interactions of all 4 stages during spheroid formation with cell-cell interactions and F-actin distribution comparable with those of the 3D hepatocyte spheroids. The prespheroid monolayer also exhibited better hepatocyte polarity (multidrug resistance protein 2) and tight junction (zonula occludens-1) formation, more-differentiated hepatocyte functions (albumin production and cytochrome P450 1 A activity), and higher sensitivity to hepatotoxicity than the conventional 2D hepatocyte monolayer. The transient prespheroid 3D monolayer could be stabilized on a hybrid glycine-arginine-glycine-aspartic acid-serine (GRGDS)/galactose-PET substratum for up to 1 week and destabilized to form 3D spheroids in excess soluble GRGDS peptide.

Differentiation of putative hepatic stem cells derived from adult rats into mature hepatocytes in the presence of epidermal growth factor and hepatocyte growth factor

Oval cells, putative hepatic stem cells, can differentiate into a wide range of cell types including hepatocytes, bile epithelial cells, pancreatic cells and intestinal epithelial cells. In this study, we used different growth factor combinations to induce oval cells to differentiate into mature hepatocytes. We isolated and purified oval cells utilizing selective enzymatic digestion and density gradient centrifugation. Oval cells were identified by their morphological characteristics and the strong expressions of OV-6, albumin, cytokeratin (CK)-19 and CK-7. Using a 2-step induction protocol, we demonstrated that oval cells first changed into small hepatocytes, then differentiated into mature hepatocytes. Small hepatocytes were distinguished from oval cells by their morphological features (e.g. round shape and nuclei) and the lack of CK-19 mRNA expression. Mature hepatocytes were identified by their ultrastructural traits and their expressions of albumin, CK-18, tyrosine aminotransferase (TAT), and alpha-1-antitrypsin (α-1-AT). Differentiated cells acquired the functional attributes of hepatocytes in that they secreted albumin and synthesized urea at a high level throughout differentiation. Oval cells can thus differentiate into cells with the morphological, phenotypic and functional characteristics of hepatocytes. This 2-step induction procedure could provide an abundant source of hepatocytes for cell transplantation and tissue engineering.