Feeder-independent continuous culture of the PICM-19 pig liver stem cell line

The PICM-19 pig liver stem cell line is a bipotent cell line, i.e., capable of forming either bile ductules or hepatocyte monolayers in vitro, that was derived from the primary culture of pig embryonic stem cells. The cell line has been strictly feeder-dependent in that cell replication, morphology, and function were lost if the cells were cultured without STO feeder cells. A method for the feeder-independent continuous culture of PICM-19 cells (FI-PICM-19) is presented. PICM-19 cells were maintained and grown without feeder cells on collagen I-coated tissue culture plastic for 26 passages (P26) with initial split ratios of 1:3 that diminished to split ratios of less than 1:2 after passage 16. Once plated, the FI-PICM-19 cells were overlaid with a 1:12 to 1:50 dilution of Matrigel or related extracellular matrix product. Growth of the cells was stimulated by daily refeedings with STO feeder-cell conditioned medium. The FI-PICM-19 cells grew to an approximate confluence of 50% prior to each passage at 2-wk intervals. Growth curve analysis showed their average cell number doubling time to be ~96 h. Morphologically, the feeder-independent cells closely resembled PICM-19 cells grown on feeder cells, and biliary canalicui were present at cell-to-cell junctions. However, no spontaneous multicellular ductules formed in the monolayers of FI-PICM-19 cells. Ultrastructural subcellular features of the FI-PICM-19 cells were similar to those of PICM-19 cells cultured on feeder cells. The FI-PICM-19 cells produced a spectrum of serum proteins and expressed many liver/hepatocyte-specific genes. Importantly, cytochrome P450 (EROD) activity, ammonia clearance, and urea production were maintained by the feeder-independent cells. This simple method for the propagation of the PICM-19 cell line without feeder cells should simplify the generation and selection of functional mutants within the population and enhances the cell line’s potential for use in toxicological/pharmacological screening assays and for use in an artificial liver device.     

The effects of space flight and microgravity on the growth and differentiation of PICM-19 pig liver stem cells

The PICM-19 pig liver stem cell line was cultured in space for nearly 16 d on the STS-126 mission to assess the effects of spaceflight on the liver’s parenchymal cells—PICM-19 cells to differentiate into either monolayers of fetal hepatocytes or 3-dimensional bile ductules (cholangiocytes). Semi-quantitative data included light microscopic assessments of final cell density, cell morphology, and response to glucagon stimulation and electron microscopic assessment of the cells’ ultrastructural features and cell-to-cell connections and physical relationships. Quantitative assessments included assays of hepatocyte detoxification functions, i.e., inducible P450 activities and urea production and quantitation of the mRNA levels of several liver-related genes. Three post-passage age groups were included: 4-d-, 10-d-, and 14-d-old cultures. In comparing flight vs. ground-control cultures 17 h after the space shuttle’s return to earth, no differences were found between the cultures with the exception being that some genes were differentially expressed. By light microscopy both young and older cultures, flight and ground, had grown and differentiated normally in the Opticell culture vessels. The PICM-19 cells had grown to approximately 75% confluency, had few signs of apoptosis or necrosis, and had either differentiated into monolayer patches of hepatocytes with biliary canaliculi visible between the cells or into 3-dimensional bile ductules with well-defined lumens. Ultrastructural features between flight and ground were similar with the PICM-19 cells displaying numerous mitochondria, Golgi apparatus, smooth and rough endoplasmic reticulum, vesicular bodies, and occasional lipid vacuoles. Cell-to-cell arrangements were typical in both flight and ground-control samples; biliary canaliculi were well-formed between the PICM-19 cells, and the cells were sandwiched between the STO feeder cells. PICM-19 cells displayed inducible P450 activities. They produced urea in a glutamine-free medium and produced more urea in response to ammonia. The experiment’s aim to gather preliminary data on the PICM-19 cell line’s suitability as an in vitro model for assessments of liver function in microgravity was demonstrated, and differences between flight and ground-control cultures were minor.     

Cytochrome P450 expression profile of the PICM-19H pig liver cell line: potential application to rapid liver toxicity assays

Liver in vitro models are needed to replace animal models for rapid assessment of drug biotransformation and toxicity. The PICM-19 pig liver stem cell line may fulfill this need since these cells have activities associated with xenobiotic phase I and II metabolism lacking in other liver cell lines. The objective of this study was to characterize phase I and II metabolic functions of a PICM-19 derivative cell line, PICM-19H, compared to the tumor-derived human HepG2 C3A cell line and primary cultures of adult porcine hepatocytes. Following exposure of PICM-19H cells to either 3-methylcholanthrene, rifampicin or phenobarbital, the induced activities of cytochrome P450 (CYP450) isozymes CYP-1A, -2, and-3A were assessed. Relative to adult porcine hepatocytes, PICM-19H cells exhibited 30% and 43%, respectively, of CYP1A and 3A activities, while HepG2 C3A cells exhibited 7% and 0% of those activities. Fluorescent metabolites were extensively conjugated, i.e., 52% and 96% of CYP450-1A and-3A metabolites were released from medium samples following treatment with β-glucuronidase/arylsulfatase. Rifampicin induction of CYP450 isozyme activities was confirmed by conversion of testosterone to 6β-OH-, 2α-OH- and 2β-OH-testosterone, as determined by mass spectrometry. Susceptibility of PICM-19H cells to acetaminophen toxicity was determined; CD₅₀ was calculated to be 14.9 ± 0.9 mM. Toxicity and bioactivation of aflatoxin B1 was determined in 3-methylcholanthrene-treated cultures and untreated controls; CD₅₀ were 1.59 μM and 31 μM, respectively. These results demonstrate the potential use of PICM-19H cells in drug biotransformation and toxicity testing and further support their use in extracorporeal artificial liver device technology.     

肝细胞生长因子基因修饰的间充质干细胞治疗肝损伤的研究进展

减轻肝脏损伤、促进肝脏修复和再生始终是肝脏疾病研究中的重点。间充质干细胞(MSCs)是众多具有组织修复和再生能力细胞中的明星细胞,合成的多种细胞因子经旁分泌途径发挥调控细胞生存,调节炎症反应,促进血管再生和减轻纤维化等多种生物学效应,肝细胞生长因子(HGF)便是重点细胞因子之一。基于HGF的信号调控作用,再结合MSCs的干细胞优势,HGF基因修饰间充质干细胞(HGF-MSCs)作为一种干细胞治疗新策略能够发挥“1+1>2”的效果。本文就HGF-MSCs在减轻和修复肝损伤中的研究进展作综述。     

兔肝细胞体外分离及培养方法的实验研究

目的：研究体外兔肝细胞分离及培养方法,比较不同培养基条件下兔肝细胞培养过程。方法：采用非灌注胶原酶消化法分离兔肝细胞,分别采用RPIM1640培养液（含10％新生牛血清）,DMEM培养液（含10％新生牛血清）,DMEM培养液（含10％胎牛血清）培养,计数法观察原代细胞增殖变化,MTF法观察传代细胞增殖情况。培养细膨采用PAS染色法鉴定,电镜观察细胞超微结构。结果：分离的肝细胞细胞活率大于85％;DMEM培养液（含10％胎牛血清）培养肝细胞生长状态较另两种培养液中的肝细胞强,DMEM培养液（含10％新生牛血清）中的细胞增殖能力较RPIM1640培养液（含10％新生牛血清）高,具有统计学意义。PAS染色和透射电镜观察培养细胞胞质中有大量糖原颗粒。结论：本实验采用的分离方法可获得较纯的肝细胞,而且操作简便实用。DMEM培养液较RPIM1640培养液更加适宜原代培养肝细胞生长,胎牛血清对培养肝细胞的生长促进作用明显高于新生牛血清。     

Intracellular signaling cascades triggered by the NK1 fragment of hepatocyte growth factor in human prostate epithelial cell line PNT1A

Hepatocyte Growth Factor (HGF)/c-MET signaling has an emerging role in promoting cell proliferation, survival, migration, wound repair and branching in a variety of cell types. HGF plays a crucial role as a mediator of stromal–epithelial interactions in the normal prostate but the precise biological function of HGF/c-Met interaction in the normal prostate and in prostate cancer is not clear. HGF has two naturally occurring splice variants and NK1, the smallest of these HGF variants, consists of the HGF amino terminus through the first kringle domain. We evaluated the intracellular signaling cascades and the morphological changes triggered by NK1 in human prostate epithelial cell line PNT1A which shows molecular and biochemical properties close to the normal prostate epithelium. We demonstrated that these cells express a functional c-MET, and cell exposure to NK1 induces the phosphorylation of tyrosines 1313/1349/1356 residues of c-MET which provide docking sites for signaling molecules. We observed an increased phosphorylation of ERK1/2, Akt, c-Src, p125FAK, SMAD2/3, and STAT3, down-regulation of the expression of epithelial cell–cell adhesion marker E-cadherin, and enhanced expression levels of mesenchymal markers vimentin, fibronectin, vinculin, α-actinin, and α-smooth muscle actin. This results in cell proliferation, in the appearance of a mesenchymal phenotype, in morphological changes resembling cell scattering and in wound healing. Our findings highlight the function of NK1 in non-tumorigenic human prostatic epithelial cells and provide a picture of the signaling pathways triggered by NK1 in a unique cell line.     

A new methodology of mesenchymal stem cell expansion using magnetic nanoparticles

Mesenchymal stem cells (MSCs), which can differentiate into multiple mesodermal tissues, may be useful for autologous cell transplantation, if MSCs, which are isolated from bone marrow in small numbers, can be expanded in vitro. We developed a combined methodological approach to enrich and proliferate MSCs in vitro using magnetic nanoparticles. Our magnetite cationic liposomes (MCLs), which have a positive surface charge in order to improve adsorption, accumulated in MSCs at a concentration of 20 pg of magnetite per cell. The MCLs exhibited no toxicity against MSCs in proliferation and differentiation to osteoblasts and adipocytes. The MSCs magnetically labeled by MCLs were enriched using magnets and then cultured, resulting in much higher density (seeding density, 1000 cells/cm²) than in ordinary culture (seeding density, 18 cells/cm²). When MSCs were seeded at high density using MCLs, there was a 5-fold increase in the number of cells, compared to culture prepared without MCLs. Our results suggest that this novel culture method using magnetic nanoparticles can be used to efficiently expand MSCs for clinical application.     

Hepatocyte growth factor and epidermal growth factor promote spheroid formation in polyurethane foam/hepatocyte culture and improve expression and maintenance of albumin production

In our previous pre-clinical study with pig hepatic failure, an artificial liver with polyurethane foam (PUF)/primary porcine hepatocyte spheroids had superior curative effect. We examined the effect of hepatocyte growth factor (HGF), also known as scatter factor, on the quick formation of hepatocyte spheroids and albumin production. Spheroids were formed in the pores of PUF within 3 days regardless of addition of growth factors. In particular, spheroids were formed within 1 day in medium containing 100 ng/ml HGF and 50 ng/ml epidermal growth factor (EGF). 10,000 ng/ml HGF was effective for albumin production, but the activity dramatically decreased after 6 days in EGF-free medium. On the other hand, 100 ng/ml HGF was effective for albumin production in EGF-containing medium. Albumin production rate with ≥1000 ng/ml HGF was about 1.5 times higher than that with 100 ng/ml HGF. Furthermore, albumin production rate at 3 weeks was about 1.5 times higher than that at 2 days with 1000 ng/ml HGF. The maintenance of albumin production rate depended on the activity of the individual cell and not cell growth. In other words, we were able to show the effectiveness of HGF for functional hepatocyte organoid formation in PUF pores.     

Delta-like 1 serves as a new target and contributor to liver fibrosis down-regulated by mesenchymal stem cell transplantation

Chronic liver injury always progresses to fibrosis and eventually to cirrhosis, a massive health care burden worldwide. Delta-like 1 (Dlk1) is well known as an inhibitor of adipocyte differentiation. However, whether it is involved in liver fibrosis remains unclear. Here, we provide the first evidence that Dlk1 is a critical contributor to liver fibrosis through promoting activation of hepatic stellate cells (HSCs) during chronic liver injury. We found that upon liver injury, Dlk1 was dramatically induced and initially expressed in hepatocytes and then into the HSCs by a paracrine manner. It leads to the activation of HSCs, which is considered to be a pivotal event in liver fibrogenesis. Two forms (～50 and ～25 kDa) of the Dlk1 protein were detected by Western blot analysis. In vitro administration of Dlk1 significantly promoted HSC activation, whereas in vivo knockdown of Dlk1 dramatically inhibited HSC activation and the subsequent fibrosis. The large soluble form (～50 kDa) of Dlk1 was shown to contribute to HSC activation. We were encouraged to find the Dlk1-promoted HSC activation and liver fibrosis can be depressed by transplantation of bone marrow-mesenchymal stem cells (BM-MSCs). Furthermore, we demonstrated that FGF2 was up-regulated in BM-MSCs under injury stimulation, and it probably participated in the inhibition of Dlk1 by BM-MSCs. Our findings provide a novel role of Dlk1 in liver fibrosis leading to a better understanding of the molecular basis in fibrosis and cirrhosis and also give insights into the cellular and molecular mechanisms of MSC biology in liver repair.     

Pregnane X receptor-dependent and -independent effects of 2-acetylaminofluorene on cytochrome P450 3A23 expression and liver cell proliferation

The arylamide 2-acetylaminofluorene (AAF) is a powerful carcinogen displaying a marked promoting activity, also known to regulate expression of liver detoxifying proteins. In this study we identified CYP3A23, a major inducible cytochrome P-450 (CYP) isoform, as an AAF target in hepatocytes. Indeed, exposure to AAF of primary rat hepatocytes resulted in a marked up-regulation of CYP3A23 expression at both mRNA and protein levels. Using CYP3A23 reporter gene constructs, we further demonstrated that AAF activated the CYP3A23 Direct Repeat 3 (DR3) promoter element interacting with the nuclear pregnane X receptor (PXR). Moreover, the PXR antagonist ecteinascidin-743 fully suppressed AAF-related CYP3A23 induction. Low doses of AAF inhibiting DNA synthesis in hepatocytes however failed to trigger PXR-related CYP3A23 induction and PXR-negative epithelial liver cells remained sensitive to the mito-inhibitory effects of AAF. Such data indicate that AAF up-regulates CYP3A23 through PXR activation but does not require PXR for exerting its carcinogenic promoting properties based on inhibition of cell growth.     

Novel bioreactors for the culture and expansion of aggregative neural stem cells

Neural stem cells have been cultured as three-dimensional aggregates in a number of different types of bioreactors. The design and configuration of the bioreactor are shown to be crucial factors for the successful propagation of the cells. A novel bioreactor with liquid re-circulation and a working volume of 200 ml has been designed, tested and shown to be able to produce a higher cell vitality compared to those produced in multi-well plates, shake flasks and stirred flasks. The novel reactor was able to produce a total density of cells of 3.5 x 10(6) cells/ml consisting of a larger number of smaller and proliferative aggregates, compared to only 1.8 x 10(6) cells/ml produced in a multi-well plate. Shake flasks and stirred flasks commonly used for facilitating mass transfer in the culture of micro-organisms are shown to be unsuitable for the propagation of neural stem cells.     

A subpopulation of bone marrow cells depleted by a novel antibody, anti-Liv8, is useful for cell therapy to repair damaged liver

We previously reported a new in vivo model named as "GFP/CCl(4) model" for monitoring the transdifferentiation of green fluorescent protein (GFP) positive bone marrow cell (BMC) into albumin-positive hepatocyte under the specific "niche" made by CCl(4) induced persistent liver damage, but the subpopulation which BMCs transdifferentiate into hepatocytes remains unknown. Here we developed a new monoclonal antibody, anti-Liv8, using mouse E 11.5 fetal liver as an antigen. Anti-Liv8 recognized both hematopoietic progenitor cells in fetal liver at E 11.5 and CD45-positive hematopoietic cells in adult bone marrow. We separated Liv8-positive and Liv8-negative cells and then transplanted these cells into a continuous liver damaged model. At 4 weeks after BMC transplantation, more efficient repopulation and transdifferentiation of BMC into hepatocytes were seen with Liv8-negative cells. These findings suggest that the subpopulation of Liv8-negative cells includes useful cells to perform cell therapy on repair damaged liver.     

Establishment of a bovine blastocyst-derived cell line collection for the comparative analysis of embryos created in vivo and by in vitro fertilization,somatic cell nuclear transfer,or parthenogenetic activation

Tools and methods for analyzing differences in embryos resulting from somatic cell nuclear transfer (NT) in comparison to those derived from normal fertilization are needed to define better the nature of the nuclear reprogramming that occurs after NT. To this end, a collection of bovine blastocyst-derived cell lines was created. In vitro expanded or hatched blastocysts, used as primary culture tissue, were from NT; in vitro maturation, fertilization, and culture (IVF); or parthenogenetic (P) activation. Also, five in vivo-fertilized and developed blastocysts were collected by uterine flushing on the eighth d postfertilization. Whole blastocysts were physically attached to STO feeder layers to initiate all of the cell lines generated. The majority of the cell lines in the collection are trophectoderm, 38 NT-derived, 6 in vivo-derived, 20 IVF-derived, and 13 P-derived. Trophectoderm identity was ascertained by morphology and, in many cases, interferon-tau production. Several visceral endoderm cell lines and putative parietal endoderm cell lines were also established. At approximately 5% efficiency, epiblast masses from NT and IVF blastocysts survived and were isolated in culture. Two epiblast masses were also isolated from P blastocysts. Spontaneous differentiation from the epiblast outgrowths resulted in the establishment of fibroblast cell lines. The use of the trophectoderm cell lines as a comparative in vitro model of bovine trophectoderm and placental function is discussed in relation to NT reprogramming.     

Molecular mechanisms of the 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced inverted U-shaped dose responsiveness in anchorage independent growth and cell proliferation of human breast epithelial cells with stem cell characteristics

Although 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has a variety of carcinogenic and noncarcinogenic effects in experimental animals, its role in human carcinogenicity remain controversial. A simian virus 40-immortalized cell line from normal human breast epithelial cells with stem cells and luminal characteristics (M13SV1) was used to study whether TCDD can induce AIG positive colony formation and cause increased cell numbers in a inverted U-shaped dose–response manner. TCDD activated Akt, ERK2, and increased the expression of CYP1A1, PAI-2, IL-lb mRNA, and ERK2 protein levels. TCDD was able to increased phosphorylation and expression of ERK2 in same dose–response manner as AIG positive colony formation. Thus, TCDD induced tumorigenicity in M13SV1, possibly through the phosphorylation of ERK2 and/or Akt. Further, cDNA microarray with 7448 sequence-verified clones was used to profile various gene expression patterns after treatment of TCDD. Three clear patterns could be delineated: genes that were dose-dependently up-regulated, genes expressed in either U-shape and/or inverted U-shape. The fact that these genes are intrinsically related to breast epithelial cell proliferation and survival clearly suggests that they may be involved in the TCDD-induced breast tumorigenesis.     

An immortalized human liver endothelial sinusoidal cell line for the study of the pathobiology of the liver endothelium

Background

The endothelium lines blood and lymph vessels and protects underlying tissues against external agents such as viruses, bacteria and parasites. Yet, microbes and particularly viruses have developed sophisticated ways to bypass the endothelium in order to gain access to inner organs. De novo infection of the liver parenchyma by many viruses and notably hepatitis viruses, is thought to occur through recruitment of virions on the sinusoidal endothelial surface and subsequent transfer to the epithelium. Furthermore, the liver endothelium undergoes profound changes with age and in inflammation or infection. However, primary human liver sinusoidal endothelial cells (LSECs) are difficult to obtain due to scarcity of liver resections. Relevant derived cell lines are needed in order to analyze in a standardized fashion the transfer of pathogens across the liver endothelium. By lentiviral transduction with hTERT only, we have immortalized human LSECs isolated from a hereditary hemorrhagic telangiectasia (HHT) patient and established the non-transformed cell line TRP3. TRP3 express mesenchymal, endothelial and liver sinusoidal markers. Functional assessment of TRP3 cells demonstrated a high capacity of endocytosis, tube formation and reactivity to immune stimulation. However, TRP3 displayed few fenestrae and expressed C-type lectins intracellularly. All these findings were confirmed in the original primary LSECs from which TRP3 were derived suggesting that these features were already present in the liver donor. We consider TRP3 as a model to investigate the functionality of the liver endothelium in hepatic inflammation in infection.     

Molecular mechanisms of the 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced inverted U-shaped dose responsiveness in anchorage independent growth and cell proliferation of human breast epithelial cells with stem cell characteristics

Although 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has a variety of carcinogenic and noncarcinogenic effects in experimental animals, its role in human carcinogenicity remain controversial. A simian virus 40-immortalized cell line from normal human breast epithelial cells with stem cells and luminal characteristics (M13SV1) was used to study whether TCDD can induce AIG positive colony formation and cause increased cell numbers in a inverted U-shaped dose–response manner. TCDD activated Akt, ERK2, and increased the expression of CYP1A1, PAI-2, IL-lb mRNA, and ERK2 protein levels. TCDD was able to increased phosphorylation and expression of ERK2 in same dose–response manner as AIG positive colony formation. Thus, TCDD induced tumorigenicity in M13SV1, possibly through the phosphorylation of ERK2 and/or Akt. Further, cDNA microarray with 7448 sequence-verified clones was used to profile various gene expression patterns after treatment of TCDD. Three clear patterns could be delineated: genes that were dose-dependently up-regulated, genes expressed in either U-shape and/or inverted U-shape. The fact that these genes are intrinsically related to breast epithelial cell proliferation and survival clearly suggests that they may be involved in the TCDD-induced breast tumorigenesis.     

Applications of patient-specific induced pluripotent stem cells; focused on disease modeling, drug screening and therapeutic potentials for liver disease

The recent advances in the induced pluripotent stem cell (iPSC) research have significantly changed our perspectives on regenerative medicine by providing researchers with a unique tool to derive disease-specific stem cells for study. In this review, we describe the human iPSC generation from developmentally diverse origins (i.e. endoderm-, mesoderm-, and ectoderm- tissue derived human iPSCs) and multistage hepatic differentiation protocols, and discuss both basic and clinical applications of these cells including disease modeling, drug toxicity screening/drug discovery, gene therapy and cell replacement therapy.     

A novel function of connexin 32: marked enhancement of liver function in a hepatoma cell line

Connexin 32 (Cx32) is the main gap junction protein in hepatocytes and plays an important role in the regulation of signal transfer and growth control in the liver by constructing gap junction channels and gap junctional intercellular communication (GJIC). In this study, the human Cx32 gene was transfected into a hepatoma cell line (HepG2) that showed aberrant expression of Cx32 and was deficient in GJIC. Cx32-transfected HepG2 not only expressed a higher level of Cx32 mRNA, but also showed increased GJIC compared with HepG2 and vector-transfected HepG2. Furthermore, the liver functions of ammonia removal and albumin secretion of HepG2 were markedly enhanced with Cx32 gene transfection. It may be expected to improve the cellular functions of the hepatoma cell line by Cx32 gene transfection and serve to develop an efficacious bioartificial liver.