ABCG5 and ABCG8 are expressed in gallbladder epithelial cells

Gallbladder epithelial cells (GBEC) are exposed to high biliary cholesterol concentrations on their apical (AP) surface. The mechanisms of cholesterol absorption and efflux by these cells are not known. We hypothesized that ABCG5 and ABCG8 are expressed in GBEC and mediate AP cholesterol efflux. Human gallbladder cDNA expressed message for ABCG5 and ABCG8. Cultured murine GBEC also expressed abcg5 and abcg8 mRNA and protein, as did cultured canine GBEC. Interestingly, treatment with model bile containing supersaturating concentrations of cholesterol, or treatment with LXRalpha/RXR ligands, did not lead to differences in expression of ABCG5 or ABCG8 in the murine or the canine cells. The subcellular localization of ABCG5 and ABCG8 did show alterations, with predominantly intracellular localization at baseline and predominantly AP localization following treatment with model bile or LXRalpha ligand. GBEC therefore express ABCG5 and ABCG8; these sterol transporters may play a role in mediating AP cholesterol efflux in the gallbladder epithelium.     

Transdifferentiation of mouse BM cells into hepatocyte-like cells

BACKGROUND: During the past few years multiple studies have revealed that adult stem cells, including BM origin stem cells, can be transdifferentiated into various cell types, including hepatocyte-like cells, under proper treatments or in a suitable microenvironment. However, little is known about the mechanism of the transdifferentiation, and the treatments employed seem to be very complicated and require simplification. It is important to determine the suitable conditions in which BM cells would be efficiently differentiated into hepatocytes. METHODS: Mouse BM cells were isolated from femurs and tibias and cultured in IMDM supplemented with 10% FBS. Hepatic differentiation was induced in a differentiation medium containing 20 ng/mL HGF, 10 ng/mL FGF-4, 10 ng/mL Oncostatin M (OSM) and different concentrations of liver-injured mouse sera. The differentiated hepatic cells were characterized by the expression of liver-associated mRNA and proteins and morphologic and functional features. RESULTS: BM cell-derived polygonal cell colonies appeared after several days of culture, and these hepatocyte-like cells expressed AFP, HNF-3beta, CK19, CK18, ALB, TAT and G-6-Pase at mRNA and protein levels, and the cells also had some hepatic cellular functions, such as glycogen storage and urea production. Interestingly, suitable concentrations of sera from liver-injured mice added to this system showed strong stimulation on the in vitro transdifferentiation of mouse BM cells into hepatocytes. DISCUSSION: In the present study we have established an effective hepatic differentiation system by a combination of HGF, FGF-4, OSM and liver-injured mouse sera in vitro. Accordingly, it will be a useful resource not only for understanding the mechanisms of transdifferentiation but also for efficient amplification of hepatocyte progenitor cells of BM origin.     

Cultured gallbladder epithelial cells synthesize apolipoproteins A-I and E

Gallbladder epithelial cells (GBEC) are exposed to high and fluctuating concentrations of biliary cholesterol on their apical (AP) surface. GBEC absorb and efflux cholesterol, but the mechanisms of cholesterol uptake, intracellular trafficking, and efflux in these cells are not known. We previously reported that ATP binding cassette (ABC)A1 mediates basolateral (BL) cholesterol efflux in cultured polarized GBEC. In addition, the nuclear hormone receptors liver X receptor (LXR)alpha and retinoid X receptor (RXR) mediate both AP and BL cholesterol efflux. An interesting finding from our previous study was that apolipoprotein (apo)A-I applied to the AP surfaces of cells elicited BL ABCA1-mediated cholesterol efflux. Because ABCA1-mediated cholesterol efflux requires the presence of a cholesterol acceptor, we hypothesized that GBEC synthesize and secrete endogenous apo into the BL compartment. Here, we demonstrate that cholesterol loading of cells with model bile and AP apoA-I treatment is associated with an increase in the synthesis of apoE mRNA and protein. Furthermore, apoE is secreted into the BL compartment. LXRalpha/RXR ligands stimulate the synthesis of endogenous apoA-I mRNA and protein, as well as apoE mRNA. BL secretion of apoA-I is elicited by LXRalpha/RXR ligands. Therefore, GBEC synthesize apoA-I and -E and efflux cholesterol using ABCA1- and non-ABCA1- mediated pathways. These processes may alter gallbladder biliary cholesterol concentrations and thereby influence gallstone formation.     

Notch1-expressing cells are indispensable for prostatic branching morphogenesis during development and re-growth following castration and androgen replacement

Notch expression is frequently associated with progenitor cells, and its function is crucial for development. Our recent work showing that Notch1 is selectively expressed in basal epithelial cells of the prostate and higher Notch1 expression during development suggests that Notch1-expressing cells may define progenitor cells in the prostate. To test this hypothesis, we have generated a transgenic mouse line in which the Notch1-expressing cells can be ablated in a controlled manner. Specific targeting was achieved by expressing the bacterial nitroreductase, an enzyme that catalyzes its substrate into a cytotoxin capable of inducing apoptosis, under the Notch1 promoter. Cell death in transgenic prostate was confirmed by histological analyses including terminal dUTP nick-end labeling and caspase 3 immunocytochemical staining. We evaluated the consequences of ablation of Notch1-expressing cells in two systems, organ culture of early postnatal prostates and re-growth of prostate in castrated mice triggered by hormone replacement. Our data show that elimination of Notch1-expressing cells inhibited the branching morphogenesis, growth, and differentiation of early postnatal prostate in culture and impaired prostate re-growth triggered by hormone replacement in castrated mice. Furthermore, we found that Notch1 expression following castration and hormone replacement was concomitant with known basal cell markers p63 and cytokeratin 14 and was high in the proliferative human prostate epithelial cells. Taken together, these data suggest that Notch1-expressing cells define the progenitor cells in the prostatic epithelial cell lineage, which are indispensable for prostatic development and re-growth.     

Persistence of a chimerical phenotype after hepatocyte differentiation of human bone marrow mesenchymal stem cells

Abstract.  Objectives : Recent studies have suggested the potential of mesenchymal stem cells (MSCs) to differentiate into a hepatocyte-like lineage. Here, we evaluate the efficacy of hepatocyte differentiation of MSCs by studying acquisition of hepatocyte-like features together with alteration of the native mesenchymal phenotype. Material and methods : In vitro , we have investigated protein and mRNA level expression of hepatocyte and mesenchymal markers of mesenchymal-derived hepatocyte-like cells (MDHLCs) and we have evaluated their functionality using metabolic assays. In vivo , we investigated co-expression of hepatocyte (albumin, α-foetoprotein, cytokeratin 18) and mesenchymal (fibronectin, vimentin) markers after transplantation of MSCs or MDHLCs into severe combined immune deficiency mice. Results : We observed that while in vitro these cells acquired some phenotypic and functional features of mature hepatocytes, they partially preserved their mesenchymal phenotype. After intrasplenic transplantation, engrafted MSCs with isolated expression of fibronectin and α-foetoprotein were observed. When these cells were injected into the liver, they expressed all analysed markers, confirming the chimaeric co-expression observed in vitro . Conversely, liver-engrafted MDHLCs conserved their hepatocyte-lineage markers but lost their chimaeric phenotype. Conclusions : Hepatocyte differentiation of MSCs predominantly allows the acquisition of phenotypic hallmarks and provides chimaeric cells that maintain expression of initial lineage markers. However, advanced maturation to the hepatocyte-like phenotype could be obtained in vivo by conditioning MSCs prior to transplantation or by infusing cells into the liver micro-environment.     

Altered T cell differentiation and Notch signaling induced by the ectopic expression of keratin K10 in the epithelial cells of the thymus

Transgenic mice expressing hK10 under the keratin K5 promoter display several alterations in the epidermis including decreased cell proliferation, and reduced susceptibility to tumor development. Given that K5 promoter is also active in the epithelial cells of the thymus, we explored the possible alterations of the thymus because of K10 transgene expression. We found severe thymic alterations, which affect not only the thymic epithelial cells (TEC), but also thymocytes. We observed altered architecture and premature thymus involution in the transgenic mice associated with increased apoptosis and reduced proliferation of the thymocytes. Interestingly, prior to the development of this detrimental phenotype, thymocytes of the transgenic mice also displayed altered differentiation, which is aggravated later on. Molecular characterization of this phenotype indicated that Akt activity is reduced in TEC, but not in thymocytes. In addition, we also observed altered expression of Notch family members and some of their ligands both in TEC and T cells. This produces reduced Notch activity in TEC but increased Notch activity in thymocytes, which is detectable prior to the disruption of the thymic architecture. In addition, we also detect altered Notch expression in the epidermis of bK5hK10 transgenic mice. Collectively the present data indicate that keratin K10 may induce severe alterations not only in a cell autonomous manner, but also in neighboring cells by the modulation of signals involved in cell-cell interactions.     

Numb modulates intestinal epithelial cells toward goblet cell phenotype by inhibiting the Notch signaling pathway

Numb was originally identified as an important cell fate determinant that is asymmetrically inherited during mitosis and controls the fate of sibling cells by inhibiting the Notch signaling pathway in neural tissue. The small intestinal epithelium originates from the division of stem cells that reside in the crypt, which further differentiate into goblet cells, absorptive cells, paneth cells, and enteroendocrine cells. However, Numb's involvement in the differentiation process of intestinal epithelium is largely unknown. In the present study, we confirm that both the Numb mRNA and protein isoforms are expressed in adult mouse intestinal mucosa. Numb protein is ubiquitously expressed throughout the crypt–villus axis of the small intestinal epithelium and is mainly localized to the cytoplasmic membrane. Down-regulation of endogenous Numb using RNA interference in cultured intestinal LS174T cells increased Notch signaling, leading to the up-regulation of Hes1 and the down-regulation of Hath1. Knockdown of Numb alleviated MUC2 protein expression and led to loss of the goblet cell phenotype in LS174Tl cells. Our results provide the first evidence that Numb, an important cell fate determinant, modulates intestinal epithelial cells towards the goblet cell phenotype by inhibiting the Notch signaling pathway.     

Absence of CFTR is associated with pleiotropic effects on mucins in mouse gallbladder epithelial cells

Mucus of cystic fibrosis patients exhibits altered biochemical composition and biophysical behavior, but the causal relationships between altered cystic fibrosis transmembrane conductance regulator (CFTR) function and the abnormal mucus seen in various organ systems remain unclear. We used cultured gallbladder epithelial cells (GBEC) from wild-type and Cftr((-/-)) mice to investigate mucin gene and protein expression, kinetics of postexocytotic mucous granule content expansion, and biochemical and ionic compositions of secreted mucins. Muc1, Muc3, Muc4, Muc5ac, and Muc5b mRNA levels were significantly lower in Cftr((-/-)) GBEC compared with wild-type cells, whereas Muc2 mRNA levels were higher in Cftr((-/-)) cells. Quantitative immunoblotting demonstrated a trend toward lower MUC1, MUC2, MUC3, MUC5AC, and MUC5B mucin levels in Cftr((-/-)) cells compared with cells from wild-type mice. In contrast, the levels of secreted MUC1, MUC3, MUC5B, and MUC6 mucins were significantly higher from Cftr((-/-)) cells; a trend toward higher levels of secreted MUC2 and MUC5AC was also noted from Cftr((-/-)) cells. Cftr((-/-)) cells demonstrated slower postexocytotic mucous granule content expansion. Calcium concentration was significantly elevated in the mucous gel secreted by Cftr((-/-)) cells compared with wild-type cells. Secreted mucins from Cftr((-/-)) cells contained higher sulfate concentrations. Thus absence of CFTR is associated with pleiotropic effects on mucins in murine GBEC.     

Differential regulation of glucose transporter 1 and 2 mRNA expression by epidermal growth factor and transforming growth factor-beta in rat hepatocytes.

We have examined by Northern blot analysis the expression of two members of the glucose transporter family of genes (GLUT-1 and GLUT-2) in regenerating liver and in hepatocytes cultured under various conditions. GLUT-1, although thought to be a growth-associated gene, is not expressed in normal or regenerating liver, whereas GLUT-2, a liver-specific gene, is abundant in normal liver and gradually up-regulated during liver regeneration. Conversely, in hepatocytes cultured conventionally on dried rat tail collagen (RTC) in the presence of EGF and insulin, which potentiate proliferation, GLUT-1 mRNA is rapidly and abundantly expressed, whereas GLUT-2 is depressed. To investigate the causes of this "switch" in glucose transporter expression seen when hepatocytes are removed from the liver and cultured under the conventional proliferative conditions, we examined the effects of specific growth factors and extracellular matrices on cultured hepatocytes. EGF, a potent liver mitogen, although causing a threefold induction of GLUT-1, was found to have no effect on GLUT-2 expression, suggesting that the increase in GLUT-2 seen in regenerating liver is not due to EGF. Inhibition of protein synthesis by cycloheximide in cultured hepatocytes does not prevent the induction of GLUT-1 mRNA. In addition, treatment of cells with cycloheximide appears to stabilize the GLUT-2 mRNA, preventing the usual down-regulation of this gene in cultured hepatocytes. The expression of the two glucose transporter mRNAs also differed when the hepatocytes were adherent to particular cell matrices. Culture of hepatocytes on a reconstituted basement membrane gel matrix (EHS) is known to restrain their growth and mediate high levels of differentiated hepatocytic functions that are lost under conventional culture conditions. Unlike cells on RTC, hepatocytes on EHS expressed low levels of GLUT-1 mRNA, and decreased GLUT-2 mRNA. TGF-beta, an attenuator of DNA synthesis, when added to cultures on RTC, substantially down-regulated GLUT-2 but had no effect on GLUT-1. We propose that the effectors, EGF, TGF-beta and basement membrane components, play a significant role in the regulation of expression of GLUT-1 and GLUT-2 in hepatocytes.     

Pluripotent stem cell-derived hepatocyte-like cells

Liver disease is an important clinical problem, impacting over 30 million Americans and over 600 million people worldwide. It is the 12th leading cause of death in the United States and the 16th worldwide. Due to a paucity of donor organs, several thousand Americans die yearly while waiting for liver transplantation. Unfortunately, alternative tissue sources such as fetal hepatocytes and hepatic cell lines are unreliable, difficult to reproduce, and do not fully recapitulate hepatocyte phenotype and functions. As a consequence, alternative cell sources that do not have these limitations have been sought. Human embryonic stem (hES) cell- and induced pluripotent stem (iPS) cell-derived hepatocyte-like cells may enable cell based therapeutics, the study of the mechanisms of human disease and human development, and provide a platform for screening the efficacy and toxicity of pharmaceuticals. iPS cells can be differentiated in a step-wise fashion with high efficiency and reproducibility into hepatocyte-like cells that exhibit morphologic and phenotypic characteristics of hepatocytes. In addition, iPS-derived hepatocyte-like cells (iHLCs) possess some functional hepatic activity as they secrete urea, alpha-1-antitrypsin, and albumin. However, the combined phenotypic and functional traits exhibited by iHLCs resemble a relatively immature hepatic phenotype that more closely resembles that of fetal hepatocytes rather than adult hepatocytes. Specifically, iHLCs express fetal markers such as alpha-fetoprotein and lack key mature hepatocyte functions, as reflected by drastically reduced activity (~ 0.1%) of important detoxification enzymes (i.e. CYP2A6, CYP3A4). These key differences between iHLCs and primary adult human hepatocytes have limited the use of stem cells as a renewable source of functional adult hepatocytes for in vitro and in vivo applications. Unfortunately, the developmental pathways that control hepatocyte maturation from a fetal into an adult hepatocyte are poorly understood, which has hampered the field in its efforts to induce further maturation of iPS-derived hepatic lineage cells. This review analyzes recent developments in the derivation of hepatocyte-like cells, and proposes important points to consider and assays to perform during their characterization. In the future, we envision that iHLCs will be used as in vitro models of human disease, and in the longer term, provide an alternative cell source for drug testing and clinical therapy.     

Targeted cell replacement with bone marrow cells for airway epithelial regeneration

It has been suggested that some adult bone marrow cells (BMC) can localize to the lung and develop tissue-specific characteristics including those of pulmonary epithelial cells. Here, we show that the combination of mild airway injury (naphthalene-induced) as a conditioning regimen to direct the site of BMC localization and transtracheal delivery of short-term cultured BMC enhances airway localization and adoption of an epithelial-like phenotype. Confocal analysis of airway and alveolar-localized BMC (fluorescently labeled) with epithelial markers shows expression of the pulmonary epithelial proteins, Clara cell secretory protein, and surfactant protein C. To confirm epithelial gene expression by BMC, we generated transgenic mice expressing green fluorescent protein (GFP) driven by the epithelial-specific cytokeratin-18 promoter and injected BMC from these mice transtracheally into wild-type recipients after naphthalene-induced airway injury. BMC retention in the lung was observed for at least 120 days following cell delivery with increasing GFP transgene expression over time. Some BMC cultured in vitro over time also expressed GFP transgene, suggesting epithelial transdifferentiation of the BMC. The results indicate that targeted delivery of BMC can promote airway regeneration.     

Hepatocyte growth factor induces differentiation of adult rat bone marrow cells into a hepatocyte lineage in vitro

Bone marrow (BM) cells originally include alpha-fetoprotein (AFP)- and c-Met [a receptor for hepatocyte growth factor (HGF)]-expressing cells. In vitro treatment of BM cells with HGF induced albumin-expressing hepatocyte-like cells. Furthermore, those hepatocyte-like cells expressed cytokeratins 8 and 18, which are typically expressed in normal adult hepatocytes. These findings demonstrate that BM cells include AFP-expressing hepatic progenitor cells that can be differentiated into hepatocytes by HGF in culture, indicating that such cultures are useful resources for cell transplantation therapy for liver diseases.     

Human amniotic fluid-derived stem cells can differentiate into hepatocyte-like cells in vitro and in vivo

Although human amniotic fluid is an attractive source of multipotent stem cells, the potential of amniotic fluid stem cells (AFSCs) to differentiate into hepatic cells has not been extensively evaluated. In this study, we examined whether human AFSCs can differentiate into a hepatic cell lineage in vitro and in vivo. After being treated with cytokines (fibroblast growth factor 4, basic fibroblast growth factor, hepatocyte growth factor, and oncostatin), AFSCs developed a morphology similar to that of hepatocytes. RT-PCR and immunofluorescence analysis showed that the treated AFSCs expressed the hepatocyte-specific markers albumin, cytokeratin 18, and alpha-fetoprotein. The differentiated cells also developed hepatocyte-specific functions, i.e., they secreted albumin, absorbed indocyanine green, and stored glycogen. When transplanted into CCl(4)-injured immunodeficient mice, undifferentiated AFSCs were integrated into the liver tissue, and they expressed markers characteristic of mature human hepatocytes. Although integration of AFSCs into the liver was limited (0.1-0.3% of hepatocytes), histological analysis showed that the recipient mice recovered more rapidly from CCl(4) injury than CCl(4)-injured mice that did not receive AFSCs. AFSCs can differentiate into hepatocyte-like cells in vitro and in vivo and can represent an easily accessible source of progenitor cells for hepatocyte regeneration and liver cell transplantation.     

Developmental regulation of claudin localization by fetal alveolar epithelial cells

Tight junction proteins in the claudin family regulate epithelial barrier function. We examined claudin expression by human fetal lung (HFL) alveolar epithelial cells cultured in medium containing dexamethasone, 8-bromo-cAMP, and isobutylmethylxanthanine (DCI), which promotes alveolar epithelial cell differentiation to a type II phenotype. At the protein level, HFL cells expressed claudin-1, claudin-3, claudin-4, claudin-5, claudin-7, and claudin-18, where levels of expression varied with culture conditions. DCI-treated differentiated HFL cells cultured on permeable supports formed tight transepithelial barriers, with transepithelial resistance (TER) >1,700 ohm/cm(2). In contrast, HFL cells cultured in control medium without DCI did not form tight barriers (TER <250 ohm/cm(2)). Consistent with this difference in barrier function, claudins expressed by HFL cells cultured in DCI medium were tightly localized to the plasma membrane; however, claudins expressed by HFL cells cultured in control medium accumulated in an intracellular compartment and showed discontinuities in claudin plasma membrane localization. In contrast to claudins, localization of other tight junction proteins, zonula occludens (ZO)-1, ZO-2, and occludin, was not sensitive to HFL cell phenotype. Intracellular claudins expressed by undifferentiated HFL cells were localized to a compartment containing early endosome antigen-1, and treatment of HFL cells with the endocytosis inhibitor monodansylcadaverine increased barrier function. This suggests that during differentiation to a type II cell phenotype, fetal alveolar epithelial cells use differential claudin expression and localization to the plasma membrane to help regulate tight junction permeability.     

Delta-like 1 expression promotes goblet cell differentiation in Notch-inactivated human colonic epithelial cells

Notch signaling has previously been implicated in the regulation of the cell fate of intestinal epithelial cells. However, the expression and function of Notch ligands in the human intestine remain largely unknown. In the present study, we showed that Notch ligands Delta-like 1 (Dll1) and Delta-like 4 (Dll4) are expressed in a goblet cell-specific manner in human colonic tissue. Additionally, we found that Dll1 and Dll4 expression was regulated in-parallel with Atoh1 and MUC2, which are both under the control of the Notch-Hes1 signaling pathway. Because knockdown of Dll1 expression completely abrogated the acquisition of the goblet cell phenotype in Notch-inactivated colonic epithelial cells, we postulate that Dll1 might function as a cis-acting regulatory element that induces undifferentiated cells to become goblet cells. Our results suggest a link between Dll1 expression and human goblet cell differentiation that might be mediated by a function that is distinct from its role as a Notch receptor ligand.     

M-CSF诱导人骨髓间充质干细胞分化为肝样细胞的分子机制

本研究主要目的是明确M-CSF诱导骨髓间充质干细胞分化为肝样细胞的分子机制,为临床中的肝移植和治疗肝病提供新思路。对取自于本院骨科治疗的患者的股骨骨髓间充质干细胞进行提取、分离、传代培养及鉴定。流式细胞仪检测BMSCs的表面表型。为了诱导BMSCs的肝分化,本研究将BMSCs加入到培养基中。骨髓间充质干细胞诱导21 d后,BMSCs表达了肝细胞特异性标志物a-蛋白(AFP)和细胞角蛋白18(CK18),通过免疫荧光染色证实了分化与为分化的BMSCs表达的差异性。分化的BMSCs还显示了肝细胞的体外功能特征,包括白蛋白产生、尿素分泌和糖原储存。本研究结果表明,BMSCs在M-CSF诱导下可分化为功能性肝细胞样细胞,可作为肝病治疗的细胞来源。