Pre-adipocytes commitment to neurogenesis 1: Preliminary localisation of cholinergic molecules

A great effort has recently been made to obtain human stem cells able to differentiate into cholinergic neurons, as a number of diseases are associated to the cholinergic neuron loss, degeneration or incorrect function (Alzheimer's disease and motor neuron disease). A stem cell population (i.e. pre-adipocytes) is present in the adipose stromal compartment. Pre-adipocytes, like the mesodermic derivative cells, retain high plasticity and potentiality to convert in vitro from one phenotype into many others, and they can be isolated from adult adipose tissue. Pre-adipocytes committed in vitro to neural differentiation were followed up to the acquisition of neural morphology. Acetylcholinesterase and choline acetyltransferase are expressed from the native cell stage, with different localisations and roles during neural commitment. Western blots show the beginning of a new synthesis of these enzymes at 4 weeks of culture of neurogenic pre-adipocytes, in parallel with neural morphology. The passage of the choline-acetyltransferase immunoreactivity from cytoplasmic to membrane localisation shows the possible onset of catalytic activity and the histochemical reaction confirms the activity of acetylcholinesterase. This explains the possibility of obtaining cholinergic-like phenotype from pre-adipocytes.     

Cytochromes P450 (CYP) in Tropical Fishes: Catalytic Activities,Expression of Multiple CYP Proteins and High Levels of Microsomal P450 in Liver of Fishes From Bermuda

Hepatic microsomes prepared from 10 fish species from Bermuda were studied to establish features of cytochrome P450 (CYP) systems in tropical marine fish. The majority (7/10) of the species had total P450 content between 0.1 and 0.5 nmol/mg, and cytochrome b₅ content between 0.025 and 0.25 nmol/mg. Ethoxycoumarin O-deethylase (ECOD) and aminopyrine N-demethylase (APND) rates in these 7 species were 0.23–2.1 nmol/min/mg and 0.5–11 nmol/min/mg, respectively, similar to rates in many temperate fish species. In contrast to those 7 species, sergeant major (Abudefduf saxatilis) and Bermuda chub (Kyphosus sectatrix) had microsomal P450 contents near 1.7 nmol/mg, among the highest values reported in untreated fish, and had greater rates of ECOD, APND, ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O-depentylase than did most of the other species. Freshly caught individuals of all species had detectable levels of EROD and aryl hydrocarbon hydroxylase (AHH) activities. Those individuals with higher rates of EROD activity had greater content of immunodetected CYP1A protein, consistent with Ah-receptor agonists acting to induce CYP1A in many fish in Bermuda waters. Injection of tomtate and blue-striped grunt with β-naphthoflavone (BNF; 50 or 100 mg/kg) induced EROD rates by 25 to 55-fold, suggesting that environmental induction in some fish was slight compared with the capacity to respond. AHH rates were induced only 3-fold in these same fish. The basis for disparity in the degree of EROD and AHH induction is not known. Rates of APND and testosterone 6β- and 16β-hydroxylase were little changed by BNF, indicating that these are not CYP1A activities in these fish. Antibodies to phenobarbital-inducible rat CYP2B1 or to scup P450B, a putative CYP2B, detected one or more proteins in several species, suggesting that CYP2B-like proteins are highly expressed in some tropical fishes. Generally, species with greater amounts of total P450 had greater amounts of proteins related to CYP2B. These species also had appreciable amounts of CYP3A-like proteins. Thus, many fishes in Bermuda appear to have induced levels of CYP1A; some also have unusually high levels of total P450 and of CYP2B-like and CYP3A-like proteins. These species may be good models for examining the structural, functional and regulatory properties of teleost CYP and the environmental or ecological factors contributing to high levels of expression of CYP in some fishes.     

Development and characterization of a rainbow trout liver cell line expressing cytochrome P450-dependent monooxygenase activity

A cell line RTL-W1, has been developed from the normal liver of an adult rainbow trout by proteolytic dissociation of liver fragments. RTL-W1 can be grown routinely in the basal medium, L-15, supplemented with 5% fetal bovine serum. In this medium, the cells have been passaged approximately 100 times over an 8-year period. The cells do not form colonies or grow in soft agar. The cultures are heteroploid. The cell shape was predominantly polygonal or epithelial-like, but as cultures became confluent, bipolar or fibroblast-like cells appeared. Among the prominent ultrastructural features of RTL-W1 were distended endoplasmic reticulum and desmosomes. Benzo[a]pyrene was cytotoxic to RTL-W1. Activity for the enzyme, 7-ethoxyresorufin O-deethylase (EROD), which is a measure of the cytochrome P4501A1 protein, increased dramatically in RTL-W1 upon their exposure to increasing concentrations of either -naphthoflavone (BNF) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). With these properties, RTL-W1 should be useful for studying the expression of the cytochrome P450 enzymes and as a tool for assessing the toxic potency of environmental contaminants.Abbreviations AHH aryl hydrocarbon hydroxylase - B[a]P benzo[a]pyrene - BNF -naphthoflavone - CHSE-214 Chinook salmon embryo cells - CYP cytochrome P450 - DMSO dimethyl sulfoxide - ECOD 7-ethoxycoumarinO-deethylase - EDTA ethylenediaminetetraacetic acid - EROD 7-ethoxyresorufinO-deethylase - FBS fetal bovine serum - HEPES N-[-hydroxyethyl]piperazine-N-[2-ethanesulfonic acid] - H4IIE rathepatoma cells - PCB polychlorinated biphenyls - PHH planar halogenated hydrocarbons - TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin - TEM transmission electron microscopy - TEFs toxic equivalent factors - TS 0.05 mM Tris 0.2M sucrose - RLE rat liver epithelial cells - RTL-W1 rainbow trout liver-Waterloo 1 cells     

Protein kinase C and CYPlAl induction in rainbow trout (Oncorhynchus mykiss) hepatocyte culture

1. The role of protein kinase C (PKC) in B-naphthoflavone (BNF) induction of CYP1A1 in rainbow trout hepatocytes was investigated.2. Primary cultures of rainbow trout hepatocytes treated with BNF for 24 hr showed an increase in microsomal 7-ethyoxyresorufm-O-deethylase (EROD) activity compared to cells treated with vehicle (DMSO) only.3. Increases in EROD activities were proportional to increased concentrations of BNF from 1 to 10 nM reaching a plateau at higher concentrations (20–100 nM) of BNF.4. Western blot analysis using specific antibody (LM4b) against CYP1A1 showed that changes in microsomal CYP1A1 protein paralleled that of EROD activity.5. The induction of EROD activity by BNF required both protein and RNA synthesis since the process was blocked by both cycloheximide and actinomycin D.6. Pretreatment of hepatocytes with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) led to a dose dependent suppression of BNF-induced EROD activity and CYP1A1 content. TPA alone had no effect on hepatic EROD activity and CYP1A1 protein level.7. Pretreatment with sn-1,2 didecanoylglycerol, a PKC activator, had no effect on BNF-induced EROD activity in these cells.8. Pretreatment of cells with staurosporine, a PKC inhibitor, effectively blocked BNF-induced EROD activity.9. PKC may play a role in the induction of CYP1A1 gene expression in fish liver by BNF.     

Importance of being Nernst: Synaptic activity and functional relevance in stem cell-derived neurons

Functional synaptogenesis and network emergence are signature endpoints of neurogenesis. These behaviors provide higher-order confirmation that biochemical and cellular processes necessary for neurotransmitter release, post-synaptic detection and network propagation of neuronal activity have been properly expressed and coordinated among cells. The development of synaptic neurotransmission can therefore be considered a defining property of neurons. Although dissociated primary neuron cultures readily form functioning synapses and network behaviors in vitro, continuously cultured neurogenic cell lines have historically failed to meet these criteria. Therefore, in vitro-derived neuron models that develop synaptic transmission are critically needed for a wide array of studies, including molecular neuroscience, developmental neurogenesis, disease research and neurotoxicology. Over the last decade, neurons derived from various stem cell lines have shown varying ability to develop into functionally mature neurons. In this review, we will discuss the neurogenic potential of various stem cells populations, addressing strengths and weaknesses of each, with particular attention to the emergence of functional behaviors. We will propose methods to functionally characterize new stem cell-derived neuron (SCN) platforms to improve their reliability as physiological relevant models. Finally, we will review how synaptically active SCNs can be applied to accelerate research in a variety of areas. Ultimately, emphasizing the critical importance of synaptic activity and network responses as a marker of neuronal maturation is anticipated to result in in vitro findings that better translate to efficacious clinical treatments.     

In Vitro Generation of Functional Liver Organoid-Like Structures Using Adult Human Cells

In this study we used differentiated adult human upcyte^® cells for the in vitro generation of liver organoids. Upcyte^® cells are genetically engineered cell strains derived from primary human cells by lenti-viral transduction of genes or gene combinations inducing transient proliferation capacity (upcyte^® process). Proliferating upcyte^® cells undergo a finite number of cell divisions, i.e., 20 to 40 population doublings, but upon withdrawal of proliferation stimulating factors, they regain most of the cell specific characteristics of primary cells. When a defined mixture of differentiated human upcyte^® cells (hepatocytes, liver sinusoidal endothelial cells (LSECs) and mesenchymal stem cells (MSCs)) was cultured in vitro on a thick layer of Matrigel™, they self-organized to form liver organoid-like structures within 24 hours. When further cultured for 10 days in a bioreactor, these liver organoids show typical functional characteristics of liver parenchyma including activity of cytochromes P450, CYP3A4, CYP2B6 and CYP2C9 as well as mRNA expression of several marker genes and other enzymes. In summary, we hereby describe that 3D functional hepatic structures composed of primary human cell strains can be generated in vitro. They can be cultured for a prolonged period of time and are potentially useful ex vivo models to study liver functions.     

Catalytic and immunochemical properties of hepatic cytochrome P450 1A in three avian species treated with β-naphthoflavone or isosafrole

Induction of cytochrome P450 1A (CYP1A) can be used as a biomarker of exposure to planar halogenated aromatic hydrocarbons (PHAHs). Our objective was to characterize the induction of CYP1A activity and protein in three avian species following in vivo treatment with β-naphthoflavone (BNF) and/or isosafrole. Alkoxyresorufin-O-dealkylase (alk-ROD) activities of hepatic microsomes from Herring Gulls (Larus argentatus) (HGs), Double-crested Cormorants (Phalacrocorax auritus) (DCCs) and chickens (Gallus domesticus) were measured using ethoxy-, methoxy-, pentoxy- and benzyloxy-resorufin, in the presence and absence of the inhibitors ellipticine or furafylline. Immunoreactivity of microsomal proteins with antibodies to several CYP1A proteins was investigated. CYP1A protein and alk-ROD activities of HGs and DCCs, but not chickens, were induced by isosafrole. Ellipticine was a potent and non-selective inhibitor of alk-ROD activity in all three species, while furafylline inhibition of alk-ROD activities varied among species and treatments. In all three species, BNF induced a protein immunoreactive with monoclonal antibody to CYP1A1 from the marine fish Stenotomus chrysops (scup), but a CYP1A2-like protein was not detected in avian microsomes probed with polyclonal antibodies to mouse CYP1A2. Variations in responses among avian species indicate that CYP1A proteins and substrate specificities should be characterized for each species used in PHAH biomonitoring programs.     

For the long run: maintaining germinal niches in the adult brain

The adult mammalian brain retains neural stem cells that continually generate new neurons within two restricted regions: the subventricular zone (SVZ) of the lateral ventricle and the dentate gyrus subgranular zone (SGZ) of the hippocampus. Though these cellular populations are spatially isolated and subserve different brain systems, common themes begin to define adult neurogenic niches: (1) astrocytes serve as both stem cell and niche cell, (2) a basal lamina and concomitant vasculogenesis may be essential components of the niche, and (3) "embryonic" molecular morphogens and signals persist in these niches and play critical roles for adult neurogenesis. The adult neurogenic niches can be viewed as "displaced" neuroepithelium, pockets of cells and local signals that preserve enough embryonic character to maintain neurogenesis for life.     

Inhibition of in vitro aflatoxin B1-DNA binding in rainbow trout by CYP1A inhibitors: α-naphthoflavone, β-naphthoflavone and trout CYP1A1 peptide antibody

Rainbow trout cytochrome P450 (CYP)1A detoxifies aflatoxin B₁ (AFB₁) to aflatoxin M₁ (AFM₁), whereas CYP2K1 activates AFB₁ to AFB₁-8,9-epoxide. We report that α-naphthoflavone (ANF) and β-naphthoflavone (BNF) both strongly inhibit CYP1A-mediated ethoxyresorufin O-deethylase (EROD) activity (K_i = 9.1 ± 0.8 and 7.6 ± 1.1 nM, respectively). These inhibitors (selective for mammalian CYP1A at low concentrations), as well as rabbit polyclonal antibody to a trout CYP1A1 peptide (residues 277–294), also strongly inhibited trout microsome-catalyzed AFB₁-DNA binding and lauric acid (ω-1) hydroxylation in vitro, reactions previously established to be CYP2K1-dependent. ANF at 0.5, 5, 50 and 500 μM inhibited liver microsome-catalyzed AFB₁-DNA binding by 22, 58, 84 and 91%, respectively, whereas BNF at the same concentrations inhibited 22, 74, 78 and 81%, respectively. The CYP1A1 peptide and CYP2K1 polyclonal antibodies (10 mg IgG/mg microsomal protein) inhibited AFB₁-DNA binding by 84 and 66%, respectively, compared with pre-immune IgG. Lauric acid (ω-1) hydroxylation was inhibited 61% by 5 μM ANF, 69% by 5 μM BNF and 100% by either antibody at 12 mg IgG/mg microsomal protein. These results demonstrate that mammalian CYP1A inhibitors also inhibit trout microsomal AFB₁-DNA binding and lauric acid (ω-1) hydroxylation, catalyzed primarily by CYP2K1. In the absence of evidence that trout CYP1A can catalyze AFB₁-DNA binding, the results suggest configuration similarities at, or near, the active sites for these two fish enzymes that result in antibody crossreaction and loss of the inhibitor specificity observed with mammalian CYP1A.     

Distinct response of adult neural stem cells to low versus high dose ionising radiation

Radiosusceptibility is the sensitivity of a biological organism to ionising radiation (IR)-induced carcinogenesis, an outcome of IR exposure relevant following low doses. The tissue response is strongly influenced by the DNA damage response (DDR) activated in stem and progenitor cells. We previously reported that in vivo exposure to 2 Gy X-rays activates apoptosis, proliferation arrest and premature differentiation in neural progenitor cells (transit amplifying cells and neuroblasts) but not in neural stem cells (NSCs) of the largest neurogenic region of the adult brain, the subventricular zone (SVZ). These responses promote adult quiescent NSC (qNSC) activation after 2 Gy. In contrast, neonatal (P5) SVZ neural progenitors continue proliferating and do not activate qNSCs. Significantly, the human and mouse neonatal brain is radiosusceptible.Here, we examine the response of stem and progenitor cells in the SVZ to low IR doses (50–500 mGy). We observe a linear dose-response for apoptosis but, in contrast, proliferation arrest and neuroblast differentiation require a threshold dose of 200 or 500 mGy, respectively. Importantly, qNSCs were not activated at doses below 500 mGy. Thus, full DDR activation in the neural stem cell compartment in vivo necessitates a threshold dose, which can be considered of significance when evaluating IR-induced cancer risk and dose extrapolation.     

Characterization of primary human hepatocytes, HepG2 cells, and HepaRG cells at the mRNA level and CYP activity in response to inducers and their predictivity for the detection of human hepatotoxins

In the pharmaceutical industry, improving the early detection of drug-induced hepatotoxicity is essential as it is one of the most important reasons for attrition of candidate drugs during the later stages of drug development. The first objective of this study was to better characterize different cellular models (i.e., HepG2, HepaRG cells, and fresh primary human hepatocytes) at the gene expression level and analyze their metabolic cytochrome P450 capabilities. The cellular models were exposed to three different CYP450 inducers; beta-naphthoflavone (BNF), phenobarbital (PB), and rifampicin (RIF). HepG2 cells responded very weakly to the different inducers at the gene expression level, and this translated generally into low CYP450 activities in the induced cells compared with the control cells. On the contrary, HepaRG cells and the three human donors were inducible after exposure to BNF, PB, and RIF according to gene expression responses and CYP450 activities. Consequently, HepaRG cells could be used in screening as a substitute and/or in complement to primary hepatocytes for CYP induction studies. The second objective was to investigate the predictivity of the different cellular models to detect hepatotoxins (16 hepatotoxic and 5 nonhepatotoxic compounds). Specificity was 100% with the different cellular models tested. Cryopreserved human hepatocytes gave the highest sensitivity, ranging from 31% to 44% (depending on the donor), followed by lower sensitivity (13%) for HepaRG and HepG2 cells (6.3%). Overall, none of the models under study gave desirable sensitivities (80–100%). Consequently, a high metabolic capacity and CYP inducibility in cell lines does not necessarily correlate with a high sensitivity for the detection of hepatotoxic drugs. Further investigations are necessary to compare different cellular models and determine those that are best suited for the detection of hepatotoxic compounds.     

Increased expression of damage-associated molecular patterns (DAMPs) in osteoarthritis of human knee joint compared to hip joint

The aryl hydrocarbon receptor (AhR) mediates a variety of biological responses to ubiquitous environmental pollutants. In this study, the effects of administration of β-naphthoflavone (BNF), a potent AhR ligand, on the expression of AhR-dependent genes were examined by microarray and qPCR analysis in both, differentiated and undifferentiated HepaRG cell lines. To prove that BNF-induced changes of investigated genes were indeed AhR-dependent, we knock down the expression of AhR by stable transfection of HepaRG cells with shRNA. Regardless of genetical identity, our results clearly demonstrate different expression profiles of AhR-dependent genes between differentiated and undifferentiated HepaRG cells. Genes involved in metabolism of xenobiotics constitute only minute fraction of all genes regulated by AhR in HepaRG cells. Participation of AhR in induction of expression of genes associated with regulation of apoptosis or involved in cell proliferation as well as AhR-dependent inhibition of genes connected to cell adhesion could support suggestion of involvement of AhR not only in initiation but also in progression of carcinogenesis. Among the AhR-dependent genes known to be involved in metabolism of xenobiotics, cytochromes P4501A1 and 1B1 belong to the most inducible by BNF. On the contrary, expression of GSTA1 and GSTA2 was significantly inhibited after BNF treatment of HepaRG cells. Among the AhR-dependent genes that are not involved in metabolism of xenobiotics SERPINB2, STC2, ARL4C, and TIPARP belong to the most inducible by BNF. Our results imply involvement of Ah receptor in regulation of CYP19A1, the gene-encoding aromatase, and an enzyme responsible for a key step in the biosynthesis of estrogens.     

Modulation by extracellular matrices of monooxygenase and CYP1A1 induction in Hep G2 cells in serum-free culture

The in vitro cellular functions of differentiated cells are influenced by culture conditions. Effects of several extracellular matrices (ECMs) on cytochrome P450-dependent monooxygenases (MFOs) induction and cytochrome P4501A1 (CYP1A1) gene expression were estimated in Hep G2 cells cultured in a serum-free medium. The cells were cultured on collagen type I- and II-, fibronectin-, and matrigel-coated dishes and MFO activities were induced by the addition of 3-methylcholanthrene (MC). The induction of ethoxycoumarin O-deethylase (ECOD) and alkoxyresorufin O-dealkylase activities as well as the expression of CYP1A1 mRNA were also determined. ECOD and methoxy- and ethoxyresorufin O-dealkylase activities in Hep G2 cells were enhanced by culturing the cells using a serum-free medium on fibronectin- or matrigel-coated dishes. ECOD activity on fibronectin-coated dishes was about 3-fold higher than that using a serum-supplemented medium on untreated dishes. Furthermore, both immobilized and soluble fibronectin enhanced the induction of MFOs. The expression of CYP1A1 mRNA using fibronectin-coated dishes was about 2-fold higher than that using a serum-supplemented medium on untreated dishes. These findings suggest that the gene expression in cultured cells is greatly influenced by ECMs. By using fibronectin-coated dishes to cell culture in a serum-free medium, reproducible and highly sensitive results can be obtained in experiments using cultured cells. This revised version was published online in August 2006 with corrections to the Cover Date.     

Adult stem cells derived from skeletal muscle — biology and potential

Skeletal muscle contains at least two distinct populations of adult stem cells — satellite cells and multipotent muscle-derived stem cells. Monopotential satellite cells are located under the basal lamina of muscle fibers. They are capable of giving rise only to cells of myogenic lineage, which play an important role in the processes of muscle regeneration. Multipotent muscle-derived stem cells are considered to be predecessors of the satellite cells. Under proper conditions, both in vitro and in vivo, they undergo myogenic, cardiogenic, chondrogenic, osteogenic and adipogenic differentiation. The main purpose of the present article is to summarize current information about adult stem cells derived from skeletal muscle, and to discuss their isolation and in vitro expansion techniques, biological properties, as well as their potential for regenerative medicine.