Hepatic 3D cultures but not 2D cultures preserve specific transporter activity for acetaminophen-induced hepatotoxicity.

Primary human hepatocytes (PHH) are the "gold standard" for in vitro toxicity tests. However, 2D PHH cultures have limitations that are due to a time-dependent dedifferentiation process visible by morphological changes closely connected to a decline of albumin production and CYP450 activity. The 3D in vitro culture corresponds to in vivo-like tissue architecture, which preserves functional characteristics of hepatocytes, and therefore can at least partially overcome the restrictions of 2D cultures. Consequently, several drug toxicities observed in vivo cannot be reproduced in 2D in vitro models, for example, the toxic effects of acetaminophen. The objective of this study was to identify molecular differences between 2D and 3D cultivation which explain the observed toxicity response. Our data demonstrated an increase in cell death after treatment with acetaminophen in 3D, but not in 2D cultures. Additionally, an acetaminophen concentration-dependent increase in the CYP2E1 expression level in 3D cultures was detected. However, during the treatment with 10 mM acetaminophen, the expression level of SOD gradually decreased in 3D cultures and was undetectable after 24 h. In line with these findings, we observed higher import/export rates in the membrane transport protein, multidrug resistance-associated protein-1, which is known to be specific for acetaminophen transport. The presented data demonstrate that PHH cultured in 3D preserve certain metabolic functions. Therefore, they have closer resemblance to the in vivo situation than PHH in 2D cultures. In consequence, 3D cultures will allow for a more accurate hepatotoxicity prediction in in vitro models in the future.

Decrease of global methylation improves significantly hepatic differentiation of Ad-MSCs: possible future application for urea detoxification.

Hepatocyte transplantation is considered to be an alternative to orthotopic liver transplantation. Cells can be used to bridge patients waiting for a donor organ, decrease mortality in acute liver failure, and support metabolic liver diseases. The limited availability of primary human hepatocytes for such applications has led to the generation of alternative hepatocyte-like cells from various adult stem or precursor cells. The aim of this study was to generate hepatocyte-like cells from adipose-derived mesenchymal stem cells (Ad-MSCs) for clinical applications, which are available "off the shelf." Epigenetic changes in hepatocyte-like cells were induced by 5-azacytidine, which, in combination with other supplements, leads to significantly improved metabolic and enzymatic activities compared to nontreated cells. Cells with sufficient hepatic features were generated with a four-step protocol: 5-azacytidine (step 1); epidermal growth factor (step 2); fibroblast growth factor-4, dexamethasone, insulin-transferrin-sodium-selenite, and nicotinamide (step 3); and hepatocyte growth factor, dexamethasone, insulin-transferrin-sodium-selenite, and nicotinamide (step 4). Generated differentiated cells had higher phase I (CYP1A1/2, CYP2E1, CYP2B6, CYP3A4) and phase II activities compared to the undifferentiated cells. A strong expression of CYP3A7 and a weak expression of 3A4, as well as the important detoxification markers α-fetoprotein and albumin, could also be detected at the mRNA level. Importantly, urea metabolism (basal, NH4-stimulated, NH4- and ornithine-stimulated) was comparable to freshly isolated human hepatocytes, and unlike cryopreserved human hepatocytes, this activity was maintained after 6 months of cryopreservation. These findings suggest that these cells may be suitable for clinical application, especially for treatment of urea cycle disorders.

Further characterization of autologous NeoHepatocytes for in vitro toxicity testing.

Gold standard for in vitro toxicity tests and drug screenings is primary human hepatocytes (hHeps). Because of their limited availability efforts have been made to provide alternatives, e.g., monocyte-derived NeoHepatocytes. In the past years it has been critically discussed if gaining hepatocyte features is associated with trans-differentiation of monocytes or their activation towards a macrophage phenotype. Generating NeoHepatocytes in the presence of six different human AB sera, fetal calf serum (FCS) or autologous serum showed that yield and quality of NeoHepatocytes is inversely correlated to macrophage activation. Using autologous serum constantly the highest amount of cells with the best metabolic capacity was obtained. Focus of this study was to further analyze bio-transformation capacity of the optimized NeoHepatocytes for use as in vitro toxicity test-system. Treatment of the optimized NeoHepatocytes with two different pro-teratogenic substances with corresponding metabolites and eight known hepatotoxins showed comparable toxicity to hHeps. Bio-transformation rates, assessed by testosterone metabolism, were comparable in both cell types. Our data reveal that use of autologous serum reduced macrophage activation which improved yield and function of NeoHepatocytes resulting in bio-transformation and toxicity profiles comparable to hHeps. Thus, their easy accessibility makes them an ideal candidate for in vitro toxicity studies.

Spongistatin 1: a new chemosensitizing marine compound that degrades XIAP.

Spongistatin 1 is a new experimental chemotherapeutic agent isolated from marine sponges. Here we show that spongistatin 1 potently induces cell death in patient primary acute leukemic cells with higher efficiency than 8/10 clinically used cytotoxic drugs and prevents long-term survival of leukemic cell lines. Spongistatin 1 triggers caspase-dependent apoptosis in Jurkat T cells by the release of cytochrome c, Smac/DIABLO and Omi/HtrA2. As caspase-9 acts as an initiator caspase and Bcl-2 and Bcl-xL overexpression suppress spongistatin 1-induced apoptosis, cell death is mediated through the mitochondrial apoptosis pathway. Importantly, spongistatin 1 leads to the degradation of the antiapoptotic X-linked inhibitor of apoptosis protein. In apoptosis-resistant leukemic tumor cells overexpressing XIAP, spongistatin 1 effectively causes cell death and potentiates cell death induction by other apoptosis-promoting factors that might be caused by spongistatin 1-mediated degradation of XIAP. Our data show that spongistatin 1 represents a promising novel therapeutic agent for the treatment of leukemic tumor cells especially in the clinically highly relevant situation of chemoresistance due to overexpression of XIAP.