Concurrent isolation of hepatic stem cells and hepatocytes from the human liver

Hepatocytes differentiated from induced pluripotent stem cells or stem cells have the potential to be representative in vitro models of the human liver for research as well as early safety assessment programs. However, up until now, there has been no definitive proof that differentiated hepatocytes recapitulate the phenotype and functional characteristics of primary hepatocytes from the same individual. Thus, a method for the concurrent isolation of hepatocytes and hepatic stem cells is presented here to provide the cells necessary for the evaluation of the required benchmarking. The method presented here generated high-quality hepatocytes with a purity of 94 ± 1% and a high percentage viability of 79 ± 2%. Furthermore, the hepatic stem cells isolated were found to be actively proliferating and have a purity of 98 ± 1%. Thus, these isolated cells can be used as a powerful tool for the validation of differentiated hepatocyte in vitro models

Characterization of a long-term mouse primary liver 3D tissue model recapitulating innate-immune responses and drug-induced liver toxicity.

Three-dimensional liver in vitro systems have recently attracted a lot of attention in drug development. These systems help to gain unprecedented insights into drug-induced liver injury (DILI), as they more closely reproduce liver biology, and as drug effects can be studied in isolated and controllable microenvironments. Many groups established human-based in vitro models but so far neglected the animal equivalent, although the availability of both models would be desirable. Animal in vitro models enable back- and forward translation of in vitro and in vivo findings, bridge the gap between rodent in vivo and human in vitro scenarios, and ultimately support the interpretation of data generated with preclinical species and humans. Since mice are often used in drug development and physiologically relevant in vitro systems are lacking, we established, for the first time, a mouse liver model that encompasses primary parenchymal and non-parenchymal cells with preserved viability and functionality over three weeks. Using our three-dimensional liver spheroids, we were able to predict the toxicity of known DILI compounds, demonstrated the interaction cascades between the different cell types and showed evidence of drug-induced steatosis and cholestasis. In summary, our mouse liver spheroids represent a valuable in vitro model that can be applied to study DILI findings, reported from mouse studies, and offers the potential to detect immune-mediated drug-induced liver toxicity

Characterization of a long-term mouse primary liver 3D tissue model recapitulating innate-immune responses and drug-induced liver toxicity

Three-dimensional liver in vitro systems have recently attracted a lot of attention in drug development. These systems help to gain unprecedented insights into drug-induced liver injury (DILI), as they more closely reproduce liver biology, and as drug effects can be studied in isolated and controllable microenvironments. Many groups established human-based in vitro models but so far neglected the animal equivalent, although the availability of both models would be desirable. Animal in vitro models enable back- and forward translation of in vitro and in vivo findings, bridge the gap between rodent in vivo and human in vitro scenarios, and ultimately support the interpretation of data generated with preclinical species and humans. Since mice are often used in drug development and physiologically relevant in vitro systems are lacking, we established, for the first time, a mouse liver model that encompasses primary parenchymal and non-parenchymal cells with preserved viability and functionality over three weeks. Using our three-dimensional liver spheroids, we were able to predict the toxicity of known DILI compounds, demonstrated the interaction cascades between the different cell types and showed evidence of drug-induced steatosis and cholestasis. In summary, our mouse liver spheroids represent a valuable in vitro model that can be applied to study DILI findings, reported from mouse studies, and offers the potential to detect immune-mediated drug-induced liver toxicity.ISSN:1932-620

Assessing single-stranded oligonucleotide drug-induced effects in vitro reveals key risk factors for thrombocytopenia.

Single-stranded oligonucleotides (ON) comprise a promising therapeutic platform that enables selective modulation of currently undruggable targets. The development of novel ON drug candidates has demonstrated excellent efficacy, but in certain cases also some safety liabilities were reported. Among them are events of thrombocytopenia, which have recently been evident in late stage trials with ON drugs. The underlying mechanisms are poorly understood and the risk for ON candidates causing such events cannot be sufficiently assessed pre-clinically. We investigated potential thrombocytopenia risk factors of ONs and implemented a set of in vitro assays to assess these risks. Our findings support previous observations that phosphorothioate (PS)-ONs can bind to platelet proteins such as platelet collagen receptor glycoprotein VI (GPVI) and activate human platelets in vitro to various extents. We also show that these PS-ONs can bind to platelet factor 4 (PF4). Binding to platelet proteins and subsequent activation correlates with ON length and connected to this, the number of PS in the backbone of the molecule. Moreover, we demonstrate that locked nucleic acid (LNA) ribosyl modifications in the wings of the PS-ONs strongly suppress binding to GPVI and PF4, paralleled by markedly reduced platelet activation. In addition, we provide evidence that PS-ONs do not directly affect hematopoietic cell differentiation in culture but at higher concentrations show a pro-inflammatory potential, which might contribute to platelet activation. Overall, our data confirm that certain molecular attributes of ONs are associated with a higher risk for thrombocytopenia. We propose that applying the in vitro assays discussed here during the lead optimization phase may aid in deprioritizing ONs with a potential to induce thrombocytopenia

Apoptosis induced by SSOs in primary mouse hepatocytes.

<p>Changes in caspase-3/7 activation in primary mouse hepatocytes after 24 and 48 h of incubation with the respective SSO at 30 μM. Data are normalized to vehicle treated cells and are means ± SD.</p

Establishment of a Predictive <i>In Vitro</i> Assay for Assessment of the Hepatotoxic Potential of Oligonucleotide Drugs

<div><p>Single stranded oligonucleotides (SSO) represent a novel therapeutic modality that opens new space to address previously undruggable targets. In spite of their proven efficacy, the development of promising SSO drug candidates has been limited by reported cases of SSO-associated hepatotoxicity. The mechanisms of SSO induced liver toxicity are poorly understood, and up to now no preclinical <i>in vitro</i> model has been established that allows prediction of the hepatotoxicity risk of a given SSO. Therefore, preclinical assessment of hepatic liability currently relies on rodent studies that require large cohorts of animals and lengthy protocols. Here, we describe the establishment and validation of an <i>in vitro</i> assay using primary hepatocytes that recapitulates the hepatotoxic profile of SSOs previously observed in rodents. <i>In vitro</i> cytotoxicity upon unassisted delivery was measured as an increase in extracellular lactate dehydrogenase (LDH) levels and concomitant reduction in intracellular glutathione and ATP levels after 3 days of treatment. Furthermore, toxic, but not safe, SSOs led to an increase in miR-122 in cell culture supernatants after 2 days of exposure, revealing the potential use of miR122 as a selective translational biomarker for detection of SSO-induced hepatotoxicity. Overall, we have developed and validated for the first time a robust <i>in vitro</i> screening assay for SSO liver safety profiling which allows rapid prioritization of candidate molecules early on in development.</p></div

Selection of tool SSOs with documented <i>in vivo</i> hepatotoxicity (ALT levels after 2 week treatment in mice with 5 x 15 mg/kg tail vein injection) that were used for the validation of <i>in vitro</i> hepatotoxicity assays.

<p>Selection of tool SSOs with documented <i>in vivo</i> hepatotoxicity (ALT levels after 2 week treatment in mice with 5 x 15 mg/kg tail vein injection) that were used for the validation of <i>in vitro</i> hepatotoxicity assays.</p

SSO induced target knock down in primary mouse hepatocytes.

<p>Normalized expression of myd88 mRNA after 48 hour treatment of mouse hepatocytes with 30 μM of the respective oligonucleotide. Data are means ± SD.</p

miR-122 release.

<p>(A) Mouse hepatocyte-NPC co-cultures or (B) hepatocyte monocultures were treated with the respective SSOs for 48 or 64 hours and cell-free supernatant was collected. The levels of miR-122 at 48 h and 64 h were assessed using real-time qPCR. miRNA levels were normalized to vehicle treated cells. Data are means ± SD.</p

SSO induced toxicity in primary mouse hepatocytes.

<p>Secreted (A) LDH and (B) albumin levels and intracellular (C) GSH and (D) ATP concentrations after 3 day treatment with a tool set of hepatotoxic and non-hepatotoxic SSOs. Data are means ± SD.</p