Characterization and Function of iPSC derived Hepatocytes for Use in Toxicity

CellTiter-Glo®

www.cellulardynamics.com Madison, WI USA (608) 310-5100

Target Identification

Target Validation

Compound Screening

Lead Optimization

Preclinical Trials

Clinical Trials

Shannon Einhorn, Giorgia Salvagiotto Jen Luebke-Wheeler, Kristin Block, David Mann

Cellular Dynamics International, Inc., Madison, WI USA

Hepatoxicity is a leading cause of drug withdrawal from the market, highlighting the fact that current preclinical models of toxicity are not universally predictive of drug effects in humans. The manifestations of hepatotoxicity are highly variable and range from intrinsic toxic effects to the enzymatic production of toxic metabolites. Development of a more predictive in vitro model system that can elucidate the underlying mechanisms of hepatotoxicity early in the drug development process is critical to subverting unanticipated drug failure in the clinic. Cellular Dynamics International (CDI) has developed human induced pluripotent stem cell (iPSC)-derived hepatocytes, iCell® Hepatocytes, that exhibit high purity (>95%) and biologically relevant functions necessary for hepatotoxicity studies. To demonstrate the functional utility of iCell Hepatocytes in elucidating mechanisms of drug-induced hepatotoxicity, a variety of functional endpoints were measured in response to known hepatotoxic compounds. Responses in iCell Hepatocytes were observed for all compounds at EC50s that were comparable to those seen with primary human hepatocytes. Mechanism-based toxicity was evaluated by analyzing cell metabolism, oxidative stress, and lysosomal phospholipase activity using multiple platforms, including luminescence-based assays (Promega), High Content Analysis (ImageXpress, Molecular Devices), and the XF96 Extracellular Flux Analyzer (Seahorse Biosciences). This multiple platform approach for functional analyses demonstrated how iCell Hepatocytes provide a biologically relevant human model system for studying drug-induced hepatotoxicity.

Abstract

CDI’s iCell Hepatocytes are a highly pure and readily available model system that can be cultured on a variety of platforms for effective use in human hepatotoxicity assays. These physiologically relevant cells exhibit functional mechanistic toxicity as shown through accumulation of phospholipids (steatosis), production of ROS resulting in oxidative stress, cell based assays showing modes of cytoxicity, and P450 mediated hepatotoxicity. iCell Hepatocytes offer a reliable and versatile way to better understand and prevent drug-induced liver injury and thereby serve to improve therapeutic development.

Summary

Platform Agonistic Mechanistic Toxicity

One advantage to screening with iCell tissue cells is that they recapitulate native biology. CDI is enabling the drug discovery community by developing assay protocols that show mechanistic toxicity in a variety of in vitro assays. Below are examples of iCell Hepatocytes treated with a set of known hepatotoxic compounds employing cells based assays to show the breakdown of cellular function leading to a toxic readout. Utilizing iCell Hepatocytes in cell based assays offers a quick and easy way to assess toxicity for compound evaluation in the drug discovery pipeline.

Drug-induced production of ROS by human hepatocytes has been shown to lead to apoptosis and necrosis. iCell Hepatocytes assayed for ROS levels offers a versatile and easy way to look at cellular and biochemical changes caused by oxidative stress. The data above highlight the functional and metabolic differences between iCell Hepatocytes and other cell types often used in early screening when treated with Menadione, highlighting the importance of physiologically relevant model systems.

XF Seahorse Flux Analysis- Oxidative Stress

Compatibility of the iCell Hepatocytes with a variety of assay kits and instrument platforms is an important factor enabling implementation in multiple applications. The example above illustrates drug-induced phospholipidosis induction upon exposure to Amiodarone in the iCell Hepatocytes as measured by the ImageXpress Micro from Molecular Devices (Sunnyvale, CA). This High Content Screening application represents an efficient assay to examine multiple compounds in miniaturized formats.

Normal hepatic functions and the extensive oxidative metabolism in hepatocytes rely on mitochondrial activity. Mitochondrial dysfunctions have been implicated in several liver diseases as well as in drug-induced hepatotoxicity, a leading cause of late stage drug withdrawal from the market. By analyzing iCell Hepatocytes on instrumentation, such as the XF Extracellular Flux Analyzer from Seahorse Biosciences (Billerica, MA), we can gain a better understanding of the mitochondrial processes by which cells produce and consume energy and how molecules can perturb these functions The data on the right illustrate the oxidative stress response of iCell Hepatocytes treated with Acetaminophen (APAP). The ability to measure and manipulate the oxygen consumption rate (OCR) offers early detection of hepatotoxic substances along with mechanistic insight. Generating similar data across patient-derived cell lines could provide insight into how genetic differences influence metabolic diseases and drug metabolism.

Cytotoxicity was measured after 48 hours exposure to the compounds listed utilizing Promega Glo assays. Data shows changes in cellular ATP levels, caspase activity, and membrane integrity that indicate induction of apoptosis and/or cell death.

High Content Imaging - Phospholipidosis

Analysis of iCell Hepatocytes was performed after 3 days in culture. The above data shows the oxygen consumption rate (OCR) after a 4hr treatment with APAP. 0.4 µM FCCP was injected for all conditions (A) to illustrate the maximum respiratory capacity for the given treatment conditions. 2.0 µM Rotenone was injected in the vehicle control (D) to show inhibition of the electron transport chain as compared to cells treated with APAP.

Cells were cultured on an 96 well plates for 24 hours prior to treatment with Aflatoxin and Menadione. Ratio of reduced to oxidized gluthanione (GSH/GSSG) was measured after 48 hr treatment with the compound.

(A) % Viability was measured after 24 hr incubation with Aflatoxin-B1 on HepG2, primary human hepatocytes, and iCell Hepatocytes. Metabolism mediated toxicity is shown on primary human hepatocytes and iCell Hepatocytes. No toxicity seen with HepG2 cells (B) Aflatoxin-B1 induced hepatotoxicity is inhibited by ketoconazole and alpha-naphthoflavone, inhibitors of P450 enzymes, after 24 hr incubation (C) A dose dependent decrease in OCR is observed after 4 hr treatment with Isoniazid (INH) on the iCell Hepatocytes as measured by the XF Seahorse Flux Analyzer.

Cytrochrome P450 (CYP) activity is at the center of understanding xenobiotic metabolism and elimination of foreign chemicals from the body. Often hepatotoxicity is the result of toxic intermediates produced through oxidation of compounds and is mediated by CYP enzymes. In the examples below, iCell Hepatocytes were treated with the compounds Aflatoxin and Isoniazid (INH) demonstrating functional p450 activity through reactive metabolite mediated toxicity, thus demonstrating the use of iCell Hepatocytes in understanding the mechanisms underlying drug sensitivity to enable the development of safer therapies.

iCell Hepatocytes were cultured in 96 well plates for 48hrs before treatment with Amiodarone. Analysis of total ATP and accumulation of phospholipids was completed 24 hours post treatment. (A) Cell Titer Glo® (Promega) shows decreased ATP production EC50=18.9 µM. (B) Fluorescent images with LipidTox Red™ staining (Life Technologies) shows increased accumulation of phospholipids EC50=18.3 µM.

Somatic cells from adult tissue are obtained via non-invasive methods and used to generate human iPSCs. Using a variety of techniques, cells are first reprogrammed to a pluripotent state, and then banked, characterized, and expanded in culture indefinitely. From the iPSC bank, terminal cell types representing mesoderm, endoderm, and ectoderm can be derived.

Shown below are cell morphology and protein markers demonstrating relevant functions native to primary human hepatocytes. iCell Hepatocytes demonstrate a cobblestone morphology with the formation of bile cananiculi for pericellular transport. They also produce albumin at similar levels as primary human hepatocytes and show intrinsic metabolic function through the production and storage of glycogen for energy and lipid production.

Physiologically Relevant

Log [Compound] M

4. PURIFY* hepatocytes using proprietary technology

3. DIFFERENTIATE iPS cells into hepatocytes

2. EXPAND iPS cell line -> 1 billion cells per day

1. CREATE iPS cell line from a single individual

Lineage-specific promoter

Antibiotic resistance gene

*proprietary CDI technology

Rat

io G

SH/G

SSG

Aflatoxin (μM) Menadione (μM)

Rat

io G

SH/G

SSG

PHH iCell Hepatocytes

P450 Mediated Reactive Metabolite Generation

Multiple Cytotoxicity Readouts

Cell Based Assays - Oxidative Stress

30 µM Amiodarone Vehicle Control

A. B.

A. B. C.

Vehicle Control

25 µM APAP

1 mM APAP

25 mM APAP

Background Corr.

Data Courtesy of Promega

Data Courtesy of Promega

Data Courtesy of Promega