Hepatocytes Derived from NASH iPSC Donors Provide a Valuable Platform for Disease Modeling and Drug DiscoveryIgor Gurevich, Sarah Burton, Christie Munn, Makiko Ohshima, Madelyn Goedland, Katherine Czysz, Deepika RajeshFUJIFILM Cellular Dynamics, Inc., Madison, WI USA

IntroductionNon-alcoholic fatty liver disease (NAFLD) affects 30 to 40% of adults and about 10% of children in the US. About 20% of people with NAFLD develop non-alcoholic steatohepatitis (NASH), which may lead to cirrhosis and liver cancer, and is projected to overtake hepatitis C as the leading cause of liver transplantation in the near future. Human induced pluripotent stem cells (iPSC) from NASH patients are useful for generating a large number of hepatocytes thus enabling better modeling of the disease and leading to more precise identification of drug targets. We developed and tested a novel defined in vitro differentiation process to generate cryoperservable hepatocytes using an iPSC panel of NASH donors obtained from the California Institute for Regenerative Medicine iPSC repository (#CW10201 [01D1] and #CW10202 [02E1]) and an apparently healthy donor (01279). iPSCs were subjected to a two dimensional stepwise differentiation by growth factors and small molecules to generate definitive endoderm (DE), assessed by quantifying purity of CXCR4 and CD117 co-expressing cells by flow cytometry and decline of pluripotency markers by qPCR. The resulting DE was differentiated further to hepatoblasts and then to mature hepatocytes as 3D aggregate cultures. Functional assessment of end-stage hepatocytes was performed by flow cytometry for hepatocyte specific markers, inducibility of CYP3A4 activity by rifampicin, and measurement of lipid accumulation in response to fatty acid (FA) treatment.

Differentiation Protocol SummaryFigure 1: Schematic of hepatocyte differentiation process. Episomally-derived iPSCs from healthy and NASH donors are maintained in E8/Matrigel and acclimatized to hypoxic conditions. To initiate differentiation, iPSCs are expanded and preconditioned prior to starting definitive endoderm (DE) differentiation for 10 days. The purity of DE cultures is assessed and DE cells are transitioned to hepatoblasts (stage 1). At the end of this stage of differentiation the cells are detached to form aggregates and differentiated further to generate mature hepatocytes. Cells can be cryopreserved at indicated points during the differentiation process, and successfully differentiated to live end-stage hepatocytes.

Summary and ConclusionsWe developed and tested a novel defined process to generate pure end-stage hepatocytes from iPSCs derived from apparently healthy normal and NASH donors. During the differentiation process, the cells demonstrated stage specific expression of relevant markers mimicking hepatocyte differentiation in vivo. The end-stage cells also demonstrated hepatocyte specific morphology and development of bile canaliculi. Importantly, both fresh and cryopreserved DE or hepatoblasts (end of stage 1 cells) successfully differentiated to pure, mature, and functional hepatocytes. End-stage hepatocytes demonstrated increased CYP3A4 activity in response to rifampicin and lipid accumulation upon fatty acid (FA) treatment. Interestingly, hepatocytes derived from one of the NASH iPSCs demonstrated spontaneous lipid accumulation in the absence of FA, displaying a hallmark of NASH hepatocytes in vivo. Thus, cryopreserved hepatocytes generated by this protocol across multiple donors will provide critical cell source to facilitate the fundamental understanding of NAFLD/NASH biology and potential high throughput screening applications for preclinical evaluation of therapeutic targets.

CYP3A4 Induction

Figure 9: Biodipy (green) and DAPI (blue) staining of intracellular lipid accumulation in response to fatty acid (FA) treatment for 24h. Note the lipid accumulation in the 02E1 (NASH) line even in the absence of added FA (red outline).

Pluripotency Shutdowna b

Figure 2: (a) qPCR analysis for pluripotency genes POU5F1 and NANOG between iPSCs and definitive endoderm (DE). (b) Flow cytometry analysis for pluripotency marker TRA1-81 at the end of DE induction in lines from healthy (2.038) and NASH (01D1 and 02E1) donors.

DE Induction

Recovery from Cryopreservation

End of process purity of hepatocytes

Line CryopreservationPoint AAT+ ALB+

01279 (AHN) End of DE 96.8% 38.1%

01D1 (NASH) End of DE 89.2% 45.1%

02E1 (NASH) End of DE 97.0% 79.5%

01279 (AHN) End of Stage 1 99.4% 87.4%

01D1 (NASH) End of Stage 1 94.6% 80.3%

Figure 7: Flow cytometry analysis of hepatic specific markers AAT and albumin (ALB) in end-stage hepatocytes derived from cells cryopreserved at the end of DE or end of stage 1. In all cases, cryopreserved cells were thawed and placed in differentiation to generate end-stage hepatocytes.

Figure 8: Induction of CYP3A4 by a 72h rifampicin treatment in end-stage hepatocytes from apparently healthy normal (01279) and NASH (01D1 and 02E1) donors measured using P450-Glo™ CYP3A4 Assay System (Promega).

Lipidosis Induction

Figure 3: Flow cytometry analysis for DE markers CXCR4 (y-axis) and CD117 (x-axis) at the end of DE induction phase in lines from healthy (2.038) and NASH (01D1 and 02E1) donors.

Stage 2 Hepatic Marker Expression

Figure 4: Flow cytometry analysis for intracellular quantification of AAT and ASGPR1 expression at end of stage 2 of the differentiation process in in lines from healthy (2.038) and NASH (01D1 and 02E1) donors.

HNF4A Subtype Expression

Figure 5: qPCR analysis of HNF4A subtypes using probes specific for promoter 1 (P1) and promoter 2 (P2) transcripts in end of stage 2 hepatocytes compared to RNA from adult human liver.

Generation of Mature Hepatocytesa b

c

Figure 6: (a) Morphology of end-stage hepatocytes plated onto collagen plates exhibiting binucleated cells, a key feature of hepatocytes, highlighted in yellow. (b) CDFDA staining of end-stage hepatocytes with bile canaliculi visualized as green lines or puncta. (c) Flow cytometry analysis for quantification of intracellular AAT and albumin (ALB) expression in end-stage hepatocytes.

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