Have you ever wondered how DefiniGEN’s proprietary OptiDIFF technology gives them a competitive advantage in rare monogenic liver disease modelling? In this interview Olly Cons speaks with lead scientist Dr Nikolaos Nikolaou to discover the benefits of OptiDIFF.



Introduce us to the OptiDIFF technology and how you are involved


The technology's name, OptiDIFF, stems from its core objective: to optimize differentiation and faithfully replicate primary human hepatocyte cells. I joined DefiniGEN as a Senior Scientist in May of 2022 progressing to Team Lead scientist in October of 2022. I focus on the project and line management of DefiniGEN’s OptiDIFF process of differentiating human Induced Pluripotent Stem Cells (hiPSCs) to Hepatocyte Like Cells (HLCs). After obtaining these HLCs, we employ them for drug screening purposes, using both diseased and Wild-Type HLCs. We have the capability to generate diseased HLCs through two avenues. Firstly, by collecting somatic cells from a diseased patient, maintaining their genetic defect through regeneration to iPSCs, and then utilizing the OptiDIFF process. Secondly, for rarer mutations, we employ gene editing tools like CRISPR/Cas9 to introduce the specific mutations into iPSCs, generating the diseased cell lines.


Long before my joining and prior to 2010, the successful differentiation of human hiPSCs into HLCs had remained elusive until Prof. Vallier our Chief Scientific Officer achieved a breakthrough. He coined the process “OptiDIFF”. The development of HLCs from iPSCs poses a well-recognized challenge in the scientific community. Existing research on this topic is scarce, with only DefiniGEN’s study highlighting an approach.


The name OptiDIFF now groups together the multiple protocols DefiniGEN has achieved and can be employed when differentiating hiPSC lines to HLCs. We found a need for multiple protocols to give a more robust, reliable, and reproducible hiPSC line. My role is to find the optimal OptiDIFF approach for each client to achieve the most mature and functional HLCs possible.


Why was differentiating to Hepatocyte Like Cells a challenge?


Having joined DefiniGEN after the successful establishment of a protocol for the differentiation of hiPSCs to HLCs, I am aware that the complexity primarily stems from the multi-step nature of differentiation coupled with the highly specialized functions of hepatocytes.


Successful replication of the intricate functions of hepatocytes in vitro demands precise mimicry of signalling cues at specific stages during the differentiation process. Prof Vallier was able to create a protocol through the replication of the natural developmental cues that differentiating cells undergo during embryonic development to successfully promote differentiation into hepatic cells.



Nik Nikolaou Scientific Team Leader DefiniGEN


Image: Dr. Nikolaos Nikolaou


Why OptiDIFF over animal models for liver disease modeling?


I think the current pre-clinical methods employing animal models have limited success in replicating liver diseases, primarily because of species-specific differences. These methods are both time-consuming and ethically challenging, as nurturing in vivo murine and primate models to develop the desired disease conditions requires significant time and effort. Furthermore, the utilization of animal models to simulate liver disease phenotypes frequently leads to limited success. In certain instances, the animals may only display mild symptoms, or the genetic knockout may not produce any noticeable effect at all. This outcome can be attributed to the activation of additional pathways, compromising the intended impact of the knockout.

When we use animal models in industry it is crucial to confirm the drug's efficacy at the cellular level before progressing to animal models. Initial screening of the isolated protein often involves using highly proliferative cell lines like Chinese Hamster Ovarian (CHO) or Human Embryonic Kidney (HEK) cells. In these cell lines, the liver proteins of interest are overexpressed, and then the protein of interest is isolated and purified for studying the drug's interaction. This approach allows us to gain insights into the drug's potential effects on the specific liver proteins before moving forward with more complex in vivo studies.


Our OptiDIFF technology offers this but at an advanced cellular level. The animal modeling can now be skipped with the recent approval through the FDA 2.0 Modernization Act. We create a more reliable approach over animal models as we can mimic the human hepatocyte cells with our HLCs reducing the uncertainty that comes with the species-specific differences. In addition to significantly shortening the timeline compared to animal models, this approach not only bypasses certain stages but also produces a more dependable disease model that closely mimics human hepatocyte cells. This improved accuracy enhances the relevance and reliability of the research findings, making it a promising alternative to animal-based studies.


Can you compare Primary Human Hepatocytes with the HLCs you generate?


In liver disease modeling primary human hepatocyte cells are often considered the gold standard when creating a model for drug screening. These cells are isolated directly from the liver of a human with the desired rare monogenic liver disease being studied. They therefore create an accurate representation of the disease and the efficacy of the drug being tested on for that specific patient.


However, human populations exhibit great genetic and phenotypic diversity, which affects how individuals respond to diseases and drugs. Primary human hepatocytes from multiple donors allows research to study this variability. However, due to the rarity of the monogenic liver diseases, it is often not possible to find enough patients to create reliability with the results and not just create a personalized medicine. The inter-patient variability in protein expression levels and the activation of specific pathways relevant to liver diseases pose significant challenges for liver disease researchers. Additionally, the limited shelf life of primary human hepatocyte cells, which typically retain their hepatic phenotype for only about 5 days before rapidly losing it, further complicates their use as a reliable model for studying liver diseases. All our hiPSC lines have been optimized over the years to yield expression of the proteins being studied and once our hiPSC lines have been differentiated out using OptiDIFF retaining their hepatocyte like phenotype for up to a month.


Why did you not generate cell lines from HepG2 cells?


I think HepG2 cells have their purpose in the liver disease modeling field however being derived from the liver tumor of a 15-yearold Caucasian male with hepatocellular carcinoma. Despite being a cancer cell line, HepG2 cells exhibit certain hepatocyte-like characteristics. An advantage HepG2 cells have is their capability to be indefinitely proliferated out allowing for large data sets to be created of the drug being screened.


However due to their cancerous nature the HepG2 carry cancerous characteristics that can completely compromise the data we are interested in, and the oncogene phenotype limits the physiological relevance of the cells. The singular donor means the HepG2 cells only retain aspects of hepatocyte function with some hepatoma lines lacking many of the key proteins essential for modeling liver disease. The OptiDIFF generated HLCs like that of HepG2 cells can proliferate indefinitely also creating large data sets but maintain greater primary hepatocyte function without compromise.


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