A considerable roadblock in the successful triaging of new therapeutics for liver and metabolic-related diseases is the reliability and effectiveness of pre-clinical disease models.
Methods employing animal models have limited success in replicating liver disease phenotypes, primarily because of species-specific differences, as the utilization of animal models to simulate liver disease frequently leads to limited success.
Primary hepatocytes represent the gold standard cell model to investigate liver functionality in vitro, however, when isolated from liver tissue, these cells maintain expression of key proteins for only a short period of time (<5 days), resulting in rapid cell de-differentiation characterised by loss of hepatic function and apoptosis.
Initial screening of therapeutics may utilize the highly proliferative Chinese Hamster Ovarian (CHO) or Human Embryonic Kidney 293 (HEK293) cell lines with the liver protein(s) of interest artificially expressed. These systems allow researchers to investigate the effect of a therapeutic on the target protein, however, the cellular environment bears little resemblance to that of the human hepatocyte, an impediment to reliable compound triaging.
Hepatocellular carcinoma-derived (hepatoma) cell lines, such as HepG2s, are less proliferative than CHO or HEK293 cells but remain readily expandable and retain aspects of hepatocyte function. However, hepatoma lines lack key proteins essential for disease modelling, demonstrate undesired cancerous characteristics, in addition to representing a single and karyotypically abnormal genotype.
An ideal model for the study of liver diseases would combine the expansion capacity and phenotypic stability of hepatoma cell lines with the functionality and healthy karyotype of primary hepatocytes. Human induced pluripotent stem cell (iPSC) -derived hepatocyte-like cells (HLCs) have the potential to fulfil this role.
In a recent webinar, hosted in association with Drug Discovery World, Professor Ludovic Vallier and Dr Chris Kirton discussed how iPSC-derived cells can be used to advance the efficiency and efficacy of disease modeling. The webinar covered topics including liver functionality, modeling disease phenotypes, therapeutic modalities, and delivery and efficacy.
In this blog post, we re-visit some of the questions asked in the webinar, including questions we did not manage to answer during the live event in the interest of time.
Question: Is there an advantage in using iPSC-derived hepatocytes over liver cell lines, like HepG2 cells?
iPSC-derived HLCs have numerous advantages over the HepG2 cell line. HepG2 cells have been used as a platform for many years and have massive implementations in the drug development pipeline. However, due to their cancerous background, they demonstrate an abnormal metabolic profile, in addition to lack of expression of key hepatic proteins. These limitations render this cell line a not-effective in vitro platform for modeling liver diseases. In contrast, iPSC-derived HLCs come from healthy donors, demonstrate functional liver metabolic pathways, and cell homogeneity. What’s more, we can generate iPSC-derived HLCs from a wide diversity of patients whereas HepG2 cells are derived from only one patient.
Question: What is the maturity and yield of your hepatocytes?
Our HLCs are able to perform most of the functions attributed to mature hepatocytes, including albumin secretion, alpha-1-antitrypsin expression, urea formation, and cytochrome P450 enzyme expression. In addition, they can accumulate glycogen and show inducible cytochrome 450 activity. The yield can vary depending on the cell line, but we can generate batches of hepatocytes ranging from hundreds of millions up to billions of cells.
Question: How can we achieve as mature as possible hepatic phenotypes in iPSC-derived hepatocytes and how advanced are the current protocols compared to 10 years ago?
The key is to be able to generate functional cells in vitro, especially when we start from human iPSCs. Those cells correspond to a very early stage of development, therefore, the only way to produce functional cells from this early stage of development in vitro is by trying to mimic as close as possible the natural development and those key stages of development.
It has been a challenge to develop fully mature liver cell models, as we do not have a great understanding of the liver development, especially during phases of maturation that are crucial in producing fully mature functional cells. Of course, we have made great progress on this question in the last 10-15 years, many driven by the need to produce those functional cells in vitro. We have generated a better basic understanding of liver development that have been transferred to in vitro application. As a result of our continuous protocol optimization, we are now getting cells that are as functional as they can be in the context of in vitro production, displaying all the key functions we require to perform a very detailed and precise disease modeling, in addition to applications such as drug development and toxicology screening.
Question: Do iPSCs derived hepatocytes forms a kind of arrangement we see inside the liver?
iPSC-derived HLCs can form similar arrangements as observed inside the liver. In addition, they can demonstrate polarity when cultured using either transwell or sandwich approaches.
Question: Cell based models have high variants, do you see this in your models and how can it be reduced?
Reducing variability in cell-based models is a common question, however, there is no single answer. When working with complicated systems, such as cell-based bioassays, there is a need to tackle a variety of small things rather than one big element during the process development. For example, using the same clonal stocks of iPSCs, applying passaging consistently, using same pipettes and making sure these are properly calibrated, as well as aliquoting reagents into single-use aliquots - are standard laboratory approaches when aiming for consistency and reproducibility. In addition, giving project ownership to individual scientists can further reduce operator-to-operator variability. DefiniGEN are ISO certified, therefore, the above approaches form a part of our quality management systems, resulting in a 10-15% Coefficient of Variation (CV) in our bioassays, which is considered pretty tight for a cell-based bioassay.
Question: Which throughput scale of drug testing is the iPSC platform currently suitable for? Are there limitations on scale-up?
Several studies have been published demonstrating the ability of iPSC-derived HLCs to be used in drug screening at different scales. One of the main advantages of these cells – compared to primary human hepatocytes – is the virtually unlimited number of cells that can be generated due to the indefinite proliferative ability of iPSCs. Therefore, limitations in this case would only depend on other practical factors, such as availability and scale of automation systems.
Question: Is it better to use an iPSC line from a patient with a disease, or is it better to use a gene edited line?
In DefiniGEN, both approaches are routinely performed. By using our parent, wild-type iPSC lines, we can generate the desired disease cell model via CRISPR-based gene editing, in addition to using iPSCs derived from people with a specific disease, correcting the mutation via CRISPR-based gene editing, and generating their isogenic control. Whilst these two sources of iPSCs provide information on different aspects of a particular disease, our initial approach usually focuses on inserting the mutation-of-interest in our wild-type iPSC line, as it is a clean, well-characterised cell system and, therefore, the experiments are better controlled. In this regard, it has been observed that patient-derived iPSC lines often come with additional unknown mutations that scientists are not aware of, downstream compromising either hepatic or metabolic pathway functionality. An additional reason behind this choice is the challenges we face when accessing patient material. DefiniGEN have secured access in patient material for a variety of monogenic liver diseases, including alpha-1-antitrypsin, glycogen storage disease 1A, or Wolman’s disease.
Question: Why do some iPSC clones differentiate better towards hepatocytes than others and is there a way to predict this?
It is well known that variability amongst different iPSC clones may result in variability in differentiation towards different lineages, including HLCs, and the reason mainly lays in the genetic background of these clones. Whilst an iPSC is highly pluripotent and can differentiate towards every cell type observed in the human body, cells respond differently to growth factors. Acknowledging these limitations, DefiniGEN’s proprietary differentiation protocol has shown success in differentiating more than 70% of the iPSC clones we have so far worked with. In addition, we have developed the OptiDIFF platform, which of consists of >3 differentiation protocols that our scientists apply when using a new, previously uncharacterised iPSC clone. This gives us confidence that our system can cover a large proportion of the human population, leading to the successful generation of highly functional HLCs. Of note, DefiniGEN have now access to a big panel of approx. 300 iPSC wild-type clones, grouped based on their demographic characteristics (e.g., age, ethnicity, sex, etc.). These clones have been pre-screened for their ability to differentiate towards HLCs, providing us with a unique tool to generate liver disease models with different genetic backgrounds to investigate drug efficacy across the human population.
Question: Are there any ethical boundaries behind the use of iPSCs?
The development of human iPSCs by Yamanaka et al. in the early ‘00s aimed to overcome the ethical boundaries when using embryonic stem cells. Indeed, the origin of iPSCs (somatic human cells, e.g., skin, blood) is associated with no ethical issues, with the exception of the patient consent from whom these somatic cells have been obtained. In this regard, all DefiniGEN iPSC lines are consented for commercial use.
Question: How do you envision using HLCs as therapeutics for cell replacement?
There has been made significant progress in using iPSC-derived tissues for cell replacement, and the prospects for the application of iPSC-derived HLCs in clinical setting are impressive. However, safety issues around the tumorigenic potential of iPSCs require further optimization of the reprogramming and differentiation processes in addition to the potential immunogenicity challenges.
Question: What are the key markers that a scientist should look for when investigating hepatocyte functionality. Which one of these markers do the iPSC derived hepatocytes demonstrate?
The gold-standard hepatocyte maturity markers include albumin (ALB), hepatocyte nuclear factor 4 Alpha (HNF4A) and alpha-1-antitrypsin (A1AT). A1AT is probably the most used marker to characterize hepatocytes in vitro. Albumin secretion is the key marker when investigating functional activity and can be easily measured by ELISA. The presence of additional hepatic-specific metabolic pathways, including urea cycle and glycogenesis (glycogen accumulation) also indicate hepatocyte functionality in iPSC-derived HLCs. DefiniGEN HLCs demonstrate expression and presence of all the above markers and metabolic pathways, respectively.