oa Derivation of Hepatocyte Like Cells from Human Pluripotent Stem Cells in GMP Compliant Conditions

The liver is the largest internal organ in the human body and performs a broad range of functions. Hepatocytes are the primary cells of liver. They perform many functions, an endocrine function, secretion of most proteins found in the blood plasma including albumin, exocrine functions and secretion of bile into the digestive tract. They are also involved in carbohydrate and lipid metabolism, drug detoxification and storage of glycogen. In addition, the liver has a unique capability of regeneration as the liver can fully regenerate itself following severe damage which leaves one-third of the cells uninjured (Michalopoulos, 1997; Taub, 2004). Liver disease and progressive loss of liver function is a major clinical challenge. Liver disease is the fifth highest cause of early mortality in the UK and the only cause of death that is increasing every year (Office for National Statics., 2008). With changes in lifestyle, new diseases such-as non-alcoholic fatty liver disease and steatohepatitis, a lack of hepatitis C vaccination and risk of hepatocellular carcinoma in hepatitis patients with increasing age are further compounding the problem of liver disease in the UK (Bosch et al., 2004; Kim et al., 2002) Currently liver transplantation is the only treatment available for end stage liver disease and a few liver based metabolic defects (Sze et al., 2009). Split liver transplantations is available and makes more transplants possible but ultimately is not sufficient to fulfill the growing need. Cell-based therapy involving the transplantation of primary hepatocytes could greatly expand the number of patients provided with effective treatment. However, primary hepatocytes are difficult to obtain in sufficient number. Indeed, they can only be obtained from organs suitable for transplantation as they cannot be expanded in vitro. These limitations can be overcome by using human Embryonic stem cells (hESCs) or induced pluripotent stem cells to differentiate into hepatocytes. Stem cells have great potential because of their self-renewable capacity differentiation potential including differentiation into hepatocytes (Hannan et al., 2013; Si-Tayeb et al., 2010; Sullivan et al., 2010). In our lab Hannan et al. have previously published an efficient and robust protocol to differentiate human stem cells to hepatocyte-like cells in a stepwise manner using defined factors. Human pluripotent stem cells (hPSCs) hold several clinical promises including the potential to produce large quantity of cells necessary for cell based therapy approaches. Hence, hPSCs provide an advantageous alternative since they can be differentiated into hepatocyte-like cells which display functional characteristics of their in vivo counterparts. However, there are two major limitations that must be overcome before this approach can go to clinical trials. Firstly current protocols are not GMP (Good Manufacturing Practice) grade. Secondly, hepatocytes-like cells derived in-vitro lack a several characteristics of adult hepatocytes and express fetal markers indicating that they are not fully functional. Here, I aim to develop a method to generate hepatocyte-like cells from hPSCs in conditions compatible with clinical applications. For that, I have developed a culture system to generate hepatocyte-like cells using culture conditions compatible with GMP. The resulting cells display characteristics of their in vivo counterpart and represent a first step toward the development of cell based therapy for liver diseases. In my study, as far as we know I am the first to have translated differentiation of hepatocytes-like cells into GMP-compliant conditions. Here, I report a method to translate our previously published efficient and promising step-wise hPSCs directed research grade differentiation protocol for the generation of hepatocytes-like cells under GMP-compliance conditions, demonstrating that this can be attained and hepatocytes-like cells can be produced at a standard suitable for clinical trail purposes. In general, the employment of cell-based products in clinical studies needs formal approval from the respective regulatory body (British Standards Institution, London, UK). According to the current national regulations this approval requires manufacturing, processing and testing of cellular products (Bailey et al, 2014). The manufacturing of cell product requires use of safe and pure components and materials in order to fulfil the regulatory standards. It is essential to use stable and clinical grade hESCs cell lines that first generated and preserved in GMP banks which provided effective, safe cell source as starting material. If possible, licensed and GMP-grade materials must be used, in case of no alternatives, highly purified xeno-free or animal-free research grade reagent should be used, with additional in-house testing to ensure safety and quality. The translated protocol fulfills all the above criteria. Depending on the availability in the market the translated protocol has GMP compliant materials wherever possible. When no GMP grade material was available I used preclinical, xeno-free reagents from reputed suppliers. I tested 9 cell lines: two control cell lines derived in research grade (H9 and BBHX8) and 8 fully GMP grade (mShef3, mShef4, mShef7, mShef8, mShef10, mShef11, Val9). The matrixes used for maintenance and differentiation and hESC lines are fully GMP grade. The growth factors and supplements used are GMP grade apart from OncostatinM and HGF, which are soon-to-be GMP grade as promised by the manufacturer and are currently under preclinical version. The media used are GMP grade apart from hepatozyme, which is a serum free medium. Small molecules used are all GMP grade apart from LY294002, which is highly purified HPLC grade. In Addition, freezing medium, reconstitution reagents, splitting reagents, PBS, water etc. are all GMP grade. Both GMP grade and RG HPSCs cell lines maintained their pluripotency in GMP maintenance conditions and were karyotypically normal when karyotyped every 10 passages for 40 passages. Expression of all pluripotent markers was observed. These finding showed that the GMP maintenance conditions are robust, effective and good enough that cell grown in RG condition can also be transferred and maintained. I was able to generate hepatocytes-like cells expressing hepatic markers from mshef3, mShef7, mShef11, H9 and BBHX8. When compared to H9 GMP compliant derived hepatocytes-like cells these differentiated cell lines showed similar protein and gene expression, mShef3 being an exceptionally good cell line having 10 fold increase in Cyp3A4 and tyrosine aminotransferase (TAT). When analysed for the functionality mSef7, BBHX8 showed similar level of Cyp3A4 activity while mShef3 was 7 folds higher expressed. Here, I report successful translation of HPSCs differentiation protocol in hepatocytes-like cells into GMP-compliant conditions by using HPSCs derived in a fully GMP manner and changing into defined feeder and serum free differentiation media and supplements to GMP- compliant conditions.