Mass Production of Stem Cell Derived Human Hepatocytes for Experimental Medicine

Liver disease is a significant threat to public health, affecting millions of people worldwide. Liver transplantation is the only treatment option for end stage liver disease1. However, donor organs are in short-supply, and therefore alternative approaches are being explored. This includes the transplant of the major metabolic cell type of the liver, the hepatocyte2. Hepatocyte transplantation offers an alternative to whole organ transplantation, however this approach has significant limitations, including a source of good quality hepatocytes, and therefore suffers from the same limitation as whole organ transplantation3.

In the quest for renewable sources of human hepatocytes, researchers have focused their attention on fetal and adult stem cells, as well as pluripotent stem cells (PSCs). Resident hepatoblasts and hepatic progenitor cells, during development and in the adult, have the potential to differentiate into hepatocytes and cholangiocytes4-7. Recently, bipotent ductal cells have been isolated from liver biopsy and expanded as organoids, forming functional cell types in vitro and in vivo8. Current studies are addressing how these technologies can be defined, fabricated at scale and validated in pre-clinical animal models before their application. PSCs represent another promising cell type. Notably, PSCs can be expanded and differentiated into the desired somatic cell type, representing a renewable cell type that is not dependent continual biopsy or organ donation9, 10.

Human PSCs (hPSCs) includes embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). Mammalian ESCs are derived from the inner cell mass (ICM) of blastocysts, while iPSCs are generated by reprogramming different somatic cell lineages. The first human ESCs (hESCs) lines were established in 1998 by Thomson et al from grade B embryos that were unsuitable for human implantation11. hESC culture is now routine throughout around the world. More recently, it has become possible to derive hESCs lines from single cells, biopsied from eight-cell stage embryos without destroying the embryo12. In 2007, Takahashi et al generated human iPSCs (hiPSCs) using four transcription factors: Krϋppel-like factor 4 (KLF4), octamer binding protein 4 (OCT4), SRY (sex determining region Y)-box 2 (SOX2) and cellular myelocytomatosis oncogene (c-MYC), termed as the ‘Yamanaka factors’13. Following this breakthrough, the generation of hiPSCs is now commonplace and has been achieved in numerous somatic cell types employing multiple reprogramming methodologies14. hiPSCs resemble hESCs in many ways and bypass the some of the ethical issues that surround hESCs. In addition, the ability to reprogramme somatic cells to an embryonic state allows for donor matching and tailor-making of cell based therapies and in vitro models15.

To have clinical impact, stem cell products are required to meet Good Manufacturing Practice (GMP) requirements. Scientists have derived a number of clinical-grade hESC and hiPSC lines16. To derive hESC lines, surplus grade B embryos from in vitro fertilization clinics are used. ICMs are isolated from blastocyst stage embryos by manual excision or laser-assisted-dissection. Excised ICMs are replated onto human feeder cells or GMP-grade extracellular matrix, such as recombinant laminins12, for expansion, characterization and banking17. Following this, banked hESC lines are subjected to identity checks, using short tandem repeat DNA fingerprinting and human leukocyte antigen class I and II profiling. To date, approximately fifty GMP- grade hESC lines have been derived, characterized and banked by different organizations around the world (www.mrc.ac.uk/research/facilities/stem-cell-bank; stemcells.nih.gov)18.

Several GMP-grade hiPSCs lines have also been generated. Recently, Baghbaderani et al reprogrammed cord blood-derived CD34+ hematopoietic stem cells using non- integrative episomal plasmids, expressing OCT4, SOX2, KLF4, c-MYC, LIN28 and Simian Virus 40 Large T Antigen19. Two weeks post transfection, hiPSC colonies began to appear and were expanded as clonal populations. Stem cell self-renewal and pluripotency were achieved and maintained using a proprietary extra cellular matrix in combination with a defined culture medium. GMP-grade hiPSCs lines have also been created from human foreskin fibroblast cells using the integration-free RNA- based Sendai virus expressing the four Yamanaka factors20. Xeno-free medium and human fibroblast feeder layers were used to support reprogramming and the maintenance of pluripotency, with hiPSC colonies emerging at two weeks post- infection. These reported efforts have demonstrated that it is feasible to manufacture hiPSCs at GMP and may serve as the starting material for future cell based therapy products.

The next challenge that exists is the cost effective mass production of human somatic cells from PSCs. Over the last decade, a number of differentiation procedures have been developed to derive hepatocyte-like cells (HLCs) from hPSCs. Differentiation media supplemented with essential growth factors and small molecules have been employed in combination with different extra-cellular matrices, to induce hepatic specification and scale up. HLCs exhibit similarities to primary hepatocyte morphology, gene expression, and perform multiple hepatocyte functions9, 10, 21-23, however there is some room for improvement. HLCs have been shown to model human liver diseases ‘in a dish’24, 25 and to predict and modulate drug toxicity22, 26, 39, 40 . It has also been possible to use human genome editing to correct a mutated form of α1-antitrypsin (A1AT). As a proof of concept, Yusa et al employed zinc finger nucleases and the PiggyBac targeting vector to correct a specific point mutation in the A1AT gene27. Following gene correction, improved cell phenotype was observed in vitro and in vivo. A1AT deficient cells have also been used to screen for novel drugs. In these studies Choi et al screened more than 3000 compounds, identifying 5 promising candidates26. Significant progress has also been made in drug safety testing using stem cell derived HLCs. Szkolnicka et al developed a scalable and shippable HLC based model which performed on a par with cryoplateable human primary hepatocytes within the pharmaceutical industry22. These studies provide exciting examples of the promise that stem derived HLCs have to offer. However, to facilitate the routine deployment of HLCs in the lab and the clinic, it is necessary to further improve and stabilise somatic phenotype in the dish (for reviews see 38,41). An important part of this process has been the definition of the differentiation process 28-30 with the extracellular matrix essential to those endeavors. Recently, we employed full length human recombinant laminins (laminin 521 and a laminin 521/111 blend) to expand and differentiate research-grade and clinical-grade hESCs lines. This procedure was efficient and delivered populations of polarized HLCs with improved and more stable phenotype28.

An essential part of the journey to the clinic is the pre-clinical modelling of potential therapies. To model the therapeutic potential of HLCs, in vivo studies have been conducted31-33. While the majority of studies provide proof of concept, PSC derived HLCs generally colonize and repopulate rodent livers at reduced levels when compared to human adult hepatocytes. To improve cell engraftment in the liver a number of approaches have been taken. Recently, a cell sheet-based tissue engineering approach has been used to deliver HLCs to the liver34. Another interesting study co-cultured hiPSCs-derived hepatic endoderm with umbilical vein endothelial cells and mesenchymal stem cells to generate liver buds. Following mesenteric transplantation, the liver buds improved the survival of mice following gancyclovir-induced liver failure35. In addition to immunodeficient mouse models, a recent study has adopted an immunoisolation strategy, transplanting human HLCs into immunocompetent mice following co-aggregation with stromal cells and encapsulation in a biocompatible hydrogel36. While these strategies are extremely promising, longer term investigations are necessary to alleviate safety concerns that surround the tumorigenic potential PSC derived HLCs and other somatic cell derivatives.

In conclusion, current efforts in the field to generate efficient levels of functional HLCs are encouraging. Stem cell derived HLCs which display many liver cell traits and are useful for modelling human disease ‘in a dish’. While these data have generated much excitement, there is room for improvement. This is not unique to stem cell derived HLCs, and is observed in primary hepatocytes following tissue processing. The loss of hepatocellular identity in cell culture points to serious deficiencies with in vitro cell niche37 and this is currently under investigation. References 1. Nicolas CT, Wang Y, Nyberg SL. Cell therapy in chronic liver disease. Curr Opin Gastroenterol 2016. 2. Fox IJ, Chowdhury JR, Kaufman SS, et al. Treatment of the Crigler-Najjar syndrome type I with hepatocyte transplantation. N Engl J Med 1998;338:1422-6. 3. Forbes SJ, Gupta S, Dhawan A. Cell therapy for liver disease: From liver transplantation to cell factory. J Hepatol 2015;62:S157-69. 4. 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