Mass production of stem cell derived human hepatocytes for experimental medicine

KEYWORDS:

• Pluripotent stem cell
• human embryonic stem cell
• human induced pluripotent stem cell
• hepatocyte like cell
• liver

Liver disease is a significant threat to public health, affecting millions of people worldwide. Liver transplantation is the only effective treatment option for end-stage liver disease [1]. 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 hepatocyte [2]. 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 transplantation [3].

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 stage, have the potential to differentiate into hepatocytes and cholangiocytes [4–7]. Recently, bipotent ductal cells have been isolated from liver biopsy and expanded as organoids, forming functional cell types in vitro and in vivo [8]. Current studies are addressing how these technologies can be defined, scaled, and applied to different liver disease scenarios. 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 on biopsy or organ donation [9,10].

Human PSCs (hPSCs) includes embryonic stem cells (ESCs) and 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 ESC (hESC) lines were established in 1998 by Thomson et al. from grade B embryos that were unsuitable for human implantation [11]. hESC culture is now routine throughout the world. More recently, it has become possible to derive hESC lines from single cells, biopsied from eight-cell stage embryos without destroying the embryo [12]. In 2007, Takahashi et al. generated human iPSCs (hiPSCs) using four transcription factors: Krϋppel-like factor 4 (KLF4), octamer binding protein 4 (OCT4), sex determining region Y (SRY)-box 2 (SOX2), and cellular myelocytomatosis oncogene (c-MYC), often 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 methodologies [14]. hiPSCs resemble hESCs in many ways and bypass some of the ethical issues that surround hESCs. In addition, the ability to reprogram somatic cells to an embryonic state allows for donor matching and tailor-making of cell-based therapies and in vitro models [15].

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 lines [16]. 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 laminins [12], for expansion, characterization, and banking [17]. Following this, banked hESC lines are subjected to identity checks, using short tandem repeat DNA fingerprinting and human leukocyte antigen classes I and II profiling. To date, approximately 50 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 hiPSC 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 antigen [19]. 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 hiPSC lines have also been created from human foreskin fibroblast cells using the integration-free RNA-based Sendai virus expressing the four Yamanaka factors [20]. 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 extracellular matrices, to induce hepatic specification and scale-up. HLCs exhibit similarities to primary hepatocyte morphology and gene expression and perform multiple hepatocyte functions [9,10,21–23]; however, there is room for improvement. Encouragingly, HLCs have been shown to model human liver diseases ‘in a dish’ [24,25] and to predict and modulate drug toxicity [22,26–28]. 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-based donor targeting vector to correct a specific point mutation in the A1AT gene [29]. 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. Choi et al. screened over 3000 compounds, identifying five promising candidates [26]. Significant progress has also been made in drug safety testing using 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 industry [22]. 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 stabilize somatic phenotype (for reviews, see [30,31]). An important part of this process has been the definition of the differentiation process [32–34] 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 hESC lines. This procedure was efficient and delivered populations of polarized HLCs with improved and more stable phenotype, when compared to an undefined extracellular matrix, Matrigel^TM [32].

An essential part of the journey to the clinic is the preclinical modeling of potential therapies. To model the therapeutic potential of HLCs, in vivo studies have been conducted [35–37]. While the majority of studies provide proof of concept, PSC-derived HLCs generally colonize and repopulate rodent livers at reduced levels when compared to their adult hepatocyte counterparts. 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 liver [38]. Another interesting study co-cultured hiPSC-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 failure [39]. 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 hydrogel [40]. While these strategies are 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 generate efficient levels of functional HLCs. Stem-cell-derived HLCs which display many liver cell traits and are useful for modeling human disease ‘in a dish’. While these data have generated much excitement, there is room for improving HLC phenotype and stability. This is not only an issue with stem-cell-derived cell types, but is also observed in primary cells following tissue processing. The loss of cell identity in cell culture points to serious deficiencies with in vitro cell niche [41] . The supportive nature of the niche is the focus of current investigations.

Financial & competing interests disclosure

Y Wang has received financial support from the China Scholarship Council. D C Hay has received financial support from UKRMP (grants numbers MR/L022974/1 and MR/K026666/1). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.