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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 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 laserassisted 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-cellbank; stemcells.nih.gov) [18]. Several GMP-grade hiPSC lines have also been generated. Recently, Baghbaderani et al. reprogrammed cord-bloodderived 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 posttransfection, 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. GMPgrade 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.