Liver cell therapy: is this the end of the beginning?

The prevalence of liver diseases is increasing globally. Orthotopic liver transplantation is widely used to treat liver disease upon organ failure. The complexity of this procedure and finite numbers of healthy organ donors have prompted research into alternative therapeutic options to treat liver disease. This includes the transplantation of liver cells to promote regeneration. While successful, the routine supply of good quality human liver cells is limited. Therefore, renewable and scalable sources of these cells are sought. Liver progenitor and pluripotent stem cells offer potential cell sources that could be used clinically. This review discusses recent approaches in liver cell transplantation and requirements to improve the process, with the ultimate goal being efficient organ regeneration. We also discuss the potential off-target effects of cell-based therapies, and the advantages and drawbacks of current pre-clinical animal models used to study organ senescence, repopulation and regeneration.

3D human liver tissue from pluripotent stem cells displays stable phenotype in vitro and supports compromised liver function in vivo.

Liver disease is an escalating global health issue. While liver transplantation is an effective mode of therapy, patient mortality has increased due to the shortage of donor organs. Developing renewable sources of human liver tissue is therefore attractive. Pluripotent stem cell-derived liver tissue represents a potential alternative to cadaver derived hepatocytes and whole organ transplant. At present, two-dimensional differentiation procedures deliver tissue lacking certain functions and long-term stability. Efforts to overcome these limiting factors have led to the building of three-dimensional (3D) cellular aggregates. Although enabling for the field, their widespread application is limited due to their reliance on variable biological components. Our studies focused on the development of 3D liver tissue under defined conditions. In vitro generated 3D tissues exhibited stable phenotype for over 1 year in culture, providing an attractive resource for long-term in vitro studies. Moreover, 3D derived tissue provided critical liver support in two animal models, including immunocompetent recipients. Therefore, we believe that our study provides stable human tissue to better model liver biology 'in the dish', and in the future may permit the support of compromised liver function in humans.

Fluid shear stress modulation of hepatocyte-like cell function.

Freshly isolated human adult hepatocytes are considered to be the gold standard tool for in vitro studies. However, primary hepatocyte scarcity, cell cycle arrest and the rapid loss of cell phenotype limit their widespread deployment. Human embryonic stem cells and induced pluripotent stem cells provide renewable sources of hepatocyte-like cells (HLCs). Despite the use of various differentiation methodologies, HLCs like primary human hepatocytes exhibit unstable phenotype in culture. It has been shown that the functional capacity can be improved by adding back elements of human physiology, such as cell co-culture or through the use of natural and/or synthetic surfaces. In this study, the effect of fluid shear stress on HLC performance was investigated. We studied two important liver functions, cytochrome P450 drug metabolism and serum protein secretion, in static cultures and those exposed to fluid shear stress. Our study demonstrates that fluid shear stress improved Cyp1A2 activity by approximately fivefold. This was paralleled by an approximate ninefold increase in sensitivity to a drug, primarily metabolised by Cyp2D6. In addition to metabolic capacity, fluid shear stress also improved hepatocyte phenotype with an approximate fourfold reduction in the secretion of a foetal marker, alpha-fetoprotein. We believe these studies highlight the importance of introducing physiologic cues in cell-based models to improve somatic cell phenotype.

Generation of induced pluripotent stem cells (UCLi024-A) from a patient with argininosuccinate lyase deficiency carrying a homozygous c.437G > A (p.Arg146Gln) mutation.

Argininosuccinic aciduria (ASA) is a rare inherited metabolic disease caused by argininosuccinate lyase (ASL) deficiency. Patients with ASA present with hyperammonaemia due to an impaired urea cycle pathway in the liver, and systemic disease with epileptic encephalopathy, chronic liver disease, and arterial hypertension. A human induced pluripotent stem cell (iPSC) line from the fibroblasts of a patient with ASA with homozygous pathogenic c.437G > A mutation of hASL was generated. Characterization of the cell line demonstrated pluripotency, differentiation potential and normal karyotype. This cell line, called UCLi024-A, can be utilized for in vitro disease modelling of ASA, and design of novel therapeutics.

Report on 2nd Royan Institute International Summer School on developmental biology and stem cells Tehran, Iran, 17-22nd July 2011.

The 2nd Royan Institute International Summer School was built around the topic of stem cells and grounding in the discipline of developmental biology. The meeting provided not only direct transfer of technical and intellectual information, the normal process in scientific meetings, but was also a forum for the exchange of personal ideas of science as a creative pursuit. This summer school introduced aspiring young Iranian scientists to international researchers and exposed the latter to a rich culture that highly values learning and education, attested by the confident, intelligent young men and women who asked probing questions and who were eager to participate in the workshops. Hossein Baharvand's dedication and passion for science have led to an impressive record of national and international peer-reviewed publications and an increasing number of students who pursue science in Iran, and shows how the right people can create an environment where good science, good science education and motivation will flourish. This report summarizes some of the activities of the workshop in the Royan Institute and the impressions of the visiting scientists in the wider context of the scientific and cultural heritage of Iran.

New Developments and Challenges in Liver Transplantation.

Liver disease is increasing in incidence and is the third most common cause of premature death in the United Kingdom and fourth in the United States. Liver disease accounts for 2 million deaths globally each year. Three-quarters of patients with liver disease are diagnosed at a late stage, with liver transplantation as the only definitive treatment. Thomas E. Starzl performed the first human liver transplant 60 years ago. It has since become an established treatment for end-stage liver disease, both acute and chronic, including metabolic diseases and primary and, at present piloting, secondary liver cancer. Advances in surgical and anaesthetic techniques, refined indications and contra-indications to transplantation, improved donor selection, immunosuppression and prognostic scoring have allowed the outcomes of liver transplantation to improve year on year. However, there are many limitations to liver transplantation. This review describes the milestones that have occurred in the development of liver transplantation, the current limitations and the ongoing research aimed at overcoming these challenges.

Liver Ultrasound Histotripsy: Novel Analysis of the Histotripsy Site Cell Constituents with Implications for Histotripsy Application in Cell Transplantation and Cancer Therapy.

Introduction: Allogenic hepatocyte transplantation is an attractive alternative to whole-organ transplantation, particularly for the treatment of metabolic disorders and acute liver failure. However, the shortage of human donor organs for cell isolation, the low cell yield from decellularisation regimes, and low engraftment rates from portal administration of donor cells have restricted its clinical application. Using ultrasound histotripsy to provide a nidus in the liver for direct cell transplantation offers a new approach to overcoming key limitations in current cell therapy. We have analysed the liver cavity constituents to assess their potential as a site for cell delivery and implantation. Methods: Using human organ retrieval techniques, pig livers were collected from the abattoir and transported in ice-cold storage to the laboratory. Following 2 h of cold storage, the livers were flushed with organ preservation solution and placed on an organ perfusion circuit to maintain viability. Organs were perfused with Soltran™ organ preservation solution via the portal vein at a temperature of 24-30 °C. The perfusion circuit was oxygenated through equilibration with room air. Perfused livers (n=5) were subjected to ultrasound histotripsy, producing a total of 130 lesions. Lesions were generated by applying 50 pulses at 1 Hz pulse repetition frequency and 1% duty cycle using a single element 2 MHz bowl-shaped transducer (Sonic Concepts, H-148). Following histotripsy, a focal liver lesion was produced, which had a liquid centre. The fluid from each lesion was aspirated and cultured in medium (RPMI) at 37 °C in an incubator. Cell cultures were analysed at 1 and 7 days for cell viability and a live-dead assay was performed. The histotripsy sites were excised following aspiration and H&E staining was used to characterise the liver lesions. Cell morphology was determined by histology. Results: Histotripsy created a subcapsular lesion (~5 mm below the liver capsule; size ranging from 3 to 5 mm), which contained a suspension of cells. On average, 61×104 cells per mL were isolated. Hepatocytes were present in the aspirate, were viable at 24 h post isolation and remained viable in culture for up to 1 week, as determined by phalloidin/DAPI cell viability stains. Cultures up to 21 days revealed metabolically active live hepatocyte. Live-dead assays confirmed hepatocyte viability at 1 week (Day 1: 12% to Day 7: 45% live cells; p < 0.0001), which retained metabolic activity and morphology, confirmed on assay and microscopy. Cell Titre-GloTM showed a peak metabolic activity at 1 week (average luminescence 24.6 RLU; p < 0.0001) post-culture compared with the control (culture medium alone), reduced to 1/3 of peak level (7.85 RLU) by day 21. Conclusions: Histotripsy of the liver allows isolation and culture of hepatocytes with a high rate of viability after 1 week in culture. Reproducing these findings using human livers may lead to wide clinical applications in cell therapy.

Ultrasound Histotripsy on a Viable Perfused Whole Porcine Liver: Histological Aspects, Including 3D Reconstruction of the Histotripsy Site.

Non-invasive therapeutic-focused ultrasound (US) can be used for the mechanical dissociation of tissue and is described as histotripsy. We have performed US histotripsy in viable perfused ex vivo porcine livers as a step in the development of a novel approach to hepatocyte cell transplantation. The histotripsy nidus was created with a 2 MHz single-element focused US transducer, producing 50 pulses of 10 ms duration, with peak positive and negative pressure values of P+ = 77.7 MPa and P- = -13.7 MPaat focus, respectively, and a duty cycle of 1%. Here, we present the histological analysis, including 3D reconstruction of histotripsy sites. Five whole porcine livers were retrieved fresh from the abattoir using human transplant retrieval and cold static preservation techniques and were then perfused using an organ preservation circuit. Whilst under perfusion, histotripsy was performed to randomly selected sites on the live. Fifteen lesional sites were formalin-fixed and paraffin-embedded. Sections were stained with Haematoxylin and Eosin and picro-Sirius red, and they were also stained for reticulin. Additionally, two lesion sites were used for 3D reconstruction. The core of the typical lesion consisted of eosinophilic material associated with reticulin loss, collagen damage including loss of birefringence to fibrous septa, and perilesional portal tracts, including large portal vein branches, but intact peri-lesional hepatic plates. The 3D reconstruction of two histotripsy sites was successful and confirmed the feasibility of this approach to investigate the effects of histotripsy on tissue in detail.

Differences in the pattern and regulation of mineral deposition in human cell lines of osteogenic and non-osteogenic origin.

Bone marrow-derived mesenchymal stem cells (MSCs) are widely used as a cellular model of bone formation, and can mineralize in vitro in response to osteogenic medium (OM). It is unclear, however, whether this property is specific to cells of mesenchymal origin. We analysed the OM response in 3 non-osteogenic lines, HEK293, HeLa and NTera, compared to MSCs. Whereas HEK293 cells failed to respond to OM conditions, the 2 carcinoma-derived lines NTera and HeLa deposited a calcium phosphate mineral comparable to that present in MSC cultures. However, unlike MSCs, HeLa and NTera cultures did so in the absence of dexamethasone. This discrepancy was confirmed, as bone morphogenetic protein inhibition obliterated the OM response in MSCs but not in HeLa or NTera, indicating that these 2 models can deposit mineral through a mechanism independent of established dexamethasone or bone morphogenetic protein signalling.

Serum-Free Production of Three-Dimensional Hepatospheres from Pluripotent Stem Cells.

Developing renewable human liver tissue from stem cells has been pursued as a potential source of biological material for pharmaceutical and clinical endeavors. At present, two-dimensional differentiation procedures deliver tissue lacking long-term phenotypic and functional stability. Efforts to overcome these limiting factors have led to the development of protocols to generate three-dimensional cellular aggregates. Here we describe a methodology to generate 3D hepatospheres from human pluripotent stem cells using defined and commercially available reagents.

Nuclear factor programming improves stem-cell-derived hepatocyte phenotype.

In this issue of Cell Stem Cell, Ma et al. demonstrate that the activation of the nuclear receptor thyroid hormone receptor beta (NR1A2) improves the differentiation status of hepatocyte-like cells derived from human pluripotent stem cells.

Generation of 3D retinal tissue from human pluripotent stem cells using a directed small molecule-based serum-free microwell platform.

Retinal degenerative diseases are a leading cause of blindness worldwide with debilitating life-long consequences for the affected individuals. Cell therapy is considered a potential future clinical intervention to restore and preserve sight by replacing lost photoreceptors and/or retinal pigment epithelium. Development of protocols to generate retinal tissue from human pluripotent stem cells (hPSC), reliably and at scale, can provide a platform to generate photoreceptors for cell therapy and to model retinal disease in vitro. Here, we describe an improved differentiation platform to generate retinal organoids from hPSC at scale and free from time-consuming manual microdissection steps. The scale up was achieved using an agarose mould platform enabling generation of uniform self-assembled 3D spheres from dissociated hPSC in microwells. Subsequent retinal differentiation was efficiently achieved via a stepwise differentiation protocol using a number of small molecules. To facilitate clinical translation, xeno-free approaches were developed by substituting Matrigel™ and foetal bovine serum with recombinant laminin and human platelet lysate, respectively. Generated retinal organoids exhibited important features reminiscent of retinal tissue including correct site-specific localisation of proteins involved in phototransduction.

The chick embryo: hatching a model for contemporary biomedical research.

Animal models play a crucial role in fundamental and medical research. Progress in the fields of drug discovery, regenerative medicine and cancer research among others are heavily dependent on in vivo models to validate in vitro observations, and develop new therapeutic approaches. However, conventional rodent and large animal experiments face ethical, practical and technical issues that limit their usage. The chick embryo represents an accessible and economical in vivo model, which has long been used in developmental biology, gene expression analysis and loss/gain of function experiments. It is also an established model for tissue/cell transplantation, and because of its lack of immune system in early development, the chick embryo is increasingly recognised as a model of choice for mammalian biology with new applications for stem cell and cancer research. Here, we review novel applications of the chick embryo model, and discuss future developments of this in vivo model for biomedical research.

Hydroxylation and further oxidation of delta9-tetrahydrocannabinol by alkane-degrading bacteria.

The microbial biotransformation of Delta(9)-tetrahydrocannabinol was investigated using a collection of 206 alkane-degrading strains. Fifteen percent of these strains, mainly gram-positive strains from the genera Rhodococcus, Mycobacterium, Gordonia, and Dietzia, yielded more-polar derivatives. Eight derivatives were produced on a mg scale, isolated, and purified, and their chemical structures were elucidated with the use of liquid chromatography-mass spectrometry, (1)H-nuclear magnetic resonance (1H-NMR), and two-dimensional NMR (1H-1H correlation spectroscopy and heteronuclear multiple bond coherence). All eight biotransformation products possessed modified alkyl chains, with hydroxy, carboxy, and ester functionalities. In a number of strains, beta-oxidation of the initially formed C5 carboxylic acid led to the formation of a carboxylic acid lacking two methylene groups.

Serum Free Production of Three-dimensional Human Hepatospheres from Pluripotent Stem Cells.

The development of renewable sources of liver tissue is required to improve cell-based modelling, and develop human tissue for transplantation. Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) represent promising sources of human liver spheres. We have developed a serum free and defined method of cellular differentiation to generate three-dimensional human liver spheres formed from human pluripotent stem cells. A potential limitation of the technology is the production of dense spheres with dead material inside. In order to circumvent this, we have employed agarose microwell technology at defined cell densities to control the size of the 3D spheres, preventing the generation of apoptotic and/or necrotic cores.  Notably, the spheres generated by our approach display liver function and stable phenotype, representing a valuable resource for basic and applied scientific research. We believe that our approach could be used as a platform technology to develop further tissues to model and treat human disease and in the future may permit the generation of human tissue with complex tissue architecture.

Three-dimensional cell culture: from evolution to revolution.

Recent advances in the isolation of tissue-resident adult stem cells and the identification of inductive factors that efficiently direct differentiation of human pluripotent stem cells along specific lineages have facilitated the development of high-fidelity modelling of several tissues in vitro Many of the novel approaches have employed self-organizing three-dimensional (3D) culturing of organoids, which offer several advantages over conventional two-dimensional platforms. Organoid technologies hold great promise for modelling diseases and predicting the outcome of drug responses in vitro Here, we outline the historical background and some of the recent advances in the field of three-dimensional organoids. We also highlight some of the current limitations of these systems and discuss potential avenues to further benefit biological research using three-dimensional modelling technologies.This article is part of the theme issue 'Designer human tissue: coming to a lab near you'.

Cannabis tea revisited: a systematic evaluation of the cannabinoid composition of cannabis tea.

Cannabis is one of the oldest known medicinal plants, and a large variety of biological activities have been described. The main constituents, the cannabinoids, are thought to be most important for these activities. Although smoking of cannabis is by far the most common way of consumption, a significant part of medicinal users consume it in the form of a tea. However, not much is known about the composition of cannabis tea, or the effect of different parameters during preparation, handling or storage. In this study we used the high-grade cannabis available in Dutch pharmacies to study the cannabinoid composition of tea under standardized and quantitative conditions. Experimental conditions were systematically varied in order to mimic the possible variations made by medicinal users. During analysis there was a specific focus on the cannabinoid tetrahydrocannabinol and its acidic precursor, tetrahydrocannabinolic acid. Also the role of non-psychoactive cannabinoids as components of cannabis tea are discussed. The results obtained in this study provide a clear quantitative insight in the phytochemistry of cannabis tea preparation and can contribute to a better appreciation of this mode of cannabis administration.

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells.

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the human body. The differentiation of hPSCs to human hepatocyte-like cells (HLCs) has been extensively studied, and efficient differentiation protocols have been established. The combination of extracellular matrix and biological stimuli, including growth factors, cytokines, and small molecules, have made it possible to generate HLCs that resemble primary human hepatocytes. However, the majority of procedures still employ undefined components, giving rise to batch-to-batch variation. This serves as a significant barrier to the application of the technology. To tackle this issue, we developed a defined system for hepatocyte differentiation using human recombinant laminins as extracellular matrices in combination with a serum-free differentiation process. Highly efficient hepatocyte specification was achieved, with demonstrated improvements in both HLC function and phenotype. Importantly, this system is easy to scale up using research and GMP-grade hPSC lines promising advances in cell-based modelling and therapies.