[For a good understanding and use of the term "organoids"]. / Pour une bonne compréhension et un bon usage du terme « organoïdes ¼.

Title: Pour une bonne compréhension et un bon usage du terme « organoïdes ¼. Abstract: Depuis une dizaine d'années, des progrès considérables ont été réalisés concernant les conditions qui permettent à des cellules de s'auto-organiser dans l'espace comme elles le font lors des phases précoces du développement embryonnaire ou dans certains tissus adultes. On nomme ainsi « organoïdes ¼ des structures en trois dimensions complexes, organisées et intégrant plusieurs types cellulaires, qui peuvent reproduire in vitro certaines fonctions d'un organe. Toutefois, ces organoïdes ne peuvent actuellement reproduire à l'identique une architecture anatomique et fonctionnelle complète. Bien qu'utilisé pour des raisons de simplification pour la communication, en particulier dans la presse généraliste, il est donc abusif d'utiliser le terme « mini-organes ¼ pour décrire ces structures.

Microencapsulated Hepatocytes Differentiated from Human Induced Pluripotent Stem Cells: Optimizing 3D Culture for Tissue Engineering Applications.

Liver cell therapy and in vitro models require functional human hepatocytes, the sources of which are considerably limited. Human induced pluripotent stem cells (hiPSCs) represent a promising and unlimited source of differentiated human hepatocytes. However, when obtained in two-dimensional (2D) cultures these hepatocytes are not fully mature and functional. As three-dimensional culture conditions offer advantageous strategies for differentiation, we describe here a combination of three-dimensional (3D) approaches enabling the successful differentiation of functional hepatocytes from hiPSCs by the encapsulation of hiPSC-derived hepatoblasts in alginate beads of preformed aggregates. The resulting encapsulated and differentiated hepatocytes (E-iHep-Orgs) displayed a high level of albumin synthesis associated with the disappearance of α-fetoprotein (AFP) synthesis, thus demonstrating that the E-iHep-Orgs had reached a high level of maturation, similar to that of adult hepatocytes. Gene expression analysis by RT-PCR and immunofluorescence confirmed this maturation. Further functional assessments demonstrated their enzymatic activities, including lactate and ammonia detoxification, as well as biotransformation activities of Phase I and Phase II enzymes. This study provides proof of concept regarding the benefits of combining three-dimensional techniques (guided aggregation and microencapsulation) with liver differentiation protocols as a robust approach to generate mature and functional hepatocytes that offer a permanent and unlimited source of hepatocytes. Based on these encouraging results, our combined conditions to produce mature hepatocytes from hiPSCs could be extended to liver tissue engineering and bioartificial liver (BAL) applications at the human scale for which large biomasses are mandatory.

Successful Derivation of Hepatoblasts, Cholangiocytes and Hepatocytes from Simian Induced Pluripotent Stem Cells.

The use of primary cells in human liver therapy is limited by a lack of cells. Induced pluripotent stem cells (iPSCs) represent an alternative to primary cells as they are infinitely expandable and can be differentiated into different liver cell types. The aim of our work was to demonstrate that simian iPSCs (siPSCs) could be used as a new source of liver cells to be used as a large animal model for preclinical studies. We first differentiated siPSCs into a homogenous population of hepatoblasts (siHBs). We then separately differentiated them into hepatocytes (siHeps) and cholangiocytes (siChols) expressing respective specific markers and displaying epithelial polarity. Moreover, we showed that polarized siChols can self-organize into 3D structures. These results should facilitate the deciphering of liver development and open the way to exploring co-culture systems that could be assessed during preclinical studies, including in autologous monkey donors, for regenerative medicine purposes.

Evidence of Adult Features and Functions of Hepatocytes Differentiated from Human Induced Pluripotent Stem Cells and Self-Organized as Organoids.

BACKGROUND: Human-induced pluripotent stem cell-derived hepatocytes (iHeps) have been shown to have considerable potential in liver diseases, toxicity, and pharmacological studies. However, there is a growing need to obtain iHeps that are truly similar to primary adult hepatocytes in terms of morphological features and functions. We generated such human iHeps, self-assembled as organoids (iHep-Orgs). METHODS: iPSC-derived hepatoblasts were self-assembled into spheroids and differentiated into mature hepatocytes modulating final step of differentiation. RESULTS: In about four weeks of culture, the albumin secretion levels and the complete disappearance of α-fetoprotein from iHep-Orgs suggested the acquisition of a greater degree of maturation than those previously reported. The expression of apical transporters and bile acid secretion evidenced the acquisition of complex hepatocyte polarity as well as the development of a functional and well-defined bile canalicular network confirmed by computational analysis. Activities recorded for CYP450, UGT1A1, and alcohol dehydrogenase, response to hormonal stimulation, and glucose metabolism were also remarkable. Finally, iHep-Orgs displayed a considerable ability to detoxify pathological concentrations of lactate and ammonia. CONCLUSIONS: With features similar to those of primary adult hepatocytes, the iHep-Orgs thus produced could be considered as a valuable tool for the development and optimization of preclinical and clinical applications.

In vitro recovery of FIX clotting activity as a marker of highly functional hepatocytes in a hemophilia B iPSC model.

BACKGROUND AND AIMS: Pluripotent stem cell-derived hepatocytes differentiated in monolayer culture are known to have more fetal than adult hepatocyte characteristics. If numerous studies tend to show that this immature phenotype might not necessarily be an obstacle to their use in transplantation, other applications such as drug screening, toxicological studies, or bioartificial livers are reliant on hepatocyte functionality and require full differentiation of hepatocytes. New technologies have been used to improve the differentiation process in recent years, usually evaluated by measuring the albumin production and CYP450 activity. Here we used the complex production and most importantly the activity of the coagulation factor IX (FIX) produced by mature hepatocytes to assess the differentiation of hemophilia B (HB) patient's induced pluripotent stem cells (iPSCs) in both monolayer culture and organoids. APPROACH AND RESULTS: Indeed, HB is an X-linked monogenic disease due to an impaired activity of FIX synthesized by hepatocytes in the liver. We have developed an in vitro model of HB hepatocytes using iPSCs generated from fibroblasts of a severe HB patient. We used CRISPR/Cas9 technology to target the genomic insertion of a coagulation factor 9 minigene bearing the Padua mutation to enhance FIX activity. Noncorrected and corrected iPSCs were differentiated into hepatocytes under both two-dimensional and three-dimensional differentiation protocols and deciphered the production of active FIX in vitro. Finally, we assessed the therapeutic efficacy of this approach in vivo using a mouse model of HB. CONCLUSIONS: Functional FIX, whose post-translational modifications only occur in fully mature hepatocytes, was only produced in corrected iPSCs differentiated in organoids. Immunohistochemistry analyses of mouse livers indicated a good cell engraftment, and the FIX activity detected in the plasma of transplanted animals confirmed rescue of the bleeding phenotype.

[Hepatic organoids: What are the challenges?] / Les organoïdes hépatiques - Quels sont les enjeux ?

The study and understanding of liver organogenesis have allowed the development of protocols for pluripotent stem cells differentiation to overcome the lack of primary cells, providing an almost unlimited source of liver cells. However, as their differentiation in conventional 2D culture systems has shown serious limits, hepatic organoids derived from human pluripotent stem cells represent a promising alternative. These complex and organized structures, containing one or more cell types, make it possible to recapitulate in vitro some of the organ functions, thus enabling numerous applications such as the study of the liver development, the mass production of functional liver cells for transplantation or the development of bioartificial livers, as well as the in vitro modeling of hepatic pathologies allowing high throughput applications in drug screening or toxicity studies. Economic and ethical issues must also be taken into account before using these organoids in therapeutic applications.

TITLE: Les organoïdes hépatiques - Quels sont les enjeux ? ABSTRACT: L'étude et la compréhension de l'organogenèse du foie ont permis le développement de protocoles de différenciation des cellules souches pluripotentes afin de pallier le manque de cellules primaires, offrant ainsi une source quasi illimitée de cellules hépatiques. La différenciation de ces cellules dans des systèmes de culture conventionnels en deux dimensions (2D) ayant cependant montré ses limites, des organoïdes hépatiques ont été dérivés de cellules souches pluripotentes humaines et représentent désormais une alternative prometteuse. Ces structures 3D, complexes et organisées, intégrant un ou plusieurs types cellulaires, permettent de reproduire in vitro une ou plusieurs fonctions de l'organe, et ouvrent ainsi la voie à de nombreuses applications, comme l'étude du développement du foie, la production en masse de cellules hépatiques fonctionnelles pour la transplantation ou le développement de foies bioartificiels, sans oublier la modélisation de pathologies hépatiques permettant le criblage à haut débit de médicaments ou des études de toxicité. Des enjeux économiques et éthiques doivent également être pris en considération avant une utilisation de ces organoïdes pour des applications thérapeutiques.

A versatile microfluidic tool for the 3D culture of HepaRG cells seeded at various stages of differentiation.

The development of livers-on-a-chip aims to provide pharmaceutical companies with reliable systems to perform drug screening and toxicological studies. To that end, microfluidic systems are engineered to mimic the functions and architecture of this organ. In this context we have designed a device that reproduces series of liver microarchitectures, each permitting the 3D culture of hepatocytes by confining them to a chamber that is separated from the medium conveying channel by very thin slits. We modified the structure to ensure its compatibility with the culture of hepatocytes from different sources. Our device was adapted to the migratory and adhesion properties of the human HepaRG cell line at various stages of differentiation. Using this device, it was possible to keep the cells alive for more than 14 days, during which they achieved a 3D organisation and acquired or maintained their differentiation into hepatocytes. Albumin secretion as well as functional bile canaliculi were confirmed on the liver-on-a-chip. Finally, an acetaminophen toxicological assay was performed. With its multiple micro-chambers for hepatocyte culture, this microfluidic device architecture offers a promising opportunity to provide new tools for drug screening applications.

Advanced Techniques and Awaited Clinical Applications for Human Pluripotent Stem Cell Differentiation into Hepatocytes.

Liver transplantation is currently the only curative treatment for several liver diseases such as acute liver failure, end-stage liver disorders, primary liver cancers, and certain genetic conditions. Unfortunately, despite improvements to transplantation techniques, including live donor transplantation, the number of organs available remains insufficient to meet patient needs. Hepatocyte transplantation has enabled some encouraging results as an alternative to organ transplantation, but primary hepatocytes are little available and cannot be amplified using traditional two-dimensional culture systems. Indeed, although recent studies have tended to show that three-dimensional culture enables long-term hepatocyte culture, it is still agreed that, like most adult primary cell types, hepatocytes remain refractory to in vitro expansion. Because of their exceptional properties, human pluripotent stem cells (hPSCs) can be amplified indefinitely and differentiated into any cell type, including liver cells. While many teams have worked on hepatocyte differentiation, there has been a consensus that cells obtained after hPSC differentiation have more fetal than adult hepatocyte characteristics. New technologies have been used to improve the differentiation process in recent years. This review discusses the technical improvements made to hepatocyte differentiation protocols and the clinical approaches developed to date and anticipated in the near future.

Liver Regeneration and Recanalization Time Course following Repeated Reversible Portal Vein Embolization in Swine.

INTRODUCTION: Portal vein embolization (PVE) is an accepted technique to preoperatively increase the volume of the future remnant liver before major hepatectomy. A permanent material is usually preferred since its superiority to induce liver hypertrophy over absorbable material has been demonstrated. Nevertheless, the use of an absorbable material generates a reversible PVE (RPVE) capable of inducing significant liver hypertrophy. In small animal models, the possibility to proceed to a repeated RPVE (RRPVE) has shown to boost liver hypertrophy further. The aim of this preliminary study was to assess the feasibility and the tolerance of RRPVE in a large animal model, in comparison with permanent PVE (PPVE) and single RPVE. METHODS: Six swine (2 per group) were assigned either to single RPVE group (using powdered gelatin sponge), RRPVE group (2 RPVEs separated by 14 days) or PPVE group (using N-butyl-cyanoacrylate). The feasibility and tolerance of the procedures were evaluated using portography, liver function tests and histological analysis. Evolution of liver volumes was assessed with volumetric imaging by computed tomography. RESULTS: Embolization of portal branches corresponding to 75% of total liver volume was performed successfully in all animals. Procedures were well tolerated, inducing moderate changes in portal pressure and transient aminotransferase increase. None of the animals developed portal vein thrombosis. After RPVE, complete recanalization occurred at day 11. RRPVE showed a trend for higher hypertrophy, the non-embolized liver to total liver ratio reaching 5.2 ± 1.0% in the RPVE group, 6.8 ± 0.1% in the RRPVE group and 5.0 ± 0.3% in the PPVE group. DISCUSSION/CONCLUSION: In this preliminary comparative study, RRPVE was as feasible and as well tolerated as the other procedures, and resulted in higher liver hypertrophy.

Pluripotent stem cell-derived cholangiocytes and cholangiocyte organoids.

The development of protocols for pluripotent stem cell (PSC) differentiation into cholangiocytes and cholangiocyte organoids in three-dimensional structures represent a huge advance in both research and medical fields because of the limited access to primary human cholangiocytes and the potential bias induced by animal models used to study cholangiopathies in vivo. PSC-derived cholangiocyte organoids consisting of either cysts with luminal space or branching tubular structures are composed of cells with apico-basal polarity that can fulfill cholangiocyte functions like the transport of bile salts. Several protocols of PSC differentiation have already been published but we added to the detailed protocol we describe here some notes or advice to facilitate its handling by new users. We also propose detailed protocols to carry out some of the characterization analyses using immunofluorescence to study the expression of specific markers and a functionality test to visualize bile acid transport using cholyl-lysyl-fluorescein (CLF).

Pluripotent-Stem-Cell-Derived Hepatic Cells: Hepatocytes and Organoids for Liver Therapy and Regeneration.

The liver is a very complex organ that ensures numerous functions; it is thus susceptible to multiple types of damage and dysfunction. Since 1983, orthotopic liver transplantation (OLT) has been considered the only medical solution available to patients when most of their liver function is lost. Unfortunately, the number of patients waiting for OLT is worryingly increasing, and extracorporeal liver support devices are not yet able to counteract the problem. In this review, the current and expected methodologies in liver regeneration are briefly analyzed. In particular, human pluripotent stem cells (hPSCs) as a source of hepatic cells for liver therapy and regeneration are discussed. Principles of hPSC differentiation into hepatocytes are explored, along with the current limitations that have led to the development of 3D culture systems and organoid production. Expected applications of these organoids are discussed with particular attention paid to bio artificial liver (BAL) devices and liver bio-fabrication.

HepaRG Self-Assembled Spheroids in Alginate Beads Meet the Clinical Needs for Bioartificial Liver.

In liver tissue engineering, cell culture in spheroids is now well recognized to promote the maintenance of hepatic functions. However, the process leading to spheroids formation is time consuming, costly, and not easy to scale-up for further use in human bioartificial liver (BAL) applications. In this study, we encapsulated HepaRG cells (precursors of hepatocyte-like cells) in 1.5% alginate beads without preforming spheroids. Starting from a given hepatic biomass, we analyzed cell differentiation and metabolic performance for further use in a fluidized-bed BAL. We observed that cells self-rearranged as aggregates within the beads and adequately differentiated over time, in the absence of any differentiating factors classically used. On day 14 postencapsulation, cells displayed a wide range of hepatic features necessary for the treatment of a patient in acute liver failure. These activities include albumin synthesis, ammonia and lactate detoxification, and the efficacy of the enzymes involved in the xenobiotic metabolism (such as CYP1A1/2). Impact statement It has been recognized that culturing cells in spheroids (SPHs) is advantageous as they better reproduce the three-dimensional physiological microenvironment. This approach can be exploited in bioartificial liver applications, where obtaining a functional hepatic biomass is the major challenge. Our study describes an original method for culturing hepatic cells in alginate beads that makes possible the autonomous formation of SPHs after 3 days of culture. In turn, the cells differentiate adequately and display a wide range of hepatic features. They are also capable of treating a pathological plasma model. Finally, this setup can easily be scaled-up to treat acute liver failure.

Genomic integrity of human induced pluripotent stem cells: Reprogramming, differentiation and applications.

Ten years after the initial generation of induced pluripotent stem cells (hiPSCs) from human tissues, their potential is no longer questioned, with over 15000 publications listed on PubMed, covering various fields of research; including disease modeling, cell therapy strategies, pharmacology/toxicology screening and 3D organoid systems. However, despite evidences that the presence of mutations in hiPSCs should be a concern, publications addressing genomic integrity of these cells represent less than 1% of the literature. After a first overview of the mutation types currently reported in hiPSCs, including karyotype abnormalities, copy number variations, single point mutation as well as uniparental disomy, this review will discuss the impact of reprogramming parameters such as starting cell type and reprogramming method on the maintenance of the cellular genomic integrity. Then, a specific focus will be placed on culture conditions and subsequent differentiation protocols and how their may also trigger genomic aberrations within the cell population of interest. Finally, in a last section, the impact of genomic alterations on the possible usages of hiPSCs and their derivatives will also be exemplified and discussed. We will also discuss which techniques or combination of techniques should be used to screen for genomic abnormalities with a particular focus on the necessary quality controls and the potential alternatives.

Low-density lipoprotein receptor-deficient hepatocytes differentiated from induced pluripotent stem cells allow familial hypercholesterolemia modeling, CRISPR/Cas-mediated genetic correction, and productive hepatitis C virus infection.

BACKGROUND: Familial hypercholesterolemia type IIA (FH) is due to mutations in the low-density lipoprotein receptor (LDLR) resulting in elevated levels of low-density lipoprotein cholesterol (LDL-c) in plasma and in premature cardiovascular diseases. As hepatocytes are the only cells capable of metabolizing cholesterol, they are therefore the target cells for cell/gene therapy approaches in the treatment of lipid metabolism disorders. Furthermore, the LDLR has been reported to be involved in hepatitis C virus (HCV) entry into hepatocytes; however, its role in the virus infection cycle is still disputed. METHODS: We generated induced pluripotent stem cells (iPSCs) from a homozygous LDLR-null FH-patient (FH-iPSCs). We constructed a correction cassette bearing LDLR cDNA under the control of human hepatic apolipoprotein A2 promoter that targets the adeno-associated virus integration site AAVS1. We differentiated both FH-iPSCs and corrected FH-iPSCs (corr-FH-iPSCs) into hepatocytes to study statin-mediated regulation of genes involved in cholesterol metabolism. Upon HCV particle inoculation, viral replication and production were quantified in these cells. RESULTS: We showed that FH-iPSCs displayed the disease phenotype. Using homologous recombination mediated by the CRISPR/Cas9 system, FH-iPSCs were genetically corrected by the targeted integration of a correction cassette at the AAVS1 locus. Both FH-iPSCs and corr-FH-iPSCs were then differentiated into functional polarized hepatocytes using a stepwise differentiation approach (FH-iHeps and corr-FH-iHeps). The correct insertion and expression of the correction cassette resulted in restoration of LDLR expression and function (LDL-c uptake) in corr-FH-iHeps. We next demonstrated that pravastatin treatment increased the expression of genes involved in cholesterol metabolism in both cell models. Moreover, LDLR expression and function were also enhanced in corr-FH-iHeps after pravastatin treatment. Finally, we demonstrated that both FH-iHeps and corr-FH-iHeps were as permissive to viral infection as primary human hepatocytes but that virus production in FH-iHeps was significantly decreased compared to corr-FH-iHeps, suggesting a role of the LDLR in HCV morphogenesis. CONCLUSIONS: Our work provides the first LDLR-null FH cell model and its corrected counterpart to study the regulation of cholesterol metabolism and host determinants of HCV life cycle, and a platform to screen drugs for treating dyslipidemia and HCV infection.

Improving Hepatocyte Engraftment Following Hepatocyte Transplantation Using Repeated Reversible Portal Vein Embolization in Rats.

Hepatocyte transplantation (HT) has emerged as a promising alternative to orthotopic liver transplantation, yet liver preconditioning is needed to promote hepatocyte engraftment. A method of temporary occlusion of the portal flow called reversible portal vein embolization (RPVE) has been demonstrated to be an efficient method of liver preconditioning. By providing an additional regenerative stimulus, repeated reversible portal vein embolization (RRPVE) could further boost liver engraftment. The aim of this study was to determine the efficiency of liver engraftment of transplanted hepatocytes after RPVE and RRPVE in a rat model. Green fluorescent protein-expressing hepatocytes were isolated from transgenic rats and transplanted into 3 groups of syngeneic recipient rats. HT was associated with RPVE in group 1, with RRPVE in group 2, and with sham embolization in the sham group. Liver engraftment was assessed at day 28 after HT on liver samples after immunostaining. Procedures were well tolerated in all groups. RRPVE resulted in increased engraftment rate in total liver parenchyma compared with RPVE (3.4% ± 0.81% versus 1.4% ± 0.34%; P < 0.001). In conclusion, RRPVE successfully enhanced hepatocyte engraftment after HT and could be helpful in the frame of failure of HT due to low cell engraftment.

Rapid and reliable diagnosis of Wilson disease using X-ray fluorescence.

Wilson's disease (WD) is a rare autosomal recessive disease due to mutations of the gene encoding the copper-transporter ATP7B. The diagnosis is hampered by the variability of symptoms induced by copper accumulation, the inconstancy of the pathognomonic signs and the absence of a reliable diagnostic test. We investigated the diagnostic potential of X-ray fluorescence (XRF) that allows quantitative analysis of multiple elements. Studies were performed on animal models using Wistar rats (n = 10) and Long Evans Cinnamon (LEC) rats (n = 11), and on human samples including normal livers (n = 10), alcohol cirrhosis (n = 8), haemochromatosis (n = 10), cholestasis (n = 6) and WD (n = 22). XRF experiments were first performed using synchrotron radiation to address the elemental composition at the cellular level. High-resolution mapping of tissue sections allowed measurement of the intensity and the distribution of copper, iron and zinc while preserving the morphology. Investigations were further conducted using a laboratory X-ray source for irradiating whole pieces of tissue. The sensitivity of XRF was highlighted by the discrimination of LEC rats from wild type even under a regimen using copper deficient food. XRF on whole formalin-fixed paraffin embedded needle biopsies allowed profiling of the elements in a few minutes. The intensity of copper related to iron and zinc significantly discriminated WD from other genetic or chronic liver diseases with 97.6% specificity and 100% sensitivity. This study established a definite diagnosis of Wilson's disease based on XRF. This rapid and versatile method can be easily implemented in a clinical setting.

[Human pluripotent stem cells and liver disorders]. / Cellules souches pluripotentes humaines et modélisation de maladies hépatiques.

The liver is associated with many diseases including metabolic and cholestatic diseases, cirrhosis as well as chronic and acute hepatitis. However, knowledge about the mechanisms involved in the pathophysiology of these diseases remains limited due to the restricted access to liver biopsies and the lack of cellular models derived from patients. The liver is the main organ responsible for the elimination of xenobiotics and thus hepatocytes have a key role in toxicology and pharmacokinetics. The induced pluripotent stem cells generated from patients with monogenic metabolic disorders, for which the corresponding gene is identified, are relevant in vitro models for the study of the mechanisms involved in generation of pathologies and also for drug screening. Towards this aim, robust protocols for generating liver cells, such as hepatocytes and cholangiocytes, are essential. Our study focused on familial hypercholesterolemia disease modeling, as well as on establishing a protocol for generation of functional cholangiocytes from pluripotent stem cells.

Hepatocytic Differentiation Potential of Human Fetal Liver Mesenchymal Stem Cells: In Vitro and In Vivo Evaluation.

In line with the search of effective stem cell population that would progress liver cell therapy and because the rate and differentiation potential of mesenchymal stem cells (MSC) decreases with age, the current study investigates the hepatogenic differentiation potential of human fetal liver MSCs (FL-MSCs). After isolation from 11-12 gestational weeks' human fetal livers, FL-MSCs were shown to express characteristic markers such as CD73, CD90, and CD146 and to display adipocytic and osteoblastic differentiation potential. Thereafter, we explored their hepatocytic differentiation potential using the hepatogenic protocol applied for adult human liver mesenchymal cells. FL-MSCs differentiated in this way displayed significant features of hepatocyte-like cells as demonstrated in vitro by the upregulated expression of specific hepatocytic markers and the induction of metabolic functions including CYP3A4 activity, indocyanine green uptake/release, and glucose 6-phosphatase activity. Following transplantation, naive and differentiated FL-MSC were engrafted into the hepatic parenchyma of newborn immunodeficient mice and differentiated in situ. Hence, FL-MSCs appeared to be interesting candidates to investigate the liver development at the mesenchymal compartment level. Standardization of their isolation, expansion, and differentiation may also support their use for liver cell-based therapy development.

The potential of induced pluripotent stem cell derived hepatocytes.

Orthotopic liver transplantation remains the only curative treatment for liver disease. However, the number of patients who die while on the waiting list (15%) has increased in recent years as a result of severe organ shortages; furthermore the incidence of liver disease is increasing worldwide. Clinical trials involving hepatocyte transplantation have provided encouraging results. However, transplanted cell function appears to often decline after several months, necessitating liver transplantation. The precise aetiology of the loss of cell function is not clear, but poor engraftment and immune-mediated loss appear to be important factors. Also, primary human hepatocytes (PHH) are not readily available, de-differentiate, and die rapidly in culture. Hepatocytes are available from other sources, such as tumour-derived human hepatocyte cell lines and immortalised human hepatocyte cell lines or porcine hepatocytes. However, all these cells suffer from various limitations such as reduced or differences in functions or risk of zoonotic infections. Due to their significant potential, one possible inexhaustible source of hepatocytes is through the directed differentiation of human induced pluripotent stem cells (hiPSCs). This review will discuss the potential applications and existing limitations of hiPSC-derived hepatocytes in regenerative medicine, drug screening, in vitro disease modelling and bioartificial livers.

Volumetric Portal Embolization: A New Concept to Improve Liver Regeneration and Hepatocyte Engraftment.

BACKGROUND: Hepatocyte transplantation has been proposed as an alternative to orthotopic liver transplantation to treat metabolic liver diseases. This approach requires preconditioning of the host liver to enhance engraftment of transplanted hepatocytes. Different methods are currently used in preclinical models: partial hepatectomy, portal ligature or embolization, and radiotherapy or chemotherapeutic drugs. However, these methods carry high risks of complications and are problematic for use in clinical practice. Here, we developed an innovative method called volumetric (distal, partial, and random) portal embolization (VPE), which preserves total liver volume. METHODS: Embolization was performed in the portal trunk of C57BL6 adult mice with polyester microspheres, to ensure a bilateral and distal distribution. The repartition of microspheres was studied by angiographic and histological analyses. Liver regeneration was evaluated by Ki67 labeling. Optimal conditions for VPE were determined, and the resulting regeneration was compared with that after partial hepatectomy (70%). Labeled adult hepatocytes were then transplanted, and engraftment was compared between embolized (n = 19) and nonembolized mice (n = 8). Engraftment was assessed in vivo and histologically by tracking labeled cells at day 5. RESULTS: The best volumetric embolization conditions, which resulted in the regeneration of 5% of total liver, were 8 × 10 ten-micron microspheres infused with a 29 G needle directly into the portal trunk at 3.3 µL/s. In these conditions, transplanted hepatocytes engraftment was significantly higher than that in control conditions (3 vs 0.65%). CONCLUSIONS: The VPE is a new, minimally invasive, and efficient technique to prepare the host liver for cell transplantation.