Functional Hepatocyte-like Cells Research Articles

Efficient hepatocyte differentiation of primary human hepatocyte-derived organoids using three dimensional nanofibers (HYDROX) and their possible application in hepatotoxicity research

Human liver organoids are in vitro three dimensionally (3D) cultured cells that have a bipotent stem cell phenotype. Translational research of human liver organoids for drug discovery has been limited by the challenge of their low hepatic function compared to primary human hepatocytes (PHHs). Various attempts have been made to develop functional hepatocyte-like cells from human liver organoids. However, none have achieved the same level of hepatic functions as PHHs. We here attempted to culture human liver organoids established from cryopreserved PHHs (PHH-derived organoids), using HYDROX, a chemically defined 3D nanofiber. While the proliferative capacity of PHH-derived organoids was lost by HYDROX-culture, the gene expression levels of drug-metabolizing enzymes were significantly improved. Enzymatic activities of cytochrome P450 3A4 (CYP3A4), CYP2C19, and CYP1A2 in HYDROX-cultured PHH-derived organoids (Org-HYDROX) were comparable to those in PHHs. When treated with hepatotoxic drugs such as troglitazone, amiodarone and acetaminophen, Org-HYDROX showed similar cell viability to PHHs, suggesting that Org-HYDROX could be applied to drug-induced hepatotoxicity tests. Furthermore, Org-HYDROX maintained its functions for up to 35 days and could be applied to chronic drug-induced hepatotoxicity tests using fialuridine. Our findings demonstrated that HYDROX could possibly be a novel biomaterial for differentiating human liver organoids towards hepatocytes applicable to pharmaceutical research.

Dynamic, IPSC-derived hepatic tissue tri-culture system for the evaluation of liver physiology in vitro

Availability of hepatic tissue for the investigation of metabolic processes is severely limited. While primary hepatocytes or animal models are widely used in pharmacological applications, a change in methodology towards more sustainable and ethical assays is highly desirable. Stem cell derived hepatic cells are generally regarded as a viable alternative for the above model systems, if current limitations in functionality and maturation can be overcome. By combining microfluidic organ-on-a-chip technology with individually differentiated, multicellular hepatic tissue fractions, we aim to improve overall functionality of hepatocyte-like cells, as well as evaluate cellular composition and interactions with non-parenchymal cell populations towards the formation of mature liver tissue. Utilizing a multi-omic approach, we show the improved maturation profiles of hepatocyte-like cells maintained in a dynamic microenvironment compared to standard tissue culture setups without continuous perfusion. In order to evaluate the resulting tissue, we employ single cell sequencing to distinguish formed subpopulations and spatial localization. While cellular input was strictly defined based on established differentiation protocols of parenchyma, endothelial and stellate cell fractions, resulting hepatic tissue was shown to comprise a complex mixture of epithelial and non-parenchymal fractions with specific local enrichment of phenotypes along the microchannel. Following this approach, we show the importance of passive, paracrine developmental processes in tissue formation. Using such complex tissue models is a crucial first step to develop stem cell-derived in vitro systems that can compare functionally with currently used pharmacological and toxicological applications.

Extracellular matrix modulates the spatial hepatic features in hepatocyte-like cells derived from human embryonic stem cells

BackgroundHuman pluripotent stem cell (hPSC)-derived hepatocyte-like cells (HLCs) can provide a valuable in vitro model for disease modelling and drug development. However, generating HLCs with characteristics comparable to hepatocytes in vivo is challenging. Extracellular matrix (ECM) plays an important role in supporting liver development and hepatocyte functions, but their impact on hepatocyte differentiation and maturation during hPSC differentiation remains unclear. Here, we investigate the effects of two ECM components—Matrigel and type I collagen on hepatic differentiation of human embryonic stem cells (hESCs).MethodshESC-derived HLCs were generated through multistage differentiation in two-dimensional (2D) and three-dimensional (3D) cultures, incorporating either type I collagen or Matrigel during hepatic specification and maturation. The resulting HLCs was characterized for their gene expression and functionality using various molecular and cellular techniques.ResultsOur results showed that HLCs cultured with collagen exhibited a significant increase in albumin and alpha-1 anti-trypsin expression with reduced AFP compared to HLCs cultured with Matrigel. They also secreted more urea than Matrigel cultures. However, these HLCs exhibited lower CYP3A4 activity and glycogen storage than those cultured with Matrigel. These functional differences in HLCs between collagen and Matrigel cultures closely resembled the hepatocytes of periportal and pericentral zones, respectively.ConclusionOur study demonstrates that Matrigel and collagen have differential effects on the differentiation and functionality of HLCs, which resemble, to an extent, hepatic zonation in the liver lobules. Our finding has an important impact on the generation of hPSC-HLCs for biomedical and medical applications.

336.1: Perfusion bioreactor and decellularized liver matrix in support of human amnion epithelial cell maturation into functional hepatocyte-like cells

336.1: Perfusion bioreactor and decellularized liver matrix in support of human amnion epithelial cell maturation into functional hepatocyte-like cells

LIGHT (TNFSF14) promotes the differentiation of human bone marrow-derived mesenchymal stem cells into functional hepatocyte-like cells.

Liver transplantation is the most effective treatment option for patients with acute or chronic liver failure. However, the applicability and effectiveness of this modality are often limited by a shortage of donors, surgical complications, high medical costs, and the need for continuing immunosuppressive therapy. An alternative approach is liver cell transplantation. LIGHT (a member of the tumor necrosis factor superfamily) could be a promising candidate for promoting the differentiation of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) into hepatocyte-like cells. In this study, we investigated the effect of LIGHT on hBM-MSC differentiation into hepatocyte-like cells. Our previous results showed that LIGHT receptor lymphotoxin-β receptor (LTβR) is constitutively expressed on the surface of hBM-MSCs. Upon treatment with recombinant human LIGHT (rhLIGHT), the phenotype of hBM-MSCs changed to round or polygonal cells. In addition, the cells exhibited high levels of hepatocyte-specific markers, including albumin, cytokeratin-18 (CK-18), CK-19, cytochrome P450 family 1 subfamily A member 1 (CYP1A1), CYP1A2, CYP3A4, SRY-box transcription factor 17 (SOX17), and forkhead box A2 (FOXA2). These results indicate that rhLIGHT enhances the differentiation of hBM-MSCs into functional hepatocyte-like cells. Furthermore, rhLIGHT-induced hepatocyte-like cells showed a higher ability to store glycogen and uptake indocyanine green compared with control cells, indicating functional progression. Additionally, treatment with rhLIGHT increased the number, viability, and proliferation of cells by inducing the S/G2/M phase and upregulating the expression of various cyclin and cyclin dependent kinase (CDK) proteins. We also found that the hepatogenic differentiation of hBM-MSCs induced by rhLIGHT was mediated by the activation of signal transducer and activator of transcription 3 (STAT3) and STAT5 pathways. Overall, our findings suggest that LIGHT plays an essential role in promoting the hepatogenic differentiation of hBM-MSCs. Hence, LIGHT may be a valuable factor for stem cell therapy.

Pluripotent Stem Cell-Derived Hepatocyte-like Cells: Induction Methods and Applications.

The development of regenerative medicine provides new options for the treatment of end-stage liver diseases. Stem cells, such as bone marrow mesenchymal stem cells, embryonic stem cells, and induced pluripotent stem cells (iPSCs), are effective tools for tissue repair in regenerative medicine. iPSCs are an appropriate source of hepatocytes for the treatment of liver disease due to their unlimited multiplication capacity, their coverage of the entire range of genetics required to simulate human disease, and their evasion of ethical implications. iPSCs have the ability to gradually produce hepatocyte-like cells (HLCs) with homologous phenotypes and physiological functions. However, how to induce iPSCs to differentiate into HLCs efficiently and accurately is still a hot topic. This review describes the existing approaches for inducing the differentiation of iPSCs into HLCs, as well as some challenges faced, and summarizes various parameters for determining the quality and functionality of HLCs. Furthermore, the application of iPSCs for in vitro hepatoprotective drug screening and modeling of liver disease is discussed. In conclusion, iPSCs will be a dependable source of cells for stem-cell therapy to treat end-stage liver disease and are anticipated to facilitate individualized treatment for liver disease in the future.

MicroRNA-122-functionalized DNA tetrahedron stimulate hepatic differentiation of human mesenchymal stem cells for acute liver failure therapy

As the most abundant liver-specific microRNA, microRNA-122 (miR122) played a crucial role in the differentiation of stem cells into hepatocytes. However, highly efficient miR122 delivery still confronts challenges including poor cellular uptake and easy biodegradation. Herein, we for the first time demonstrated that the tetrahedral DNA (TDN) nanoplatform had great potential in inducing the differentiation of human mesenchymal stem cells (hMSCs) into functional hepatocyte-like cells (HLCs) by transferring the liver-specific miR122 to hMSCs efficiently without any extrinsic factors. As compared with miR122, miR122-functionalized TDN (TDN-miR122) could significantly up-regulate the protein expression levels of mature hepatocyte markers and hepatocyte-specific marker genes in hMSCs, indicating that TDN-miR122 could particularly activate the hepatocyte-specific properties of hMSCs for developing cell-based therapies in vitro. The transcriptomic analysis further indicated the potential mechanism that TDN-miR122 assisted hMSCs differentiated into functional HLCs. The TDN-miR122-hMSCs exhibited hepatic cell morphology phenotype, significantly up-regulated specific hepatocyte genes and hepatic biofunctions in comparison with the undifferentiated MSCs. Preclinical in vivo transplantation appeared that TDN-miR122-hMSCs in combination with or without TDN could efficiently rescue acute liver failure injury through hepatocyte function supplement, anti-apoptosis, cellular proliferation promotion, and anti-inflammatory. Collectively, our findings may provide a new and facile approach for hepatic differentiation of hMSCs for acute liver failure therapy. Further large animal model explorations are needed to study their potential in clinical translation in the future.

Direct chemical induction of hepatocyte-like cells with capacity for liver repopulation.

Cell fate can be directly reprogrammed from accessible cell types (e.g., fibroblasts) into functional cell types by exposure to small molecule stimuli. However, no chemical reprogramming method has been reported to date that successfully generates functional hepatocyte-like cells that can repopulate liver tissue, casting doubt over the feasibility of chemical reprogramming approaches to obtain desirable cell types for therapeutic applications. Here, through chemical induction of phenotypic plasticity, we provide a proof-of-concept demonstration of the direct chemical reprogramming of mouse fibroblasts into functional hepatocyte-like cells using exposure to small molecule cocktails in culture medium to successively stimulate endogenous expression of master transcription factors associated with hepatocyte development, such as hepatocyte nuclear factor 4a, nuclear receptor subfamily 1, group I, member 2, and nuclear receptor subfamily 1, group H, member 4. RNA sequencing analysis, metabolic assays, and in vivo physiological experiments show that chemically induced hepatocytes (CiHeps) exhibit comparable activity and function to primary hepatocytes, especially in liver repopulation to rescue liver failure in fumarylacetoacetate hydrolase -/- recombination activating gene 2 -/- interleukin 2 receptor, gamma chain -/- mice in vivo . Single-cell RNA-seq further revealed that gastrointestinal-like and keratinocyte-like cells were induced along with CiHeps, resembling the activation of an intestinal program within hepatic reprogramming as described in transgenic approaches. Our findings show that direct chemical reprogramming can generate hepatocyte-like cells with high-quality physiological properties, providing a paradigm for establishing hepatocyte identity in fibroblasts and demonstrating the potential for chemical reprogramming in organ/tissue repair and regeneration therapies.

Combinational Overexpression of Foxa3 and Hnf4α Enhance the Proliferation and Prolong the Functional Maintenance of Primary Hepatocytes

In an in vitro culture system, primary hepatocytes usually display a low proliferation capacity, accompanied with a decrease of viability and a loss of hepatocyte-specific functions. Previous studies have demonstrated that the combination introductions of certain hepatocyte-specific transcription factors are able to convert fibroblasts into functional hepatocyte-like cells. However, such combinational usage of transcription factors in primary hepatocytes culture has not yet sufficiently studied. The forkhead box protein A3 (FoxA3) and hepatocyte nuclear factor 4α (Hnf4α) are liver-enriched transcription factors that play vital roles in the differentiation, and maintenance of hepatocytes. Thus, we simultaneously overexpressed the two genes, Foxa3 and Hnf4α, in rat hepatocytes and observed that the combinational augmentation of these two transcription factors have enhanced the proliferation and stabilized the hepatocyte-specific functions of primary hepatocytes over a long-term culture period.

Rat liver extracellular matrix and perfusion bioreactor culture promote human amnion epithelial cell differentiation towards hepatocyte-like cells

Congenital and chronic liver diseases have a substantial health burden worldwide. The most effective treatment available for these patients is whole organ transplantation; however, due to the severely limited supply of donor livers and the side effects associated with the immunosuppressive regimen required to accept allograft, the mortality rate in patients with end-stage liver disease is annually rising. Stem cell-based therapy aims to provide alternative treatments by either cell transplantation or bioengineered construct transplantation. Human amnion epithelial cells (AEC) are a widely available, ethically neutral source of cells with the plasticity and potential of multipotent stem cells and immunomodulatory properties of perinatal cells. AEC have been proven to be able to achieve functional improvement towards hepatocyte-like cells, capable of rescuing animals with metabolic disorders; however, they showed limited metabolic activities in vitro. Decellularised extracellular matrix (ECM) scaffolds have gained recognition as adjunct biological support. Decellularised scaffolds maintain native ECM components and the 3D architecture instrumental of the organ, necessary to support cells’ maturation and function. We combined ECM-scaffold technology with primary human AEC, which we demonstrated being equipped with essential ECM-adhesion proteins, and evaluated the effects on AEC differentiation into functional hepatocyte-like cells (HLC). This novel approach included the use of a custom 4D bioreactor to provide constant oxygenation and media perfusion to cells in 3D cultures over time. We successfully generated HLC positive for hepatic markers such as ALB, CYP3A4 and CK18. AEC-derived HLC displayed early signs of hepatocyte phenotype, secreted albumin and urea, and expressed Phase-1 and -2 enzymes. The combination of liver-specific ECM and bioreactor provides a system able to aid differentiation into HLC, indicating that the innovative perfusion ECM-scaffold technology may support the functional improvement of multipotent and pluripotent stem cells, with important repercussions in the bioengineering of constructs for transplantation.

MicroRNAs: Crucial Players in the Differentiation of Human Pluripotent and Multipotent Stem Cells into Functional Hepatocyte-Like Cells.

Hepatic disease negatively impacts liver function and metabolism. Primary human hepatocytes are the gold standard for the prediction and successful treatment of liver disease. However, the sources of hepatocytes for drug toxicity testing and disease modeling are limited. To overcome this issue, pluripotent stem cells (PSCs) have emerged as an alternative strategy for liver disease therapy. Human PSCs, including embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) can self-renew and give rise to all cells of the body. Human PSCs are attractive cell sources for regenerative medicine, tissue engineering, drug discovery, and developmental studies. Several recent studies have shown that mesenchymal stem cells (MSCs) can also differentiate (or trans-differentiate) into hepatocytes. Differentiation of human PSCs and MSCs into functional hepatocytelike cells (HLCs) opens new strategies to study genetic diseases, hepatotoxicity, infection of hepatotropic viruses, and analyze hepatic biology. Numerous in vitro and in vivo differentiation protocols have been established to obtain human PSCs/MSCs-derived HLCs and mimic their characteristics. It was recently discovered that microRNAs (miRNAs) play a critical role in controlling the ectopic expression of transcription factors and governing the hepatocyte differentiation of human PSCs and MSCs. In this review, we focused on the role of miRNAs in the differentiation of human PSCs and MSCs into hepatocytes.

Direct Differentiation of Human Embryonic Stem Cells to 3D Functional Hepatocyte-like Cells in Alginate Microencapsulation Sphere.

Background: The lack of a stable source of hepatocytes is one of major limitations in hepatocyte transplantation and clinical applications of a bioartificial liver. Human embryonic stem cells (hESCs) with a high degree of self-renewal and totipotency are a potentially limitless source of a variety of cell lineages, including hepatocytes. Many techniques have been developed for effective differentiation of hESCs into functional hepatocyte-like cells. However, the application of hESC-derived hepatocyte-like cells (hESC-Heps) in the clinic has been constrained by the low yield of fully differentiated cells, small-scale culture, difficulties in harvesting, and immunologic graft rejection. To resolve these shortcomings, we developed a novel 3D differentiation system involving alginate-microencapsulated spheres to improve current hepatic differentiation, providing ready-to-use hESC-Heps. Methods: In this study, we used alginate microencapsulation technology to differentiate human embryonic stem cells into hepatocyte-like cells (hESC-Heps). Hepatic markers of hESC-Heps were examined by qPCR and Western blotting, and hepatic functions of hESC-Heps were evaluated by indocyanine-green uptake and release, and ammonia removal. Results: The maturity and hepatic functions of the hESC-Heps derived from this 3D system were better than those derived from 2D culture. Hepatocyte-enriched genes, such as HNF4α, AFP, and ALB, were expressed at higher levels in 3D hESC-Heps than in 2D hESC-Heps. 3D hESC-Heps could metabolize indocyanine green and had better capacity to scavenge ammonia. In addition, the 3D sodium alginate hydrogel microspheres could block viral entry into the microspheres, and thus protect hESC-Heps in 3D microspheres from viral infection. Conclusion: We developed a novel 3D differentiation system for differentiating hESCs into hepatocyte-like cells by using alginate microcapsules.

Single-cell atlas of human liver development reveals pathways directing hepatic cell fates.

The liver has been studied extensively due to the broad number of diseases affecting its vital functions. However, therapeutic advances have been hampered by the lack of knowledge concerning human hepatic development. Here, we addressed this limitation by describing the developmental trajectories of different cell types that make up the human liver at single-cell resolution. These transcriptomic analyses revealed that sequential cell-to-cell interactions direct functional maturation of hepatocytes, with non-parenchymal cells playing essential roles during organogenesis. We utilized this information to derive bipotential hepatoblast organoids and then exploited this model system to validate the importance of signalling pathways in hepatocyte and cholangiocyte specification. Further insights into hepatic maturation also enabled the identification of stage-specific transcription factors to improve the functionality of hepatocyte-like cells generated from human pluripotent stem cells. Thus, our study establishes a platform to investigate the basic mechanisms directing human liver development and to produce cell types for clinical applications.

SAT153 - Three-dimensional conditions in a perfusion bioreactor to support maturation of human amnion epithelial stem cells into functional hepatocyte-like cells

SAT153 - Three-dimensional conditions in a perfusion bioreactor to support maturation of human amnion epithelial stem cells into functional hepatocyte-like cells

Optimisation of the HepaRG cell line model for drug toxicity studies using two different cultivation conditions: advantages and limitations.

The HepaRG cell line represents a successful model for hepatotoxicity studies. These cells are of human origin and are differentiated in vitro into mature and functional hepatocyte-like cells. The objective of this research was to compare two different culture protocols, Sison-Young et al. 2017 (hereinafter referred as Sison) and Gripon et al. 2002 (hereinafter referred as Biopredic) for HepaRG cells in order to optimise this model for drug metabolism and toxicity testing studies. HepaRG cells obtained from the same batch were cultured according to the described protocols. Using both protocols, differentiated HepaRG cells retained their drug metabolic capacity (major phase I/II enzymes) and transporters, as well as their morphological characteristics. Morphologically, HepaRG cells cultured after the Biopredic protocol formed more apical membranes and small ductular-like structures, than those cultivated using the Sison protocol. Also, the efflux activity of multidrug resistance protein 1 (MDR1) and multidrug resistance-associated protein 1 (MRP1) as well as the activity of uridine-glucuronosyltransferase (UGT) and glutathione S-transferase (GST) were significantly reduced in HepaRG cultured using the Sison protocol. Applying well-established drug cocktails to measure cytochrome P450 (CYPs) activity, we found that production of the corresponding metabolites was hampered in Sison-cultured HepaRG cells, indicating that the activity of CYP1A2, CYP2C9, CYP3A4, CYP2B6 and CYP2C19 was significantly reduced. Moreover, HepaRG sensitivity to well-known drugs, namely diclofenac, amiodarone, imipramine and paracetamol, revealed some differences between the two culture protocols. Furthermore, the HepaRG cells can be maintained with higher viability and sufficient CYPs activity and expression (i.e. CYP3A4, CYP1A2 and CYP2B6) as well as liver-specific functions, using Biopredic compared with the Sison culture protocol. These maintained liver-specific functions might be dependent on the prolongation of the culture conditions in the case of the Biopredic protocol. In conclusion, based on the metabolic activity of HepaRG cells using the standard protocol from Biopredic, we believe that this protocol is optimal for investigating drug metabolism and pharmacokinetic screening studies.

Improved hepatoblast differentiation of human pluripotent stem cells by coffee bean derived graphene quantum dots

Graphene quantum dots (GQDs) have been found to promote the efficient differentiation of stem cells into a variety of cell types. However, their bioactivity and signaling mechanisms in the hepatic lineage have not been fully investigated. Here, we report that the hepatoblast (HB) differentiation rate can be promoted by using Ca2+-rich coffee bean (CB)-derived GQDs, where the incorporated calcium ions activate the signaling of insulin-like growth factor 1 receptor (IGF1R)-protein kinase B (AKT). We also found that the human pluripotent stem cell (hPSC)-derived HBs from the CB-GQD treatment tend to upregulate the maturation into hepatocyte-like cells and downregulate the differentiation towards cholangiocytes, thereby successfully obtaining a large number of functional hepatocyte-like cells. Moreover, the activation of IGF1R from the CB-GQD treatment enhances the HB differentiation efficiency regardless of growth factors, implying that chelated calcium may play a key role in activating IGF1R. Our findings suggest that CB-GQDs with independent bioactivity can be widely used in place of IGF1 and represent a cost-effective growth factor as well as a potential differentiation factor.

Efficiently generate functional hepatic cells from human pluripotent stem cells by complete small-molecule strategy

BackgroundVarious methods have been developed to generate hepatic cells from human pluripotent stem cells (hPSCs) that rely on the combined use of multiple expensive growth factors, limiting industrial-scale production and widespread applications. Small molecules offer an attractive alternative to growth factors for producing hepatic cells since they are more economical and relatively stable.MethodsWe dissect small-molecule combinations and identify the ideal cocktails to achieve an optimally efficient and cost-effective strategy for hepatic cells differentiation, expansion, and maturation.ResultsWe demonstrated that small-molecule cocktail CIP (including CHIR99021, IDE1, and PD0332991) efficiently induced definitive endoderm (DE) formation via increased endogenous TGF-β/Nodal signaling. Furthermore, we identified that combining Vitamin C, Dihexa, and Forskolin (VDF) could substitute growth factors to induce hepatic specification. The obtained hepatoblasts (HBs) could subsequently expand and mature into functional hepatocyte-like cells (HLCs) by the established chemical formulas. Thus, we established a stepwise strategy with complete small molecules for efficiently producing scalable HBs and functionally matured HLCs. The small-molecule-derived HLCs displayed typical functional characteristics as mature hepatocytes in vitro and repopulating injured liver in vivo.ConclusionOur current small-molecule-based hepatic generation protocol presents an efficient and cost-effective platform for the large-scale production of functional human hepatic cells for cell-based therapy and drug discovery using.

A Comparison of Mesenchymal Stem Cell-derived Hepatocyte-like Cells and HepG2 Cells for Use in Drug-Induced Liver Injury Studies.

Liver cell lines obtained from hepatomas, for example, HepG2 cells, are commonly used in drug toxicity studies. However, functional hepatocyte-like cells derived from mesenchymal stem cells (MSCs) could be a better option for use in the study of drug metabolism and toxicity. Overdose of acetaminophen (APAP) and excess alcohol consumption are common causes of liver damage. The objective of the present study was to investigate the use of MSC-derived hepatocyte-like cells (MSCdH) in the assessment of drug-induced liver injury (by using APAP and ethanol), and to compare the toxic effects observed in the MSCdH with those exhibited by HepG2 cells. MSCs were isolated from umbilical cord and their functionality confirmed by their ability to differentiate into adipocytes, osteocytes and hepatocyte-like cells. It was shown that the MSCs successfully differentiated into hepatocyte-like cells, and these cells were further characterised by using various enzyme assays and by assessing albumin secretion and urea synthesis. Cytotoxicity was evaluated in the HepG2 and MSCdH after exposure to ethanol and APAP, with cell viability being determined by using the MTT assay. After exposure to ethanol and to APAP, cell viability decreased in a concentration-dependent manner for both types of hepatocytes. The respective EC50 values of ethanol-induced toxicity for HepG2 and MSCdH cells were 2.5% and 1.3% v/v (p < 0.001); for APAP-induced toxicity they were 19.1mM and 12.6mM (p < 0.001). These findings show that there is a distinct difference between the two types of hepatocytes in terms of APAP-induced and ethanol-induced liver injury.

5-Aminolevulinate improves metabolic recovery and cell survival of the liver following cold preservation.

Rationale: Hibernating thirteen-lined ground squirrels (GS; Ictidomys tridecemlineatus) are naturally adapted to prolonged periods of ultraprofound hypothermia (body temperature < 5 ºC) during torpor, and drastic oscillations of body temperature and ischemia/reperfusion-like stress during their short euthermic interbout arousals. Thus, their superior adaptability may hold tremendous promise for the advancement of donor organ cold preservation and subsequent organ transplantation. However, bridging hibernation research and translational medicine has been impeded by a dearth of in vitro research tools, till the recent establishment of the GS induced pluripotent stem cells (iPSCs). In this study, we reported the generation of functional hepatocyte-like cells (HLCs) from GS iPSCs. As temperature and oxygen supply affect cellular metabolism, we hypothesized that the GS HLCs can metabolically counter drastic temperature and oxygen supply changes. Differentially regulated metabolites can be evaluated and included into the preservation solution to mitigate temperature and ischemia/reperfusion-associated damage to donor livers.Methods: A protocol has been developed to produce GS iPSCs-derived HLCs. Comparative metabolomic analysis on GS HLCs and human donor liver samples revealed changes in metabolites caused by cold storage and rewarming. Human embryonic stem cell (ESC)-derived HLCs and ex vivo cold preservation and reperfusion of isolated rat livers were used to assess candidate metabolites that may have protective effects against preservation-related injuries.Results: GS iPSCs were efficiently differentiated into expandable, cryopreservation-compatible and functional HLCs. Metabolomic analysis unveiled distinct changes of mitochondrial metabolites between GS and human cells following cold storage and rewarming. GS and human HLC-based experiments indicated that the metabolism of 5-aminolevulinate (5-ALA) is key to restricting free radical production during rewarming. Survival of human HLCs was significantly increased following cold exposure and rewarming, as supplemented 5-ALA enhanced Complex III activity and improved mitochondrial respiration. Further, 5-ALA mitigated damage in rat livers following 48-h cold preservation and ex vivo reperfusion. Metabolomic and transcriptomic analyses revealed that supplemented 5-ALA promoted both anabolic and catabolic activities while alleviating cell death, inflammation, hypoxia and other stress responses in isolated perfused rat livers.Conclusion: In the liver, rewarming from ultraprofound hypothermia imposes complex metabolic challenges and stresses on the mitochondria. Metabolites such as 5-ALA can help alleviate mitochondrial stress. Supplementing 5-ALA to the liver preservation solution can substantially improve the functional recovery of rat livers following prolonged cold preservation, rewarming and reperfusion.

Liver injury associated with acute respiratory distress syndrome and the prospects of mesenchymal stem cells therapy for liver failure

The pathogenesis of acute respiratory distress syndrome (ARDS) includes neutrophilic alveolitis, alteration of alveolar epithelium and endothelium, formation of hyaline membranes and microvascular thrombosis, which results in an acute hypoxemic respiratory failure. ARDS results in major structural and cellular changes in organs and organ systems. It causes liver dysfunction in critical patients through paracrine action of cytokines and other pro-inflammatory mediators as well as hypoxemia, oxidative stress, toxins and hypoperfusion. Coronavirus disease 2019 (COVID-19)-associated ARDS affects liver through the development of systemic inflammatory response syndrome and hypoxia as well as cytokine storm. Liver injury manifests itself as increased plasma levels of hepatic transaminases and cholestatic liver enzymes. Stem cell therapy is one of the promising modern methods for treating ARDS-induced liver failure. Many studies showed the ability of multipotent mesenchymal stem cells (MMSCs) to differentiate into functional hepatocyte-like cells, which were then successfully used for liver regeneration. MMSCs were proven to be able to prevent the apoptosis of hepatocytes, as well as have anti-fibrotic and anti-inflammatory activity which allows their successful use in the treatment of ARDS-induced liver injury.