Mature Hepatocytes Research Articles

A two-step strategy to expand primary human hepatocytes in vitro with efficient metabolic and regenerative capacities

BackgroundPrimary human hepatocytes (PHHs) are highly valuable for drug-metabolism evaluation, liver disease modeling and hepatocyte transplantation. However, their availability is significantly restricted due to limited donor sources, alongside their constrained proliferation capabilities and reduced functionality when cultured in vitro. To address this challenge, we aimed to develop a novel method to efficiently expand PHHs in vitro without a loss of function.MethodsBy mimicking the in vivo liver regeneration route, we developed a two-step strategy involving the de-differentiation/expansion and subsequent maturation of PHHs to generate abundant functional hepatocytes in vitro. Initially, we applied SiPer, a prediction algorithm, to identify candidate small molecules capable of activating liver regenerative transcription factors, thereby formulating a novel hepatic expansion medium to de-differentiate PHHs into proliferative human hepatic progenitor-like cells (ProHPLCs). These ProHPLCs were then re-differentiated into functionally mature hepatocytes using a new hepatocyte maturation condition. Additionally, we investigated the underlying mechanism of PHHs expansion under our new conditions.ResultsThe novel hepatic expansion medium containing hydrocortisone facilitated the de-differentiation of PHHs into ProHPLCs, which exhibited key hepatic progenitor characteristics and demonstrated a marked increase in proliferation capacity compared to cells cultivated in previously established expansion conditions. Remarkably, these subsequent matured hepatocytes rivaled PHHs in terms of transcriptome profiles, drug metabolizing activities and in vivo engraftment capabilities. Importantly, our findings suggest that the enhanced expansion of PHHs by hydrocortisone may be mediated through the PPARα signaling pathway and regenerative transcription factors.ConclusionsThis study presents a two-step strategy that initially induces PHHs into a proliferative state (ProHPLCs) to ensure sufficient cell quantity, followed by the maturation of ProHPLCs into fully functional hepatocytes to guarantee optimal cell quality. This approach offers a promising means of producing large numbers of seeding cells for hepatocyte-based applications.

Genome Engineering of Primary and Pluripotent Stem Cell-Derived Hepatocytes for Modeling Liver Tumor Formation.

Genome editing has demonstrated its utility in generating isogenic cell-based disease models, enabling the precise introduction of genetic alterations into wild-type cells to mimic disease phenotypes and explore underlying mechanisms. However, its application in liver-related diseases has been limited by challenges in genetic modification of mature hepatocytes in a dish. Here, we conducted a systematic comparison of various methods for primary hepatocyte culture and gene delivery to achieve robust genome editing of hepatocytes ex vivo. Our efforts yielded editing efficiencies of up to 80% in primary murine hepatocytes cultured in monolayer and 20% in organoids. To model human hepatic tumorigenesis, we utilized hepatocytes differentiated from human pluripotent stem cells (hPSCs) as an alternative human hepatocyte source. We developed a series of cellular models by introducing various single or combined oncogenic alterations into hPSC-derived hepatocytes. Our findings demonstrated that distinct mutational patterns led to phenotypic variances, affecting both overgrowth and transcriptional profiles. Notably, we discovered that the PI3KCA E542K mutant, whether alone or in combination with exogenous c-MYC, significantly impaired hepatocyte functions and facilitated cancer metabolic reprogramming, highlighting the critical roles of these frequently mutated genes in driving liver neoplasia. In conclusion, our study demonstrates genome-engineered hepatocytes as valuable cellular models of hepatocarcinoma, providing insights into early tumorigenesis mechanisms.

P02-28 Microfluidics technology as a tool to improve maturation and drug metabolism capacity of stem cell-derived hepatocytes in vitro

P02-28 Microfluidics technology as a tool to improve maturation and drug metabolism capacity of stem cell-derived hepatocytes in vitro

Framework nucleic Acid-MicroRNA mediated hepatic differentiation and functional hepatic spheroid development for treating acute liver failure

The specific induction of hepatic differentiation presents a significant challenge in developing alternative liver cell sources and viable strategies for clinical therapy of acute liver failure (ALF). The past decade has witnessed the blossom of microRNAs in regenerative medicine. Herein, microRNA 122-functionalized tetrahedral framework nucleic acid (FNA-miR-122) has emerged as an unprecedented and potential platform for directing the hepatic differentiation of adipose-derived mesenchymal stem cells (ADMSCs), which offers a straightforward and cost-effective method for generating functional hepatocyte-like cells (FNA-miR-122-iHep). Additionally, we have successfully established a liver organoid synthesis strategy by optimizing the co-culture of FNA-miR-122-iHep with endothelial cells (HUVECs), resulting in functional Hep:HUE-liver spheroids. Transcriptome analysis not only uncovered the potential molecular mechanisms through which miR-122 influences hepatic differentiation in ADMSCs, but also clarified that Hep:HUE-liver spheroids could further facilitate hepatocyte maturation and improved tissue-specific functions, which may provide new hints to be used to develop a hepatic organoid platform. Notably, compared to transplanted ADMSCs and Hep-liver spheroid, respectively, both FNA-miR-122-iHep-based single cell therapy and Hep:HUE-liver spheroid-based therapy showed high efficacy in treating ALF in vivo. Collectively, this research establishes a robust system using microRNA to induce ADMSCs into functional hepatocyte-like cells and to generate hepatic organoids in vitro, promising a highly efficient therapeutic approach for ALF.

Generation of human hepatobiliary organoids with a functional bile duct from chemically induced liver progenitor cells

BackgroundLiver disease imposes a significant medical burden that persists due to a shortage of liver donors and an incomplete understanding of liver disease progression. Hepatobiliary organoids (HBOs) could provide an in vitro mini-organ model to increase the understanding of the liver and may benefit the development of regenerative medicine.MethodsIn this study, we aimed to establish HBOs with bile duct (BD) structures and mature hepatocytes (MHs) using human chemically induced liver progenitor cells (hCLiPs). hCLiPs were induced in mature cryo-hepatocytes using a small-molecule cocktail of TGF-β inhibitor (A-83-01, A), GSK3 inhibitor (CHIR99021, C), and 10% FBS (FAC). HBOs were then formed by seeding hCLiPs into ultralow attachment plates and culturing them with a combination of small molecules of Rock-inhibitor (Y-27632) and AC (YAC).ResultsThese HBOs exhibited bile canaliculi of MHs connected to BD structures, mimicking bile secretion and transportation functions of the liver. The organoids showed gene expression patterns consistent with both MHs and BD structures, and functional assays confirmed their ability to transport the bile analogs of rhodamine-123 and CLF. Functional patient-specific HBOs were also successfully created from hCLiPs sourced from cirrhotic liver tissues.ConclusionsThis study demonstrated the potential of human HBOs as an efficient model for studying hepatobiliary diseases, drug discovery, and personalized medicine.

Efficacy of chemically induced human hepatic progenitor cells from diseased liver against nonalcoholic steatohepatitis model.

Numerous chemical reprogramming techniques have been reported, rendering them applicable to regenerative medicine research. The aim of our study was to evaluate the therapeutic potential of human CLiP derived from clinical specimens transplanted into a nonalcoholic steatohepatitis (NASH) mouse model of liver fibrosis. We successfully generated chemically induced liver progenitor (CLiP), which exhibited progenitor-like characteristics, through stimulation with low-molecular-weight compounds. We elucidated their cell differentiation ability and therapeutic effects. However, the therapeutic efficacy of human CLiP generated from clinical samples on liver fibrosis, such as liver cirrhosis, remains unproven. Following a 4 week period, transplanted human CLiP in the NASH model differentiated into mature hepatocytes and demonstrated suppressive effects on liver injury markers (i.e., aspartate transaminase and alanine transaminase). Although genes related to inflammation and fat deposition did not change in the human CLiP transplantation group, liver fibrosis-related factors (Acta2 and Col1A1) showed suppressive effects on gene expression following transplantation, with approximately a 60% reduction in collagen fibers. Importantly, human CLiP could be efficiently induced from hepatocytes isolated from the cirrhotic liver, underscoring the feasibility of using autologous hepatocytes to produce human CLiP. Our findings demonstrate the effectiveness of human CLiP transplantation as a viable cellular therapy for liver fibrosis, including NASH liver. These results hold promise for the development of liver antifibrosis therapy utilizing human CLiP within the field of liver regenerative medicine.

ESRP2-microRNA-122 axis directs the postnatal onset of liver polyploidization and maturation.

Hepatocyte polyploidy and maturity are critical to acquiring specialized liver functions. Multiple intra- and extracellular factors influence ploidy, but how they cooperate temporally to steer liver polyploidization and maturation or how post-transcriptional mechanisms integrate into these paradigms is unknown. Here, we identified an important regulatory hierarchy in which postnatal activation of Epithelial-Splicing-Regulatory-Protein-2 (ESRP2) stimulates biogenesis of liver-specific microRNA (miR-122), thereby facilitating polyploidization, maturation, and functional competence of hepatocytes. By determining transcriptome-wide protein-RNA interactions in vivo and integrating them with single-cell and bulk hepatocyte RNA-seq datasets, we delineate an ESRP2-driven RNA processing program that drives sequential replacement of fetal-to-adult transcript isoforms. Specifically, ESRP2 binds the primary miR-122 host gene transcript to promote its processing/biogenesis. Combining constitutive and inducible ESRP2 gain- and loss-of-function mice models with miR-122 rescue experiments, we demonstrate that timed activation of ESRP2 augments miR-122-driven program of cytokinesis failure, ensuring proper onset and extent of hepatocyte polyploidization.

HDAC inhibitors support long-term expansion of porcine hepatocytes in vitro

Hepatocyte transplantation is an effective treatment for end-stage liver disease. However, due to the limited supply of human hepatocytes, porcine hepatocytes have garnered attention as a potential alternative source. Nonetheless, traditional primary porcine hepatocytes exhibit certain limitations in function maintenance and in vitro proliferation. This study has discovered that by using histone deacetylase inhibitors (HDACi), primary porcine hepatocytes can be successfully reprogrammed into liver progenitor cells with high proliferative potential. This method enables porcine hepatocytes to proliferate over an extended period in vitro and exhibit increased susceptibility in lentivirus-mediated gene modification. These liver progenitor cells can readily differentiate into mature hepatocytes and, upon microencapsulation transplantation into mice with acute liver failure, significantly improve the survival rate. This research provides new possibilities for the application of porcine hepatocytes in the treatment of end-stage liver disease.

Aberrant differentiation and proliferation of hepatocytes in chronic liver injury and liver tumors.

Chronic liver injury induces liver cirrhosis and facilitates hepatocarcinogenesis. However, the effects of this condition on hepatocyte proliferation and differentiation are unclear. We showed that rodent hepatocytes display a ductular phenotype when they are cultured within a collagenous matrix. This process involves transdifferentiation without the emergence of hepatoblastic features and is at least partially reversible. During the ductular reaction in chronic liver diseases with progressive fibrosis, some hepatocytes, especially those adjacent to ectopic ductules, demonstrate ductular transdifferentiation, but the majority of increased ductules originate from the existing bile ductular system that undergoes extensive remodeling. In chronic injury, hepatocyte proliferation is weak but sustained, and most regenerative nodules in liver cirrhosis are composed of clonally proliferating hepatocytes, suggesting that a small fraction of hepatocytes maintain their proliferative capacity in chronic injury. In mouse hepatocarcinogenesis models, hepatocytes activate the expression of various fetal/neonatal genes, indicating that these cells undergo dedifferentiation. Hepatocyte-specific somatic integration of various oncogenes in mice demonstrated that hepatocytes may be the cells of origin for a broad spectrum of liver tumors through transdifferentiation and dedifferentiation. In conclusion, the phenotypic plasticity and heterogeneity of mature hepatocytes are important for understanding the pathogenesis of chronic liver diseases and liver tumors.

Hepatocytes differentiate into intestinal epithelial cells through a hybrid epithelial/mesenchymal cell state in culture

Hepatocytes play important roles in the liver, but in culture, they immediately lose function and dedifferentiate into progenitor-like cells. Although this unique feature is well-known, the dynamics and mechanisms of hepatocyte dedifferentiation and the differentiation potential of dedifferentiated hepatocytes (dediHeps) require further investigation. Here, we employ a culture system specifically established for hepatic progenitor cells to study hepatocyte dedifferentiation. We found that hepatocytes dedifferentiate with a hybrid epithelial/mesenchymal phenotype, which is required for the induction and maintenance of dediHeps, and exhibit Vimentin-dependent propagation, upon inhibition of the Hippo signaling pathway. The dediHeps re-differentiate into mature hepatocytes by forming aggregates, enabling reconstitution of hepatic tissues in vivo. Moreover, dediHeps have an unexpected differentiation potential into intestinal epithelial cells that can form organoids in three-dimensional culture and reconstitute colonic epithelia after transplantation. This remarkable plasticity will be useful in the study and treatment of intestinal metaplasia and related diseases in the liver.

Generation and characterization of mature hepatocyte organoids for liver metabolic studies.

Hepatocyte organoids (HOs) generated in vitro are powerful tools for liver regeneration. However, previously reported HOs have mostly been fetal in nature with low expression levels of metabolic genes characteristic of adult liver functions, hampering their application in studies of metabolic regulation and therapeutic testing for liver disorders. Here, we report development of novel culture conditions that combine optimized levels of triiodothyronine (T3) with the removal of growth factors to enable successful generation of mature hepatocyte organoids (MHOs) of both mouse and human origin with metabolic functions characteristic of adult livers. We show that the MHOs can be used to study various metabolic functions including bile and urea production, zonal metabolic gene expression, and metabolic alterations in both alcoholic liver disease and non-alcoholic fatty liver disease, as well as hepatocyte proliferation, injury and cell fate changes. Notably, MHOs derived from human fetal hepatocytes also show improved hepatitis B virus infection. Therefore, these MHOs provide a powerful in vitro model for studies of human liver physiology and diseases. The human MHOs are potentially also a robust research tool for therapeutic development.

Mapping of mitogen and metabolic sensitivity in organoids defines requirements for human hepatocyte growth

Mechanisms underlying human hepatocyte growth in development and regeneration are incompletely understood. In vitro, human fetal hepatocytes (FH) can be robustly grown as organoids, while adult primary human hepatocyte (PHH) organoids remain difficult to expand, suggesting different growth requirements between fetal and adult hepatocytes. Here, we characterize hepatocyte organoid outgrowth using temporal transcriptomic and phenotypic approaches. FHs initiate reciprocal transcriptional programs involving increased proliferation and repressed lipid metabolism upon initiation of organoid growth. We exploit these insights to design maturation conditions for FH organoids, resulting in acquisition of mature hepatocyte morphological traits and increased expression of functional markers. During PHH organoid outgrowth in the same culture condition as for FHs, the adult transcriptomes initially mimic the fetal transcriptomic signatures, but PHHs rapidly acquire disbalanced proliferation-lipid metabolism dynamics, resulting in steatosis and halted organoid growth. IL6 supplementation, as emerged from the fetal dataset, and simultaneous activation of the metabolic regulator FXR, prevents steatosis and promotes PHH proliferation, resulting in improved expansion of the derived organoids. Single-cell RNA sequencing analyses reveal preservation of their fetal and adult hepatocyte identities in the respective organoid cultures. Our findings uncover mitogen requirements and metabolic differences determining proliferation of hepatocytes changing from development to adulthood.

Direct reprogramming of fibroblasts into functional hepatocytes via CRISPRa activation of endogenous Gata4 and Foxa3.

The ability to generate functional hepatocytes without relying on donor liver organs holds significant therapeutic promise in the fields of regenerative medicine and potential liver disease treatments. Clustered regularly interspaced short palindromic repeats (CRISPR) activator (CRISPRa) is a powerful tool that can conveniently and efficiently activate the expression of multiple endogenous genes simultaneously, providing a new strategy for cell fate determination. The main purpose of this study is to explore the feasibility of applying CRISPRa for hepatocyte reprogramming and its application in the treatment of mouse liver fibrosis. The differentiation of mouse embryonic fibroblasts (MEFs) into functional induced hepatocyte-like cells (iHeps) was achieved by utilizing the CRISPRa synergistic activation mediator (SAM) system, which drove the combined expression of three endogenous transcription factors- Gata4, Foxa3 , and Hnf1a -or alternatively, the expression of two transcription factors, Gata4 and Foxa3 . In vivo , we injected adeno-associated virus serotype 6 (AAV6) carrying the CRISPRa SAM system into liver fibrotic Col1a1-CreER ; Cas9fl/fl mice, effectively activating the expression of endogenous Gata4 and Foxa3 in fibroblasts. The endogenous transcriptional activation of genes was confirmed using real-time quantitative polymerase chain reaction (RT-qPCR) and RNA-seq, and the morphology and characteristics of the induced hepatocytes were observed through microscopy. The level of hepatocyte reprogramming in vivo is detected by immunofluorescence staining, while the improvement of liver fibrosis is evaluated through Sirius red staining, alpha-smooth muscle actin (α-SMA) immunofluorescence staining, and blood alanine aminotransferase (ALT) examination. Activation of only two factors, Gata4 and Foxa3 , via CRISPRa was sufficient to successfully induce the transformation of MEFs into iHeps. These iHeps could be expanded in vitro and displayed functional characteristics similar to those of mature hepatocytes, such as drug metabolism and glycogen storage. Additionally, AAV6-based delivery of the CRISPRa SAM system effectively induced the hepatic reprogramming from fibroblasts in mice with live fibrosis. After 8 weeks of induction, the reprogrammed hepatocytes comprised 0.87% of the total hepatocyte population in the mice, significantly reducing liver fibrosis. CRISPRa-induced hepatocyte reprogramming may be a promising strategy for generating functional hepatocytes and treating liver fibrosis caused by hepatic diseases.

Deletion of Tgm2 suppresses BMP-mediated hepatocyte-to-cholangiocyte metaplasia in ductular reaction.

Transglutaminase 2 (Tgm2) plays an essential role in hepatic repair following prolonged toxic injury. During cholestatic liver injury, the intrahepatic cholangiocytes undergo dynamic tissue expansion and remodelling, referred to as ductular reaction (DR), which is crucial for liver regeneration. However, the molecular mechanisms governing the dynamics of active cells in DR are still largely unclear. Here, we generated Tgm2-knockout mice (Tgm2-/-) and Tgm2-CreERT2-Rosa26-mTmG flox/flox (Tgm2CreERT2-R26T/Gf/f) mice and performed a three-dimensional (3D) collagen gel culture of mouse hepatocytes to demonstrate how Tgm2 signalling is involved in DR to remodel intrahepatic cholangiocytes. Our results showed that the deletion of Tgm2 adversely affected the functionality and maturity of the proliferative cholangiocytes in DR, thus leading to more severe cholestasis during DDC-induced liver injury. Additionally, Tgm2 hepatocytes played a crucial role in the regulation of DR through metaplasia. We unveiled that Tgm2 regulated H3K4me3Q5ser via serotonin to promote BMP signalling activation to participate in DR. Besides, we revealed that the activation or inhibition of BMP signalling could promote or suppress the development and maturation of cholangiocytes in DDC-induced DR. Furthermore, our 3D collagen gel culture assay indicated that Tgm2 was vital for the development of cholangiocytes in vitro. Our results uncovered a considerable role of BMP signalling in controlling metaplasia of Tgm2 hepatocytes in DR and revealed the phenotypic plasticity of mature hepatocytes.

Improvement in aged liver regeneration using cell transplantation with chemically induced liver progenitors.

A decrease in the regenerative capacity of age-damaged liver tissue has been reported. Liver progenitor cells may play an important role in the regeneration of injured livers. In the present study we aimed to investigate improvements in the regenerative capacity of age-damaged livers using chemically induced liver progenitors (CLiPs) derived from mature hepatocytes. Old (>90 weeks) and young (<20 weeks) mice underwent 70% hepatectomy, with or without trans-splenic CLiP administration. The residual liver/bodyweight (LW/BW) ratio was measured on postoperative days 1 and 7, and changes in liver regeneration and histology were evaluated. At 7 days post-hepatectomy, LW/BW ratios were significantly better in CLiP-treated old mice than in untreated old mice (p = .02). By contrast, no effect of CLiP transplantation was observed in young mice (p = .62). Immunofluorescence staining of liver tissue after CLiP administration showed an increase in Ki67-positive cells (p < .01). Flow cytometry analysis of green fluorescent protein-labeled CLiPs indicated that transplanted CLiPs differentiated into mature hepatocytes and were present in the recipient liver. CLiP transplantation appears to ameliorate the age-related decline in liver regeneration in mice.

Evaluation of different media compositions promoting hepatocyte differentiation in the canine liver organoid model

Organoids are 3-dimensional (3D) self-assembled structures capable of replicating the microanatomy and physiology of the epithelial components of their organ of origin. Adult stem cell (ASC) derived organoids from the liver have previously been shown to differentiate into primarily mature cholangiocytes, and their partial differentiation into functional hepatocytes can be promoted using specific media compositions. While full morphological differentiation of mature hepatocytes from ASCs has not yet been reported for any species, the functional differentiation can be approximated using various media compositions.Six differentiation media formulations from published studies on hepatic organoids were used for the differentiation protocol. Target species for these protocols were humans, mice, cats, and dogs, and encompassed various combinations and concentrations of four major hepatocyte media components: Bone morphogenetic protein 7 (BMP7), Fibroblast Growth Factor 19 (FGF19), Dexamethasone (Dex), and Gamma-Secretase Inhibitor IX (DAPT). Additionally, removing R-spondin from basic organoid media has previously been shown to drive the differentiation of ASC into mature hepatocytes. Differentiation media (N = 20) were designed to encompass combinations of the four major hepatocyte media components. The preferred differentiation of ASC-derived organoids from liver tissue into mature hepatocytes over cholangiocytes was confirmed by albumin production in the culture supernatant.Out of the twenty media compositions tested, six media resulted in the production of the highest amounts of albumin in the supernatant of the organoids. The cell lines cultured using these six media were further characterized via histological staining, transmission electron microscopy, RNA in situ hybridization, analysis of gene expression patterns, immunofluorescence, and label-free proteomics. The results indicate that preferential hepatocyte maturation from canine ADC-derived organoids from liver tissue is mainly driven by Dexamethasone and DAPT components. FGF19 did not enhance organoid differentiation but improved cell culture survival. Furthermore, we confirm that removing R-spondin from the media is crucial for establishing mature hepatic organoid cultures.

Abstract 5437: Chemoproteomics-enabled discovery of covalent inhibitors targeted at AGPAT4: Unravelling tumor lineage plasticity to overcome drug resistance in hepatocellular carcinoma

Abstract The liver is a unique organ that is responsible for many metabolic functions. During hepatocellular carcinoma (HCC) development, these metabolic machineries are extensively reprogrammed to support the insatiable nutrient requirements of HCC. Tumor lineage plasticity, a recognized hallmark of cancer, is a phenomenon in which tumor cells co-opt developmental pathways to attain cellular plasticity, enabling them to evade targeted therapeutic interventions. Cancer cells can reprogram their metabolic pathways to match their increased metabolic needs for cancer cell survival under adverse conditions. Identifying novel metabolic targets related to stemness can offer promising strategies for targeting cancer stemness roots and hence to kill and control their growth. Through pathway enrichment analysis of genes that linked metabolism and stemness, we identified an aberrant glycerophospholipid metabolism signature, with AGPAT4 ranking as the top-hit. AGPAT4 upregulation in HCC is strongly correlated with aggressive clinical features, including survival, metastasis, and stemness signatures. AGPAT4 expression peaked during early liver progenitor development, decreased during hepatocyte maturation and progressively increased from well-differentiated to poorly differentiated HCCs. Enrichment of AGPAT4 in HCC is mediated by promoter binding of SOX9 to drive AGPAT4 transcriptional activity. AGPAT4 inhibition can mitigate tumor initiation, self-renewal, metastasis, and sorafenib resistance. Mechanistic studies revealed an AGPAT4-mediated phosphatidic acid production axis that promotes HCC through the regulation of mTOR signaling. Inhibition of Agpat4 by AAV8 shRNA reduced tumorigenicity and stemness, and sensitized HCC tumors to sorafenib. AGPAT4 overexpression can predict sorafenib response in clinical settings. Through chemoproteomics screening of a cysteine-reacting compound library using activity-based protein profiling, a cysteine-reacting compound with high binding affinity and selectivity towards AGPAT4 was identified and found to work synergistically with sorafenib to suppress HCC, as demonstrated in HCC patient-derived tumor xenograft (PDTX) models. Toxicity analysis through histological examination of organs, body weight measurements, and biochemical tests revealed minimal toxicity associated with the covalent inhibitor. In conclusion, AGPAT4 is a novel metabolic driver of oncogenic stemness, dedifferentiation, and metastasis in HCC. AGPAT4-induced tumor lineage plasticity may represent an Achilles heel for HCC treatment, and inhibition of AGPAT4 may widen the therapeutic window for sorafenib treatment in the clinic. Citation Format: Kai-Yu Ng, Tin-Yan Koo, Tsz-Lok Fong, Ya Gao, Tin-Lok Wong, Yuan Gao, Jing-Ping Yun, Xin-Yuan Guan, Ming Liu, Clive YS Chung, Stephanie Ma. Chemoproteomics-enabled discovery of covalent inhibitors targeted at AGPAT4: Unravelling tumor lineage plasticity to overcome drug resistance in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5437.

In vitro characterization of 3D culture-based differentiation of human liver stem cells.

Introduction: The lack of functional hepatocytes poses a significant challenge for drug safety testing and therapeutic applications due to the inability of mature hepatocytes to expand and their tendency to lose functionality in vitro. Previous studies have demonstrated the potential of Human Liver Stem Cells (HLSCs) to differentiate into hepatocyte-like cells within an in vitro rotary cell culture system, guided by a combination of growth factors and molecules known to regulate hepatocyte maturation. In this study, we employed a matrix multi-assay approach to comprehensively characterize HLSC differentiation. Methods: We evaluated the expression of hepatic markers using qRT-PCR, immunofluorescence, and Western blot analysis. Additionally, we measured urea and FVIII secretion into the supernatant and developed an updated indocyanine green in vitro assay to assess hepatocyte functionality. Results: Molecular analyses of differentiated HLSC aggregates revealed significant upregulation of hepatic genes, including CYP450, urea cycle enzymes, and uptake transporters exclusively expressed on the sinusoidal side of mature hepatocytes, evident as early as 1day post-differentiation. Interestingly, HLSCs transiently upregulated stem cell markers during differentiation, followed by downregulation after 7days. Furthermore, differentiated aggregates demonstrated the ability to release urea and FVIII into the supernatant as early as the first 24h, with accumulation over time. Discussion: These findings suggest that a 3D rotation culture system may facilitate rapid hepatic differentiation of HLSCs. Despite the limitations of this rotary culture system, its unique advantages hold promise for characterizing HLSC GMP batches for clinical applications.

A Novel Approach to Orthotopic Hepatocyte Transplantation Engineered With Liver Hydrogel for Fibrotic Livers, Enhancing Cell-Cell Interaction and Angiogenesis.

Hepatocyte transplantation (HCT) is a potential bridging therapy or an alternative to liver transplantation. Conventionally, single-cell hepatocytes are injected via the portal vein. This strategy, however, has yet to overcome poor cell engraftment and function. Therefore, we developed an orthotopic HCT method using a liver-derived extracellular matrix (L-ECM) gel. PXB cells (flesh mature human hepatocytes) were dispersed into the hydrogel solution in vitro, and the gel solution was immediately gelated in 37°C incubators to investigate the affinity between mature human hepatocyte and the L-ECM gel. During the 3-day cultivation in hepatocyte medium, PXB cells formed cell aggregates via cell-cell interactions. Quantitative analysis revealed human albumin production in culture supernatants. For the in vivo assay, PXB cells were encapsulated in the L-ECM gel and transplanted between the liver lobes of normal rats. Pathologically, the L-ECM gel was localized at the transplant site and retained PXB cells. Cell survival and hepatic function marker expression were verified in another rat model wherein thioacetamide was administered to induce liver fibrosis. Moreover, cell-cell interactions and angiogenesis were enhanced in the L-ECM gel compared with that in the collagen gel. Our results indicate that L-ECM gels can help engraft transplanted hepatocytes and express hepatic function as a scaffold for cell transplantation.

Role of connexin 32 in the directional differentiation of induced pluripotent stem cells into hepatocytes.

This study aimed to explore the role of connexin 32 (Cx32) in the directional differentiation of induced pluripotent stem cells (iPSCs) into hepatocytes. Urine-derived epithelial cells were collected from the fresh urine of a healthy donor and transducted with reprogramming plasmid mixture to generate iPSCs. The iPSCs were then directionally differentiated into hepatocytes. During the differentiation, the upregulated and downregulated groups were treated with vitamin K2 (VK2) and 2-aminoethoxyboronate diphenylester (2-APB) to increase and inhibit Cx32 expression, respectively. The control group was not treated with the regulatory factor. Expression of Cx32 and hepatocyte-specific markers, including AFP, hepatocyte nuclear factor 4α (HNF-4α), albumin (ALB) and cytokeratin 18 (CK18) were detected. It indicated that Cx32 expression was not observed in iPSCs, but gradually increased during the process of hepatic differentiation from iPSCs. Upregulation of Cx32 expression by VK2 treatment promoted hepatocyte maturation and enhanced the expression of the aforementioned hepatic specific markers, whereas downregulation of Cx32 expression by 2-APB treatment had the opposite effects. In conclusion, urine-derived iPSCs could be directionally differentiated into hepatocytes. Up-regulation of Cx32 improves the efficiency and maturity of differentiation of iPSCs into hepatocytes, and Cx32 may be a promoting factor during the process of hepatic differentiation from iPSCs.