Hepatocyte-like Cells Research Articles

Modeling metabolic-associated steatohepatitis with human pluripotent stem cell-derived liver organoids.

Metabolic-associated steatohepatitis (MASH) is one of the most prevalent liver diseases worldwide, with a global prevalence estimated between 3% and 5%, posing a significant health burden. Human liver organoids (HLOs) have previously been generated to model steatohepatitis, offering a potential cellular disease model for studying MASH. However, the current HLO model lacks detailed molecular characterizations and requires further improvement. HLOs derived from human pluripotent stem cells were treated with oleic acid and TGFβ to mimic the MASH progression. Treated HLOs were then analyzed using both bulk and single-cell RNA sequencing. Functional characterization was performed through staining with BODIPY, TMRM, CellROX, and Collagen I, as well as terminal deoxynucleotidyl transferase dUTP nick end labeling and ELISA assays. In addition, a test using the MASH HLO model to validate the hepatoprotective effects of several herb extracts was also conducted. Both RNA-seq and single-cell RNA sequencing demonstrated a close resemblance of multiple molecular signatures and key intercellular communications in and between hepatocyte-like cells and stellate-like cells in the MASH HLO model, compared to human MASH. Furthermore, functional characterizations revealed progressive features of human MASH in the MASH HLO model, including severe steatosis, oxidative stress, mitochondrial dysfunction, inflammation, and fibrosis. In addition, the Schisandra extracts have been demonstrated to have significant antioxidative, anti-inflammatory, and antifibrotic properties in the context of MASH. This study offers an improved HLO disease model of human MASH, which can be potentially applied to facilitate the understanding of the MASH pathogenesis and the discovery of effective treatments.

Statin-associated regulation of hepatic PNPLA3 in patients without known liver disease.

Statins are used for metabolic dysfunction-associated steatotic liver disease (MASLD) (NAFLD) treatment, but their role in this context is unclear. Genetic variants of patatin-like phospholipase domain containing 3 (PNPLA3) are associated with MASLD susceptibility and statin treatment efficacy. Access to liver biopsies before established MASLD is limited, and statins and PNPLA3 in early liver steatosis are thus difficult to study. Liver biopsies were collected from 261 patients without known liver disease at surgery and stratified based on statin use and criteria for the metabolic syndrome (MS). Genotypes and transcript levels were measured using Illumina and Affymetrix arrays, and metabolic and lipoprotein profiles by clinical assays. Statin effects on PNPLA3, de novo lipogenesis (DNL), and lipid accumulation were further studied in vitro. The PNPLA3I148M genetic variant was associated with significantly lower hepatic levels of cholesterol synthesis-associated transcripts. Patients with MS had significantly higher hepatic levels of MASLD and lipogenesis-associated transcripts than non-MS patients. Patients with MS on statin therapy had significantly higher hepatic levels of PNPLA3, acetyl-CoA carboxylase alpha, and ATP citrate lyase, and statin use was associated with higher plasma fasting glucose, insulin, and HbA1c. Exposure of hepatocyte-like HepG2 cells to atorvastatin promoted intracellular accumulation of triglycerides and lipogenesis-associated transcripts. Atorvastatin-exposure of HepG2, sterol O-acyltransferase (SOAT) 2-only-HepG2, primary human hepatic stellate, and hepatic stellate cell-like LX2 cells significantly increased levels of PNPLA3 and SREBF2-target genes, whereas knockdown of SREBF2 attenuated this effect. Collectively, these observations suggest statin-associated regulation of PNPLA3 and DNL in liver. The potential interaction between PNPLA3 genotype and metabolic status should be considered in future studies in the context of statin therapy for MASLD.

Palmitic Acid Induced a Dedifferentiation Profile at the Transcriptome Level: A Collagen Synthesis but no Triglyceride Accumulation in Hepatocyte-Like Cells Derived From Human-Induced Pluripotent Stem Cells Cultivated Inside Organ on a Chip.

Nonalcoholic fatty liver disease (NAFLD) is one of the main causes of critical liver diseases leading to steatosis, steatohepatitis, fibrosis, and ultimately to liver cirrhosis and hepatic carcinoma. In this study, the effect of palmitic acid (PA), one of the most abundant dietary fatty acids, was investigated using an organ-on-a-chip (OoC) technology on hepatocyte-like cells derived from human-induced pluripotent stem cells (hiPSCs). After 1 week of hepatic maturation, followed by 1 week of exposure, the transcriptomic analysis showed lower liver transcription factor activity. It also revealed that 318 genes were differentially expressed between the control and 0.5-mM PA conditions. The 0.5-mM PA conditions were characterized by the downregulation of hepatic markers (liver transcription factors, phase I and phase II metabolism genes) of lipidic genes (metabolism and transport). In parallel, the 0.5-mM PA treatment upregulated several extracellular matrix genes (such as collagen genes). The physiopathological staining demonstrated no lipid accumulation in our model and confirmed the secretion of collagen in the 0.5-mM PA conditions. However, the production of albumin, the metabolic biotransformation by the cytochrome P450 enzymes, and the biliary acid concentrations were not altered by the PA treatments. Overall, our data illustrated the response to PA characterized by an early stage of dedifferentiation observed at the transcriptomic levels associated with a modification of the collagenic profile but without lipid accumulation. We believe that our model provides new insight of the onset of palmitic lipotoxicity in the early stage of NAFLD.

A Study on iPSC-Associated Factors in the Generation of Hepatocytes.

Hepatocytes are an attractive cell source in hepatic tissue engineering because they are the primary cells of the liver, maintaining liver homeostasis through their intrinsic function. Due to the increasing demand for liver donors, a wide range of methods are being studied to obtain functionally active hepatocytes. iPSCs are one of the alternative cell sources, which shows great promise as a tool for generating hepatocytes. This study determined whether factors associated with iPSCs contributed to variation in hepatocyte-like cells derived from iPSCs. The factors of concern for the iPSCs included the culture system, the source of iPSCs, and cell seeding density for initiating the differentiation. Our results found iPSC-dependent variances among differentiated hepatocyte-like cells. The matrix used in culturing iPSCs significantly impacts cell morphologies, characteristics, and the expression of pluripotent genes, such as OCT4 and SOX2, varied in iPSCs derived from different sources. These characteristics, in turn, play a consequential role in determining the functional activity of the iPSC-derived hepatocyte-like cells. In addition, cell seeding density was observed to be an essential factor for the efficient generation of iPSC-derived hepatocyte-like cells, with 2- 4 × 10 cells/cm of seeding density resulting in good morphology and functionality. This study provides the baseline of effective differentiation protocols for iPSC-derived hepatocyte-like cells with the appropriate conditions, including cell culture media, iPSC source, and the seeding density of iPSCs.

Accelerated production of human epithelial organoids in a miniaturized spinning bioreactor

Conventional static culture of organoids necessitates weekly manual passaging and results in nonhomogeneous exposure of organoids to nutrients, oxygen, and toxic metabolites. Here, we developed a miniaturized spinning bioreactor, RPMotion, specifically optimized for accelerated and cost-effective culture of epithelial organoids under homogeneous conditions. We established tissue-specific RPMotion settings and standard operating protocols for the expansion of human epithelial organoids derived from the liver, intestine, and pancreas. All organoid types proliferated faster in the bioreactor (5.2-fold, 3-fold, and 4-fold, respectively) compared to static culture while keeping their organ-specific phenotypes. We confirmed that the bioreactor is suitable for organoid establishment directly from biopsies and for long-term expansion of liver organoids. Furthermore, we showed that after accelerated expansion, liver organoids can be differentiated into hepatocyte-like cells in the RPMotion bioreactor. In conclusion, this miniaturized bioreactor enables work-, time-, and cost-efficient organoid culture, holding great promise for organoid-based fundamental and translational research and development.

Induced hepatocyte-like cells derived from adipose-derived stem cells alleviates liver injury in mice infected with Echinococcus Multilocularis

Accumulating evidence has shown that adipose-derived stem cells (ADSCs) have the potential to differentiate into hepatic lineages, which are ideal engraftments for tissue-engineered repair. In this study, we investigated the potential of transplanted induced hepatocyte-like cells (iHEPs) in treating hepatic alveolar echinococcosis and describe an efficient three-step protocol for the generation of iHEPs in vitro from ADSCs. The expression of hepatocyte lineage markers was assessed and iHEPs function was evaluated by Periodic acid-Schiff staining. iHEPs were intravenously transplanted into mice infected with Echinococcus multilocularis. Histopathological analysis and liver function tests were used to assess therapeutic effects. The iHEPs exhibit morphological features and a glycogen storage function similar to those of mature hepatocytes and demonstrate an upregulation in hepatic gene programs with increasing induction time. Following transplantation, iHEPs were observed surrounding the metacestode lesions in the liver parenchyma of E. multilocularis-infected mice. iHEPs transplantation effectively restored liver function and improved liver injury in the infected mice. Additionally, we observed significant activation of the Wnt/β-catenin signaling pathway in the livers of infected mice transplanted with iHEPs. Our results provide evidence that iHEPs transplantation can alleviate E. multilocularis-induced liver injury, potentially creating new avenues for treating liver injury in end-stage hepatic alveolar echinococcosis.

Oxygen control in bioreactor drives high yield production of functional hiPSC-like hepatocytes for advanced liver disease modelling

Hepatocytes-like cells (HLC) derived from human induced pluripotent stem cells show great promise for cell-based liver therapies and disease modelling. However, their application is currently hindered by the low production yields of existing protocols. We aim to develop a bioprocess able to generate high numbers of HLC. We used stirred-tank bioreactors with a rational control of dissolved oxygen concentration (DO) for the optimization of HLC production as 3D aggregates. We evaluated the impact of controlling DO at physiological levels (4%O2) during hepatic progenitors’ stage on cell proliferation and differentiation efficiency. Whole transcriptome analysis and biochemical assays were performed to provide a detailed characterization of HLC quality attributes. When DO was controlled at 4%O2 during the hepatic progenitors’ stage, cells presented an upregulation of genes associated with hypoxia-inducible factor pathway and a downregulation of oxidative stress genes. This condition promoted higher HLC production (maximum cell concentration: 2 × 106 cell/mL) and improved differentiation efficiencies (80% Albumin-positive cells) when compared to the bioreactor operated under atmospheric oxygen levels (21%O2, 0.6 × 106 cell/mL, 43% Albumin positive cells). These HLC exhibited functional characteristics of hepatocytes: capacity to metabolize drugs, ability to synthesize hepatic metabolites, and inducible cytochrome P450 activity. Bioprocess robustness was confirmed with HLC derived from different donors, including a primary hyperoxaluria type 1 (PH1) patient. The generated PH1.HLC showed metabolic features of PH1 disease with higher secretion of oxalate compared with HLC generated from healthy individuals. This work reports a reproducible bioprocess, that shows the importance of controlling DO at physiological levels to increase HLC production, and the HLC capability to display PH1 disease features.

Innovative Strategies for Liver Transplantation: The Role of Mesenchymal Stem Cells and Their Cell-Free Derivatives.

Despite being the standard treatment for end-stage liver disease, liver transplantation has limitations like donor scarcity, high surgical costs, and immune rejection risks. Mesenchymal stem cells (MSCs) and their derivatives offer potential for liver regeneration and transplantation. MSCs, known for their multipotency, low immunogenicity, and ease of obtainability, can differentiate into hepatocyte-like cells and secrete bioactive factors that promote liver repair and reduce immune rejection. However, the clinical application of MSCs is limited by risks such as aberrant differentiation and low engraftment rates. As a safer alternative, MSC-derived secretomes and extracellular vesicles (EVs) offer promising therapeutic benefits, including enhanced graft survival, immunomodulation, and reduced ischemia-reperfusion injury. Current research highlights the efficacy of MSC-derived therapies in improving liver transplant outcomes, but further studies are necessary to standardize clinical applications. This review highlights the potential of MSCs and EVs to address key challenges in liver transplantation, paving the way for innovative therapeutic strategies.

Long-term expanded hepatic progenitor cells ameliorate D-GalN/LPS-induced acute liver failure through repolarizing M1 macrophage to M2-Like phenotype via activation of the IL-10/JAK2/STAT3 signaling pathway

Acute liver failure (ALF) is a devastating liver disease characterized by the rapid deterioration of hepatocytes, which causes a series of clinical complications, including hepatic dysfunction, coagulopathy, encephalopathy, and multiorgan failure. Cell-based therapy is a promising alternative as it can bridge patients until their livers regenerate, releasing immunomodulatory molecules to suppress inflammation. This study reports an iPSCs-derived long-term expanded hepatic progenitor cell (LTHepPCs), which can differentiate into hepatocyte-like cells (HLCs) in vivo. When introduced into drug-induced ALF models, LTHepPCs mitigate liver damage by modulating the local immune microenvironment. This is achieved by shifting macrophages/Kupffer cells towards an anti-inflammatory state, resulting in a decrease in the expression of inflammatory cytokines such as TNF-a, IL-1β, and IL-8, and an increase in the expression of anti-inflammatory cytokines such as IL-10 and ARG-1. In vitro co-culturing of THP-1 or mBMDMs with LTHepPCs suggested that LTHepPCs could activate the anti-inflammatory state of macrophages/Kupffer cells via the IL-10/JAK2/STAT3 signaling pathway. Therefore, LTHepPC transplantation is a promising therapy for ALF patients.

Small Molecule-Mediated Stage-Specific Reprogramming of MSCs to Hepatocyte-Like Cells and Hepatic Tissue for Liver Injury Treatment

BackgroundDerivation of hepatocytes from stem cells has been established through various protocols involving growth factor (GF) and small molecule (SM) agents, among others. However, mesenchymal stem cell-based derivation of hepatocytes still remains expensive due to the use of a cocktail of growth factors, and a long duration of differentiation is needed, thus limiting its potential clinical application.MethodsIn this study, we developed a chemically defined differentiation strategy that is exclusively based on SM and takes 14 days, while the GF-based protocol requires 23–28 days.ResultsWe optimized a stage-specific differentiation protocol for the differentiation of rat bone marrow-derived mesenchymal stem cells (MSCs) into functional hepatocyte-like cells (dHeps) that involved four stages, i.e., definitive endoderm (DE), hepatic competence (HC), hepatic specification (HS) and hepatic differentiation and growth. We further generated hepatic tissue using human decellularized liver extracellular matrix and compared it with hepatic tissue derived from the growth factor-based protocol at the transcriptional level. dHep, upon transplantation in a rat model of acute liver injury (ALI), was capable of ameliorating liver injury in rats and improving liver function and tissue damage compared to those in the ALI model.ConclusionsIn summary, this is the first study in which hepatocytes and hepatic tissue were derived from MSCs utilizing a stage-specific strategy by exclusively using SM as a differentiation factor.Graphical

Apoptotic vesicles (apoVs) derived from fibroblast-converted hepatocyte-like cells effectively ameliorate liver fibrosis

Liver fibrosis is a serious global health issue for which effective treatment remains elusive. Chemical-induced hepatocyte-like cells (ciHeps) have emerged as an appealing source for cell transplantation therapy, although they present several challenges such as the risk of lung thromboembolism or hemorrhage. Apoptotic vesicles (apoVs), small membrane vesicles generated during the apoptosis process, have gained attention for their role in regulating various physiological and pathological processes. In this study, we generated ciHep-derived apoVs (ciHep-apoVs) and investigated their therapeutic potential in alleviating liver fibrosis. Our findings revealed that ciHep-apoVs induced the transformation of macrophages into an anti-inflammatory phenotype, effectively suppressed the activity of activated hepatic stellate cells (aHSCs), and enhanced the survival of hepatocytes. When intravenously administered to mice with liver fibrosis, ciHep-apoVs were primarily engulfed by macrophages and myofibroblasts, leading to a reduction in liver inflammation and fibrosis. Proteomic and miRNA analyses showed that ciHep-apoVs were enriched in various functional molecules that modulate crucial cellular processes, including metabolism, signaling transduction, and ECM-receptor interactions. ciHep-apoVs effectively suppressed aHSCs activity through the synergistic inhibition of glycolysis, the PI3K/AKT/mTOR pathway, and epithelial-to-mesenchymal transition (EMT) cascades. These findings highlight the potential of ciHep-apoVs as multifunctional nanotherapeutics for liver fibrosis and provide insights into the treatment of other liver diseases and fibrosis in other organs.Graphical abstract

An hepatitis B and D virus infection model using human pluripotent stem cell-derived hepatocytes

Current culture systems available for studying hepatitis D virus (HDV) are suboptimal. In this study, we demonstrate that hepatocyte-like cells (HLCs) derived from human pluripotent stem cells (hPSCs) are fully permissive to HDV infection across various tested genotypes. When co-infected with the helper hepatitis B virus (HBV) or transduced to express the HBV envelope protein HBsAg, HLCs effectively release infectious progeny virions. We also show that HBsAg-expressing HLCs support the extracellular spread of HDV, thus providing a valuable platform for testing available anti-HDV regimens. By challenging the cells along the differentiation with HDV infection, we have identified CD63 as a potential HDV co-entry factor that was rate-limiting for HDV infection in immature hepatocytes. Given their renewable source and the potential to derive hPSCs from individual patients, we propose HLCs as a promising model for investigating HDV biology. Our findings offer new insights into HDV infection and expand the repertoire of research tools available for the development of therapeutic interventions.

The antifibrotic potential of IMT504: modulation of GLAST + Wnt1 + bone marrow stromal progenitors and hepatic microenvironment

BackgroundThe immunomodulatory oligodeoxynucleotide (ODN) IMT504 might harbor antifibrotic properties within the liver.MethodsFibrosis models were induced in mice through thioacetamide (TAA) administration and bile-duct ligation. Cre-loxP mice were utilized to identify GLAST + Wnt1 + bone marrow stromal progenitors (BMSPs) and to examine their contribution with cells in the liver. In vivo and in vitro assays; flow-cytometry, immunohistochemistry, and qPCR were conducted.ResultsIMT504 demonstrated significant inhibition of liver fibrogenesis progression and reversal of established fibrosis. Early responses to IMT504 involved the suppression of profibrogenic and proinflammatory markers, coupled with an augmentation of hepatocyte proliferation. Additionally, this ODN stimulated the proliferation and mobilization of GLAST + Wnt1 + BMSPs, likely amplifying their contribution with endothelial- and hepatocytes-like cells. Moreover, IMT504 significantly modulated the expression levels of Wnt ligands and signaling pathway/target genes specifically within GLAST + Wnt1 + BMSPs, with minimal impact on other BMSPs. Intriguingly, both IMT504 and conditioned media from IMT504-pre-treated GLAST + Wnt1 + BMSPs shifted the phenotype of fibrotic macrophages, hepatic stellate cells, and hepatocytes, consistent with the potent antifibrotic effects observed.ConclusionIn summary, our findings identify IMT504 as a promising candidate molecule with potent antifibrotic properties, operating through both direct and indirect mechanisms, including the activation of GLAST + Wnt1 + BMSPs.

P21-58 AhR-activating polycyclic aromatic hydrocarbons disrupt metabolic pathways in human hepatocyte-like cells

P21-58 AhR-activating polycyclic aromatic hydrocarbons disrupt metabolic pathways in human hepatocyte-like cells

Self-Assembled Generation of Multi-zonal Liver Organoids from Human Pluripotent Stem Cells.

Distinct hepatocyte subpopulations are spatially segregated along the portal-central axis and critical to understanding metabolic homeostasis and liver injury. While several bioactive molecules have been described to play a role in directing zonal fates, including ascorbate and bilirubin, in vitro replication of zonal liver architecture has not been achieved to date. In order to evaluate hepatic zonal polarity, we developed a self-assembling zone-specific liver organoid culture by co-culturing ascorbate and bilirubin enriched hepatic progenitors derived from human induced pluripotent stem cells. We found that preconditioned hepatocyte-like cells exhibited zone-specific functions associated with urea cycle, glutathione synthesis and glutamate synthesis. Single nucleus RNA sequencing analysis of these zonally patterned organoids identifies hepatoblast differentiation trajectory that mimics periportal-, interzonal-, and pericentral human hepatocytes. Epigenetic and transcriptomic analysis showed that zonal identity is orchestrated by ascorbate or bilirubin dependent binding of histone acetyltransferase p300 (EP300) to methylcytosine dioxygenase TET1 or hypoxia-inducible factor 1-alpha (HIF1α). Transplantation of the self-assembled zonally patterned human organoids improved survival of immunodeficient rats who underwent bile duct ligation by ameliorating the hyperammonemia and hyperbilirubinemia. Overall, this multi-zonal organoid system serves as an in vitro human model to better recapitulate hepatic architecture relevant to liver development and disease.

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 induced pluripotent stem cells carrying albumin-sfGFP reporter

Current methodologies for hepatocyte induction from human induced pluripotent stem cells (hiPSCs) have limited efficacy due to lack of a functional hepatocyte reporter. To address this, we developed an endogenous albumin (ALB)-sfGFP reporter system in hiPSCs using homologous directed recombination (HDR)-mediated knock-in. The hiPSCs maintained the characteristic morphology, pluripotency, and normal karyotype while demonstrating successful differentiation into all three germ layers both in vitro and in vivo. Co-expression of EGFP and ALB was observed in the derived hepatocyte-like cells (HLCs). This reporter system holds promise for functional hepatocyte induction.

Impact of triiodothyronine (T3) on differentiation and T3-responsive characteristics in ipsc-derived hepatocyte-like cells to study energy and lipid metabolism

Impact of triiodothyronine (T3) on differentiation and T3-responsive characteristics in ipsc-derived hepatocyte-like cells to study energy and lipid metabolism

Hyaluronic acid-alginate hydrogel stimulates the differentiation of neonatal mouse testicular cells into hepatocyte-like and other cell lineages in three-dimensional culture.

Extracellular matrix (ECM)-derived hydrogels are frequently used in three-dimensional (3D) cell culture and organoid formation in several tissues. However, in the 3D cultivation of testicular cells, the hyaluronic acid (HA) hydrogel has not received as much attention. This study examined the effects of three distinct composites, including HA-alginate (HA-Alg), HA-alginate-collagen (HA-Alg-Col), and HA-alginate-decellularized ECM (HA-Alg-dECM), on mouse testicular cell culture and in vitro spermatogenesis. For the creation of composites, the concentration of biomaterials used was 0.5% HA, 1% alginate, 2.5mg/mL collagen, and 25mg/mL dECM derived from the testicles of Rams. After 3D culture of 5 days post-partum (dpp) mouse testicular cells for 14 days, HA-Alg was selected as a superior composite due to the greater number and size of the produced organoids. Then, cell culture was rerun by HA-Alg for 14 days, which was later extended for an additional 28 days. In addition, the 3D culture of 10 dpp mouse testicular cells was used to compare with 5 dpp mice on day 14. The morphology and gene expression were analyzed using appropriate techniques. On day 14, the HA-Alg hydrogel showed significantly more organoids in terms of size and number than the other two groups (p<0.05); nevertheless, none of the groups showed the expected signs of testis organoids. Remarkably, on day 14, the histology and immunostaining tests revealed features of hepatocyte-like cells (HLCs) and albumin production as a marker of HLC functionality. Furthermore, the analysis of gene expression verified the significant expression of angiogenesis markers (p<0.01). After the extended culture to 28 days, 5 dpp testicular cells once more differentiated into erythrocytes and HLCs, while a small number of organoids showed the characteristic of renal cells. Cell culture of 10 dpp mice for 14 days showed a wide range of cell lineages, including renal, glandular, chondrocyte, and hepatocyte-like cells in comparison to the 5 dpp mice. While the HA-Alg composite did not support spermatogenesis in the 3D culture of mouse testicular cells, it demonstrated an unpredicted potential for promoting the differentiation of neonate mouse testicular cells into HLC, erythrocytes, and other cell lineages.

Correlations of Long Noncoding RNA HNF4A-AS1 Alternative Transcripts with Liver Diseases and Drug Metabolism.

Hepatocyte nuclear factor 4 alpha antisense 1 (HNF4A-AS1) is a long noncoding RNA (lncRNA) gene physically located next to the transcription factor HNF4A gene in the human genome. Its transcription products have been reported to inhibit the progression of hepatocellular carcinoma (HCC) and negatively regulate the expression of cytochrome P450s (CYPs), including CYP1A2, 2B6, 2C9, 2C19, 2E1, and 3A4. By altering CYP expression, lncRNA HNF4A-AS1 also contributes to the susceptibility of drug-induced liver injury. Thus, HNF4A-AS1 lncRNA is a promising target for controlling HCC and modulating drug metabolism. However, HNF4A-AS1 has four annotated alternative transcripts in the human genome browsers, and it is unclear which transcripts the small interfering RNAs or small hairpin RNAs used in the previous studies are silenced and which transcripts should be used as the target. In this study, four annotated and two newly identified transcripts were confirmed. These six transcripts showed different expression levels in different liver disease conditions, including metabolic dysfunction-associated steatotic liver disease, alcohol-associated liver disease, and obesity. The expression patterns of all HNF4A-AS1 transcripts were further investigated in liver cell growth from human embryonic stem cells to matured hepatocyte-like cells, HepaRG differentiation, and exposure to rifampicin treatment. Several HNF4A-AS1 transcripts highly displayed correlations with these situations. In addition, some of the HNF4A-AS1 transcripts also showed a strong correlation with CYP3A4 during HepaRG maturation and rifampicin exposure. Our findings provide valuable insights into the specific roles of HNF4A-AS1 transcripts, paving the way for more targeted therapeutic strategies for liver diseases and drug metabolism. SIGNIFICANCE STATEMENT: This study explores the alternative transcripts of HNF4A-AS1, showing how their expression changes in different biological conditions, from various liver diseases to the growth and differentiation of hepatocytes and drug metabolism. The generated knowledge is essential for understanding the independent roles of different transcripts from the same lncRNA in different liver diseases and drug metabolism situations.