Author Correction: Treatment of liver cirrhosis using hepatocyte-derived liver progenitor-like cells: a prospective, open-label, single-arm, safety trial

Liver transplantation remains constrained by the scarcity of donor organs and the risks inherent in the procedure, underscoring the urgent need for novel cirrhosis therapies. We developed a protocol to convert human primary hepatocytes into expandable hepatocyte-derived liver progenitor-like cells (HepLPCs), which secrete high levels of matrix metalloproteinases and hepatocyte growth factor. In a thioacetamide-induced rat model of cirrhosis, human HepLPCs demonstrated potent anti-fibrotic properties and promoted liver regeneration. Biodistribution studies revealed that most xenogenic HepLPCs were cleared from the body within one week, suggesting that their therapeutic benefits likely arise from paracrine signaling rather than long-term engraftment. We initiated a first-in-human clinical trial involving nine patients with cirrhosis to evaluate the feasibility and safety of HepLPCs. Preclinical toxicity assessments in 36 crab-eating macaques confirmed the safety of HepLPC treatment. In the clinical trial, nine patients (mean age: 53 years), primarily with HBV-related cirrhosis, received HepLPCs via trans-hepatic arterial infusion without immunosuppressants. No serious adverse event was observed, and the minor adverse events were consistent with those commonly seen in cirrhosis patients. The treatment was well tolerated, with no transfusion reactions or dose-limiting toxicities. While significant changes in Child-Pugh and MELD scores were not observed, some patients showed improvements in liver biochemical parameters, coagulation profiles, and portal hypertension indicators during the six-month follow-up. These findings indicate that HepLPC therapy is safe and feasible, offering a promising new strategy for treating cirrhosis. Further clinical trials are needed to assess its efficacy in patients with decompensated cirrhosis and acute-on-chronic liver failure.

Cell therapy demonstrates promising potential as a substitute therapeutic approach for liver cirrhosis. We have developed a strategy to effectively expand murine and human hepatocyte-derived liver progenitor-like cells (HepLPCs) in vitro. The primary objective of the present study was to apply HepLPCs to the treatment of liver cirrhosis and to elucidate the underlying mechanisms responsible for their therapeutic efficacy. The effects of allogeneic or xenogeneic HepLPC transplantation were investigated in rat model of liver cirrhosis. Liver tissues were collected and subjected to immunostaining to assess changes in histology. In vitro experiments used HSCs to explore the antifibrotic properties of HepLPC-secretomes and their underlying molecular mechanisms. Additionally, proteomic analysis was conducted to characterize the protein composition of HepLPC-secretomes. Transplantation of HepLPCs resulted in decreased active fibrogenesis and net fibrosis in cirrhosis models. Apoptosis of HSCs was observed in vivo after HepLPC treatment. HepLPC-secretomes exhibited potent inhibition of TGF-β1-induced HSC activation and promoted apoptosis through signal transducer and activator of transcription (STAT)1-mediated pathways in vitro. Furthermore, synergistic effects between amphiregulin and FGF19 within HepLPC-secretomes were identified, contributing to HSC apoptosis and exerting antifibrotic effects via activation of the janus kinase-STAT1 pathway. HepLPCs have the potential to ameliorate liver cirrhosis by inducing STAT1-dependent apoptosis in HSCs. Amphiregulin and FGF19 are key factors responsible for STAT1 activation, representing promising novel therapeutic targets for the treatment of liver cirrhosis.

Rationale: We developed a cocktail of soluble molecules mimicking the in vivo milieu supporting liver regeneration that could convert mature hepatocytes to expandable liver progenitor-like cells in vitro. This study aimed to induce endogenous liver progenitor cells by the administration of the soluble molecules to provide an alternative approach for the resolution of liver fibrosis.Methods:In vitro cultured hepatocyte-derived liver progenitor-like cells (HepLPCs) were transplanted into CCL4-treated mice to investigate the therapeutic effect against liver fibrosis. Next, we used HGF in combination with a cocktail of small molecules (Y-27632, A-83-01, and CHIR99021 (HACY)) to induce endogenous CD24+ liver progenitor cells and to inhibit the activation of hepatic stellate cells (HSCs) during CCL4-induced hepatic injury. RNA sequencing was performed to further clarify the features of HACY-induced CD24+ cells compared with CCL4-induced CD24+ cells and in vitro derived HepLPCs. Finally, we evaluated the expansion of HACY-induced CD24+ cells in human hepatocyte-spheroids from fibrotic liver tissues.Results: HepLPCs exhibited the capacity to alleviate liver fibrosis after transplantation into CCL4-treated mice. The in vivo administration of HACY not only induced the conversion of mature hepatocytes (MHs) to CD24+ progenitor cells but prevented the activation of HSCs, thus leading to enhanced improvement of liver fibrosis in CCL4-treated mice. Compared to CD24+ cells induced by CCL4 alone, HACY-induced CD24+ cells retained an enhanced level of hepatic function and could promote the restoration of liver function that exhibited comparable gene expression profiles with HepLPCs. CD24+ cells were also observed in human liver fibrotic tissues and were expanded in three-dimensional (3D) hepatic spheroids in the presence of HACY in vitro.Conclusions: Hepatocyte-derived liver progenitor-like cells are crucial for liver regeneration during chronic hepatic injuries. The administration of HACY, which allowed the induction of endogenous CD24+ progenitor cells and the inactivation of HSCs, exerts beneficial effects in the treatment of liver fibrosis by re-establishing a balance favoring liver regeneration while preventing fibrotic responses.

The origin of fibrogenic cells in liver fibrosis remains controversial. We assessed the emerging concept that hepatocytes contribute to production of extracellular matrix (ECM) in liver fibrosis through epithelial-mesenchymal transition (EMT). We bred triple transgenic mice expressing ROSA26 stop beta-galactosidase (beta-gal), albumin Cre, and collagen alpha1(I) green fluorescent protein (GFP), in which hepatocyte-derived cells are permanently labeled by beta-gal and type I collagen-expressing cells are labeled by GFP. We induced liver fibrosis by repetitive carbon tetrachloride (CCl(4)) injections. Liver sections and isolated cells were evaluated for GFP and beta-gal as well as expression of alpha-smooth muscle actin (alpha-SMA) and fibroblast-specific protein 1 (FSP-1). Upon stimulation with transforming growth factor beta-1, cultured hepatocytes isolated from untreated liver expressed both GFP and beta-gal with a fibroblast-like morphological change but lacked expression of other mesenchymal markers. Cells from CCl(4)-treated livers never showed double-positivity for GFP and beta-gal. All beta-gal-positive cells exhibited abundant cytoplasm, a typical morphology of hepatocytes, and expressed none of the mesenchymal markers including alpha-SMA, FSP-1, desmin, and vimentin. In liver sections of CCl(4)-treated mice, GFP-positive areas were coincident with fibrotic septa and never overlapped X-gal-positive areas. Type I collagen-producing cells do not originate from hepatocytes. Hepatocytes in vivo neither acquire mesenchymal marker expression nor exhibit a morphological change clearly distinguishable from normal hepatocytes. Our results strongly challenge the concept that hepatocytes in vivo acquire a mesenchymal phenotype through EMT to produce the ECM in liver fibrosis.

Research Highlights

The remarkable plasticity of hepatocytes underlies diverse roles in distinct patterns of liver injury. Specifically, hepatocyte-derived progenitor cells play dominant roles in driving the regenerative response during chronic liver injury. However, the potential for hepatocytes to transdifferentiate into progenitor-like cells following partial hepatectomy (PHx) remains debated. In this study, we generate single-nucleus Assay for Transposase Accessible Chromatin using sequencing (snATAC-seq) data from mice subjected to the 2/3 PHx, aiming to characterize the dynamics of hepatocyte chromatin accessibility and the existence of hepatic progenitor cells. Our findings reveal that PHx induces dynamic changes in hepatocyte chromatin accessibility, giving rise to distinct hepatocyte subtypes balancing metabolic functions and proliferative activities. Notably, a subset of hepatocytes displays enhanced accessibility of progenitor cell-associated genes (e.g., Sox9 and Tnfrsf12a), along with increased accessibility to reprogramming-related genes (RRGs). By integrating with transcriptome data, we identify a hepatocyte population exhibiting progenitor-like traits. However, these progenitor-like cells show limited contribution to liver regeneration in the PHx model. Mechanistically, our results suggest that disrupted critical crosstalks (e.g., Pdgfa-Pdgfra signaling) with immune cells are attributed to impaired hepatic progenitor cells activation. Moreover, concurrent activation of growth signals in hepatocytes and other non-parenchymal cells (NPCs) suppresses the activation of progenitor cells.

Chronic liver injury can result in fibrosis that may progress over years to end-stage liver disease. The most effective anti-fibrotic therapy is treatment of the underlying disease, however when not possible, interventions to reverse or slow fibrosis progression are needed. The aim of this study was to study the safety and tolerability of simtuzumab, a monoclonal antibody directed against lysyl oxidase-like 2 (LOXL2) enzyme, in subjects with hepatitis C virus (HCV), human immunodeficiency virus (HIV), or HCV-HIV co-infection and advanced liver disease. Eighteen subjects with advanced liver fibrosis received simtuzumab 700 mg intravenously every 2 weeks for 22 weeks. Transjugular liver biopsies were performed during screening and at the end of treatment to measure hepatic venous pressure gradient (HVPG) and to stage fibrosis. Treatment was well-tolerated with no discontinuations due to adverse events. No significant changes were seen in HVPG or liver biopsy fibrosis score after treatment. Exploratory transcriptional and protein profiling using paired pre- and post-treatment liver biopsy and serum samples suggested up-regulation of TGF-β3 and IL-10 pathways with treatment. In this open-label, pilot clinical trial, simtuzumab treatment was well-tolerated in HCV- and HIV-infected subjects with advanced liver disease. Putative modulation of TGF-β3 and IL-10 pathways during simtuzumab treatment merits investigation in future trials.

Background & AimsAdvanced hepatic fibrosis is the main risk factor of liver-related morbidity and mortality in patients with chronic liver disease. In this study, we assessed the potential role of bone morphogenetic protein 8A (BMP8A) as a novel target involved in liver fibrosis progression.MethodsHistological assessment and BMP8A expression were determined in different murine models of hepatic fibrosis. Furthermore, serum BMP8A was measured in mice with bile duct ligation (BDL), in 36 subjects with histologically normal liver (NL) and in 85 patients with biopsy-proven non-alcoholic steatohepatitis (NASH): 52 with non- or mild fibrosis (F0-F2) and 33 with advanced fibrosis (F3-F4). BMP8A expression and secretion was also determined in cultured human hepatocyte-derived (Huh7) and human hepatic stellate (LX2) cells stimulated with transforming growth factor ꞵ (TGFꞵ).ResultsBmp8a mRNA levels were significantly upregulated in livers from fibrotic mice compared to control animals. Notably, serum BMP8A levels were also elevated in BDL mice. In addition, in vitro experiments showed increased expression and secretion to the culture supernatant of BMP8A in both Huh7 and LX2 cells treated with TGFꞵ. Noteworthy, we found that serum BMP8A levels were significantly higher in NASH patients with advanced fibrosis than in those with non- or mild fibrosis. In fact, the AUROC of circulating BMP8A concentrations to identify patients with advanced fibrosis (F3-F4) was 0.74 (p˂0.0001). Moreover, we developed an algorithm based on serum BMP8A levels that showed an AUROC of 0.818 (p˂0.0001) to predict advanced fibrosis in NASH patients.ConclusionThis study provides experimental and clinical evidence indicating that BMP8A is a novel molecular target linked to liver fibrosis and introduces an efficient algorithm based on serum BMP8A levels to screen patients at risk for advanced hepatic fibrosis.

BackgroundHuman primary hepatocytes (PHCs) are considered to be the best cell source for cell-based therapies for the treatment of end-stage liver disease and acute liver failure. To obtain sufficient and high-quality functional human hepatocytes, we have established a strategy to dedifferentiate human PHCs into expandable hepatocyte-derived liver progenitor-like cells (HepLPCs) through in vitro chemical reprogramming. However, the reduced proliferative capacity of HepLPCs after long-term culture still limits their utility. Therefore, in this study, we attempted to explore the potential mechanism related to the proliferative ability of HepLPCs in vitro culture.ResultsIn this study, analysis of assay for transposase accessible chromatin using sequencing (ATAC-seq) and RNA sequencing (RNA-seq) were performed for PHCs, proliferative HepLPCs (pro-HepLPCs) and late-passage HepLPCs (lp-HepLPCs). Genome-wide transcriptional and chromatin accessibility changes during the conversion and long-term culture of HepLPCs were studied. We found that lp-HepLPCs exhibited an aged phenotype characterized by the activation of inflammatory factors. Epigenetic changes were found to be consistent with our gene expression findings, with promoter and distal regions of many inflammatory-related genes showing increased accessibility in the lp-HepLPCs. FOSL2, a member of the AP-1 family, was found to be highly enriched in the distal regions with increased accessibility in lp-HepLPCs. Its depletion attenuated the expression of aging- and senescence-associated secretory phenotype (SASP)-related genes and resulted in a partial improvement of the aging phenotype in lp-HepLPCs.ConclusionsFOSL2 may drive the aging of HepLPCs by regulating inflammatory factors and its depletion may attenuate this phenotypic shift. This study provides a novel and promising approach for the long-term in vitro culture of HepLPCs.

Rationale: The idiosyncratic drug-induced liver injury (iDILI) is a major cause of acute liver injury and a key challenge in late-stage drug development. Individual heterogeneity is considered to be an essential factor of iDILI. However, few in vitro model can predict heterogeneity in iDILI. We have previously shown that mouse and human hepatocytes can be converted to expandable liver progenitor-like cells in vitro (HepLPCs). However, the limited proliferation potential of human HepLPCs confines its industrial application. Here, we reported the generation of a novel hepatocyte model not only to provide unlimited cell sources for human hepatocytes but also to establish a tool for studying iDILI in vitro.Methods: Human primary hepatocytes were isolated by modified two-step perfusion technique. The chemical reprogramming culture condition together with gene-transfer were then used to generate the immortalized HepLPC cell lines (iHepLPCs). Growth curve, doubling time, and karyotype were analyzed to evaluate the proliferation characteristics of iHepLPCs. Modified Hepatocyte Maturation Medium and 3D spheroid culture were applied to re-differentiate iHepLPCs.Results: iHepLPCs exhibited efficient expansion for at least 40 population doublings, with a stable proliferative ability. They could easily differentiate back into metabolically functional hepatocytes in vitro within 10 days. Furthermore, under three-dimensional culture conditions, the formed hepatic spheroids showed multiple liver functions and toxicity profiles close to those of primary human hepatocytes. Importantly, we established a hepatocyte bank by generating a specific number of such cell lines. Screening for population heterogeneity allowed us to analyze the in vitro heterogeneous responses to hepatotoxicity induced by molecular targeted drugs.Conclusions: In light of the proliferative capacity and the heterogeneity they represented, these iHepLPCs cell lines may offer assistance in studying xenobiotic metabolism as well as liver diseases in vitro.

Hepatocyte-derived liver progenitor-like cells (HepLPCs) exhibit a remarkable capacity to support liver function by detoxifying ammonia, promoting native liver regeneration, and suppressing inflammation, which leads to improvements in the recovery and survival of animals with acute liver failure (ALF). However, the mechanism through which HepLPCs promote liver regeneration is unclear. Here, we isolated HepLPC-derived extracellular vesicles (HepLPC-EVs) from conditioned media and performed microRNA sequencing analysis. Our results showed HepLPC-EVs promoted liver regeneration in mice with carbon tetrachloride or acetaminophen induced ALF. Cell cycle progression and proliferation of primary human hepatocytes were promoted after coculture with HepLPC-EVs. Exosomal miRNA sequencing confirmed that HepLPC-EVs were enriched with miR-183-5p, which played an essential role in ameliorating ALF. Mechanistically, HepLPC-derived exosomal miR-183-5p negatively regulated the expression of the target gene FoxO1, activated the Akt/GSK3β/β-catenin signaling pathway, and thereby promoted liver regeneration and restoration of normal liver function. These results indicate that during ALF, HepLPC-Exos mediate liver regeneration mainly through a paracrine exosome-dependent mechanism and these effects accelerate liver regeneration and lead to the restoration of normal liver function.Graphical

The study of pathophysiological mechanisms in human liver disease has been constrained by the inability to expand primary hepatocytes in vitro while maintaining proliferative capacity and metabolic function. We and others have previously shown that mouse mature hepatocytes can be converted to liver progenitor-like cells in vitro with defined chemical factors. Here we describe a protocol achieving efficient conversion of human primary hepatocytes into liver progenitor-like cells (HepLPCs) through delivery of developmentally relevant cues, including NAD + -dependent deacetylase SIRT1 signaling. These HepLPCs could be expanded significantly during in vitro passage. The expanded cells can readily be converted back into metabolically functional hepatocytes in vitro and upon transplantation in vivo. Under three-dimensional culture conditions, differentiated cells generated from HepLPCs regained the ability to support infection or reactivation of hepatitis B virus (HBV). Our work demonstrates the utility of the conversion between hepatocyte and liver progenitor-like cells for studying HBV biology and antiviral therapies. These findings will facilitate the study of liver diseases and regenerative medicine.

Globally, about two million people die from liver diseases every year. Liver transplantation is the only reliable therapy for severe end-stage liver disease, however, the shortage of organ donors is a huge limitation. Human hepatocytes derived liver progenitor-like cells (HepLPCs) have been reported as a novel source of liver cells for development of in vitro models, cell therapies, and tissue-engineering applications, but their functionality as transplantation donors is unclear. Here, a 3-dimensional (3D) co-culture system using HepLPCs and human umbilical vein endothelial cells (HUVECs) was developed. These HepLPC spheroids mimicked the cellular interactions and architecture of mature hepatocytes, as confirmed through ultrastructure morphology, gene expression profile and functional assays. HepLPCs encapsulated in alginate beads are able to mitigate liver injury in mice treated with carbon tetrachloride (CCL4), while alginate coating protects the cells from immune attack. We confirmed these phenomena due to HUVECs producing glial cell line-derived neurotrophic factor (GDNF) to promote HepLPCs maturation and enhance HepLPCs tight junction through MET phosphorylation. Our results display the efficacy and safety of the alginate microencapsulated spheroids in animal model with acute liver injury (ALF), which may suggest a new strategy for cell therapy.

Constructing an engineered hepatic lobule-mimetic model is challenging owing to complicated lobular architecture and crucial hepatic functionality. Our previous study has demonstrated the feasibility of using silk fibroin (SF) scaffolds as functional templates for engineering hepatic lobule-like constructs. But the unsatisfactory chemical and physical performances of the SF-only scaffold and the inherent defect in the functional activity of the carcinoma-derived seeding cells remain to be addressed to satisfy the downstream application demand. In this study, SF-collagen I (SFC) composite scaffolds with improved physical and chemical properties were fabricated, and their utilization for bioengineering a more hepatic lobule-like construct was explored using the immortalized human hepatocyte-derived liver progenitor-like cells (iHepLPCs) and endothelial cells incorporated in the dynamic culture system. The SFC scaffolds prepared through the directional lyophilization process showed radially aligned porous structures with increased swelling ratio and porosity, ameliorative mechanical stiffness that resembled the normal liver matrix more closely, and improved biocompatibility. The iHepLPCs displayed a hepatic plate-like distribution and differentiated into matured hepatocytes with improved hepatic function in vitro and in vivo. Moreover, hepatocyte–endothelial cell interphase arrangement was generated in the co-culture compartment with improved polarity, bile capillary formation, and enhanced liver functions compared with the monocultures. Thus, a more biomimetic hepatic lobule-like model was established and could provide a valuable and robust platform for various applications, including bioartificial liver and drug screening.

Splenectomy attenuates murine liver fibrosis with hypersplenism stimulating hepatic accumulation of Ly-6Clo macrophages