Editor’s Note: At the 32nd Asia-Pacific Association for the Study of the Liver (APASL) annual conference, Professor Hu Jin-Hua from the Department of Hepatology at the Fifth Medical Center of the PLA General Hospital in China was invited to present a special report on liver cell regeneration and its application in the treatment of liver failure. This publication has invited Professor Hu to write on this topic for the benefit of its readers.
The study of liver regeneration often employs animal models, as the regenerative processes in human and rodent livers are similar. The partial hepatectomy model (PHx) remains the preferred method for investigating the mechanisms of liver regeneration. Clinical liver diseases, often caused by toxins, alcohol, viruses, or immune factors, have led to the development of chemical injury models. These include liver injury animal models induced by substances such as CCl4, D-GlaN, TAA, and APAP, which are common in the study of liver regeneration mechanisms and treatments under disease conditions.
However, the complexity of the causes and clinical manifestations of liver diseases makes it challenging for current animal models to accurately mimic specific human diseases. This discrepancy often results in variations between the outcomes of animal experiments and actual clinical treatment effectiveness.
Transgenic animals, used as new models for liver regeneration, offer significant advantages. For example, mice with the fah gene knocked out can host transplanted human liver cells, effectively turning them into “mice with human livers.” These chimeric animals, containing human liver tissues and cells with unique biological functions, are more suitable for studying human liver damage and regeneration.
Research on Liver Regeneration Mechanisms
In models of extensive liver resection, the mechanisms of liver regeneration have been well elucidated. Classical liver regeneration comprises three important stages: a) initiation or priming phase, b) proliferation phase, and c) termination phase. Each stage is regulated and interconnected by a variety of signaling pathways and factors, including the Wnt/β-Catenin signaling, Notch signaling, mTOR signaling, and factors like TNF-α, HGF, EGF, TGF-β. Recent studies have highlighted the critical role of the Hippo signaling pathway in controlling the size of liver regeneration during the termination phase. In 2020, George’s team published an article in the Nature Reviews sub-journal Gastroenterology & Hepatology, suggesting that the mitotic process of liver cell regeneration is directly regulated by the MET and EGFR pathways. Other factors like TNF and TNFR1, IL-6, VEGF, Serotonin, Leptin, Insulin, PPARγ, and complex signaling pathways including Hedgehog, WNT and β-catenin, Hippo and Yap, play regulatory roles in mitosis, as blocking these pathways only causes a delay in liver regeneration.
Liver cell regeneration exhibits significant differences between normal and damaged livers. In normal or slightly damaged livers, regeneration primarily follows the classical liver cell regeneration model, where hepatocytes and cholangiocytes are renewed from the same body, a phenomenon termed “phenotypic fidelity.” This type of regeneration leaves almost no trace in the newly formed liver tissue. However, when hepatocytes are severely damaged or their proliferation is inhibited, progenitor cells originating from cholangiocytes with hepato-biliary characteristics transform into hepatocytes for regeneration. Similarly, when cholangiocytes are severely damaged or their proliferation is inhibited, hepatocytes around the portal vein transform into cholangiocytes. But such repair often leads to ineffective or erroneous proliferation, such as the formation of liver cirrhosis nodules. Hence, liver tissue remodeling is a critical aspect of liver regeneration, especially in severe liver injuries, including liver failure, but is currently under-researched, and the specific mechanisms are unclear. When the liver is damaged, liver endothelial cells and immune cells directly participate in tissue repair and remodeling. Other factors like lipid metabolism, the autonomic nerve network, and the complement system might also directly or indirectly impact liver regeneration.
Research on the application of liver regeneration in liver failure treatment can be broadly divided into the following aspects:
Pharmacological Intervention:
The goal here is to promote liver regeneration and repair by activating liver regeneration pathways or blocking liver necrosis pathways. Currently, numerous chemical drugs and cytokines have entered animal experiments and clinical trials. Randomized controlled clinical studies of granulocyte colony-stimulating factor (G-CSF), pentoxifylline, and omega-3 fatty acids have confirmed their efficacy in promoting liver regeneration. G-CSF, in particular, is a promising drug targeting liver regeneration post-acute severe injury. Multiple independent clinical studies have shown that G-CSF can improve the survival rate of patients with HBV and alcoholic steatohepatitis leading to acute-on-chronic liver failure (ACLF). However, a multicenter clinical study from Europe reported that G-CSF did not improve the overall 90-day survival rate of ACLF patients. Further analysis revealed that over 60% of the patients in the European study had severe complications such as infections, acute kidney injury, and hepatic encephalopathy at the time of enrollment.
Our team has recently conducted experimental research on the mechanism of action of G-CSF. Dr. Tong Jingjing’s thesis (published in Front. Immunol., 16 May 2022) indicated that G-CSF could promote the transformation of monocytes to M2 (anti-inflammatory/pro-recovery) type, which may aid in the recovery of ACLF. Dr. Liu Zifeng, in her research paper (Molecular Biology Reports. 04 July 2022), through in vitro cell experiments and in vivo animal model studies, has substantiated that G-CSF can directly promote the expression and proliferation of injured liver cells’ Ki67 and VEGF-A, as well as liver angiogenesis. The mechanism may involve regulation through the Akt and Erk signaling pathways.
Liver Regeneration in the Treatment of Liver Failure:
Research on the application of liver regeneration in treating liver failure can be categorized into several areas:
- Pharmacological Interventions :
The aim is to enhance liver regeneration and repair by stimulating liver regeneration pathways or inhibiting liver necrosis pathways. Currently, a variety of chemical drugs and cytokines are in animal testing and clinical trials. Randomized controlled clinical studies have confirmed the efficacy of granulocyte colony-stimulating factor (G-CSF), pentoxifylline, and omega-3 fatty acids in promoting liver regeneration. However, G-CSF’s effectiveness varies, as shown in different studies. For instance, while it improved survival rates in HBV and alcohol-induced ACLF patients, a European multicenter clinical study found it did not enhance the overall 90-day survival rate for ACLF patients. Our team’s recent studies suggest that G-CSF might aid in ACLF recovery by promoting monocytes’ transformation into an anti-inflammatory state. Another study showed that G-CSF directly enhances the expression and proliferation of Ki67 and VEGF-A in injured liver cells and promotes liver angiogenesis, potentially through the Akt and Erk signaling pathways.
Concurrently, research by Corn. E and others showed that combining G-CSF with TLR-4 inhibitors in ACLF mouse models significantly reduced liver inflammation and promoted regeneration. Notably, while G-CSF alone had similar effects in GLaN/CCL4 ACLF mice, it worsened inflammation in LPS/CCL4 ACLF mice.
These findings imply that future clinical applications of G-CSF should be optimized, possibly by combining it with other drugs like TLR-4 inhibitors, to maximize efficacy. Current research on autophagy and hepatic cell senescence also offers potential new targets for liver regeneration treatment in acute severe liver injuries.
- Stem Cell Transplantation :
This includes bone marrow stem cells and mesenchymal stem cell transplantation. Clinical studies on stem cell reinfusion for treating liver failure and end-stage liver diseases have been underway for years. Teams led by academicians like Wang Fusheng and professors Gao Zhiliang and Lin Bingliang have used mesenchymal stem cells to treat ACLF, improving patient survival and liver function. However, challenges remain in directing stem cells to differentiate appropriately and remodel liver tissue in vivo, necessitating further large-scale validation.
- Liver Organoids :
In 2013, a major breakthrough in liver regeneration biology was reported in Nature, where 4 mm “liver buds” constructed from human iPSCs (induced pluripotent stem cells) were transplanted into mice, successfully saving a patient with fatal liver failure. Recent years have seen a focus on culturing “liver-like” organoids in vitro. Researchers have created “liver-like tissues” comprising hepatocytes, cholangiocytes, and hepatic stromal cells, and successfully integrated these with artificial liver support systems, achieving success in in vivo experiments. Engineered liver organoids using stem cells are seen as a solution for developing complex bioartificial livers.
With modern technological advancements, 3D bioprinting is emerging as a promising technique. It can rapidly and reliably create bioartificial livers with more physiological functions, using patients’ medical imaging and liver data. However, many challenges, such as combining six unique types of liver cells and constructing layered vascular and bile duct networks, need to be overcome. Once these challenges are addressed, printing fully functional bioartificial livers will become a reality.
In Conclusion:
Liver regeneration is a crucial pathway for treating severe liver injury and failure. The concept has evolved beyond just regenerating the original liver tissue. Using liver regeneration principles, stem cell culture, and engineering techniques to create “liver-like organs,” or fabricating bioactive, physiologically functional human livers through 3D bioprinting, is no longer a distant dream. Future liver regeneration technologies promise to bring new hope to patients with liver failure and end-stage liver diseases.
TAG: APASL 2023, Master Class, Liver failure and end-stage liver diseases
