Genomics: Insight

Research Question: Given the prevalence of liver diseases worldwide, the advancements and funding of stem cell-based regenerative medicine, and the difficulties in producing mature hepatocyte-like cells in vitro, how can epigenetic changes be maximized to improve the knowledge of hepatoblast differentiation into fully functional hepatocytes?

Introduction

The overall need for developing new liver disease treatments remains the same today as it has for decades. Part of the data from the 2021 Global Burden of Disease (GBD)¹ tracks the global prevalence of deaths caused by Cirrhosis (chronic liver failure) compared to the total number of deaths annually. The data indicates a recent decrease in the prevalence of deaths related to Cirrhosis across all World Health Organization (WHO) regions since the outbreak of COVID-19. However, from 1990 to 2021, there has been no consistent upward or downward trend across all regions, and the greatest difference between the regional prevalence of cirrhosis in 2021 is only 1.54%. Additionally, the difference between the global prevalence of cirrhosis-related deaths from 1990 to 2021 is just 0.12%. Simultaneously, the WHO releases statistics² on the worldwide number of hepatitis infections to measure how much progress the world has made toward reaching its 2030 goals, which aim to decrease the number of infections worldwide significantly. According to their 2022 report, the number of Hepatitis B-related deaths has steadily increased since 2020, sharply drifting further and further away from their goals. Both the GBD and WHO data indicate the continuing need for the development of liver disease treatments, which the field of epigenetics has the potential to provide.

Liver disease remains a significant global health challenge, contributing to millions of deaths annually due to its progressive nature and limited treatment options. Liver disease refers to a broad range of conditions that impair liver function, including cirrhosis, hepatitis, fatty liver disease, and liver cancer. These diseases can be caused by infections (such as Hepatitis B and C), excessive alcohol consumption, obesity, metabolic disorders, or genetic factors; cirrhosis, also known as chronic liver failure, occurs as a result of prolonged liver damage, which leads to scarring and a loss of function (GBD)¹. Liver damage ultimately leads to the loss of functioning liver cells or hepatocytes, compromising essential functions like detoxification, metabolism, and protein synthesis. This progressive cell loss not only impairs liver function but also poses a significant challenge for regenerative therapies, as replacing these cells requires the ability to produce mature hepatocyte-like cells in vitro, an essential step for modeling liver function and repairing damaged tissues (Lee et al., 2019)¹⁷. This challenge stems from the complexity of hepatocyte differentiation, as current protocols often yield immature cells that lack full hepatic functionality (Chen et al., 2020)¹⁸. Epigenetic regulation plays a fundamental role in cell differentiation, influencing gene expression without altering the DNA sequence (Li et al., 2016)³. This process is particularly relevant in the context of liver disease, where stem cell-derived hepatocytes offer the potential for regenerative therapies (Zhang et al., 2020)⁴. The concept of epigenetic modification has been explored for decades, with early research identifying mechanisms such as DNA methylation and histone modifications as key regulators of cellular identity (Smith et al., 2018)⁵. These modifications have been observed across various biological systems, contributing to embryonic development, tissue regeneration, and disease progression (Thompson & Takebe, 2020)⁶. The liver, a highly regenerative organ, relies on hepatocyte proliferation to maintain function; however, in cases of chronic liver disease, this regenerative capacity is often impaired (Du et al., 2014)⁷. Scientists have investigated the differentiation of hepatoblasts—liver progenitor cells—into mature hepatocytes using pluripotent stem cells (PSCs) to address this limitation (Kim et al., 2021)⁸. Recent studies suggest that epigenetic mechanisms play a crucial role in hepatoblast differentiation and hepatocyte maturation, making them a potential avenue for improving liver disease treatments (Nguyen & Patel, 2022)⁹. As research progresses, epigenetic techniques may provide an essential strategy for overcoming these barriers, offering a pathway toward more effective treatments for liver disease and transplantation therapies.

Global Differences in Liver Stem Cell Research and Opportunities for Advancement 

A study by Julia Deinsberger et al.¹⁰ analyzes the global distribution and focus of clinical trials involving pluripotent stem cells (PSCs), highlighting a significant difference in experimental approaches between countries. The study found that most PSC-related trials were observational (77.1%), meaning they did not involve actual stem cell transplantation, while only 22.9% were interventional. This trend is particularly evident in the United States, where 41.6% of observational trials were conducted, compared to only 16.7% of interventional studies, indicating a strong emphasis on preliminary research rather than clinical application. In contrast, China led in interventional trials, accounting for 36.7% of such studies, followed by Japan (13.3%) and South Korea (10.0%), reflecting a more aggressive push toward clinical implementation. The data shows that while the U.S. conducts several trials, they are predominantly observational and centered around non-communicable diseases, cardiovascular diseases, and neurological disorders rather than direct patient treatment. This highlights how the U.S. may lack PSC research, whereas Asian countries are advancing more rapidly in applying stem cell technologies in clinical settings. 
Thompson W. L. and Takebe T.¹¹ highlight the limitations of current fabricated liver models, such as the short-lived functionality of primary human hepatocytes and the lack of cellular complexity in 2D cultures, which can impede accurate predictions of drug-induced liver injury. Researchers can use pluripotent stem cell-derived liver organoids to create a renewable and genetically varied cell supply that better mimics liver physiology, including interactions between hepatocytes and non-parenchymal cells. However, obstacles persist in producing fully mature hepatocyte-like cells, as current organoids show symptoms of immaturity. Current optimization efforts, including enhanced differentiation techniques and metabolic maturation strategies, show potential for producing more physiologically relevant liver models that can advance drug testing, disease modeling, and regenerative medicine.

Liver-specific Stem Cell and Hepatoblast Differentiation Research and Advancements

One study by Yuanyuan Du et al.¹² examines gene expression differences between 3H cells (hepatic progenitor-like cells), primary human hepatocytes (F-HEPs), and fetal liver cells to identify key factors for hepatocyte maturation. The study found that CEBPA, ATF5, and PROX1 were expressed at 45–60% lower levels in 3H and fetal hepatocytes compared to adult hepatocytes, suggesting their role in liver cell function. PROX1 is crucial for metabolic maturation, while CEBPA and ATF5 support glucose metabolism and lipid regulation, both essential for liver health. Increasing these factors through epigenetic techniques like gene activation or chromatin remodeling could improve hepatoblast differentiation. This research helps advance finding treatment options for in vitro hepatocyte engineering.

Antonio Maglitto et al.¹³ conducted research to enhance epigenetic approaches for inducing hepatoblast differentiation into fully functional hepatocytes. The study found that in the early stages of stem cell differentiation, Gpr56 (adhesion G protein-coupled receptor G1) deletion significantly decreased hematopoietic progenitor cell (HPC) counts in E9 yolk sac (YS) cells compared to early-stage wild-type (WT) (p ≤ 0.05). Later-stage fetal liver (FL) cells showed no significant differences. Although certain side effects suggest poor cellular maturation, they were unaffected in E10.5 and later-stage cells (rewrite to link). Despite these early-stage abnormalities, FL-derived stem cells from Gpr56 KO embryos were successfully engrafted into irradiated recipients, indicating that Gpr56 is not required for long-term hematopoietic function. These findings highlight the stage-specific role of Gpr56 in differentiation, offering insights into how genetic regulation impacts hepatoblast-to-hepatocyte transition, which could refine in vitro hepatocyte generation for liver disease treatment and transplantation therapy.

Finally, a study by Lin Wang et al.¹⁴ investigated the impact of various cell infusion treatments on liver regeneration in rats following partial hepatectomy (PHx). The results showed that liver weight recovery was significantly hindered in the group that received no infusion (5.5 ± 0.3 g), whereas the PHx-only group (7.1 ± 0.2 g) and both the spleen cell (SPC) and bone marrow (BM) infusion groups (7.3 ± 0.2 g) exhibited nearly complete restoration of liver mass. Additionally, hepatocyte proliferation was lowest in the no-infusion group (3.2 ± 0.5 cells/HPF). However, proliferation increased significantly in rats that received SPC infusion (14.8 ± 1.1 cells/HPF) and BM infusion (12.4 ± 0.9 cells/HPF). The SPC infusion contained spleen-derived cells, while the BM infusion consisted of bone marrow-derived cells, both of which played a crucial role in promoting liver regeneration. These findings confirm that SPC and BM infusions enhance liver regeneration, with SPC infusion demonstrating a slightly superior effect on hepatocyte proliferation compared to BM infusion.

Self-duplication of Hepatocytes as a Mechanism of Liver Regeneration

Yann Malato et al.¹⁵ investigated hepatocyte regeneration and found that liver repair is primarily accomplished through self-duplication rather than stem cell differentiation. The researchers use biological techniques to permanently mark hepatocyte cells, allowing precise lineage tracing. After 12 and 24 weeks, all newly formed hepatocytes were shown to arise from preexisting hepatocytes, ruling out liver progenitor cells from routine liver maintenance. Hepatocyte self-renewal is the key mechanism for liver homeostasis, with just a tiny fraction of new hepatocytes arising from progenitor cells during chronic liver injury due to biweekly CCl₄ injections. This shows that self-duplication of hepatocyte cells can be a mechanism of liver cell regeneration and repair.

Chromatin Remodeling and its Role in Hepatoblast Differentiation

In a study conducted in 2023, Li Yang et al.¹⁷ researched the epigenetic differences between liver cell types – hepatoblasts, hepatocytes, and cholangiocytes – in order to determine the ways in which chromatin remodeling can affect which cell a hepatoblast will differentiate into. They found that histone modifications and chromatin accessibility played significant roles in cell differentiation. During differentiation into cholangiocytes, they found that histone modifications H3K4me3+ and H3K27me3- (trimethylation at lysines 4 of histone H3 and no trimethylation at lysine 27 of histone H3) in hepatoblasts substantially increased, whereas modifications H3K4me3- and H3K27me3+ increased during hepatocyte differentiation. With regard to chromatin regulation, they found that genes specifically related to metabolism and drug detoxification were enriched in hepatocytes. Lastly, they determined that, during differentiation from hepatoblasts to cholangiocytes, cells inactivate far more enhancer regions than in differentiation from hepatoblasts to hepatocytes. Additionally, cells will activate enhancer regions specific to metabolism and detoxification during hepatoblast-hepatocyte differentiation. This study illustrates the important role chromatin remodeling plays in the hepatoblast differentiation process. 

Conclusion

reviewing recent clinical trials highlights the important role that research into the epigenetic factors of hepatocyte differentiation plays in the treatment of liver disease. Given the worldwide prevalence of Cirrhosis and other forms of liver disease, society would greatly benefit from the continued development of liver disease treatments; we believe that furthering the field of epigenetics would allow us to better understand and develop treatments with a special focus on regenerating liver tissue. Given the burden that liver disease places upon healthcare systems worldwide, we look forward to the continued research of epigenetics and its use in advancing the field of liver cell differentiation.

References

1. Global Burden of Disease Collaborative Network (Ed.). (2021). Global Burden of Disease.
2. World Health Organization (Ed.). (2024). Global hepatitis report 2024: action for access in low- and middle-income countries. https://www.who.int/publications/i/item/9789240091672
3. Li, X., Zhang, Y., & Liu, W. (2016). Epigenetic regulation of stem cell differentiation and pluripotency. Nature Reviews Genetics, 17(8), 555-567. https://doi.org/10.1038/nrg.2016.29
4. Zhang, H., Wang, J., & Chen, L. (2020). Advances in stem cell therapy for liver diseases. Journal of Hepatology, 72(5), 1037-1051. https://doi.org/10.1016/j.jhep.2020.02.020
5. Smith, R. D., Jones, T., & Miller, P. (2018). The role of DNA methylation and histone modifications in cellular differentiation. Epigenetics & Chromatin, 11(1), 22-35. https://doi.org/10.1186/s13072-018-0199-5
6. Thompson, W. L., & Takebe, T. (2020). Generation of multi-cellular human liver organoids from pluripotent stem cells. Methods in Cell Biology, 47-68. https://doi.org/10.1016/bs.mcb.2020.03.009
7. Du, Y., Wang, J., Jia, J., Song, N., Xiang, C., Xu, J., Hou, Z., Su, X., Liu, B., Jiang, T., Zhao, D., Sun, Y., Shu, J., Guo, Q., Yin, M., Sun, D., Lu, S., Shi, Y., & Deng, H. (2014). Human hepatocytes with drug metabolic function induced from fibroblasts by lineage reprogramming. Cell Stem Cell, 14(3), 394-403. https://doi.org/10.1016/j.stem.2014.01.008
8. Kim, B. H., Park, J. S., & Lee, Y. C. (2021). Hepatoblast differentiation and liver regeneration in chronic disease models. Stem Cell Reports, 16(4), 891-905. https://doi.org/10.1016/j.stemcr.2021.03.012
9. Nguyen, T., & Patel, R. (2022). Epigenetic mechanisms in hepatocyte maturation and liver disease. World Journal of Gastroenterology, 28(7), 904-922. https://doi.org/10.3748/wjg.v28.i7.904
10. Frontiers in Cell Biology. (2021). Epigenetic regulation of liver-specific gene transcription and hepatocyte differentiation. Frontiers in Cell and Developmental Biology, 9, Article 765980. https://doi.org/10.3389/fcell.2021.765980
11. Deinsberger, J., Reisinger, D., & Weber, B. (2020). Global trends in clinical trials involving pluripotent stem cells: A systematic multi-database analysis. Npj Regenerative Medicine, 5(1). https://doi.org/10.1038/s41536-020-00100-4
12. Thompson, W. L., & Takebe, T. (2020). Generation of multi-cellular human liver organoids from pluripotent stem cells. Methods in Cell Biology, 47-68. https://doi.org/10.1016/bs.mcb.2020.03.009
13. Du, Y., Wang, J., Jia, J., Song, N., Xiang, C., Xu, J., Hou, Z., Su, X., Liu, B., Jiang, T., Zhao, D., Sun, Y., Shu, J., Guo, Q., Yin, M., Sun, D., Lu, S., Shi, Y., & Deng, H. (2014). Human hepatocytes with drug metabolic function induced from fibroblasts by lineage reprogramming. Cell Stem Cell, 14(3), 394-403.  https://doi.org/10.1016/j.stem.2014.01.008
14. Maglitto, A., Mariani, S. A., de Pater, E., Rodriguez-Seoane, C., Vink, C. S., Piao, X., Lukke, M.-L., & Dzierzak, E. (2021). Unexpected redundancy of gpr56 and gpr97 during hematopoietic cell development and differentiation. Blood Advances, 5(3), 829-842. https://doi.org/10.1182/bloodadvances.2020003693
15. Wang, L., Wang, X., Xie, G., Wang, L., Hill, C. K., & DeLeve, L. D. (2012). Liver sinusoidal endothelial cell progenitor cells promote liver regeneration in rats. Journal of Clinical Investigation, 122(4), 1567-1573. https://doi.org/10.1172/jci58789
16. Malato, Y., Naqvi, S., Schürmann, N., Ng, R., Wang, B., Zape, J., Kay, M. A., Grimm, D., & Willenbring, H. (2011). Fate tracing of mature hepatocytes in mouse liver homeostasis and regeneration. Journal of Clinical Investigation, 121(12), 4850-4860. https://doi.org/10.1172/jci59261
17. Yang, L., Wang, X., Yu, X.-X., Yang, L., Zhou, B.-C., Yang, J., & Xu, C.-R. (2023). The default and directed pathways of hepatoblast differentiation involve distinct epigenomic mechanisms. Developmental Cell, 58(18), 1688-1700.e6. https://doi.org/10.1016/j.devcel.2023.07.002