Liver Regeneration

Figure 1. Changes in hepatocytes during liver regeneration. Cells exit G0 and enter G1 in the priming phase (A), progress through the rest of the cell cycle to divide in the proliferative phase (B), and exit in the termination phase (C) (Kiseleva et al., 2021).

What is liver regeneration?

The liver is the only organ capable of regeneration without intervention. After resection or decreases in cell numbers caused by chemicals, viruses, or other means, the liver mass to body mass ratio can be restored by building up tissue and the liver can function normally (Fausto, 2000).

Liver regeneration consists of three phases (Figure 1.). Firstly, cytokines induce quiescent hepatocytes to enter the cell cycle at the G1 phase. This is called the priming phase. Next, hepatocytes progress through the cell cycle as mitogens stimulate DNA synthesis and mitosis. This is the proliferative phase. Finally, once the liver has reached the appropriate mass, inhibitory molecules stop proliferation in the termination phase (Kiseleva et al., 2021).

Issues with Current Liver Disease Treatments: Liver Transplants

Unfortunately, when damage from liver disease becomes extremely severe, the liver's regeneration capacity may not be sufficient to restore normal mass and function. As a result, exogenous solutions may be required to prevent liver failure. Currently, liver transplants are the only treatment for liver diseases. However, there are a couple of issues with this. Firstly, there is a shortage of organ donors. Secondly, complications can arise post-transplant. For example, patients need to take immunosuppressant agents (which have side effects) for the rest of their lives to help prevent their immune systems from rejecting the new liver. Furthermore, liver rejection still occurs in some patients and results in the development of graft-vs-host disease. Therefore, researchers have been investigating stem cell-based approaches as alternative treatments (Cao et al., 2019).

Figure 2. Pathway of fibroblast-derived iMPC differentiation into hepatocytes. iMPCs are reprogrammed into iMPC-EPCs and proliferate before differentiation into iMPC-Hepatocytes. Factors that influence progression into subsequent stages are shown at the bottom of the figure (Zhu et al., 2014).

Future Liver Disease Treatments: Stem Cell Transplants

Many studies exploring stem cell-based treatments focus on human induced pluripotent stem cells (iPSCs) derived from human fibroblasts. Unfortunately, while capable of differentiating into hepatocytes, iPSCs are incapable of expansion (proliferation). Luckily, induced multipotent progenitor cells (iMPCs) may be more successful (Zhu et al., 2014).

Unlike iPSCs, iMPCs do not have a pluripotent stage before differentiating into hepatocytes. This is evidenced by the absence of OCT4 and NANOG expression, which occur during pluripotency. Instead, iMPCs are reprogrammed into endoderm progenitor cells (iMPC-EPC) as endoderm-specific gene expression can be observed approximately 14 days after reprogramming starts. Then, they can differentiate into iMPC-Hepatocytes when stimulated by the same growth factors that induce hepatic differentiation of iPSC-definitive endoderm cells, like epidermal growth factor (Figure 2.) (Zhu et al., 2014).

iMPC-Hepatocytes have been transplanted into mice with human tyrosinaemia type I, which causes liver injury. Three to nine months later, repopulation nodules that grow significantly can be observed. As a result, iMPC-Hepatocytes are capable of proliferation post-transplant. However, iMPC-Hepatocytes are less mature than iPSC-Hepatocytes. Therefore, they need to mature after transplantation. Luckily, there seems to be very little difference in gene expression in the nodules and adult hepatocytes, indicating iMPC-Hepatocytes are also capable of maturation (Zhu et al., 2014).

Issues with Stem Cell Transplants

iMPC-Hepatocytes’ ability to proliferate and mature post-transplant makes cell-based approaches promising potential therapies for liver diseases. Unfortunately, there are problems associated with these types treatments as well. For example, the patient's immune system could reject and attack the stem cells. Also, the methods used to harvest stem cells could injure the donor. Therefore, researchers have also been investigating cell-free treatments, which are much safer (Cao et al., 2019).

Figure 3. Micrographs showing differences in proliferation and apoptosis between 70% hepatectomized rats treated with 30 μg MVs and 70% hepatectomized rats treated with vehicle. Significant differences in BrdU uptake at 24, 48, and 72 hours indicates enhanced proliferation in MV-treated rats (A). TUNEL assay shows significant reduction of apoptosis at 72 hours in 70% hepatectomized rats treated with 30 μg MVs compared to 70% hepatectomized rats treated with vehicle (B) (Herrera et al., 2010).

Future Liver Disease Treatments: Stem Cell-derived Extracellular Vesicles

Fortunately, researchers have found that whole cells may not be needed for liver disease treatment. Instead, the contents of extracellular vesicles (EVs) released by stem cells may be sufficient to induce liver regeneration (Herrera et al., 2010).

One of example of an EV is a microvesicle (MV). These are 80-1000 nm structures that form when endosomal membranes fuse with plasma membranes. Within the MVs released by human liver stem cells (HLSC), there are mRNA molecules that code for proteins known to be important in proliferation and liver regeneration. This includes MATK, MRE11A, CHECK2, MYH11, VASP and CDK2. On the HLSC plasma membrane there are adhesion molecules, like α4-integrin (Herrera et al., 2010).

Interestingly, anti-α4-integrin blocking antibody prevents internalization of HLSC-MVs by hepatocytes, suggesting the mechanism of uptake is dependent on α4-integrin. After entering the cell, the mRNA in the MV is translated. Consequently, the behaviour of the hepatocyte changes. This is evidenced by the enhanced proliferation and inhibited apoptosis observed in MV-treated hepatocytes in in vitro analyses. This enhanced proliferation and decreased apoptosis are also observed in vivo in 70% hepatectomy rats (Figure 3.). Furthermore, 70% hepatectomy rats treated with HLSC-derived MVs have also shown enhancement of the liver/body ratio and reduction of the lesions and steatosis typically seen in 70% hepatectomy rats. Based on these findings, HLSC-derived MVs seem to support liver regeneration (Herrera et al., 2010).

Figure 4. Results of qRT-PCR analysis of pro- and anti-inflammatory gene expression in the liver tissue. There was significant downregulation of pro-inflammatory gene (TNF-α and IL-1β) expression in AIE-EV-treated ALI mice compared to untreated ALI mice (PBS and AIEgens). However, expression of anti-inflammatory genes (IL-10 and IL-4) is significantly upregulated in the AIE-EV treatment (Cao et al., 2019).

Similarly, EVs from mesenchymal stem cells (MSCs) have also shown therapeutic potential. By tracking them with DPA-SCP, an aggregation-induced emission luminogen (AIEgen) that fluoresces red, researchers have been able to elucidate the journeys and effects of MSC-EVs (Cao et al., 2019).

When injected in mice, AIE-EVs (MSC-EVs tagged with DPA-SCP) have shown localization at the liver, with detectable fluorescence at the region after one hour. Ex vivo analyses confirm that the highest fluorescence, and therefore the greatest presence of EVs, occurs at the liver. As a result, AIE-EVs target the liver with high specificity. In acute liver injury (ALI) mice, AIE-EVs enhance proliferation, as evidenced by the upregulation of proliferation-associated genes HGF, EGF, IGF-1, and FGF-2. Also, apoptosis-associated genes Bad, Bax, Caspase 8, and Fas are downregulated, suggesting inhibition of apoptosis. Furthermore, AIE-EVs reduce inflammation. Expression of proinflammatory cytokines TNF-α and IL-1β is decreased (Figure 4.). Concurrently, expression of anti-inflammatory cytokines IL-10 and IL-4 is increased (Cao et al., 2019).

Fortunately, AIE-EVs also seem to have no effect on the function of numerous important organs, including the heart, liver, spleen, lungs, and kidneys. Therefore, MSC-EVs may be capable of producing major changes in the liver without causing many side effects (Cao et al., 2019).

Figure 5. The formation and release of MVs and exosomes. Endosomes fuse with the plasma membrane and are released into the extracellular environment as MVs (i). Endosomes fuse with CCVs to form MVEs before fusing with the plasma membrane to be released as exosomes (ii) (Psaraki et al., 2021).

Exosomes (another group of EVs) also seem to support liver regeneration (Psaraki et al., 2021). Unlike MVs, there are two steps in exosome formation (Figure 5.). First, exosomes fuse with clathrin-coated vesicles (CCVs) to form multivesicular endosomes (MVEs). Then, MVEs fuse with the plasma membrane to form exosomes. These structures are smaller than MVs (45-150nm), but also carry RNA. This includes noncoding RNA (ncRNA) which are found in MVs as well and are known to be associated with mechanisms in liver disease. Consequently, exosomes have been found to enhance proliferation, inhibit apoptosis, reduce inflammation, and promote blood vessel formation in the liver. For example, in mice wth acute liver failure/injury, bone marrow (BM)-MSC-exosomes have been found to inhibit apoptosis by downregulating Bax and cleaved caspase-3, which are proapoptotic factors. Concurrently, the expression of autophagy markers is increased. Furthermore, HLSC-derived exosomes have therapeutic effects for mice with chronic liver damage. In these cases, exosomes may inhibit the progression of conditions like cirrhosis (Psaraki et al., 2021).

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