In our study, administration of hydrogel-encapsulated MSC-CM to the intramesenteric space significantly promoted liver regeneration after partial hepatectomy even until the termination stage of liver regeneration in rodent MASLD models. Additionally, the mode of regeneration was dominated by cellular hypertrophy, with a suppression of cell division. Furthermore, this regeneration was accompanied by maintenance of mitochondrial function, such as ATP production and β-oxidation of fatty acids, seemingly the energy produced in the mitochondria fueled liver regeneration.
In the present study, rats fed the MCD diet were used as a model of MASLD. The MCD diet induces systemic lipodystrophy that increases the influx of free fatty acids to the liver, resulting in oxidative stress and hepatocyte apoptosis. In mice fed the MCD diet, glucose storage was significantly reduced compared to mice fed standard diet22. Additionally, mice with MASLD induced by a methionine-choline deficient diet had poorer post-surgical outcomes following hepatectomy compared to their healthier counterparts23, reflecting the increased complications and death rates observed in MASLD patients undergoing partial hepatic resection24.
Hepatocyte hypertrophic regeneration after partial hepatectomy has been considered to occur only when proliferative potential of hepatocytes is compromised, such as hepatocytes deficient in specific genes related to cell cycle25, bleeding26 during hepatectomy or administration of dexamethasone27. Interestingly, Miyaoka et al. reported that hypertrophy and proliferation almost equally contribute to liver regeneration after 70% partial hepatectomy in normal murine liver28, which is contrary to the notion that proliferation dominantly contribute to regeneration. In the present study, expression of PCNA which is a marker of cell cycle entry increased in the MSC-CM group compared to controls. In parallel, cyclin D1 expression and mitosis were depressed in the MSC-CM group. The rate of Ki-67-positive nuclei were comparable between the groups, suggesting that there was no difference in proliferation. Concurrently, liver regeneration rate was significantly increased in the MSC-CM group compared to controls at 30h, which correspond to the progression/maintenance phase after hepatectomy. Above mentioned results indicate that MSC-CM-induced cell cycle arrest in fact promoted liver regeneration in a hepatocyte hypertrophic manner instead of hindering it.
Cytokine array analysis revealed that MSC-CM used in this study contained substantial amount of Activin-A. Activin-A plays the most crucial role in maintaining the homeostasis of liver weight. In a normal liver of quiescent state, moderate expression of Activin-A is observed. However, following partial hepatectomy, its expression dramatically decreases immediately. As regeneration progresses, its expression significantly increases, reaching its peak around the time when liver weight is restored to its original level29. Hepatocytes are the primary cells responsible for the synthesis of Activin-A and exhibit a strong expression of type I and II activin receptors. Activin-A orchestrates the inhibition of hepatocyte expansion via autocrine/paracrine pathways, highlighted by alterations in the expression of genes governing proliferation and senescence, a reduced propensity for cell division30.
Moreover, functional analysis identified that hepatocyte nuclear factor (HNF)-4α might be critical targets of activin A signaling, implicated in the regulation of hepatocyte proliferation and lipid homeostasis. Meanwhile activin-HNF4α-coagulation axis plays a crucial role in determining the clinical outcome of severe acute liver failure31. Thus, Activin-A does not simply inhibit the proliferation of hepatocytes but also promotes their survival and affects lipid metabolism, which aligns with the findings of this study. To the best of our knowledge, the effect of exogenous Activin-A administration after liver resection has not been reported previously. This unique pattern of hepatocyte hypertrophic regeneration demonstrated in the present study through MSC-CM might be partially attributed to the action of Activin-A.
Previous study reported that impairments in oxidative phosphorylation, such as decreased liver ATP synthesis and increased production of ROS in MASH patients32. Therefore, mitochondrial dysfunction is supposed to play a significant role in the progression of MASH. Acetyl-CoA is normally generated through the sugar and fatty acids (β-oxidation) metabolism and converted to ketone bodies in the liver under metabolic disturbances. Since acetyl-CoA produces ATP in the tricarboxylic acid (TCA) cycle, energy shortage occurs due to defective utilization of acetyl-CoA in starvation state and thereby enhancement of β-oxidation to generate ketone bodies is required. Intrahepatic glycogen and free glucose storage were significantly reduced in mice fed the MCD diet33, indicating that supply shortage and impaired utilization of acetyl-CoA coexist in this animal model of MCD diet induced MASLD-like features in the context of leanness, leading to ATP deficit in liver. Analogous to our results, the reduction in phosphatase and tensin homolog (PTEN) expression following hepatectomy enhances the utilization of transient regeneration-associated steatosis (TRAS) -derived accumulated liver lipids by β-oxidation in mitochondria34, providing energy for hypertrophic liver regeneration. Our results demonstrated that MSC-CM treatment improved liver regeneration rate as well as increased ATP and β-hydroxybutyrate content and decreased acetyl CoA and triglyceride in remnant liver until the termination phase of regeneration after hepatectomy. This implies that MSC-CM promoted liver regeneration not only in liver volume, but also in functionality by affecting lipid metabolism as fuel sources to support regeneration. Further studies are needed to clarify the functional link among MSC-CM, hepatocyte hypertrophic regeneration and β-oxidation.
The present study showed that the serum level of IL-10 at 9h and 30h after hepatectomy were significantly higher in the MSC-CM group. IL-10 is a cytokine with anti-inflammatory properties that plays a pivotal role in modulating immune responses. It is produced by almost all subsets of leukocytes, including dendritic cells, macrophages, T cells, natural killer cells, B cells35 and MSCs. IL-10 secreted by transplanted MSCs activated the mammalian target of rapamycin (mTOR) pathway and thereby enhanced mitochondrial function as well as correct abnormal lipid metabolism in the treatment of post-hepatectomy liver failure36. Although MSC-CM used in our model contained IL-10, its concentration was not high compared to serum IL-10 levels. It is possible that the other factors in MSC-CM acted on IL-10 producing cells to enhance the production of IL-10.
Olfactory receptors (ORs) are seven-transmembrane G protein-coupled receptors (GPCRs) mainly located in the olfactory sensory neurons of the olfactory epithelium. GCPRs play a crucial role as chemosensors by detecting and decoding chemical signals in their environment37. According to the previous studies, ORs are ectopically expressed throughout the body including the liver38, heart39, kidney40, and pancreas41. The liver is the largest metabolic organ which regulates whole-body homeostasis by sensing and detoxifying drugs, alcohol, and environmental toxins, metabolizing nutrients, synthesizing and secreting bile acids, and removing toxins and other waste products from the blood. Thus, the liver appears to be primed to utilize these ORs.
Recently, several groups have identified distinctive metabolic functions for hepatic ORs. Olfr734 responds to Asprosin and serves as a fasting-induced glucogenic hormone42. Asprosin is encoded by FBN1 gene whose mRNA expression is confirmed to be abundant in MSCs. In addition, Olfr544 can trigger lipolysis upon activation in diabetic mice43. Notably, GO enrichment analysis showed that the expression of functional category sensory perception of smell genes were enhanced following hepatectomy (the control group) compared with the MCD-fed alone group whereas those genes were suppressed in the MSC-CM group compared with the control group. For the brain to identify a wide variety of odors, it is crucial that each sensor carries only one type of olfactory receptor, a principle known as the 'one sensor, one receptor' rule in the olfactory system. Once a specific set of ORs is activated, it becomes necessary to inhibit the activation of other OR genes in that cell. This is ensured by a negative feedback mechanism that deactivates additional specific control regions known as locus control regions and their associated protein factors, thereby upholding the principle of one neuron per receptor44. This perception led us to speculate that MSC-CM might contain factors that serve as ligands for certain olfactory receptors, and their delivery subsequently suppressed the expression of other olfactory gene receptors. ORs in the liver could potentially function as chemical sensors, altering liver metabolism. Further detailed research is deemed necessary to determine whether MSC-CM contains ligands for specific ORs and the impact of activating those ORs on liver regeneration.
Previous research has shown that pharmacological alteration of macrophage polarization towards an M2 phenotype partially reversed liver steatosis45. Moreover, in animal model of acute liver failure (ALF), MCP-1 and Siglec-9 secreted from mesenchymal stem cells derived from human exfoliated deciduous teeth synergistically have been shown to promote the M2 differentiation of bone marrow-derived macrophages via CCR2 and thereby improved the survival of ALF rats46. In our model, MSC-CM containing certain amounts of MCP-1 did not alter the number of CD163-positive cells, one of the specific markers of M2-type macrophage, implying that regeneration effect of MSC-CM was not M2-type macrophage-mediated.
In liver regeneration after partial hepatectomy, vascular remodeling through autocrine and paracrine control mediated by angiogenic growth factors and their receptors is known to be involved. It has been demonstrated that hepatocyte produce VEGF within 48h-72h after PHx, promoting sinusoidal endothelial cell proliferation and hepatocyte proliferation47. Additionally, other research has revealed that VEGF promotes hepatocyte proliferation by inducing HGF production through VEGFR1 in LSECs48. The MSC-CM used in this study contained certain amount of VEGF, thus we assessed the angiogenesis during regeneration by CD31 immunohistochemistry. Unexpectedly, our results revealed that MSC-CM did not seem to affect angiogenesis in this model.