Peripheral Injection of hUC-MSCs in the Treatment of Acute Liver Failure: A Pre-Clinical Cohort Study in Rhesus Monkeys

Background Using a toxin-induced lethal acute liver failure (ALF) monkey model, we have recently shown that early peripheral infusion of human umbilical cord mesenchymal stem cells (hUC-MSCs) can alleviate liver damage and improve animal survival by suppressing the activation of circulating monocytes and the subsequent cytokine storm. Here, we explored whether the administration of hUC-MSCs could still improve ALF when the cytokine storm is fully developed. Method We treated ALF monkeys with peripheral delivery of hUC-MSCs at 48 hr after toxin challenge. Liver indices, histology, imaging, and animal survival were recorded and analyzed. Results In our cohort study, we conducted and demonstrated that the infusion of hUC-MSCs significantly improved liver histology, effectively controlled inflammatory cytokine storms, and increased survival rates. Additionally, the administration of a higher dose of hUC-MSCs (2 × 107/monkey) yielded superior outcomes compared to a lower dose (1 × 107/monkey). Conclusion Treatment of hUC-MSCs can significantly improve the pathological and survival outcomes of ALF even when the cytokine storm has been fully developed, indicating a promising clinical solution for ALF.


Introduction
Acute liver failure (ALF) [1] is characterized by rapid deterioration of hepatic function resulting from hepatocyte damage caused by various etiological factors, such as infection with hepatitis virus, major hepatectomy, toxic drugs, and nonpharmacological intoxication (e.g., mushroom poisoning).Severe liver injury-induced coagulopathy and altered mention, namely, hepatic encephalopathy, are required for the diagnosis of this rare and life-threatening clinical syndrome, according to the European Association for the Study of the Liver (EASL) [2].Hepatocyte transplantation (HCT) and liver transplantation (LT) are regarded as effective therapies for ALF.However, HCT has limitations, such as an unknown source of hepatocytes, a high demand for cells, and the inability to proliferate in vitro.Meanwhile, LT is unavailable for most patients due to the shortage of donor livers and the limited therapy window.A reported 71%-85% of 1-year survival rate was observed in patients who underwent emergency LT [3,4], while 82% of ALF patients died within a week of diagnosis while awaiting an organ [3].Thus, new feasible and reliable alternative treatments are greatly needed to improve the outcomes of ALF patients.
Amanitin-containing species are responsible for over 90% of fatalities caused by mushroom intoxications worldwide [5], and ALF is the featured clinical syndrome in amanitin-poisoned patients.In particular, α-amanitin is believed to be the main cause of ALF by leading to hepatocyte dysfunction and, ultimately, cell death through inhibition of polymerase II and protein synthesis [6].In addition, the reabsorption of α-amanitin via the enterohepatic circulation further prolongs and exacerbates the hepatotoxic effect [7].
In our previous work, low-dose α-amanitin extracted from Amanita phalloides (AP; "death cap") and lipopolysaccharide (LPS) were used to establish rhesus monkey models of ALF [8].Although a mouse model of ALF has been used to study the related mechanism and potential medical therapy [9], our large primate model more effectively simulated human cases with the similar metabolic and physiopathological process.The monkeys exhibited typical symptoms and physiopathological changes observed in ALF cases, including dynamic changes in serology, histology, imaging, and clinical behaviors.Based on monkey models, we uncovered the essential role of activated circulating monocytes (c-Mos) in initiating ALF rather than Kupffer cells that are specific to the liver [10,11].A broad screening of serology and cytokines indicated that activated monocytes were critical in the development and exacerbation of hepatocyte injuries through massive secretion of interleukin 6 (IL-6).The subsequent excessive systemic inflammation triggered by IL-6 was the main factor promoting hepatocyte necrosis, loss of systemic homeostasis, and eventual fatality.Our findings revealed that c-Mos were a promising therapeutic target, and its activation should be inhibited prior to uncontrolled systemic inflammation.
Mesenchymal stem cells (MSCs), a group of cells with the ability to self-renew and differentiate, garnered significant interest over the past decade due to their therapeutic potential in various diseases [12,13,14].In particular, human umbilical cord mesenchymal stem cells (hUC-MSC) [15,16,17] and hUC-MSC-derived exosomes [18] have shown protective effects against acute hepatic injury, fibrosis, and other disorders.We recently demonstrated that peripheral delivery of hUC-MSCs significantly improved the survival of monkeys with ALF [19].Our previous report [10] identified that IL-6 secreted by c-Mos was the key initiator of the inflammatory cascade, and hUC-MSCs simultaneously suppressed the activation of c-Mos and its aggregation in the liver [19].The monkeys treated with hUC-MSCs had profoundly ameliorated clinical manifestations, physiopathology, and serology.The suppression of c-Mos possibly resulted from the inhibitory miRNA in the exosome released by hUC-MSCs after toxin stimulation [18], which involved factors that inhibited the inflammatory reaction and immune response.This finding suggests that hUC-MSCs and their derivatives may be potential therapeutic options for ALF and other liver disorders.
Compared to many other tissues and organs, the human umbilical cord is a rich source of MSCs without risk to donors, and hUC-MSCs possess a great capacity for proliferation and differentiation into hepatocyte-like cells [20].The low immunogenicity of hUC-MSCs allows for allogeneic transplantation and even xenogeneic transplantation, both of which have been demonstrated to be feasible in monkeys [19,21].Moreover, we did not detect carcinogenesis in the recipient monkeys during follow-ups of over 5 years.Therefore, hUC-MSC-based treatment is anticipated to be a safe, feasible, and effective solution for ALF that meets the prerequisites for translation to clinical application.In this study, we conducted a cohort study to further validate the efficacy and safety of hUC-MSCs in the rhesus monkey model of toxin-induced ALF.

Peripheral Infusion of hUC-MSCs Significantly Improves
Survival in Monkeys with ALF.For the establishment of the toxin-induced ALF model, a total of 37 monkeys were recruited and randomly divided into two dose groups: low dose (α-amanitin at 20 μg/kg, n = 18) and high dose (α-amanitin at 40 μg/kg, n = 19).After the toxin infusion, the monkeys in each dose group were further randomly assigned to three subgroups, receiving either 1 U or 2 U of hUC-MSCs or an identical volume of saline at 48 hr post α-amanitin infusion (Figure 1(a)).Liver biopsies and blood collection were conducted at indicated time points (Figure 1(b)).
Nearly all monkeys demonstrated progressive poor appetite, mental indifference, and fatigue 24-48 hr after toxin injection.The clinical symptoms and signs were more common and severe in animals injected with a high dose of α-amanitin.The monkeys were euthanized when an irreversible hepatic coma developed.All 18 monkeys who received a low dose of αamanitin survived, regardless of treatment.While no monkey that received a high dose of α-amanitin died before 48 hr following toxin exposure, six (85.7%), four (66.7%), and three (50.0%)monkeys died within the first week, with median survival times of 68, 86, and 108 hr in the saline, 1-U and 2-U hUC-MSC-treated group, respectively.The hUC-MSC therapy obviously improved the overall survival of animals with severe ALF (P ¼ 0:036; Figure 1(c) and Additional file 1: Table S1).All the monkeys that survived for the first week have achieved long-term survival and have been followed up for 5 years so far.

hUC-MSCs Improve Liver Histology and Hepatic Indices.
In spite of the fact that all the monkeys survived when they received a low dose of amanitin injection, a significant increase in most hepatic indices, including bilirubin and liver enzymes, was observed 48 hr after toxin exposure.In saline-treated monkeys, these indices began to soar and peaked at 72 or 96 hr (Figure 2(a)).A similar variation was observed in 1-U hUC-MSC-treated monkeys, but all their parameters showed lower peak values and began to decrease earlier than in the saline group.In addition, monkeys treated with 2 U of hUC-MSCs displayed much lower peak values even than those in 1-U hUC-MSC-treated monkeys.We observed a 14-fold increase in the peak value of total bilirubin (TBIL) to 31 μmol/L in the saline-treated group, which decreased to 20 and 4.5 μmol/L after different doses of hUC-MSC intervention.More incredible fold changes were also observed in alanine aminotransferase (ALT) and aspartate aminotransferase (AST), a 186-fold increase in ALT to 5,211 U/L and a 119-fold increase in AST to 4,460 U/L.Then ALT decreased to 3,058 U/L in the 1-U hUC-MSC group and 1,807 U/L in the

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The biopsies at 48 hr after toxin exposure showed mild to severe steatosis, focal necrosis, and infiltration of inflammatory cells in the liver.Then another 48 hr later, hUC-MSC therapy seemed to more noticeably improve the histology compared to saline-treated ones, although the histology of each group recovered to some extent (Figure 2(b)).
After a high dose of amanitin challenge, the monkeys without hUC-MSC infusion exhibited a sharp and dramatic increase in hepatic indices.All the indices peaked at around 120 hr, reaching hundreds of times the normal upper limit (Figure 3(a)).TBIL increased 42-fold to 97 μmol/L, ALT increased 72-fold to 3,917 U/L, and AST increased 110-fold to 4,190 U/L.Because only one monkey survived, we did not analyze its parameters further.Infusion of hUC-MSCs profoundly ameliorated the hepatic indices; all the parameters showed much lower peaks at a similar time point to salinetreated monkeys and then gradually returned to normal levels with 1-2 weeks (Figure 3(a)).TBIL decreased to 49.5 μmol/L in 1-U hUC-MSC-treated monkeys and 14.12 μmol/L in 2-U hUC-MSC-treated monkeys; ALT decreased to 1,860 U/L and 1,994 U/L in monkeys treated with two different doses of hUC-MSCs, respectively; and AST decreased to 1,202 and 1,015 U/L.The five indices of DBIL, IBIL, ALP, GGT, and total bile acid (TBA) were significantly lower in the hUC-MSCtreated groups (1 U and 2 U) compared to the saline-treated group (Figure S2).
The biopsy showed that 48 hr after the high dose of amanitin challenge, the livers developed mild-to-moderate steatosis and focal necrosis.Then, lethal patchy necrosis and sinusoid hyperemia appeared at later times if hUC-MSC infusion was unavailable.For monkeys that received hUC-MSC therapy, hepatic histology also showed apparent steatosis, piecemeal necrosis, and infiltration of inflammatory cells (Figures 3(b) and 3(c)).The autopsy of the deceased monkeys showed a shrunken appearance and visible ecchymosis on the surface of the failed liver (Figure 3(d)).However, the treatment succeeded in remarkably alleviating hepatic necrosis and bleeding and inhibiting the disintegration of hepatic lobules.

hUC-MSCs Inhibit the Activation of Circulating Monocytes
and Suppress Systemic Inflammation.Our previous study showed that c-Mos are critical in accelerating ALF by producing IL-6.Early infusion of hUC-MSCs could inhibit the activation of c-Mos and thereby prevent the liver from excessive inflammatory injury [19].We explored whether the infusion of hUC-MSCs has a role in regulating c-Mos activation and systemic inflammation when c-Mos and a cytokine storm were robustly activated.Flow cytometry assay revealed that the proportion of CD14+ CD16+ CCR2+ monocytes in peripheral blood was approximately halved at 24 hr after hUC-MSC intervention (Figures 4(a IL-6 is considered both an inflammatory cytokine and a regenerative mediator in liver conditions.The secretion of IL-6 in ALF affects the complete recovery and reconstitution of liver parenchyma and liver function.In animals injected with either dose of toxin, IL-6 began to increase at 12-24 hr.Additionally, other circulating inflammatory factors, such as TNF-α and IL-1β, showed similar trends.They peaked between 24 and 48 hr after toxin induction.Although the fluctuation of data might have impaired the statistical difference, we could see that hUC-MSC infusion played a role in suppressing the serum levels of inflammatory cytokines but increasing some anti-inflammatory and protective factors, such as IL-10, IL-4, and IL-1RA.This effect was more obviously observed in monkeys that received a high dose of toxin.Six out of seven saline-treated monkeys were sacrificed without a noticeable decline in these cytokines, while it took the only surviving monkey another week to control systemic inflammation.hUC-MSCs, particularly in a higher concentration, remarkably ameliorated systemic disarrangement around 48 hr after infusion (Figure 5).

hUC-MSC Infusion
Facilitates Liver Regeneration.Active liver regeneration, indexed by Ki67 immunohistochemistry staining, was observed in all monkeys during the first 4 days after toxin injection, except that very rare proliferating hepatocytes were detected in the autopsied livers.Notably, infusion of hUC-MSCs enhanced the Ki67 indexes two-to threefold at each indicated time point (Figures 6(a) and 6(b)).Proliferation was detectable up to 4 days after toxin injection in all survived monkeys.Interestingly, the only surviving monkey treated with saline experienced a delayed but significantly more prolonged regeneration period than those treated with hUC-MSCs.

Monkeys That Survived ALF Remain Healthy in the
Long-Term Follow-Ups.To further confirm the safety of hUC-MSC transplantation, clinicopathologic and imaging evaluations were applied in the six surviving monkeys every year for 5 years.Most serum parameters were kept at normal levels, including blood counts, hepatic indices, renal function, and clotting times.No tumoral lesion, fibrosis, or thrombosis was detected in ultrasonography, MRI, or liver biopsies.In addition, no immune disorders, including those related to immune cells, antibodies, and complements, were detected.Our results demonstrated that hUC-MSC treatment was not associated with carcinogenesis or other secondary complications.

Discussion
There is still no robust nonsurgical strategy for ALF, partially due to its unclear pathophysiological mechanism.In the current study, we established primate animal models of ALF using amanitin alone for the first time to further evaluate the therapeutic effect of hUC-MSCs.Compared to our previous preliminary study, the following modifications were made for certain reasons: (1) Only α-amanitin was used to establish the ALF model, rather than a combination of αamanitin and LPS [8,10].All saline-treated animals died after infusion of α-amanitin and LPS in previous studies [19,22], where the mortality was significantly higher than in a clinical setting [7].Besides, the intraperitoneal injection of LPS was supposed to activate peritoneal macrophages before being absorbed by the peritoneum and entering circulation [23], making it extremely difficult to determine the role of the c-Mos in ALF pathogenesis.The addition of LPS (2) Early hUC-MSC therapy is critical, as c-Mos-derived IL-6 increases dramatically within 24 hr after the toxin challenge [10].We previously treated monkeys with hUC-MSCs only 2 hr after toxin injection.However, human cases caused by a variety of etiological factors, not limited to poisonous mushroom intake, are often admitted with apparent manifestation after the full development of symptoms and signs.Here, we set out to explore whether hepatic indices and prognosis could be improved by hUC-MSCs at 48 hr after toxin injection when the cytokine storm is fully developed.
(3) Preliminary experiments suggested that an increased number of hUC-MSCs was more effective in improving liver histology and hepatic function.In the current study, an infusion of 1 × 10 7 (1 U) or 2 × 10 7 (2 U) stem cells per monkey was applied to reveal the dose effect of hUC-MSCs, which was ambiguous before.(4) Last but not least, we explored the potential role of hUC-MSCs in ameliorating cytokine storm when the c-Mos are fully activated.We found that hUC-MSCtreatment significantly improved toxin-induced hepatic indices, liver histology, systemic inflammation, and survival.In our previous work, all monkeys who received a lethal dose of toxins were rescued by immediate peripheral infusion of hUC-MSCs, which prevented the activation of c-Mos and IL-6 secretion and the secondary cytokine storms [19].However, in routine clinical settings, symptoms of ALF from various causes, such as initial jaundice, lethargy, and mental alterations, are hardly noted, and the best and most effective time for treatment is always missed [2].It is, therefore, worth exploring whether there is still a role for hUC-MSC therapy when c-Mos have been already fully activated [2].Herein, our study suggested that delayed hUC-MSC treatment could also significantly improve the liver indices and histology in nonfatal ALF and survival outcomes of monkeys with fatal ALF.We noted that a doubled dose of hUC-MSC therapy was related to an earlier recovery from ALF in surviving monkeys according to earlier ameliorated liver function, serum BA, and clinical manifestations.Although neither the survival rate (50.0% vs. 33.3%)nor the median survival time (108 hr vs. 86 hr) was significantly improved in animals with severe ALF treated with 2-U hUC-MSCs compared with those that received 1-U cell therapies, though tendencies were observed.It is undoubtedly that the efficiency of MSC therapy is determined by their potential and infused cell number.Our data recommend a high dose of cell infusion if available.
The survival benefit from hUC-MSCs in ALF models was exerted mainly through an inhibitory effect on the activation and proliferation of immune cells and severe acute inflammation [16,19].Though hepatocyte injury is the initial event, it is widely believed that uncontrolled immune-driven inflammation in response to liver damage is central to the pathogenesis of liver and extrahepatic organ failures [10,24,25].In ALF, systemic inflammation results in hepatic dysfunction, thereby leading to diminished ammonia detoxification and hyperammonemia, which is directly associated with hepatic encephalopathy and ALF-related mortality [26].As for the therapeutic effect of hUC-MSCs, their multiple immunomodulatory properties are involved: (1) suppressing proliferation or activation of immune cells, such as peripheral blood mononuclear cells (PBMCs) [27] and monocytes [19]; (2) inhibiting expression of pro-inflammatory factors, such as TNF-α, IL-1β, IL-6, and IL-8, thereby controlling inflammation and hepatocyte apoptosis [19,28]; and (3) promoting secretion of anti-inflammatory cytokines [15,19], such as IL-4 and IL-10, which prevent excessive inflammation.Although we infused hUC-MSCs when the c-Mos were activated and the cytokine storm was inflamed, the number of activated c-Mos and the serum levels of proinflammatory factors could be decreased.Our findings further support the immunoregulatory role of hUC-MSCs played in ALF, and delayed cell infusion is also recommended to ameliorate sustained overactivation of inflammatory cells or promote the secretion of inhibitory factors.
Apart from the protective effect on liver function and hepatocyte necrosis, we suspect that hUC-MSCs were associated with more activated hepatocyte proliferation and shortened time required for the completion of liver repair in surviving monkeys.After a steady rise in the first 2 or 3 days, proliferation in the saline-treated animals showed a sharp decline along with a phenomenal increase in inflammatory cytokines.In contrast, reparative ability continued to increase after hUC-MSC administration before a gradual decrease in the survived monkeys.Immune cells exert dual roles at different phases of ALF, from promoting to inhibiting hepatocyte proliferation [29].TNF-α, IL-1, and IL-6 initially generated by liver-specific macrophages (KCs) were found to induce liver proliferation in the early stage in response to liver injury [30].Furthermore, circulating macrophages also contributed to hepatocyte regeneration and vascularization [31,32].However, the subsequent systemic inflammation due to the   Stem Cells International massive activation of macrophages disrupted hepatic homeostasis and parenchymal repair [22], which was ameliorated by hUC-MSC therapy in our study.As xenogeneic cells in preclinical settings, hUC-MSCs were well-tolerated in our primate models [19].hUC-MSC activation was not supposed to result in allogeneic immune rejection according to their minimal expression of human leukocyte antigen DR (HLA-DR) after activation [27].hUC-MSCs have been increasingly used to treat a variety of human diseases, for example, systemic lupus erythematosus (SLE) [33], arthritis [34], spinal cord injury (SCI) [35], and severe COVID-19 [35].All these instances demonstrated the long-term safety and efficacy of hUC-MSC therapy.Another safety concern of translating hUC-MSC therapy to clinical practice is the potential for tumorigenicity.Previous studies reported no tumors either in vitro or in mice [36,37].Likewise, none of our monkeys developed tumor with follow-up of more than 5 years.Therefore, based on current evidence, hUC-MSC transplantation is expected to be safe.[19].Cultured MSCs were examined for quality control by flow cytometry checking the expression of surface biomarkers.To check the multipotent differentiation potential, osteogenic and adipogenic differentiation of hUC-MSCs were induced by culturing in a certain medium.Viral factors (hepatitis B surface antigen, hepatitis C antibody, HIV, syphilis, and cytomegalovirus), bacteria (aerobes, anaerobes, and fungi), and endotoxin were monitored.All test results were provided by the company, while hUC-MSCs with viability of over 95% at p3-5 were applied in the subsequent process.4.6.Statistical Analysis.All data were presented as mean (AE SEM).Student's t-test was used to compare selected parameters between two groups or compare with the data at −48 hr.Survival outcomes were plotted by Kaplan-Meier curves, while the difference was determined by log-rank analysis.Data processing was performed by SPSS 24.0 and GraphPad 9.0.0 analysis software.P value < 0.05 was considered as statistically significant.

Conclusion
Treatment of hUC-MSCs significantly improved pathologic and survival outcomes in monkeys when the systemic inflammation was fully developed by ameliorating the activation of c-Mos, decreasing inflammatory factors, and improving liver repair.A higher dose of hUC-MSCs tended to have a better therapeutic effect.Nevertheless, we are still unable to trace the distribution, metabolism, and function of hUC-MSCs in vivo, and the underlying mechanisms need further investigation.In summary, using a nonhuman primate model, our cohort study indicates that hUC-MSCs exhibit great potential for prevention and recovery from ALF, providing a clinical solution for this lethal syndrome.

FIGURE 1 :
FIGURE 1: hUC-MSCs improve survival in monkeys with ALF.(a) Schematic representation of the experimental grouping.(b) Schematic representation of the experimental design.(c) Survival curves for the monkeys submitted to different treatments (Kaplan-Meier method with log-rank test) P <0:001.

4. 5 .
Transplantation of hUC-MSCs to Rescue ALF Monkeys.Animals were randomly assigned to an intervention group (MSC+) and control group (MSC−).For MSC+ group, 1 U (1 × 10 7 ) or 2 U (2 × 10 7 ) of MSCs was suspended in 100 mL saline before slow peripheral infusion at 48 hr after toxin challenge.It took about 30-50 min to complete the cell transplantation with an infusion rate of 15-30 drops/min.For MSC− group, an equal volume of saline was applied at the same time point.
[10]rmination of Toxin.In the previous study, we established the ALF monkey model with α-amanitin (25 μg/kg bodyweight) and LPS (1 μg/kg)[19].To eliminate the impact of the LPS, in the initial pre-experiment, 20 and 40 μg/kg bodyweight of α-amanitin without LPS were administrated in two monkeys, respectively.The monkey that received 40 μg/kg αamanitin died within 96 hr, while the other one survived but displayed robustly increased hepatic indices and noticeable histological changes.Thus, we decided to establish monkey models of mild and severe acute liver injury by slow infusion of 20 and 40 μg/kg bodyweight of α-amanitin, respectively, which was modified on the basis of our previous report[10].