Introduction

Hepatocyte sheet (HS) transplantation is expected to be an effective therapeutic strategy as an alternative to liver transplantation. We have developed a unique HS formation technique and have conducted study on the improvement of liver function (Miyamoto et al., Regen Ther. 2021). However, although HS has shown improvement in liver function for a short period of time, long-term functional improvement has not been achieved. This may be due to the lack of multidimensional structures in existing hepatocyte sheets, although the liver maintains various functions by possessing unique structures such as sinusoids and biliary ducts. In fact, the lack of biliary structures in the HS may induce liver damage due to bile stasis. (Fujii et al., J Tissue EngRegen Med, 2018). In other words, the formation of bile drainage mechanism is considered an important issue to maintain long-term liver function. In parallel, we have established a biliary drainage system with a luminal structure in vitro using chemically induced hepatic progenitor cells (CLiPs) (Huang et al., Biotechnol Bioeng. 2021).

Aims & Methods

In this study, we attempted to develop a new novel Hepato-Biliary Sheet (HBS) based on our biliary drainage system and evaluated its structure and hepatic function.Mouse embryonic fibroblasts (MEFs) and GFP-positive rat-derived CLiPs were sequentially seeded in temperature-responsive culture dishes and cultured for 2 weeks to induce the biliary drainage system. HBS was formed by seeding and culturing primary mature rat hepatocytes. The HBS was then implanted on the liver surface of a mouse model of liver failure, which was created by administration of Retrorsine and 70% hepatectomy, and its structure and function were evaluated in vivo.

Results

First, immunofluorescence image analysis of the prepared HBS revealed a structure consisting of CLiP-derived bile ducts (GFP-positive) and mature hepatocytes (Alb-positive, Glutamine-positive). To evaluate the incorporation of bile into the HBS, a fluorescent reagent (Cell Tracker Orange) as a marker substance was added to culture medium and bile excretion via hepatocytes bile duct cells was observed 24 hours later. Compared to HS prepared from mature rat hepatocytes alone, drug metabolism (CYP3A4 activity) and albumin production were significantly improved. CYP3A4 activities in HBS and HS were 139.3±59.9 and 75.4±47.1 (LUM/hr) (p<0.0001) and albumin productions were 1.5±0.5×104 and 1.1±0.1 x104 (ng /day) (p=0.005), respectively. Next, when the HBS was transplanted to the liver surface in non-liver failure mouse, it was confirmed that a bile duct-like structure (CK7-positive) was maintained and that mature hepatocytes (ALB-positive) were adjacent to the bile duct-like structure, and that vascular components (CD34-positive) derived from mice were infiltrated, confirming long-term cell viability, at least 1 month. Finally, to evaluate the effect of transplantation in a mouse model of liver failure, rat albumin concentration in blood was measured, and the HBS transplantation group showed higher albumin concentration than the HS group at 7 days (9.5×10^5±8.2×10^5 ng/mL vs 5.8×10^5±6.1×10^4ng/mL, P<0.05) and 28 days (1.1×10^6±9.2×10^5 ng/mL vs 3.2×10^5±3.2×10^5 ng/mL, P<0.05),respectively.

Conclusion

We have observed formations of hepatocyte and bile duct structure in HBS which is confirmed to have biliary drainage and drug metabolizing activities. HBS are expected to improve liver function, and to play a valuable role in the treatment of liver diseases through regenerative medicine.

References

Miyamoto D, Sakai Y, Huang Y, et al. Functional changes of cocultured hepatocyte sheets subjected to continuous liver regeneration stimulation in cDNA-uPA/SCID mouse: Differences in transplantation sites. Regen Ther. 2021;18:7-11.
Fujii M, Yamanouchi K, Sakai Y, et al. In vivo construction of liver tissue by implantation of a hepatic non-parenchymal/adipose-derived stem cell sheet. J Tissue Eng Regen Med. 2018;12:287-295.
Huang Y, Sakai Y, Hara T, et al. Bioengineering of a CLiP‐derived tubular biliary‐duct‐like structure for bile transport in vitro. Biotechnol Bioeng. 2021;118:2572-2584

Disclosure

This study was supported in part by the research grant from Eisai Co., Ltd.

Introduction

Chronic liver disease (CLD) is a disease of the liver that involves progressive destruction and regeneration of the liver parenchyma, leading to fibrosis and cirrhosis. Although liver fibrosis and cirrhosis have been considered irreversible, it has recently been suggested that even advanced fibrosis and cirrhosis may be reversible. The use of platelet-rich plasma (PRP) in tissue regeneration is a developing area of research for clinicians and specialists. Platelets contain proteins required for hemostasis as well as numerous growth factors. They have a prominent role in suppressing the progression of liver fibrosis in vitro and in vivo. Elevating growth factors induced by platelet transfusion can enhance liver function in patients with CLD and cirrhosis. However, PRP could increase the risk of rejection or immune response in allogenic transfusion; therefore, searching for effective therapeutic strategies is much needed.

Aims & Methods

This study evaluated the possible regenerative effect of purified platelet-derived growth factors on an experimentally induced model of liver cirrhosis. Purified Platelet Derived Growth Factors were prepared using a patented method (Gooris & Fahmy, 2019). Twenty-eight mice were included and categorized into four groups. The 1^st group acted as a normal control group. The 2^nd group acted as a Purified Platelet Derived Growth Factors control group and was injected subcutaneously with Purified Platelet Derived Growth Factors only twice weekly for eight weeks. The 3^rd group acted as a pathological control group and was injected intraperitoneally with thioacetamide (TAA) three times a week for eight weeks. At the same time, the 4^th group was injected with both Purified Platelet Derived Growth Factors and TAA for eight weeks. After eight weeks, animals were euthanized, and liver specimens and blood samples were collected for pathological and biochemical assessment.

Results

Our results showed that Purified Platelet Derived Growth Factors administration could improve the hepatic status of the mice, as cirrhotic livers were regenerated back into normal-appearing parenchyma when compared to the pathological control group. In addition, the levels of ALT, AST, and total bilirubin decreased significantly (P<0.001), and the Relative expression of hepatic Alpha-SMA was significantly down-regulated (P<0.001) after Purified Platelet Derived Growth Factors administration.

Conclusion

Purified Platelet Derived Growth Factors can be used as a new therapeutic modality for liver cirrhosis.

References

• Gooris T and Fahmy HM (2019): A method for preparing a growth factors containing platelet releasate. United States patent application US 16/462,452. Oct 24.

Introduction

Liver transplant is the only definitive treatment for chronic liver disease (CLD). Referral to a hepatologist for consideration for liver transplant should be made when a patient with CLD experiences a hepatic decompensation or has a MELD score ≥ 15. Failure to refer in a timely manner can result needing a liver transplant evaluation (LTE) in the inpatient setting while a patient is experiencing acute decompensation.

Aims & Methods

We aimed to compare the clinical presentation of patients undergoing LTE in the inpatient setting versus the outpatient setting to identify potential risk factors for requiring urgent evaluation. A list of all LTEs performed at our institution between 10/2017-8/2021 was obtained. Medical records were preliminarily reviewed for evaluation setting (Inpatient vs. Outpatients); evaluations for re-transplantation and acute liver failure were excluded. After all Inpatients (n=159) were identified, Outpatients (n=159) were selected at random to create a sample matched for age, gender, race, and ethnicity (n=318). Data were collected using our institutions electronic medical record and outside health records as obtained during LTE process. “Prior decompensations” refers to the number of the following clinical decompensations experienced prior to the time of LTE: ascites, hepatic encephalopathy (HE), variceal/acute UGIB, SBP and HRS. Categorical variables and continuous were compared between using Fisher’s Exact test, and student’s T test, respectively.

Results

Alcohol was the most common CLD etiology (42.1% Inpatient, 48.4% Outpatient). Inpatients tended to live further from our center than Outpatients (p=0.079). Inpatients had significantly higher MELD-Na scores (27.1 ± 8 vs. 13.3 ± 6; p<0.0001). Malnutrition was more common in 5.6 times more common in inpatients than outpatients (OR 5.6, 95% CI 2.9-10.8, p<0.001).. A similar proportion of patients had 1 hospitalization. But inpatients were 3 times as likely to have had ≥ 1 liver-related hospital admission prior to LTE (p<0.0001). Inpatients had significantly more prior decompensations than Outpatients prior to inpatient LTE (3.2 ± 1.4 vs. 1.8 ± 1.1, p<0.001), with the greatest differences in SBP and HRS.

Conclusion

Multiple liver-related hospitalizations and malnutrition may be considered a risk factor for ultimately needing inpatient LTE and should be taken seriously by health care providers caring for patients with CLD. Episodes of SBP and HRS should prompt providers to consider immediate or urgent LTE referral regardless of other factors.

References

Multiple liver-related hospitalizations may be considered a risk factor for ultimately needing inpatient LTE and should be taken seriously by health care providers caring for patients with CLD. Episodes of SBP and HRS should prompt providers to consider immediate or urgent LTE referral regardless of other factors.

Disclosure

D Devuni has received grant funding from Sequana Medical and receives grant support from the NIAAA (AA017986-11), both for research unrelated to this work.

Introduction

Transplantation techniques and immunosuppression treatments have been increasing notably in the past years, particularly when considering liver transplant (LT). LT is the preferred treatment for early-stage hepatocellular carcinoma (HCC). Despite the use of the Milan criteria, recurrence remains a major concern, with an estimated incidence ranging from 5% to 20%. Therefore, our objective is to assess potential risk factors, including clinical, laboratory and pathological parameters, as well as prognosis of HCC recurrence in LT recipients.

Aims & Methods

Retrospective evaluation of medical records from a Brazilian tertiary hospital, including adults who were submitted to LT due to HCC, during the period of 01/01/2002 up to 31/12/2021. Patients who had a LT for other causes, who presented a follow-up less than 1 year or with incidental detection of HCC in the liver explants were excluded. Data collection included demographic information, immunosuppressive treatments, clinical, radiological and histhopathological findings related to the HCC, as well as characteristics associated with the post-transplant follow-up and tumoral recurrence, including death. Primary outcome was recurrence of HCC, whereas secondary outcomes were survival and adopted treatments after cancer recurrence, as well as time elapsed until HCC recrudescence.

Results

150 patients were included (males 95 [63.3%], median age 60 years-old [IQR 56-66]), 130 (86.6%) were classified as Milan-in. The main cirrhosis etiologies were viral (88%) and metALD (10%). Half of patients presented an A CHILD score (50%) and a median MELDNa score of 12 (IQR 9-14). Main locoregional therapies included transarterial chemoembolization (TACE) and percutaneous ethanol injection (PEI), accounting for 48 (32%) and 45 (30%) respectively. 47 (31.33%) patients underwent two or more locoregional therapy modalities, and downstaging was achieved in 20 patients (13.3%). Explant analysis with microvascular invasion and Milan-out were detected in 18 (12%) and 30 (20.1%) individuals, respectively. HCC recurrence occurred in 21/150 patients (14%), while non-HCC neoplasias in 27/150 (18.1%) after LT. The median time to recurrence was 30 months (IQR 20-43), and systemic therapy to HCC with sorafenib was used in 12/21 patients (57.14%). Among those who presented recurrence, 19/21 died (90.4%), and prognosis after recurrence was dismal (median survival of 14.5 months, IQR 3.25-33.5). Evaluation of possible associations between HCC recurrence and clinical, laboratorial, imaging and histopathological parameters was performed, but none showed impact on HCC recurrence after LT. However, there was a trend to an association between recurrence and CHILD score (p = 0.06) and smoking (p = 0.08). Also, when evaluating patients who presented early recurrence (less than 24 months after LT), a trend of an association with the number of involved liver segments was found (p = 0.07).

Conclusion

Identifying patients at risk of HCC recurrence after LT remains as a great challenge. There are some parameters previously suggested as possible predictors, but, in accordance to literature findings, our study didn't identify significant associations. Nonetheless, the possible association between early recurrence and the number of affected liver segments hasn't been studied yet, and this is the first study that suggests an association. Thus, evaluating the number of liver segments affected by HCC might help physicians to identify patients with higher risk of recurrence, as well as giving another possible parameter to investigate in further studies.