Naoko Kamo

Transplantation of Embryonic Stem Cell‐Derived Endodermal Cells into Mice with Induced Lethal Liver Damage

ESCs are a potential cell source for cell therapy. However, there is no evidence that cell transplantation using ESC‐derived hepatocytes is therapeutically effective. The main objective of this study was to assess the therapeutic efficacy of the transplantation of ESC‐derived endodermal cells into a liver injury model. The β‐galactosidase‐labeled mouse ESCs were differentiated into α‐fetoprotein (AFP)‐producing endodermal cells. AFP‐producing cells or ESCs were transplanted into transgenic mice that expressed diphtheria toxin (DT) receptors under the control of an albumin enhancer/promoter. Selective damage was induced in the recipient hepatocytes by the administration of DT. Although the transplanted AFP‐producing cells had repopulated only 3.4% of the total liver mass 7 days after cell transplantation, they replaced 32.8% of the liver by day 35. However, these engrafted cells decreased (18.3% at day 40 and 7.9% at day 50) after the cessation of DT administration, and few donor cells were observed by days 60–90. The survival rate of the AFP‐producing cell‐transplanted group (66.7%) was significantly higher in comparison with that of the sham‐operated group (17.6%). No tumors were detected by day 50 in the AFP‐producing cell‐transplanted group; however, splenic teratomas did form 60 days or more after transplantation. ESC transplantation had no effect on survival rates; furthermore, there was a high frequency of tumors in the ESC‐transplanted group 35 days after transplantation. In conclusion, this study demonstrates, for the first time, that ESC‐derived endodermal cells improve the survival rates after transplantation into mice with induced hepatocellular injury.

Improvement of the survival rate by fetal liver cell transplantation in a mice lethal liver failure model.

Background. The use of cell transplantation as an alternative therapy for orthotopic liver transplantation has been widely anticipated due to a chronic donor shortage. We previously reported the method used to enrich hepatic progenitor cells (HPCs) forming cell aggregations. In this study, we transplanted HPCs into the liver injury model mice to determine whether HPC transplantation may improve the liver dysfunction. Methods. We obtained donor cells from E13.5 fetal livers of green fluorescent protein (GFP) transgenic mice. We transplanted GFP-positive fetal liver cells into the transgenic mice which express diphtheria toxin (DT) receptors under the control of an albumin enhancer/promoter. Subsequently, we induced selective liver injury to recipient mice by DT administration. We then evaluated the engraftment of the transplanted cells and their effect on survivorship. Results. The low dose of DT induced sublethal liver injury and the high dose of DT was lethal to the liver injury model mice. The transplanted GFP-positive cells were engrafted into the recipient livers and expressed albumin, resembling mature hepatocytes. They continued to proliferate, forming clusters. The survival rate at 25 days after transplantation of the cell-transplanted group (8 of 20; 40.0%) was improved significantly (P=0.0047) in comparison to that of the sham-operated group (0 of 20; 0%). Conclusions. The transplanted cells were engrafted and repopulated the liver of recipient mice, resulting in the improvement of the survival rate of the liver injury model mice. We therefore propose that HPCs are a desirable cell source for cell transplantation.

Impact of skeletal muscle mass index, intramuscular adipose tissue content, and visceral to subcutaneous adipose tissue area ratio on early mortality of living donor liver transplantation.

Background Skeletal muscle depletion has been shown to be an independent risk factor for poor survival in various diseases. However, in surgery, the significance of other body components including visceral and subcutaneous adipose tissue remains unclear. Methods This retrospective study included 250 adult patients undergoing living donor liver transplantation (LDLT) between January 2008 and April 2015. Using preoperative plain computed tomography imaging at the third lumbar vertebra level, skeletal muscle mass, muscle quality, and visceral adiposity were evaluated by the skeletal muscle mass index (SMI), intramuscular adipose tissue content (IMAC), and visceral to subcutaneous adipose tissue area ratio (VSR), respectively. The cutoff values of these parameters were determined for men and women separately using the data of 657 healthy donors for LDLT between 2005 and 2016. Impact of these parameters on outcomes after LDLT was analyzed. Results VSR was significantly correlated with patient age (P = 0.041), neutrophil-lymphocyte ratio (P < 0.001), body mass index (P < 0.001), and SMI (P = 0.001). The overall survival probability was significantly lower in patients with low SMI (P < 0.001), high IMAC (P < 0.001), and high VSR (P < 0.001) than in each respective normal group. On multivariate analysis, low SMI (hazard ratio [HR], 2.367, P = 0.002), high IMAC (HR, 2.096, P = 0.004), and high VSR (HR, 2.213, P = 0.003) were identified as independent risk factors for death after LDLT. Conclusions Preoperative visceral adiposity, as well as low muscularity, was closely involved with posttransplant mortality.

Impact of sarcopenic overweight on the outcomes after living donor liver transplantation

Background The effect of body composition disturbances has been recently in focus. Sarcopenic obesity, a co-occurrence of low muscle mass and high body fat was reportedly predictive of high mortality in patients with cirrhosis. However, the impact of the interacting sarcopenia and overweight on the outcomes after liver transplantation is still unclear. Methods We evaluated 200 patients undergoing adult-to-adult living donor liver transplantation at our institution between January 2008 and November 2013 classified according to BMI and psoas muscle index (PMI) on admission to transplant into 4 subgroups; sarcopenic overweight (SO), sarcopenic non-overweight (SN), non-sarcopenic overweight and non-sarcopenic non-overweight (NN). Short-term outcomes and overall post-transplant survival were compared among the four subgroups. Results Sarcopenic patients with preoperative low PMI had higher incidence of postoperative bacteremia and major postoperative complications, and poorer overall post-transplant survival than non-sarcopenic patients with normal/high PMI (P<0.001, respectively). Overweight recipients had a significantly higher overall survival (OS) rate than non-overweight patients (P=0.021). SO subgroup (low PMI and BMI ≥25) had statistically indifferent incidence of postoperative bacteremia, major postoperative complications or overall post-transplant survival than other recipients. In contrast, SN subgroup (low PMI and BMI <25) had higher incidence of postoperative bacteremia (P<0.001), major postoperative complications (P<0.001) than the SO subgroup and possessed the poorest OS among the four recipient subgroups (P=0.001). Conclusions In living donor liver transplantation, preoperative SO did not confer added significant morbidity or mortality risks than the stand-alone sarcopenia.

A transmembrane glycoprotein, gp38, is a novel marker for immature hepatic progenitor cells in fetal mouse livers

Previously, we clarified the surface antigen profiles of hepatic progenitor cells (HPCs) in fetal liver tissue as the CD49f+CD45−Thy1− cell fraction. However, these cells were a heterogeneous cell population containing various stages of differentiation. This study aimed to detect more immature HPCs, using a novel surface antigen, gp38. After the collagenase digestion of fetal livers harvested from E13.5 to E18.5 fetal mice, HPCs were obtained and divided into two subpopulations using flow cytometry: gp38-positive HPCs, and gp38-negative HPCs. Both types of HPCs were characterized by immunocytochemistry and RT-PCR. The proliferative activity was compared by BrdU incorporation and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTS) assay. Furthermore, the comprehensive gene expression was investigated by DNA microarray. Both types of HPCs expressed alpha-fetoprotein. However, the gp38-positive HPCs derived from E13.5 fetal livers did not express albumin or cytokeratin 19, while the gp38-negative HPCs did. DNA microarray revealed that some genes related to the Wnt signal pathway were up-regulated in the gp38-positive HPCs. Furthermore, Wnt3a had a proliferative effect on the gp38-positive HPCs. In conclusion, the gp38-positive HPCs derived from fetal liver tissue until E13.5 could therefore be candidates for hepatic stem cells in the fetal liver.

Preoperative Low Muscle Mass and Low Muscle Quality Negatively Impact on Pulmonary Function in Patients Undergoing Hepatectomy for Hepatocellular Carcinoma

Background: Sarcopenia is a prognostic factor for mortality in digestive surgery. However, the correlation between preoperative cardiopulmonary function and sarcopenia in patients undergoing hepatectomy for hepatocellular carcinoma (HCC) remains unclear. Methods: The present study investigated the impact of preoperative sarcopenia on cardiopulmonary function in 402 patients who underwent first hepatectomy for HCC between April 2005 and April 2015. The quantity and quality of skeletal muscle were evaluated using psoas muscle index (PMI) and intramuscular adipose tissue content (IMAC), respectively, as determined from preoperative computed tomography imaging. Correlations between preoperative cardiopulmonary function and sarcopenic factors (PMI and IMAC) were evaluated. Results: No significant correlations were found between left ventricular ejection fraction and the two sarcopenic factors. On the other hand, preoperative vital capacity (VC) and forced expiratory volume in 1 s (FEV1) correlated significantly with PMI (p < 0.001 each) in males and with IMAC (p < 0.001 each) in females. Moreover, VC and FEV1 in the preoperative low PMI (p < 0.001 each) and high IMAC (p = 0.002 and p < 0.001, respectively) groups were significantly lower than in the normal group in males. In females, VC and FEV1 were significantly lower in the preoperative high IMAC group than in the normal group (p < 0.001 each). Conclusion: Preoperative low muscle mass in males and low muscle quality in males and females were significantly associated with pulmonary dysfunction.

Impact of Sarcopenic Obesity on Outcomes after Liver Transplantation; Does Sarcopenic Obesity cause Poor Prognosis?

Introduction We have reported that sarcopenia is a poor prognostic factor after liver transplantation (LT). However, the significance of sarcopenic obesity is unclear. In this study, we examined the impact of sarcopenic obesity on outcomes after living donor LT (LDLT). Methods We retrospectively analyzed 277 adult patients who underwent LDLT at our institute between January 2008 and June 2016. Using preoperative plain computed tomography imaging at the third lumbar vertebra level, skeletal muscle mass, muscle quality, and visceral adiposity were evaluated by the skeletal muscle mass index (SMI), intramuscular adipose tissue content (IMAC), and visceral to subcutaneous adipose tissue area ratio (VSR), respectively. In this study, we defined low skeletal muscle mass (SMI: male <40.31cm2/m2, female <30.88cm2/m2) and visceral fat area >=100cm2 as sarcopenic obesity. Using this definition, we divided all patients into 4 groups, non-sarcopenic/non-obesity (NN), non-sarcopenic/obesity (NO), sarcopenic/non-obesity (SN), and sarcopenic/obestiy (SO) groups. Overall survivals after LT liver transplantation were compared among the 4 groups Independent risk factors after LT were also analyzed. Results One/five-y OS in patients with of NN group (n=167) were 86%/80%. Compared with NN group, 1/5-y OS in patients of NO (n=55), SN (n=46), and SO (n=9) was 84%/75% (P=0.505), 59%/46% (P<0.001), and 56%/56% (P=0.056), respectively. Multivariate analysis identified low SMI (P=0.020), high IMAC (muscle steatosis) (P<0.001), high VSR (visceral adiposity) (P=0.001), and post-transplant bacteremia (P<0.001) as independent risk factores for death after LT. Conclusion Patients with sarcopenic obesity had worse survival after LDLT. Low muscle mass, muscle steatosis, and visceral adiposity were independent risk factors after LDLT.

Proposal for New Selection Criteria Considering Pre-Transplant Muscularity and Visceral Adiposity in Living Donor Liver Transplantation

Introduction The significance of preoperative body composition has recently attracted much attention in various diseases. However, cut-off values for these parameters remain undetermined, and these factors are not currently included in selection criteria for recipients of living donor liver transplantation (LDLT). The present study aimed to establish sex-specific cut-offs for body composition using data from healthy general population, and to develop new selection criteria for recipients of LDLT considering pre-transplant nutritional and physical statuses. Methods Using computed tomography of 657 donors for LDLT between April 2005 and July 2016 in our institution, skeletal muscle mass, muscle quality, and visceral adiposity were evaluated using skeletal muscle mass index (SMI), intramuscular adipose tissue content (IMAC), and visceral-to-subcutaneous adipose tissue area ratio (VSR). On the basis of younger donor data, we determined sex-specific cut-off values for low SMI, high IMAC, and high VSR (mean ± 2 standard deviations). To evaluate the validity of cut-offs for body composition parameters, we examined data from 277 patients who underwent adult-to-adult LDLT between January 2008 and July 2016. The impact of body composition on outcomes after LDLT was investigated with the aim of establishing new selection criteria for LDLT. Results Among the 277 LDLT recipients, 55 (20.0%), 121 (43.7%), and 83 (30.0%) patients exhibited low SMI, high IMAC, and high VSR, respectively. The overall survival rate was significantly lower for each group of patients with low SMI (P < 0.001), high IMAC (P < 0.001), or high VSR (P < 0.001) compared to the respective normal groups. In addition, low SMI, high IMAC, and high VSR contributed to an increased risk of post-LDLT mortality in an additive manner. Patients with all three factors showed the lowest survival rate after LDLT (1-year survival rate, 41.2%; P < 0.001). On multivariate analysis, low SMI (P = 0.002), high IMAC (P = 0.002), and high VSR (P = 0.001) were identified as independent risk factors for mortality after LDLT. Based on these findings, we have excluded patients showing all 3 factors (low SMI, high IMAC and high VSR) as candidates for LDLT since October 2016. Conclusion Using cut-off values determined from healthy donors, we investigated risk factors for post-LDLT mortality, and extracted the group that exhibited the poorest prognosis after LT, leading to the establishment of our new selection criteria for recipients of LDLT.

Is Muscle MELD a More Promising Predictor for Mortality After Living Donor Liver Transplantation

Background: To improve the outcome of living donor liver transplantation (LDLT), a scoring system that could predict accurately the patient and graft survival posttransplant is necessary. The aim of this study is to evaluate our previously proposed Muscle-model for end-stage liver disease (M-MELD) score and to compare it with the other available scores to find the best system that correlates with postoperative outcome after liver transplant. Methods: We retrospectively reviewed the data of 199 patients who underwent LDLT from January 2010 to July 2016 and calculated the preoperative MELD, MELD Na, the product of donor age and MELD (D-MELD), M-MELD, integrated MELD, and the balance of risk (BAR) score in all patients. The area under the receiver operating characteristics curves (AUCs) of each score was computed and compared at 3-, 6-months, and 1-year after LDLT. Results: The M-MELD, D-MELD, and integrated MELD had a good discriminative performance in predicting 3-month mortality after LDLT with AUCs > 0.7, while the M-MELD was the only score that showed a good discriminative performance in predicting 6-month and 1-year mortality after LDLT with AUCs > 0.7. Conclusion: Muscle-MELD score is a simple and useful predictor of patient survival after LDLT which showed a better predictive performance than other available scores.

Liver Transplantation for Intermediate-Stage Hepatocellular Carcinoma

Transarterial chemoembolization is the standard treatment for patients with intermediate-stage hepatocellular carcinoma (HCC) according to the Barcelona Clinic Liver Cancer staging system. However, in Japan, not a few patients with intermediate-stage HCC undergo liver transplantation (LT). The present study investigated characteristics and outcomes of LT for intermediate-stage HCC. Between February 1999 and November 2016, a total of 226 patients underwent LT for HCC at our institute. Among these, 56 patients showed intermediate-stage HCC (24.8%). We examined overall survival and recurrence rate after LT according to our extended criteria (maximum size ≤5 cm, number ≤10, des-gamma-carboxy prothrombin ≤400 mAU/mL) and pretreatment. One-, 3-, and 5-year overall survival and recurrence rates of LT for intermediate-stage HCC were 88/64/58% and 22/34/44%, respectively. One-, 3-, and 5-year overall survival and recurrence rates in patients within (n = 35) the criteria (94/80/80% and 9/15/22%, respectively) were significantly better than those in patients beyond (n = 21) the criteria (81/43/29%, p = 0.002 and 39/41/66%, p = 0.001, respectively). Forty-nine cases (88%) had a history of pretreatment. In patients within our extended criteria, overall survival and recurrence rates did not differ significantly between patients with (n = 31) and without (n = 4) pretreatment. In conclusion, outcomes after LT for intermediate-stage HCC are more favorable if patients meet our extended criteria.