Keisuke Harada

Morphological changes induced by extracellular matrix are correlated with maturation of rat small hepatocytes

Small hepatocytes (SHs), which are known to be hepatic progenitor cells, were isolated from an adult rat liver. SHs in a colony sometimes change their shape from small to large and from flat to rising/piled‐up. The aim of the present study is to clarify whether the alteration of cell shape is correlated with the maturation of SHs and whether extracellular matrix (ECM) can induce the morphological changes of SHs. We used liver‐enriched transcription factors (LETFs) such as hepatocyte nuclear factor (HNF) 4α, HNF6, CCAAT/enhancer binding proteins (C/EBP) α, and C/EBPβ, tryptophan 2,3‐dioxygenase (TO), and serine dehydratase (SDH) as markers of hepatic maturation. To enrich the number of SH colonies, the colonies were isolated from dishes and replated. Replated colonies proliferated and the average number of cells per colony was about five times larger at day 9 than at day 1. When the cells were treated with laminin, type IV collagen, a mixture of laminin and type IV collagen, Matrigel™ or collagen gel (CG), only the cells treated with Matrigel dramatically changed their shape within several days and had reduced growth activity, whereas the cells treated with other ECM did not. HNF4α, HNF6, C/EBPα, C/EBPβ, and TO were well expressed in the cells treated with Matrigel. Furthermore, addition of both glucagon and dexamethasone dramatically induced the expression of SDH mRNA and protein in the cells treated with Matrigel. In conclusion, morphological changes of SHs may be correlated with hepatic maturation and basement membrane (BM)‐like structure may induce the morphological changes of SHs. J. Cell. Biochem. 87: 16–28, 2002.

Rapid formation of hepatic organoid in collagen sponge by rat small hepatocytes and hepatic nonparenchymal cells

BACKGROUND/AIMS Hybrid bioartificial liver devices supporting a large mass of metabolically active hepatocytes are thought to be necessary for the successful treatment of patients with severe acute liver failure. However, it is very difficult to obtain cells with both growth activity and differentiated functions. Rat small hepatocytes (SHs), which are hepatic progenitor cells, can differentiate into mature hepatocytes and reconstruct a hepatic organoid by interacting with hepatic nonparenchymal cells (NPCs). METHODS Colonies of SHs were collected and replated on a collagen sponge. Hepatic functions were examined by ELISA, immunoblotting, and Northern blotting. Cells in the sponge were characterized by immunocytochemistry and transmission electron microscopy. Urea synthesis was measured and metabolization of fluorescein diacetate was examined. RESULTS SHs could proliferate and expand to form a hepatic organoid in the sponge. Albumin secretion and other hepatic protein production of the cells in the sponge increased with time in culture and the amounts were much larger than for those obtained from cells grown on dishes. Morphologically and functionally differentiated hepatocytes were observed and some CK19-positive cells formed duct-like structures within the sponge. Excretion of fluorescein was observed in bile canaliculi. CONCLUSIONS Hepatic organoids can be rapidly reconstructed in a collagen sponge by rat SHs and NPCs.

Proliferation of rat small hepatocytes after long-term cryopreservation

BACKGROUND/AIMS The demand for clinical use of hepatocytes is escalating because cell transplantation will be an alternative to orthotopic liver transplantation and the shortage of liver donors is a serious problem throughout the world. However, the supply of fresh differentiated hepatocytes is limited and methods for cryopreservation of hepatocytes that can proliferate with hepatic functions are not satisfactorily established. METHODS Colonies of small hepatocytes were collected and then maintained at -80 degrees C for more than 6 months. Albumin secretion and mRNA expression of thawed cells were measured by enzyme linked immunosorbent assay and Northern blotting, respectively, and the expression of hepatic functions was examined by immunoblotting. The ultrastructure of cryopreserved cells was also examined. RESULTS About 60% of the cryopreserved colonies attached on dishes and then proliferated. The average area of small hepatocyte colonies was about 7.5 times larger at day 15 than at day 1. Albumin production increased with time in culture. In addition, the cells produced other serum proteins such as transferrin and fibrinogen, and expressed carbamoyl phosphate synthetase I and tryptophan 2,3-dioxygenase. CONCLUSIONS Small hepatocytes maintain growth ability and hepatic differentiated functions even after long-term cryopreservation.

Bile canalicular formation in hepatic organoid reconstructed by rat small hepatocytes and nonparenchymal cells

The morphogenesis and movement of bile canaliculi (BC) are not well understood. This is because culture of hepatocytes that maintain polarity of cell membranes and possess highly differentiated functions has never been successful. We found that small hepatocytes (SHs), which are known to be hepatic progenitor cells, could proliferate and differentiate into mature hepatocytes and that BC‐like structures developed between rising/piled‐up cells. We investigated how BC‐like structures developed with maturation of SHs and whether the structures were functionally active as BC. Hepatic cells, including SHs, were isolated from an adult rat liver and cultured. Immunocytochemistry and immunoblotting for BC proteins, such as ectoATPase, 5′‐nucleotidase, dipeptidylpeptidase IV, and multidrug‐resistance associated protein 2, were examined and time‐lapse microscopy was used for the observation of BC contractions. Secretion of bilirubin into the reconstructed BC was also observed. The results of immunocytochemistry, immunoblots, and immunoelectron micrographs revealed that BC proteins were localized in the intercellular space that coincided with BC‐like structures reconstructed between rising/piled‐up cells. Tight junction‐associated protein ZO‐1 was also expressed along the BC‐like structures. Bilirubin added to the medium were secreted into BC‐like structure and accumulated without leakage. Time‐lapse microscopy showed continuous contractions of reconstructed BC. In conclusion, BC‐like structures reconstructed by SHs may be functional with membrane polarity, secretory ability, and motility. These results show that this culture system may suitable for investigating the mechanism of the formation of BC and their functions. J. Cell. Physiol. 199: 252–261, 2004© 2003 Wiley‐Liss, Inc.

Expression of cytochrome P450 enzymes in hepatic organoid reconstructed by rat small hepatocytes

Background and Aims:  Small hepatocytes (SH), which are hepatic progenitor cells, were isolated from an adult rat liver. SH in a colony sometimes change their shape from small to large and from flat to rising/piled‐up. The morphological changes of SH may be correlated with hepatic maturation. Cytochrome P450s (CYP) are drug‐metabolizing enzymes and the expression is one of hepatic differentiated functions. However, it is well known that the re‐expression and maintenance of CYP activity are very difficult in cultured hepatocytes. We investigated the expression of CYP and the enzymatic activities in long‐term cultured SH.

Hyperbaric oxygen stimulates cell proliferation and normalizes multidrug resistance protein-2 protein localization in primary rat hepatocytes

Hyperbaric oxygen therapy (HBO) has been used for many clinical treatments, including primary liver nonfunction. However, the cellular mechanism by which HBO treatment ameliorates liver function is not understood. Therefore, the purpose of this study was to elucidate this cellular mechanism using primary cultured rat hepatocytes in in vitro studies. Hepatocytes were treated with HBO at 1 day after plating, and the morphological and functional characteristics of bile canaliculi formed in cultured hepatocytes were observed by time‐lapse microscopy. Multidrug resistance protein‐2 localization was observed by confocal laser microscopy. In cultured hepatocytes, the labeling index in the HBO group at 2 days after treatment was significantly higher than that in the control group. In addition, the proliferating cellular nuclear antigen level in the HBO group was significantly higher than that in the control group. The contraction of the bile canaliculi in the HBO group was slower than in the control group and the dilatation of bile canaliculi in the HBO group was much larger than in the control group. Multidrug resistance protein‐2 in the HBO group was localized at the apical membrane. These results show that HBO stimulates hepatocytes to proliferate and HBO normalizes Multidrug resistance protein‐2 localization to the apical membrane, which could dilate bile canaliculi.

How to manage tension gastrothorax: a case report of tension gastrothorax with multiple trauma due to traumatic diaphragmatic rupture

BackgroundTension gastrothorax is a kind of obstructive shock with prolapse and distention of the stomach into the thoracic cavity. Progressive gastric distension leads to mediastinal shift, reduced venous return, decreased cardiac output, and ultimately cardiac arrest. Therefore, it is crucial to decompress the stomach distension for the initial resuscitation of tension gastrothorax.Case presentationA 75-year-old female was transported to our resuscitation bay due to motor vehicle crash. At the time of arrival to our hospital, the patient developed cardiac arrest. While undergoing cardiopulmonary resuscitation, an unstable pelvic ring was recognized, so we performed a resuscitative thoracotomy to control hemorrhage and to perform direct cardiac massage. Once we performed the thoracotomy, the stomach and omentum prolapsed out of the thoracotomy site and through the diaphragm rupture site and spontaneous circulation was recovered. Neither the descending aorta nor the heart was collapsed. Although we had continued the treatment for severe pelvic fracture (including blood transufusions), the patient died. Given that (1) the stomach prolapsed out of the body at the time of the thoracotomy; (2) at the same timing, spontaneous circulation returned; and (3) the descending aorta and heart did not collapse, we hypothesized that the main cause of the initial cardiac arrest was tension gastrothorax.ConclusionsRecognition of tension gastrothorax pathophysiology, which is a form of obstructive shock, makes it possible to manage this injury correctly.

Risk factors for acute pancreatitis in patients with accidental hypothermia

Background: Pancreatic damage is commonly observed as a consequence of accidental hypothermia (core body temperature below 35°C). We aimed to investigate the risk factors for pancreatic damage and the causal relationship in patients with accidental hypothermia. Methods: This retrospective, single‐center, observational case‐control study was conducted in the emergency department of a tertiary care medical center. We investigated patients who were admitted for accidental hypothermia over a course of ten years (January 2008 to December 2017). Results: Of the 138 enrolled patients, 70 had elevated serum amylase levels (51%). We observed a correlation between initial core body temperature and serum amylase level (Spearmans rank correlation coefficient −0.302, p<0.001). Patients who developed acute pancreatitis had a significantly lower initial core body temperature than those who did not develop it (odds ratio=0.76; 95% confidence interval [CI]=0.61–0.94; p=0.011). Receiver operating characteristic analysis showed that a body temperature lower than 28.5°C at the time of visit was predictive of acute pancreatitis (area under the curve=0.71, 95% CI=0.54–0.88, sensitivity=0.67, specificity=0.69, p=0.017). Conclusions: We concluded that an initial core body temperature lower than 28.5°C was a risk factor for acute pancreatitis in accidental hypothermia cases. In such situations, careful follow‐up is necessary. HIGHLIGHTSPancreatic damage is commonly associated with accidental hypothermia.We investigated the risk factors for pancreatic damage in such cases.An initial core body temperature lower than 28.5°C was a risk factor.This finding can enable the early diagnosis of acute pancreatitis.