Masaya Saito

Lecithin: retinol acyltransferase protein is distributed in both hepatic stellate cells and endothelial cells of normal rodent and human liver.

Background: To determine the extent to which hepatic stellate cell (HSC) activation contributes to liver fibrosis, it was found necessary to develop an alternative structural and functional stellate cell marker for in situ studies. Although several HSC markers have been reported, none of those are associated with particular HSC functions.

Transplantation of Liver Organoids in the Omentum and Kidney

Liver organoids were reconstructed by mouse-immortalized hepatocytes and nonparenchymal cells (sinusoidal endothelial cells and hepatic stellate cells) in a radial-flow bioreactor (RFB). A biodegradable apatite-fiber scaffold (AFS) was used as a scaffold packed in the RFB, which enables three-dimensional cell cultures. The organoids cocultured in the RFB showed a liver-like structure with high-density layers of hepatocytes and the formation of vessel-like structures. A liver organoid consisting of three cocultured cells was transplanted under the kidney capsule (kidney group) or into the omentum (omentum group) using BALB/c nude mice. Transplanted liver organoids survived in the kidney or omentum. The expression of mRNAs of albumin, connexin 26 and 32, hepatocyte nuclear factor 4α, and glucose-6-phosphatase was increased in both groups at 8 weeks after transplantation in comparison to the pretransplant status. Tyrosine aminotransferase appeared only in the omentum group. The results suggested that the functions of liver organoids differed depending on the transplanted site in the recipient animals.

The Functional Interrelationship between Gap Junctions and Fenestrae in Endothelial Cells of the Liver Organoid

Functional intact liver organoid can be reconstructed in a radial-flow bioreactor when human hepatocellular carcinoma (FLC-5), mouse immortalized sinusoidal endothelial M1 (SEC) and A7 (HSC) hepatic stellate cell lines are cocultured. The structural and functional characteristics of the reconstructed organoid closely resemble the in vivo liver situation. Previous liver organoid studies indicated that cell-to-cell communications might be an important factor for the functional and structural integrity of the reconstructed organoid, including the expression of fenestrae. Therefore, we examined the possible relationship between functional intact gap junctional intercellular communication (GJIC) and fenestrae dynamics in M1-SEC cells. The fine morphology of liver organoid was studied in the presence of (1) irsogladine maleate (IM), (2) oleamide and (3) oleamide followed by IM treatment. Fine ultrastructural changes were studied by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) and compared with control liver organoid data. TEM revealed that oleamide affected the integrity of cell-to-cell contacts predominantly in FLC-5 hepatocytes. SEM observation showed the presence of fenestrae on M1-SEC cells; however, oleamide inhibited fenestrae expression on the surface of endothelial cells. Interestingly, fenestrae reappeared when IM was added after initial oleamide exposure. GJIC mediates the number of fenestrae in endothelial cells of the liver organoid.

Study of the reappearance of sieve plate-like pores in immortalized sinusoidal endothelial cells – Effect of actin inhibitor in mixed perfusion cultures

We previously reported that when the high-functioning human hepatoma cell line, FLC-5, immortalized sinusoidal endothelial cell line, M1, and immortalized hepatic stellate cell line, A7, were cultured in the 3-dimensional filled type bioreactor, tissue reorganization resembling that seen in the live liver occurred, with the appearance of pores in the sinusoidal endothelial cells (SECs) [1]. The process and mechanism of formation of these pores remain unclarified. The presence of actin at the margin of these pores has been demonstrated by electron microscopic study [2]. Swinholide-A, which is actin inhibitor synthesized from Okinawa sponge, increase the number of pores on primary culture on SECs derived from the rat [3]. In present study, we examine whether or not the pores on SECs under three-dimensional perfusion co-culture treatment with Swinholide-A behave like those in primary culture cells.

Hepatic stellate cells that coexpress LRAT and CRBP-1 partially contribute to portal fibrogenesis in patients with human viral hepatitis.

Precisely what type of cells mainly contributes to portal fibrosis, especially in chronic viral hepatitis, such as hepatic stellate cells (HSCs) in the parenchyma or myofibroblasts in the portal area, still remains unclear. It is necessary to clarify the characteristics of cells that contribute to portal fibrosis in order to determine the mechanism of portal fibrogenesis and to develop a therapeutic target for portal fibrosis. This study was undertaken to examine whether LRAT+/CRBP‐1+ HSCs contribute to portal fibrosis on viral hepatitis.

Noninvasive assessment of insulin resistance in the liver using the fasting 13C-glucose breath test

Evaluating hepatic insulin resistance (IR) is the key to making a sensitive an accurate diagnosis of glucose intolerance. However, there is currently no suitable method to perform this procedure. This study was conducted to investigate whether the fasting (13)C-glucose breath test (FGBT) is useful as a convenient and highly sensitive clinical test for evaluating hepatic IR. Healthy nonobese subjects and a disease group consisting of patients with mild glucose intolerance were administered 100 mg (13)C-glucose after an overnight fast. A series of breath samples was collected until 360 minutes after ingestion, and the (13)CO2-to-(12)CO2 ratio was measured using an infrared spectrometer and was plotted as a kinetic curve of (13)C excretion. The area under the curve until 360 minutes (AUC360) of the (13)C excretion kinetic curve of the FGBT reflects the efficiency of energy production in the liver. First, we assessed the correlations between the AUC360 (or the (13)C excretion rate at 120 minutes) and the HOMA-IR and HbA1c levels as standard measurements of IR and diabetes mellitus (DM). There were relatively strong correlation coefficients (r = -0.49 to -0.81, r(2) = 0.24-0.66, P < 0.01; n = 35 males, n = 33 females). Second, we compared the AUC360 of healthy subjects and that of the patients with mild glucose intolerance. The AUC360 of the healthy subjects was consistently higher than that of the patients with mild glucose intolerance. The presence of IR or DM in males and females was diagnosed using cutoff values. The FGBT is a novel glucose metabolism test that can be used conveniently and safely to evaluate the balance of glucose metabolism in the liver. This test has excellent sensitivity for diagnosing alterations in hepatic glucose metabolism, particularly hepatic IR.

The fine structure of bioreactor liver tissue seen through the eyes of X-ray micro-computed tomography

X-ray micro-computed tomography (m-CT) is increasingly becoming a standard method to investigate soft biological material in 3-D. Numerous recent studies have been published outlining its use in investigating the fine structure of different organs such as heart, kidney and lung tissue [1]. In previous studies, we successfully imaged intact liver tissue and its associated vasculature via X-ray m-CT [2,3]. The urgency to facilitate patients with severe life treating liver conditions like cirrhosis and hepatitis has resulted in researchers investigating methods to increase recovery rate and delay transplantation by developing artificial livers grown in bioreactors [4]. In line, several studies indicate that the choice of cell culture substrate is central in generating fully functional bioreactor liver tissue. Therefore, we aimed to image and model liver tissue grown on different scaffolds in a bioreactor via X-ray m-CT. Classical SEM and TEM imaging were performed to accumulate cross-correlative structural evidence [5].