Are Adipose-Derived Stem Cells From Liver Falciform Ligaments Another Possible Source of Mesenchymal Stem Cells?

Falciform ligaments in the liver are surrounded by adipose tissue. We investigated the capability of adipose-derived stem cells from human liver falciform ligaments (hLF-ADSCs) to differentiate into hepatic-type cells and confirmed the functional capacity of the cells. Mesenchymal stem cells (MSCs) were isolated from the liver falciform ligament and abdominal subcutaneous adipose tissue in patients undergoing partial hepatectomy for liver disease. Cells were cultivated in MSC culture medium. Properties of MSCs were confirmed by flow cytometry, RT-PCR analysis, immunocytochemistry assays, and multilineage differentiation. Hepatic induction was performed using a three-step differentiation protocol with various growth factors. Morphology, capacity for expansion, and characteristics were similar between hLF-ADSCs and adipose-derived stem cells from human abdominal subcutaneous adipose tissue (hAS-ADSCs). However, hematopoietic- and mesenchymal-epithelial transition (MET)-related surface markers (CD133, CD34, CD45, and E-cadherin) had a higher expression in hLF-ADSCs. The hepatic induction marker genes had a higher expression in hLF-ADSCs on days 7 and 10 after the hepatic induction. Albumin secretion was similar between hLF-ADSCs and hAS-ADSCs at 20 days after the hepatic induction. The hLF-ADSCs had a different pattern of surface marker expression relative to hAS-ADSCs. However, proliferation, multilineage capacity, and hepatic induction were similar between the cell types. Accordingly, it may be a useful source of MSCs for patients with liver disease.

Ideal sphere-forming culture conditions to maintain pluripotency in a hepatocellular carcinoma cell lines.

BACKGROUND: Cancer stem cells (CSCs) constitute 1-2 % of cancer tissue and are a major cause of tumor metastasis and recurrence. The culture environment is important in maintaining CSCs in vitro. Sphere formation is one method of culturing CSCs. In this study, we identified and validated optimal culture conditions for sphere formation in hepatocellular carcinoma cells. METHODS: Huh7 and HepG2 cells were plated in three different media types and were allowed to form spheres. To confirm the pluripotency of sphere cells, the expression of stem cell markers was evaluated. EpCAM, Connexin 32, and Connexin 43 expression was evaluated using reverse transcription-polymerase chain reaction (RT-PCR). The expression of E-cadherin, ß-catenin, CD90, and CD133 was evaluated using immunocytochemistry. Flow cytometry was performed to confirm the presence of stem cell markers CD133 and Connexin 32. RESULTS: Cells maintained in medium containing growth factors ((DMEM(+))GF) showed greater cell proliferation than cells in media with fetal bovine serum (FBS) (DMEM(+)FBS) or without FBS (DMEM(-)FBS). Cells cultured in DMEM(+)FBS medium exhibited greater proliferation after 20 days in culture than cells cultured under the other two conditions. Cells cultured in DMEM(-)FBS medium did not proliferate; therefore, this condition was removed from further analysis. RT-PCR and flow cytometry showed that sphere-forming cells cultured in DMEM(+)GF and DMEM(+)FBS media had similar expression of stem cell markers. CONCLUSION: Therefore, growth factor-free medium is an adaptable, efficient, and cost-effective tool for in vitro cultivation of CSCs.

Efficient endodermal induction of human adipose stem cells using various concentrations of Activin A for hepatic differentiation.

UNLABELLED: Activin A, which is a signaling molecule similar to Nodal, rapidly promotes endoderm induction of both embryonic stem (ES) cells and MSCs. Protocols for hepatic induction exhibit differences in efficiency and reproducibility depending on the specific methods or sources of MSCs. We characterized the effects of Activin A concentration on induction efficiency during hepatic differentiation of MSCs. Human MSCs (hMSCs) were differentiated into a hepatic lineage via a three-step protocol. Cells were first cultured in fetal bovine serum-free MSCs conditioned medium supplemented with Activin A (20, 50, or 100 ng/mL) for 3 days followed by treatment with additional agents. RT-PCR analysis, immunocytochemistry assays, periodic acid and Schiff's solution staining, and ELISAs were performed to confirm hepatic induction of hMSCs. Expression of genes related to the primitive foregut endoderm was observed in cells treated with higher concentration of Activin A. Gene expression related to functional primitive hepatocytes was observed in the early phases of hepatic differentiation. During the early period of the differentiation protocol, greater albumin secretion was observed when cells were treated with higher concentrations of Activin A. CONCLUSION: Thus, Activin A concentration affects the rate of endoderm induction of hMSCs, and at higher concentrations in vitro.

Toward angiogenesis of implanted bio-artificial liver using scaffolds with type I collagen and adipose tissue-derived stem cells.

BACKGROUNDS/AIMS: Stem cell therapies for liver disease are being studied by many researchers worldwide, but scientific evidence to demonstrate the endocrinologic effects of implanted cells is insufficient, and it is unknown whether implanted cells can function as liver cells. Achieving angiogenesis, arguably the most important characteristic of the liver, is known to be quite difficult, and no practical attempts have been made to achieve this outcome. We carried out this study to observe the possibility of angiogenesis of implanted bio-artificial liver using scaffolds. METHODS: This study used adipose tissue-derived stem cells that were collected from adult patients with liver diseases with conditions similar to the liver parenchyma. Specifically, microfilaments were used to create an artificial membrane and maintain the structure of an artificial organ. After scratching the stomach surface of severe combined immunocompromised (SCID) mice (n=4), artificial scaffolds with adipose tissue-derived stem cells and type I collagen were implanted. Expression levels of angiogenesis markers including vascular endothelial growth factor (VEGF), CD34, and CD105 were immunohistochemically assessed after 30 days. RESULTS: Grossly, the artificial scaffolds showed adhesion to the stomach and surrounding organs; however, there was no evidence of angiogenesis within the scaffolds; and VEGF, CD34, and CD105 expressions were not detected after 30 days. CONCLUSIONS: Although implantation of cells into artificial scaffolds did not facilitate angiogenesis, the artificial scaffolds made with type I collagen helped maintain implanted cells, and surrounding tissue reactions were rare. Our findings indicate that type I collagen artificial scaffolds can be considered as a possible implantable biomaterial.

Immediately transcripted genes in various hepatic ischemia models.

PURPOSE: To elucidate the characteristic gene transcription profiles among various hepatic ischemia conditions, immediately transcribed genes and the degree of ischemic injury were compared among total ischemia (TI), intermittent clamping (IC), and ischemic preconditioning (IPC). METHODS: Sprague-Dawley rats were equally divided into control (C, sham-operated), TI (ischemia for 90 minutes), IC (ischemia for 15 minutes and reperfusion for 5 minutes, repeated six times), and IPC (ischemia for 15 minutes, reperfusion for 5 minutes, and ischemia again for 90 minutes) groups. A cDNA microarray analysis was performed using hepatic tissues obtained by partial hepatectomy after occluding hepatic inflow. RESULTS: THE CDNA MICROARRAY REVEALED THE FOLLOWING: interleukin (IL)-1ß expression was 2-fold greater in the TI group than in the C group. In the IC group, IL-1α/ß expression increased by 2.5-fold, and Na+/K+ ATPase ß1 expression decreased by 2.4-fold. In the IPC group, interferon regulatory factor-1, osteoprotegerin, and retinoblastoma-1 expression increased by approximately 2-fold compared to that in the C group, but the expression of Na+/K+ ATPase ß1 decreased 3-fold. CONCLUSION: The current findings revealed characteristic gene expression profiles under various ischemic conditions. However, additional studies are needed to clarify the mechanism of protection against IPC.

The effect of preconditioning on liver regeneration after hepatic resection in cirrhotic rats.

BACKGROUND/AIMS: Ischemic preconditioning (IP) decreases severity of liver necrosis and has anti-apoptotic effects in previous studies using liver regeneration in normal rats. This study assessed the effect of IP on liver regeneration after hepatic resection in cirrhotic rats. METHODS: To induce liver cirrhosis, thioacetamide (300 mg/kg) was injected intraperitoneally into Sprague-Dawley rats twice per week for 16 weeks. Animals were divided into four groups: non-clamping (NC), total clamping (TC), IP, and intermittent clamping (IC). Ischemic injury was induced by clamping the left portal pedicle including the portal vein and hepatic artery. Liver enzymes alanine transaminase (ALT) and aspartate aminotransferase (AST) were measured to assess liver damage. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining for apoptosis and proliferating cell nuclear antigen (PCNA) staining for cell replication were also performed. RESULTS: Day-1 ALT and AST were highest in IP, however, levels in NC and IC were comparably low on days 1-7. There was no significant correlation of AST or ALT with experimental groups (P=0.615 and P=0.186). On TUNEL, numbers of apoptotic cells at 100× magnification (cells/field) were 31.8±24.2 in NC, 69.0±72.3 in TC, 80.2±63.1 in IP, and 21.2±20.8 in IC (P<0.05). When regeneration capacity was assessed by PCNA staining, PCNA-positive cells (cells/field) at 400× were 3.4±6.0 in NC, 16.9±69 in TC, 17.0±7.8 in IP and 7.4±7.6 in IC (P<0.05). CONCLUSIONS: Although regeneration capacity in IP is higher than IC, the liver is vulnerable to ischemic damage in cirrhotic rats. Careful consideration is needed in applying IP in the clinical setting.

Ideal Experimental Rat Models for Liver Diseases.

There are many limitations for conducting liver disease research in human beings due to the high cost and potential ethical issues. For this reason, conducting a study that is difficult to perform in humans using appropriate animal models, can be beneficial in ascertaining the pathological physiology, and in developing new treatment modalities. However, it is difficult to determine the appropriate animal model which is suitable for research purposes, since every patient has different and diverse clinical symptoms, adverse reactions, and complications due to the pathological physiology. Also, it is not easy to reproduce identically various clinical situations in animal models. Recently, the Guide for the Care and Use of Laboratory Animals has tightened up the regulations, and therefore it is advisable to select the appropriate animals and decide upon the appropriate quantities through scientific and systemic considerations before conducting animal testing. Therefore, in this review article the authors examined various white rat animal testing models and determined the appropriate usable rat model, and the pros and cons of its application in liver disease research. The authors believe that this review will be beneficial in selecting proper laboratory animals for research purposes.