Takumi Teratani

Adipose tissue-derived mesenchymal stem cells as a source of human hepatocytes

Recent observations indicate that several stem cells can differentiate into hepatocytes; thus, cell‐based therapy is a potential alternative to liver transplantation. The goal of the present study was to examine the in vitro hepatic differentiation potential of adipose tissue‐derived mesenchymal stem cells (AT‐MSCs). We used AT‐MSCs from different age patients and found that, after incubation with specific growth factors (hepatocyte growth factor [HGF], fibroblast growth factor [FGF1], FGF4) the CD105+ fraction of AT‐MSCs exhibited high hepatic differentiation ability in an adherent monoculture condition. CD105+ AT‐MSC‐derived hepatocyte‐like cells revealed several liver‐specific markers and functions, such as albumin production, low‐density lipoprotein uptake, and ammonia detoxification. More importantly, CD105+ AT‐MSC‐derived hepatocyte‐like cells, after transplantation into mice incorporated into the parenchyma of the liver. Conclusion: Adipose tissue is a source of multipotent stem cells that can be easily isolated, selected, and induced into mature, transplantable hepatocytes. The fact that they are easy to procure ex vivo in large numbers makes them an attractive tool for clinical studies in the context of establishing an alternative therapy for liver dysfunction. (HEPATOLOGY 2007;46:219–228.)

IFATS Collection: In Vivo Therapeutic Potential of Human Adipose Tissue Mesenchymal Stem Cells After Transplantation into Mice with Liver Injury

Mesenchymal stem cells (MSCs), largely present in the adult human body, represent an attractive tool for the establishment of a stem cell‐based therapy for liver diseases. Recently, the therapeutic potential and immunomodulatory activity of MSCs have been revealed. Adipose tissue‐derived mesenchymal stem cells (AT‐MSCs), so‐called adipose‐derived stem cells or adipose stromal cells, because of their high accessibility with minimal invasiveness, are especially attractive in the context of future clinical applications. The goal of the present study was to evaluate the therapeutic potential of AT‐MSCs by their transplantation into nude mice with CCl4‐caused liver injury. We observed that after transplantation, AT‐MSCs can improve liver functions, which we verified by changes in the levels of biochemical parameters. Ammonia, uric acid, glutamic‐pyruvic transaminase, and glutamic‐oxaloacetic transaminase concentrations returned to a nearly normal level after AT‐MSC transplantation. These results raised the question of how AT‐MSCs can achieve this. To discover the possible mechanisms involved in this therapeutic ability of AT‐MSCs, in vitro production of cytokines and growth factors was analyzed and compared with MSCs from bone marrow (BM‐MSCs) and normal human dermal fibroblasts (NHDFs). As a result we observed that AT‐MSCs secrete interleukin 1 receptor α (IL‐1Rα), IL‐6, IL‐8, granulocyte colony‐stimulating factor (G‐CSF), granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), monocyte chemotactic protein 1, nerve growth factor, and hepatocyte growth factor in a volume higher than both BM‐MSCs and NHDFs. Thus, our findings suggest that AT‐MSCs may account for their broad therapeutic efficacy in animal models of liver diseases and in the clinical settings for liver disease treatment.

Rapid hepatic fate specification of adipose‐derived stem cells and their therapeutic potential for liver failure

Background and Aim:  Multipotential mesenchymal stem cells (MSC), present in many organs and tissues, represent an attractive tool for the establishment of a successful stem cell‐based therapy in the field of regeneration medicine. Adipose tissue mesenchymal stem cells (AT‐MSC), known as adipose‐derived stem cells (ASC) are especially attractive in the context of future clinical applications because of their high accessibility and minimal invasiveness during the procedure to obtain them. The goal of the present study was to induce human ASC into functional hepatocytes in vitro within a very short period of time and to check their therapeutic potential in vivo.

Direct hepatic fate specification from mouse embryonic stem cells

The molecules responsible for hepatic differentiation from embryonic stem (ES) cells have yet to be elucidated. Here we have identified growth factors that allow direct hepatic fate‐specification from ES cells by using simple adherent monolayer culture conditions. ES cell–derived hepatocytes showed liver‐specific characteristics, including several metabolic activities, suggesting that ES cells can differentiate into functional hepatocytes without the requirement for embryoid body (EB) formation, in vivo transplantation, or a coculture system. Most importantly, transplantation of ES cell–derived hepatocytes in mice with cirrhosis showed significant therapeutic effects. In conclusion, this novel system for hepatic fate specification will help elucidate the precise molecular mechanisms of hepatic differentiation in vitro and could represent an attractive approach for developing stem cell therapies for treatment of hepatic disease in humans. Supplementary material for this article can be found on the HEPATOLOGY website ( http://www.interscience.wiley.com/jpages/0270‐9139/suppmat/index.html). (HEPATOLOGY 2005.)

Establishment of Rat Embryonic Stem Cells and Making of Chimera Rats

The rat is a reference animal model for physiological studies and for the analysis of multigenic human diseases such as hypertension, diabetes, neurological disorders, and cancer. The rats have long been used in extensive chemical carcinogenesis studies. Thus, the rat embryonic stem (rES) cell is an important resource for the study of disease models. Attempts to derive ES cells from various mammals, including the rat, have not succeeded. Here we have established two independent rES cells from Wister rat blastocysts that have undifferentiated characters such as Nanog and Oct3/4 genes expression and they have stage-specific embryonic antigen (SSEA) -1, -3, -4, and TRA-1-81 expression. The cells were successfully cultured in an undifferentiated state and can be possible over 18 passages with maintaining more than 40% of normal karyotype. Their pluripotent potential was confirmed by the differentiation into derivatives of the endoderm, mesoderm, and ectoderm. Most importantly, the rES cells are capable of producing chimera rats. Therefore, we established pluripotent rES cell lines that are widely used to produce genetically modified experimental rats for study of human diseases.

A comparative analysis of the transcriptome and signal pathways in hepatic differentiation of human adipose mesenchymal stem cells

The specific features of the plasticity of adult stem cells are largely unknown. Recently, we demonstrated the hepatic differentiation of human adipose tissue‐derived mesenchymal stem cells (AT‐MSCs). To identify the genes responsible for hepatic differentiation, we examined the gene expression profiles of AT‐MSC‐derived hepatocytes (AT‐MSC‐Hepa) using several microarray methods. The resulting sets of differentially expressed genes (1639 clones) were comprehensively analyzed to identify the pathways expressed in AT‐MSC‐Hepa. Clustering analysis revealed a striking similarity of gene clusters between AT‐MSC‐Hepa and the whole liver, indicating that AT‐MSC‐Hepa were similar to liver with regard to gene expression. Further analysis showed that enriched categories of genes and signaling pathways such as complementary activation and the blood clotting cascade in the AT‐MSC‐Hepa were relevant to liver‐specific functions. Notably, decreases in Twist and Snail expression indicated that mesenchymal‐to‐epithelial transition occurred in the differentiation of AT‐MSCs into hepatocytes. Our data show a similarity between AT‐MSC‐Hepa and the liver, suggesting that AT‐MSCs are modulated by their environmental conditions, and that AT‐MSC‐Hepa may be useful in basic studies of liver function as well as in the development of stem cell‐based therapy.

Stem cell plasticity: Learning from hepatogenic differentiation strategies

Many studies on stem cell plasticity are challenging the concept that stem cells contain an intrinsically predefined, unidirectional differentiation program. This means that the developmental fate of a stem cell is dependent on the general potential of the cell (pre‐determined stem cell fate) as well as on microenvironmental cues, such as stimuli from growth factors (stem cell niche). Here, we reviewed reports that examined the hepatocyte differentiation ability of stem cells from two different sources: embryonic stem cells and adult stem cells. All of those stem cells revealed the ability to give rise to hepatocyte‐like cells using different induction strategies. However, it is still not clear which of those stem cells would be the best source for hepatocyte replacement or which would be the best protocol. We herein present the current knowledge regarding available protocols and factors used in order to obtain functional hepatocytes from stem cells. Developmental Dynamics 236:3228–3241, 2007.

Recapitulation of in vivo gene expression during hepatic differentiation from murine embryonic stem cells

Hepatic differentiation at the molecular level is poorly understood, mainly because of the lack of a suitable model. Recently, using adherent monoculture conditions, we demonstrated the direct differentiation of hepatocytes from embryonic stem (ES) cells. In this study, we exploited the direct differentiation model to compare the gene expression profiles of ES cell–derived hepatocytes with adult mouse liver using DNA microarray technology. The results showed that the ES cell–derived hepatocyte gene expression pattern is very similar to adult mouse liver. Through further analysis of gene ontology categories for the 232 most radically altered genes, we found that the significant categories related to hepatic function. Furthermore, through the use of small interfering RNA technology in vitro, hepatocyte nuclear factor 3β/FoxA2 was identified as having an essential role in hepatic differentiation. These results demonstrate that ES cell–derived hepatocytes recapitulate the gene expression profile of adult mouse liver to a significant degree and indicate that our direct induction system progresses via endoderm differentiation. In conclusion, our system closely mimics in vivo hepatic differentiation at the transcriptional level and could, therefore, be useful for studying the molecular basis of hepatocyte differentiation per se. (HEPATOLOGY 2005.)

Transplantation of a fetal liver cell-loaded hyaluronic acid sponge onto the mesentery recovers a Wilson's disease model rat

An auxiliary liver represents a promising alternative for liver transplantation. The use of a large amount of mature hepatocytes, however, despite their high function, is limited in a clinical setting. Here, we propose a novel transplantation system that dramatically improved a diseased animal by incorporating fetal liver cells (FLCs) as a cell source, the mesentery as a transplantation site and a hyaluronic acid (HA) sponge as a cell scaffold. We transplanted wild-type Long Evans Agouti rat FLCs embedded in HA sponges onto the mesentery of Long Evans Cinnamon (LEC) rats, an animal model for Wilsons disease. The FLC-loaded HA sponges successfully grafted and consequently prevented jaundice. Accordingly, the treated animals showed a significant reduction in blood copper concentration, which consequently led to significant decreases in serum total bilirubin and direct bilirubin, and to a significant increase in albumin productivity. Furthermore, haematoxylin and eosin staining of the host livers demonstrated that fibrosis at the periportal area was moderated in the treated animals. In conclusion, we transplanted FLC-loaded HA sponges onto the mesenteric blood vessels, leading to thick, liver-like tissue possessing blood vessels, and the liver tissue engineered thus exhibited a remarkable therapeutic effect on the copper metabolism deficiency of LEC rats.

Regenerative cells for transplantation in hepatic failure.

Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells have an enormous potential; however, their potential clinical application is being arrested due to various limitations such as teratoma formation followed by tumorigenesis, emergent usage, and the quality control of cells, as well as safety issues regarding long-term culture are also delaying their clinical application. In addition, human ES cells have two crucial issues: immunogenicity and ethical issues associated with their clinical application. The efficient generation of human iPS cells requires gene transfer, yet the mechanism underlying pluripotent stem cell induction has not yet been fully elucidated. Otherwise, although human adult regenerative cells including mesenchymal stem cells have a limited capacity for differentiation, they are nevertheless promising candidates for tissue regeneration in a clinical setting. This review highlights the use of regenerative cells for transplantation in hepatic failure.