Hitoshi Okochi

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.

Regulatory B cells (B10 cells) have a suppressive role in murine lupus: CD19 and B10 cell deficiency exacerbates systemic autoimmunity.

B cells play critical roles in the pathogenesis of lupus. To examine the influence of B cells on disease pathogenesis in a murine lupus model, New Zealand Black and New Zealand White F1 hybrid (NZB/W) mice were generated that were deficient for CD19 (CD19−/− NZB/W mice), a B cell-specific cell surface molecule that is essential for optimal B cell signal transduction. The emergence of anti-nuclear Abs was significantly delayed in CD19−/− NZB/W mice compared with wild type NZB/W mice. However, the pathologic manifestations of nephritis appeared significantly earlier, and survival was significantly reduced in CD19−/− NZB/W mice compared with wild type mice. These results demonstrate both disease-promoting and protective roles for B cells in lupus pathogenesis. Recent studies have identified a potent regulatory B cell subset (B10 cells) within the rare CD1dhiCD5+ B cell subset of the spleen that regulates acute inflammation and autoimmunity through the production of IL-10. In wild type NZB/W mice, the CD1dhiCD5+B220+ B cell subset that includes B10 cells was increased by 2.5-fold during the disease course, whereas CD19−/− NZB/W mice lacked this CD1dhiCD5+ regulatory B cell subset. However, the transfer of splenic CD1dhiCD5+ B cells from wild type NZB/W mice into CD19−/− NZB/W recipients significantly prolonged their survival. Furthermore, regulatory T cells were significantly decreased in CD19−/− NZB/W mice, but the transfer of wild type CD1dhiCD5+ B cells induced T regulatory cell expansion in CD19−/− NZB/W mice. These results demonstrate an important protective role for regulatory B10 cells in this systemic autoimmune disease.

Protective and Pathogenic Roles for B Cells during Systemic Autoimmunity in NZB/W F1 Mice

Delineating the relative contributions of B lymphocytes during the course of autoimmune disease has been difficult. Therefore, the effects of depleting all mature B cells using a potent CD20 mAb, or of depleting circulating and marginal zone B cells using a ligand-blocking CD22 mAb, were compared in NZB/W F1 mice, a model for human systemic lupus erythematosus. Single low-dose mAb treatments depleted B cells efficiently in both NZB/W F1 and C57BL/6 mice. Prophylactic B cell depletion by repeated CD20 mAb treatments prolonged survival during pristane-accelerated lupus in NZB/W F1 mice, whereas CD22 mAb had little effect. Despite effective B cell depletion, neither mAb treatment prevented autoantibody generation. In addition, CD20, CD22, and control mAb-treated NZB/W F1 mice developed anti-mouse IgG autoantibodies in contrast to parental NZB and NZW strains, which may have reduced the effectiveness of B cell depletion. Despite this, low-dose CD20 mAb treatment initiated in 12–28-wk-old mice, and administered every 4 wk thereafter, significantly delayed spontaneous disease in NZB/W F1 mice. By contrast, B cell depletion initiated in 4-wk-old mice hastened disease onset, which paralleled depletion of the IL-10–producing regulatory B cell subset called B10 cells. B10 cells were phenotypically similar in NZB/W F1 and C57BL/6 mice, but were expanded significantly in young NZB/W F1 mice. Thus, B cell depletion had significant effects on NZB/W F1 mouse survival that were dependent on the timing of treatment initiation. Therefore, distinct B cell populations can have opposing protective and pathogenic roles during lupus progression.

Characterization and Localization of Side Population Cells in Mouse Skin

Recently, the detection of side population (SP) cells, which have the ability to strongly efflux Hoechst 33342 fluorescence dye, has attracted attention as a method of stem cell isolation. We identified SP cells from mouse skin using the same method as from bone marrow. This population almost completely disappeared after treatment with the calcium channel blocker verapamil. SP cells were mainly localized in the epidermis, with a few in the dermis. The ratio of SP cells decreased as the mouse became older. Surface marker analysis revealed that the sorted SP cells expressed α6‐integrin, β1‐integrin, Sca‐1, keratin 14, and keratin 19, which are proliferating and progenitor cell markers, at levels higher than in non‐SP cells, while they expressed E‐cadherin, CD34, and CD71 at lower levels. The expression of breast cancer resistance protein 1 (BCRP1), which participates in dye efflux, was expressed at high levels at both the protein and mRNA level in sorted SP cells. Immunohistochemical analysis showed that BCRP1 was expressed in the basal layers and hair bulge regions of mouse skin. BCRP1 mRNA was found in basal layers and hair follicles of newborn skin by in situ hybridization. These results indicate that the localization of BCRP1‐positive cells is compatible with that of keratinocyte stem cells. Based on the close relationship between BCRP1 and the SP cell phenotype, we conclude that keratinocyte stem cells are closely related to the SP‐ or BCRP1‐positive cells.

B Lymphocyte signaling established by the CD19/CD22 loop regulates autoimmunity in the tight-skin mouse.

Systemic sclerosis (SSc) is characterized by fibrosis and autoimmmunity. Peripheral blood B cells from SSc patients specifically overexpress CD19, a critical cell-surface signal transduction molecule in B cells. CD19 deficiency in B cells also attenuates skin fibrosis in the tight-skin (TSK/+) mouse, a genetic model for SSc. Herein we analyzed two transgenic mouse lines that overexpress CD19. Remarkably, 20% increase of CD19 expression in mice spontaneously induced SSc-specific anti-DNA topoisomerase I (topo I) antibody (Ab) production, which was further augmented by 200% overexpression. In TSK/+ mice overexpressing CD19, skin thickness did not increase, although anti-topo I Ab levels were significantly augmented, indicating that abnormal CD19 signaling influences autoimmunity in TSK/+ mice and also that anti-topo I Ab does not have a pathogenic role. The molecular mechanisms for abnormal CD19 signaling were further assessed. B-cell antigen receptor crosslinking induced exaggerated calcium responses and augmented activation of extracellular signal-regulated kinase in TSK/+ B cells. CD22 function was specifically impaired in TSK/+ B cells. Consistently, CD19, a major target of CD22-negative regulation, was hyperphosphorylated in TSK/+ B cells. These findings indicate that reduced inhibitory signal provided by CD22 results in abnormal activation of signaling pathways including CD19 in TSK/+ mice and also suggest that this disrupted B cell signaling contribute to specific autoantibody production.

LEUKEMIA INHIBITORY FACTOR AS AN ANTI-APOPTOTIC MITOGEN FOR PLURIPOTENT MOUSE EMBRYONIC STEM CELLS IN A SERUM-FREE MEDIUM WITHOUT FEEDER CELLS

SummaryWe have developed a serum-free medium, designated ESF7, in which leukemia inhibitory factor (LIF) clearly stimulated murine embryonic stem (ES) cell proliferation accompanied by increased expression of nanog and Rex-1 and decreased FGF-5 expression. These effects were dependent on the concentration of LIF. The ES cells maintained in ESF7 medium for more than 2 yr retained an undifferentiated phenotype, as manifested by the expression of the transcription factor Oct-3/4, the stem cell marker SSEA-1, and alkaline phosphatase. Withdrawal of LIF from ESF7 medium resulted in ES cell apoptosis. Addition of serum to ESF7 medium promoted ES cell differentiation. Addition of MBP4 promoted ES cell differentiation into simple epithelial-like cells. In contrast, FGF-2 promoted ES cell differentiation into neuronal and glial-like cells. Under serum-free culture conditions, LIF was sufficient to stimulate cell proliferation, it inhibited cell differentiation, and it maintained self-renewal of ES cells. Because this simple serum-free adherent monoculture system supports the long-term propagation of pluripotent ES cells in vitro, it will allow the elucidation of ES cell responses to growth factors under defined conditions.

Elevated levels of eotaxin and interleukin‐5 in blister fluid of bullous pemphigoid: correlation with tissue eosinophilia

Background Bullous pemphigoid (BP) often provokes blood and tissue eosinophilia, which suggests that some chemoattractants modulate the eosinophil infiltration in BP. Eotaxin, a CC chemokine, strongly attracts eosinophils, and interleukin (IL)‐5 induces eosinophil differentiation, proliferation and colony formation in vitro. Objectives To examine the correlation between levels of eotaxin and IL‐5 and the number of lesional eosinophils, and the expression of eotaxin in BP lesions. Patients/methods In this study we measured eotaxin and IL‐5 levels in blister fluid of BP by enzyme‐linked immunosorbent assay. We also examined the expression of eotaxin in BP lesions by immunohistochemistry. Results Both eotaxin and IL‐5 were detected at high levels in BP blister fluid. Blister fluid eotaxin, but not IL‐5 levels, correlated significantly with the number of dermal infiltrating eosinophils. By immunohistochemistry, eotaxin was strongly expressed in epidermal keratinocytes around BP blisters. Conclusions These findings suggest that eotaxin and IL‐5 are strongly associated with the tissue eosinophilia of BP. Therapies which aim to inhibit production of eotaxin and IL‐5 may improve the inflammation and blister formation in BP.

Pdx1-transfected adipose tissue-derived stem cells differentiate into insulin-producing cells in vivo and reduce hyperglycemia in diabetic mice

Insulin-dependent diabetes mellitus (IDDM) is characterized by the rapid development of potentially severe metabolic abnormalities resulting from insulin deficiency. The transplantation of insulin-producing cells is a promising approach for the treatment of IDDM. The transcription factor pancreatic duodenal homeobox 1 (Pdx1) plays an important role in the differentiation of pancreatic beta cells. In this study, the human Pdx1 gene was transduced and expressed in murine adipose tissue-derived stem cells (ASCs). To evaluate pancreatic repair, we used a mouse model of pancreatic damage resulting in hyperglycemia, which involves injection of mice with streptozotocin (STZ). STZ-treated mice transplanted with Pdx1-transduced ASCs (Pdx1-ASCs) showed significantly decreased blood glucose levels and increased survival, when compared with control mice. While stable expression of Pdx1 in ASCs did not induce the pancreatic phenotype in vitro in our experiment, the transplanted stem cells became engrafted in the pancreas, wherein they expressed insulin and C-peptide, which is a marker of insulin-producing cells. These results suggest that Pdx1-ASCs are stably engrafted in the pancreas, acquire a functional beta-cell phenotype, and partially restore pancreatic function in vivo. The ease and safety associated with extirpating high numbers of cells from adipose tissues support the applicability of this system to developing a new cell therapy for IDDM.