Yuichi Hasebe

Age-related changes of p75 Neurotrophin receptor-positive adipose-derived stem cells

BACKGROUND The existence of multipotent stem cells in subcutaneous adipose tissue has been reported. We previously confirmed that p75 neurotrophin receptor (p75NTR; CD271)-positive cells in subcutaneous adipose tissue possessed multipotency, although changes of the characteristics in p75NTR-positive adipose-derived stem cells (ASCs) with aging remain unclear. OBJECTIVE To investigate the effect of aging on p75NTR-positive ASCs. METHODS The number of p75NTR-positive ASCs in subcutaneous adipose tissue of ICR mice aged 3-24 weeks was analyzed by immunostaining and flow cytometry. Subsequently, the cells were isolated and their ability to attach to the cell culture dish, proliferation rate (doubling time) and the expression of senescence-associated beta-galactosidase (SA-beta gal), a cellular senescence marker, were assessed. Age-related changes in the differentiation potential of p75NTR-positive cells in adipogenic, osteogenic, chondrogenic and myogenic lineage were also investigated. RESULTS The number of ASCs per unit of tissue weight in adipose tissue and the attachment rate of isolated cells decreased with aging. No difference in the cell proliferation rate and the percentage of SA-beta gal-positive cells was detected. Although the efficacy of differentiation into adipogenic and osteogenic lineages slightly decreased with aging, the differentiation potential into chondrogenic and myogenic lineages was not changed. CONCLUSION The number of ASCs per unit of tissue weight decreased in aged mice. However, the cells possessed proliferation and differentiation potentials almost equal to those of young mice even though the differentiation potentials showed a tendency of decrease. These results raise the possibility that stem cell functions, self-renewal and multipotency, are maintained regardless of aging.

Plasminogen activator-plasmin system potentiates the proliferation of hepatocytes in primary culture

BACKGROUND/AIMS Liver regeneration after partial hepatectomy is thought to be regulated by various molecules including the components of the plasminogen activator (PA)-plasmin system. We have examined the role of fibrinolytic factors, i.e., tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), and their substrate, plasminogen, in the proliferation of hepatocytes in primary culture. METHODS Hepatocyte and nonparenchymal liver cells were isolated from Wistar strain rat by a method perfusing the liver with collagenase. DNA synthesis was assessed by measuring the incorporation of [3H]-thymidine into cellular DNA fraction. tPA, uPA and type-1 plasminogen activator inhibitor (PAI-1) gene expressions were measured by Northern blotting. PA activity was measured by fibrin/agarose plate method. RESULTS Cellular density-dependent DNA synthesis was observed in the primary cultured hepatocytes; DNA synthesis was lower at high cell density (1.0 x 10(5) cells/cm(2)) than that at low cell density (0.2 x 10(5) cells/cm(2)). DNA synthesis in the hepatocytes cultured at a low cell density was increased by co-culture with nonparenchymal liver cells. Under these growth-stimulated culture conditions, tPA and uPA mRNAs were induced and up-regulated. On the contrary, the PAI-1 mRNA level was decreased under these conditions, and total PA activity was augmented accordingly. The synthetic plasmin inhibitor tranexamic acid, a competitive inhibitor for the plasmin molecule, and PASI-535, a plasmin active center-directed inhibitor, both suppressed hepatocyte proliferation in a dose-dependent fashion. Anti-plasmin antibody also suppressed hepatocyte proliferation. CONCLUSIONS The up-regulation of PA activity for ensuring plasmin activity should be an important mechanism in the proliferation of hepatocytes.

Bleomycin inhibits adipogenesis and accelerates fibrosis in the subcutaneous adipose layer through TGF‐β1

Systemic sclerosis [scleroderma (SSc)]‐associated skin fibrosis is characterized by increased fibrosis in the dermis and a reduction in the thickness of the subcutaneous adipose tissue layer. Although many studies have examined fibrosis in SSc, only a few studies have focused on the associated reduction in the thickness of the subcutaneous adipose tissue layer. In this study, we investigated the effects of SSc‐induced fibrosis on adipose tissue. We found that bleomycin suppresses adipogenesis in adipose‐derived stem cells (ASCs) and stimulates ASCs to express transforming growth factor β1 (TGF‐β1), which suppresses adipogenesis and promotes fibrosis. Furthermore, we found that adipocyte‐conditioned medium suppressed collagen synthesis by fibroblasts in fibrosis‐like conditions. We concluded that in the skin affected by bleomycin‐induced fibrosis, increased TGF‐β1 expression suppresses adipogenesis and promotes adipocyte fibrosis. It was also suggested that adipocytes have an inhibitory effect on the progression of fibrosis.

Plasminogen activator/plasmin system regulates formation of the hepatocyte spheroids

The isolated rat hepatocytes inoculated onto the surface of positively charged culture dishes are anchored initially and then begin to migrate and aggregate gradually to form multicellular spheroids detached from the dish. We studied the roles of fibrinolytic factors in the spheroid formation. The fibrinolytic factors, tissue-type plasminogen activator (tPA), and urokinase-type plasminogen activator (uPA), were increased in the course of spheroid formation. Then, we introduced fibrinolytic inhibitors into the spheroid cultures to determine functions of fibrinolytic factors. Plasmin inhibitor inhibited markedly the spheroid formation. Interestingly, the anti-plasmin antibody showed different effect depending on the timing of its administration. In summary, we demonstrated for the first time that induction of PAs and ensuing plasmin generation on the cell surface play important roles in hepatocyte spheroid formation, and that plasmin is involved in the different processes such as cell migration and cell detachment in the formation of hepatocyte spheroid.

Analysis of cell characterization using cell surface markers in the dermis

BACKGROUND In recent years, it has been reported that stem cells exist in the mesenchymal tissues of the bone marrow and adipose. These stem cells are thought to express specific cell surface markers such as CD44, CD54, CD105, CD90, and CD271 and have been confirmed to be pluripotent. Furthermore, although it has been reported that stem cells are also present in the dermis, their cell surface markers and characteristics are not fully understood. OBJECTIVE To confirm the presence of stem cells in the dermis and their ability, employing the mesenchymal stem cell markers which have previously been reported as an indication. METHODS We analyzed the percentages of CD44 (+), CD54 (+), CD90 (+), CD105 (+), and CD271 (+) cells in the dermis of neonatal mice (HR-1 mouse) by performing immunostaining and FACS. Secondly, we isolated each type of marker-positive and -negative cells from dermal tissues and evaluated their proliferation potential and their ability to differentiate into adipocytes, osteoblasts, and chondrocytes. RESULTS According to the immunostaining and FACS results, we confirmed that stem cells that express CD44, CD54, CD90, CD105, and CD271 are present in the dermal tissues of neonatal mice. In addition, when we measured the proliferation and differentiation potentials of each type of marker-positive cells, it was revealed that cells expressing CD54 or CD271 have a high proliferation potential and are able to differentiate into adipocytes, osteoblasts, and chondrocytes. CONCLUSIONS These results indicated that dermal tissues contain stem cells that express CD44, CD54, CD90, CD105, and CD271 which are stem cell markers. More precisely, it was suggested that both CD54 (+) and CD271 (+) stem cells have high proliferation and differentiation potentials.

A Novel Function of Thrombin-activatable Fibrinolysis Inhibitor during Rat Liver Regeneration and in Growth-promoted Hepatocytes in Primary Culture

Thrombin-activatable fibrinolysis inhibitor (TAFI) exhibits anti-fibrinolytic activity by removing C-terminal lysine residues from fibrin or plasminogen receptor proteins on the cellular surface, and plays an important role in the regulation of fibrinolysis. In this study, we examined the regulation of TAFI in hepatocytes during liver regeneration, and revealed its pivotal role in hepatocyte proliferation. In rat models, partial hepatectomy or carbon tetrachloride (CCl4)-induced acute liver injury suppressed the levels of plasma TAFI activity and hepatic TAFI mRNA, whereas this operation markedly increased both the hepatic plasmin activity and the level of proliferating cell nuclear antigen. In primary cultures of rat hepatocytes, the TAFI mRNA level was decreased under growth-promoting culture conditions. Treatment of the hepatocytes with TAFI siRNA increased the amount of plasmin on the hepatocytes and promoted hepatocyte proliferation. We concluded that TAFI regulates plasmin activity through its enzymatic activity whereby it reduces the plasminogen-binding capacity of the hepatocytes. The TAFI gene expression is down-regulated in hepatocyte proliferation for producing a fibrinolytic microenvironment suitable for cell growth. This is the first report on the role of TAFI in the pericellular fibrinolysis necessary for cellular proliferation.

Dermal CD271+ Cells are Closely Associated with Regeneration of the Dermis in the Wound Healing Process

Stem cells have recently been shown to play important roles in wound healing. The aim of this study was to investigate the role of dermal CD271+ cells in wound healing. Full-thickness wounds were produced on the backs of 5-year-old and 24-week-old mice, and time-course of wound closure, CD271+ cell counts, and gene expression levels were compared. Delayed wound healing was observed in 24-week-old mice. The peak of CD271+ cell increase was delayed in 24-week-old mice, and gene expression levels of growth factors in wounded tissue were significantly increased in 5-year-old mice. Dermal CD271+ cells purified by fluorescence-activated cell sorting (FACS) expressed higher growth factors than CD271- cells, suggesting that CD271+ cells play important roles by producing growth factors. This study also investigated dermal CD271+ cells in patients with chronic skin ulcers. Dermal CD271+ cells in patients were significantly reduced compared with in healthy controls. Thus, dermal CD271+ cells are closely associated with wound healing.

Senescent dermal fibroblasts enhance stem cell migration through CCL2/CCR2 axis

During aging, increases in the number of senescent cells are seen in various tissues. On the other hand, stem cells play crucial roles in tissue repair and homeostasis. Therefore, it is likely that stem cells give rise to new cells that replace senescent cells. However, how stem cells contribute to homeostasis in the dermis has not been elucidated. Here, we investigated the effects of factors secreted from senescent fibroblasts on stem cells. We found that senescent human dermal fibroblast (HDF) conditioned medium (CM) significantly enhanced stem cell migration compared with young HDF CM. The senescent HDF CM strongly secreted chemokine ligand 2 (CCL2). Furthermore, CCL2 was found to enhance stem cell migration, and the inhibition of CCR2, a receptor for CCL2, reduced stem cell migration. These results suggest that senescent fibroblasts recruit stem cells by secreting various factors and that the CCL2/CCR2 axis is one of the mechanisms underlying this phenomenon.

Skin-resident stem cells and wound healing

CD271 is common stem cell marker for the epidermis and dermis. We assessed a kinetic movement of epidermal and dermal CD271+ cells in the wound healing process to elucidate the possible involvement with chronic skin ulcers. Epidermal CD271+ cells were proliferated and migrated from 3 days after wounding. Purified epidermal CD271+ cells expressed higher TGFβ2 and VEGFα transcripts than CD271- cells. Delayed wound healing was observed in the aged mice compared with young mice. During the wound healing process, the peak of dermal CD271+ cell accumulation was delayed in aged mice compared with young mice. The expression levels of collagen-1, -3, -5, F4-80, EGF, FGF2, TGFβ1, and IL-1α were significantly increased in young mice compared with aged mice. Furthermore, purified dermal CD271+ cells expressed higher FGF2, EGF, PDGFB, and TGFβ1 gene transcripts than CD271- cells. These results suggested that epidermal and dermal CD271+ cells were closely associated with wound healing process by producing various growth factors. Epidermal and dermal CD271+ cells in chronic skin ulcer patients were significantly reduced compared with healthy controls. Thus, both epidermal and dermal stem cells can play an important role in wound healing process.

The Role of Plasminogen in the Proliferation of Murine Hepatocytes in Primary Culture

Liver regeneration after partial hepatectomy is thought to be regulated by various molecules including the components of the plasminogen activator (PA)-plasmin system [1]. This system has pleiotropic effects, being active in fibrin and ECM degradation, in growth factor activation, and in the integrin signaling in cell proliferation [2,3].