Participation of Somatic Stem Cells, Labeled by a Unique Antibody (A3) Recognizing both N-glycan and Peptide, to Hair Follicle Cycle and Cutaneous Wound Healing in Rats.

A monoclonal antibody (A3) was generated by using rat malignant fibrous histiocytoma (MFH) cells as the antigen. Generally, MFH is considered to be a sarcoma derived from undifferentiated mesenchymal cells. Molecular biological analyses using the lysate of rat MFH cells revealed that A3 is a conformation specific antibody recognizing both N-glycan and peptide. A3-labeled cells in bone marrow were regarded as somatic stem cells, because the cells partly coexpressed CD90 and CD105 (both immature mesenchymal markers). In the hair follicle cycle, particularly the anagen, the immature epithelial cells (suprabasal cells) near the bulge and some immature mesenchymal cells in the disassembling dermal papilla and regenerating connective tissue sheath/hair papilla reacted to A3. In the cutaneous wound-healing process, A3-labeled epithelial cells participated in re-epithelialization in the wound bed, and apparently, the labeled cells were derived from the hair bulge; in addition, A3-labeled immature mesenchymal cells in the connective tissue sheath of hair follicles at the wound edge showed the expansion of the A3 immunolabeling. A3-labeled immature epithelial and mesenchymal cells contributed to morphogenesis in the hair cycle and tissue repair after a cutaneous wound. A3 could become a unique antibody to identify somatic stem cells capable of differentiating both epithelial and mesenchymal cells in rat tissues.

Participation of Somatic Stem Cells, Recognized by a Unique A3 Antibody, in Mucosal Epithelial Regeneration in Dextran Sulfate Sodium (DSS)-Induced Rat Colonic Lesions.

A3, generated as a monoclonal antibody against rat malignant fibrous histiocytoma cells, recognizes somatic stem cells in rats. We analyzed the distribution of A3-positive cells in dextran sulfate sodium (DSS)-induced colonic lesions consisting of regenerating mucosa and fibrosis. Male 6-week-old F344 rats were administered 5% DSS in drinking water for 5 to 7 days, and lesions at recovery stage were also examined. In untreated control adult colons, A3-positive cells are localized around the crypts where stem cell niche is formed. Histopathologically, in colons of DSS-administered rats, mucosal atrophy, inflammatory cell infiltration, and fibrosis were observed in the lamina propria; thereafter, mucosal epithelia were desquamated, and crypts were decreased gradually with decrease in surrounding A3-positive cells. At the early recovery stage, crypts showed regeneration with reappearance of A3-positive cells. Interestingly, A3-positive cells aggregated in desquamated mucosa surface of fibrosis. Aggregated A3-positive cells coexpressed with vimentin, Thy-1, and partly CK19 but did not react simultaneously with α-SMA. Likely, aggregated A3-positive cells may be rescue cells with nature of both mesenchymal and epithelial cells to maintain self-renewal after injury in the colon. A3 antibody would become a useful tool to investigate the participation of stem cells in rat colonic lesions.

The localization and distribution of cells labeled by a somatic stem cell-recognizing antibody (A3) in rat colon development; possible presence of a new cell type forming the intestinal stem cell niche.

A3, generated as a monoclonal antibody against rat malignant fibrous histiocytoma (MFH)-derived cloned cells, recognizes somatic stem cells (bone-marrow/hair follicle stem cells). We investigated the distribution of cells immunoreactive to A3 in the developing rat intestine (particularly, the colon), focusing on the ontogenic kinetics of A3-positive cells. In the rat intestine, A3 labeled spindle-shaped stromal cells localized in the submucosa and labeled endothelial cells of capillaries in the lamina propria forming villi in the early development stage. With development progression, A3-positive cells were exclusively localized around the crypts of the colon. Double immunofluorescence revealed that A3-positive cells around the crypts reacted to vimentin (for mesenchymal cells) and Thy-1 (for mesenchymal stromal cells) but not to α-SMA (for mesenchymal myofibroblastic cells) or CD34 (for hematopoietic stem cells), indicating that A3-positive cells around the crypts may have characteristics of immature mesenchymal cells. In addition, A3 labeled a few epithelial cells at the base of colon crypts. Furthermore, immunoelectron microscopy revealed that A3-positive cells lay inside myofibroblasts adjacent to the epithelium of the crypts. A3-positive cells were regarded as a new type of immature mesenchymal cells around the crypts. Collectively, A3-positive cells might take part in the stem cell niche in the colon, which is formed through epithelial-mesenchymal interaction.

Establishment and characterization of a transplantable tumor line (RMM) and cell line (RMM-C) from a malignant amelanotic melanoma in the F344 rat, with particular reference to galectin-3 expression in vivo and in vitro.

To investigate characteristics of malignant melanomas with various pathobiological features, a homotransplantable tumor line (RMM) was established from a spontaneous amelanotic melanoma found in the pinna of an aged F344 rat. RMM tumors were transplanted in syngeneic rats by serial subcutaneous implantation with 100% intake. The original and RMM tumors consisted of spindle-shaped cells arranged mainly in interlacing bundles. Immunohistochemically, the neoplastic cells were positive to PNL-2 (melanocytes), nestin (neuroectodermal stem cells), S-100 (neurogenic cells) and vimentin (mesenchymal cells). Electron microscopically, tumor cells possessed single membrane-bound pre-melanosomes. Further, a cell line (RMM-C) was induced from an RMM tumor. RMM-C cells and the induced tumors in syngeneic rats showed immunohistochemical reactions similar to the original and RMM tumors. Interestingly, serum level of galectin-3 expression was increased with growing RMM tumors, and the expression was influenced by TNF-α (increase) or TGF-ß1 (decrease), indicating a possible biomarker of amelanotic melanomas. The RMM tumors and RMM-C cell line could become useful tools for studies on the pathobiology, including tumor immunity, and development of therapeutic strategies against this malignancy. These tools are the first tumor lines of amelanotic melanomas in the rat.

Rat malignant fibrous histiocytoma (MFH)-derived cloned cell lines (MT-8 and MT-9) show different differentiation in mesenchymal stem cell lineage.

Malignant fibrous histiocytomas (MFHs) show a storiform growth pattern consisting of fibroblastic, histiocytic and undifferentiated mesenchymal cells with possible multipotency. Because MFH-like tumors are induced experimentally by some chemicals and materials, it is important to know the histogenesis of MFHs. We analyzed in vitro and in vivo characteristics of two cloned cell lines (MT-8 and MT-9) established from a spontaneous MFH found in an aged F344 rat. MT-8 and MT-9 cultured cells and their tumors induced in syngeneic rats by injection were investigated morphologically, and their tumors were evaluated by immunohistochemistry. Gene expression profiles of their cultures and induced tumors were analyzed by the comprehensive gene analysis. MT-8 cells had less developed organelles and the induced tumors represented histological characteristics of undifferentiated sarcoma (sarcoma not otherwise specified (NOS)), whereas MT-9 cells had relatively well-developed intracytoplasmic organelles such as endoplasmic reticulum, mitochondria and lysosomes and the tumors showed a storiform growth pattern typical of MFHs. MT-8 and MT-9 tumors were immuno-positive for vimentin, and the reactivity for stem cell markers (nestin, CD90, CD34, and A3) appeared to be greater in MT-9 tumor cells, and their tumor cells did not react to markers for well-differentiated cells of epithelial, myogenic and neurogenic tissues except for faint reaction for S-100 protein in MT-9 tumors. The gene analyses revealed that genes relating to "cell differentiation" were more activated in MT-9 than MT-8 tumors, whereas those involved in "cell cycle" were greater in MT-8 than MT-9 tumors. In MT-8 and MT-9, additionally, genes involved in "cell differentiation" were much greater in their tumors than in their cultures. These findings indicate that MT-8 cells are poorly differentiated mesenchymal stem cells which induce sarcomas NOS, whereas MT-9 cells, which can develop typical MFHs, have more differentiated stem cell nature with greater multipotential differentiation. In MFHs, collectively, MT-8 and MT-9 cells are regarded as "tumor stem cells" and "tumor precursors" in the stem cell lineage, respectively, according to the concept of "cancer stem cell theory".

Analysis of glial fibrillary acidic protein (GFAP)-expressing ductular cells in a rat liver cirrhosis model induced by repeated injections of thioacetamide (TAA).

Glial fibrillary acidic protein (GFAP), a type III intermediate filament protein, is expressed in hepatic stellate cells (HSCs), the principal fibrogenic cell type in the liver. Further, GFAP could be a marker for hepatic progenitor cells (HPCs). In this study, the participation of GFAP-expressing cells in HPC expansion/ductular reaction was investigated in a rat model of liver cirrhosis. Six-week-old male F344 rats were injected intraperitoneally with thioacetamide (100mg/kg BW, twice a week) and examined at post-first injection weeks 5, 10, 15, 20 and 25. Fibrosis-related proliferation of ductular cells was observed as demonstrated by CK19 immunostaining. Some of these cells were stained with GFAP. No co-staining was observed between CK19 and α-smooth muscle actin (α-SMA; myofibroblast marker). There were proliferating ductular cells stained with α-fetoprotein or ß-catenin; the ductular reaction was related to increased expression of hepatocarcinogenesis-related factors (Wnt2, Wnt4 and glypican-3). These results for the first time show the participation of GFAP-positive HPCs in ductular reaction in a chemically induced rodent model. Though the ductular cells were chaperoned by myofibroblasts, they show no direct evidence for epithelial to mesenchymal transition. These findings shed new light in understanding the roles of GFAP-expressing HPCs in liver cirrhosis and provide further evidence of interaction between newly-formed bile ductules and HSCs, suggesting that both cells could be in the common lineage of HPCs.

Immunohistochemical characterization of glial fibrillary acidic protein (GFAP)-expressing cells in a rat liver cirrhosis model induced by repeated injections of thioacetamide (TAA).

Hepatic stellate cells, the principal fibrogenic cell type in the liver, are known to express the astrocyte marker glial fibrillary acidic protein (GFAP). However, the exact role of GFAP-expressing cells in liver fibrosis remains to be elucidated. In this study, cellular properties of GFAP-expressing cells were investigated in a rat model of liver cirrhosis. Six-week-old male F344 rats were injected intraperitoneally with thioacetamide (100 mg/kg BW, twice a week) and examined at post first injection weeks 5, 10, 15, 20 and 25. Appearance of GFAP-expressing myofibroblasts peaked at week 15, associated with fibrosis progression. The majority of GFAP-expressing myofibroblasts co-expressed vimentin, desmin and alpha-smooth muscle actin. Some GFAP-positive myofibroblasts co-expressed nestin (neural stem cell marker), while a few co-expressed A3 (mesenchymal stem cell marker) and Thy-1 (immature mesenchymal cell marker). A few GFAP expressing cells underwent both mitosis and apoptosis. These results indicate that there is a dynamic participation of GFAP-expressing myofibroblasts in rat liver cirrhosis, and that they are mainly derived from hepatic stellate cells, and partly from cells in the stem cell lineage. These findings, which were shown for the first time in detail, would be useful to understand the role of GFAP-expressing myofibroblasts in the pathogenesis of chemically induced liver cirrhosis.

M1- and M2-macrophage polarization in rat liver cirrhosis induced by thioacetamide (TAA), focusing on Iba1 and galectin-3.

INTRODUCTION: Resident and exudate macrophages play an important role in the development of liver cirrhosis. Ionized calcium binding adaptor molecule 1(+) (Iba1(+)) and galectin-3(+) (Gal-3(+)) macrophages regulate liver fibrosis probably through pro-inflammatory and pro-fibrotic factors. Macrophages show polarized functions in liver fibrosis; however, M1-/M2-polarization of Iba1(+) and Gal-3(+) macrophages remains obscured. This study investigated the M1-/M2-polarized properties of Iba1(+) and Gal-3(+) macrophages in chemical-induced liver cirrhosis. MATERIALS AND METHODS: Cirrhosis was induced in F344 rats by repeated injections of thioacetamide (100mg/kg BW, twice a week for 25 weeks). Liver samples were collected from post-first-injection (PFI) week 5 to 25. Macrophage immunophenotypes and myofibroblasts in the fibrous bridges (FBs) and pseudolobules (PLs) were analyzed by immunohistochemistry. Expressions of M1- and M2-related factors were analyzed with RT-PCR, separately in FBs and PLs. RESULTS: Activation of myofibroblasts was most pronounced in livers at week 15. CD68(+) (M1), CD204(+) (M2), Iba1(+) and Gal-3(+) macrophages in the FBs increased gradually and peaked at week 15, consistent with the upregulation of both M1-(MCP-1, IFN-γ, IL-1ß, IL-6, and TNF-α) and M2-(TGF-ß1, IL-4, and IL-10) related factors. Iba1(+) and Gal-3(+) macrophages showed both M1- and M2-immunophenotypes. CD163(+) macrophages showed a persistent increase, consistent with TGF-ß1 upregulation. MHC class II(+) macrophages increased in the developing fibrotic lesions, and then reduced in the advanced stage cirrhosis. CONCLUSION: Both M1- and M2-macrophage polarizations occur during development of liver cirrhosis. Iba1(+) and Gal-3(+) macrophages participate in liver cirrhosis through production of both M1- and M2-related factors.