Human Fibroblast-like Cells Research Articles

Anti-rheumatic effects of Aconitum leucostomum Worosch. on human fibroblast-like synoviocyte rheumatoid arthritis cells.

The aim of the present study was to investigate the effects of Aconitum leucostomum Worosch. crude drug, processed products and monomer components on human fibroblast-like synoviocyte rheumatoid arthritis (HFLS-RA) cells, and its associated mechanisms. Following drug treatment, cell proliferation was assessed using a Cell Counting Kit-8 assay. Cellular apoptosis and cell cycle were evaluated using flow cytometry. Levels of hypoxia-inducible factor 1α (HIF-1α), vascular endothelial growth factor (VEGF) and toll-like receptor 4 (TLR4) mRNA and protein were evaluated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis, respectively. Levels of pro-inflammatory cytokines were evaluated using ELISA. Analysis of cell proliferation indicated that crude drug and processed products markedly inhibited the cell proliferation. Compared with the control group, the apoptosis rates were significantly elevated in all treatment groups (all P<0.05). Furthermore, the proportion of cells in G0/G1 phase was significantly decreased in all treatment groups compared with the control group (all P<0.05). RT-qPCR and western blotting indicated that, compared with the control group, mRNA and protein expression levels of HIF-1α, and TLR4 were significantly downregulated in all treatment groups (P<0.05). The mRNA and protein expression levels of VEGF in all treatment groups were decreased compared with those in the control group, but the difference was not significant. Results from ELISA demonstrated that the levels of interleukin (IL)-6, IL-1β and tumor necrosis factor-α in the cell culture supernatant were all significantly decreased following drug treatment in HFLS-RA cells (all P<0.05). Therefore, A. leucostomum Worosch. crude drug, processed products and monomer components may exert anti-rheumatic effects on HFLS-RA cells, inhibiting cell proliferation and enhancing cellular apoptosis. These effects may be attributable to the downregulated expression of HIF-1α and TLR4, as well as decreased levels of pro-inflammatory cytokines.

Cytotoxic, Anti-Proliferative and Apoptotic Effects of Tribulus terrestris L. Fruit Extract on Human Prostate Cancer Lncap and Colon Cancer HT-29 Cell Lines

Background: The medicinal herb Tribulus terrestris L. (Zygophyllaceae) has been used for a long time to treat various kinds of diseases including hepatocellular carcinoma. Objectives: The aim of the present study was to investigate the anticancer activity of hydroalcoholic extract of T. terrestris fruit on prostate and colon cancerous cell lines. Materials and Methods: The activity of the extract was studied at seven different concentrations on three cell lines, human colon adenocarcinoma (HT29), prostate carcinoma (LNCap-FGC-10) and fibroblast-like cells (HSkMC). MTT, bromo-2’-deoxyuridine(BrdU) and terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assays were used for measuring cytotoxicity, cell proliferation and apoptotic cell death, respectively. Results: The IC50 index of the extract of T. terrestris was obtained as 0.3 µg/mL, 7.1 µg/mL and 8.7 µg/mL for prostate, colon and fibroblast-like cell lines, respectively. Tribulus terrestris L. was significantly more toxic on prostate cancer cell line than on colon cancer cell and fibroblast-like cells (P value < 1%). Quantitating BrdU incorporation at the highest concentration was determined as 0.097 and 0.018. Furthermore, the extract induced 74% and 46% apoptosis in prostate and colon cancer cells, respectively. Conclusions: These data indicate that T. terrestris extract has cytotoxic, anti-proliferative and pro-apoptotic activities. It was less toxic against normal human fibroblast-like cells in comparison to cancer cell lines. Further in vivo research would help explore and interpret the potential properties of T. terrestris extract and its components as an anticancer supplement.

Cytotoxic, Anti-Proliferative and Apoptotic Effects of Tribulus terrestris L. Fruit Extract on Human Prostate Cancer Lncap and Colon Cancer HT-29 Cell Lines

Background: The medicinal herb Tribulus terrestris L. (Zygophyllaceae) has been used for a long time to treat various kinds of diseases including hepatocellular carcinoma. Objectives: The aim of the present study was to investigate the anticancer activity of hydroalcoholic extract of T. terrestris fruit on prostate and colon cancerous cell lines. Materials and Methods: The activity of the extract was studied at seven different concentrations on three cell lines, human colon adenocarcinoma (HT29), prostate carcinoma (LNCap-FGC-10) and fibroblast-like cells (HSkMC). MTT, bromo-2’-deoxyuridine(BrdU) and terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assays were used for measuring cytotoxicity, cell proliferation and apoptotic cell death, respectively. Results: The IC50 index of the extract of T. terrestris was obtained as 0.3 µg/mL, 7.1 µg/mL and 8.7 µg/mL for prostate, colon and fibroblast-like cell lines, respectively. Tribulus terrestris L. was significantly more toxic on prostate cancer cell line than on colon cancer cell and fibroblast-like cells (P value < 1%). Quantitating BrdU incorporation at the highest concentration was determined as 0.097 and 0.018. Furthermore, the extract induced 74% and 46% apoptosis in prostate and colon cancer cells, respectively. Conclusions: These data indicate that T. terrestris extract has cytotoxic, anti-proliferative and pro-apoptotic activities. It was less toxic against normal human fibroblast-like cells in comparison to cancer cell lines. Further in vivo research would help explore and interpret the potential properties of T. terrestris extract and its components as an anticancer supplement.

Anti-inflammatory effects of interleukin-23 receptor cytokine-binding homology region rebalance T cell distribution in rodent collagen-induced arthritis

IL-23 is an important cytokine to regulate Th17 cell differentiation and promote the proliferation of inflammatory cells in Th17-mediated autoimmune diseases. The collagen-induced arthritis (CIA) in rat is a model of rheumatoid arthritis characterized by pronounced inflammatory auto-responses from B and T cells, especially Th17 cells in lesions. In the present study, we used rhIL23R-CHR to block the IL-23 signaling pathway to probe the importance of IL-23 in misbalancing the ratio of Th17/Th9/Treg cells in CIA rats. After treatments with rhIL23R-CHR, the CIA rats showed a significant decrease of secretions of IL-17 and IL-9, whereas FoxP3 was activated in the process, indicating that IL-23 can manipulate the balance of Th17/Th9/Treg cells. Similar to the animal model, IL-23 also possessed remarkable proinflammatory effects on human fibroblast-like synoviocyte cells (HFLS), showing synergetic outcomes with TNF-α. Together, IL-23 could act as a modulator to imbalance the ratio of Th17/Th9/Treg cells, and rhIL23R-CHR could serve as a potential therapeutic agent for RA patients.

Effects of cranberry components on IL-1β-stimulated production of IL-6, IL-8 and VEGF by human TMJ synovial fibroblasts

ObjectiveOsteoarthritis (OA) in the TMJ is characterized by deterioration of articular cartilage and secondary inflammatory changes. Interleukin-1β (IL-1β) stimulates IL-6, IL-8, and vascular endothelial growth factor (VEGF) in synovial fluid of TMJ with internal derangement and bony changes. The cranberry (Vaccinium macrocarpon) contains polyphenolic compounds that inhibit production of pro-inflammatory molecules by gingival cells in response to several stimulators. This study examined effects of cranberry components on IL-1β-stimulated IL-6, IL-8, and VEGF production by human TMJ synovial fibroblast-like cells. DesignCranberry high molecular weight non-dialyzable material (NDM) was derived from cranberry juice. Human TMJ synovial fibroblast-like cells from joints with degenerative OA and an ankylosed TMJ without degeneration were incubated with IL-1β (0.001–1nM)±NDM (25–250μg/ml) (2h preincubation). Viability was assessed via activity of a mitochondrial enzyme. IL-6, IL-8, and VEGF in culture supernatants were measured by ELISA; NF-κB and AP-1 transcription factors were measured in nuclear extracts via binding to specific oligonucleotides. Data analysis: ANOVA and Scheffe’s F procedure for post hoc comparisons. ResultsNDM did not affect cell viability but inhibited IL-1β stimulated IL-6, IL-8, and VEGF production in all cell lines (p<0.05). NDM partially reduced nuclear levels of NF-κB and AP-1 (p<0.04), depending upon cell line and time of exposure to IL-1β+NDM. ConclusionCranberry NDM inhibition of IL-1β-stimulated IL- 6, IL-8, and VEGF production by TMJ synovial fibroblast-like cells suggests that cranberry components may be useful as a host modulatory therapeutic agent to prevent or treat inflammatory arthropathies of the TMJ.

Ochratoxin A and T-2 Toxin Induce Clonogenicity and Cell Migration in Human Colon Carcinoma and Fetal Lung Fibroblast Cell Lines.

T-2 toxin and Ochratoxin A (OTA) are toxic secondary metabolites produced by various fungi, and together they contaminate feedstuffs worldwide. T-2 toxin and OTA may exert carcinogenic action in rodent. Despite the various in vivo experiments, carcinogenicity of these two mycotoxins has not yet been proven for human. In this current study, we proposed to investigate, in Human colon carcinoma cells and fetal lung fibroblast-like cells transfected with MYC, the effect of T-2 toxin and OTA on cell clonogenicity and cell migration. Results of the present investigation showed that T2-toxin as well as OTA has an important clonogenic effect in all cell lines, suggesting that these mycotoxins could promote the transcription of c-myc gene. Furthermore, T-2 toxin and OTA enhanced the migration effect of HCT116 cells at very low concentrations, proposing that these mycotoxins may exhibit carcinogenesis-like properties in the studied cells.

Imatinib mesylate induces mitochondria-dependent apoptosis and inhibits invasion of human pigmented villonodular synovitis fibroblast-like synovial cells.

Pigmented villonodular synovitis (PVNS) is a rare sarcoma-like disorder characterized by synovial lesions proliferation and invasion to articular cartilage for which no effective treatments are available. Imatinib mesylate (IM) is known to exert antitumor activity in some tumors, but its effects on PVNS fibroblast-like synoviocytes (PVNS-FLS) and the specific mechanism involved remain to be established. In the present study, the in vitro effects of IM on cell proliferation and survival rates were investigated in PVNS-FLS. Apoptosis induction was assessed via acridine orange/ethidium bromide (AO)/(EB) and Annexin V/PI staining as well as western blotting. The invasion ability of PVNS-FLS was evaluated by Transwell invasion chambers. IM significantly inhibited survival and invasion ability of PVNS-FLS in a dose- and time-dependent manner. The drug-treated cell groups exhibited markedly higher apoptosis, which was blocked upon pretreatment with the specific caspase-9 inhibitor Z-LEHD-FMK. Expression of cleaved caspase-9 was significantly increased and the Bcl-2 family and caspase-3 were activated following treatment with IM. Our results collectively demonstrated that IM has a strong antiproliferative effect on PVNS-FLS in vitro, attributable to induction of mitochondrial-dependent apoptosis in association with activation of caspase-9/-3 and the Bcl-2/Bax family, and exhibits significant inhibition on the invasion ability of PVNS-FLS, suggesting that IM may be useful as a novel treatment of this disease.

Anti-DR5 mAb inhibits proliferation of human fibroblast-like synovial cells and reduces their cytokine secretion in vitro.

BackgroundWe have previously reported that anti-death receptor 5 (DR5) monoclonal antibody (mAb) is therapeutically effective in the treatment of rheumatoid arthritis (RA) in a collagen-induced arthritis rat model. However, the molecular mechanism and the effect of anti-DR5 mAb on proapoptotic genes and cytokine secretion in the human fibroblast-like synovial cells (FLS) requires further clarification. This study may provide new evidence for the application of anti-DR5 mAb as a treatment for RA.MethodsHuman FLS were isolated from patients with RA and were treated with anti-DR5 mAb. An MTT assay and flow cytometry were used to detect the induction of apoptosis in vitro. Cytokine secretion by the FLS was detected using the enzyme-linked immunosorbent assay. The mRNA expression was assessed by reverse transcription polymerase chain reaction, and the protein expression was analyzed by Western blot. The apoptotic pathway was investigated further using a caspase inhibition assay.ResultsAnti-DR5 mAb-induced apoptosis in human RA FLS in vitro. The protein expressions of caspase-8, -3, and -9 were decreased in human anti-DR5 mAb-treated FLS in a dose-dependent manner through exposure to a caspase inhibitor, indicating that anti-DR5 mAb induction of apoptosis is through the caspase pathway. Decreased levels of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) were detected after treatment with anti-DR5 mAb in vitro.ConclusionAnti-DR5 mAb may induce apoptosis in human FLS through the caspase pathway and through decreased secretions of TNF-α and IFN-γ.

PP39 - Indole- and Chromane-type heterocycles: Playing with molecules to defeat ageing

Heterocyclic compounds with chromane- and indole-type skeleton represent two fairly large groups of substances with proved beneficial antioxidant effects even beyond the nutritional base. Although these compounds have usual origin in the nature, a rational chemical synthesis might offer an efficient tool for “playing” with their physico-chemical properties so as to optimize their biological effects. In the present study, on using the cellular models, we tried to evaluate the potential of chemically modified flavonoids and synthetic hexahydropyridoindoles to ameliorate ageing-related degenerative processes. Introduction of electron-accepting quinonoid substituent into quercetin increased its prooxidant capacity and toxicity, however, at lower concentration, the effects encompassing hormesis were observed. These properties involved enhanced protection of human fibroblast-like cells from oxidative insult as well as anti-inflammatory effects. 3´-O-Acylation of quercetin (ensuring its higher lipophilicity) yielded in enhanced protection of adult rat microglial cells based on suppression of their protein oxidation, downregulation of the cellular senescence markers and induction of a resting, ramified morphology. On the other hand, weakly basic piperidine moiety in pyridoindoles appeared to steer these substances into lysosomal compartments via a proton-trap mechanism, this being followed by their enhanced accumulation. This mechanism yielded in increased staining of the cells, induction of osmotic processes and enhanced protection of the macrophageous cells from oxidative damage. Thus, rational synthetic variations of indole- and chromane-type compounds can facilitate the search for the substances showing enhanced interference in ageing-related pathologies. This is to say, there are numerous modifications susceptible to optimize either specific targeting, bioavailability or electron properties of these hopeful biological molecules [VEGA2/0031/12].

Effect of interleukin-1beta and dehydroepiandrosterone on the expression of lumican and fibromodulin in fibroblast-like synovial cells of the human temporomandibular joint.

Several epidemiological studies have reported that temporomandibular disorders (TMDs) are more prevalent in women than in men. It has recently been proposed that sex hormones such as estrogen, testosterone and dehydroepiandrosterone (DHEA) are involved with the pathogenesis of TMDs. Although studies have investigated the relationship between estrogen and testosterone and the restoration of TMDs, the relationship between DHEA and TMDs is unknown. The synovial tissue of the temporomandibular joint (TMJ) is made up of connective tissue with an extracellular matrix (ECM) composed of collagen and proteoglycan. One proteoglycan family, comprised of small leucine-rich repeat proteoglycans (SLRPs), was found to be involved in collagen fibril formation and interaction. In recent years, the participation of SLRPs such as lumican and fibromodulin in the internal derangement of TMJ has been suggested. Although these SLRPs may contribute to the restoration of the synovium, their effect is still unclear. The purpose of this study was to investigate the effect of DHEA, a sex hormone, on the expression of lumican and fibromodulin in human temporomandibular specimens and in cultured human TMJ fibroblast-like synovial cells in the presence or absence of the pro-inflammatory cytokine interleukin-1beta (IL-1beta). In the in vivo study, both normal and osteoarthritic (OA) human temporomandibular synovial tissues were immunohistochemically examined. In the in vitro study, five fibroblast-like synoviocyte (FLS) cell lines were established from human TMJ synovial tissue of patients with osteoarthritis. The subcultured cells were then incubated for 3, 6, 12 or 24 h with/without IL-1beta (1 ng/mL) in the presence or absence of DHEA (10 μM). The gene expression of lumican and fibromodulin was examined using the real-time polymerase chain reaction (PCR) and their protein expression was examined using immunofluorescent staining. We demonstrated that the expression of lumican differs from that of fibromodulin in synovial tissue and furthermore, that IL-1beta induced a significant increase in lumican mRNA and immunofluorescent staining in FLS compared to cells without IL-1beta. DHEA plus IL-1beta induced a significant increase in fibromodulin, but not in lumican mRNA, compared to DHEA alone, IL-1beta alone and in the absence of DHEA and IL-1beta. In immunofluorescent staining, weaker fibromodulin staining of FLS cells was observed in cells cultured in the absence of both DHEA and IL-1beta compared to fibromodulin staining of cells cultured with DHEA alone, with DHEA plus IL-1beta, or with IL-1beta alone. These results indicate that DHEA may have a protective effect on synovial tissue in TMJ by enhancing fibromodulin formation after IL-1beta induced inflammation. DHEA enhancement of fibromodulin expression may also exert a protective effect against the hyperplasia of fibrous tissue that TGF-beta1 induces. In addition lumican and fibromodulin are differentially expressed under different cell stimulation conditions and lumican and fibromodulin may promote regeneration of the TMJ after degeneration and deformation induced by IL-1beta.

Human iPS cell-derived fibroblast-like cells as feeder layers for iPS cell derivation and expansion

Mouse embryonic fibroblasts (MEFs) are commonly used as feeder cells for the generation of human induced pluripotent stem cells (hiPSCs). However, medical applications of cell derivatives of hiPSCs generated with a MEF feeder system run the risk of having xeno-factor contamination due to long-term cell culturing under an animal factor-containing environment. We developed a new method for the derivation of human fibroblast-like cells (FLCs) from a previously established hiPSC line in an FLC differentiation medium. The method was based on direct differentiation of hiPSCs seeded on Matrigel followed by expansion of differentiating cells on gelatin. Using inactivated FLCs as feeder layers, primary human foreskin fibroblasts were successfully reprogrammed into a state of pluripotency by Oct4, Sox2 Klf4, and c-Myc (OSKM) transcription factor genes, with a reprogramming efficiency under an optimized condition superior to that obtained on MEF feeder layers. Furthermore, the FLCs were more effective in supporting the growth of human pluripotent stem cells. The pluripotency and differentiation capability of the cells cultured on FLC feeder layers were well retained. Our results suggest that FLCs are a safe alternative to MEFs for hiPSC generation and expansion, especially in the clinical settings wherein hiPSC derivatives will be used for medical treatment.

Effects of JNK1/2 on the inflammation cytokine TNF-α-enhanced production of MMP-3 in human dental pulp fibroblast-like cells.

To investigate the effects of the c-Jun N-terminal kinase (JNK1/2) on the inflammation cytokine tumour necrosis factor-alpha (TNF-α)-enhanced production of matrix metalloproteinase-3 (MMP-3) in human dental pulp fibroblast-like cells (HPFs). HPFs were grown from pulp explants from healthy donors. Primary cultures were established by culturing the cells for 20 to 30days. The experiments with HPFs were performed between passages 3 and 10. The HPFs were incubated in serum-free medium containing TNF-α for 24h. The medium in each well was prepared in SDS sample buffer and was analysed for MMP-3 by Western blotting. JNK inhibitor SP601245 markedly inhibited the production of MMP-3 in TNF-α-stimulated human dental pulp fibroblasts. MMP-3 production was enhanced by TNF-α in HPFs; silencing JNK1 and JNK2 expression inhibited this activation. cAMP response element-binding protein (CREB) was activated by TNF-α in HPFs; silencing JNK1 and JNK2 expression inhibited this activation. The activation of CREB via JNK pathways in the presence of TNF-α occurred with enhancement of MMP-3 production in dental pulp fibroblasts.

Helium generated cold plasma finely regulates activation of human fibroblast-like primary cells.

Non-thermal atmospheric pressure plasmas are being developed for a wide range of health care applications, including wound healing. However in order to exploit the potential of plasma for clinical applications, the understanding of the mechanisms involved in plasma-induced activation of fibroblasts, the cells active in the healing process, is mandatory. In this study, the role of helium generated plasma in the tissue repairing process was investigated in cultured human fibroblast-like primary cells, and specifically in hepatic stellate cells and intestinal subepithelial myofibroblasts. Five minutes after treatment, plasma induced formation of reactive oxygen species (ROS) in cultured cells, as assessed by flow cytometric analysis of fluorescence-activated 2′,7′-dichlorofluorescein diacetate probe. Plasma-induced intracellular ROS were characterized by lower concentrations and shorter half-lives with respect to hydrogen peroxide-induced ROS. Moreover ROS generated by plasma treatment increased the expression of peroxisome proliferator activated receptor (PPAR)-γ, nuclear receptor that modulates the inflammatory responses. Plasma exposure promoted wound healing in an in vitro model and induced fibroblast migration and proliferation, as demonstrated, respectively, by trans-well assay and partitioning between daughter cells of carboxyfluorescein diacetate succinimidyl ester fluorescent dye. Plasma-induced fibroblast migration and proliferation were found to be ROS-dependent as cellular incubation with antioxidant agents (e.g. N-acetyl L-cysteine) cancelled the biological effects. This study provides evidence that helium generated plasma promotes proliferation and migration in liver and intestinal fibroblast-like primary cells mainly by increasing intracellular ROS levels. Since plasma-evoked ROS are time-restricted and elicit the PPAR-γ anti-inflammatory molecular pathway, this strategy ensures precise regulation of human fibroblast activation and can be considered a valid therapeutic approach for liver and gut lesions.

Hempseed oil induces reactive oxygen species- and C/EBP homologous protein-mediated apoptosis in MH7A human rheumatoid arthritis fibroblast-like synovial cells

Ethnopharmacological relevanceThe medicinal efficacy of hempseed (Cannabis sativa L.), which is rich in polyunsaturated fatty acids, in atopic dermatitis, inflammation, and rheumatoid arthritis (RA) has been suggested for centuries. Hempseed has been used as a treatment for these diseases in Korean and Chinese folk medicine. The aim of the study is to investigate the effects of hempseed oil (HO) on MH7A human RA fibroblast-like synovial cells. Materials and methodsMH7A cells were used to study the anti-rheumatoid effects of hempseed (Cannabis sativa L., cv. Cheungsam/Cannabaceae) oil by investigating cell viability, apoptosis, lipid accumulation, oxidative stress, and endoplasmic reticulum (ER) stress-induced apoptosis. ResultsHO treatment reduced the survival rate of MH7A cells and promoted apoptotic cell death in a time- and dose-dependent manner. Both lipid accumulation and the level of intracellular reactive oxygen species (ROS) increased in HO-treated MH7A cells. Co-treatment with the antioxidant Tiron effectively abrogated the cytotoxic effects of HO; the ROS level was reduced, cell viability was recovered, and apoptotic cell death was significantly diminished. Moreover, HO-treated cells exhibited increased expression of the major ER stress markers, glucose-regulated protein 78 and C/EBP homologous protein (CHOP). The siRNA-mediated knockdown of CHOP prevented HO-induced apoptosis. ConclusionsOur results suggest that HO treatment induced lipid accumulation, ROS production, CHOP expression, and apoptosis in MH7A cells, and that CHOP functions as an anti-rheumatoid factor downstream of HO in MH7A cells.

Human Fibroblast–Like Cultures in the Presence of Platelet-Rich Plasma as a Single Growth Factor Source

The purpose of this study was to compare the proliferation, morphology, and antigenic expression of human fibroblast-like cells between primary cultures treated with platelet-rich plasma (PRP) or fetal bovine serum (FBS) as the growth factor source. Cells from human gingival tissue samples obtained from healthy volunteers during oral surgery were studied. Isolated cells were cultured in media supplemented with 10% PRP or FBS. Platelet-rich plasma was prepared from the venous blood of each patient. The authors studied short- and long-term cell cultures in the presence of PRP or FBS as the sole growth factor source in order to determine (a) cell growth rate, by MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay; (b) cell morphology, by electronic microscopy; and (c) antigenic expression, by flow cytometry and confocal microscopy. In short-term cultures, the cell growth rate was higher with PRP versus FBS treatment. No differences in morphology or expression of vimentin, fibronectin, or α-actin antigens were observed between PRP and FBS cultures. In long-term cultures, PRP and FBS did not significantly differ in cell growth rate but differed in morphology and in the expression of vimentin, fibronectin, and α-actin. The PRP enhances cell proliferation over the short term and induces cell differentiation of fibroblast-like cells to myofibroblast-like cells over the long term, suggesting that fibroblast differentiation to myofibroblasts may underlie the action mechanism of PRP in soft tissue regeneration.

Ex Vivo Expansion of Functional Human UCB-HSCs/HPCs by Coculture with AFT024-hkirreCells

Kiaa1867 (human Kirre, hKirre) has a critical role in brain development and/or maintenance of the glomerular slit diaphragm in kidneys. Murine homolog of this gene, mKirre expressed in OP9 and AFT024 cells could support hematopoietic stem cells/hematopoietic progenitor cells (HSC/HPC) expansion in vitro. HKirre is also expressed in human FBMOB-hTERT cell line and fetal liver fibroblast-like cells but its function has remained unclear. In this paper, we cloned a hKirre gene from human fetal liver fibroblast-like cells and established a stably overexpressing hKirre-AFT024 cell line. Resultant cells could promote self-renewal and ex vivo expansion of HSCs/HPCs significantly higher than AFT024-control cells transformed with mock plasmid. The Expanded human umbilical cord blood (hUCB) CD34+ cells retained the capacity of multipotent differentiation as long as 8 weeks and successfully repopulated the bone marrow of sublethally irradiated NOD/SCID mice, which demonstrated the expansion of long-term primitive transplantable HSCs/HPCs. Importantly, hkirre could upregulate the expressions of Wnt-5A, BMP4, and SDF-1 and downregulate TGF-β with other hematopoietic growth factors. By SDS-PAGE and Western Blot analysis, a ~89 kDa protein in total lysate of AFT024-hKirre was identified. Supernatants from AFT024-hkirre could also support CD34+CD38− cells expansion. These results demonstrated that the AFT024-hKirre cells have the ability to efficiently expand HSCs/HPCs.

3 Effects of Rac1 on the production of MMP-3 in human dental pulp fibroblast-like cells

3 Effects of Rac1 on the production of MMP-3 in human dental pulp fibroblast-like cells

Er:ヒト歯髄由来線維芽細胞におけるMMP‐3産生に及ぼすRac1の影響

Er:ヒト歯髄由来線維芽細胞におけるMMP‐3産生に及ぼすRac1の影響

Extracellular Matrix Remodeling by Bone Marrow Fibroblast-like Cells Correlates with Disease Progression in Multiple Myeloma

The pathogenesis of multiple myeloma (MM) is regarded as a multistep process, in which an asymptomatic stage of monoclonal gammopathy of undetermined significance (MGUS) precedes virtually all cases of MM. Molecular events characteristic for the transition from MGUS to MM are still poorly defined. We hypothesized that fibroblast-like cells in the tumor microenvironment are critically involved in the pathogenesis of MM. Therefore, we performed a comparative proteome profiling study, analyzing primary human fibroblast-like cells isolated from the bone marrow of MM, of MGUS, as well as of non-neoplastic control patients. Thereby, a group of extracellular matrix (ECM) proteins, ECM receptors, and ECM-modulating enzymes turned out to be progressively up-regulated in MGUS and MM. These proteins include laminin α4, lysyl-hydroxylase 2, prolyl 4-hydroxylase 1, nidogen-2, integrin α5β5, c-type mannose receptor 2, PAI-1, basigin, and MMP-2, in addition to PDGF-receptor β and the growth factor periostin, which are likewise involved in ECM activities. Our results indicate that ECM remodeling by fibroblast-like cells may take place already at the level of MGUS and may become even more pronounced in MM. The identified proteins which indicate the stepwise progression from MGUS to MM may offer new tools for therapeutic strategies.

ヒト歯髄由来線維芽細胞におけるIL-1β刺激によるMMP-3 産生に及ぼすJNK の影響

ヒト歯髄由来線維芽細胞におけるIL-1β刺激によるMMP-3 産生に及ぼすJNK の影響