Bone morphogenetic proteins (BMPs) belong to TGF-β superfamily and are a group of important cytokines involved in cell differentiation, proliferation and embryonic development. Multiple BMPs play important roles in several functions of vertebrates. Signaling pathway of BMPs is known to be mediated by Smad proteins, which include 8 members while Smad1, Smad5 and Smad8 are involved in BMPs signal transduction while Smad2 and Smad3 are mediated TGF-β signal transduction. Although several BMPs such as BMP4 and BMP9 have been documented in the liver, BMP13 has not been examined in the liver. BMP13 also known as growth differentiation factor (GDF)-6 or cartilage-derived morphogenetic protein (CDMP)-2 is one of the BMPs family members. Function of BMP13 has been investigated in bone and tendon repair. It can stimulate tendon-like cell proliferation. However, our recent findings revealed that there was expression of BMP13 in the liver and its expression was modulated during metabolic disorders. The current article is to understand biological function of BMP13 especially in the liver.
Bone Morphogenetic Proteins ; metabolism ; physiology ; Growth Differentiation Factor 6 ; metabolism ; physiology ; Humans ; Liver ; metabolism ; Liver Diseases ; metabolism ; Smad Proteins ; metabolism

BACKGROUND/AIMS: Therapies involving bone-marrow-derived mesenchymal stem cells (BM-MSCs) have considerable potential in the management of hepatic disease. BM-MSCs have been investigated in regenerative medicine due to their ability to secrete various growth factors and cytokines that regress hepatic fibrosis and enhance hepatocyte functionality. The aim of this study was to determine the antifibrosis effect of BM-MSCs on activated hepatic stellate cells (HSCs) and the mechanism underlying how BM-MSCs modulate the function of activated HSCs. METHODS: We used HSCs in both direct and indirect co-culture systems with BM-MSCs to evaluate the antifibrosis effect of BM-MSCs. The cell viability and apoptosis were evaluated by a direct co-culture system of activated HSCs with BM-MSCs. The activations of both HSCs alone and HSCs with BM-MSCs in the direct co-culture system were observed by immunocytochemistry for alpha-smooth muscle actin (alpha-SMA). The levels of growth factors and cytokines were evaluated by an indirect co-culture system of activated HSCs with BM-MSCs. RESULTS: The BM-MSCs in the direct co-culture system significantly decreased the production of alpha-SMA and the viability of activated HSCs, whereas they induced the apoptosis of activated HSCs. The BM-MSCs in the indirect co-culture system decreased the production of transforming growth factor-beta1 and interleukin (IL)-6, whereas they increased the production of hepatocyte growth factor and IL-10. These results confirmed that the juxtacrine and paracrine effects of BM-MSCs can inhibit the proliferative, fibrogenic function of activated HSCs and have the potential to reverse the fibrotic process by inhibiting the production of alpha-SMA and inducing the apoptosis of HSCs. CONCLUSIONS: These results have demonstrated that BM-MSCs may exert an antifibrosis effect by modulating the function of activated HSCs.
Apoptosis ; Bone Marrow Cells/*cytology ; Cell Differentiation ; Coculture Techniques ; Hepatic Stellate Cells/*cytology/metabolism ; Hepatocyte Growth Factor/metabolism ; Humans ; Immunophenotyping ; Interleukin-10/metabolism ; Interleukin-6/metabolism ; Liver Cirrhosis ; Mesenchymal Stromal Cells/*cytology/metabolism ; Transforming Growth Factor beta1/metabolism

BACKGROUNDRecent studies showed that bone marrow-derived mesenchymal stem cells (BMSCs) had risk of ectopic bone formation. In this study, we aimed to investigate the effect of growth and differentiation factor 6 (GDF-6) on the tenogenic differentiation of BMSCs in vitro, and then combined with small intestine submucous (SIS) to promote tendon regeneration in vivo.

METHODSThe BMSCs were isolated from the green fluorescent protein (GFP) rats, and were characterized by multi-differentiation assays following our previous study protocol. BMSCs cultured with different concentrations of GDF-6, without growth factors served as control. After 2 weeks, mRNA expression and protein expression of tendon specific markers were examined by qRT-PCR and Western blotting to define an optimal concentration of GDF-6. Mann-Whitney U-test was used to compare the difference in relative mRNA expression among all groups; P ≤ 0.05 was regarded as statistically significant. The GDF-6 treated BMSCs combined with SIS were implanted in nude mice and SD rat acute patellar tendon injury model, the BMSCs combined with SIS served as control. After 12 and 4 weeks in nude mice and tendon injury model, the samples were collected for histology.

RESULTSAfter the BMSCs were treated with different concentration of GDF-6 for 2 weeks, the fold changes of the specific markers (Tenomodulin and Scleraxis) mRNA expression were significantly higher in GDF-6 (20 ng/ml) group (P ≤ 0.05), which was also confirmed by Western blotting result. The BMSCs became parallel in orientation after GDF-6 (20 ng/ml) treatment, but the BMSCs in control group were randomly oriented. The GDF-6 (20 ng/ml) treated BMSCs were combined with SIS, and were implanted in nude mice for 12 weeks, the histology showed neo-tendon formation. In the SD rat patellar tendon window injury model, the histology also indicated the GDF-6 (20 ng/ml) treated BMSCs combined with SIS could promote tendon regeneration.

CONCLUSIONSGDF-6 has tenogenic effect on the tenogenic differentiation of BMSCs, and GDF-6 (20 ng/ml) has better tenogenic effect compared to other concentrations. The GDF-6 (20 ng/ml) treated BMSCs combined with SIS can form neo-tendons and promote tendon regeneration.

Animals ; Cell Differentiation ; drug effects ; Growth Differentiation Factor 6 ; pharmacology ; Male ; Membrane Proteins ; genetics ; Mesenchymal Stromal Cells ; cytology ; drug effects ; Mice ; Mice, Nude ; Rats ; Rats, Sprague-Dawley ; Regeneration ; drug effects ; Tendons ; drug effects ; physiology

BACKGROUND: A living skin equivalent(LSE) is useful as a skin replacement and as a model system for basic studies such as skin physiology and pathology. This system attempts to reproduce in vitro the cell-cell and cell-matrix interactions responsible for cell differentiation. Recently, there have been many studies that cytokines(IL-1, IL-6, IL-8, TGF-beta, TNF-alpha) play an important role in proliferating skin disease, especially psoriasis. Hesides, many cytokines can influence the proliferation and differentiation of normal human keratinocytes. OBJECTIVE AND METHODS: In the present study, the author investigated the proliferation and differentiation of cytokines in LSE as compared with control skin using histopathological and immunohistochemical staining methods. The author also investigated the proper concentration of cytokines on the proliferation of cultured human keratinocytes. RESULTS: Morphological analysis of LSE showed reorganization of epidermal layers with the appearance of a distinct basal laer and of a hyperkeratotic horny layer. We demonstrated that the fine granules resembling keratchyaline granules were observed in the control and cytokine treated groups(IL-l alpha, IL-2, IL-6, TNF-alpha). We observed significant epidermal proliferation in the PBMC (25%), IL 6(10ng), TGF-beta(20ng) treated groups than in the control group(p<0.01). Immunohistochemical analysis demonstrated that the terminal differentiation marker involucrin was expressed at the level of the prickle cell layer in the cytokine treated groups. Also the cellular proliferation marker PCNA was expressed at the level of the basal layer in the PBMC(25%), IL-2(100ng) and IL-6(10ng) treated groups. CONCLUSION: The results indicate that cytokine IL-6 and TGF-beta can induce a proliferation and differentiation of the epidermis in this in vitro model.
Cell Differentiation ; Cell Proliferation ; Cytokines* ; Epidermis ; Humans* ; Interleukin-2 ; Interleukin-6 ; Interleukin-8 ; Keratinocytes ; Lymphocytes* ; Pathology ; Proliferating Cell Nuclear Antigen ; Psoriasis ; Skin Diseases ; Skin Physiological Phenomena ; Skin* ; Transforming Growth Factor beta

OBJECTIVETo investigate the modulatory effect of the MSC derived from low attaching culture systems (suspending MSC) on T lymphocytes and the related mechanisms.

METHODSThe suspending MSC were generated from mouse compact bones by using low attaching plates and adherent cell culture flasks, respectively. The morphology of suspending MSC was observed under the inverted microscope and the cells were induced to differentiate into osteoblasts and adipocytes. Further, the surface antigen profile of MSC was analyzed with flow cytometry. In addition, the culture medium (CM) of suspending MSC and adherent MSC was collected and added into the activated T cell cultures before detection of the proliferation by CFSE assay. Moreover, the modulaory effects of the CM on the T cell-derived cytokines were detected by quantitative PCR. Also, the mRNA expression of cytokines of MSC was detected.

RESULTSThe suspending MSC grew in floating cell spheres and differentiated into osteoblasts and adipocytes in the induction medium. Furthermore, the suspending MSC shared the typical immuno-phenotype with their adherent counterparts. In addition, the results of CFSE assay demonstrated that suspending MSC derived CM suppressed ConA induced T cell proliferation. The results of quantitative PCR revealed that suspending MSC expressed transforming factor β1 and interleukin-6 at a higher level and suppressed the T cell expressing interferon γ and interleukine-17A.

CONCLUSIONThe suspending MSC exerted an unique modulatoy effect on T cells, which is quite different to adherent MSC.

Adipocytes ; cytology ; Animals ; Cell Adhesion ; Cell Culture Techniques ; Cell Differentiation ; Cell Proliferation ; Culture Media, Conditioned ; Flow Cytometry ; Immunophenotyping ; Interleukin-6 ; metabolism ; Lymphocyte Activation ; Mesenchymal Stromal Cells ; cytology ; Mice ; Osteoblasts ; cytology ; T-Lymphocytes ; cytology ; metabolism ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVETo explore an optional condition to induce mouse embryonic stem (ES) cells to differentiate into endothelial cells and to establish in vitro models of vasculogenesis and angiogenesis.

METHODSMouse ES cells were cultured in differentiation medium containing a cocktail of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), interleukin-6 (IL-6) and erythropoietin (EPO) in 1% methylcellulose to induce formation of embryoid bodies (EBs). At day 11, EBs were harvested and suspended in rat-tail collagen type I with the same cocktail of cytokines cultured for three additional days. The differentiation of ES cells into endothelial cells, processes of vasculogenesis and angiogenesis were examined using immunostaining of EBs slices and whole-mount immunocytochemistry of EBs with monoclonal antibodies (mAbs) against platelet endothelial cell adhesion molecule-1 (PECAM-1) and alpha-smooth muscle actin (SMA).

RESULTSUnder appropriate culture conditions; ES cells spontaneously differentiated and formed EBs containing vascular structures and tubular channels, which were positive for PECAM-1 co-differentiated with smooth muscle. When not treated with angiogenic growth factors, PECAM-1-positive cells could not organize into vascular structures of 11-day-old EBs. In the presence of angiogenic factors 11-day old EBs embedded into type I collagen, and rapidly developed an endothelial networks. Whole-mount immunocytochemistry of collagen gel with anti-PECAM-1 antibody showed the formation of primary vascular structures sprouting from EBs. Quantitative analysis revealed that 100 microg/ml thalidomide significantly reduced the number and length of EBs endothelial sprouting.

CONCLUSIONSMouse ES cells can differentiate into endothelial cells combined with smooth muscle differentiation during EBs formation and further develop endothelial outgrowths after EBs embedded into collagen, which respectively recapitulate vasculogenesis, angiogenesis, and arteriogenesis processes in vivo. The models provide a useful tool to investigate vasculogenesis, angiogenesis, and arteriogenesis mechanisms and evaluate the effects of angiogenic and angiostatic agents.

Animals ; Cell Culture Techniques ; Cell Differentiation ; Collagen ; pharmacology ; Culture Media ; Embryo, Mammalian ; cytology ; physiology ; Endothelial Cells ; cytology ; physiology ; Erythropoietin ; pharmacology ; Fibroblast Growth Factor 2 ; pharmacology ; Interleukin-6 ; pharmacology ; Mice ; Neovascularization, Physiologic ; physiology ; Stem Cells ; cytology ; physiology ; Vascular Endothelial Growth Factor A ; pharmacology

OBJECTIVE: To explore the effect of nerve growth factor (NGF) on the  differentiation of murine bone marrow-derived dendritic cells (DCs) in vitro.
 METHODS: The bone marrow cells of femur and tibia from healthy C57B -L/6 mice were isolated and divided into 4 groups: a phosphate buffered saline (PBS) group (PBS group), a NGF group, a granulocyte monocyte colony stimulating factor (GM-CSF) plus interleukin 4 (IL-4) group (GM-CSF+IL-4 group), and a GM-CSF plus IL-4 and NGF group (n=6 in each group). The positive rate of CD11c+ and the proportion of CD8a- were compared at the 7th day among the different groups by flow cytometry. The immature DCs were acquired by classic methods with GM-CSF and IL-4. The purified DCs were obtained by magnetic bead positive selection for CD11c+ cells. The immature DCs were divided into 4 groups: a PBS group, a NGF group, a LPS group, and a NGF+LPS group (n=6 in each group), which were incubated with PBS, NGF, LPS and NGF+LPS, respectively. Cytokine levels of IL-6, IL-10 and IL-12 were detected by ELISA after 24 hours..
 RESULTS: 1) the percentage of CD11c+ DCs in the NGF group were more than that in the PBS group, and lower than that in the the GM- CSF+IL-4 group (both P<0.05). There was no difference between the GM-CSF + IL-4 group and the NGF+GM-CSF+IL-4 group (P>0.05). CD8a- DCs were dominant in these four groups; 2) NGF could further up-regulate the LPS-induced cytokine secretion from DCs, such as IL-6, IL-10, and IL-12 (all P<0.05), but NGF alone had no such effect (all P<0.05).
 CONCLUSION NGF can promote the murine bone-marrow cells differentiation into CD11c+ DCs, with CD8a-subset; NGF could enhance LPS-induced cytokine secretion from DCs (IL-6, IL-10 and IL-12).
Animals ; Bone Marrow Cells ; cytology ; drug effects ; Cell Differentiation ; drug effects ; Cells, Cultured ; Dendritic Cells ; cytology ; drug effects ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Interleukin-10 ; analysis ; Interleukin-12 ; analysis ; Interleukin-4 ; pharmacology ; Interleukin-6 ; analysis ; Mice ; Mice, Inbred C57BL ; Nerve Growth Factor ; pharmacology

OBJECTIVE: To explore the mechanism by which estradiol modulates the immunophenotype of macrophages through the endoplasmic reticulum stress pathway. METHODS: Peritoneal macrophages isolated from C57 mice were cultured in the presence of 60 ng/mL interferon-γ (IFN-γ) followed by treatment with estradiol (1.0 nmol/L) alone, estradiol with estrogen receptor antagonist (Acolbifene, 4 nmol/L), estradiol with IRE1α inhibitor (4 μ 8 C), or estradiol with IRE1α agonist. After the treatments, the expression levels of MHC-Ⅱ, iNOS and endoplasmic reticulum stress marker proteins IRE1α, eIF2α and ATF6 in the macrophages were detected with Western blotting, and the mRNA levels of TGF-β, IL-6, IL-10 and TNF-α were detected with RT-PCR. RESULTS: Estrogen treatment of the macrophages significantly decreased the expressions of M1-related proteins MHC-Ⅱ (P=0.021) and iNOS (P < 0.001) and the mRNA expressions of TNF-α (P=0.003) and IL-6 (P=0.004), increased the mRNA expression of TGF-β (P=0.002) and IL-10 (P=0.008), and up-regulated the protein expressions of IRE1α (P < 0.001) and its downstream transcription factor XBP-1 (P < 0.001). Addition of the estrogen inhibitor obviously blocked the effect of estrogen. Compared with estrogen treatment alone, combined treatment of the macrophages with estrogen and the IRE1α inhibitor 4 μ 8 C significantly up-regulated the protein expressions of MHC-Ⅱ (P=0.002) and iNOS (P=0.003) and the mRNA expressions of TNF-α (P=0.003) and IL-6 (P=0.024), and obviously down-regulated the mRNA expression of TGF-β (P < 0.001) and IL-10 (P < 0.001); these changes were not observed in cells treated with estrogen and the IRE1α agonist. CONCLUSION Estrogen can inhibit the differentiation of murine macrophages into a pro-inflammatory phenotype by up-regulating the IRE1α-XBP-1 signaling axis, thereby producing an inhibitory effect on inflammatory response.
Animals ; Cell Differentiation/drug effects* ; Endoribonucleases/metabolism* ; Estradiol/pharmacology* ; Estrogens/metabolism* ; Interleukin-10 ; Interleukin-6/metabolism* ; Macrophages, Peritoneal/metabolism* ; Mice ; Phenotype ; Protein Serine-Threonine Kinases/metabolism* ; RNA, Messenger/metabolism* ; Signal Transduction/drug effects* ; Transforming Growth Factor beta/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Up-Regulation/drug effects* ; X-Box Binding Protein 1/metabolism*

The periodontal ligament-derived mesenchymal stem cell is regarded as a source of adult stem cells due to its multipotency. However, the proof of chondrogenic potential of the cells is scarce. Therefore, we investigated the chondrogenic differentiation capacity of periodontal ligament derived mesenchymal stem cells induced by transforming growth factor (TGF)-β3 and bone morphogenetic protein (BMP)-6. After isolation of periodontal ligament stem cells (PDLSCs) from human periodontal ligament, the cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 20% fetal bovine serum (FBS). A mechanical force initiated chondrogenic differentiation of the cells. For chondrogenic differentiation, 10 µg·L⁻¹ TGF-β3 or 100 µg∙L⁻¹ BMP-6 and the combination treating group for synergistic effect of the growth factors. We analyzed the PDLSCs by fluorescence-activated cell sorting and chondrogenesis were evaluated by glycosaminoglycans assay, histology, immunohistochemistry and genetic analysis. PDLSCs showed mesenchymal stem cell properties proved by FACS analysis. Glycosaminoglycans contents were increased 217% by TGF-β3 and 220% by BMP-6. The synergetic effect of TGF-β3 and BMP-6 were shown up to 281% compared to control. The combination treatment increased Sox9, aggrecan and collagen II expression compared with not only controls, but also TGF-β3 or BMP-6 single treatment dramatically. The histological analysis also indicated the chondrogenic differentiation of PDLSCs in our conditions. The results of the present study demonstrate the potential of the dental stem cell as a valuable cell source for chondrogenesis, which may be applicable for regeneration of cartilage and bone fracture in the field of cell therapy.
Adult Stem Cells ; physiology ; Aggrecans ; analysis ; Bone Morphogenetic Protein 6 ; pharmacology ; Cell Culture Techniques ; Cell Differentiation ; drug effects ; Cell Separation ; Chondrogenesis ; drug effects ; physiology ; Collagen Type II ; analysis ; Flow Cytometry ; Glycosaminoglycans ; analysis ; Humans ; Immunohistochemistry ; Mesenchymal Stromal Cells ; drug effects ; physiology ; Molar, Third ; cytology ; Periodontal Ligament ; cytology ; drug effects ; Reverse Transcriptase Polymerase Chain Reaction ; SOX9 Transcription Factor ; analysis ; Stress, Mechanical ; Tooth, Impacted ; pathology ; Transforming Growth Factor beta3 ; pharmacology