Mesenchymal stem cells isolated from peripheral blood and umbilical cord Wharton's jelly.

INTRODUCTION: Mesenchymal stem cells (MSCs) are a promising tool for regenerative medicine, but due to the heterogeneity of their populations, different sources and isolation techniques, the characteristics defining MSCs are inconsistent. OBJECTIVE: The aim of this study was to compare the characteristics of MSCs derived from two different human tissues: peripheral blood (PB-MSCs) and umbilical cord Wharton's Jelly (UC-MSCs). METHODS: The PB-MSC and UC-MSC were isolated by adherence to plastic after gradient-density separation or an explant culture method, respectively, and compared regarding their morphology, clonogenic efficiency, proliferating rates, immunophenotype and differentiation potential. RESULTS: MSCs derived from both sources exhibit similar morphology, proliferation capacity and multilineage (osteogenic, chondrogenic, adipogenic and myogenic) differentiation potential. Differences were observed in the clonogenic capacity and the immunophenotype, since UC-MSCs showed higher CFU-F (colony-forming units-fibroblastic) cloning efficiency, as well as higher embryonic markers (Na-nog, Sox2, SSEA4) expression. When additional surface antigens were analyzed by flow cytometry (CD44, CD90, CD105, CD33, CD34, CD45, CD11b, CD235a) or immunofluorescent labeling (vimentin, STRO-1 and alpha-smooth muscle actin), most appeared to have similar epitope profiles irrespective of MSC source. CONCLUSION: The results obtained demonstrated that both MSCs represent good alternative sources of adult MSCs that could be used in cell therapy applications.

Skip Regulates TGF- ß 1-Induced Extracellular Matrix Degrading Proteases Expression in Human PC-3 Prostate Cancer Cells.

Purpose. To determine whether Ski-interacting protein (SKIP) regulates TGF- ß 1-stimulated expression of urokinase-type plasminogen activator (uPA), matrix metalloproteinase-9 (MMP-9), and uPA Inhibitor (PAI-1) in the androgen-independent human prostate cancer cell model. Materials and Methods. PC-3 prostate cancer cell line was used. The role of SKIP was evaluated using synthetic small interference RNA (siRNA) compounds. The expression of uPA, MMP-9, and PAI-1 was evaluated by zymography assays, RT-PCR, and promoter transactivation analysis. Results. In PC-3 cells TGF- ß 1 treatment stimulated uPA, PAI-1, and MMP-9 expressions. The knockdown of SKIP in PC-3 cells enhanced the basal level of uPA, and TGF- ß 1 treatment inhibited uPA production. Both PAI-1 and MMP-9 production levels were increased in response to TGF- ß 1. The ectopic expression of SKIP inhibited both TGF- ß 1-induced uPA and MMP-9 promoter transactivation, while PAI-1 promoter response to the factor was unaffected. Conclusions. SKIP regulates the expression of uPA, PAI-1, and MMP-9 stimulated by TGF- ß 1 in PC-3 cells. Thus, SKIP is implicated in the regulation of extracellular matrix degradation and can therefore be suggested as a novel therapeutic target in prostate cancer treatment.

SMAD3 is essential for transforming growth factor-ß1-induced urokinase type plasminogen activator expression and migration in transformed keratinocytes.

Transforming growth factor-ß1 (TGF-ß1) stimulates the extracellular matrix degrading proteases expression and cell migration in order to enhance cancer cells malignancy. In the present study, we analysed the role of TGF-ß1-induced Smad3 activation in the urokinase type plasminogen activator (uPA) production, as well as in cell migration and E-cadherin downregulation in transformed PDV keratinocyte cell line. TGF-ß1 signalling was interfered by the chemical inhibitor of the TGF-ß1-receptor 1 (ALK5), SB505124, and the specific Smad3 inhibitor, SiS3. Our results showed that TGF-ß1 stimulates uPA expression directly through ALK5 activation. The inhibition of Smad3 strongly reduced the capacity of TGF-ß1 to stimulate uPA expression, in parallel decreasing the uPA inhibitor plasminogen activator inhibitor type 1 (PAI-1) expression. In addition, the transient expression of dominant negative Smad3 mutant inhibited the TGF-ß1-induced uPA promoter transactivation. Moreover, Smad3-/- mouse embryonic fibroblasts were refractory to the induction of uPA by TGF-ß1. The inhibition of both ALK5 and Smad3 dramatically blocked the TGF-ß1-stimulated E-cadherin downregulation, F-actin reorganisation and migration of PDV cells. Taken together, our results suggest that the TGF-ß1-induced activation of Smad3 is the critical step for the uPA upregulation and E-cadherin downregulation, which are the key events preceding the induction of cell migration by TGF-ß1 in transformed cells.

IL-17 and FGF signaling involved in mouse mesenchymal stem cell proliferation.

The mouse is a suitable experimental model to study the biology of mesenchymal stem cells (MSCs), as well as to be used in biocompatibility studies and tissue engineering models. However, the isolation and purification of murine MSCs is far more challenging than their counterparts from other species. In this study, we isolated, expanded and characterized mouse MSCs from bone marrow (BM-MSCs). Additionally, we analyzed the effects of two regulatory molecules, interleukin 17 (IL-17) and basic fibroblast growth factor (bFGF), on BM-MSCs growth and elucidated the signaling pathways involved. The results revealed that IL-17 increased the frequency of colony-forming units fibroblast (CFU-F) as well as the BM-MSCs proliferation in a dose-dependent manner, while bFGF supplementation had no significant effect on CFU-F frequency but induced an increase in cell proliferation. Their combined usage did not produce additive effects on BM-MSCs proliferation and even induced reduction in the number of CFU-F. Also, the involvement of both p38 and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases (MAPKs) signaling in proliferative activity of IL-17 and bFGF on murine BM-MSCs and, moreover, the increased co-activation of a common signaling molecule, p38 MAPK, were demonstrated. Together, the data presented highlighted the role of IL-17 and bFGF in murine BM-MSCs proliferation and pointed to the complexity and specificity of the signaling networks leading to MSCs proliferation in response to different regulatory molecules.

SKIP is required for TGF-ß1-induced epithelial mesenchymal transition and migration in transformed keratinocytes.

Transforming growth factor-ß1 (TGF-ß1) potently induces the epithelial-mesenchymal transition (EMT) during tumoral progression. Although Sky-interacting protein (SKIP) regulates TGF-ß1-induced Smad activation, its role in the induction of cell malignance remains uncertain. We found that TGF-ß1 increases SKIP expression in PDV cells. In cells stably transfected with SKIP antisense, AS-S, Smad3 activation decreased, along with an inhibition of TGF-ß1-induced EMT, and the cells were sensitized to the TGF-ß1-dependent inhibition of proliferation. Also, AS-S cells showed a weaker migration and invasion response. Moreover, TGF-ß1-induced urokinase-type plasminogen activator expression was inhibited, concomitantly with a TGF-ß1-independent increment of the plasminogen-activator inhibitor-1 expression. Thus, these results suggest that SKIP is required for EMT and invasiveness induced by TGF-ß1 in transformed cells.

New ruthenium(II) complexes with N-alkylphenothiazines: synthesis, structure, in vivo activity as free radical scavengers and in vitro cytotoxicity.

Three new complexes of the general formula L[RuCl(3)(DMSO)(3)] (1-3), where L = chlorpromazine hydrochloride, trifluoroperazine dihydrochloride or thioridazine hydrochloride, were prepared and characterized by elemental analysis and spectroscopic methods (FT-IR, UV-Vis, (1)H NMR and (13)C NMR). In addition, the crystal structure of the complex 2 containing trifluoroperazine dihydrochloride was solved by single crystal X-ray diffraction. The complex crystallizes in the monoclinic system, space group P2(1)/n, with a = 10.4935(7) A, b = 18.6836(12) A, c = 19.9250(13) A, beta = 98.448(2) degrees, V = 3864.0(4) A(3). The structure was refined to the agreement factors of R = 4.79%, R(w) = 11.23%. The effect of three different doses (0.4, 4.5 and 90.4 microM/kg bw) of complex 2 on superoxide dismutase (SOD) and catalase (CAT) activity was investigated under physiological conditions. Influence on nitrite production (NO(2)(-)) and the level of erythrocytes malondialdehyde (MDA) in rats blood was also evaluated. Complex 2 did not affect the CAT enzyme activity in vivo and did not cause the hydroxyl radicals production. In the 0.4 and 4.5 microM/kg bw doses it showed almost the same or lower SOD activity and nitrite levels, while the dose of 90.4 microM/kg bw significantly increased these parameters. Finally, the cytotoxicity of complexes were assayed in four human carcinoma cell lines MCF-7, MDA-MB-453 (breast carcinoma), SW-480 (colon adenocarcinoma) and IM9 (myeloma multiple cells). Antiproliferative activity in vitro with low IC(50) during 48 h of treatment was observed.

Rac1 modulates TGF-beta1-mediated epithelial cell plasticity and MMP9 production in transformed keratinocytes.

Transforming growth factor-beta1 (TGF-beta1) activates Rac1 GTPase in mouse transformed keratinocytes. Expression of a constitutively active Q61LRac1 mutant induced an epithelial to mesenchymal transition (EMT) linked to stimulation of cell migration and invasion. On the contrary, expression of a dominant-negative N17TRac1 abolished TGF-beta1-induced cell scattering, migration and invasion. Moreover, Q61LRac1 enhanced metalloproteinase-9 (MMP9) production to levels comparable to those induced by TGF-beta1, while N17TRac1 was inhibitory. TGF-beta1-mediated EMT involves the expression of the E-cadherin repressor Snail1, regulated by the Rac1 and mitogen-activated protein kinase (MAPK) pathways. Furthermore, MMP9 production was MAPK-dependent, as the MEK inhibitor PD98059 decreased TGF-beta1-induced MMP9 expression and secretion in Q61LRac1 expressing cells. We propose that regulation of TGF-beta1-mediated plasticity of transformed keratinocytes requires the cooperation between the Rac1 and MAPK signalling pathways.

Spred2 inhibits TGF-beta1-induced urokinase type plasminogen activator expression, cell motility and epithelial mesenchymal transition.

TGF-beta1 is a potent inductor of malignance in cancer cells. TGF-beta1 stimulates the expression of extracellular matrix degrading proteases, cell migration and it is also involved in the epithelial-mesenchymal transition (EMT). In the present work, we analyzed the role of Spred2 in the urokinase-type plasminogen activator (uPA) stimulation, EMT and cell migration by TGF-beta1. We found that both the expression of mRNA and the protein level of Spred2 were lower in transformed keratinocytes PDV compared with immortalized keratinocytes MCA-3D. The transient ectopic expression of Spred2 in PDV cells inhibited the TGF-beta1-transactivated SRE-Luc reporter which is related with the ERK1,2 signal. The stable ectopic expression of Spred2 in PDV cells (SP cells) led to the loss of ERK 1,2 activation by TGF-beta1, although Smad2 activation was not affected, and the knockdown of Spred2 enhanced the activation of ERK1,2 signal by TGF-beta1. The increment of uPA expression induced by TGF-beta1 was suppressed in SP cells. In contrast, the stimulus on PAI-1 expression was not affected and comparable to parental PDV cells. SP cells under TGF-beta1 treatment were unable to display the EMT, since the overexpression of Spred2 abolished the TGF-beta1-induced disruption of the E-cadherin cell to cell interactions, reorganization of the actin cytoskeleton and upregulation of the mesenchymal marker vimentin. Finally, SP cells could not respond to the TGF-beta1 stimulus on cell migration. Taken together, the data in the present study suggests that Spred2 is a regulator of TGF-beta1-induced malignance in transformed keratinocytes.