OBJECTIVETo analyze the differential proteomics in human umbilical cord mesenchymal stem cells (MSC) induced by chemical hypoxia-mimetic agent cobalt chloride (CoCl(2)) by two-dimensional gel electrophoresis (2-DE) and mass-spectrometry.

METHODS2-DE was performed to separate proteins from treated and untreated human umbilical cord MSC with CoCl(2). 2-DE images were analyzed by ImageMaster 2D Platinum software 6.0. The differential expressed proteins was identified by MALDI-TOF-MS. The differential proteins were classified based on their functions.

RESULTS2-DE reference patterns of CoCl(2) treated human umbilical cord MSC were established. A total of twenty-six differential proteins were identified, of them eleven proteins were up-regulated and fifteen down-regulated. Their biological functions involved in carbohydrate metabolism, protein metabolism and modification, lipid metabolism, coenzyme and prosthetic group metabolism, cell cycle, immunity and defense, cell structure and motility, signal transduction, protein targeting and localization, neuronal activities, muscle contraction, etc. Peroxiredoxin1 (Prdx) was down-regulated, whereas alpha-enolase (ENO1) and vesicle amine transport protein 1 homolog (VAT1) up-regulated.

CONCLUSIONThe effects of hypoxia on human umbilical cord MSC were participated by multiple proteins and involved in multiple functional pathways.

Cobalt ; pharmacology ; Humans ; Mesenchymal Stromal Cells ; drug effects ; metabolism ; Proteome ; analysis ; Proteomics ; Umbilical Cord ; cytology ; drug effects

This study was purposed to investigate the effects of interferon (IFN)-γ on expression of adhesion molecules in mesenchymal stromal cells derived from human umbilical cord tissue (UC-MSC). The UC-MSC were isolated from human umbilical cord by tissue culture. The expressions of specific markers on UC-MSC were detected by flow cytometry in the physiological condition. The adipogenic and osteogenic induction of UC-MSC was detected by alizarin and Oil red O staining. UC-MSC were exposed to IFN-γ (100, 1 000, 10 000 U/ml) for 24 h, the expressions of CD54, CD58, CD44, CD49d, CD62p, CD62L, CD102 and CD106 on cell surface were detected using flow cytometry. The results showed that in physiological condition, UC-MSC extremely low expressed CD102, CD106, CD62P, CD62L, while the expression of CD54 was relatively high (41.58 ± 0.83)%. When stimulated by IFN-γ, the expression of CD102, CD106, CD62P, CD62L increased slightly, but still low (< 5%), while CD54 and CD58 upregulated concentration-dependently up to (59.66 ± 1.36)% and (43.96 ± 0.62)% respectively. The expression of CD49d upregulated to (51.33 ± 0.74)% when UC-MSC exposed to IFN-γ 100 U/ml. CD44 increased to (73.22 ± 1.93)% when UC-MSC exposed to IFN-γ 1 000 U/ml. It is concluded that IFN-γ can elevate significantly the expression of CD54, CD49d, CD44 and CD58, but has no significant effect on CD102, CD106, CD62P and CD62L expression on the surface of UC-MSC.
Cell Adhesion Molecules ; metabolism ; Cells, Cultured ; Humans ; Interferon-gamma ; pharmacology ; Mesenchymal Stromal Cells ; cytology ; drug effects ; metabolism ; Umbilical Cord ; cytology

Amniotic Fluid ; drug effects ; metabolism ; Female ; Fetus ; drug effects ; metabolism ; Gene Expression Regulation, Developmental ; drug effects ; genetics ; Humans ; MicroRNAs ; genetics ; pharmacology ; Placenta ; metabolism ; Pregnancy ; RNA, Plant ; genetics ; pharmacology ; Umbilical Cord ; drug effects ; metabolism

This study was purposed to investigate the effects of transforming growth factor-β1 (TGF-β1) on proliferation and extracellular matrix (ECM) and gene expressions of human umbilical cord derived mesenchymal stem cells (hUC-MSC). The hUC-MSCs were isolated and cultured by transplantation technique from umbilical cords. Surface antigens of the fifth passage cultured hUC-MSCs were detected by flow cytometry. The effects of TGF-β1 0.1 - 20.0 ng/ml on hUC-MSC proliferation were determined by Cell Counting Kit-8. The mRNA expression of ECM components and the components involved in the mechanism of ECM production regulation were detected by qRT-PCR. Expressions of collagen I (Col-I), collagen IV (Col-IV), fibronectin (FN) and laminin (LN) were observed by immunocytochemistry. The gene expression profiles of hUC-MSC in response to TGF-β1 were examined by microarray. The results showed that no significant changes of the OD values were observed with the increasing doses of TGF-β1, while all OD values of 0.1 ng/ml group showed relative peaks at 24, 48 and 72 hour time points. The mRNA expression level of Col-I, MMP-2, MMP-9 and TIMP-2 increased to maximum in 0.5 ng/ml group, and the mRNA expression of Col-IV, FN, LN, Integrin, Tenascin-C, MMP-1 and TIMP-1 reached the peak level in 0.1 ng/ml group. Immunocytochemical analysis for Col-I and Col-IV revealed that their production increased slightly in 0.5 and 0.1 ng/ml groups, respectively, while immunostaining for FN showed diffuse cell membrane and cytoplasmic staining randomly distributed, but LN staining was absent in both groups. Microarray analysis showed that 9 genes closely related to cell movement and migration were up-regulated. Analysis by Pathway and Go indicated that differentially expressed genes were involved in transcription, translation, biosynthesis, metabolism, signal transduction, migration and adhering junction etc. It is concluded that TGF-β1 0.1 ng/ml promotes the proliferation of hUC-MSC. TGF-β1 of different concentrations upregulates the expression of different ECM components. TGF-β1 may play important roles in various aspects of hUC-MSC including transcription, translation, biosynthesis, metabolism, signal transduction, migration and adherens junction and so on.
Cell Proliferation ; drug effects ; Cells, Cultured ; Extracellular Matrix ; drug effects ; metabolism ; Gene Expression ; Humans ; Mesenchymal Stromal Cells ; drug effects ; metabolism ; Oligonucleotide Array Sequence Analysis ; RNA, Messenger ; genetics ; Transcriptome ; Transforming Growth Factor beta1 ; pharmacology ; Umbilical Cord ; cytology ; metabolism

The aim of this study was to investigate the effects of interferon (IFN)-γ on biological characteristics and immunomodulatory property of human umbilical cord-derived mesenchymal stem cells (hUC-MSC). hUC-MSC were treated with IFN-γ 10 ng/ml (IFN-γ group) or without IFN-γ (control group). The phenotype of hUC-MSC was detected by flow cytometry. The proliferation status was detected by CCK-8 method, and its differentiation ability was assessed by oil red O and von Kossa staining. The production of PGE-2 was measured by ELISA, and the mRNA expression levels of COX-2, IDO-1 and IDO-2 in hUC-MSC were detected by real-time quantitative PCR. Furthermore, the proliferation of human peripheral blood mononuclear cells (hPBMNC) was evaluated after co-culture with hUC-MSC, IFN-γ pretreatment or not. The results showed that after IFN-γ stimulation, the expression of SSEA-4 on hUC-MSC decreased significantly [(8.15 ± 2.94) vs (16.42 ± 8.5), P < 0.05], and the expression of CD54 increased [(96.64 ± 3.29) vs (84.12 ± 10.73), P = 0.051]. The immunomodulatory property of hUC-MSC on the proliferation of hPBMNC was enhanced (P < 0.05). All the above mentioned effects were IFN-γ concentration-dependent. When hUC-MSC were stimulated by IFN-γ for 24 h, the production of PGE-2 secreted by hUC-MSC decreased significantly (P < 0.01). The mRNA expression level of COX-2 also decreased though the difference did not reach to statistically significant level. Compared with control group, IDO-1 expression level in IFN-γ group increased significantly (P < 0.01), and the mRNA expression level of IDO-2 remained unchanged. It is concluded that IFN-γ can influence the phenotype of hUC-MSC and enhance the immunomodulatory property of hUC-MSC.
Cells, Cultured ; Cyclooxygenase 2 ; metabolism ; Flow Cytometry ; Humans ; Indoleamine-Pyrrole 2,3,-Dioxygenase ; metabolism ; Interferon-gamma ; pharmacology ; Mesenchymal Stromal Cells ; cytology ; drug effects ; immunology ; Umbilical Cord ; cytology

Coumarins comprise a group of natural phenolic compounds found in a variety of plant sources. In view of the established low toxicity, relative cheapness, presence in the diet and occurrence in various herbal remedies of coumarins, it appears prudent to evaluate their properties and applications further. The purpose of this study is to investigate cellular protective activity of coumarin compound, fraxin extracted from Weigela florida var. glabbra, under oxidative stress, to identify genes expressed differentially by fraxin and to compare antioxidative effect of fraxin with its structurally related chemicals. Of the coumarins, protective effects of fraxin against cytotoxicity induced by H2O2 were examined in human umbilical vein endothelial cells (HUVECs). Fraxin showed free radical scavenging effect at high concentration (0.5 mM) and cell protective effect against H2O2-mediated oxidative stress. Fraxin recovered viability of HUVECs damaged by H2O2- treatment and reduced the lipid peroxidation and the internal reactive oxygen species level elevated by H2O2 treatment. Differential display reverse transcription-PCR revealed that fraxin upregulated antiapoptotic genes (clusterin and apoptosis inhibitor 5) and tumor suppressor gene (ST13). Based on structural similarity comparing with fraxin, seven chemicals, fraxidin methyl ether (29.4% enhancement of viability), prenyletin (26.4%), methoxsalen (20.8 %), diffratic acid (19.9%), rutoside (19.1%), xanthyletin (18.4%), and kuhlmannin (18.2%), enhanced more potent cell viability in the order in comparison with fraxin, which showed only 9.3% enhancement of cell viability. These results suggest that fraxin and fraxin-related chemicals protect HUVECs from oxidative stress.
Catalase/metabolism ; Cell Survival/drug effects ; Cells, Cultured ; Coumarins/*chemistry/*pharmacology ; Endothelial Cells/drug effects/metabolism ; Humans ; Hydrogen Peroxide/pharmacology ; Lipid Peroxidation/drug effects ; Molecular Structure ; Oxidative Stress/*drug effects ; Reactive Oxygen Species/metabolism ; Research Support, Non-U.S. Gov't ; Structure-Activity Relationship ; Superoxide Dismutase/metabolism ; Umbilical Cord/drug effects/metabolism

OBJECTIVETo investigate the protection of silymarin against the human mesenchymal stem cell (MSC) apoptosis induced by serum deprivation and its underlying mechanism.

METHODSHuman umbilical cord MSCs were cultured in the absence of serum, and the silymain of different concentration (1-10 µg/ml) was added into the medium. MTT test was performed to observe the cell proliferation status. After being cultured for 72 hours, the cells were collected, and flow cytometry with Annexin-V-PI double-staining was used to detect the apoptotic cells from the control and silymarin-treated groups. Furthermore, the intracellular contents of BAX and BCL-2 were detected by Western blot for exploring the potential mechanism.

RESULTSThe silymarin promoted the proliferation of human UC-MSCs in a dose-dependent manner, reaching its maximal at a dose of 5 µg/ml. Moreover, silymarin could inhibit the serum deprivation-induced apoptosis of MSCs and, the inhibitory rate reached up to 30% when it was added at a concentration of 5 µg/ml. The content of intracellular BAX was obviously elevated after serum-deprivation treatment, and this increase could be blunted by the addition of silymarin. Meanwhile, the content of BCL-2 was not obviously changed.

CONCLUSIONThe silymarin can stimulate MSC growth and inhibit the apoptosis of MSCs probably by the mitochondria pathway.

Apoptosis ; drug effects ; Cell Proliferation ; Culture Media, Serum-Free ; Humans ; Mesenchymal Stromal Cells ; drug effects ; Mitochondria ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Silymarin ; pharmacology ; Umbilical Cord ; cytology ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo investigate effects of retinol on the expressions of epidermal growth factor (EGF), stem cell factor (SCF), colony-stimulating factor 1 (CSF1) and leukemia inhibitory factor (LIF) in cultured human umbilical-derived mesenchymal stem cells (UCMSCs).

METHODSHuman UCMSCs were isolated from human umbilical cord and identified for immunophenotypes. The cells were then cultured in DMEM/F12 media supplemented with 12% fetal bovine serum (FBS), 12% FBS+1 µmol/L retinol, 15% knockout serum replacement (KSR) and 15% KSR+ 1 µmol/L retinol. The expressions of the cytokines EGF, SCF, CSF1 and LIF in the cells were detected using RT-PCR and ELISA.

RESULTSThe isolated cells exhibited characteristic immunophenotypes of human UCMSCs and expressed EGF, CSF1 and SCF at both mRNA and protein levels but not LIF protein. Retinol (1 µmol/L) significantly promoted the expressions of SCF and CSF1 at both mRNA and protein levels but did not result in changes of EGF and LIF expressions in human UCMSCs.

CONCLUSIONRetinol at the concentration of 1 µmol/L can promote expression of SCF and CSF1 in human UCMSCs in vitro.

Cell Differentiation ; Cells, Cultured ; EGF Family of Proteins ; metabolism ; Humans ; Immunophenotyping ; Leukemia Inhibitory Factor ; metabolism ; Macrophage Colony-Stimulating Factor ; metabolism ; Mesenchymal Stromal Cells ; drug effects ; metabolism ; Stem Cell Factor ; metabolism ; Umbilical Cord ; cytology ; Vitamin A ; pharmacology

To study the effect of sphingosine-1-phosphate (S1P) on the cardiomyogenic differentiation of human umbilical cord mesenchymal stem cells (UC-MSCs) and human adipose-derived mesenchymal stem cells (AD-MSCs), we seeded the cells in the culture plates and used cardiomyocyte culture medium (CMCM) combining with different concentration of S1P to induce UC-MSCs and AD-MSCs in vitro for 7, 14 and 28 days. Cardiomyogenic differentiations were identified through immunofluorescence staining, and the results were observed with fluorescence microscopy and confocal microscopy. The effects of S1P and CMCM on cell activity were evaluated by the methyl thiazolyl tetrazolium assay. The functional characteristic similar to cardiomyocytes was evaluated through detecting calcium transient. Our results showed that cardiomyogenic differentiation of UC-MSCs or AD-MSCs were enhanced with S1P concentration increasing, but cell activities declined. Results showed that the suitable differentiation time was 14 days, and the optimal concentration of S1P was 0.5 micromol/L. When working together with CMCM, S1P could promote the differentiation of UC-MSCs or AD-MSCs into functional cardiomyocytes, giving rise to specific electrophysiological properties (the calcium transient). Taken together, our results suggested that S1P could promote the differentiation of UC-MSCs or AD-MSCs into functional cardiomyocytes when being cultured in CMCM.
Adipose Tissue ; cytology ; metabolism ; Cell Differentiation ; drug effects ; Cells, Cultured ; Culture Media ; Humans ; Lysophospholipids ; pharmacology ; Mesenchymal Stromal Cells ; cytology ; drug effects ; Myocytes, Cardiac ; cytology ; Sphingosine ; analogs & derivatives ; pharmacology ; Umbilical Cord ; cytology

OBJECTIVE: To investigate the effect of hypoxia on cell viability and the endothelial differentiation potential in human umbilical cord derived mesenchymal stem cells (UC-MSCs), and to assess the in vitro protective role of VEGF under low oxygen tension. METHODS: MSCs were isolated from human umbilical cords and cultured in vitro. The morphological and phenotypic characterizations of human UC-MSCs were analyzed. The hypoxia induction was performed with or without the presence of 50 ng/mL of VEGF for different lengths of time. The cell proliferation, apoptosis, and reactive oxygen species (ROS) generation were assessed. Meanwhile, the endothelial differentiation potential of the UC-MSCs was measured. RESULTS: An increased apoptosis and ROS generation but reduced proliferation rate were observed at early stages (6, 12 h) after transferring the UC-MSCs from the atmospheric condition to the hypoxia condition. However, the UC-MSCs presented equal proliferation and apoptosis levels under hypoxic condition as compared with those under the atmospheric condition at the later stages (24, 72 h). A high concentration of exogenous VEGF (50 ng/mL) attenuated the increased apoptosis and inhibited the proliferation of UC-MSCs, induced by a short-term hypoxia treatment. After 14 days of exogenous VEGF induction under the hypoxia condition, the UC-MSCs acquired an early endothelial phenotype consisting of a mature endothelial molecule and some endothelial functions. CONCLUSION UC-MSCs progressively adapt to hypoxia in a step-by-step manner and maintain differentiation potential under hypoxia condition. VEGF can protect the UC-MSCs from cell damage and induce a differentiation of UC-MSCs toward endothelial lineage under hypoxic conditions.
Apoptosis ; drug effects ; Cell Differentiation ; drug effects ; Cell Hypoxia ; Cell Proliferation ; Cell Survival ; Cells, Cultured ; Humans ; Mesenchymal Stem Cells ; cytology ; Protective Agents ; pharmacology ; Reactive Oxygen Species ; metabolism ; Umbilical Cord ; cytology ; Vascular Endothelial Growth Factor A ; pharmacology