1950 MHz射频电磁场对人脐带间充质干细胞增殖和成骨分化的影响研究

目的:间充质干细胞(Mesenchymal stem cells,MSCs)具有广阔的临床应用前景,但由于其体外增殖和定向分化等问题,制约了其进一步应用。本研究拟探讨1950MHz射频电磁场(Radio-frequency electromagnetic fields,RF-EMF)对人脐带间充质干细胞(Human umbilical cord mesenchymal stem cells,hUC-MSCs)增殖和成骨方向分化的影响,以期为MSCs的体外增殖和定向分化提供一条新途径。方法:华通氏胶组织块法分离培养人脐带间充质干细胞,流式细胞仪检测间充质干细胞特异性标志物。选择鉴定后的第3至第6代(P3-P6)hUC-MSCs用于实验。将hUC-MSCs细胞暴露或假暴露于频率为1950 MHz,比吸收率(Specific absorption rate,SAR)分别为0.5,1.0和2.0 W/kg的RF-EMF中,每天暴露1 h(5 min开,10 min关),连续暴露7 d。暴露结束后,流式细胞仪检测细胞周期,免疫荧光检测增殖相关蛋白Ki67表达,连续6天用CCK-8方法检测细胞数。在成骨分化研究中,将P3代的hUC-MSCs随机分为假暴露(sham)组,射频辐射暴露(RF)组,成骨诱导培养基组(Induction medium,OM)和成骨诱导培养基联合射频辐射暴露(OM+RF)组,暴露SAR值为2.0 W/kg,其它参数不变。暴露结束后立即检测细胞的碱性磷酸酶(Alkaline phosphatase,ALP)活性。结果:原代培养的细胞具有MSC典型外观,且表达MSCs特异性表面抗原。与sham组相比,不同SAR值RF暴露后,hUC-MSCs的增殖能力无明显变化,S期细胞比例及Ki67蛋白水平也无显著改变。此外,hUC-MSCs经SAR值为2.0W/kg的RF暴露7 d,与sham组相比其ALP活性无显著变化。与OM组相比,OM+RF组的ALP活性亦无显著改变。结论:华通氏胶组织块法能够培养出纯度较高的间充质干细胞,本实验条件下的1950 MHz射频电磁场对hUC-MSCs的增殖和成骨分化均无显著影响。     

The role of albumin and PPAR‐α in differentiation‐dependent change of fatty acid profile during differentiation of mesenchymal stem cells to hepatocyte‐like cells

Differentiation of mesenchymal stem cells (MSCs) to hepatocyte‐like cells is associated with morphological and biological changes. In this study, the effect of hepatogenic differentiation on fatty acid profile and the expression of proliferator‐activated receptors‐α (PPAR‐α) have been studied. For this purpose, MSCs isolated from human umbilical cord were differentiated into hepatocyte‐like cells on selective culture media. The morphological and biochemical changes, PPAR‐α expression and reactive oxygen species (ROS) levels were studied during the differentiation process. Besides, the cells were processed to determine changes in fatty acid profile using gas chromatography analysis. The results showed that hepatic differentiation of the MSCs is associated with a decrease in major polyunsaturated fatty acids in mature hepatocytes, whereas there was an increase in the saturated fatty acid (SFA) levels during hepatocyte maturation. The differentiation‐dependent shift in the ratio of SFA/USFA was associated with changes in albumin and PPAR‐α expression, whereas changes in fatty acid profile were independent of ROS production and lipid peroxidation in differentiating cells. In conclusion, these data may suggest that hepatocyte formation during the stem cell differentiation is associated with a shift in the fatty acid profile that is probably a normal phenomenon in hepatogenic differentiation of the MSCs. Copyright © 2014 John Wiley & Sons, Ltd.     

Umbilical cord-derived mesenchymal stem cells preconditioned with isorhamnetin: potential therapy for burn wounds

BACKGROUNDImpaired wound healing can be associated with different pathological states. Burn wounds are the most common and detrimental injuries and remain a major health issue worldwide. Mesenchymal stem cells (MSCs) possess the ability to regenerate tissues by secreting factors involved in promoting cell migration, proliferation and differentiation, while suppressing immune reactions. Preconditioning of MSCs with small molecules having cytoprotective properties can enhance the potential of these cells for their use in cell-based therapeutics.AIMTo enhance the therapeutic potential of MSCs by preconditioning them with isorhamnetin for second degree burn wounds in rats.METHODSHuman umbilical cord MSCs (hU-MSCs) were isolated and characterized by surface markers, CD105, vimentin and CD90. For preconditioning, hU-MSCs were treated with isorhamnetin after selection of the optimized concentration (5 µmol/L) by cytotoxicity analysis. The migration potential of these MSCs was analyzed by the in vitro scratch assay. The healing potential of normal, and preconditioned hU-MSCs was compared by transplanting these MSCs in a rat model of a second degree burn wound. Normal, and preconditioned MSCs (IH + MSCs) were transplanted after 72 h of burn injury and observed for 2 wk. Histological and gene expression analyses were performed on day 7 and 14 after cell transplantation to determine complete wound healing.RESULTSThe scratch assay analysis showed a significant reduction in the scratch area in the case of IH + MSCs compared to the normal untreated MSCs at 24 h, while complete closure of the scratch area was observed at 48 h. Histological analysis showed reduced inflammation, completely remodeled epidermis and dermis without scar formation and regeneration of hair follicles in the group that received IH + MSCs. Gene expression analysis was time dependent and more pronounced in the case of IH + MSCs. Interleukin (IL)-1β, IL-6 and Bcl-2 associated X genes showed significant downregulation, while transforming growth factor β, vascular endothelial growth factor, Bcl-2 and matrix metallopeptidase 9 showed significant upregulation compared to the burn wound, showing increased angiogenesis and reduced inflammation and apoptosis.CONCLUSIONPreconditioning of hU-MSCs with isorhamnetin decreases wound progression by reducing inflammation, and improving tissue architecture and wound healing. The study outcome is expected to lead to an improved cell-based therapeutic approach for burn wounds.     

Neural differentiation of umbilical cord mesenchymal stem cells by sub-sonic vibration

AimsAdult stem cells, such as umbilical cord-derived mesenchymal stem cells (UC-MSCs), have the potential to differentiate into various types of cells, including neurons. Research has shown that mechanical stimulation induces a response in MSCs, specifically, low and high intensity sub-sonic vibration (SSV) has been shown to facilitate wound healing. In this study, the effects of SSV were examined by assessing the proliferation and differentiation properties of MSCs.Main methodshUC-MSCs were isolated from Wharton's jelly, including the smooth muscle layer of the umbilical cord. During subculture, the cells were passaged every 5–6 days using nonhematopoietic stem cell media. To measure the effect of sonic vibration, SSV was applied to these cells continuously for 5 days.Key findingsIn this study, the morphology of hUC-MSCs was altered to resemble neurons by SSV. Further, the mRNA and protein levels of neuron-specific markers, including MAP2, NF-L, and NeuroD1, increased. In addition, other neural cell markers, such as GFAP and O4, were increased. These results suggest that hUC-MSCs differentiated into neural cells upon SSV nonselectively. In a mechanism study, the ERK level increased in a time-dependent manner upon SSV for 12 h.SignificanceThe results of this study suggest that SSV caused hUC-MSCs to differentiate into neural cells via ERK activation.     

How important is differentiation in the therapeutic effect of mesenchymal stromal cells in liver disease?

Background aimsThe protocols for differentiation of hepatocyte-like cells (HLCs) from mesenchymal stromal cells (MSCs) have been well established. Previous data have shown that MSCs and their derived HLCs were able to engraft injured liver and alleviate injuries induced by carbon tetrachloride. The goal of the current study was to determine the differences of MSCs and their derived HLCs in terms of therapeutic functions in liver diseases.MethodsAfter hepatic differentiation of umbilical cord–derived MSCs in vitro, we detected both MSC and HLC expressions of adhesion molecules and chemokine receptor CXCR4 by flow cytometry; immunosuppressive potential and hepatocyte growth factor expression were determined by means of enzyme-linked immunosorbent assay. We compared the therapeutic effect for fulminant hepatic failure in a mouse model.ResultsMSC-derived-HLCs expressed lower levels of hepatocyte growth factor, accompanied by impaired immunosuppression in comparison with MSCs. Furthermore, undifferentiated MSCs showed rescuing potentials superior to those in HLCs for the treatment of fulminant hepatic failure.ConclusionsAfter differentiation, HLCs lost several major properties in comparison with undifferentiated MSCs, which are beneficial for their application in liver diseases. Undifferentiated MSCs may be more appropriate than are HLCs for the treatment of liver diseases.     

人滑膜间充质干细胞与人脐带间充质干细胞的生物学性状比较

该文旨在比较人滑膜间充质干细胞(human synovial mesenchymal stem cells,hSMSCs)与人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUC-MSCs)的生物学性状.流式细胞仪鉴定hSMSCs和hUC-MSCs.比较两种间...     

Synthesis and immunomodulating activity of new glycopeptides of glycyrrhizic acid containing residues of L-glutamic acid

New glycopeptides of glycyrrhizic acid (GA) containing Glu residues and their α-methyl esters, γ-methyl esters, and α,γ-dimethyl esters were synthesized using N,N′-dicyclohexylcarbodiimide in the presence of N-hydroxybenzotriazole or N-hydroxysuccinimide. Formation of amide bonds was observed on all the three COOH groups of GA, or selectively on the COOH groups of the GA carbohydrate part in dependence on the ratio of reagents and the reaction conditions. The GA glycopeptide with three residues of Glu(OH)-OMe at a dose of 2 mg/kg stimulated the production of antibody-forming cells in mouse spleen in comparison with the control. The GA glycopeptide containing Glu residues only in the GA carbohydrate part turned out to be an immunosuppressor. The glycopeptide of the 30-methyl ester of GA with residues of free Glu in its carbohydrate part increased the hemagglutinine titer at oral doses of 2 and 10 mg/kg. All the studied compounds had practically no effect on the delayed-type hypersensitivity in mice.     

Nanofiber-expanded stem cells mitigate liver fibrosis: Experimental study

ObjectivesThis study examines a pretreatment strategy to strengthen the hepatic lineage divergence of mesenchymal stem cells (MSCs).Design and methodsBMSCs were expanded in the presence or absence of nanofiber (NF) and treated with growth factors (GF) prior to transplantation. Thioacetamide (TA) was used for liver fibrosis induction and transplantation of NF-expanded BMSCs was compared biochemically and histologically to the cells expanded without NF scaffold.ResultsThe ultraweb NF caused better proliferation and characterization of MSCs. MSCs transplantation significantly improved liver functions, increased hepatic HGF and Bcl-2 levels, whereas decreased serum fibronectin, hepatic TNF-α and TGF-β1 levels. Hepatic HNF4α, FOXa2, CYP7a1 genes expression were enhanced while β-5-Tub and AFP genes expression were depressed. Histological study documented these results. Differentiated NF-MSCs showed pronounced enhancement of the aforementioned parameters as compared to differentiated MSCs in the absence of NF.Conclusionpretreatment with growth factors in the presence of NF augment homing, repopulation and hepatic differentiation abilities of MSCs and proves to be a promising approach for the treatment of liver fibrosis.     

Induction of hepatocyte‐like cells from human umbilical cord‐derived mesenchymal stem cells by defined microRNAs

Generating functional hepatocyte‐like cells (HLCs) from mesenchymal stem cells (MSCs) is of great urgency for bio‐artificial liver support system (BALSS). Previously, we obtained HLCs from human umbilical cord‐derived MSCs by overexpressing seven microRNAs (HLC‐7) and characterized their liver functions in vitro and in vivo. Here, we aimed to screen out the optimal miRNA candidates for hepatic differentiation. We sequentially removed individual miRNAs from the pool and examined the effect of transfection with remainder using RT‐PCR, periodic acid—Schiff (PAS) staining and low‐density lipoprotein (LDL) uptake assays and by assessing their function in liver injury models. Surprisingly, miR‐30a and miR‐1290 were dispensable for hepatic differentiation. The remaining five miRNAs (miR‐122, miR‐148a, miR‐424, miR‐542‐5p and miR‐1246) are essential for this process, because omitting any one from the five‐miRNA combination prevented hepatic trans‐differentiation. We found that HLCs trans‐differentiated from five microRNAs (HLC‐5) expressed high level of hepatic markers and functioned similar to hepatocytes. Intravenous transplantation of HLC‐5 into nude mice with CCl₄‐induced fulminant liver failure and acute liver injury not only improved serum parameters and their liver histology, but also improved survival rate of mice in severe hepatic failure. These data indicated that HLC‐5 functioned similar to HLC‐7 in vitro and in vivo, which have been shown to resemble hepatocytes. Instead of using seven‐miRNA combination, a simplified five‐miRNA combination can be used to obtain functional HLCs in only 7 days. Our study demonstrated an optimized and efficient method for generating functional MSC‐derived HLCs that may serve as an attractive cell alternative for BALSS.     

Synthesis and evaluation of triazole linked glycosylated 18β-glycyrrhetinic acid derivatives as anticancer agents

A series of glycosyl triazol linked 18β-glycyrrhetinic acid (GA) derivatives have been synthesized using 1,3-dipolar cycloaddition reaction of per-O-acetylated glycosyl azide derivatives (4a–h) with propargyl ester of 18β-glycyrrhetinic acid (GA) (2 and 3) following the concept of ‘Click chemistry’. The synthesized triazole derivatives were de-O-acetylated to furnish compounds (7a–h and 8a–c) with free hydroxyl groups in the carbohydrate moieties, which were evaluated for their anticancer potential against human cervical cancer cells (HeLa) and normal kidney epithelial (NKE) cells. GA (1), compound 7d, compound 7g and compound 8c showed promising anticancer activities.     

Valproic acid promotes differentiation of hepatocyte-like cells from whole human umbilical cord-derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) are mesoderm-derived cells that are considered a good source of somatic cells for treatment of many degenerative diseases. Previous studies have reported the differentiation of mesodermal MSCs into endodermal and ectodermal cell types beyond their embryonic lineages, including hepatocytes and neurons. However, the molecular pathways responsible for the direct or indirect cell type conversion and the functional ability of the differentiated cells remain unclear and need further research. In the present study, we demonstrated that valproic acid (VPA), which is a histone deacetylase inhibitor, induced an increase in the expression of endodermal genes including CXCR4, SOX17, FOXA1, FOXA2, GSC, c-MET, EOMES, and HNF-1β in human umbilical cord derived MSCs (hUCMSCs). In addition, we found that VPA is able to increase these endodermal genes in hUCMSCs by activating signal transduction of AKT and ERK. VPA pretreatment increased hepatic differentiation at the expense of adipogenic differentiation. The effects of VPA on modulating hUCMSCs fate were diminished by blocking AKT and ERK activation using specific signaling inhibitors. Together, our results suggest that VPA contributes to the lineage conversion of hUCMSCs to hepatic cell fate by upregulating the expression of endodermal genes through AKT and ERK activation.     

Protective effects of hepatocyte-specific glycyrrhetic derivatives against carbon tetrachloride-induced liver damage in mice

Glycyrrhetic acid (GA), the main hydrolysate of glycyrrhizic acid extracted from the roots of the Chinese herb Glycyrrhiza glabra, was reported to be accumulated in hepatocytes due to the extensive distribution of GA receptors in liver. A series of hepatocyte-specific derivatives on the basis of anetholtrithione and glycyrrhizic were designed and synthesized. The potential beneficial effect was evaluated in carbon tetrachloride (CCl₄)-induced liver injury model. In addition, the hepatoprotective activity of these derivatives was assessed by measuring levels of serum marker enzymes, including serum glutamate oxaloacetate transaminase (GOT), serum glutamate pyruvate transaminase (GPT), alkaline phosphatase (AKP), lactate dehydrogenase (LDH) and the ratio of GSH to GSSG. Gratifyingly, compounds 5a–c (100 mg/kg, p.o.) markedly prevented CCl₄-induced elevation of levels of serum GPT, GOT. A comparative histopathological study of liver exhibited almost a normal liver lobular architecture and cell structure of the livers, as compared to CCl₄-treated group. These findings were confirmed with the histopathological observations, where hepatocyte-specific glycyrrhetic acid derivatives 5a–c were capable of reversing the toxic effects of CCl₄ on hepatocytes.     

Glycyrrhizin and its derivatives promote hepatic differentiation via sweet receptor,Wnt, and Notch signaling

The acute liver disease is involved in aberrant release of high-mobility group box 1 (HMGB1). Glycyrrhizin (GL), a traditional Chinese medicine for liver disease, binds to HMGB1, thereby inhibits tissue injury. However the mode of action of GL for chronic liver disease remains unclear.We investigated the effects of glycyrrhizin (GL) and its derivatives on liver differentiation using human iPS cells by using a flow cytometric analysis.GL promoted hepatic differentiation at the hepatoblast formation stage. The GL derivatives, 3-O-mono-glucuronyl 18β-glycyrrhetinic acid (Mono) and 3-O-[glucosyl (1 → 2)-glucuronyl] 18β-glycyrrhetinic acid increased AFP⁺ cell counts and albumin⁺ cell counts. Glucuronate conjugation seemed to be a requirement for hepatic differentiation. Mono exhibited the most significant hepatic differentiation effect.We evaluated the effects of (±)-2-(2,4-dichlorophenoxy) propionic acid (DP), a T1R3 antagonist, and sucralose, a T1R3 agonist, on hepatic differentiation, and found that DP suppressed Mono-induced hepatic differentiation, while sucralose promoted hepatic differentiation. Thus, GL promoted hepatic differentiation via T1R3 signaling. In addition, Mono increased β-catenin⁺ cell count and decreased Hes5⁺ cell count suggesting the involvement of Wnt and Notch signaling in GL-induced hepatic differentiation.In conclusion, GL exerted a hepatic differentiation effect via sweet receptor (T1R3), canonical Wnt, and Notch signaling.     

BMP-2 promotes osteogenic differentiation of mesenchymal stem cells by enhancing mitochondrial activity

Objectives:Mesenchymal stem cells (MSCs) have become seed cells and basic elements for bone regeneration and bone tissue engineering. The aim of the present study was to investigate the roles and mechanisms of bone morphogenetic protein 2 (BMP-2) on osteogenic differentiation of MSCs.Methods:Primary MSCs were isolated from the femur and tibia bone of rats and then transfected with BMP-2 and PGC-1α adenovirus vectors. Alkaline phosphatase (ALP) activity and alizarin red staining were used to measure osteogenic differentiation of MSCs. Real-time PCR and western blot assays were performed to assess osteogenic differentiation-related proteins levels. The activities of mitochondrial respiratory chain complexes I and II and mitochondrial fluorescence intensity were used to explore mitochondria status during osteogenic differentiation of MSCs.Results:We found that the ability of BMP-2 overexpressed (OE) group osteogenic differentiation was significantly improved, compared with the negative control (NC) group. The results also indicated that BMP-2 can promote the activity of mitochondria. We further used the gain- and loss-of-function approaches to demonstrate that BMP-2 promotes mitochondrial activity by up-regulating PGC-1α to promote osteogenic differentiation of MSCs.Conclusions:These results explored the important role of BMP-2 in the osteoblast differentiation of MSCs from a new perspective, providing a theoretical and experimental basis for bone defect and repair.     

Effects of chlorogenic acid,caffeine, and coffee on behavioral and biochemical parameters of diabetic rats

Mesenchymal stem cells (MSCs) represent a potential therapeutic target for glioma. We determined the molecular mechanism of inhibitory effect of human umbilical cord-derived MSCs (hUC-MSCs) on the growth of C6 glioma cells. We demonstrated that hUC-MSCs inhibited C6 cell growth and modulated the cell cycle to G0/G1 phase. The expression of β-catenin and c-Myc was downregulated in C6 cells by conditioned media from hUC-MSCs, and the levels of secreted DKK1 were positively correlated with concentrations of hUCMSCs-CM. The inhibitory effect of hUC-MSCs on C6 cell proliferation was enhanced as the concentration of DKK1 in hUCMSCs-CM increased. When DKK1 was neutralized by anti-DKK1 antibody, the inhibitory effect of hUC-MSCs on C6 cells was attenuated. Furthermore, we found that conditioned media from hUC-MSCs transfection with siRNA targeting DKK1 mRNA or pEGFPN1-DKK1 plasmid lost or enhanced the abilities to regulate the Wnt signaling in C6 cells. Therefore, hUC-MSCs inhibited C6 glioma cell growth via secreting DKK1, an inhibitor of Wnt pathway, may represent a novel therapeutic strategy for malignant glioma.