Investigation of the Effective Action Distance Between Hematopoietic Stem/Progenitor Cells and Human Adipose-Derived Stem Cells During Their In Vitro Co-culture

The in vitro suitable action distance between umbilical cord blood-derived hematopoietic stem/progenitor cells and its feeder cell, human adipose-derived stem cells, during their co-culture, was investigated through a novel transwell co-culture protocol, in which the distance between the two culture chambers where each cell type is growing can be adjusted from 10 to 450 μm. The total cell number was determined with a hemacytometer, and the cell morphology was observed under an inverted microscope each day. After 7 days of co-culture, the fold-expansion, surface antigen expression of CD34(+) and CFU-GM assay of the hematopoietic mononuclear cells (MNCs) were analyzed. The results showed that there was an optimal communication distance at around 350 μm between both types of stem cells during their in vitro co-culture. By using this distance, the UCB-MNCs and CD34(+) cells were expanded by 15.1?±?0.2 and 5.0?±?0.1-fold, respectively. It can therefore be concluded that the optimal action distance between stem cells and their supportive cells, when cultured together for 7 days, is of around 350 μm.     

The Impact of Long-Term In Vitro Expansion on the Senescence-Associated Markers of Human Adipose-Derived Stem Cells

Human adipose-derived stem cells (ASCs) have generated a great deal of excitement in regenerative medicine. However, their safety and efficacy issue remain a major concern especially after long-term in vitro expansion. The aim of this study was to investigate the fundamental changes of ASCs in long-term culture by studying the morphological feature, growth kinetic, surface marker expressions, expression level of the senescence-associated genes, cell cycle distribution and ß-galactosidase activity. Human ASCs were harvested from lipoaspirate obtained from 6 patients. All the parameters mentioned above were measured at P5, P10, P15 and P20. Data were subjected to one-way analysis of variance with a Tukey post hoc test to determine significance difference (P?相似文献   

Growth Characteristics of Human Adipose-Derived Stem Cells During Long Time Culture Regulated by Cyclin A and Cyclin D1

Abundant and less passaged cells are highly expected in clinical application since repeated subculture reduces stem cell characteristics. Long time culture of stem cells without passage is therefore needed. The growth and cell viability of human adipose-derived stem cells (hADSCs) were investigated by live/dead staining, cck-8 kits, and hemocytometer every day in 30?days of culture. The stem cell characteristics of hADSCs at the beginning and the end of culture were detected by flow cytometry and histochemical staining. hADSCs can be cultured up to the 30th day in one passage while maintaining high level cell viability and their stem cell characteristics. In addition, the cells displayed two plateau phases and three logarithmic phases during 1?month of culture. Increasing expression of cyclin A at protein level resulted in an increase in the percentage of hADSCs in the S and G₂/M phases, while decreasing protein level of cyclin D1 induced a decline in the proportion of hADSCs in the G₀/G₁ phase, regulating cells to move into rapid proliferation. This study demonstrates that a great quantity of hADSCs can be obtained in vitro by prolonging the culture time of each passage. And cyclin A and cyclin D1 affect the distribution of cell cycle and regulate the growth of hADSCs.     

EGCG Inhibits Adipose-Derived Mesenchymal Stem Cells Differentiation into Adipocytes and Prevents a STAT3-Mediated Paracrine Oncogenic Control of Triple-Negative Breast Cancer Cell Invasive Phenotype

Obese subjects have an increased risk of developing triple-negative breast cancer (TNBC), in part associated with the chronic low-grade inflammation state. On the other hand, epidemiological data indicates that increased consumption of polyphenol-rich fruits and vegetables plays a key role in reducing incidence of some cancer types. Here, we tested whether green tea-derived epigallocatechin-3-gallate (EGCG) could alter adipose-derived mesenchymal stem cell differentiation into adipocytes, and how this impacts the secretome profile and paracrine regulation of the TNBC invasive phenotype. Here, cell differentiation was performed and conditioned media (CM) from preadipocytes and mature adipocytes harvested. Human TNBC-derived MDA-MB-231 real-time cell migration was performed using the exCELLigence system. Differential gene arrays and RT-qPCR were used to assess gene expression levels. Western blotting was used to assess protein expression and phosphorylation status levels. In vitro vasculogenic mimicry (VM) was assessed with Matrigel. EGCG was found to inhibit the induction of key adipogenic biomarkers, including lipoprotein lipase, adiponectin, leptin, fatty acid synthase, and fatty acid binding protein 4. Increased TNBC-derived MDA-MB-231 cell chemotaxis and vasculogenic mimicry were observed in response to mature adipocytes secretome, and this was correlated with increased STAT3 phosphorylation status. This invasive phenotype was prevented by EGCG, the JAK/STAT inhibitors Tofacitinib and AG490, as well as upon STAT3 gene silencing. In conclusion, dietary catechin-mediated interventions could, in part through the inhibition of adipogenesis and modulation of adipocytes secretome profile, prevent the onset of an obesogenic environment that favors TNBC development.     

Isoliquiritigenin Enhances the Beige Adipocyte Potential of Adipose-Derived Stem Cells by JNK Inhibition

Human adipose-derived stem cells (hASCs) can be isolated from fat tissue and have attracted interest for their potential therapeutic applications in metabolic disease. hASCs can be induced to undergo adipogenic differentiation in vitro by exposure to chemical agents or inductive growth factors. We investigated the effects and mechanism of differentiating hASC-derived white adipocytes into functional beige and brown adipocytes with isoliquiritigenin (ILG) treatment. Here, we showed that hASC-derived white adipocytes could promote brown adipogenesis by expressing both uncoupling protein 1 (UCP1) and PR/SET Domain 16 (PRDM16) following low-dose ILG treatments. ILG treatment of white adipocytes enhanced the expression of brown fat-specific markers, while the expression levels of c-Jun N-terminal kinase (JNK) signaling pathway proteins were downregulated. Furthermore, we showed that the inhibition of JNK phosphorylation contributed to white adipocyte differentiation into beige adipocytes, which was validated by the use of SP600125. We identified distinct regulatory effects of ILG dose responses and suggested that low-dose ILG induced the beige adipocyte potential of hASCs via JNK inhibition.     

In Vitro Culture, Determination, and Directed Differentiation of Adult Adipose-Derived Stem Cells Towards Cardiomyocyte-Like Cells Induced by Angiotensin II

The in vitro basic biological characteristics and directed differentiation potential towards cardiomyocytes of adult adipose-derived stem cells (ADSCs) induced by angiotensin II were both investigated. ADSCs were isolated from adult adipose tissue and cultured in vitro, and were subsequently induced into adipocytes, chondrocytes, and osteoblasts for assays of multipotential differentiation. The morphological characteristics of ADSCs were observed under an inverted microscope in bright field and phase-contrast ways and a confocal laser scanning microscopy. Moreover, the directional differentiation potential was observed by Oil Red, alkaline phosphatase, von Kossa, and toluidine blue stainings, respectively. The expressions of CD34, CD44, CD45, CD105, and HLA-DR were also detected via flow cytometry. Following to this, ADSCs were induced by angiotensin II and basic fibroblast growth factor for the purpose of directional differentiation towards cardiomyocyte-like cells, and the cells treated with 5-azacytidine were regarded as the control. The results showed that the isolated and cultured ADSCs presented a typical morphology of fusiform shape and also expressed CD44, CD105, but not CD34, CD45, and HLA-DR with assays of flow cytometry. The multi-differentiations to adipocytes, chondrocytes, and osteoblasts confirmed that the isolated cells maintained the stem characteristics generating from adipose tissues. After 4 weeks of induction by angiotensin II, the cells expressed myosin heavy chain, troponin I, and connexin43 by immunocytochemistry staining, but without beating of the cells. This current study indicated that ADSCs possessed the characteristics of mesenchymal stem cells and angiotensin II could induce ADSCs into cardiomyocyte-like cells.     

Modified Hyaluronan Hydrogels Support the Maintenance of Mouse Embryonic Stem Cells and Human Induced Pluripotent Stem Cells

These studies provide evidence for the ability of a commercially available, defined, hyaluronan‐gelatin hydrogel, HyStem‐C?, to maintain both mouse embryonic stem cells (mESCs) and human induced pluripotent stem cells (hiPSCs) in culture while retaining their growth and pluripotent characteristics. Growth curve and doubling time analysis show that mESCs and hiPSCs grow at similar rates on HyStem‐C? hydrogels and mouse embryonic fibroblasts and Matrigel?, respectively. Immunocytochemistry, flow cytometry, gene expression and karyotyping reveal that both human and murine pluripotent cells retain a high level of pluripotency on the hydrogels after multiple passages. The addition of fibronectin to HyStem‐C? enabled the attachment of hiPSCs in a xeno‐free, fully defined medium.

     

Eudesmane and Eremophilane Sesquiterpenes from the Fruits of Alpinia oxyphylla with Protective Effects against Oxidative Stress in Adipose-Derived Mesenchymal Stem Cells

Alpinia oxyphylla Miquel (Zingiberaceae) has been reported to show antioxidant, anti-inflammatory, and neuroprotective effects. In this study, two new eudesmane sesquiterpenes, 7α-hydroperoxy eudesma-3,11-diene-2-one (1) and 7β-hydroperoxy eudesma-3,11-diene-2-one (2), and a new eremophilane sesquiterpene, 3α-hydroxynootkatone (3), were isolated from the MeOH extract of dried fruits of A. oxyphylla along with eleven known sesquiterpenes (4–14). The structures were elucidated by the analysis of 1D/2D NMR, high-resolution electrospray ionization mass spectrometry (HRESIMS), and optical rotation data. Compounds (1–3, 5–14) were evaluated for their protective effects against tert-butyl hydroperoxide (tBHP)-induced oxidative stress in adipose-derived mesenchymal stem cells (ADMSCs). As a result, treatment with isolated compounds, especially compounds 11 and 12, effectively reverted the damage of tBHP on ADMSCs in a dose-dependent manner. In particular, 11 and 12 at 50 µM improved the viability of tBHP-toxified ADMSCs by 1.69 ± 0.05-fold and 1.61 ± 0.03-fold, respectively.     

用于人类脂肪干细胞的纯化和体外长期培养的聚合物涂层

采用紫外固化法制备了基于丙烯酸酯类水凝胶的聚合物涂层(PC),并用X射线光电子能谱(XPS)、水接触角(WCA)和原子力显微镜(AFM)分别对PC进行了化学组成和表面性能的表征.在PC表面进行了人类脂肪干细胞(h ASC)的体外长期培养扩增,得到的第3代细胞的生物学表征结果表明,干细胞在PC表面能正常黏附生长,流式细胞仪检测发现干细胞对特征标记物CD49d,CD73,CD105的阳性显性比例较高,对HLA-DR和CD31几乎不显性,说明扩增的干细胞具有h ASC特征.对PC上扩增的干细胞进行诱导分化,并用油红O、茜素红和阿利新蓝分别进行染色分析,结果表明,该干细胞保留了h ASC的多能特性:能分化为成脂、成骨和成软骨细胞.含有单体甲基丙烯酰氧乙基三甲基氯化铵(DMC)、甲基丙烯酸环己酯(CHMA)和甲基丙烯酸-2-(二乙氨基)乙酯(DEAEMA)的PC2(质量比为3∶1∶2)在用于h ASC体外长期培养时,比其它PC和TCP更有利于细胞的黏附和增殖,纯化细胞,保持其多能性.实时荧光定量PCR(RT-q PCR)的分析表明PC2上得到的细胞更容易向成骨和成软骨细胞分化.     

Effect of Ceramide on Mesenchymal Stem Cell Differentiation Toward Adipocytes

Proinflammatory cytokines such as tumor necrosis factor (TNF) α are well known to inhibit adipocyte differentiation. TNF-α triggers ceramide synthesis through binding of TNF-α to its p55 receptor. Therefore, ceramide is implicated in many of the multiple signaling pathways initiated by TNF-α. In breast tissue engineering, it is important to know how to modulate adipocyte differentiation of the stem cells with exogenous additives like ceramide in vitro. We hypothesized that stem cell adipogenesis could be retained in TNF-α-treated preadipocytes in which ceramide synthesis was blocked and that exogenous ceramide could inhibit adipocyte differentiation. We first studied the effect of ceramide synthase inhibitor, Fumonisin B2, on the adipogenesis of murine mesenchymal stem cells (D1 cells), treated with TNF-α. We then studied the effect of specific exogenous C6-ceramide on D1 cell viability and differentiation. It was found that 1 ng/ml of TNF-α significantly inhibited D1 cell adipogenesis. Cells treated with 5 μM of Fumonisin B2 were able to undergo adipogenesis, even when treated with TNF-α. High concentrations of exogenous C6-ceramide (>50 μM) had an inhibitory effect, not only on the pre-confluent proliferation of the D1 cells but also on the post-confluent cell viability. High concentrations of C6-ceramide (>50 μM) also inhibited mitotic clonal expansion when D1 cell differentiation was induced by the addition of an adipogenic hormonal cocktail. C6-ceramide at low concentrations (10–25 μM) inhibited lipid production in D1 cells, demonstrated by decreased levels of both total triglyceride content and specific fatty acid composition percentages. Genetic expression of peroxisome proliferator-activated receptor (PPAR) γ and aP2 in D1 cells was reduced by C6-ceramide treatment. CCAAT/enhancer-binding protein (C/EBP) β levels in D1 cells were reduced by C6-ceramide treatment during early differentiation; PPARγ and aP2 protein levels were reduced at terminal differentiation. C6-ceramide at lower concentrations also decreased lipid accumulation of differentiating D1 cells. Our results suggest that ceramide synthase inhibitor retains the adipogenic potential of TNF-α-treated mesenchymal stem cells, while exogenous ceramide at lower concentrations inhibit the adipogenesis of mesenchymal stem cells. Ceramide, therefore, could be a modulator candidate in breast tissue engineering strategies.     

Ex Vitro Expansion of Human Placenta-Derived Mesenchymal Stem Cells in Stirred Bioreactor

The high demand of human placenta-derived mesenchymal stem cells (hPDMSCs) for therapeutic applications requires reproducible production of large numbers of well-characterized cells under well-controlled conditions. However, no method for fast hPDMSCs proliferation has yet been reported. In the present study, the feasibility of using a stirred bioreactor system to expand hPDMSCs was examined. hPDMSCs were cultured either in stirred bioreactors or in tissue culture flasks (T-flasks) for 5 days. Total cell density and several parameters of physical microenvironments were monitored in the two culture systems every 24 h. The maintenance of the antigenic phenotype of hPDMSCs before and after culturing in the stirred bioreactor system was cytometrically assessed. Data suggested that the physical microenvironment in the stirred bioreactors was much more favorable than that of the T-flasks. At the end of 144 h culturing, the total cell number was increased 1.73 times from the T-flasks to the stirred bioreactors. In addition, hPDMSCs could maintain their antigenic phenotype when cultured in stirred bioreactors. These results provide the initial assessment for large-scale hPDMSCs production using suspension culture bioreactors.     

Propagation and Purification of Human Induced Pluripotent Stem Cells with Selective Homopolymer Release Surfaces

The selective detachment of undifferentiated human induced pluripotent stem (iPS) cells from a thermal release coating, fabricated from a tailored poly(di(ethylene glycol) methyl ether methacrylate) (PDEGMA) homopolymer layer on gold, is reported. By exploiting the mild, thermally triggered release of iPS cell colonies in the absence of any releasing reagent, pluripotent iPS cells are shown to be selectively separated from spontaneously differentiated cells. The maintained pluripotency and high cell viability of detached and reseeded iPS cell colonies were confirmed and suggest the feasibility of a generally applicable platform approach for cell separation and purification in the context of iPS cell culture, differentiation of pathologically altered cells and normal cells, as well as isolation of different cell types derived from certain tissues, for example, from biopsies.     

Electrical Stimulation of Human Mesenchymal Stem Cells on Conductive Substrates Promotes Neural Priming

Electrical stimulation (ES) within a conductive scaffold is potentially beneficial in encouraging the differentiation of stem cells toward a neuronal phenotype. To improve stem cell-based regenerative therapies, it is essential to use electroconductive scaffolds with appropriate stiffnesses to regulate the amount and location of ES delivery. Herein, biodegradable electroconductive substrates with different stiffnesses are fabricated from chitosan-grafted-polyaniline (CS-g-PANI) copolymers. Human mesenchymal stem cells (hMSCs) cultured on soft conductive scaffolds show a morphological change with significant filopodial elongation after electrically stimulated culture along with upregulation of neuronal markers and downregulation of glial markers. Compared to stiff conductive scaffolds and non-conductive CS scaffolds, soft conductive CS-g-PANI scaffolds promote increased expression of microtubule-associated protein 2 (MAP2) and neurofilament heavy chain (NF-H) after application of ES. At the same time, there is a decrease in the expression of the glial markers glial fibrillary acidic protein (GFAP) and vimentin after ES. Furthermore, the elevation of intracellular calcium [Ca²⁺] during spontaneous, cell-generated Ca²⁺ transients further suggests that electric field stimulation of hMSCs cultured on conductive substrates can promote a neural-like phenotype. The findings suggest that the combination of the soft conductive CS-g-PANI substrate and ES is a promising new tool for enhancing neuronal tissue engineering outcomes.     

Bmp 2 and Bmp 7 Induce Odonto- And Osteogenesis of Human Tooth Germ Stem Cells

Bone morphogenetic proteins (BMPs) initiate, promote, and maintain odontogenesis and osteogenesis. In this study, we studied the effect of bone morphogenic protein 2 (BMP 2) and bone morphogenic protein 7 (BMP 7) as differentiation inducers in tooth and bone regeneration. We compared the effect of BMP 2 and BMP 7 on odontogenic and osteogenic differentiation of human tooth germ stem cells (hTGSCs). Third molar-derived hTGSCs were characterized with mesenchymal stem cell surface markers by flow cytometry. BMP 2 and BMP 7 were transfected into hTGSCs and the cells were seeded onto six-well plates. One day after the transfection, hTGSCs were treated with odontogenic and osteogenic mediums for 14 days. For confirmation of odontogenic and osteogenic differentiation, mRNA levels of BMP2, BMP 7, collagen type 1 (COL1A), osteocalsin (OCN), and dentin sialophosphoprotein (DSPP) genes were measured by quantitative real-time PCR. In addition to this, immunocytochemistry was performed by odontogenic and osteogenic antibodies and mineralization obtained by von Kossa staining. Our results showed that the BMP 2 and BMP 7 both promoted odontogenic and osteogenic differentiation of hTGSCs. Data indicated that BMP 2 treatment and BMP 7 treatment induce odontogenic differentiation without affecting each other, whereas they induce osteogenic differentiation by triggering expression of each other. These findings provide a feasible tool for tooth and bone tissue engineering.