共查询到20条相似文献,搜索用时 31 毫秒
1.
Gupta MK Walthall JM Venkataraman R Crowder SW Jung DK Yu SS Feaster TK Wang X Giorgio TD Hong CC Baudenbacher FJ Hatzopoulos AK Sung HJ 《PloS one》2011,6(12):e28935
Myocardial infarction results in extensive cardiomyocyte death which can lead to fatal arrhythmias or congestive heart failure. Delivery of stem cells to repopulate damaged cardiac tissue may be an attractive and innovative solution for repairing the damaged heart. Instructive polymer scaffolds with a wide range of properties have been used extensively to direct the differentiation of stem cells. In this study, we have optimized the chemical and mechanical properties of an electrospun polymer mesh for directed differentiation of embryonic stem cells (ESCs) towards a cardiomyogenic lineage. A combinatorial polymer library was prepared by copolymerizing three distinct subunits at varying molar ratios to tune the physicochemical properties of the resulting polymer: hydrophilic polyethylene glycol (PEG), hydrophobic poly(ε-caprolactone) (PCL), and negatively-charged, carboxylated PCL (CPCL). Murine ESCs were cultured on electrospun polymeric scaffolds and their differentiation to cardiomyocytes was assessed through measurements of viability, intracellular reactive oxygen species (ROS), α-myosin heavy chain expression (α-MHC), and intracellular Ca(2+) signaling dynamics. Interestingly, ESCs on the most compliant substrate, 4%PEG-86%PCL-10%CPCL, exhibited the highest α-MHC expression as well as the most mature Ca(2+) signaling dynamics. To investigate the role of scaffold modulus in ESC differentiation, the scaffold fiber density was reduced by altering the electrospinning parameters. The reduced modulus was found to enhance α-MHC gene expression, and promote maturation of myocyte Ca(2+) handling. These data indicate that ESC-derived cardiomyocyte differentiation and maturation can be promoted by tuning the mechanical and chemical properties of polymer scaffold via copolymerization and electrospinning techniques. 相似文献
2.
Yu Suk Choi Gregory J. Dusting Samantha Stubbs Sandeep Arunothayaraj Xiao Lian Han Philippe Collas Wayne A. Morrison Rodney J. Dilley 《Journal of cellular and molecular medicine》2010,14(4):878-889
Human adipose‐derived stem cells (ASCs) may differentiate into cardiomyocytes and this provides a source of donor cells for tissue engineering. In this study, we evaluated cardiomyogenic differentiation protocols using a DNA demethylating agent 5‐azacytidine (5‐aza), a modified cardiomyogenic medium (MCM), a histone deacetylase inhibitor trichostatin A (TSA) and co‐culture with neonatal rat cardiomyocytes. 5‐aza treatment reduced both cardiac actin and TropT mRNA expression. Incubation in MCM only slightly increased gene expression (1.5‐ to 1.9‐fold) and the number of cells co‐expressing nkx2.5/sarcomeric α‐actin (27.2%versus 0.2% in control). TSA treatment increased cardiac actin mRNA expression 11‐fold after 1 week, which could be sustained for 2 weeks by culturing cells in cardiomyocyte culture medium. TSA‐treated cells also stained positively for cardiac myosin heavy chain, α‐actin, TropI and connexin43; however, none of these treatments produced beating cells. ASCs in non‐contact co‐culture showed no cardiac differentiation; however, ASCs co‐cultured in direct contact co‐culture exhibited a time‐dependent increase in cardiac actin mRNA expression (up to 33‐fold) between days 3 and 14. Immunocytochemistry revealed co‐expression of GATA4 and Nkx2.5, α‐actin, TropI and cardiac myosin heavy chain in CM‐DiI labelled ASCs. Most importantly, many of these cells showed spontaneous contractions accompanied by calcium transients in culture. Human ASC (hASC) showed synchronous Ca2+ transient and contraction synchronous with surrounding rat cardiomyocytes (106 beats/min.). Gap junctions also formed between them as observed by dye transfer. In conclusion, cell‐to‐cell interaction was identified as a key inducer for cardiomyogenic differentiation of hASCs. This method was optimized by co‐culture with contracting cardiomyocytes and provides a potential cardiac differentiation system to progress applications for cardiac cell therapy or tissue engineering. 相似文献
3.
目的 探讨黄芪甲苷对心肌干细胞分化的促进作用。方法 采用磁珠分选法,分离小鼠Sca-1+心肌干细胞,通过免疫组化方法观察黄芪甲甙处理后心肌细胞表面标志蛋白desmin、α-sarcomeric?actin和C-TnT表达的变化,以判断是否对心肌干细胞分化有促进作用。结果 250 mg/L的黄芪甲甙诱导4周后免疫组化染色显示心肌干细胞明显表达desmin、α-sarcomeric actin和C-TnT。而未诱导的细胞desmin、α-sarcomeric actin、C-TnT 均为阴性。因此黄芪甲甙可以促进小鼠Sca-1+心肌干细胞分化为心肌样细胞,这些细胞表达心肌特异性的蛋白。结论 黄芪甲苷对心肌干细胞分化的促进作用表明其在心肌损伤性疾病的康复中有潜在的治疗价值,值得进一步研究。 相似文献
4.
Stem cells in cell based therapy for cardiac injury is being potentially considered. However, genetic regulatory networks involved in cardiac differentiation are not clearly understood. Among stem cell differentiation models, mouse P19 embryonic carcinoma (EC) cells, are employed for studying (epi)genetic regulation of cardiomyocyte differentiation. Here, we comprehensively assessed cardiogenic differentiation potential of 5-azacytidine (Aza) on P19 EC-cells, associated gene expression profiles and the changes in DNA methylation, histone acetylation and activated-ERK signaling status during differentiation. Initial exposure of Aza to cultured EC-cells leads to an efficient (55%) differentiation to cardiomyocyte-rich embryoid bodies with a threefold (16.8%) increase in the cTnI+ cardiomyocytes. Expression levels of cardiac-specific gene markers i.e., Isl-1, BMP-2, GATA-4, and α-MHC were up-regulated following Aza induction, accompanied by differential changes in their methylation status particularly that of BMP-2 and α-MHC. Additionally, increases in the levels of acetylated-H3 and pERK were observed during Aza-induced cardiac differentiation. These studies demonstrate that Aza is a potent cardiac inducer when treated during the initial phase of differentiation of mouse P19 EC-cells and its effect is brought about epigenetically and co-ordinatedly by hypo-methylation and histone acetylation-mediated hyper-expression of cardiogenesis-associated genes and involving activation of ERK signaling. 相似文献
5.
目的探讨不同浓度全反式维甲酸(all-trans retinoic acid,atRA)诱导P19细胞向心肌分化的效力。方法细胞分成P19细胞组,2nm/L atRA诱导组,5nm/L atRA诱导组,8nm/L atRA诱导组。各组细胞经过诱导、聚集培养、聚集体贴壁培养10天后,RT-PCR检测GATA-4,α-肌球蛋白重链(α-myosin heavychain,α-MHC)的mRNA表达,免疫荧光双标检测α-sarcomeric actin和cTnT蛋白共表达,Western blot检测cTnT的蛋白表达。结果 atRA可诱导聚集P19细胞表达GA-TA-4、a-MHC mRNA;α-sarcomeric actin和cTnT的表达和共表达增加;5nm/L atRA组,8nm/L atRA组GATA-4、a-MHCmRNA的表达量显著高于P19细胞组;5nm/L atRA组,8nm/L atRA组两种蛋白的表达和共表达量显著高于P19细胞组,以5nm/L atRA组最高,显著高于其它组。结论 atRA可诱导聚集P19细胞向心肌分化,其中5nm/L atRA组效果最好。 相似文献
6.
Bone marrow multipotent stromal cells (BMSCs) have the ability to transdifferentiate into various cell types, including: osteoblasts, chondrocytes, adipocytes, neurons, and cardiomyocytes. This study aimed to differentiate the BMSCs into cardiomyocyte. BMSCs were exposed to 5-azacytidine for 24 h. Seven days after the induction of cell differentiation by 5-azacytidine, the cardiomyogenic cells were stained by fushin and binucleated cells were counted and compared with the neonate cardiomyocyte as positive control. In addition, immunofluorescence analysis and western blot were performed using the antibodies against α-actinin, desmin, troponin T, and β-myosin heavy chain. Our results showed that there was no significant difference between the number of binucleated cells within the cardiomyogenic cell group and positive control group; however, a statistically significant difference was observed between both of these groups and undifferentiated cell group (P < 0.005). In addition, after 5-azacytidine treatment, BMSCs had a higher expression of cardiac-specific markers such as desmin, α-actinin, troponin T and β-myosin heavy chain compared with the untreated groups (P < 0.005). We concluded that 5-azacytidine is an effective inducer for the differentiation of BMSCs into cardiomyocytes and could produce a population of binucleated cells, which express α-actinin, desmin, troponin T, and β-myosin heavy chain, four markers of cardiomyocytes. 相似文献
7.
8.
9.
So‐Jung Gwak Suk H. Bhang Hee S. Yang Sang‐Soo Kim Dae‐Hee Lee Soo‐Hong Lee Byung‐Soo Kim 《Cell biochemistry and function》2009,27(3):148-154
Transplanting stem cells differentiated towards a cardiac lineage can regenerate cardiac muscle tissues to treat myocardial infarction. In this study, we tested the hypothesis that transforming growth factor‐β1 (TGF‐β1) induces cardiomyogenic differentiation of adipose‐ derived stromal cells (ADSCs) in vitro. Rat ADSCs were cultured with TGF‐β1 (10 ng ml?1) for 2 weeks in vitro. ADSCs cultured without TGF‐β1 served as a control. The mRNA expression of cardiac‐specific gene was induced by TGF‐β1, while the control culture did not show cardiac‐specific gene expression. Immunocytochemical analyses showed that a small fraction of ADSCs cultured with TGF‐β1 for 2 weeks stained positively for cardiac myosin heavy chain (MHC) and α‐sarcomeric actin. Flow cytometric analyses showed that the proportion of cells expressing cardiac MHC increased with TGF‐β1. However, no mesenchymal differentiation (e.g., osteogenic and adipogenic differentiation) was detected other than cardiomyogenic differentiation. These results showed that TGF‐β1 induce ADSC cardiomyogenic differentiation in vitro, which could be useful for myocardial infarction stem cell therapy. Copyright © 2009 John Wiley & Sons, Ltd. 相似文献
10.
The unmet clinical need for myocardial repair after irreversible ischemic injury requires a better understanding of the biological properties of cardiac stem cells (CSCs). Using a primary culture of neonatal rat myocardial cells, we describe the formation and maturation of contracting cardiomyocyte colonies stemming from c-kit+, Sca+, or Isl1+ CSCs, which occurs in parallel to the hypertrophy of the major cardiac myocyte population. The contracting cardiomyocyte colonies (~1–2 colonies per 1 × 105 of myocardial cells) were identified starting from eighth day of culturing. At first, spontaneous weak, asynchronous, and arrhythmic contractions of the colonies at a rate of 2–3 beats/min were registered. Over time, the contractions of the colonies became more synchronous and frequent, with a contraction rate of 58–60 beats/min by the 30th day of culturing. The colonies were characterized by the CSCs subtype-specific pattern of growth and structure. The cells of the colonies were capable of spontaneous cardiomyogenic differentiation, demonstrating expression of both sarcomeric α-actinin and α-sarcomeric actin as well as the maturation of contractile machinery and typical Ca2+ responses to caffeine (5 mМ) and K+ (120 mМ). Electromechanical coupling, characterized by cardiac muscle-specific Ca2+-induced Ca2+ release, was evident at 3 weeks of culturing. Thus, the co-cultivation of CSCs with mature cardiac cells resulted in the formation of contracting cardiomyocyte colonies, resembling the characteristics of in vivo cardiomyogenesis. The proposed model can be used for the investigation of fundamental mechanisms underlying cardiomyogenic differentiation of CSCs as well as for drug testing and/or other applications. 相似文献
11.
探讨大鼠脂肪间充质干细胞(adipose-derived mesenchymal stem cells,AMSC)体外分化成心肌样细胞的潜能,为自体干细胞移植治疗心肌梗死提供理论基础.采用消化法分离大鼠AMSC,培养于RPMI1640生长培养基中,倒置相差显微镜观察细胞形态发现,随着培养时间的延长,细胞形态趋向于心肌细胞,SQ RT-PCR检测表达心肌特异性基因:β-肌球蛋白重链(β-MHC)、α-肌球蛋白重链(α-MHC)、心房利钠肽(ANP)、心肌肌钙蛋白(cTnT)、心肌肌动蛋白、肌肉增强因子和GATA-4;免疫细胞化学和免疫荧光染色检测表达心肌细胞特异性蛋白:结蛋白、横纹肌辅肌动蛋白、心肌肌动蛋白和间隙连接蛋白45(connexin 45);Western印迹检测表达心肌特异性蛋白Nkx2.5. 实验表明,大鼠AMSC在体外培养条件下能分化成心肌样细胞,在组织工程学及干细胞移植领域有着良好的应用前景. 相似文献
12.
Campan M Lionetti V Aquaro GD Forini F Matteucci M Vannucci L Chiuppesi F Di Cristofano C Faggioni M Maioli M Barile L Messina E Lombardi M Pucci A Pistello M Recchia FA 《American journal of physiology. Heart and circulatory physiology》2011,300(6):H2238-H2250
The methods currently utilized to track stem cells by cardiac MRI are affected by important limitations, and new solutions are needed. We tested human ferritin heavy chain (hFTH) as a reporter gene for in vivo tracking of stem cells by cardiac MRI. Swine cardiac stem/progenitor cells were transduced with a lentiviral vector to overexpress hFTH and cultured to obtain cardiospheres (Cs). Myocardial infarction was induced in rats, and, after 45 min, the animals were subjected to intramyocardial injection of ~200 hFTH-Cs or nontransduced Cs or saline solution in the border zone. By employing clinical standard 1.5-Tesla MRI scanner and a multiecho T2* gradient echo sequence, we localized iron-accumulating tissue only in hearts treated with hFTH-Cs. This signal was detectable at 1 wk after infarction, and its size did not change significantly after 4 wk (6.33 ± 3.05 vs. 4.41 ± 4.38 mm(2)). Cs transduction did not affect their cardioreparative potential, as indicated by the significantly better preserved left ventricular global and regional function and the 36% reduction in infarct size in both groups that received Cs compared with control infarcts. Prussian blue staining confirmed the presence of differentiated, iron-accumulating cells containing mitochondria of porcine origin. Cs-derived cells displayed CD31, α-smooth muscle, and α-sarcomeric actin antigens, indicating that the differentiation into endothelial, smooth muscle and cardiac muscle lineage was not affected by ferritin overexpression. In conclusion, hFTH can be used as a MRI reporter gene to track dividing/differentiating stem cells in the beating heart, while simultaneously monitoring cardiac morpho-functional changes. 相似文献
13.
Zhen Zhu Zhihui Liu Jingyue Liu Ming Bi Taoyuan Yang Jingyun Wang 《Cytotechnology》2015,67(2):285-297
Human placenta-derived mesenchymal stem cells (hPDMSCs) can differentiate into different types of cells and thus have tremendous potential for cell therapy and tissue engineering. LIM mineralization protein-1 (LMP-1) plays an important role in osteoblast differentiation, maturation and bone formation. To determine a global effect of LMP-1 on hPDMSCs, we designed a study using a proteomic approach combined with adenovirus-mediated gene transfer of LMP-1 to identify LMP-1-induced changes in hPDMSCs on proteome level. We have generated proteome maps of undifferentiated hPDMSCs and LMP-1 induced hPDMSCs. Two dimensional gel electrophoresis revealed 22 spots with at least 2.0-fold changes in expression and 15 differently expressed proteins were successfully identified by MALDI-TOF-MS. The proteins regulated by LMP-1 included cytoskeletal proteins, cadmium-binding proteins, and metabolic proteins, etc. The expression of some identified proteins was confirmed by further Western blot analyses. Our results will play an important role in better elucidating the underlying molecular mechanism in LMP-1 included hPDMSCs differentiation into osteoblasts. 相似文献
14.
15.
Nuclear actin - which is immunologically distinct from cytoplasmic actin - has been documented in a number of differentiated cell types, and cardiac isoforms of troponin I (cTnI) and troponin T (cTnT) have been detected in association with nuclei of adult human cardiac myocytes. It is not known whether these and related proteins are present in undifferentiated stem cells, or when they appear in cardiomyogenic cells following differentiation. We first tested the hypothesis that nuclear actin and cardiac isoforms of troponin C (cTnC) and tropomyosin (cTm) are present along with cTnI and cTnT in nuclei of isolated, neonatal rat cardiomyocytes in culture. We also tested the hypothesis that of these five proteins, only actin is present in nuclei of multipotent, bone marrow-derived mesenchymal stem cells (BM-MSCs) from adult rats in culture, but that cTnC, cTnI, cTnT and cTm appear early and uniquely following cardiomyogenic differentiation. Here we show that nuclear actin is present within nuclei of both ventricular cardiomyocytes and undifferentiated, multipotent BM-MSCs. We furthermore show that cTnC, cTnI, cTnT and cTm are not only present in myofilaments of ventricular cardiomyocytes in culture but are also within their nuclei; significantly, these four proteins appear between days 3 and 5 in both myofilaments and nuclei of BM-MSCs treated to differentiate into cardiomyogenic cells. These observations indicate that cardiac troponin and tropomyosin could have important cellular function(s) beyond Ca(2+)-regulation of contraction. While the roles of nuclear-associated actin, troponin subunits and tropomyosin in cardiomyocytes are not known, we anticipate that the BM-MSC culture system described here will be useful for elucidating their function(s), which likely involve cardiac-specific, Ca(2+)-dependent signaling in the nucleus. 相似文献
16.
Tae-Jin Lee Subeom Park Suk Ho Bhang Jeong-Kee Yoon Insu Jo Gun-Jae Jeong Byung Hee Hong Byung-Soo Kim 《Biochemical and biophysical research communications》2014
Graphene has drawn attention as a substrate for stem cell culture and has been reported to stimulate the differentiation of multipotent adult stem cells. Here, we report that graphene enhances the cardiomyogenic differentiation of human embryonic stem cells (hESCs) at least in part, due to nanoroughness of graphene. Large-area graphene on glass coverslips was prepared via the chemical vapor deposition method. The coating of the graphene with vitronectin (VN) was required to ensure high viability of the hESCs cultured on the graphene. hESCs were cultured on either VN-coated glass (glass group) or VN-coated graphene (graphene group) for 21 days. The cells were also cultured on glass coated with Matrigel (Matrigel group), which is a substrate used in conventional, directed cardiomyogenic differentiation systems. The culture of hESCs on graphene promoted the expression of genes involved in the stepwise differentiation into mesodermal and endodermal lineage cells and subsequently cardiomyogenic differentiation compared with the culture on glass or Matrigel. In addition, the culture on graphene enhanced the gene expression of cardiac-specific extracellular matrices. Culture on graphene may provide a new platform for the development of stem cell therapies for ischemic heart diseases by enhancing the cardiomyogenic differentiation of hESCs. 相似文献
17.
本研究构建了心肌特异性α-肌球蛋白重链(α-MHC)启动子表达载体,并对其功能进行了鉴定。以小鼠基因组DNA为模板,通过PCR扩增得到α-MHC启动子,插入pGEM-TEasy载体,酶切后回收目的片段,置换pcDNA3.1(+)-EGFP-hygro中的CMV启动子,成功构建出α-MHC-EGFP表达载体。对其进行酶切鉴定后,通过电穿孔法转染原代小鼠心肌细胞,转染阳性的心肌细胞出现绿色荧光,而非心肌细胞无荧光出现。α-MHC-EGFP表达载体具有心肌特异性表达特性,可用于纯化胚胎干细胞来源的心肌细胞。 相似文献
18.
Yan Huang Zhong-Quan Dai Shu-Kuan Ling Hong-Yu Zhang Yu-Min Wan Ying-Hui Li 《Journal of biomedical science》2009,16(1):87-14
Background
Stem cell therapy has emerged as a potential therapeutic option for tissue engineering and regenerative medicine, but many issues remain to be resolved, such as the amount of seed cells, committed differentiation and the efficiency. Several previous studies have focused on the study of chemical inducement microenvironments. In the present study, we investigated the effects of gravity on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into force-sensitive or force-insensitive cells.Methods and results
Rat BMSCs (rBMSCs) were cultured under hypergravity or simulated microgravity (SMG) conditions with or without inducement medium. The expression levels of the characteristic proteins were measured and analyzed using immunocytochemical, RT-PCR and Western-blot analyses. After treatment with 5-azacytidine and hypergravity, rBMSCs expressed more characteristic proteins of cardiomyocytes such as cTnT, GATA4 and β-MHC; however, fewer such proteins were seen with SMG. After treating rBMSCs with osteogenic inducer and hypergravity, there were marked increases in the expression levels of ColIA1, Cbfa1 and ALP. Reverse results were obtained with SMG. rBMSCs treated with adipogenic inducer and SMG expressed greater levels of PPARgamma. Greater levels of Cbfa1- or cTnT-positive cells were observed under hypergravity without inducer, as shown by FACS analysis. These results indicate that hypergravity induces differentiation of rBMSCs into force-sensitive cells (cardiomyocytes and osteoblasts), whereas SMG induces force-insensitive cells (adipocytes).Conclusion
Taken together, we conclude that gravity is an important factor affecting the differentiation of rBMSCs; this provides a new avenue for mechanistic studies of stem cell differentiation and a new approach to obtain more committed differentiated or undifferentiated cells. 相似文献19.
20.
人羊膜间充质细胞具有向心肌样细胞分化的特性 总被引:1,自引:0,他引:1
摘 要 探讨人羊膜间充质细胞(human amniotic mesenchymal cells,hAMCs)向心肌细胞分化的能力。采用胶原酶消化法分离hAMCs,用流式细胞仪进行表型鉴定;用5-氮杂胞苷和碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)诱导hAMCs向心肌细胞分化,免疫荧光染色法检测诱导后细胞中特异蛋白结蛋白和α-辅肌动蛋白的表达,RT-PCR检测心肌特异性转录因子Nkx2.5 、GATA-4和心肌特异性收缩蛋白α-肌球蛋白重链(α-myosin heavy chain,α-MHC)mRNA的表达。结果显示:①hAMCs原代培养至第6 d,贴壁细胞汇合度可达80%,呈漩涡状生长。传代后hAMCs增殖迅速,3~4 d细胞汇合度可达100%,细胞呈梭形或多角形。②hAMCs表达CD44和波形蛋白,不表达CK19。③hAMCs经诱导分化8~10 d后细胞排列紧密,多为长梭形。③hAMCs诱导2 w和4 w表达α-辅肌动蛋白和心肌特异性转录因子Nkx2.5。④诱导前后的hAMCs均表达结蛋白和GATA-4,但均未见α-MHC表达。说明hAMCs具有向心肌样细胞分化的能力,可望成为细胞心肌成形术(cellular cardiomyoplasty,CCM)的候选细胞。 相似文献