胎儿肺脏来源间充质干细胞的鉴定与损伤修复的实验研究

目的 :为研究胎儿肺脏来源间充质干细胞的生物学性状 ,表型和多向分化能力。方法 :取胎龄为 4～ 5个月水囊引产胎儿 ,将肺脏细胞在SF(含 2 %FBS)培养基中培养。测定生长曲线、利用流式细胞仪对培养细胞进行表型测定 ,细胞周期分析 ,体外诱导分化实验。NOD SCID鼠放射损伤后 ,尾静脉输入经PKH2 6染色的间充质干细胞 ,两个月后检测外源细胞在肺脏的定植情况。结果 :从胎儿肺脏可培养出间充质干细胞 ,并可诱导成骨、软骨和脂肪细胞分化 ;移植两个月后可以检测到外源细胞在肺脏的定植。结论 :从胎儿肺脏可分离培养出间充质干细胞 ,在体外有效扩增且保持其低分化状态 ;间充质干细胞可以在肺脏长时间定植。     

人尿源性干细胞及人脐带间充质干细胞的生物学性状比较

该研究探讨人尿源性干细胞(human urine-derived stem cells,hUSCs)及人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUC-MSCs)的生物学性状差异。分离培养hUSCs及hUC-MSCs,显微镜下观察细胞形态,流式细胞术检测干细胞表面标记物,锥虫蓝拒染实验及克隆形成实验检测细胞增殖能力,划痕实验及Transwell迁移实验检测细胞迁移能力,碱性磷酸酶(alkaline phosphatase,ALP)染色、茜素红染色、油红O染色及阿利新蓝染色评估多向分化潜能。hUSCs为米粒状贴壁生长细胞,hUC-MSCs为长梭形贴壁细胞,呈旋涡状排列生长,两种细胞表型分析相似,均表达多种间充质干细胞标志物,但CD24在hUC-MSCs表达阳性,而CD105在hUSCs表达阳性。hUC-MSCs的增殖及迁移能力优于hUSCs,但后者的克隆形成能力更强。hUSCs及hUCMSCs都具有成骨、成脂、成软骨分化能力,hUC-MSCs的成骨能力强而hUSCs的成脂能力强。该研究成功分离培养出增殖能力强并具有多向分化潜能的hUSCs,该细胞与hUC-MSCs相比具有相似的生物学性状,可作为再生医学自体移植的理想种子细胞来源。     

诱导人脐带间充质干细胞分化为视网膜色素上皮样细胞的研究

目的：探讨用猪视网膜色素上皮细胞（RPE）条件培养基诱导hUCMSCs分化为RPE样细胞的方法。方法通过流式细胞技术鉴定hUCMSCs的表面标记分子;通过诱导hUCMSCs分化成为脂肪、骨和软骨细胞确定其多系分化能力;将hUCMSCs培养在猪RPE的条件培养液中并添加诱导因子来诱导hUCMSCs向RPE细胞分化。对照组与处理组Q-PCR结果采用t检验比较。结果 hUCMSCs表达CD105、CD90、CD73、CD44和CD29,但不表达CD34,CD45和MHCII等分子标记,在成脂、成骨、成软骨分化培养基中可分化为脂肪、骨和软骨细胞。单独使用猪RPE条件培养液不能有效诱导hUCMSCs向RPE细胞分化,但联合应用猪RPE条件培养液和诱导因子（视黄酸,activin-A和人重组骨形成蛋白-7）可有效诱导hUCMSCs分化为RPE样细胞,RPE细胞标记分子RPE65、Mitf和Ck8/18的基因表达量分别提高了2.1±0.4、6.8±1.3和2.5±0.3倍（P〈0.05）。诱导产生的RPE样细胞呈多边形,但不含色素颗粒。结论猪RPE条件培养液联合诱导因子可有效诱导hUCMSCs分化为RPE样细胞,可能会为治疗视网膜变性疾病提供合适的种子细胞。     

大鼠脂肪间充质干细胞的鉴定及肝内移植的研究

目的:从脂肪组织中获取间充质干细胞(ADMSCs)并验证其多向分化潜能,探讨ADMSCs在肝再生中的作用。方法:获取大鼠脂肪组织,用胶原酶消化法获取干细胞,并进行体外扩增、传代,取第3代细胞分别用不同诱导培养液进行成骨、成脂诱导,诱导后通过细胞形态学和特殊染色观察诱导效果。用PKH26标记细胞,制作部分肝切除模型,将标记的自体ADMSCs经门静脉植入体内,2周后切下取肝脏制成冰冻切片,荧光显微镜观察植入细胞在肝脏的定位,免疫荧光染色观察其白蛋白的表达。结果:从脂肪组织中分离出的细胞能在体外大量扩增,能被诱导分化为成骨细胞、脂肪细胞,ADMSCs移植2周后,可见PKH26标记细胞散在分布于肝内,免疫荧光染色显示标记细胞白蛋白染色阳性。结论:大鼠脂肪组织中可以获取具有多向分化潜能的间充质干细胞,该细胞在肝再生环境中能向肝细胞分化,参与肝再生。     

成体心脏干细胞经静脉移植后在心梗小鼠体内的分布研究

目的：观察心肌梗死小鼠静脉移植成体心脏干细胞后,细胞在小鼠体内各器官的分布情况。明确心梗后静脉移植成体心脏干 细胞在小鼠体内的分布和归巢情况。方法：分离培养小鼠心脏成体干细胞,采用流式细胞仪鉴定细胞,通过亲脂性染料CM-DiI标 记细胞后行小鼠急性心肌梗死模型建立和细胞移植,分别在细胞移植后7、14、8 天取小鼠心脏、肝脏、脑、脊髓、肺脏,行冰冻切 片,在荧光显微镜下观察移植细胞在各组织器官存活和分布情况。结果：成体心脏干细胞分离培养后呈贴壁生长,流式细胞仪检 测显示细胞纯度>80%。CM-DiI标记后荧光显微镜下观察可见标记的细胞胞浆胞核均被染成呈明亮的红色。心肌梗死后经静脉 移植成体心脏干细胞,细胞在各组织中分布呈变化过程,7 天时,在肺脏和肝脏分布较多,至14 天和28 天时,肺脏和肝脏分布减 少,心脏分布逐渐增多,表现出向心脏的" 归巢" 现象,而脑和脊髓在28 天的观察时间内分布较少。结论：采用CM-DiI标记心 脏成体干细胞,操作简单,标记效果好,可用于短期的细胞体内追踪。小鼠心肌梗死后行经静脉成体心脏干细胞移植,28 天后细胞 在心脏的分布逐渐增多,表现出向心脏的" 归巢" 现象。     

长期培养人脐带间充质干细胞分化能力改变的研究

目的:分析体外传代培养至23代,人脐带间充质干细胞(human umbilical cord-MSCs, hUC-MSCs)多向分化能力的改变,以探索hUC-MSCs体外诱导分化的最佳时间窗。方法:采用胶原酶消化法提取hUC-MSCs,并用胰酶消化传代;收集不同代细胞,用流式细胞术检测细胞表面抗原及细胞周期;MTT法检测不同代细胞的增殖活性;取不同代细胞进行成骨、成软骨及成脂肪诱导鉴定;并利用实时定量PCR分别检测OCT-4、SOX-2、Nanog的mRNA表达水平。结果:用胶原酶消化法可获得形态均一,可稳定传代23代以上的hUC-MSCs;体外传代培养至23代,细胞表面标志物表达率无明显改变;细胞生长曲线形态相似;细胞周期亦无明显差异,(73.04±1.15)%的细胞处于G0/G1期;细胞均可被诱导成骨细胞、软骨细胞和脂肪细胞;不同代细胞OCT-4、SOX-2、Nanog 的mRNA表达无显著差异。结论:体外培养至23代,随着传代次数的增加,hUC-MSCs的多向分化能力并未受影响。     

PD-1/PD-L1参与骨髓间充质干细胞归巢修复慢阻肺大鼠的肺损伤

目的 研究程序性死亡蛋白1(programmed death-1, PD-1)/程序性死亡蛋白配体1(programmed death ligand-1,PD-L1)在间充质干细胞（mesenchymal stem cells, MSCs）归巢修复慢性阻塞性肺疾病（chronic obstructive pulmonary disease,COPD）中的影响。方法 分离纯化骨髓来源MSCs,流式细胞术检测细胞表面分子表达,油红O染色、碱性磷酸酶染色分别观察MSCs成脂及成骨分化情况。采用烟气暴露法构建COPD大鼠模型,造模完成后将绿色荧光蛋白（GFP）标记的MSCs经尾静脉分别注射入正常及COPD大鼠模型中,通过观察肺部荧光评价MSCs的归巢效果;经尾静脉向COPD模型大鼠注射MSCs作为MSCs治疗组,模型组和正常对照组注射等体积生理盐水,HE染色观察各组大鼠肺脏组织病理改变,qRT-PCR比较各组大鼠肺脏组织PD-1的表达情况;体外qRT-PCR检测PD-L1在MSCs的表达,Transwell实验法观察PD-1及PD-L1抗体对MSCs定向迁移（归巢）的影响。结果 大鼠骨髓来源M...     

人体皮肤成纤维细胞的快速分离及多向分化能力鉴定

目的建立快速分离人皮肤成纤维细胞的方法,并探讨成纤维细胞在成脂、成骨、成软骨和成神经的多向分化潜能。 方法利用组织块培养法分离人体皮肤成纤维细胞,通过形态学观察、流式分析、Vimentin蛋白染色鉴定成纤维细胞;再利用生长曲线、核型分析、线粒体染色分析不同传代细胞的增殖速度,线粒体及染色体形态的改变;最后进行成纤维细胞成脂、成骨、成软骨和成神经的诱导分化实验,鉴定其多向分化潜能。两代细胞增殖速度及线料体相对量的比较采用t检验统计分析。 结果分离的皮肤成纤维细胞呈典型梭状及多角形;高表达细胞表面标记物CD90 （NCF1,NCF2占比分别为99.9%,98.7%）和CD73 （NCF1,NCF2占比分别为98.2%,85.6%）,但极少表达造血干细胞标记物CD34 （NCF1,NCF2占比分别为1.8%,2.6%）;细胞Vimentin蛋白表达呈阳性,阳性率为100%;对细胞生长曲线进行分析,表明分离后不同代次细胞增殖差异无统计学意义（t?= 1.586,P?= 0.1567）;线粒体相对含量统计分析,同一株细胞系第5代（相对荧光强度值：6876±577.8）与第10代（相对荧光强度值：7371±471.9）之间的差异无统计学意义（t?= 0.664,P?= 0.543）;核型分析分别显示传代后保持染色体数目为正常46条且形态无明显异常;经诱导后成纤维细胞可向成脂、成骨、成软骨和类神经分化。 结论利用组织块培养法分离出的人体皮肤成纤维细胞状态稳定,增殖能力强,具有成脂、成骨、成软骨和成神经多向分化诱导潜能,为细胞移植修复骨损伤、软骨损伤和神经损伤性疾病的临床研究提供细胞来源及实验依据。     

脐带间充质干细胞恶性分化并促进肺癌A549细胞移植瘤生长及转移

目的探讨人脐带来源的间充质干细胞(human umbilical cord mesenchymal stem cells,hUCMSCs)对裸鼠肺癌移植瘤生长及转移的影响。方法将24只裸鼠随机分为4组,分别为A549细胞单独注射组(A549组)、hUCMSCs单独注射组(MSCs组)、A549细胞与正常人皮肤成纤维细胞(skin fibroblasts,SKs)共同注射组(A549+SKs组)、hUCMSCs与A549细胞共注射组(A549+MSCs组)。HE染色和免疫组织化学染色确定hUCMSCs注射裸鼠皮下结节性质;流式细胞分选技术分选出各组移植瘤瘤组织中的A549细胞;实时荧光定量PCR和Westernblot检测A549细胞中上皮-间质转化(epithelial-mesenchymal transition,EMT)相关分子标志物的表达差异。结果 hUCMSCs注射后,hUCMSCs发生恶性分化,hUCMSCs与A549细胞共注射后肺癌移植瘤体积明显大于单独A549注射组和A549与SKs共注射组;2只A549与hUCMSCs共注射组裸鼠出现了肺癌肝脏转移,而A549组与A549+SKs组的全部6只裸鼠均没有发现肝脏转移。A549与MSCs共注射组A549细胞中上皮表型特征分子E-cadherin、β-catenin mRNA相对表达量和β-catenin蛋白表达水平高于A549组与A549+SKs组,E-cadherin蛋白表达水平明显高于A549+SKs组,间质表型特征分子N-cadherin和vimentin蛋白表达水平明显低于A549组与A549+SKs组,EMT转录调控因子twist蛋白表达水平低于A549组与A549+SKs组。结论 hUCMSCs可以自我恶性分化;hUCMSCs能促进肺癌A549细胞移植瘤的生长与转移;hUCMSCs增强A549细胞侵袭、转移的机制不是通过诱导A549细胞发生EMT(上皮-间质转化)。     

比较两种不同来源间充质干细胞的生物学特性及多向分化潜能

该文旨在比较人脂肪间充质干细胞(hADSCs)和脐带间充质干细胞(hUMSCs)的生物学特性,并鉴定其多向分化潜能。体外扩增培养hADSCs和hUMSCs,绘制细胞生长曲线并计算群体倍增时间,通过细胞集落实验比较两种细胞的增殖能力,流式细胞仪和RT-PCR方法分别检测细胞表面抗原和多能性相关基因的表达,采用成脂、成骨诱导分化试剂盒比较两种细胞的分化潜能。hADSCs和hUMSCs表达CD34、CD44、CD45、CD105的比例分别为2.7% vs 6.2%、92.3% vs 93.4%、1.3% vs 3.1%、99.4% vs 98.0%,均表达Oct4和Nanog基因;生长曲线均呈"S"型,两种细胞的群体倍增时间差异不显著(P0.05);随着代数的增加,两种细胞的增殖能力均变弱,但P7的hADSCs的增殖能力要显著优于hUMSCs(P0.05);经油红O、茜素红染色及RT-PCR和细胞免疫荧光方法检测特异基因的表达,表明两种细胞均具备成脂、成骨分化的能力,hADSCs的成脂能力优于hUMSCs,但两种间充质干细胞的成骨分化能力没有显著性差异。hADSCs和hUMSCs具有相似的生物学特性,但hADSCs可能具备更强的增殖能力和成脂分化潜能。     

Live cell imaging of highly activated natural killer cells against human hepatocellular carcinoma in vivo

Background aimsTracking administered natural killer (NK) cells in vivo is critical for developing an effective NK cell-based immunotherapy against human hepatocellular carcinoma (HCC). Here the authors established a new molecular imaging using ex vivo-activated NK cells and investigated real-time biodistribution of administered NK cells during HCC progression.MethodsEx vivo-expanded NK cells from healthy donors were labeled with a near-infrared lipophilic cytoplasmic dye, and their proliferation, surface receptor expression and cytotoxicity activity were evaluated. Human HCC HepG2 cells were implanted into the livers of NOD.Cg-Prkdc^scid IL2rg^tm1Wjl/SzJ (NSG) mice. The authors administered 1,1’-dioctadecyltetramethyl indotricarbocyanine iodide (DiR)-labeled NK cells intravenously to non-tumor-bearing and intrahepatic HCC tumor-bearing NSG mice. Fluorescent imaging was performed using a fluorescence-labeled organism bioimaging instrument. Single cell suspensions from the resected organs were analyzed using flow cytometry.ResultsThe fluorescent DiR dye was nontoxic and did not affect the proliferation or surface receptor expression levels of the NK cells, even at high doses. The administered DiR-labeled NK cells immediately migrated to the lungs of the non-tumor-bearing NSG mice, with increased NK cell signals evident in the liver and spleen after 4 h. NK cells migrated to the intrahepatic tumor-bearing livers of both early- and late-stage HCC mice within 1 h of injection. In early-stage intrahepatic tumor-bearing mice, the fluorescence signal increased in the liver until 48 h post-injection and decreased 7 days after NK injection. In late-stage HCC, the NK cell fluorescence signal was the highest in the liver for 7 days after NK injection and persisted for 14 days. The purity of long-term persistent CD45⁺CD56⁺CD3⁻ NK cells was highest in early- and late-stage HepG2-bearing liver compared with normal liver 2 weeks after NK injection, whereas highest purity was still observed in the lungs of non-tumor-bearing mice. In addition, Ki-67 expression was detected in migrated human NK cells in the liver and lung up to 72 h after administration. With HepG2 tumor progression, NK cells reduced the expression of NKp30 and NKG2D.ConclusionsAdministered NK cells were successfully tracked in vivo by labeling the NK cells with near-infrared DiR dye. Highly expanded, activated NK cells migrated rapidly to the tumor-bearing liver, where they persisted for 14 days after administration, with high purity of CD45⁺CD56⁺CD3⁻ NK cells. Liver biodistribution and persistence of administered NK cells showed significantly different accumulation patterns during HCC progression.     

Evaluation of different routes of administration and biodistribution of human amnion epithelial cells in mice

Placenta is a non-controversial and promising source of cells for the treatment of several liver diseases. We previously reported that transplanted human amnion epithelial cells (hAECs) differentiate into hepatocyte-like cells, resulting in correction of mouse models of metabolic liver disease or acute hepatic failure. As part of preclinical safety studies, we investigated the distribution of hAECs using two routes of administration to efficiently deliver hAECs to the liver. Optical imaging is commonly used because it can provide fast, high-throughput, whole-body imaging, thus DiR-labeled hAECs were injected into immunodeficient mice, via the spleen or the tail vein. The cell distribution was monitored using an in vivo imaging system over the next 24 h. After splenic injection, the DiR signal was detected in liver and spleen at 1, 3 and 24 h post-transplant. The distribution was confirmed by analysis of human DNA content at 24 h post-transplant and human-specific cytokeratin 8/18 staining. Tail vein infusion resulted in cell engraftment mainly in the lungs, with minimal detection in the liver. Delivery of cells to the portal vein, via the spleen, resulted in efficient delivery of hAECs to the liver, with minimal, off-target distribution to lungs or other organs.     

Near-Infrared Imaging of Adoptive Immune Cell Therapy in Breast Cancer Model Using Cell Membrane Labeling

The overall objective of this study is to non-invasively image and assess tumor targeting and retention of directly labeled T-lymphocytes following their adoptive transfer in mice. T-lymphocytes obtained from draining lymph nodes of 4T1 (murine breast cancer cell) sensitized BALB/C mice were activated in-vitro with Bryostatin/Ionomycin for 18 hours, and were grown in the presence of Interleukin-2 for 6 days. T-lymphocytes were then directly labeled with 1,1-dioctadecyltetramethyl indotricarbocyanine Iodide (DiR), a lipophilic near infrared fluorescent dye that labels the cell membrane. Assays for viability, proliferation, and function of labeled T-lymphocytes showed that they were unaffected by DiR labeling. The DiR labeled cells were injected via tail vein in mice bearing 4T1 tumors in the flank. In some cases labeled 4T1 specific T-lymphocytes were injected a week before 4T1 tumor cell implantation. Multi-spectral in vivo fluorescence imaging was done to subtract the autofluorescence and isolate the near infrared signal carried by the T-lymphocytes. In recipient mice with established 4T1 tumors, labeled 4T1 specific T-lymphocytes showed marked tumor retention, which peaked 6 days post infusion and persisted at the tumor site for up to 3 weeks. When 4T1 tumor cells were implanted 1-week post-infusion of labeled T-lymphocytes, T-lymphocytes responded to the immunologic challenge and accumulated at the site of 4T1 cell implantation within two hours and the signal persisted for 2 more weeks. Tumor accumulation of labeled 4T1 specific T-lymphocytes was absent in mice bearing Meth A sarcoma tumors. When lysate of 4T1 specific labeled T-lymphocytes was injected into 4T1 tumor bearing mice the near infrared signal was not detected at the tumor site. In conclusion, our validated results confirm that the near infrared signal detected at the tumor site represents the DiR labeled 4T1 specific viable T-lymphocytes and their response to immunologic challenge can be imaged in vivo.     

Applications of mesenchymal stem cells labeled with Tat peptide conjugated quantum dots to cell tracking in mouse body

Fluorescent quantum dots have great potential in cellular labeling and tracking. Here, PEG encapsulated CdSe/ZnS quantum dots have been conjugated with Tat peptide, and introduced into living mesenchymal stem cells. The Tat peptide conjugated quantum dots in mesenchymal stem cells were assessed by fluorescent microscopy, laser confocal microscope and. flow cytometry. The result shows that Tat peptide conjugated quantum dots could enter mesenchymal stem cells efficiently. The Tat-quantum dots labeled stem cells were further injected into the tail veins of NOD/SCID beta2 M null mice, and the tissue distribution of these labeled cells in nude mice were examined with fluorescence microscope. The result shows that characteristic fluorescence of quantum dots was observed primarily in the liver, the lung and the spleen, with little or no quantum dots accumulation in the brain, the heart, or the kidney.     

Ultrasound-Triggered Phase Transition Sensitive Magnetic Fluorescent Nanodroplets as a Multimodal Imaging Contrast Agent in Rat and Mouse Model

Ultrasound-triggered phase transition sensitive nanodroplets with multimodal imaging functionality were prepared via premix Shirasu porous glass (SPG) membrane emulsification method. The nanodroplets with fluorescence dye DiR and SPIO nanoparticles (DiR-SPIO-NDs) had a polymer shell and a liquid perfluoropentane (PFP) core. The as-formed DiR-SPIO-NDs have a uniform size of 385±5.0 nm with PDI of 0.169±0.011. The TEM and microscopy imaging showed that the DiR-SPIO-NDs existed as core-shell spheres, and DiR and SPIO nanoparticles dispersed in the shell or core. The MTT and hemolysis studies demonstrated that the nanodroplets were biocompatible and safe. Moreover, the proposed nanodroplets exhibited significant ultrasound-triggered phase transition property under clinical diagnostic ultrasound irradiation due to the vaporization of PFP inside. Meanwhile, the high stability and R₂ relaxivity of the DiR-SPIO-NDs suggested its applicability in MRI. The in vivo T₂-weighted images of MRI and fluorescence images both showed that the image contrast in liver and spleen of rats and mice model were enhanced after the intravenous injection of DiR-SPIO-NDs. Furthermore, the ultrasound imaging (US) in mice tumor as well as MRI and fluorescence imaging in liver of rats and mice showed that the DiR-SPIO-NDs had long-lasting contrast ability in vivo. These in vitro and in vivo findings suggested that DiR-SPIO-NDs could potentially be a great MRI/US/fluorescence multimodal imaging contrast agent in the diagnosis of liver tissue diseases.     

Heterogeneity of protein labeling with a fluorogenic reagent, 3-(2-furoyl)quinoline-2-carboxaldehyde

Fluorogenic reagents are used for protein labeling when high-sensitivity fluorescence detection is required. Similar to traditional labeling with activated fluorescent dyes, such as fluorescein isothiocyanate, a fluorogenic reaction is expected to change the physical-chemical properties of proteins. Knowledge of these changes may be essential for efficient separation and identification of labeled proteins. Here we studied the effect of labeling of myoglobin with a fluorogenic reagent on the acid-base properties of the protein. The fluorogenic reagent used was 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ). In slab-gel isoelectric focusing, we found that the labeling reaction generated at least six species with pI values lower than that of non-labeled myoglobin. These species can be identified as products of progressive labeling of myoglobin with one to six FQ molecules. The same series of FQ-labeled species were observed when the reaction products were analyzed by capillary zone electrophoresis. The comparison of experimental and theoretical pI values allowed us to elucidate the labeling pattern--the number of FQ molecules corresponding to each labeled product detected by isoelectric focusing.     

Stem-like and non-stem human pancreatic cancer cells distinguished by morphology and metastatic behavior

We report here that XPA1 human pancreatic cancer cells are dimorphic. After injection in the spleen, XPA1 cells isolated from the primary tumor in the spleen were predominantly round; while cells isolated from the resulting liver metastasis and ascites were comprised of both round- and spindle-shaped cell types. Cancer cells previously grown in the spleen and re-implanted in the spleen developed large primary tumors in the spleen only. Cancer cells isolated from liver metastasis and re-transplanted to the spleen resulted in a primary tumor in the spleen and liver metastasis. Cancer cells derived from ascites and re-transplanted to the spleen developed primary tumors in the spleen and distant metastasis in the liver, lung, and diaphragm in addition to ascites formation. Spindle and round cells were differentially labeled with fluorescent proteins of different colors. After co-injection of the two cell types in the spleen, cells were isolated from the primary tumors, liver metastasis, and ascites and analyzed by color-coded fluorescence microscopy and fluorescence-activated cell sorting (FACS). No significant differences between the percentages of spindle-shaped and round cancer cells in the primary tumor and the liver metastasis were observed. However, spindle-shaped cancer cells were enriched in the ascites. One hundred percent of the spindle-shaped and round cancer cells expressed CD44, suggesting that morphology and metastatic behavior rather than CD44 expression can distinguish the stem-like cells of the XPA1 pancreatic cancer cell line. The spindle-shaped cancer cells had the greater capability for distant metastasis and ascites formation, suggesting they are stem-like cells, which can be readily targeted for therapy.     

Synergistic effect of bioactive lipid and condition medium on cardiac differentiation of human mesenchymal stem cells from different tissues

Human umbilical cord mesenchymal stem cells (hUCMSCs) and human adipose tissue mesenchymal stem cells (hATMSCs) have the potential to differentiate into cardiomyocytes, making them promising therapeutic candidates for treating damaged cardiac tissues. Currently, however, the differentiated cells induced from hUCMSCs or hATMSCs can hardly display functional characteristics similar to cardiomyocytes. In this study, we have investigated the effects of bioactive lipid sphingosine‐1‐phosphate (S1P) on cardiac differentiations of hUCMSCs and hATMSCs in condition medium composed of cardiac myocytes culture medium or 5‐azacytidine. Cardiac differentiations were identified through immunofluorescence staining, and the results were observed with fluorescence microscopy and confocal microscopy. Synergistic effects of S1P and condition medium on cell viability were evaluated by MTT assays. Functional characteristics similar to cardiomyocytes were evaluated through detecting calcium transient. The differentiated hUCMSCs or hATMSCs in each group into cardiomyocytes showed positive expressions of cardiac specific proteins, including α‐actin, connexin‐43 and myosin heavy chain‐6 (MYH‐6). MTT assays showed that suitable differentiation time was 14 days and that the optimal concentration of S1P was 0.5 μM. Moreover, incorporation of S1P and cardiac myocytes culture medium gave rise to calcium transients, an important marker for displaying in vivo electrophysiological properties. This feature was not observed in the S1P‐5‐azacytidine group, indicating the possible lack of cellular stimuli such as transforming growth factor‐beta, TGF‐β. © 2016 The Authors. Cell Biochemistry and Function published by John Wiley & Sons, Ltd.     

Retinol-binding protein 4 is expressed in chondrocytes of developing mouse long bones: implications for a local role in formation of the secondary ossification center

Retinol-binding protein 4 (Rbp4) is the major carrier of retinol in the bloodstream, a retinoid whose metabolites influence osteogenesis, chondrogenesis and adipogenesis. Rbp4 is mainly produced in the liver where it mobilizes hepatic retinol stores to supply other tissues. However, Rbp4 is also expressed in several extrahepatic tissues, including limbs, where its role is largely unknown. This study aimed to identify the cellular localization of Rbp4 to gain insight into its involvement in limb development and bone growth. Using immunohistochemistry, we discovered that Rbp4 was present in a variety of locations in developing embryonic and postnatal mouse hindlimbs. Rbp4 was present in a restricted population of epiphyseal chondrocytes and perichondral cells correlating to the future region of secondary ossification. With the onset of secondary ossification, Rbp4 was detected in chondrocytes of the resting zone and in chondrocytes that bordered invading cartilage canals and the expanding front of ossification. Rbp4 was less abundant in proliferating chondrocytes involved in primary ossification. Our data implicate the involvement of chondrocytic Rbp4 in bone growth, particularly in the formation of the secondary ossification center of the limb.