β-ME和BHA体外诱导入胎肝CD34^＋细胞向神经组织细胞分化研究

目的：建立巯基乙醇(β-mercaptoethanol,β-ME)和丁羟回醚(butylated hydroxyanisole,BHA)体外诱导入胎肝(fetal liver,FL)干细胞向神经细胞分化模型。方法：采用MACS试剂盒分离人胚胎肝CD34^ 细胞,以DMEM 10％胎牛血清培养液培养;第五代细胞待细胞融合达80％后,用DMEM 10％胎牛血清 1mmol／Lβ-ME 0．2mmol／L BHA诱导24h,PBS洗涤。然后在无血清培养基中培养5h～5d。用免疫细胞化学方法分析诱导前后的细胞表型特点。结果：经β-ME BHA诱导处理后,细胞表现神经元样细胞形态,表达神经组织细胞特异蛋白,如neustin、NeuN、NF-M、TuJ-1和NSE。统计显示81％细胞NeuN染色阳性,75％细胞TuJ-1染色阳性,47％染色NF-M阳性,90％染色NSE阳性。结论：β-ME和BHA能够诱导体外培养的人FL CD34^ 细胞分化为具有神经细胞特异性抗原和成分的神经样细胞;胚胎肝细胞具有向神经组织分化的潜能。     

PI3K活性与BHA诱导小鼠胎肝细胞表达神经组织细胞特异基因有关

目的：了解丁羟回醚（BHA）对小鼠胎肝（FL）细胞神经组织特异基因表达的影响及其信号途径。方法：小鼠胎肝细胞,以DMEM／F12＋10％胎牛血清培养液培养;第4d后,去悬浮细胞,留黏附细胞,加入或者不加入磷酸肌醇3羟基激酶（PI3K）抑制剂LY294002（20μmol／L）处理24h,再加入BHA至终浓度0．2mmol／L,然后继续培养5d。用Western blot和半定量RT—PCR方法分析BHA处理前后神经组织细胞特异基因表达。结果：胎肝细胞本身表达神经组织特异基因水平较低或者不表达。BHA则促进了胎肝细胞内神经组织特异基因表达：NF-L mRNA增加5．8倍、NF—H mRNA增加8．0倍、TH mRNA增加30倍、BF-1 mRNA增加2．68倍;NF-L蛋白增加11．29倍、NF—H蛋白增加5,5倍、BF-1蛋白增加2．53倍、TH蛋白增加4．76倍。而LY294002能明显抑制BHA诱导的神经组织细胞特异蛋白NF—L、NF-H、BF-1和TH的表达。结论：PI3K活性与BHA诱导小鼠胎肝细胞表达神经组织细胞特异结构和功能基因有关。     

大鼠骨髓基质干细胞的分离培养与向神经元样细胞诱导分化的实验研究

目的 探讨大鼠骨髓基质干细胞的提取、分离培养和体外扩增的最佳条件,研究其在体外培养中定向诱导分化为神经元样细胞的可能。方法 通过密度梯度离心和贴壁培养法从成年大鼠骨髓中分离骨髓基质干细胞,进行培养扩增,观察其生长特性;用2-巯基乙醇(β-mercaptoethanol,β-ME)对传代细胞诱导分化,并通过免疫细胞化学染色鉴定分化细胞的类型。结果 原代培养时形成由基质干细胞组成的细胞集落,细胞集落14d时接近融合,传代后,细胞体积变大,约5～7d传代一次。β-ME诱导后,70％以上的细胞在形态上呈神经元样,免疫细胞化学染色呈NSE阳性,GFAP阴性,说明诱导分化的细胞为神经元,而不是星形胶质细胞。结论 骨髓基质干细胞在体外培养条件下生长良好,并可连续传代;在β-ME作用下可被诱导分化为神经元样细胞。     

器官型脑片培养液对BMSCs向神经元样细胞分化的诱导作用

目的在体外培养大鼠骨髓间充质干细胞(bone mesenchymalstem cells,BMSCs),观察BMSCs的形态特征和分类,并诱导其向神经细胞分化,探讨BMSCs向神经细胞诱导分化的可能性和条件。方法取大鼠骨髓,用直接贴壁法培养、传代,取第3代细胞(P3)进行CD34,CD71免疫细胞化学染色鉴定。BMSCs(P3)诱导前24h加1ug.L-1碱性成纤维细胞生长因子(bFGF)以促分裂。再以脑片培养液作为条件培养液进行诱导,随后进行Nissl、神经元特异性烯醇化酶(NSE),胶质原纤维酸性蛋白(GFAP)免疫细胞化学染色。结果BMSCs中含有较小的长梭形细胞,较大的扁平细胞及中等大小的圆形细胞,经鉴定CD34阴性,CD71弱阳性;诱导后BMSCs中长梭形细胞形态呈神经元样外形,Nissl的表达上调,NSE阳性,GFAP阴性。较大的扁平细胞形态变化不明显。结论BMSCs中含有多种细胞,细胞放置于脑片培养液微环境中可以分化为神经细胞,具有治疗神经系统疾病的潜能。     

神经上皮干细胞的分离培养及其体外分化特性的观察

目的探讨大鼠胚胎神经管神经上皮干细胞的分离培养条件,并观察其在体外的分化特性.方法采用显微解剖、机械吹打、无血清悬浮培养方法分离培养神经上皮干细胞,采用巢蛋白(nestin)免疫细胞化学染色技术检测神经上皮干细胞,用NSE和GFAP免疫组化染色检测并计数神经细胞和神经胶质细胞.结果大鼠胚胎神经管神经上皮干细胞在无血清培养基中可形成大量呈nestin抗原阳性细胞构成的神经球,经传代有血清培养后分化为NSE阳性和GFAP阳性细胞,其中NSE阳性细胞占细胞总数的47.7%,GFAP阳性细胞占细胞总数的39.8%.结论胎鼠神经管神经上皮干细胞在无血清培养中可增殖和传代,在有血清培养中可分化为神经细胞和神经胶质细胞,两者之比为47.7∶39.8.     

大鼠脂肪干细胞向雪旺细胞诱导分化能力的研究

目的：研究脂肪干细胞（ADSCs）向雪旺细胞的诱导分化,为神经组织工程提供新的种子细胞。方法：取SD大鼠项背处的皮下脂肪,分离出脂肪干细胞并培养传代,流式细胞仪检测细胞表面特异标记CD29,CD34,CD44,CD45,CD90,以评价干细胞的生物学特性;采用b-FGF和forskolin等诱导脂肪干细胞向雪旺细胞分化,光镜观察诱导后细胞形态的变化;免疫荧光染色鉴定雪旺细胞特异性标记物S100、P75和GFAP的表达;PCR检测诱导前后雪旺细胞特异性标记物S100、P75的表达。结果：分离培养的鼠脂肪干细胞CD29、CB90表达呈阳性,而CD34、CD44和CD45表达呈阴性,具有脂肪干细胞的生物学特性;脂肪干细胞经过胶质细胞生长因子的作用,光镜下发现诱导的细胞形态与雪旺细胞相似;免疫荧光染色S100、P75和GFAP阳性;RT-PCR结果显示诱导的雪旺细胞标记物S100和P75表达上调。结论：脂肪干细胞可诱导分化成雪旺细胞,其表型和分子特征与雪旺细胞相似,诱导分化的脂肪干细胞是一种理想的神经组织工程的种子细胞。     

化学方法体外诱导大鼠骨髓间充质干细胞成神经样细胞分化

目的:研究化学方法体外诱导大鼠骨髓间充质干细胞向神经样细胞的可持续性分化。方法:体外培养大鼠骨髓间充质干细胞至P5代,流式细胞术检测细胞表面标志物,将细胞分为丁羟茴醚(BHA)诱导组、β巯基乙醇(BME)诱导组和对照组,分别进行化学诱导分化,通过荧光定量PCR、Western印迹和免疫细胞化学法分别检测巢蛋白、神经元特异性烯醇化酶(NSE)、胶质原纤维酸性蛋白(GFAP)等神经细胞标志分子的表达情况。结果:BHA诱导组高表达GFAP,而BME诱导组高表达巢蛋白,2组NSE表达均较弱;同时在诱导结束后继续培养的过程中,2组神经标志分子表达量持续降低。结论:2种方法都能诱导骨髓间充质干细胞向神经样细胞分化,但分化结局不同,BHA诱导分化后细胞主要表达星形胶质细胞表面标志GFAP,而BME诱导分化后细胞主要表达神经前体细胞表面标志巢蛋白;此外,通过化学方法诱导分化的效果是不可持续的,在诱导结束后继续培养的过程中神经标志分子表达逐渐下调。     

一种简便的小鼠精原干细胞分离培养方法

采用一步酶消化法分离小鼠精原干细胞,比较α-MEM、DMEM培养基对体外培养的精原干细胞生长状态的影响,对精原干细胞集落进行形态观察、碱性磷酸酶(alkaline phosphatase,AKP)染色和免疫组化鉴定,并诱导精原干细胞向精子细胞分化。结果显示,以小鼠胚胎成纤维细胞作为饲养层,用α-MEM培养的精原干细胞集落较大且呈葡萄串状或念珠状,细胞状态较好;小鼠精原干细胞集落的AKP染色阳性呈紫红色;在红色荧光下精原干细胞集落的Oct-4核蛋白表达为阳性、膜蛋白c-Kit、β_1-integrin和Gfrα-1表达为阳性;精原干细胞经维甲酸(all-trans-retinoic-acid,RA)诱导可初步分化成精子样细胞。因此,采用一步酶消化法能够分离小鼠精原干细胞,α-MEM更适合小鼠精原干细胞体外培养。     

单克隆人胰腺干细胞分化特性的研究

研究1例来源于4月龄男性流产胎儿胰腺组织的单克隆人胰腺干细胞（monoclonal human pancreatic stem cell,mhPSC）系的体内外分化特性。将mhPSCs接种在铺有0．1％明胶的培养皿内,扩增培养3d后,加高糖DMEM诱导液诱导培养25d。相差显微镜下．观察细胞生长状况。采用双硫腙染色法、RT—PCR及葡萄糖刺激释放胰岛素和C肽实验．对体外定向诱导mhPSCs分化为功能性胰岛进行检测。将mhPSCs悬液注射在成年雄性裸鼠腹股沟皮下．注射30d时,取出移植物,采用SP法进行免疫组织化学反应,以检测mhPSCs的体内自然分化潜能。体外扩增培养,mhPSCs贴壁生长,呈多角形上皮样。生长至单层．呈“铺路石”状。体外定向诱导,细胞逐渐由多角形变成圆形,并聚集成类胰岛。诱导培养15d时．形成的类胰岛中少数细胞分化为B细胞,双硫腙染色阳性。诱导培养25d时,多数细胞分化为8细胞,双硫腙染色阳性,转录表达胰岛素的mRNA。用不同浓度葡萄糖刺激．诱导胰岛不仅释放胰岛素和C肽,而且其释放量随糖刺激浓度升高显著增加（0．01〈P〈0．05）。体内分化实验显示,mhPSCs在裸鼠背部形成类畸胎瘤。类畸胎瘤易与裸鼠分离,色白,血管丰富。显著表达pdx1、胰岛素、胰高血糖素、CK、MBP及NF蛋白。该研究结果证实单克隆人胰腺干细胞系体外定向诱导分化为包含大量β细胞的功能性类胰岛,在体内自然分化为胰岛、上皮及神经组织细胞。     

分离与培养人脂肪组织间充质干细胞的研究

目的：探索人脂肪组织源性间充质干细胞（ASCs）的分离、体外培养,为其广泛应用提供实验依据。方法：无菌条件下获取腹部手术病人皮下脂肪组织,酶消化法分离、培养ASCs,观察细胞形态并绘制细胞生长曲线,计算细胞群体倍增时间;对第2代细胞进行免疫组织化学染色,鉴定其表面分子CD44表达;取2—4代细胞用含体积分数为10％胎牛血清、1％青链霉素原液、1μmmol／L地塞米松、10μmmol／L胰岛素、0．5mmmol／LIBMX的高糖DMEM培养基中诱导培养一周,观察细胞形态变化,并用油红“O”染色定性。结果：人脂肪组织中含有大量间充质干细胞,呈成纤维细胞样贴壁生长,细胞群体倍增时间为55h左右;免疫化学染色鉴定CD44阳性;成脂诱导分化一周,可见细胞内有大量脂滴,油红“0”染色可见胞浆内有大量红染颗粒。结论：建立了一种自人体脂肪组织分离,培养ASCs经济简便的方法,为其能够作为组织工程理想的种子细胞及广泛应用于临床提供实验依据。     

Th17细胞和Treg细胞与动脉粥样硬化

动脉粥样硬化从脂质条纹的形成到更复杂的病变和斑块破裂的进程是由多种不同类型的细胞和细胞因子网络共同参与作用的,其中最主要的是Th17细胞和Treg细胞及它们分泌的细胞因子。大量研究显示,Th17细胞对动脉粥样硬化的作用仍存在争议,但大部分研究仍认为其具有促动脉粥样硬化的作用。Treg细胞具有抗动脉粥样硬化的作用,Th17／Treg平衡对动脉粥样硬化的发生和发展具有重要的调节作用。本文将对Th17细胞、Treg细胞的生物学特性以及Th17细胞、Treg细胞和Th17／Treg平衡对动脉粥样硬化影响的最新研究进展做一综述。     

Induction of oocyte-like cells from mouse embryonic stem cells by co-culture with ovarian granulosa cells

Abstract In vitro derivation of oocytes from embryonic stem (ES) cells has the potential to be an important tool for studying oogenesis as well as advancing the field of therapeutic cloning by providing an alternative source of oocytes. Here, we demonstrate a novel, two-step method for inducing mouse ES cells to differentiate into oocyte-like cells using mouse ovarian granulosa cells. First, primordial germ cells (PGCs) were differentiated within the embryonic body (EB) cells around day 4 as defined by the expression of PGC-specific markers and were distinguished from undifferentiated ES cells. Second, day 4 EB cells were co-cultured with ovarian granulosa cells. After 10 days, these cells formed germ cell colonies as indicated by the expression of the two germ cell markers Mvh and SCP3. These cells also expressed the oocyte-specific genes Fig α, GDF-9 , and ZP1-3 but not any testis-specific genes by RT-PCR analysis. EB cultured alone or cultured in granulosa cell-conditioned medium did not express any of these oocyte-specific markers. In addition, EB co-cultured with Chinese hamster ovary (CHO) cells or cultured in CHO cell-conditioned medium did not express all of these oocyte-specific markers. Immunocytochemistry analysis using Mvh and GDF-9 antibodies confirmed that some Mvh and GDF-9 double-positive oocyte-like cells were generated within the germ cell colonies. Our results demonstrate that granulosa cells were effective in inducing the differentiation of ES cell-derived PGCs into oocyte-like cells through direct cell-to-cell contacts. Our method offers a novel in vitro system for studying oogenesis; in particular, for studying the interactions between PGCs and granulosa cells.     

Early-outgrowth of endothelial progenitor cells can function as antigen-presenting cells

Endothelial progenitor cells (EPCs) have been recently found to exist circulating in peripheral blood of adults, and home to sites of neovascularization in peripheral tissues. They can also be differentiated from peripheral blood mononuclear cells (PBMNCs). In tumor tissues, EPCs are found in highly vascularized lesions. Few reports exist in the literature concerning the characteristics of EPCs, especially related to their surface antigen expressions, except for endothelial markers. Here, we aimed to investigate the surface expression of differentiation markers, and the functional activities of early-outgrowth of EPCs (EO-EPCs), especially focusing on their antigen-presenting ability. EO-EPCs were generated from PBMNCs, by culture in the presence of angiogenic factors. These EO-EPCs had the morphological and functional features of endothelial cells and, additionally, they shared antigen-presenting ability. They induced the proliferation of allogeneic lymphocytes in a mixed-lymphocyte reaction, and could generate cytotoxic lymphocytes, with the ability to lyze tumor cells in an antigen-specific manner. The antigen-presenting ability of EO-EPCs, however, was weaker than that of monocyte-derived dendritic cells, but stronger than peripheral blood monocytes. Since EO-EPCs play an important role in the development of tumor angiogenesis, targeting EPCs would be an effective anti-angiogenic strategy. Alternatively, due to their antigen-presenting ability, EO-EPCs can be used as the effectors of anti-tumor immunotherapy. Since they share endothelial antigens, the activation of a cellular immunity against angiogenic vessels can be expected. In conclusion, EO-EPCs should be an interesting alternative for the development of new therapeutic strategies to combat cancer, either as the effectors or as the targets of cancer immunotherapy.     

Identification of stage-specific markers during differentiation of hair cells from mouse inner ear stem cells or progenitor cells in vitro

The induction of inner ear hair cells from stem cells or progenitor cells in the inner ear proceeds through a committed inner ear sensory progenitor cell stage prior to hair cell differentiation. To increase the efficacy of inducing inner ear hair cell differentiation from the stem cells or progenitor cells, it is essential to identify comprehensive markers for the stem cells/progenitor cells from the inner ear, the committed inner ear sensory progenitor cells and the differentiating hair cells to optimize induction conditions. Here, we report that we efficiently isolated and expanded the stem cells or progenitor cells from postnatal mouse cochleae, and induced the generation of inner ear progenitor cells and subsequent differentiation of hair cells. We profiled the gene expression of the stem cells or progenitor cells, the inner ear progenitor cells, and hair cells using aRNA microarray analysis. The pathway and gene ontology (GO) analysis of differentially expressed genes was performed. Analysis of genes exclusively detected in one particular cellular population revealed 30, 38, and 31 genes specific for inner ear stem cells, inner ear progenitor cells, and hair cells, respectively. We further examined the expression of these genes in vivo and determined that Gdf10+Ccdc121, Tmprss9+Orm1, and Chrna9+Espnl are marker genes specific for inner ear stem cells, inner ear progenitor cells, and differentiating hair cells, respectively. The identification of these marker genes will likely help the effort to increase the efficacy of hair cell induction from the stem cells or progenitor cells.     

Autophagy in stem cells

Autophagy is a highly conserved cellular process by which cytoplasmic components are sequestered in autophagosomes and delivered to lysosomes for degradation. As a major intracellular degradation and recycling pathway, autophagy is crucial for maintaining cellular homeostasis as well as remodeling during normal development, and dysfunctions in autophagy have been associated with a variety of pathologies including cancer, inflammatory bowel disease and neurodegenerative disease. Stem cells are unique in their ability to self-renew and differentiate into various cells in the body, which are important in development, tissue renewal and a range of disease processes. Therefore, it is predicted that autophagy would be crucial for the quality control mechanisms and maintenance of cellular homeostasis in various stem cells given their relatively long life in the organisms. In contrast to the extensive body of knowledge available for somatic cells, the role of autophagy in the maintenance and function of stem cells is only beginning to be revealed as a result of recent studies. Here we provide a comprehensive review of the current understanding of the mechanisms and regulation of autophagy in embryonic stem cells, several tissue stem cells (particularly hematopoietic stem cells), as well as a number of cancer stem cells. We discuss how recent studies of different knockout mice models have defined the roles of various autophagy genes and related pathways in the regulation of the maintenance, expansion and differentiation of various stem cells. We also highlight the many unanswered questions that will help to drive further research at the intersection of autophagy and stem cell biology in the near future.     

Lysis of endothelial cells by autologous lymphokine-activated killer cells

The mechanisms of lysis of endothelial cells derived from human umbilical vein (HUVEC) by autologous lymphokine-activated killer (LAK) cells, generated from cord blood lymphocytes of the same donor, were investigated. Freshly isolated HUVEC as well as HUVEC cultured for several passages were efficiently lysed by autologous LAK cells, and their susceptibility to the LAK cells was almost the some as that of allogenic HUVEC. Complement-depletion experiments revealed that the lysis was mainly dependent on CD16-natural killer (NK) LAK cells. Pretreatment of HUVEC with recombinant interferon (rIFN) for 24 h made them resistant to lysis by autologous LAK cells, while pretreatment with either rIL-1. rTNF, or acidic or basic fibroblast growth factor did not alter the lytic sensitivity of HUVEC. The resistance of rIFN-treated HUVEC was specific to lysis by CD16⁺ NK LAK cells, and their lysis by CD3⁺ T-LAK cells was not significantly altered. Moreover, in comparison with control HUVEC or rIL-1-treated HUVEC, rIFN-treated HUVEC had a significantly less potent inhibitory effect on the lysis of untreated HUVEC, when used as an unlabeled target. This suggests that rIFN treatment may down-regulate the recognition of some molecules on HUVEC by rIL-2-activated NK cells. These data suggest that damage of the endothelium during LAK therapy is mainly dependent on LAK cells with a NK phenotype that can specifically recognize a certain molecule on autologous endothelial cells.     

Adipose-derived stem cells and cancer cells fuse to generate cancer stem cell-like cells with increased tumorigenicity

Adipose-derived stem cells (ADSCs) are a type of mesenchymal stem cells isolated from adipose tissue and have the ability to differentiate into adipogenic, osteogenic, and chondrogenic lineages. Despite their great therapeutic potentials, previous studies showed that ADSCs could enhance the proliferation and metastatic potential of breast cancer cells (BCCs). In this study, we found that ADSCs fused with BCCs spontaneously, while breast cancer stem cell (CSC) markers CD44⁺CD24^-/lowEpCAM⁺ were enriched in this fusion population. We further assessed the fusion hybrid by multicolor DNA FISH and mouse xenograft assays. Only single nucleus was observed in the fusion hybrid, confirming that it was a synkaryon. In vivo mouse xenograft assay indicated that the tumorigenic potential of the fusion hybrid was significantly higher than that of the parent tumorigenic triple-negative BCC line MDA-MB-231. We had compared the fusion efficiency between two BCC lines, the CD44-rich MDA-MB-231 and the CD44-poor MCF-7, with ADSCs. Interestingly, we found that the fusion efficiency was much higher between MDA-MB-231 and ADSCs, suggesting that a potential mechanism of cell fusion may lie in the dissimilarity between these two cell lines. The cell fusion efficiency was hampered by knocking down the CD44. Altogether, our findings suggest that CD44-mediated cell fusion could be a potential mechanism for generating CSCs.     

Human umbilical cord-derived cells can often serve as feeder cells to maintain primate embryonic stem cells in a state capable of producing hematopoietic cells

Clinical application of human embryonic stem (ES) cells will require the establishment of methods for their culture, either in the presence or absence of human-derived feeder cells. We have tested the ability of non-immortalized cultured cells derived from human umbilical cord (HUC cells) to support ES cell culture. A primate ES cell line that had been established and maintained with mouse embryonic fibroblasts was cultured on HUC cells for >3 months (HUC-maintained ES cells). These cells retained their expression of alkaline phosphatase, SSEA-4, Oct-3/4, and to a lesser extent Nanog, but did not express Rex-1. Nevertheless, HUC-maintained ES cells could produce ectoderm-, mesoderm- and endoderm-derived cells in teratomata that they formed in immunodeficient mice. We show that HUC-maintained ES cells could give rise to hematopoietic cells, although this ability of HUC cells varied among HUC cell populations derived from different neonates. HUC cells are promising as human material with which to maintain ES cells in a state that retains their ability to produce mature cells, including hematopoietic cells.