胚胎干细胞向心肌细胞诱导分化的研究进展

胚胎干细胞具有多向分化潜能,在器官移植及组织修复治疗方面具有广阔的应用前景。近来研究表明,胚胎干细胞在相关基因、转录因子调控以及体内外细胞因子、激素、微环境等作用下能够诱导分化为心肌细胞。但临床应用胚胎干细胞进行心脏修复治疗还有许多问题需要研究和解决。     

胚胎干细胞向心肌细胞诱导分化的研究进展

胚胎干细胞具有多向分化潜能,在器官移植及组织修复治疗方面具有广阔的应用前景.近来研究表明,胚胎干细胞在相关基因、转录因子调控以及体内外细胞因子、激素、微环境等作用下能够诱导分化为心肌细胞.但临床应用胚胎干细胞进行心脏修复治疗还有许多问题需要研究和解决.     

不同诱导条件对小鼠胚胎干细胞分化为心肌细胞的实验研究

目的 :比较小鼠胚胎干细胞 （ESC）在不同诱导条件下分化为心肌细胞的分化比率。方法 :用鼠胚成纤维细胞（MEF）作为饲养层促进 ESC增殖并抑制其分化 ,用维甲酸 （RA ）、二甲基亚砜 （DMSO）、转化生长因子β1 （TGF-β1 ）、激活素 - A（activin- A）为分化诱导剂 ,采用三步法诱导 ESC分化为心肌样细胞 ,并比较各组分化比率。结果 :各组实验用的各种诱导剂均能诱导 ESC分化为心肌样细胞 ,尤以 TGF- β1 （2 ng/m l）、activin- A（2 0 ng/ml）及 2 0 %胎牛血清 （FCS）组成的分化培养基可以使其分化比率高达 88% ,显著高于其它各组 （P<0 .0 1）。结论 :以 MEF饲养层培养 ,TGF- β1 、activin- A作分化诱导剂可作为一种比较简便、稳定、高效的 ESC分化条件。     

Ghrelin以非经典受体依赖性方式促进人胚胎干细胞定向分化为心肌细胞的研究

目的 探讨Ghrelin对人胚胎干（hES）细胞定向分化为心肌细胞的影响. 方法 以不同浓度Ghrelin诱导hES细胞定向分化为心肌细胞,显微镜下计数搏动细胞团的比例,应用实时PCR检测心肌特异性标志物的表达.应用RT-PCR、Western blot及免疫荧光染色检测分化细胞中生长激素促分泌物受体1α（GHSR1α）的表达,添加GHS-R1α拮抗剂[D-lys3]-GHRP-6,观察其对Ghrelin促分化作用的影响. 结果 与对照组比较,10-10、10-9及10-8 mol/L Ghrelin均可增加搏动细胞团的比例[搏动比例分别为（12.9±1.4）％％,（19.5±2.2）％及（13.5±3.1）％vs（10.3±2.2）％,P＜0.05].RT-PCR分析显示,Ghrelin可上调α-肌球蛋白重链（α-MHC）和心肌肌钙蛋白I（cTnI）的表达[α-MHC：100％ vs （40.1±13.2）％,P＜0.001;cTnI：100％ vs （52.6±9.8）％,P＜0.001].RT-PCR、Western blot及免疫荧光染色均显示GHS-R1α在不同阶段的分化细胞中表达,但酰基化和非酰基化Ghrelin的促分化作用相似,且添加GHS-R1α拮抗剂不能阻断Ghrelin的促分化作用. 结论 Ghrelin可促进hES细胞定向分化为心肌细胞,该作用可能由GHS-R1α以外的信号途径所介导.     

胚胎干细胞及其心肌细胞定向分化研究进展

近年来 ,胚胎干细胞已成为生命科学研究的热点之一。作者在回顾胚胎干细胞的生物学特性、获取和培养方法的基础上 ,着重综述在胚胎干细胞分化为心肌细胞后 ,心肌细胞特异性基因表达的变化、电生理特征、离子通道出现、形态学特征和治疗心肌梗死实验的初步结果     

鼠胚胎干细胞体外分化的单个心肌细胞获取方法

用鼠胚胎干细胞在体外分化成心肌细胞是研究心肌细胞进化和生理学实验的新途径 ,胚胎干细胞最初是从鼠胚胎在胚泡阶段或 8个细胞的胚胎阶段的内部没分化的细胞中得到。其可以定向诱导分化为几乎所有种类的细胞 ,此文介绍了由鼠胚胎干细胞分化成为心肌细胞及单个心肌细胞的获取方法。     

催产素联合乳鼠心肌细胞条件培养液诱导胚胎干细胞分化的作用

目的:探讨安全高效体外诱导胚胎干细胞(ESCs)向心肌细胞(CMs)分化的途径。方法:ESCs经三步法形成拟胚体(embryoid bodies,EBs)后,按培养基的不同分为4组:乳鼠CMs条件培养液诱导组;催产素(oxytocin,OT)诱导组;混合添加诱导组(乳鼠CMs条件培养液加催产素);对照组(无任何添加)。利用免疫组化染色法检测心肌特异蛋白、RT-PCR检测细胞特殊因子的表达、变时性反应观察细胞对心脏药物的反应及观察细胞的超微结构,对比各组分化后细胞的结构及功能。结果:对照组未观察到细胞跳动。混合添加诱导组、OT诱导组和乳鼠CMs条件培养液诱导组早期分别有(90.30±3.43)%、(21.53±2.69)%和(22.37±6.31)%EBs搏动;中期分别有(92.34±2.65)%、(22.36±2.52)%和(24.15±5.12)%EBs搏动;晚期分别有(83.65±6.27)%、(11.35±2.14)%和(10±4.25)%EBs搏动。免疫组化染色法检测、RT-PCR、变时性反应及电镜观察等的结果提示,混合添加诱导组诱导ESCs分化的效率远高于其余各组。结论:OT、乳鼠CMs条件培养液、OT加乳鼠CMs的条件培养液均具有诱导ESCs分化为CMs的作用,且后者的诱导更具有高效性。     

胚胎干细胞的定向心肌分化及调控

胚胎干细胞是具有分化为三个胚层来源的各种类型组织细胞潜能的多能干细胞,可在体外无限增殖。信号分子、化学诱导剂、激素和细胞内转录因子可诱导和调控胚胎干细胞进行心肌细胞分化,为干细胞移植用于心肌再生和改善心功能提供了潜在的细胞来源。     

内脏内胚层样END-2细胞体外诱导鼠胚胎干细胞分化为心肌细胞的实验研究

目的探讨内脏内胚层样END-2细胞体外诱导胚胎干细胞（embryon ic stem cells,ESCs）分化为心肌细胞的特征。方法用鼠胚胎成纤维细胞（mouse embryon ic fibrob lasts,MEF）作为饲养层促进ESCs增殖并抑制其分化,先将ESCs悬浮培养形成23 d的拟胚体（embryoid bod ies,EBs）,再和END-2细胞共培养诱导向心肌细胞分化。实验分4组。第1,2组EBs分别和END-2细胞或END-2细胞条件培养液共培养;第3组EBs和表面铺有一层琼脂糖的END-2细胞共培养;第4组自然分化组为对照组。相差显微镜下观察分化细胞的形态学变化,免疫细胞荧光技术检测心肌细胞特异性肌钙蛋白T（TnT）的表达;透射电镜观察分化心肌细胞的超微结构。结果各实验组均可见自发节律性收缩的拟胚体。随着培养的延长,自发节律性收缩的拟胚体数目也增加,均表达心肌细胞特异性蛋白TnT,以及观察到心肌样超微结构。在和END-2细胞直接接触的诱导条件下,分化的细胞形态较单一。结论END-2细胞通过分泌可溶性细胞因子可诱导ESCs向心肌细胞分化,直接接触在END-2细胞诱导作用中并不是必要的,但可诱导出较单一的细胞。     

骨髓间充质干细胞诱导分化为心肌细胞的研究进展

<正>骨髓间充质干细胞(BMSCs)又称骨髓基质干细胞,是骨髓内的一类非造血干细胞,它能够分化为多种中胚层来源的间质细胞。心肌细胞不具备再生能力,故损伤的心肌细胞无法通过自身的增殖和分化来修复,心肌梗死、心衰、心肌重构等均为传统医学方法难以医治的顽疾。近年研究发现,BMSCs在一定的诱导条件下,可以分化为心肌细胞。BMSCs具备易获得性、多     

Ghrelin induces cardiac lineage differentiation of human embryonic stem cells through ERK1/2 pathway

Background

Ghrelin, an endogenous ligand for growth hormone secretagogue receptor (GHS-R), shows cardioprotective activity and regulates the differentiation of several mesoderm-derived cells, including myocytes, adipocytes and osteoblasts. The effect of ghrelin on cardiogenesis and its underlying mechanism, however, have not been studied in detail.

Methods

The effects of ghrelin on cardiomyocyte differentiation were tested both in human embryonic stem cells (hESCs) cultured in embryoid body (EB)-based differentiation protocol, and in hESCs transplanted into rat hearts. The signaling mechanisms of ghrelin were further investigated under the EB-based culture condition.

Results

The generation of beating EBs and the expression of cardiac-specific markers including cardiac troponin I (cTnI) and α-myosin heavy chain (α-MHC) were 2 to 3-fold upregulated by ghrelin. Although GHS-R1α protein was expressed in differentiated EBs, the effects of exogenous ghrelin were unchanged by D-[lys³]-GHRP-6, a specific GHS-R1α antagonist. Moreover, des-acyl ghrelin, which does not bind to GHS-R1α, displayed similar effects with ghrelin. Importantly, activation of ERK1/2, but not Akt, was induced by ghrelin in the newly-formed EBs, and the ghrelin-induced effects of cardiomyocyte differentiation were abolished by adding specific ERK1/2 inhibitor PD98059, but not specific PI3K inhibitor Wortmannin. In addition, ghrelin promoted the differentiation of grafted hESCs into Sox9- and Flk1-positive mesodermal/cardiac progenitor cells in rat hearts.

Conclusions

These results suggest that ghrelin induces cardiomyocyte differentiation from hESCs via the activation of the ERK1/2 signaling pathway. Our study, therefore, indicates that using ghrelin may be an effective strategy to promote the differentiation of hESCs into cardiomyocytes.     

Survival and maturation of human embryonic stem cell-derived cardiomyocytes in rat hearts

Human embryonic stem cell (hESC)-derived cardiomyocytes are a promising cell source for cardiac repair. Whether these cells can be transported long distance, survive, and mature in hearts subjected to ischemia/reperfusion with minimal infarction is unknown. Taking advantage of a constitutively GFP-expressing hESC line we investigated whether hESC-derived cardiomyocytes could be shipped and subsequently form grafts when transplanted into the left ventricular wall of athymic nude rats subjected to ischemia/reperfusion with minimal infarction. Co-localization of GFP-epifluorescence and cardiomyocyte-specific marker staining was utilized to analyze hESC-derived cardiomyocyte fate in a rat ischemia/reperfused myocardium. Differentiated, constitutively green fluorescent protein (GFP)-expressing hESCs (hES3-GFP; Envy) containing about 13% cardiomyocytes were differentiated in Singapore, and shipped in culture medium at 4 degrees C to Los Angeles (shipping time approximately 3 days). The cells were dissociated and a cell suspension (2 x 10(6) cells for each rat, n=10) or medium (n=10) was injected directly into the myocardium within the ischemic risk area 5 min after left coronary artery occlusion in athymic nude rats. After 15 min of ischemia, the coronary artery was reperfused. The hearts were harvested at various time points later and processed for histology, immunohistochemical staining, and fluorescence microscopy. In order to assess whether the hESC-derived cardiomyocytes might evade immune surveillance, 2 x 10(6) cells were injected into immune competent Sprague-Dawley rat hearts (n=2), and the hearts were harvested at 4 weeks after cell injection and examined as in the previous procedures. Even following 3 days of shipping, the hESC-derived cardiomyocytes within embryoid bodies (EBs) showed active and rhythmic contraction after incubation in the presence of 5% CO(2) at 37 degrees C. In the nude rats, following cell implantation, H&E, immunohistochemical staining and GFP epifluorescence demonstrated grafts in 9 out of 10 hearts. Cells that demonstrated GFP epifluorescence also stained positive (co-localized) for the muscle marker alpha-actinin and exhibited cross striations (sarcomeres). Furthermore, cells that stained positive for the antibody to GFP (immunohistochemistry) also stained positive for the muscle marker sarcomeric actin and demonstrated cross striations. At 4 weeks engrafted hESCs expressed connexin 43, suggesting the presence of nascent gap junctions between donor and host cells. No evidence of rejection was observed in nude rats as determined by inspection for lymphocytic infiltrate and/or giant cells. In contrast, hESC-derived cardiomyocytes injected into immune competent Sprague-Dawley rats resulted in an overt lymphocytic infiltrate. hESCs-derived cardiomyocytes can survive several days of shipping. Grafted cells survived up to 4 weeks after transplantation in hearts of nude rats subjected to ischemia/reperfusion with minimal infarction. They continued to express cardiac muscle markers and exhibit sarcomeric structure and they were well interspersed with the endogenous myocardium. However, hESC-derived cells did not escape immune surveillance in the xenograft setting in that they elicited a rejection phenomenon in immune competent rats.     

机械牵张对诱导多能干细胞向心肌细胞分化的研究

目的研究机械牵张对诱导多能干细胞向心肌细胞分化效率的影响。方法诱导多能干细胞形成拟胚体后将拟胚体分为四组:对照组(未行牵张处理),牵张组1(5-6天行24小时牵张),牵张组2(5-6天行24小时牵张,7-8天再行24小时牵张),牵张组3(5-6天行24小时牵张,7-10天再行72小时牵张),通过对小鼠诱导多能干细胞施加20%形变率的机械牵张力后,于第15天,分别计数每组跳动克隆数目从而初步在上述四组中选出诱导效率最高组,此后用荧光免疫染色、Westernblot、RT-PCR和激光共聚焦法,进一步鉴定对照组和初步筛选出的牵张组最终分化效率和细胞成熟度差异。结果机械牵张刺激下,分化15天时,牵张组2的跳动克隆数上升(P<0.05),初步筛选出牵张组2可以提高分化效率;统计α-MHC免疫荧光染色面积发现牵张组2是对照组的2.1倍(P<0.05);牵张组2TroponinI的蛋白表达量为对照组的1.7倍(P<0.05);半定量PCR结果发现,心肌细胞标志基因β-MHC,MLC-2v及心肌细胞早期转录因子Nkx2.5的表达量分别提高了6.7倍、4.4倍和11.4倍(P值均<0.05);激光扫描共聚焦显微镜对分化来的单个心肌细胞进行α-actinin观察发现,牵张组2有利于心肌细胞的伸展和成熟。结论初步验证机械牵张力作为一种刺激诱导因素,20%拉伸形变率,牵张组2(贴壁的拟胚体5-6天行24小时牵张,7-8天再行24小时牵张)的处理方法可以显著促进诱导多能干细胞向心肌细胞的分化效率,为以后的深入研究和临床应用提供了实验基础。     

人胚胎肝干细胞的形态特点

目的:观察胚胎发育早期肝干细胞的形态特征、时空分布及分化,以探讨肝干细胞的生物学特征．方法:运用发育第3-12 wk人胚标本47例(其中 3．5 wk各8例;6-8 wk各5例,9-12 wk各2例),石蜡切片,连续切片,免疫组化染色,光镜下观察人胚肝及肝干细胞的发育及其AFP、c-Met和 CK19的时空表达．结果:发育第3 wk,肝芽形成,第4 wk形成肝索,第5 wk出现原始肝血窦．第3-5 wk人胚肝芽和肝索细胞排列紧密,较小,形态不规则,核圆形或卵圆形,核质比例大,核深染,胞质颜色较淡,偏蓝色,显示出幼稚细胞的形态学特征,并呈甲胎蛋白(α-Fetoprotein,AFP)、c-Met 阳性反应．第6 wk,肝索内出现了体积大、核大、淡染的细胞,呈AFP、c-Met阴性反应．随胚龄增加,这类细胞数量增加．10-12 wk． AFP、c-Met阳性细胞主要分布于汇管区周围． CK19阳性反应在7 wk时开始出现于一些与 AFP、c-Met阳性反应的细胞形态类似的肝索细胞中．10-11 wk时,CK19阳性反应主要位于汇管区附近的肝索细胞、胆管板细胞及胆管上皮细胞,12 wk时,CK19阳性信号仅见于胆管板和胆管上皮细胞．此时所有的胆管板细胞及胆管上皮细胞均呈AFP、c-Met和CK19阳性．结论:人胚发育3-5 wk肝实质由肝干细胞组成,其表型为AFP /c-Met ．6 wk,肝干细胞开始向肝细胞系分化,7 wk向胆管系分化,10-12 wk,肝干细胞主要局限于汇管区周围的肝索, 与成年肝中卵圆细胞(成年肝干细胞)的分布一致．AFP ／c-Met／ CK19 细胞可能为胆管祖细胞．     

鼠胚胎干细胞早期分化来源的心肌细胞同种异体移植后的电生理特性

目的 探讨小鼠胚胎干细胞早期分化来源的心肌细胞进行同种异体移植后的电生理特性.方法 首先通过电转染构建a-MHC-EGFP-ESc系[将心肌细胞特异的OL-MHC启动子与表达基因--绿色荧光蛋白(EGFP)基因融合,构建成真核表达载体],悬滴法诱导胚胎干细胞分化,在分化早期(7+4)d利用流式细胞仪筛选带绿色荧光的心肌细胞,将纯化的心肌细胞(5×106个/ml)移植到小鼠心室壁,对照组注入等体积培养基,移植前3 d开始应用环孢素A(静脉注射,5 mg·kg-1·d-1)和泼尼松龙(静脉注射,2.5 mg·kg-1·d-1)抑制免疫排斥反应.移植后2周分离两侧颈迷走神经行电刺激抑制窦房结与房室结,记录刺激前后的体表心电图,然后分别行免疫荧光显像和膜片钳研究.结果 刺激迷走神经前,移植组和对照组均呈正常的窦性心律,两组无室性心律失常的发生;刺激迷走神经后,两组动物均出现异位的心室起搏心律,细胞移植组与对照组心室频率无差异.移植区冰冻切片免疫荧光分析可见EGFP标记的移植细胞具有肌钙蛋白I(cTnI)表达,说明分化细胞移植后仍具有心肌特性,且移植细胞与宿主心肌细胞间有连接蛋白43的表达,表明移植细胞与宿主心肌细胞间形成电偶联通道.膜片钳分析,绿色荧光细胞移植前后其具有起搏细胞动作电位的比例分别为(85.1%vs 1 1.4%,P＜0.05),该类细胞在移植前后的起搏电流强度有所增强((11.2±2.4)pA/pF vs(15.5±1.9)pA/pF,P＜0.05].移植区分离的绿色荧光细胞中48%具有心室肌动作电位.结论 胚胎干细胞早期分化来源的心肌细胞在体移植后能进一步分化为成熟的心室肌细胞及起搏细胞.     

Wnt3a对小鼠胚胎干细胞心肌细胞分化的影响

目的：观察Wnt3a信号对小鼠胚胎干细胞（embryonicstemcell,ESC）心肌细胞分化的影响。方法：用悬滴培养法促进ESCs形成拟胚体（embryoidbodies,EBs）。用免疫荧光染色法检测心肌特异性蛋白cTnT的表达。在不同分化时间加入Wnt3a及Wnt信号通路抑制剂Dkk一1观察对搏动EBs百分率的影响。用RT—PCR检测Nk同源异型盒5（Nkx2．5）、锌指转录因子-4（GATA．4）、B．肌球蛋白重链（[~-MHC）及心房钠尿肽（ANP）基因表达水平的变化。用Westernblot检测cTnT表达水平的变化。将不加入Wnt3a,分化第0—5天及5～lO天加入Wnt3a的组分别命名为对照组,D0-5组及D5．10组。加入Dkk．1的组命名为Dkk．1组。结果：通过悬滴培养法形成的EBs能够出现自发性搏动并且有TnT表达。EBs形成过程中即分化第0—5天加入Wnt3a与对照组相比具有更高的搏动EBs百分率,Nkx2．5、GATA4、B．MHC和ANP基因表达的水平,以及cTnT蛋白表达水平,而EBs形成后即分化第5—10天加入Wnt3a的结果相反。分化第5—10天加入Dkk．1与对照组相比具有更高的搏动EBs百分率及cTnT蛋白表达水平,并且在分化第0—5天分别加入Wnt3a及Dkk．1与单独加入Wnt3a及Dkk-1相比,搏动EBs百分率及cTnT蛋白表达水平更高。结论：Wnt3a对ESCs向心肌细胞分化的调控呈时间依耐性,EBs形成过程中激活Wnt3a信号及EBs形成后阻断Wnt3a信号能够获得更多的心肌细胞。     

Therapeutic potential of embryonic stem cells

Nearly 20 years after murine embryonic stem cells (mESC) were isolated, the first report of the derivation of human embryonic stem cells (hESC) in 1998 spawned the field of hESC research [Evans MJ, Kaufman MH, Establishment in culture of pluripotential cells from mouse embryos. Nature 1981; 292 (5819): 154-6; Thomson JA, Itskovitz-Eldor J, Shapiro SS, et al. Embryonic stem cell lines derived from human blastocysts. Science 1998; 282 (5391): 1145-7.]. Although this field is only in its infancy, hESC represent a theoretically inexhaustible source of precursor cells that could be differentiated into any cell type to treat degenerative, malignant, or genetic diseases, or injury due to inflammation, infection, and trauma. This pluripotent, endlessly dividing cell has been hailed as a possible means for treating diabetes, Parkinson's disease, Alzheimer's, spinal cord injury, heart failure, and bone marrow failure. But the regenerative medicine applications of embryonic stem cells are only one facet of hESC therapeutic potential. Human ESC are an invaluable research tool to study development, both normal and abnormal, and can serve as a platform to develop and test new therapies. In addition to discussing the therapeutic potential of hESC, this chapter will cover limitations to using hESC for replacement cell therapy, strategies to overcome these limitations, and alternative methods of deriving hESC.