体外诱导人胚胎干细胞向神经细胞分化的研究进展

研究表明胚胎干细胞（embryonic stemcells,ES细胞）在体外可以发育分化出神经组织细胞,这对于了解神经发育的机制和研究神经组织退化性或脑组织损伤性疾病的治疗具有重要的意义。本文就胚胎干细胞分化为神经组织细胞的常用方法和机制做一综述。     

GDF-5诱导hADSCs分化为软骨样细胞

目的分离培养人脂肪干细胞(hADSCs),用不同浓度GDF-5进行诱导,观察细胞形态变化及检测相关蛋白的表达,评价其是否具有软骨样细胞的特性。方法取人抽脂术后的无菌脂肪组织,酶消化法分离培养hADSCs,观察细胞形态,MTT法检测细胞增殖能力。用含0、50、100ng/mlGDF-5的软骨诱导液培养,观察形态变化,检测Ⅱ型胶原及GAG的表达。结果 hADSCs呈长梭形,P10以后仍具有较强的增殖能力。经GDF-5诱导的hADSCs逐渐变为圆形、多角形,部分细胞聚集生长,形成软骨样结节;诱导28d的细胞Ⅱ型胶原表达阳性,甲苯胺蓝将GAG染成蓝色,但100ng/mlGDF-5诱导组阳性表达明显高于50ng/ml诱导组。结论 hADSCs在100ng/mlGDF-5的诱导下可分化为软骨样细胞。     

丹参注射液诱导大鼠骨髓间充质干细胞分化为神经元样细胞的研究

目的:探讨丹参注射液在体外诱导大鼠骨髓间充质干细胞(MSCs)向神经元样细胞分化的作用。方法:采用差速贴壁法体外分离培养大鼠MSCs,流式细胞仪检测其表面标志,经碱性成纤维细胞生长因子(bFGF)预诱导24h,采用含丹参注射液的无血清L-DMEM培养液诱导MSCs,倒置显微镜下观察细胞形态的变化,免疫荧光细胞化学检测巢蛋白、神经元特异性烯醇化酶(NSE)、神经丝蛋白(NF-M)、胶质纤维酸性蛋白(GFAP)的表达。结果:体外培养的大鼠MSCs表达CD29、CD44、CD105、CD166。丹参注射液诱导后MSCs胞体收缩,突起伸出,较密的部分神经元拉成网状,免疫细胞化学显示巢蛋白、NSE、NF-M表达阳性,阳性率分别为(58.68±4.50)%、(61.23±5.72)%、(63.47±6.38)%,GFAP阳性细胞率(1.47±0.23)%。结论:丹参注射液在体外可以将大鼠MSCs诱导分化为神经元样细胞。     

小鼠诱导多能干细胞向雄性生殖细胞样细胞诱导分化的研究

目的：对小鼠诱导多能干细胞向原始生殖细胞样细胞进行诱导培养,并对获得的原始生殖细胞样细胞进行鉴定。方法：在诱导干细胞培养基中对小鼠诱导多能干细胞进行培养,用蛋白质印迹法(Western blot)对Oct-4及C-kit蛋白表达水平进行鉴定,用定时定量PCR对Mvh, Fragilis, Stella的mRNA水平进行鉴定。结果：由小鼠诱导多能干细胞体外诱导分化获得的原始生殖细胞样细胞特异性表达Oct-4蛋白、C-kit蛋白以及Mv hmRNA,Fragilis RNA,Stella RNA。结论：由小鼠诱导多能干细胞成功诱导培养出原始生殖细胞样细胞,为进一步研究其向精子细胞的转化奠定了较好的基础。     

人脂肪间充质干细胞体外定向诱导分化为胰岛样细胞的实验研究

目的体外定向诱导人脂肪间充质干细胞向胰岛样细胞的分化。方法人脂肪间充质干细胞分3个阶段进行诱导,第一阶段培养在含适当浓度的2-巯基乙醇的高糖-达氏修正依氏培养基(HG-DMEM)培养2d,第二阶段在含适当浓度的B27、碱性成纤维细胞生长因子(bFGF)及表皮生长因子(EGF)的HG-DMEM培养基中诱导6d,第三阶段在含适当浓度的2-巯基乙醇和B27和尼克酰胺高糖无血清DMEM培养基诱导细胞向胰岛样细胞分化。对照组用HG-DMEM培养。在相差显微镜下观察细胞的形态;用反转录-聚合酶链反应(RT-PCR)法检测诱导前后nestin、胰岛素基因的表达;用免疫荧光染色法检测诱导前后nestin、胰岛素的表达;诱导第三阶段进行双硫腙染色鉴定胰岛B样细胞团。结果未经诱导的脂肪间充质干细胞呈长梭形贴壁生长,诱导后细胞逐渐变圆,并聚集成团。诱导8d细胞nestin基因呈阳性表达,诱导14d细胞nestin基因表达量下降,胰岛素基因表达呈阳性。诱导后的细胞团双硫腙染色呈棕红色。结论2-巯基乙醇、EGF、bFGF及尼克酰胺等可在体外诱导人脂肪间充质干细胞分化为具有分泌胰岛素功能的胰岛样细胞。     

成年大鼠脑膜原代组织干细胞特性细胞的分离及神经诱导分化研究

目的 分离并检测成年大鼠脑膜组织中具有干细胞特性的细胞亚群,探讨其诱导成神经细胞的能力.方法 自成年大鼠活体分离、剪取脑膜经胰酶消化制成细胞悬液,接种于培养皿,用无血清的特殊培养基培养,动态观察脑膜细胞克隆球的形成及分化过程,并用巢蛋白（Nestin）、表面抗原CD133抗体进行免疫荧光染色,对阳性细胞进行表观遗传学鉴定.分离的脑膜细胞经曲古抑菌素A（TSA）诱导培养基分别诱导分化后检测脑膜细胞向神经细胞分化成熟程度,用免疫印迹法（westen blotting）检测诱导分化后脑膜细胞内成熟神经细胞相关标志性蛋白--高分子量神经丝蛋白（NF-200）、神经元蛋白（BM88）的表达情况.结果 成年大鼠脑膜组织体外培养时不同类型细胞的贴壁时间有差异,具有干细胞特性细胞呈球形,黏着在首先贴壁的扁平基底细胞上分裂形成克隆球,克隆球细胞Nestin、CD133免疫荧光染色阳性.诱导分化后NF-200、BM88有明显表达,表明经诱导分化后脑膜细胞可分化为神经细胞.结论 活体分离成年大鼠脑膜组织部分细胞体外培养具有干细胞特性,并可向神经细胞方向分化.     

骨髓间质干细胞向心肌样细胞分化的研究

目的：诱导骨髓间质干细胞（BMSCs）向心肌样细胞分化,为 BMSCs 心肌再生移植治疗提供基础。方法取第5代的 BMSCs,5-氮杂胞苷诱导24 h,显微镜下进行形态学观察,3周后通过 RT - PCR 检测心肌蛋白 desmin 和αMHC 的表达。结果 BMSCs 经5-氮杂胞苷诱导后,细胞形态发生变化,出现肌管结构,RT - PC R 检测结蛋白（desmin ）和αMHC 的表达阳性。结论在体外成功诱导 BMSCs 分化成为心肌样细胞。     

胚胎干细胞/诱导多能干细胞向间充质干细胞转化的研究进展

胚胎干细胞(ESCs)/诱导多能干细胞(iPSCs)因其有潜在成瘤性,制约了它的临床应用.间充质干细胞(MSCs)具有多向分化能力、免疫调节功能以及低免疫原性,且仍未发现它有成瘤性,因而成为干细胞治疗颇具临床应用价值的种子细胞.近年来研究发现应用合适的诱导方案可以从ESCs/iPSCs获取MSCs,结合ESCs/iPSCs的无限自我更新能力,ESCs/iPSCs向MSCs转化将可能是获取MSCs的一种数量充足且稳定的新来源.本文将对目前ESCs/iPSCs向MSCs转化的方法 、机制以及新衍生的MSCs的免疫调节功能、干性特征做一综述.     

神经干细胞诱导分化调控新进展

长期以来 ,人们认为中枢神经细胞是终末分化的细胞 ,近年已经从胚胎及成年中枢神经系统不同部位分离出并证实了神经干细胞的存在。神经干细胞具有以下两个基本特性 :(1 )自我复制能力 ;(2 )多向分化潜能。神经干细胞的发现彻底改变了以往认为中枢神经系统神经元不能再生的认识 ,还为从事神经损伤修复的研究者提供了一种新的治疗策略。但神经干细胞的临床应用目前尚有时日 ,这期间首要解决的难题便是如何控制神经干细胞的定向分化。目前 ,神经干细胞增殖、分化的机制的研究已成为世界神经科学界关注的热点。本文简介 2 0 0 1年在美国圣地亚哥…     

人脐血MSCs的分离培养及向神经的诱导分化

目的探讨人脐血中分离所得到间充质干细胞（MSCs）向神经组织细胞诱导分化的实验条件。方法淋巴细胞分离液分离正常人脐血单个核细胞,利用差速消化法通过多次传代得到MSCs,流式细胞仪测定细胞表型。取2至4代的MSCs加入含有维甲酸（RA）与神经生长因子（NGF）的诱导分化培养基将其定向诱导,免疫细胞化学染色检测诱导后的MSCs神经元和神经胶质细胞标志的表达。结果脐血MSCs每传一代,细胞数量增加约2～3倍。流式细胞仪检测,脐血MSCs不表达CD34,表达CD25、CD29、CD105、CD106。诱导后的细胞不仅形态类似神经元和神经胶质细胞,而且表达神经丝蛋白200（NF）、神经元特异性烯醇化酶（NSE）和胶质原纤维酸性蛋白（GFAP）。结论从人脐血中可以分离得到与骨髓相似的MSCs,并且能够在体外分化为神经元样和神经胶质细胞样细胞。     

Ghrelin promotes differentiation of human embryonic stem cells into cardiomyocytes

Aim:

Ghrelin is involved in regulating the differentiation of mesoderm-derived precursor cells. The aim of this study was to investigate whether ghrelin modulated the differentiation of human embryonic stem (hES) cells into cardiomyocytes and, if so, whether the effect was mediated by growth hormone secretagogue receptor 1α (GHS-R1α).

Methods:

Cardiomyocyte differentiation from hES cells was performed according to an embryoid body (EB)-based protocol. The cumulative percentage of beating EBs was calculated. The expression of cardiac-specific markers including cardiac troponin I (cTnI) and α-myosin heavy chain (α-MHC) was detected using RT-PCR, real-time PCR and Western blot. The dispersed beating EBs were examined using immunofluorescent staining.

Results:

The percentage of beating EBs and the expression of cTnI were significantly increased after ghrelin (0.1 and 1 nmol/L) added into the differentiation medium. From 6 to 18 d of differentiation, the increased expression of cTnI and α-MHC by ghrelin (1 nmol/L) was time-dependent, and in line with the alteration of the percentages of beating EBs. Furthermore, the dispersed beating EBs were double-positively immunostained with antibodies against cTnI and α-actinin. However, blockage of GHS-R1α with its specific antagonist D-[lys³]-GHRP-6 (1 μmol/L) did not alter the effects of ghrelin on cardiomyocyte differentiation.

Conclusion:

Our data show that ghrelin enhances the generation of cardiomyocytes from hES cells, which is not mediated via GHS-R1α.     

Evaluation of developmental toxicity using undifferentiated human embryonic stem cells

An embryonic stem cell test (EST) has been developed to evaluate the embryotoxic potential of chemicals with an in vitro system. In the present study, novel methods to screen toxic chemicals during the developmental process were evaluated using undifferentiated human embryonic stem (hES) cells. By using surface marker antigens (SSEA‐4, TRA‐1‐60 and TRA‐1‐81), we confirmed undifferentiated conditions of the used hES cells by immunocytochemistry. We assessed the developmental toxicity of embryotoxic chemicals, 5‐fluorouracil, indomethacin and non‐embryotoxic penicillin G in different concentrations for up to 7 days. While expressions of the surface markers were not significantly affected, the embryotoxic chemicals influenced their response to pluripotent ES cell markers, such as OCT‐4, NANOG, endothelin receptor type B (EDNRB), secreted frizzled related protein 2 (SFRP2), teratocarcinoma‐derived growth factor 1 (TDGF1), and phosphatase and tensin homolog (PTEN). Most of the pluripotent ES cell markers were down‐regulated in a dose‐dependent manner after treatment with embryotoxic chemicals. After treatment with 5‐fluorouracil, indomethacin and penicillin G, we observed a remarkable convergence in the degree of up‐regulation of development, cell cycle and apoptosis‐related genes by gene expression profiles using an Affymetrix GeneChips. Taken together, these results suggest that embryotoxic chemicals have cytotoxic effects, and modulate the expression of ES cell markers as well as development‐, cell cycle‐ and apoptosis‐related genes that have pivotal roles in undifferentiated hES cells. Therefore, we suggest that hES cells may be useful for testing the toxic effects of chemicals that could impact the embryonic developmental stage. Copyright © 2014 John Wiley & Sons, Ltd.     

Ouabain facilitates cardiac differentiation of mouse embryonic stem cells through ERK1/2 pathway

Aim:To investigate the effects of the cardiotonic steroid, ouabain, on cardiac differentiation of murine embyronic stem cells (mESCs).Methods:Cardiac differentiation of murine ESCs was enhanced by standard hanging drop method in the presence of ouabain (20 μmol/L) for 7 d. The dissociated ES derived cardiomyocytes were examined by flow cytometry, RT-PCR and confocal calcium imaging.Results:Compared with control, mESCs treated with ouabain (20 μmol/L) yielded a significantly higher percentage of cardiomyocytes, and significantly increased expression of a panel of cardiac markers including Nkx 2.5, α-MHC, and β-MHC. The α1 and 2- isoforms Na(+)/K(+)-ATPase, on which ouabain acted, were also increased in mESCs during differentiation. Among the three MAPKs involved in the cardiac hypertrophy pathway, ouabain enhanced ERK1/2 activation. Blockage of the Erk1/2 pathway by U0126 (10 μmol/L) inhibited cardiac differentiation while ouabain (20 μmol/L) rescued the effect. Interestingly, the expression of calcium handling proteins, including ryanodine receptor (RyR2) and sacroplasmic recticulum Ca(2+) ATPase (SERCA2a) was also upregulated in ouabain-treated mESCs. ESC-derived cardiomyocyes (CM) treated with ouabain appeared to have more mature calcium handling. As demonstrated by confocal Ca(2+) imaging, cardiomyocytes isolated from ouabain-treated mESCs exhibited higher maximum upstroke velocity (P<0.01) and maximum decay velocity (P<0.05), as well as a higher amplitude of caffeine induced Ca(2+) transient (P<0.05), suggesting more mature sarcoplasmic reticulum (SR).Conclusion:Ouabain induces cardiac differentiation and maturation of mESC-derived cardiomyocytes via activation of Erk1/2 and more mature SR for calcium handling.     

人胚胎干细胞在药物毒性风险评估中的研究进展

人胚胎干细胞（hESCs）是来源于胚胎发育早期内细胞团的一类未分化的多能干细胞,具有自我更新和多向分化的生物学特性,在发育生物学、再生医学以及细胞治疗领域等方面已开展了广泛的研究。与常规药物毒性评估所采用的癌细胞或原代细胞不同,干细胞不仅能满足基础毒性评估,由于其多向分化的特点,可经诱导产生各种正常人体器官或组织细胞,为药物临床前评估提供多方面数据。与动物实验模型相比,hESCs不仅避免了种属差异,还具备高通量、低成本的优势,在预测药物毒性效应方面具有广阔的前景。对目前hESCs在药物毒性风险评估中的研究进展进行总结,为体外药物毒性筛选试验提供新思路。     

葡萄糖对小鼠胚胎干细胞生长的影响

目的：目前小鼠胚胎干细胞（mouse embryonic stem cells,mESC）常规应用高浓度葡萄糖（25mmol／L）培养基培养,但是在应用干细胞向糖尿病胰岛beta细胞分化的研究中发现,慢性高糖培养可促进干细胞的凋亡、降低细胞分化的效率及分化后胰岛beta细胞对葡萄糖的反应性,因此本研究拟选择合适的较低浓度的葡萄糖以优化胚胎干细胞的培养基、提高胚胎干细胞的生长、分化效率。方法：mESC传代4或12h后,将传统的25mmol／L葡萄糖培养基分别换为5、10、15、25mmol／L葡萄糖培养基培养,均用碱性磷酸酶（AP）染色计数细胞集落形成情况、台盼兰染色测定细胞数目,MTT法测定细胞活力及用4’,6-联脒-2-苯基吲哚（4’,6-diamidino-2-phenylindole,DAPI）染色检测细胞凋亡情况。结果：mESC传代4h后即调换葡萄糖浓度：（1）各葡萄糖浓度组（5、10、15mmol／L）与25mmol／L组相比,集落形成明显减少。（2）各葡萄糖浓度组与25mmol／L组相比,mESC增殖及细胞活力均受到不同程度明显影响。mESC传代12h后调换葡萄糖浓度：（1）各葡萄糖浓度组（5、10、15mmol／L）与25mmol／L组相比,集落形成无明显变化,且AP染色呈强阳性。（2）15mmol／L葡萄糖组对mESC增殖及细胞活力均无明显影响。（3）15mmol／L葡萄糖组与25mmol／L葡萄糖组细胞核形态正常,均未见明显凋亡。结论：ESC传代12h后将培养基中传统的高糖（25mmol／L）降低为15mmol／L,不影响mESC细胞活力及多能分化潜能和未分化状态。     

Disruption of cardiogenesis in human embryonic stem cells exposed to trichloroethylene

Trichloroethylene (TCE) is ubiquitous in our living environment, and prenatal exposure to TCE is reported to cause congenital heart disease in humans. Although multiple studies have been performed using animal models, they have limited value in predicting effects on humans due to the unknown species‐specific toxicological effects. To test whether exposure to low doses of TCE induces developmental toxicity in humans, we investigated the effect of TCE on human embryonic stem cells (hESCs) and cardiomyocytes (derived from the hESCs). In the current study, hESCs cardiac differentiation was achieved by using differentiation medium consisting of StemPro‐34. We examined the effects of TCE on cell viability by cell growth assay and cardiac inhibition by analysis of spontaneously beating cluster. The expression levels of genes associated with cardiac differentiation and Ca²⁺ channel pathways were measured by immunofluorescence and qPCR. The overall data indicated the following: (1) significant cardiac inhibition, which was characterized by decreased beating clusters and beating rates, following treatment with low doses of TCE; (2) significant up‐regulation of the Nkx2.5/Hand1 gene in cardiac progenitors and down regulation of the Mhc‐7/cTnT gene in cardiac cells; and (3) significant interference with Ca²⁺ channel pathways in cardiomyocytes, which contributes to the adverse effect of TCE on cardiac differentiation during early embryo development. Our results confirmed the involvement of Ca²⁺ turnover network in TCE cardiotoxicity as reported in animal models, while the inhibition effect of TCE on the transition of cardiac progenitors to cardiomyocytes is unique to hESCs, indicating a species‐specific effect of TCE on heart development. This study provides new insight into TCE biology in humans, which may help explain the development of congenital heart defects after TCE exposure. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1372–1380, 2016.     

胚胎干细胞体外分化为肝细胞和心肌细胞的研究进展

胚胎干细胞是从早期胚胎或原始性腺分离后能在体外长期传代培养的全能细胞系。胚胎干细胞体外可以诱导分化为各种体细胞,并且用这些体细胞治疗相应的疾病,一直以来是生物医药领域的研究焦点。目前,在该领域用于诱导胚胎干细胞分化的方法很多,其中的一些方法就用到化合物和部分中药提取物。整理近5年来胚胎干细胞体外通过拟胚体途径、细胞单层诱导分化为肝细胞和心肌细胞的有关文献,做一简要综述。     

Human embryonic stem cells and lung regeneration

Human embryonic stem cells are pluripotent cells derived from the inner cell mass of preimplantation stage embryos. Their unique potential to give rise to all differentiated cell types has generated great interest in stem cell research and the potential that it may have in developmental biology, medicine and pharmacology. The main focus of stem cell research has been on cell therapy for pathological conditions with no current methods of treatment, such as neurodegenerative diseases, cardiac pathology, retinal dysfunction and lung and liver disease. The overall aim is to develop methods of application either of pure cell populations or of whole tissue parts to the diseased organ under investigation. In the field of pulmonary research, studies using human embryonic stem cells have succeeded in generating enriched cultures of type II pneumocytes in vitro. On account of their potential of indefinite proliferation in vitro, embryonic stem cells could be a source of an unlimited supply of cells available for transplantation and for use in gene therapy. Uncovering the ability to generate such cell types will expand our understanding of biological processes to such a degree that disease understanding and management could change dramatically.     

烟酰胺磷酸核糖转移酶基因编辑对人胚胎干细胞生长影响的研究

目的 研究烟酰胺磷酸核糖转移酶（nicotinamide phosphoribosyl transferase,Nampt）基因在人胚胎干细胞生长中的作用。方法 采用CRISPR/Cas9技术对人胚胎干细胞H9细胞系的Nampt基因进行移码突变;随后,筛选Nampt基因突变细胞系进行基因测序,同时在显微镜下观察其生长状态。另外,给予H9细胞不同浓度的Nampt抑制剂FK866进行干预,采用CCK-8试剂盒检测细胞活力。结果 Nampt基因发生突变后,人胚胎干细胞会逐渐死亡。同样,Nampt抑制剂FK866也会抑制胚胎干细胞的活力,使细胞逐渐死亡。结论 Nampt基因对维持人胚胎干细胞生长具有不可或缺的重要作用。