Isolation and differentiation of embryonic stem cells from BALB/c mouse

Objective To invest the efficient method which can culture and induce embryonic stem cells to neuroeyte in vitro. Methods Isolate the blastula o f 3.5 d from BALB/c species mouse. Culture the cells from inner cell mass （inner cell mass, ICM） which were isolated by mechanical method on the mouse embryonic fibroblaste cell （MEF） feeder layer or 0.1% gelatin coated dishes. The stem ceils were identified by characterized morphology, alkaline phosphatase stain, differential potency in vivo and immunoehemistry stain. The isolated cells were differentiated by serial induction method that mimicking the intrinsic developmental process of the neural system. Results The isolated cells were positive for alkaline phosphatatse and SSEA-1 （ stage specific embryonic antigen 1 ）. Moreover they were identified pluripotent by differentiation in vivo. Therefore the isolated ceils presented the characters of ESCs. Then the isolated cells were able to differentiate into neuroeytes in vitro. Conclusion Mouse embryonic stem ceils isolation, culture and differentiation system has been established.     

Fluoxetine promotes gliogenesis during neural differentiation in mouse embryonic stem cells

Selective serotonin reuptake inhibitors (SSRIs) are commonly prescribed for treatment of mood disorders and depression, even during pregnancy and lactation. SSRIs are thought to be much safer than tricyclic antidepressants, with a low risk of embryonic toxicity. Several recent studies, however, have reported that fetal exposure to SSRIs increases the risk of adverse effects during fetal and neonatal development. This is consistent with our previous finding that fluoxetine, a prototypical SSRI, profoundly affected the viability of cultured embryonic stem (ES) cells as well as their ability to differentiate into cardiomyocytes. Furthermore, we found that fluoxetine induced fluctuations in ectodermal marker gene expression during ES cell differentiation, which suggests that fluoxetine may affect neural development. In the present study, we investigated the effects of fluoxetine on the process of differentiation from ES cells into neural cells using the stromal cell‐derived inducing activity (SDIA) method. Fluoxetine treatment was found to enhance the expression of glial marker genes following neural differentiation, as observed by immunocytochemical analysis or quantitative RT‐PCR. The promoter activity of glial marker genes was also significantly enhanced when cells were treated with fluoxetine, as observed by luciferase reporter assay. The expression of neuronal markers during ES cell differentiation into neural cells, on the other hand, was inhibited by fluoxetine treatment. In addition, FACS analysis revealed an increased population of glial cells in the differentiating ES cells treated with fluoxetine. These results suggest that fluoxetine could facilitate the differentiation of mouse ES cells into glial cell lineage, which may affect fetal neural development. © 2010 Wiley‐Liss, Inc.     

Efficient generation of mature cerebellar Purkinje cells from mouse embryonic stem cells

Mouse embryonic stem cells (ESCs) can generate cerebellar neurons, including Purkinje cells (PCs) and their precursor cells, in a floating culture system called serum‐free culture of embryoid body‐like aggregates (SFEB) treated with BMP4, Fgf8b, and Wnt3a. Here we successfully established a coculture system that induced the maturation of PCs in ESC‐derived Purkinje cell (EDPC) precursors in SFEB, using as a feeder layer a cerebellum dissociation culture prepared from mice at postnatal day (P) 6–8. PC maturation was incomplete or abnormal when the adherent culture did not include feeder cells or when the feeder layer was from neonatal cerebellum. In contrast, EDPCs exhibited the morphology of mature PCs and synaptogenesis with other cerebellar neurons when grown for 4 weeks in coculture system with the postnatal cerebellar feeder. Furthermore, the electrophysiological properties of these EDPCs were compatible with those of native mature PCs in vitro, such as Na⁺ or Ca²⁺ spikes elicited by current injections and excitatory or inhibitory postsynaptic currents, which were assessed by whole‐cell patch‐clamp recordings. Thus, EDPC precursors in SFEB can mature into PCs whose properties are comparable with those of native PCs in vitro. © 2009 Wiley‐Liss, Inc.     

Differentiation of neuron-like cells from mouse parthenogenetic embryonic stem cells

Parthenogenetic embryonic stem cells have pluripotent differentiation potentials,akin to fertilized embryo-derived embryonic stem cells.The aim of this study was to compare the neuronal differentiation potential of parthenogenetic and fertilized embryo-derived embryonic stem cells.Before differentiation,karyotype analysis was performed,with normal karyotypes detected in both parthenogenetic and fertilized embryo-derived embryonic stem cells.Sex chromosomes were identified as XX.Immunocytochemistry and quantitative real-time PCR detected high expression of the pluripotent gene,Oct4,at both the mRNA and protein levels,indicating pluripotent differentiation potential of the two embryonic stem cell subtypes.Embryonic stem cells were induced with retinoic acid to form embryoid bodies,and then dispersed into single cells.Single cells were differentiated in N2 differentiation medium for 9 days.Immunocytochemistry showed parthenogenetic and fertilized embryo-derived embryonic stem cells both express the neuronal cell markers nestin,βIII-tubulin and myelin basic protein.Quantitative real-time PCR found expression of neurogenesis related genes(Sox-1,Nestin,GABA,Pax6,Zic5 and Pitx1) in both types of embryonic stem cells,and Oct4 expression was significantly decreased.Nestin and Pax6 expression in parthenogenetic embryonic stem cells was significantly higher than that in fertilized embryo-derived embryonic stem cells.Thus,our experimental findings indicate that parthenogenetic embryonic stem cells have stronger neuronal differentiation potential than fertilized embryo-derived embryonic stem cells.     

昆明小鼠胚胎干细胞最佳分离方法及培养液选择

背景：到目前为止已经建立数百个小鼠胚胎干细胞系,昆明小鼠是中国应用最多的实验小鼠,其建系成功报道极少。 目的：建立一简单有效的昆明小鼠胚胎干细胞分离培养体系,以提高昆明鼠胚胎干细胞建系成功率。 方法：用添加血清替代物或胎牛血清培养液的小鼠胚胎成纤维细胞制备饲养层。昆明小鼠经孕马血清促性腺激素和人绒毛膜促性腺激素促排卵,交配后3.5 d冲洗子宫取胚胎,将胚胎接种在饲养层中培养,观察胚胎贴壁、内细胞团集落形成率。4~ 6 d后在0.05%trypsin-0.02%EDTA消化液中用切割法处理增殖的内细胞团块,再接种到新的饲养层上。倒置显微镜下观察形成的胚胎干细胞样集落形态。 结果与结论：妊娠3.5 d孕鼠适合胚胎干细胞的分离培养;在0.05%trypsin-0.02%EDTA消化液中采用切割法进行内细胞团集落的分离,集落增殖快、分化低;在含胎牛血清培养液中进行干细胞培养较血清替代物培养液中传代次数多,此培养液更适宜进行分离培养昆明鼠胚胎干细胞。     

Culture method for the induction of neurospheres from mouse embryonic stem cells by coculture with PA6 stromal cells

Embryonic stem (ES) cells proliferate and maintain their pluripotency for over 1 year in vitro and may therefore provide a sufficient source for cell therapies. However, most of the previously reported methods for obtaining a source for cell therapies have not been simple. We describe here a novel method for induction of neurospheres from mouse ES cells by coculturing on PA6 cells instead of the formation of embryoid bodies. The ES cells cocultured with the PA6 stromal cell line for at least 3 days were capable of differentiating into spheres. The cells in the spheres were all green fluorescent protein (GFP) positive, showing that they were derived from GFP-expressing D3-ES cells. The spheres contained nestin-positive cells. The number of spheres increased when they were cocultured with PA6 for a longer period. Sphere formation was observed even after 10 mechanical dissociations and subculturings, showing its self-renewal ability. The cells differentiated into microtubule-associated protein-2 (MAP2)-positive neuronal cells and glial fibrillary acidic protein (GFAP)-positive glial cells. gamma-Aminobutyric acid-positive cells and tyrosine hydroxylase-positive cells were also observed in the spheres. The percentages of the MAP2- or GFAP-positive cells in the sphere changed according to the period of coculture on PA6 cells. At an early stage of coculture, more neurons were generated and, at a later period, more glial cells were generated. These results suggested that neurosphere could be generated from ES cells by coculturing with PA6, and that these cells resembled neural stem cells derived from mouse fetal brain tissue.     

Analysis of the expression and function of BRINP family genes during neuronal differentiation in mouse embryonic stem cell‐derived neural stem cells

We previously identified a novel family of genes, BRINP1, 2, and 3, that are predominantly and widely expressed in both the central nervous system (CNS) and peripheral nervous system (PNS). In the present study, we analyzed the expression pattern of three BRINP genes during differentiation of mouse embryonic stem (ES) cell‐derived neural stem cells (NSCs) and their effects on the cell‐cycle regulation of NSCs. While there was no significant expression of any BRINP‐mRNA expressed in mouse ES cells, BRINP 1 and 2‐mRNAs was expressed at high levels in the ES cell‐derived neural stem cells. Upon differentiation into neuronal cells in the presence of retinoic acid and BDNF, all three types of BRINP‐mRNA were induced with a similar time course peaking at day three of treatment. Upon differentiation into astroglial cells in the presence of serum, BRINP1‐mRNA was slightly up‐regulated, while BRINP2‐ and BRINP3‐mRNAs were almost abolished in the astrocytes. While 69.2, 26.1, and 7.7% of cells in a population of NSCs in the exponentially growing phase were in the G1, S and G2 phases, respectively, over‐expression of any one of the three BRINP genes completely abolished cells in the G2 phase and significantly reduced the cells in S phase to 11.8–13.8%. Based on these results, the physiological roles of induced BRINP genes in the cell‐cycle suppression of terminally differentiated post‐mitotic neurons are discussed. © 2009 Wiley‐Liss, Inc.     

In vitro culture and differentiation of rat embryonic midbrain-derived neural stem cells

BACKGROUND: Midbrain-derived neural stem cells (mNSCs) can differentiate into functional mature dopamincrgic neurons. The mNSCs are considered the ideal choice for cell therapy of Parkinson's disease. OBJECTIVE: To isolate rat embryonic mNSCs and to observe the differentiation characteristics of mNSCs induced by cell growth-promoting factors. DESIGN, TIME AND SETTING: An in vitro cell culture study based on the molecular biology of nerve cells was carried out at the Institute of Clinical Medicine, China-Japan Friendship Hospital (China) from March to November 2007. MATERIALS: Sprague Dawley rats at embryonic day 14 were used in this study. Nestin antibody, β-Ⅲ tubulin antibody, glial fibrillary acidic protein (GFAP) antibody and cyclic nucleotide 3'-phosphohydrolase (CNPase) antibody were provided by Abeam; DMEM/F12 medium and N2 supplement were provided by Invitrogen; epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF2) were provided by R＆D Systems. METHODS: The ventral mesencephalon was dissected from embryonic day 14 rat embryos. By trypsin digestion and mechanical separation, the brain tissue was triturated into a fine single-cell suspension. The cells were cultured in 5 mL serum-free medium containing DMEM/Fl2, 1% N2 supplement, 20 ng/mL EGF and FGF2. The mNSCs at the third generation were coated with 10 μg/mL polylysine and induced to differentiate in the DMEM/Fl2 supplemented with 1% fetal bovine serum and 1% N2. MAIN OUTCOME MEASURES: The neural spheres of the third passage were identified by nestin immunofluorescence; at the same time, the cells were induced to differentiate, and the types of differentiated cell were identified by immunofluorescence for βⅢ tubulin, GFAP and CNPase. RESULTS: Seven days after primary culture, a great many neurospheres could be obtained by successive pasage. Immunofluorescence assays showed that the neurospheres were nestin positive, and after differentiation, the cells expressed GFAP, CNPase and β -Ⅲ-tubulin. CONCLUSION: Embryonic day 14 rat mNSCs can differentiate into neuron-like cells and glial cells following induction by EGF, FGF2 and N2 additive.     

In vitro culture and differentiation of rat embryonic midbrain-derived neural stem cells*☆

BACKGROUND： Midbrain-derived neural stem cells （mNSCs） can differentiate into functional mature dopaminergic neurons. The mNSCs are considered the ideal choice for cell therapy of Parkinson＇s disease. OBJECTIVE： To isolate rat embryonic mNSCs and to observe the differentiation characteristics of mNSCs induced by cell growth-promoting factors. DESIGN, TIME AND SETTING： An in vitro cell culture study based on the molecular biology of nerve cells was carried out at the Institute of Clinical Medicine, China-Japan Friendship Hospital （China） from March to November 2007. MATERIALS： Sprague Dawley rats at embryonic day 14 were used in this study. Nestin antibody, β-Ⅲ tubulin antibody, glial fibrillary acidic protein （GFAP） antibody and cyclic nucleotide 3＇-phosphohydrolase （CNPase） antibody were provided by Abcam; DMEM/F12 medium and N2 supplement were provided by Invitrogen; epidermal growth factor （EGF） and fibroblast growth factor-2 （FGF2） were provided by R＆D Systems. METHODS： The ventral mesencephalon was dissected from embryonic day 14 rat embryos. By trypsin digestion and mechanical separation, the brain tissue was triturated into a fine single-cell suspension. The cells were cultured in 5 mL serum-free medium containing DMEM/FI 2, 1% N： supplement, 20 ng/mL EGF and FGF2. The mNSCs at the third generation were coated with 10ug/mL polylysine and induced to differentiate in the DMEM/F12 supplemented with 1% fetal bovine serum and 1% N2. MAIN OUTCOME MEASURES： The neural spheres of the third passage were identified by nestin immunofluorescence; at the same time, the cells were induced to differentiate, and the types of differentiated cell were identified by immunofluorescence for β Ⅲ tubulin, GFAP and CNPase. RESULTS： Seven days after primary culture, a great many neurospheres could be obtained by successive pasage. Immunofluorescence assays showed that the neurospheres were nestin positive, and after differentiation, the cells expressed GFAP, CNPase and β -Ⅲ-tu     

Folic acid deficiency inhibits neural rosette formation and neuronal differentiation from rhesus monkey embryonic stem cells

Evidence from epidemiological studies has proved that periconceptional use of folic acid (FA) can significantly reduce the risk of neural tube defects (NTDs). However, it is hard to explore when and how FA plays roles in neurogenesis and brain development in vivo, especially in human or other nonhuman primate systems. Primate embryonic stem cell (ESC) lines are ideal models for studying cell differentiation and organogenesis in vitro. In the present study, the roles of FA in neural differentiation were assessed in a rhesus monkey ESC system in vitro. The results showed no significant difference in the expression of neural precursor markers, such as nestin, Sox-1, or Pax-6, among neural progenitors obtained from different FA concentrations or with the FA antagonist methotrexate (MTX). However, FA depletion decreased cell proliferation and affected embryoid body (EB) and neural rosette formation, as well as neuronal but not neuroglia differentiation. Our data imply that the ESC system is a suitable model for further exploring the mechanism of how FA works in prevention of NTDs in primates.     

体外羊膜间充质干细胞分离培养及向神经元样细胞的分化

背景：胎盘组织作为寻找人类间充质干细胞的新来源,已成为备受人们关注的研究热点。从胎盘组织中羊膜层分离、培养细胞,因其来源广泛、不受伦理限制等优越性而具有广阔的应用前景。 目的：观察人羊膜间充质干细胞体外分离培养的方法,及向神经元样细胞分化的能力。 方法：用酶消化法从羊膜中分离和培养间充质干细胞,并通过形态的均一性及流式细胞术检测其表面标志以鉴定纯度。应用DMEM-LG+20 g/L DMSO+100 µmol/L BHA+10 μg/L碱性成纤维细胞生长因子诱导分化,通过免疫荧光染色鉴定诱导后的细胞。 结果与结论：可从羊膜组织成功分离培养出间充质干细胞,细胞贴壁生长,在体外短期内可以稳定增殖和传代。具有与骨髓间充质干细胞相似的表面标志,体外可以诱导分化为神经元样细胞,胶质纤维酸性蛋白与神经元特异性烯醇化酶免疫荧光染色均为阳性。提示羊膜间充质干细胞可以在体外分离培养并诱导分化为神经元样细胞,羊膜组织可以作为干细胞研究以及神经组织疾病细胞治疗的一个全新的细胞来源。     

不同培养体系可逆改变人类胚胎干细胞的分化倾向

背景：人类胚胎干细胞可以在饲养细胞依赖性培养体系和化学限定性培养体系下维持未分化状态,能够在体内外诱导分化成三胚层来源的细胞类型。 目的：比较饲养细胞和化学限定性培养体系对人类胚胎干细胞特性的影响。 方法：将在饲养细胞培养体系下培养27代的人类胚胎干细胞转入到化学限定性培养基体系中培养56代,然后再将其转回到饲养细胞培养体系中,将3种培养条件下的人类胚胎干细胞(饲养细胞培养体系培养70代、化学限定性培养体系培养56代、化学限定性培养体系下培养70代后转回饲养细胞培养体系下培养13代和20代)进行多能性分子标记SSEA4流式分析等检测分析,同时对3种培养条件下人类胚胎干细胞经拟胚体诱导分化后分别检测多能性基因和三胚层分化基因的表达。 结果与结论：人类胚胎干细胞在饲养细胞和化学限定性培养体系下表现出不同的诱导分化倾向,在化学限定性培养体系下表现出向神经诱导分化抑制,这种不同的诱导分化倾向可发生可逆性转换,当人类胚胎干细胞由化学限定性培养体系转回到饲养细胞培养体系时,诱导分化倾向表现出与其在饲养细胞下诱导分化一致的模式。在拟胚体分化中,多能性基因Nanog高可能对诱导分化倾向起着重要作用。与此同时,人类胚胎干细胞SSEA4细胞亚群发生相应的变化,人类胚胎干细胞在饲养细胞和化学限定下培养体系下表现的分化倾向与人类胚胎干细胞亚群所占的比例存在关联。     

人胚胎干细胞在无血清mTeSR®1培养基中维持培养和向内皮细胞的诱导分化

背景：传统的人胚胎干细胞培养扩增方法中应用含动物血清培养基,并依赖饲养层细胞培养,这种培养方法显著制约了干细胞的体外培养规模;另外异源动物血清成分介入,使病原污染及免疫排斥的概率显著增加。目的：明确应用无血清培养基mTeSR®1对人胚胎干细胞进行长期体外培养的可行性,并建立诱导人胚胎干细胞分化为血管内皮细胞的相关技术平台。方法：采用无血清培养基mTeSR®1以非饲养层细胞依赖的方式体外培养、扩增人胚胎干细胞株H9。经过40余次体外传代后,于倒置显微镜下观察其生长形态,并利用免疫荧光染色方法评估其细胞表型。此外,应用条件培养基诱导H9细胞株向内皮细胞方向分化。利用免疫荧光染色技术,定量RT-PCR以及低密度脂蛋白摄取实验对该胚胎干细胞源内皮细胞的表型及功能进行评价、分析。结果与结论：mTeSR®1培养基能够支持H9细胞株在体外以非饲养层依赖的方式进行长期扩增,同时维持其未分化的干细胞潜能。添加血管内皮细胞的条件培养基能够定向诱导H9细胞向内皮细胞方向分化。该胚胎干细胞源内皮细胞不但表达内皮细胞的标志基因(kdr,pecam)和标记蛋白CD31,而且还能够摄取低密度脂蛋白,形成类似微血管结构。提示实验中所提供的培养及诱导分化体系能够支持胚胎干细胞的增殖与分化行为。     

Time-sensitive effects of hypoxia on differentiation of neural stem cells derived from mouse embryonic stem cells in vitro

Abstract

Objectives:

Oxygen tension is an important component of microenvironment for the differentiation of embryonic stem cells including neural lineage. However, the comprehensive influence of hypoxia on neural differentiation during embryonic neural development has not yet been examined.

Methods:

In this study, we investigated the effect of low oxygen levels (5% O₂), or hypoxia, in two stages of neural differentiation in vitro: (1) inducing mouse embryonic stem cells into neural stem cells (NSCs); and then (2) inducing NSCs into neural progenitor cells in neurospheres.

Results:

In the first stage, NSCs generation was reduced under hypoxia. Less mature morphological changes (including neural marker) of NSCs were observed, suggesting the prevention of early differentiation under hypoxic conditions. Thus undifferentiated stem cells were maintained in this stage. However, in the second stage, hypoxia induced neural differentiation in neurospheres. Nevertheless, non-neural progenitor cell formation, such as mesoderm progenitor cell lines or epithelial cell lines, was restricted by low oxygen tension.

Discussions:

Our results demonstrate that hypoxia is essential for regulating neural differentiation and show the different effects on NSC differentiation dependent on the time-course of NSC development. In the early stage of NSCs induction, hypoxia inhibits neural differentiation and maintains the undifferentiated state; in the later stage of NSCs induction, hypoxia induces neural differentiation. Our study may contribute to the development of new insights for expansion and control of neural differentiation.     

胚胎干细胞定向分化运动神经元的基因调控

本文着重就胚胎干细胞 (ESCs)定向诱导分化为脊髓运动神经元过程中两个重要阶段及相关基因的表达调控进行综述。运动神经元分化的基因调控研究将有利于阐明其发育和分化的模式 ,为其应用于脊髓损伤修复和运动神经元退行性疾病的替代治疗提供理论依据     

胚胎小鼠脊髓源性与纹状体源性神经干细胞的培养及分化特点

目的 探讨小鼠脊髓源性神经干细胞与纹状体源性神经干细胞的分离培养方法 及增殖特点,比较两种来源的神经干细胞发育时期上的异同,寻找更有利于脊髓损伤修复的种子细胞.方法 利用显微解剖、无血清培养和单细胞克隆技术在孕14 d小鼠的胎鼠的脊髓及纹状体中分离培养具有单细胞克隆能力的细胞,免疫荧光染色检测克隆细胞的神经巢蛋白(nestin)抗原和诱导分化后特异性成熟神经细胞抗原的表达,并比较两种来源的干细胞在培养及分化方向上的异同点.结果从胎鼠的脊髓和纹状体中成功分离出神经干细胞.两种来源的干细胞均具有连续克隆能力可传代培养,表达nestin.脊髓血清诱导分化后脊髓源性神经干细胞β-tubulinⅢ阳性细胞(13.5±0.8)较纹状体源性神经干细胞(17.4±1.1)减少,而nestin、GFAP阳性细胞明显增多(45.7±0.3vs 39.2±1.2;25.2±1.3 vs 18.8±0.9),差异均有统计学意义(P<0.05). 结论 依据细胞增殖特点和分化结果的区别,证实纹状体源性神经干细胞更适合用于移植修复脊髓损伤.     

The "brainy side" of human embryonic stem cells

The recent isolation of human embryonic stem (ES) cells is evoking great hopes for their future utilization in cell-replacement therapies and human development research. The hallmarks of ES cells, pluripotency and self-renewal capacity, suggest an infinite source for tissues of virtually all desired types. Specifically, human ES cells may potentially be the basis for effective treatments of a wide range of human neurodegenerative disorders. To enable the translation of this novel biomedical field into the clinic, mechanisms that control the differentiation of human embryonic stem cells into fully functional neuronal cells should be analyzed and controlled.     

小鼠胚胎干细胞诱导的神经前体细胞纹状体移植对PD大鼠的治疗作用

目的 观察来源于小鼠胚胎干细胞的神经前体细胞移植PD大鼠纹状体后的存活、分化以及细胞移植对PD大鼠的治疗作用。方法 采用无血清方法将小鼠胚胎干细胞定向诱导为神经前体细胞，免疫组化技术观察移植细胞的存活、分化。结果 胚胎体在N2选择性培养基选择生长5d后，85％以上的小鼠胚胎干细胞分化为nestin阳性的神经前体细胞。移植到PD大鼠纹状体后大部分神经前体细胞存活良好，移植细胞分别保持为未分化的nestin阳性的神经前体细胞和TH阳性的神经元。移植后3周，PD大鼠的旋转次数明显减少。结论 胚胎干细胞来源的神经前体细胞移植PD大鼠纹状体后能分化为TH阳性的神经细胞，对PD有治疗作用。