外胚层细胞诱导后的间隙连接

用电镜观察了经受诱导作用之后胚胎细胞的冷冻蚀刻复型膜。和未经诱导作用的对照组比较,早期和中期神经胚的神经上皮细胞以及经过豚鼠骨髓粗提液(BME)——一种有效的异源中胚层诱导物——处理过的早期原肠胚外胚层,它们的间隙连接都处于活跃的动态状态。用图像分析仪测得的间隙连接连接子的排列密度,指出经受过诱导作用的三组分别和未经受诱导作用的对照组比较,计算求出P值,神经上皮两组和对照组的差别为非常显著,BME处理过的细胞和对照组的差别为显著。结合对照组与诱导后胚胎细胞间隙连接连接子的变化讨论了它们在信息传递上可能起的作用。     

Self-Contained Induction of Neurons from Human Embryonic Stem Cells

Background

Neurons and glial cells can be efficiently induced from mouse embryonic stem (ES) cells in a conditioned medium collected from rat primary-cultured astrocytes (P-ACM). However, the use of rodent primary cells for clinical applications may be hampered by limited supply and risk of contamination with xeno-proteins.

Methodology/Principal Findings

We have developed an alternative method for unimpeded production of human neurons under xeno-free conditions. Initially, neural stem cells in sphere-like clusters were induced from human ES (hES) cells after being cultured in P-ACM under free-floating conditions. The resultant neural stem cells could circumferentially proliferate under subsequent adhesive culture, and selectively differentiate into neurons or astrocytes by changing the medium to P-ACM or G5, respectively. These hES cell-derived neurons and astrocytes could procure functions similar to those of primary cells. Interestingly, a conditioned medium obtained from the hES cell-derived astrocytes (ES-ACM) could successfully be used to substitute P-ACM for induction of neurons. Neurons made by this method could survive in mice brain after xeno-transplantation.

Conclusion/Significance

By inducing astrocytes from hES cells in a chemically defined medium, we could produce human neurons without the use of P-ACM. This self-serving method provides an unlimited source of human neural cells and may facilitate clinical applications of hES cells for neurological diseases.     

杂交狼尾草胚性愈伤组织的诱导与植株再生

以杂交狼尾草初级愈伤为材料,运用方差分析的方法研究不同因素对杂交狼尾草胚性愈伤诱导率与植株再生率的影响。结果发现,2,4-D和TDZ对杂交狼尾草胚性愈伤诱导影响显著,最佳的胚性愈伤诱导培养基为MS+3.0 mg·L-12,4-D+0.4 mg·L-16-BA+0.2 mg·L-1 TDZ,诱导率为54%;愈伤分化培养基以附加0.1 mg·L-1 TDZ+0.2 mg·L-16-BA+3.2 mg·L-1 CuSO4的 MS培养基为最佳,再生率为68.33%,褐化率为8.33%;分化培养基中添加0.8~3.2mg·L-1的CuSO4均能促进杂交狼尾草愈伤组织分化,而添加AgNO3对杂交狼尾草愈伤分化无显著影响。该技术为离体诱变获得杂交狼尾草低温种质材料奠定了基础。     

A Rapid Experimental Method to Study Primary Embryonic Induction

The technique described here is a combination of the sandwich-method and cell culture. It allows one to know quickly whether induction occurred or not (48 h if cell spreading is considered, 4–5 days if morphological differentiation is observed). The dissociation of the explants soon after induction does not disturb their inductive pattern. Moreover, this method allows a daily observation of the morphological events and study at the cellular and/or molecular level.     

Mechanisms of Cell Interaction during Primary Embryonic Induction Studied in Transfilter Experiments

The transmission mechanisms operative at different stages of neutralisation during primary embryonic induction of the newt Triturus vulgaris were studied in experiments employing Nuclepore filters placed between interactive tissue explants. The transmission time of the neuralising effect was determined with 0.2 μm Nuclepore filter. In another series of experiments the transformation of neuralised ectoderm by archenteron roof mesoderm into other parts of the CNS was studied. Although sufficiently long induction times were used no transformation into hindbrain structures could be induced across filters with pore sizes from 0.1 μm to 1.0 μm. However, electron microscopy demonstrated cytoplasmic penetration into 0.6 μm filters at 15 h of induction. The results speak against free long-range diffusion of inductive material at the stage of transformation of the neuralised ectoderm to more caudal parts of CSN and warrant a more detailed structural study of the transmission phenomenon in question.     

诱导胚胎干细胞向神经细胞分化方法的研究与探讨

胚胎干细胞(ES细胞)是一种能够在体外进行不断自我更新,并具有多种分化潜能的细胞。胚胎干细胞向神经细胞诱导分化的研究进展迅速,相关实验技术和理论也不断发展。总结了近年来各国研究者诱导小鼠和人胚胎干细胞向神经细胞分化的方法,分析了一些方法的原理并初步探讨其相关的分子机制,并提出一些可行性新方法。胚胎干细胞向神经细胞诱导分化因其体外的可操作性、来源的广泛性及质量可控性将有可能成为临床上治疗神经系统疾病的有效方法。     

Induction of Cancerous Stem Cells during Embryonic Stem Cell Differentiation

Stem cell maintenance depends on their surrounding microenvironment, and aberrancies in the environment have been associated with tumorigenesis. However, it remains to be elucidated whether an environmental aberrancy can act as a carcinogenic stress for cellular transformation of differentiating stem cells into cancer stem cells. Here, utilizing mouse embryonic stem cells as a model, it was illustrated that environmental aberrancy during differentiation leads to the emergence of pluripotent cells showing cancerous characteristics. Analogous to precancerous stages, DNA lesions were spontaneously accumulated during embryonic stem cell differentiation under aberrational environments, which activates barrier responses such as senescence and apoptosis. However, overwhelming such barrier responses, piled-up spheres were subsequently induced from the previously senescent cells. The sphere cells exhibit aneuploidy and dysfunction of the Arf-p53 module as well as enhanced tumorigenicity and a strong self-renewal capacity, suggesting development of cancerous stem cells. Our current study suggests that stem cells differentiating in an aberrational environment are at risk of cellular transformation into malignant counterparts.     

食蟹猴胚胎干细胞向间充质前体细胞诱导分化及鉴定

间充质干细胞(mesenchymal stem cells,MSCs)可以诱导分化成脂肪、软骨、骨骼和骨骼肌细胞,并可作为骨骼、软骨或肌肉移植中的再生干细胞,广泛应用于细胞治疗和组织工程。胚胎干细胞(embryonic stem cells,ESCs)具有体外培养无限增殖和多向分化的特性,能被诱导分化为机体几乎所有的细胞类型。该研究通过无血清条件下诱导食蟹猴ESCs形成类胚体(embryoid bodies,EBs),然后在血清条件下贴壁分化EBs成间充质前体细胞(mesenchymal precursor cells,MPCs),再经过长期体外培养,纯化和扩增MPCs。结果显示,纯化后的MPCs具有MSCs生物学特征,并能在体外诱导分化成脂肪细胞和骨细胞。将这些细胞皮下注射给SCID小鼠,并未发现形成肿瘤,提示食蟹猴ESCs来源的MPCs具有一定的安全性。     

Induction of Glutamine Synthetase in Rat Astrocytes by Co-Cultivation with Embryonic Chick Neurons

Co-cultivation of confluent rat astrocyte cultures with embryonic chick neurons resulted in induction of glutamine synthetase activity in the astrocytes. This induction of glutamine synthetase in astrocytes by neurons was independent of induction by hydrocortisone and forskolin, but was dependent on the length of co-cultivation and the number of neurons present in the co-culture. Cycloheximide and actinomycin D inhibited the induction of glutamine synthetase in astrocytes by neurons, whereas cytosine arabinoside had no apparent effect. Results suggest that this induction of glutamine synthetase in astrocytes is mediated by cell contact with neurons and may represent a specific neuronal and glial interaction.     

Germ Layer Interactions in Pattern Formation of Amphibian Mesoderm during Primary Embryonic Induction

Mesodermal differentiation of dorsal marginal zone (DMZ) before and after invagination was analyzed by a series of combination experiments with different kinds of ectoderm.
Lower DMZ of early gastrula didn't show any axial-mesoderm (notochord and somitic mesoderm) but lateral mesoderm (mesenchyme, mesothelium, or blood cells) in combinant with non-competent ventral ectoderm, while combinant with competent ectoderm was found to have well-differentiated axial-mesoderm with deutero-spinocaudal neurals. The axial-mesoderms have origin in the ectoderm. Uninvaginated DMZ of middle gastrula also showed difference in mesodermal differentiation between competent and non-competent ectoderms; axial-mesoderm differentiation was much better in competent than in non-competent. The axial-mesoderm originated from the uninvaginated DMZ. Archenteron roof of late gastrula showed regional difference in mesodermal differentiation in both combinants with competent and non-competent. The present study further demonstrated that there was regionality in promoting effect of induced neurectoderm on axial-mesoderm differentiation of invaginated archenteron roof.
The present experiments suggest that the cranio-caudal and dorso-ventral axis formations of amphibian mesoderm are finally determined by sequential and reciprocal interactions between the mesodermal anlage and the overlying ectoderm. It should be also shown that lower DMZ acts to trigger a series of the sequential interactions during primary embryonic induction.     

不同人胚胎干细胞系向限定性内胚层分化能力存在差异

研究不同的人胚胎干细胞系向限定性内胚层细胞分化能力是否存在差异,并尝试寻找造成差异的原因.基于已建立的人胚胎干细胞库资源和限定性内胚层定向诱导分化体系,流式检测诱导分化3天后10株细胞系Sox17的阳性比率发现其值在60%到80%之间波动,而SSEA4的阳性比率在人胚胎干细胞系中不存在明显差异.基因表达谱结果显示像Sox17,Foxa2等内胚层标记在这些细胞之间不存在显著的差异,而像MEG3和SNORD114-3在差异细胞系之间和同株细胞早晚期代数之间存在表达差异.结果提示不同的人胚胎干细胞系向限定性内胚层细胞分化能力存在着差异,推测这些差异可能与MEG3和SNORD114-3的差异表达相关.     

Induction of Specific Chromosomal Aberrations by Adenovirus Type 12 in Human Embryonic Kidney Cells

Nonrandom chromosomal breaks in chromosomes 1 and 17 were provoked in human embryonic kidney cells 24 hr after infection with adenovirus type 12. These chromosomal changes disappeared in persistently infected cultures. Neutralization of the virus with type-specific antiviral serum prior to infection prevented the occurrence of chromosomal aberrations. No viral deoxyribonucleic acid (DNA) synthesis, as determined by autoradiography, was seen in metaphases containing adenovirus type 12-induced chromosomal aberrations. Ultraviolet irradiation of the virus reduced chromosomal aberrations linearly. This reduction in aberrations was fourfold slower than the inactivation of viral infectivity. At 24 hr after infection of cells with purified (3)H-labeled adenovirus type 12, the isotope was found to be associated with the nuclei. The uptake of isotope was reduced ninefold when the labeled virus was neutralized with type-specific antiviral serum. This difference is considered to account for neutralization of labeled virions. In metaphases infected with labeled viruses, most of the clustered grains were seen only on one arm of the chromatid, even after 72 hr. Isochromatid labeling was found, however, in a small percentage of chromosomes, and increased with time after infection. This increase was threefold between 24 and 72 hr after infection, whereas the mean grain counts decreased twofold during the same period. This has been tentatively interpreted to mean that most of the viral DNA molecules or parts thereof are merely attached to cellular chromatin, but a small fraction of them becomes gradually integrated as time proceeds. Certain chromosomal sites appeared to be preferentially labeled when chromosome 2 was used as a model for evaluation.     

Nuclear Binding of Hydrocortisone-Receptors in the Embryonic Chick Retina and its Relationship to Glutamine Synthetase Induction

Binding of hydrocortisone (HC) to cytoplasmic receptors andof the resulting receptor-hydrocortisone complex (R-HC) to nuclearacceptor sites has been studied in neural retina cells of thechick embryo, in which this hormone induces glutamine synthetase(GS). These studies were done in a cell-free system, as wellas in intact retina tissue in culture. Optimal conditions, specificity, and the quantitative aspectsof R-HC binding to nuclei in the cell-free system were determined.Isolated nuclei retained their binding specificity for R-HCprepared from retina cytosol; at saturation, the total numberof nuclear acceptor sites for R-HC was estimated to be in therange of 1500 per nucleus. These sites were resistant to RNAsebut sensitive to DNAse. When retina tissue was cultured in the presence of progressivelyhigher doses (0–90 nM) of HC, increasing amounts of receptorswere translocated from the cytoplasm to the nuclei. Followingincubation of retina with 9–90 nM HC, the cytoplasm wasalmost completely depleted of receptors and the nuclei becamesaturated with R-HC. From the amount of R-HC bound to nucleiat saturation, the estimated total number of R-HC acceptor sitesper nucleus was comparable to that derived from the cell-freesystem. Increases in the level of GS induction in the retinacorrelated well with amounts of R-HC complex bound by nuclei.     

Wls Is Expressed in the Epidermis and Regulates Embryonic Hair Follicle Induction in Mice

Wnt proteins are secreted molecules that play multiple roles during hair follicle development and postnatal hair cycling. Wntless (Wls) is a cargo protein required for the secretion of various Wnt ligands. However, its role during hair follicle development and hair cycling remains unclear. Here, we examined the expression of Wls during hair follicle induction and postnatal hair cycling. We also conditionally deleted Wls with K14-cre to investigate its role in hair follicle induction. K14-cre;Wls^c/c mice exhibited abnormal hair follicle development, which is possibly caused by impaired canonical Wnt signaling. Meanwhile, Wnt5a is also expressed in embryonic epidermis, but Wnt5a null mice showed no significant defect in embryonic hair follicle morphogenesis. Therefore, Wls may regulate hair follicle induction by mediating the Wnt/β-catenin pathway.