Quality of embryonic bodies and seeding density effects on neural differentiation of mouse embryonic stem cells

Mouse embryonic stem (ES) cells can be differentiated into neural lineage cells, but the differentiation efficiency remains low. This study revealed two important factors that influence the neural differentiation efficiency of mouse ES cells: the first is the quality of embryonic bodies (EBs); good quality of EBs consistently originated from a suspension culture of 1 × 10⁵ ES cells/ml serum-free chemically defined neural inducing medium and they exhibited a smooth round shape, with a dark central region surrounded by a light band. Such EBs are capable of attaining high neural differentiation efficiency. However, poor quality EBs originated from a suspension culture of 1 × 10⁶ ES cells/ml serum-free chemically defined neural inducing medium and exhibited an irregular shape or adhered to the bottom of the dish; they displayed low neural differentiation efficiency. The second factor is the seeding density of EBs: a low seeding density (5 EBs/cm²) induced cells to differentiate into a more caudalized subtypes compared to the cells obtained from high seeding density (20 EBs/cm²). These findings provided fresh insight into the neural induction of mouse ES cells.     

Differentiation of rhesus embryonic stem cells to neural progenitors and neurons

Embryonic stem (ES) cells are pluripotent cells capable of differentiating into cell lineages derived from all primary germ layers including neural cells. In this study we describe an efficient method for differentiating rhesus monkey ES cells to neural lineages and the subsequent isolation of an enriched population of Nestin and Musashi positive neural progenitor (NP) cells. Upon differentiation, these cells exhibit electrophysiological characteristics resembling cultured primary neurons. Embryoid bodies (EBs) were formed in ES growth medium supplemented with 50% MEDII. After 7 days in suspension culture, EBs were transferred to adherent culture and either differentiated in serum containing medium or expanded in serum free medium. Immunocytochemistry on differentiating cells derived from EBs revealed large networks of MAP-2 and NF200 positive neurons. DAPI staining showed that the center of the MEDII-treated EBs was filled with rosettes. NPs isolated from adherent EB cultures expanded in serum free medium were passaged and maintained in an undifferentiated state by culture in serum free N2 with 50% MEDII and bFGF. Differentiating neurons derived from NPs fired action potentials in response to depolarizing current injection and expressed functional ionotropic receptors for the neurotransmitters glutamate and gamma-aminobutyric acid (GABA). NPs derived in this way could serve as models for cellular replacement therapy in primate models of neurodegenerative disease, a source of neural cells for toxicity and drug testing, and as a model of the developing primate nervous system.     

Chemical inhibition of sulfation accelerates neural differentiation of mouse embryonic stem cells and human induced pluripotent stem cells

Pluripotency of embryonic stem cells (ESCs) is maintained by the balancing of several signaling pathways, such as Wnt, BMP, and FGF, and differentiation of ESCs into a specific lineage is induced by the disruption of this balance. Sulfated glycans are considered to play important roles in lineage choice of ESC differentiation by regulating several signalings. We examined whether reduction of sulfation by treatment with the chemical inhibitor chlorate can affect differentiation of ESCs. Chlorate treatment inhibited mesodermal differentiation of mouse ESCs, and then induced ectodermal differentiation and accelerated further neural differentiation. This could be explained by the finding that several signaling pathways involved in the induction of mesodermal differentiation (Wnt, BMP, and FGF) or inhibition of neural differentiation (Wnt and BMP) were inhibited in chlorate-treated embryoid bodies, presumably due to reduced sulfation on heparan sulfate and chondroitin sulfate. Furthermore, neural differentiation of human induced pluripotent stem cells (hiPSCs) was also accelerated by chlorate treatment. We propose that chlorate could be used to induce efficient neural differentiation of hiPSCs instead of specific signaling inhibitors, such as Noggin.     

Restricted expression of LUZP in neural lineage cells: A study in embryonic stem cells

A novel protein LUZP with 3 leucine zipper motifs at its amino terminus is predominantly expressed in the adult brain. A modified gene targeting approach was employed in an attempt to establish in vitro and in vivo models in which Luzp is knock-out (KO) for phenotype assessment and a reporter gene lacZ is knock-in (KI) for tracing its expression. We report in this study the molecular cloning of the Luzp gene, its targeting vector construction and Luzp-KO/lacZ-KI embryonic stem (ES) clone selection. Since LUZP is also expressed in ES cells, the possibility of embryonic lethality cannot be excluded when attempting to establish Luzp-null mutant mice. We have therefore examined the development of homozygous Luzp-KO/lacZ-KI clones in nude mice. Tissue types derived from all three embryonic germ layers were observed in teratomas developed in nude mice. In situ X-gal staining further revealed restricted expression of LUZP in neural lineage cells.     

Culturing and differentiation of murine embryonic stem cells in a three-dimensional fibrous matrix

Embryonic stem (ES) cells have indefinite self-renewal ability and pluripotency, and can provide a novel cell source for tissue engineering applications. In this study, a murine CCE ES cell line was used to derive hematopoietic cells in a 3-D fibrous matrix. The 3-D matrix was found to maintain the phenotypes of undifferentiated ES cells as indicated by alkaline phosphatase (ALP) activity and stage specific embryonic antigen-1 (SSEA-1) expression. In hematopoietic differentiation, cells from 3-D culture exhibited similar cell cycle distribution and SSEA-1 expression to those in the initial cell population. The Oct-4 expression was significantly down-regulated, which indicated the occurrence of differentiation, although the level was slightly higher than that in Petri dish culture. The expression of c-kit, cell surface marker for hematopoietic progenitor, was higher in the 3-D culture, suggesting a better-directed hematopoietic differentiation. Cells in the 3-D matrix tended to form large aggregates associated with fibers. For large-scale processes, a perfusion bioreactor can be used for both maintenance and differentiation cultures. As compared to the static culture, a higher growth rate and final cell density were resulted from the perfusion bioreactor due to better control of the reactor environment. At the same time, the differentiation capacity of ES cells was preserved in the perfusion culture. The ES cell culture in the fibrous matrix thus can be used as a 3-D model system to study effects of extracellular environment and associated physico-chemical parameters on ES cell maintenance and differentiation.     

Efficient suspension bioreactor expansion of murine embryonic stem cells on microcarriers in serum-free medium

Large numbers of cells will be required for successful embryonic stem cell (ESC)-based cellular therapies or drug discovery, thus raising the need to develop scaled-up bioprocesses for production of ESCs and their derived progeny. Traditionally, ESCs have been propagated in adherent cultures in static flasks on fibroblasts layers in serum-containing medium. Direct translation of two-dimensional flatbed cultures to large-scale production of the quantities of cells required for therapy simply by increasing the number of dishes or flasks is not practical or economical. Here, we describe successful scaled-up production of ESCs on microcarriers in a stirred culture system in a serum-free medium. Cells expanded on CultiSpher S, Cytodex 3, and Collagen microcarriers showed superior cell-fold expansions of 439, 193, and 68, respectively, without excessive agglomeration, compared with 27 in static culture. In addition, the ESCs maintained their pluripotency after long-term culture (28 days) in serum-free medium. This is the first time mESCs have been cultured on microcarriers without prior exposure to serum and/or fibroblasts, while also eliminating the excessive agglomeration plaguing earlier studies. These protocols provide an economical, practical, serum-free means for expanding ESCs in a stirred suspension bioprocess.     

Embryonic stem cells in non-human primates: An overview of neural differentiation potential

Non-human primate (NHP) embryonic stem (ES) cells show unlimited proliferative capacities and a great potential to generate multiple cell lineages. These properties make them an ideal resource both for investigating early developmental processes and for assessing their therapeutic potential in numerous models of degenerative diseases. They share the same markers and the same properties with human ES cells, and thus provide an invaluable transitional model that can be used to address the safety issues related to the clinical use of human ES cells. Here, we review the available information on the derivation and the specific features of monkey ES cells. We comment on the capacity of primate ES cells to differentiate into neural lineages and the current protocols to generate self-renewing neural stem cells. We also highlight the signalling pathways involved in the maintenance of these neural cell types. Finally, we discuss the potential of monkey ES cells for neuronal differentiation.     

Effects of iron oxide nanoparticles on cardiac differentiation of embryonic stem cells

The therapeutic potential of transplantation of embryonic stem cells (ESCs) in animal model of myocardial infarction has been consistently demonstrated. The development of superparamagnetic iron oxide (SPIO) nanoparticles labeling and cardiac magnetic resonance imaging (MRI) have been increasingly used to track the migration of transplanted cells in vivo allowing cell fate determination. However, the impact of SPIO- labeling on cell phenotype and cardiac differentiation capacity of ESCs remains unclear. In this study, we demonstrated that ESCs labeled with SPIO compared to their unlabeled counterparts had similar cardiogenic capacity, and SPIO-labeling did not affect calcium-handling property of ESC-derived cardiomyocytes. Moreover, transplantation of SPIO-labeled ESCs via direct intra-myocardial injection to infarct myocardium resulted in significant improvement in heart function. These findings demonstrated the feasibility of in vivo ESC tracking using SPIO-labeling and cardiac MRI without affecting the cardiac differentiation potential and functional properties of ESCs.     

人胚胎神经干细胞的基础及应用研究进展

神经干细胞是中枢神经系统中具有增殖、自我更新能力以及多种分化潜能的细胞,对它的研究已经成为神经生物学、发育生物学以及脑科学研究的一个热点。随着神经干细胞（特别是胚胎神经干细胞）的分离、培养成功,神经干细胞移植已被尝试用于神经系统损伤等疾病的治疗。但是,关于胚胎神经干细胞的研究尚处于初级阶段,特别是人胚胎神经干细胞的研究、报道还比较少。本文对国内、外近几年来关于人胚胎神经干细胞的基础及应用研究进展作了综述。     

Proteomic analysis of neural differentiation of mouse embryonic stem cells

Mouse embryonic stem cells (mESCs) can differentiate into different types of cells, and serve as a good model system to study human embryonic stem cells (hESCs). We showed that mESCs differentiated into two types of neurons with different time courses. To determine the global protein expression changes after neural differentiation, we employed a proteomic strategy to analyze the differences between the proteomes of ES cells (E14) and neurons. Using 2-DE plus LC/MS/MS, we have generated proteome reference maps of E14 cells and derived dopaminergic neurons. Around 23 proteins with an increase or decrease in expression or phosphorylation after differentiation have been identified. We confirmed the downregulation of translationally controlled tumor protein (TCTP) and upregulation of alpha-tubulin by Western blotting. We also showed that TCTP was further downregulated in derived motor neurons than in dopaminergic neurons, and its expression level was independent of extracellular Ca(2+) concentration during neural differentiation. Potential roles of TCTP in modulating neural differentiation through binding to Ca(2+), tubulin and Na,K-ATPase, as well as the functional significance of regulation of other proteins such as actin-related protein 3 (Arp3) and Ran GTPase are discussed. This study demonstrates that proteomic tools are valuable in studying stem cell differentiation and elucidating the underlying molecular mechanisms.     

组蛋白修饰对胚胎干细胞分化过程[]的调控机制

选用人类胚胎干细胞系和由人类胚胎干细胞系分化来的神经干细胞系为研究对象,分析组蛋白修饰对胚胎干细胞分化过程的调控作用。得到了两种细胞系差异表达基因转录起始位点侧翼区域内八种组蛋白修饰的分布模式,以及组蛋白修饰功能簇。研究表明在两类细胞系中,八种组蛋白修饰谱分布模式一致,且呈现两种分布类型; H3K27ac,H3K4me3和H3K9ac组成的功能簇是保守的;H3K27me3,H3K36me3和H3K79me1组成的功能簇以及H3K9me3和H3K27me3组成的功能簇在胚胎干细胞向神经干细胞分化的过程中消失。结果揭示了组蛋白修饰对胚胎干细胞系向神经干细胞系分化过程的部分调控机制,为该分化过程分子调控机制的研究提供部分重要的理论基础。     

Retinoic-acid-concentration-dependent acquisition of neural cell identity during in vitro differentiation of mouse embryonic stem cells

Retinoic acid (RA) is one of the most important morphogens, and its embryonic distribution correlates with neural differentiation and positional specification in the developing central nervous system. To investigate the concentration-dependent effects of RA on neural differentiation of mouse embryonic stem cells (ES cells), we investigated the precise expression profiles of neural and regional specific genes by ES cells aggregated into embryoid bodies (EBs) exposed to various concentrations of RA or the BMP antagonist Noggin. RA promoted both neural differentiation and caudalization in a concentration-dependent manner, and the concentration of RA was found to regulate dorso-ventral identity, i.e., higher concentrations of RA induced a dorsal phenotype, and lower concentrations of RA induced a more ventral phenotype. The induction of the more ventral phenotype was due to the higher expression level of the N-terminus of sonic hedgehog protein (Shh-N) when treated with low concentration RA, as it was abrogated by an inhibitor of Shh signaling, cyclopamine. These findings suggest that the concentration of RA strictly and simultaneously regulates the neuralization and positional specification during differentiation of mouse ES cells and that it may be possible to use it to establish a strategy for controlling the identity of ES-cell-derived neural cells.     

体外诱导鸡胚胎生殖细胞分化为神经干细胞

本研究探讨体外诱导鸡胚胎生殖细胞(EGCs)分化为神经干细胞(NSCs)的可能性.EGCs经类胚体(EB)阶段,以维生素A酸(RA)等进行诱导,在NSCs选择性培养基中筛培养扩增7 d,观察形态变化;采用RT-PCR法检测nestin基因表达及免疫细胞化学法检测nestin等NSCs特异性标志物,并对其扩增及分化能力进行观察.结果显示:EGCs经初级诱导,NSCs选择性培养基筛选培养7 d后,形成大量神经球样结构,可扩增传代;绝大部分神经球样结构呈nestin抗原阳性,表达nestin基因,且可分化为神经上皮样及少突胶质细胞.研究结果表明:RA等诱导的EGCs,经选择性培养基筛选培养可获得NSCs,有望为眼部神经变性疾病的治疗提供新的技术参考.     

Derivation of cranial neural crest-like cells from human embryonic stem cells

The neural crest is a transient population of multipotent progenitors contributing to a diverse array of tissues throughout the vertebrate embryo. Embryonic stem (ES) cells are able to form embryoid body and spontaneously differentiate to various lineages, following a reproducible temporal pattern of development that recapitulates early embryogenesis. Embryoid bodies were triturated and the dissociated cells were processed for fluorescence-activated cell sorting (FACS), and more than 1% of cells were identified as frizzled-3⁺/cadherin-11⁺. Expression of marker genes associated with various terminal fates was detected for chondrocytes, glia, neurons, osteoblasts and smooth muscles, indicating that the FACS-sorted frizzled-3⁺/cadherin-11⁺ cells were multipotent progenitor cells capable of differentiating to fates associated with cranial neural crest. Moreover, the sorted cells were able to self-renew and maintain multipotent differentiation potential. The derivation of cranial neural crest-like multipotent progenitor cells from ES cells provides a new tool for cell lineage analysis of neural crest in vitro.     

Comparison of different culture modes for long-term expansion of neural stem cells

Neural stem cells (NSCs) can be cultured in two modes of suspension and monolayer in vitro. The cultured cells are different in both the ability to proliferate and heterogeneity. In order to find the appropriate methods for large-scale expansion of NSCs, we systematically compared the NSCs cultured in suspension with those cultured in monolayer. The forebrain tissue was removed from embryonic day 14 (E14) mice, then the tissue was dissociated into single-cell suspension by Accutase and mechanical trituration. The cells were cultured in both suspension and monolayer. The NSCs cultured in suspension and in monolayer were compared on viability, ability to proliferate and heterogeneity by fluorescent dyes, immunofluorescence and flow cytometry on DIV21 (21 days in vitro), DIV56 and DIV112, respectively. The results indicated that the NSCs cultured in both suspension and monolayer represented good viability in long-term cultures. But they displayed a distinct ability to proliferate in long-term cultures. The NSCs cultured in monolayer preceded those cultured in suspension on the ability to proliferate on DIV21 and DIV56, but no obvious difference on DIV112. The NSCs population cultured in suspension displayed more nestin-positive cells than those in monolayer during the whole process of culture. The NSCs population cultured in monolayer, however, displayed more βIII tubulin-positive cells than those in suspension in the same period. The suspension culture mode excels the monolayer culture mode for large-scale expansion of NSCs.     

Neuronal differentiation of NTE-deficient embryonic stem cells

Organophosphates induce neurological disorders. One of the enzymes inhibited by these compounds is neuropathy target esterase (NTE). In vitro, inhibition of NTE activity by organophosphates is correlated with inhibition of neurite initiation and reduction of neurite length, supporting the hypothesis that organophosphate-induced neurological disorders are caused by inhibition of NTE activity. However, there is no direct evidence for the involvement of NTE in organophosphate-induced impairment of neurites in vitro. To examine the role of NTE, we have generated NTE-deficient mouse embryonic stem cells. These cells can differentiate into neuron-like cells. Although NTE-deficient cells exhibited a delay in neurite initiation in vitro, both the proportion of neuron-like cells which initiated neurites and the elongation of these neurites occurred at the normal rate. These results demonstrate that NTE activity is not required for neurite initiation or elongation per se, but is essential for the optimal rate of neurite initiation.     

ES cell neural differentiation reveals a substantial number of novel ESTs

We have used a method for synchronously differentiating murine embryonic stem (ES) cells into functional neurons and glia in culture. Using subtractive hybridization we isolated approximately 1200 cDNA clones from ES cell cultures at the neural precursor stage of neural differentiation. Pilot studies indicated that this library is a good source of novel neuro-embryonic cDNA clones. We therefore screened the entire library by single-pass sequencing. Characterization of 604 non-redundant cDNA clones by BLAST revealed 96 novel expressed sequence tags (ESTs) and an additional 197 matching uncharacterized ESTs or genomic clones derived from genome sequencing projects. With the exception of a handful of genes, whose functions are still unclear, most of the 311 known genes identified in this screen are expressed in embryonic development and/or the nervous system. At least 80 of these genes are implicated in disorders of differentiation, neural development and/or neural function. This study provides an initial snapshot of gene expression during early neural differentiation of ES cell cultures. Given the recent identification of human ES cells, further characterization of these novel and uncharacterized ESTs has the potential to identify genes that may be important in nervous system development, physiology and disease. Electronic Publication