Culture of cells from zebrafish (Brachydanio rerio) blastula-stage embryos

The zebrafish has become a popular model for studies of vertebrate development and toxicology. However,in vitro approaches utilizing this organism have not been fully exploited due to the absence of suitable cell culture systems. Previously, we developed methods for the culture of cells derived from zebrafish blastula-stage embryos. One of these cultures, ZEM-2, was derived in a complex medium containing trout embryo extract, trout serum and medium conditioned by buffalo rat liver cells. In this study we describe a zebrafish embryo cell line, ZEM-2A, derived from ZEM-2 following selection for growth in a simplified medium. Optimal growth of ZEM-2A cells is attained in nutrient medium supplemented with 5% fetal bovine serum.     

Osmolarity and composition of cell culture media affect further development and survival in zebrafish embryos

With the aim of carrying out chimaerism and somatic cell-midblastula transition (MBT) embryos co-culture experiments in freshwater fish species, we evaluated the effect of osmolarity and composition of two media commonly used in cell fish culture on MBT zebrafish embryos and their further development and survival. To this end, wild zebrafish dechorionated embryos in midblastula stage were cultured for 6 days (Experiment 1: 189 embryos) or 1 h (Experiment 2: 150 embryos) in three different media: Hanks' 10% (H-10), 35 mOsm; Hanks' 100% (CH), 315 mOsm; and L-15 with serum (L-15: 315 mOsm). High osmolarity affected the survival rate (6 days: L-15: 45.1% v. CH: 72.34% v. H-10: 100%, P < 0.05; after 6 days: 0% both in L-15 and CH) and slowed their developmental timing. Embryos showed tail deformation (curly) as well as body paralysis at 48 h when they showed tail movements at 28 h. Differences in tail deformation were observed between high-osmolarity groups (CH: 85.10% v. L-15: 98.04%; P < 0.05). In Experiment 2, no effects on survival rate were observed. Teratogenic effects were only observed in L-15 (L-15: 12.98% v. CH: 0%; P< 0.05). Loss of motility was not detected in any group at 48 h. Optimum osmolar condition for cultured cells and also embryonic cells is around 315 mOsm and so, during chimaerism experiments (usually practised at MBT stage), present results indicate that midblastula embryos can acceptably bear the effects caused by 315 mOsm (CH) for 1 h, even though this involves a certain delay in developmental timing.     

Direct visualization of replication dynamics in early zebrafish embryos

We analyzed DNA replication in early zebrafish embryos. The replicating DNA of whole embryos was labeled with the thymidine analog 5-ethynyl-2?-deoxyuridine (EdU), and spatial regulation of replication sites was visualized in single embryo-derived cells. The results unveiled uncharacterized replication dynamics during zebrafish early embryogenesis.     

Cell cultures derived from embryos and melanoma of poeciliid fish

Summary In poeciliid fish, melanoma of different degrees of malignancy can be produced by crossing specific genotypes. For a detailed investigation of the processes leading to proliferation or differentiation of the melanoma cells, it is necessary to establish cell cultures. The aim of the present study was to find out the optimal conditions for initiating and culturing poeciliid fish cells for the purpose of establishing cell cultures of melanoma. The optimal method was developed by using small pieces of late embryos as starting material and includes: (a) dispersion of tissue by mild stepwise treatment with a trypsin-EDTA mixture at low temperature; (b) culture of cells in the complex medium 199; (c) supplementation of medium with high percentage (20%) of fetal bovine serum; and (d) stabilization of pH by buffering the medium with HEPES. Under these conditions, primary and secondary cultures of embryonic cells have been initiated. An epithelial-like cell line has been subcultured for more than 80 passages. The method developed for embryonic tissues was used to start cell cultures from melanoma of platyfish-swordtail hybrids. Until now, only cells of rapidly growing malignant albino melanoma could be maintained in primary cultures. Secondary cultures could not be initiated since the melanoma cells tended to differentiate and stopped growing before a confluent monolayer was formed. Dedicated to Professor Dr. C. Kosswig on the occasion of his 75th birthday. This work was supported by grants from the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 103, Marburg, Zellenergetik und Zelldifferenzierung), and is part of the thesis of C. K.     

Characterization of a cell line derived from zebrafish (brachydanio rerio) embryos

Summary During the last decade, zebrafish (Brachydanio rerio) have emerged as a novel and attractive system to study embryogenesis and organogenesis in vertebrates. The main reason is that both extensive genetic studies and detailed embryologic analysis are possible using this small tropical fresh water teleost. However, in vitro analysis using cell culture or molecular genetics are still far less advanced than in other vertebrate systems. Here we report the generation and characterization of a fibroblast like cell line, ZF4, derived from 1-day-old zebrafish embryos. The hyperploid cell line has been stable in multiple passages for more than 2 yr now and is the first zebrafish cell line that can be maintained in conventional medium containing mammalian serum. Using a series of plasmids for expression of a marker gene, we evaluate in ZF4 cells the relative strength of expression from several different viral, fish, and mammalian promoters. Stable integration can be obtained by using G418 selection. We hope that our cell line will be a useful tool for the analysis of gene regulation in zebrafish.     

Cell junctions in explanted tissues from early chick embryos

Summary Hypoblast and definitive endoblast derived from young chick embryos were explanted and grown for 24 h in culture. The junctional complexes which characterise these tissues were studied on freeze-fracture replicas and thin sections. Cell membranes of the hypoblast displayed tight junctions only, disposed in randomly arranged strands or narrow belts which included many discontinuous strands. The definitive endoblast showed tight and gap junctions as well as desmosomes in close association with the tight junctions. It is suggested that the differences between the two types of tissue may be related to cell cohesiveness, which appears to be relatively low in the hypoblast and high in the definitive endoblast.     

Incidence of chromosomal anomalies in early bovine embryos derived from in vitro fertilization

The incidence of chromosomal anomalies in early bovine embryos derived from follicular oocytes fertilized in vitro using sperm separated by Percoll density gradient centrifugation was investigated. Overall, chromosomal anomalies were observed in 13.7% (138/1005) of embryos. There were 14 haploids (1.4%), 2 hypodiploids (0.2%), 6 hyperdiploids (0.6%), 101 triploids (10.0%), 12 tetraploids (1.2%), 2 diploid/triploid mosaics (0.2%), and 1 diploid/tetraploid mosaic (0.1%). The frequency of triploidy was caused mainly by polyspermy. There was a significant difference in the frequency of embryos with abnormal chromosomes between the two bulls used (P < 0.005), but Percoll centrifugation did not affect the observed incidence of anomalies. The frequency of chromosomal anomalies in embryos at each stage increased with delay or arrest of development. These results suggest that the incidence of chromosomal anomalies depended on the conditions of in vitro fertilization and the arrest of development.     

In vitro developmental toxicology assays: A review of the state of the science of rodent and zebrafish whole embryo culture and embryonic stem cell assays

In vitro developmental model systems have been an important tool for advancing basic research in the embryology and teratology fields. The rat and zebrafish embryo models have had broad utility in both fields for many decades. Furthermore embryonic stem cells, applied as a basic research tool, have broad applications across the development fields and many other fields including cancer, regeneration and epigenetic research. These models have historically been applied in mechanistic studies but were also considered promising for evaluating teratogenic potential of test substances. In recent years, in vitro teratogenicity assays have become an area of interest for supporting the 3 Rs (reduction, refinement, and replacement of animal use). Generation of such assays also provides a means to facilitate early assessment of test agents at a higher throughput without excessive use of animals. In this review, the three models are described with an emphasis of how they are being developed and/or refined to support teratogenicity assessment as screening tools. An overview of the state of the science and future directions are described. Birth Defects Research (Part C) 90:87–98, 2010. © 2010 Wiley‐Liss, Inc.     

Human neural progenitor cells derived from embryonic stem cells in feeder-free cultures

Derivation of human neural progenitors (hNP) from human embryonic stem (hES) cells in culture has been reported with the use of feeder cells or conditioned media. This introduces undefined components into the system, limiting the ability to precisely investigate the requirement for factors that control the process. Also, the use of feeder cells of non-human origin introduces the potential for zoonotic transmission, limiting its clinical usefulness. Here we report a feeder-free system to produce hNP from hES cells and test the effects of various media components involved in the process. Five protocols using defined media components were compared for efficiency of hNP generation. Based on this analysis, we discuss the role of basic fibroblast growth factor (FGF2), N2 supplement, non-essential amino acids (NEAA), and knock-out serum replacement (KSR) on the process of hNP generation. All protocols led to down-regulation of Oct4/POU5F1 expression (from 90.5% to <3%), and up-regulation of neural progenitor markers to varying degrees. Media with N2 but not KSR and NEAA produced cultures with significantly higher (p<0.05) expression of the neural progenitor marker Musashi 1 (MSI1). Approximately 89% of these cells were Nestin (NES)+ after 3 weeks, but they did not proliferate. In contrast, differentiation media supplemented with KSR and NEAA produced fewer NES+ (75%) cells, but these cells were proliferative, and by five passages the culture consisted of >97% NES+ cells. This suggests that KSR and NEAA supplements did not enhance early differentiation but did promote proliferating of hNP cell cultures. This resulted in an efficient, robust, repeatable differentiation system suitable for generating large populations of hNP cells. This will facilitate further study of molecular and biochemical mechanisms in early human neural differentiation and potentially produce uniform neuronal cells for therapeutic uses without concern of zoonotic transmission from feeder layers.     

Developmental toxicity of triclocarban in zebrafish (Danio rerio) embryos

Triclocarban (TCC), which is used as an antimicrobial agent in personal care products, has been widely detected in aquatic ecosystems. However, the consequence of TCC exposure on embryo development is still elusive. Here, by using zebrafish embryos, we aimed to understand the developmental defects caused by TCC exposure. After exposure to 0.3, 30, and 300 μg/L TCC from 4‐hour postfertilization (hpf) to 120 hpf, we observed that TCC exposure significantly increased the mortality and malformation, delayed hatching, and reduced body length. Exposure to TCC also affected the heart rate and expressions of cardiac development–related genes in zebrafish embryos. In addition, TCC exposure altered the expressions of the genes involved in hormonal pathways, indicating its endocrine disrupting effects. In sum, our data highlight the impact of TCC on embryo development and its interference with the hormone system of zebrafish.     

缺失mir122抑制斑马鱼肝脏前体细胞向肝细胞分化

作为机体内最大的脏器之一, 肝脏对于机体的新陈代谢、解毒作用以及内环境的稳定有着重要的作用。FGF、BMP以及WNT信号通路及众多转录因子形成的基因调节网络精密调控着肝脏的发育, 然而小分子RNA在肝脏形成中的功能却所知甚少。近年来的研究发现, mir122在肝细胞中高表达, 对于维持肝脏的代谢功能起着至关重要的作用, 但是其在肝脏发育中的功能还不清楚。为了深入探讨mir122在斑马鱼(Danio rerio)胚胎发育中的作用, 文章首先检测了mir122在发育过程中的时空表达, 发现mir122特异表达在肝脏, 并且在肝脏前体细胞向肝细胞分化过程中表达水平显著增高。文章还通过用反义核苷酸Morpholino (MO)敲降mir122的表达, 发现敲降mir122对肝脏前体细胞的起源、肝原基的形成以及肝脏前体细胞的增殖没有明显的影响, 但是, mir122的缺失导致肝脏前体细胞不能分化为肝细胞。因此, mir122不仅参与了成体肝脏代谢功能的调节, 还在肝脏前体细胞向肝细胞分化过程中不可或缺。     

Peripheral sensory neurons differentiate from neural precursors derived from human embryonic stem cells

Abstract Neural precursors have been derived from human embryonic stem cells (hESC) using the bone morphogenetic protein antagonist noggin. These neural precursors can be further differentiated to produce neural cells that express central nervous system (CNS) markers. We have recently shown that naïve hESC can be directed to differentiate into peripheral sensory (PS) neuron-like cells and putative neural crest precursors by co-culturing with PA6 stromal cells. In the present study, we examine whether hESC-derived neural precursors (NPC) can differentiate into the peripheral nervous system, as well as CNS cells. As little as 1 week after co-culture with PA6 cells, cells with the molecular characteristics of PS neurons and neural crest are observed in the cultures. With increased time in culture, more PS-like neurons appear, in parallel with a reduction in the neural crest-like cells. These results provide the first evidence that neural precursors derived from hESC have the potential to develop into PS neurons-like as well as CNS-like neuronal cells. About 10% of the cells in NPC-PA6 co-cultures express PS neuron markers after 3 weeks, compared with <1% of hESC cultured on PA6. This enrichment for peripheral neurons makes this an attractive system for generation of peripheral neurons for pathophysiology study and drug development for diseases of the peripheral nervous system such as Familial Dysautonomia and varicella virus infection.     

Cell cultures derived from embryos and melanoma of poeciliid fish

In poeciliid fish, melanoma of different degrees of malignancy can be produced by crossing specific genotypes. For a detailed investigation of the processes leading to proliferation or differentiation of the melanoma cells, it is necessary to establish cell cultures. The aim of the present study was to find out the optimal conditions for initiating and culturing poeciliid fish cells for the purpose of establishing cell cultures of melanoma. The optimal method was developed by using small pieces of late embryos as starting material and includes: (a) dispersion of tissue by mild stepwise treatment with a trypsin-EDTA mixture at low temperature; (b) culture of cells in the complex medium 199; (c) supplementation of medium with high percentage (20%) of fetal bovine serum; and (d) stabilization of pH by buffering the medium with HEPES. Under these conditions, primary and secondary cultures of embryonic cells have been initiated. An epithelial-like cell line has been subcultured for more than 80 passages. The method developed for embryonic tissues was used to start cell cultures from melanoma of platyfish-swordtail hybrids. Until now, only cells of rapidly growing malignant albino melanoma could be maintained in primary cultures. Secondary cultures could not be initiated since the melanoma cells tended to differentiate and stopped growing before a confluent monolayer was formed.     

Protoplasmic astrocytes enhance the ability of neural stem cells to differentiate into neurons in vitro

Protoplasmic astrocytes have been reported to exhibit neuroprotective effects on neurons, but there has been no direct evidence for a functional relationship between protoplasmic astrocytes and neural stem cells (NSCs). In this study, we examined neuronal differentiation of NSCs induced by protoplasmic astrocytes in a co-culture model. Protoplasmic astrocytes were isolated from new-born and NSCs from the E13-15 cortex of rats respectively. The differentiated cells labeled with neuron-specific marker β-tubulin III, were dramatically increased at 7 days in the co-culture condition. Blocking the effects of brain-derived neurotrophic factor (BDNF) with an anti-BDNF antibody reduced the number of neurons differentiated from NSCs when co-cultured with protoplasmic astrocytes. In fact, the content of BDNF in the supernatant obtained from protoplasmic astrocytes and NSCs co-culture media was significantly greater than that from control media conditions. These results indicate that protoplasmic astrocytes promote neuronal differentiation of NSCs, which is driven, at least in part, by BDNF.     

DNA fingerprinting with oligonucleotides can differentiate cell lines derived from the same tumor

Summary Oligonucleotide fingerprinting was applied to investigate the relatedness of several cell lines that were established between 1973 and 1977 from a teratocarcinoma. We were able to distinguish cell lines derived at different times. In addition, sublines from one cell line (PYS-2) could be discriminated by using a combination of different probes. Therefore multilocus fingerprinting with oligonucleotides is a useful method for monitoring changes in cell lines kept in culture for many generations. This work was supported by the Deutsche Forschungsgemeinschaft (OB 66/2-1) and by the VW-Stiftung.     

Transgenic zebrafish expressing fluorescent proteins in central nervous system neurons

Zebrafish is a powerful model system for investigations of vertebrate neural development. The animal has also become an important model for studies of neuronal function. Both in developmental and functional studies, transgenic zebrafish expressing fluorescent proteins in central nervous system neurons have been playing important roles. We review here the methods for producing transgenic zebrafish. Recent advances in transposon- or bacterial artificial chromosome-based transgenesis greatly facilitate the creation of useful lines. We also present our study on alx -positive neurons to reveal how transgenic zebrafish expressing fluorescent proteins in a specific class of neurons can be used to investigate their development and function.     

Mature endothelium and neurons are simultaneously derived from embryonic stem cells by 2D in vitro culture system

The connections existing between vessels and nerves go beyond the structural architecture of vascular and nervous systems to comprise cell fate determination. The analysis of functional/molecular links that interconnect endothelial and neural commitments requires a model in which the two differentiation programs take place at the same time in an artificial controllable environment. To this regard, this work presents an in vitro model to differentiate embryonic stem (ES) cells simultaneously into mature neurons and endothelial cells. Murine ES cells are differentiated within an artificial environment composed of PA6 stromal cells and a serum-free medium. Upon these basal culture conditions ES cells preferentially differentiate into neurons. The addition of basic fibroblast growth factor (FGF2) to the medium allows the simultaneous maturation of neurons and endothelial cells, whereas bone morphogenetic protein (BMP)4 drives endothelial differentiation to the disadvantage of neural commitment. The responsiveness of the system to exogenous cytokines was confirmed by genes expression analysis that revealed a significant up-regulation of endothelial genes in presence of FGF2 and a massive down-regulation of the neural markers in response to BMP4. Furthermore, the role played by single genes in determining endothelial and neural fate can be easily explored by knocking down the expression of the target gene with lentiviruses carrying the corresponding shRNA sequence. The possibility to address the neural and the endothelial fate separately or simultaneously by exogenous stimuli combined with an efficient gene silencing strategy make this model an optimal tool to identify environmental signals and genes pathways involved in both endothelial and neural specification.     

Musashi 1‐positive cells derived from mouse embryonic stem cells can differentiate into neural and intestinal epithelial‐like cells in vivo

Msi1 (Musashi 1) is regarded as a marker for neural and intestinal epithelial stem cells. However, it is still unclear whether Msi1‐positive cells derived from mouse embryonic stem cells have the ability to differentiate into neural or intestinal epithelial cells. A pMsi1–GFP (green fluorescent protein) reporter plasmid was constructed in order to sort Msi1‐positive cells out of the differentiated cell population. The GFP‐positive cells (i.e. Msi1‐positive cells) were sorted by FACS and were hypodermically engrafted into the backs of NOD/SCID (non‐obese diabetic/severe combined immunodeficient) mice. The presence of neural and intestinal epithelial cells in the grafts was detected. Msi1 was highly expressed in the GFP‐positive cells, but not in the GFP‐negative cells. The markers for neural cells (Nestin and Tubulin β III) and intestinal epithelial cells [FABP2 (fatty acid binding protein 2), Lyz (lysozyme) and ChA (chromogranin A)] were more highly expressed in the grafts from Msi1‐positive cells than those from Msi1‐negative cells (P<0.05). The grafts from the Msi1‐negative cells contained more mesodermal‐like tissues than those from the Msi1‐positive cells. The pMsi1–GFP vector can be used to sort Msi1‐positive cells from a cell population derived from mouse embryonic stem cells. The Msi1‐positive cells can differentiate into neural and intestinal epithelial‐like cells in vivo.