Enhanced apoptosis during early neuronal differentiation in mouse ES cells with autosomal imbalance

Although particular chromosomal syndromes are phenotypicaUy and clinically distinct, the majority of individuals with autosomal imbalance, such as aneuploidy, manifest mental retardation. A common abnormal phenotype of Down syndrome （DS）, the most prevalent autosomal aneuploidy, shows a reduction in both the number and the density of neurons in the brain. As a DS model, we have recently created chimeric mice from ES cells containing a single human chromosome 21. The mice mimicked the characteristic phenotypic features of DS, and ES cells showed a higher incidence of apoptosis during early neuronal differentiation in vitro. In this study, we examined the induction of anomalous early neural development by aneuploidy in mouse ES cells by transferring various human chromosomes or additional mouse chromosomes. Results showed an elevated incidence of apoptosis in all autosome-aneuploid clones examined during early neuronal differentiation in vitro. Further, cDNA microarray analysis revealed a common cluster of down-regulated genes, of which eight known genes are related to cell proliferation, neurite outgrowth and differentiation. Importantly, targeting of these genes by siRNA knockdown in normal mouse ES cells led to enhanced apoptosis during early neuronal differentiation. These findings strongly suggest that autosomal imbalance is associated with general neuronal loss through a common molecular mechanism for apoptosis.

LincRNA1230 inhibits the differentiation of mouse ES cells towards neural progenitors

In vitro, mouse embryonic stem （ES） cells can differentiate into many somatic cell types, including neurons and glial cells. When cultured in serum-free medium, ES cells convert spontaneously and efficiently to a neural fate. Previous studies have shown that the neural conversion of mouse ES cells includes both the participation of neural-specific transcription factors and the regulation of epigenetic modifications. However, the intracellular mechanism underlying this intrinsic transition still re- mains to be further elucidated. Herein, we describe a long intergenic non-coding RNA, LincRNA1230, which participates in the regulation of the neural lineage specification of mouse ES cells. The ectopic forced expression of LincRNAI230 dramatically inhibited mouse ES cells from adopting a neural cell fate, while LincRNA1230 knockdown promoted the conversion of mouse ES cells towards neural progenitors. Mechanistic studies have shown that LincRNA1230 inhibits the activation of early neural genes, such as Pax6 and Soxl, through the modulation of bivalent modifications （tri-methylation of histone3 lysine4 and his- tone3 lysine27） at the promoters of these genes. The interaction of LincRNA1230 with Wdr5 blocked the localization of Wdr5 at the promoters of early neural genes, thereby inhibiting the enrichment of H3K4me3 modifications at these loci. Collectively, these findings revealed a crucial role for LincRNA1230 in the regulation of the neural differentiation of mouse ES cells.

Embryonic stem cells generated by nuclear transfer of human somatic nuclei into rabbit oocytes

To solve the problem of immune incompatibility, nuclear transplantation has been envisaged as a means to produce cells or tissues for human autologous transplantation. Here we have derived embryonic stem cells by the transfer of human somatic nuclei into rabbit oocytes. The number of blastocysts that developed from the fused nuclear transfer was comparable among nuclear donors at ages of 5, 42, 52 and 60 years, and nuclear transfer (NT) embryonic stem cells (ntES cells) were subsequently derived from each of the four age groups. These results suggest that human somatic nuclei can form ntES cells independent of the age of the donor. The derived ntES cells are human based on karyotype, isogenicity, in situ hybridization, PCR and immunocytochemistry with probes that distinguish between the various species. The ntES cells maintain the capability of sustained growth in an undifferentiated state, and form embryoid bodies, which, on further induction, give rise to cell types such as neuron and muscle, as well as mixed cell populations that express markers representative of all three germ layers. Thus, ntES cells derived from human somatic cells by NT to rabbit eggs retain phenotypes similar to those of conventional human ES cells, including the ability to undergo multilineage cellular differentiation.

TGF-β receptors in mouse ES-5 cells and their differentiated derivatives

By radioreceptor binding studies with iodinated TGF-β1, it has been shown that an undifferentiated ES-5 cell expresses approximately 3270 receptors with a dissociation constant Kd=130pM, but after the induction of differenti-ation by retinoic acid and dBcAMP, the receptor number of a differentiated RA-ES-5 cell was increased about 80% and the Kd was also increased to 370 pM. Furthermore,more direct evidence supporting the expression of TGF-βtype Ⅰand type Ⅱ receptors in both ES-5 and RA-ES-5 cells has come from dot blot hybridization of cellular mRNA with cDNA probes for type Ⅰ and type Ⅱ recep-tors. Meanwhile, mRNA expression level of types Ⅰ and Ⅱreceptors in RA-ES-5 cells were higher than that in ES-5 cells. Down regulation of TGF-β receptors with a signifi-cant decrease in the rate of cell proliferation in both cells, was found by employing a pretreatment with neutralizing antibody to TGF-β1. The possible role of receptors for TGF-β in cen differentiation is discussed here.

Establishment of Embryonic Stem Cell Lines Derived from Outbred Mouse Embryos and Production of Chimeras

The aim of the present investigation was to determine if embryonic stem (ES) cells could be isolated from outbred mouse embryos (KM) and if chimeras could be producedly using outbred ES cells. Three ES cell lines,designated KE1, KE2, and KE5,were isolated from 5 Kunming albino blastocysts. Normal diploid composition of these cell lines was above 70%. By using C57BL/6J and 615 blastocysts as host embryos, one chimera was obtained in living pups. It was shown for the first time that chimeras can be produced by using outhred ES cells. This work implies that to establish ES cell lines from other animal embryos is possible. More interestingly, white color dots from ES cells on the coat of this chimeric mouse enlarged a lot after half a year, indicated that ES cells were inhibited by cells from outbred mouse or the cells of outbred mouse grew vigiously.

Effects of feeder layer and BRL conditioned medium on mouse embryonic stem cells

In vitro growth and maintenance of embryonic stem (ES) cell lines derived from ICM cells of various blastocysts of 129 strain mice, the sustenance of their pluripotency and normal karyotype depend on the feeder layer of mouse embryonic fibroblasts (MEF). Compared with the feeder layer of MEF cells, medium conditioned by Buffalo rat liver cells (BRL-CM) is able to maintain pluripotency and karyo-typic normality of ES cells only in short term cell propagation. Besides, ES cells grown in BRL-CM are also capable of aggregation with 8-cell embryos of Swiss strain and develop into germ line chimaeras. Modification to the method of aggregating ES cells with early embryos by making a hole in agar layer on the top of MEF feeder cells was shown to be more convenient and efficient than the conventional microdrop method.

Regulated expression of TATA-binding protein-related factor 3(TRF3)during early embryogenesis

RNA polymerase （Pol） Ⅱ transcription persists in TATA-box-binding protein （TBP）^-/- mutant mouse embryos, indicating TBP-independent mechanisms for Pol Ⅱ transcription in early development. TBP-related factor 3 （TRF3） has been proposed to substitute for TBP in TBP^-/- mouse embryos. We examined the expression of TRF3 in maturing oocytes and early embryos and found that TRF3 was co-expressed with TBP in the meiotic oocytes and early embryos from the late one-cell stage onward. The amounts of TBP and TRF3 changed dynamically and correlated well with transcriptional activity. Chromatin immunoprecipitation （CHIP） assay revealed that different gene promoters in mouse embryonic stem （ES） cells recruited TRF3 and TBP selectively. Comparative analyses of TRF3 and TBP during cell cycle showed that both factors proceeded through cell cycle in a similar pace, except that TRF3 was slightly delayed than TBP in entering the nucleus when cells were exiting the M-phase. Data from expression and biochemical analyses therefore support the hypothesis that TRF3 plays a role in early mouse development. In addition, results from co-localization study suggest that TRF3 may be also involved in Pol Ⅰ transcription.

Establishment of Embryonic Stem Cell Lines from C57BL/6J Mice and Generation of Chimeras

Four embryonic stem (ES) cell lines, designated CE1, CE2, CE3 and CE4, were isolated from C57BL/6J blastocysts. The ratio of normal diploid composition of these cell lines is above 70%. To examine the differentiation potential of the ES cells, the CE2 cells were injected subcutaneously into syngenic mice, and many kinds of differentiated cells were observed on the sections of the teratoma derived from this ES cell line. On the other hand, to test the chimeric ability of the ES cells, the CE2 cells were microinjected into the blastocysts of ICR mice, and a chimera was obtained among living pups. These results show that CE2 ES cells are pluripotent stem cells, which can differentiate into many kinds of cell types, and can be used as a cell system for further research.

Establishment of Germ-line Competent C57BL/6J Embryonic Stem Cell Lines

Objective To establish C57BL/6J embryonic stem （ES） cell lines with potential germ- line contribution Methods ES cells were isolated from blastocyst inner cell mass of C5 7BL/6J mice, and cultured for 15 passages, and then injected into blastococels of ICR mice blastocysts to establish chimeric mice. Results Three ES cell lines （mC57ES1,mC57ES3, mC57ES7） derived from the inner cell mass of C57BL/6J mice blastocysts were established. They were characteristic of undifferentiated state, including normal XY karyotype, expression of a specific cell surface marker “stage-specific embryonic antigen-I” and alkaline phosphatase in continuous passage. When injected into immunodeficient mice, mC57ES1 cells consistently differentiated into derivatives of all three embryonic germ layers. When mC57ES1 cells were transferred into ICR mice blastocysts, 4 chimeric mice have been obtained. One male of them revealed successful germ-line transmission. Conclussion We have obtained C57BL/6J ES cell lines with a potential germ-line contribution, which can be used to generate transgenic and gene knock-out mice.

Stem cell pluripotency and transcription factor Oct4

Mammalian cell totipotency is a subject that has fascinated scientists for generations. A long lasting question whether some of the somatic cells retains totipotency was answered by the cloning of Dolly at the end of the 20th century. The dawn of the 21st has brought forward great expectations in harnessing the power of totipotentcy in medicine. Through stem cell biology, it is possible to generate any parts of the human body by stem cell engineering. Considerable resources will be devoted to harness the untapped potentials of stem cells in the foreseeable future which may transform medicine as we know today. At the molecular level, totipotency has been linked to a singular transcription factor and its expression appears to define whether a cell should be totipotent. Named Oct4, it can activate or repress the expression of various genes. Curiously, very little is known about Oct4 beyond its ability to regulate gene expression. The mechanism by which Oct4 specifies totipotency remains entirely unresolved. In this review, we summarize the structure and function of Oct4 and address issues related to Oct4 function in maintaining totipotency or pluripotency of embryonic stem cells.

Embryoid bodies formation and differentiation from mouse embryonic stem cells in collagen/Matrigel scaffolds

Embryonic stem （ES） cells have the potential to develop into any type of tissue and are considered as a promising source of seeding cells for tissue engineering and transplantation therapy.The main catalyst for ES cells differentiation is the growth into embryoid bodies （EBs）,which are utilized widely as the trigger of in vitro differentiation.In this study,a novel method for generating EBs from mouse ES cells through culture in collagen/Matrigel scaffolds was successfully established.When single ES cells were seeded in three dimensional collagen/Matrigel scaffolds,they grew into aggregates gradually and formed simple EBs with circular structures.After 7 days＇ culture,they formed into cystic EBs that would eventually differentiate into the three embryonic germ layers.Evaluation of the EBs in terms of morphology and potential to differentiate indicated that they were typical in structure and could generate various cell types;they were also able to form into tissue-like structures.Moreover,with introduction of ascorbic acid,ES cells differentiated into cardiomyocytes efficiently and started contracting synchronously at day 19.The results demonstrated that collagen/Matrigel scaffolds supported EBs formation and their subsequent differentiation in a single three dimensional environment.

Regulation beyond genome sequences: DNA and histone methylation in embryonic stem cells

Embryonic stem(ES)cells distinct themselves from other cell type populations by their pluripotent ability.The unique features of ES cells are controlled by both genetic and epigenetic factors.Studies have shown that the methylation status of DNA and histones in ES cells is quite different from that of differentiated cells and somatic stem cells.Herein,we summarized recent advances in DNA and histone methylation studies of mammalian ES cells.The methylation status of several key pluripotent regulatory genes is also discussed.

Rapid and Cost-Effective Gene Targeting in Rat Embryonic Stem Cells by TALENs

The rat is the preferred animal model in many areas of biomedical research and drug development. Genetic manipulation in rats has lagged behind that in mice due to the lack of efficient gene targeting tools. Previously, we generated a knockout rat via conventional homologous recombination in rat embryonic stern （ES） cells. Here, we show that efficient gene targeting in rat ES cells can be achieved quickly through transcription activator-like effector nuclease （TALEN）-mediated DNA double-strand breaks. Using the Golden Gate cloning technique, we constructed a pair of TALEN targeting vectors for the gene of interest in 5 days. After gene transfection, the targeted rat ES cell colonies were isolated, screened, and confirmed by PCR without the need of drug selection. Our results suggest that TALEN-mediated gene targeting is a superior means of establishing genetically modified rat ES cell lines with high efficiency and short turnaround time.

Germ layer formation during Xenopus embryogenesis: the balance between pluripotency and differentiation

The African clawed frog, Xenopus laevis, has long been a model animal for the studies in the fields of animal cloning, developmental biology, biochemistry, cell biology, and physiology. With the aid of Xenopus, major molecular mechanisms that are involved in embryonic development have been understood. Germ layer formation is the first event of embryonic cellular differentiation, which is induced by a few key maternal factors and subsequently by zygotic signals. Meanwhile, another type of signals, the pluripotency factors in ES cells, which maintain the undifferentiated state, are also present during early embryonic cells. In this review, the functions of the pluripotency factors during Xenopus germ layer formation and the regulatory relationship between the signals that promote differentiation and pluripotency factors are discussed.

Establishment of novel embryonic stem(ES) cell lines from OG2/rtTA blastocysts

Embryonic stem （ES） cells derived from the pre-implantation blastocyst-stage embryos have been widely used to investigate the molecular events determining pluripotency and cell lineage differentiation. As the first discovered ES-specific transcription factor, Oct4 has been considered as the core pluripotency factor of ES cells. In the present study, we successfully established seven ES lines from the blastocysts collected from female OG2 （Oct4-GFP transgenic） mice, which have been crossed with male rtTA transgenic mice. The pluripotency of the ES cell lines can be visualized by the expression of Oct4-GFP under fluorescent microscopy and germ-line transmission capability has been further confirmed. More importantly, the presence of rtTA could induce transgene＇s expression with the help of doxycycline. Therefore, these ES cell lines provide an excellent tool to further discover novel factors affecting pluripotency and to investigate the molecular mechanism of reprogramming in defined transcription factors mediated nuclear reprogramming.

Subcellular redistribution and sequential recruitment of macromolecular components during SGIV assembly

Virus infection consists of entry, synthesis of macro- molecular components, virus assembly and release. Understanding of the mechanisms underlying each event is necessary for the intervention of virus infection in human healthcare and agriculture. Here we report the visualization of Singapore grouper iridovirus （SGIV） assembly in the medaka haploid embryonic stem （ES） cell line HX1. SGIV is a highly infectious DNA virus that causes a massive loss in marine aquaculture. Ectopic expression of VP88GFP, a fusion between green fluo- rescent protein and the envelope protein VP088, did not compromise the ES cell properties and susceptibility to SGIV infection. Although VP88GFP disperses evenly in the cytoplasm of non-infected cells, it undergoes aggregation and redistribution in SGIV-infected cells. Real-time visualization revealed multiple key stages of VP88GFP redistribution and the dynamics of viral assembly site （VAS）. Specifically, VP88GFP entry into and condensation in the VAS occurred within a 6-h duration, a similar duration was observed also for the release of VP88GFP-containing SGIV out of the cell, Taken together, VP088 is an excellent marker for visu- alizing the SGIV infection process. Our results provide new insight into macromolecular component recruit- ment and SGIV assembly.