A cDNA-based random RNA interference library for functional genetic screens in embryonic stem cells

To facilitate high-throughput functional genetic screens in embryonic stem cells, a simple and efficient system to construct cDNA-based random RNA interference (RNAi) library was developed in the study. Previous studies have demonstrated that sequence-specific gene silencing could be induced by long double-stranded RNA (dsRNA) in mouse embryos, mouse oocytes, embryonic stem cells, and other mammalian cells. Based on these findings, a dsRNA-expressing RNAi vector system was designed. This study provided evidence that the vector design could induce efficient knockdown of expression of both exogenous egfp gene and endogenous MTM1 gene in mouse embryonic stem cells. A random RNAi library was established by cloning enzyme-digested cDNA of mouse embryonic stem (ES) cells into the BamHI site of the convergent dual promoter RNAi vector. Sequencing of 20 randomly selected clones from the library showed that 17 contained inserts and that all of them were unique sequences. A functional genetic screen of genes involving in self-renewal and differentiation with the random RNAi library identified ubiquitin. The ubiquitin knockdown ES cell line generated 20%-30% of undifferentiated colonies in the absence of leukemia inhibitor factor, whereas parental ES cells and control vector pDCont transfectants produced less than 5% of colonies of undifferentiated cells, suggesting that ubiquitin plays a role in ES cell differentiation. The random RNAi library provides a useful tool for investigation of molecular mechanisms of cellular development and differentiation.     

Screening for genes essential for mouse embryonic stem cell self-renewal using a subtractive RNA interference library

The pluripotency of mouse embryonic stem (ES) cells is maintained by self-renewal. To screen for genes essential for this process, we constructed an RNA interference (RNAi) library by inserting subtracted ES cell cDNA fragments into plasmid containing two opposing cytomegalovirus promoters. ES cells were transfected with individual RNAi plasmids and levels of the pluripotency marker Oct-4 were monitored 48 hours later by real time RT-PCR. Of the first 89 RNAi plasmids characterized, 12 downregulated Oct-4 expression to less than 50% of the normal level and 7 of them upregulated Oct-4 expression to more than 150% of the normal level. To investigate their long-term effect on self-renewal, ES cells were transfected by these 19 RNAi plasmids individually and G418-resistant colonies were subjected to alkaline phosphatase (AP) staining after 7 days selection. Except for 4 plasmids that caused cell death, the ratio of AP positive colonies was repressed to less than 60% of the control group by the other 15 plasmids and even below 20% by 10 plasmids. The cDNA fragments in these 10 plasmids correspond to eight genes, including Zfp42/Rex-1, which was chosen for further functional analysis. RNAi knockdown of Zfp42 induced ES cells differentiate to endoderm and mesoderm lineages, and overexpression of Zfp42 also caused ES cells to lose the capacity of self-renewal. Our results indicate that RNAi screen is a feasible and efficient approach to identify genes involved in ES cells self-renewal. Further functional characterization of these genes will promote our understanding of the complex regulatory networks in ES cells.     

Neural induction and neural stem cell development

Embryonic stem (ES) cells are a pluripotent and renewable cellular resource with tremendous potential for broad applications in regenerative medicine. Arguably the most important consideration for stem cell-based therapies is the ability to precisely direct the differentiation of stem cells along a preferred cellular lineage. During development, lineage commitment is a multistep process requiring the activation and repression of sets of genes at various stages, from an ES cell identity to a tissue-specific stem cell identity and beyond. Thus, the challenge is to ensure that the pattern of genomic regulation is recapitulated during the in vitro differentiation of ES cells into stem/progenitor cells of the appropriate tissue in a robust, predictable and stable manner. To address this issue, we must understand the ontogeny of tissue-specific stem cells during normal embryogenesis and compare the ontogeny of tissue-specific stem cells in ES cell models. Here, we discuss the issue of directed differentiation of pluripotent ES cells into neural stem cells, which is fundamentally linked to two early events in the development of the mammalian nervous system: the 'decision' of the ectoderm to acquire a neural identity (neural determination) and the origin of neural stem cells within this neural-committed population of cells. A clearer understanding of the molecular and cellular mechanisms that govern mammalian neural cell fate determination will lead to improved ES technology applications in neural regeneration.     

Multilineage differentiation from human embryonic stem cell lines

Stem cells are unique cell populations with the ability to undergo both self-renewal and differentiation. A wide variety of adult mammalian tissues harbors stem cells, yet "adult" stem cells may be capable of developing into only a limited number of cell types. In contrast, embryonic stem (ES) cells, derived from blastocyst-stage early mammalian embryos, have the ability to form any fully differentiated cell of the body. Human ES cells have a normal karyotype, maintain high telomerase activity, and exhibit remarkable long-term proliferative potential, providing the possibility for unlimited expansion in culture. Furthermore, they can differentiate into derivatives of all three embryonic germ layers when transferred to an in vivo environment. Data are now emerging that demonstrate human ES cells can initiate lineage-specific differentiation programs of many tissue and cell types in vitro. Based on this property, it is likely that human ES cells will provide a useful differentiation culture system to study the mechanisms underlying many facets of human development. Because they have the dual ability to proliferate indefinitely and differentiate into multiple tissue types, human ES cells could potentially provide an unlimited supply of tissue for human transplantation. Though human ES cell-based transplantation therapy holds great promise to successfully treat a variety of diseases (e.g., Parkinson's disease, diabetes, and heart failure) many barriers remain in the way of successful clinical trials.     

Potential application of embryonic stem cells in Parkinson's disease: drug screening and cell therapy

Embryonic stem (ES) cells are genetically normal, continuous cell lines that can give rise to a variety of somatic cells in culture. These include the midbrain dopaminergic neurons, a major cell type lost in Parkinson's disease. With the promising outcome of mesencephalic fetal transplantation in some Parkinson's disease patients, the establishment of human ES cells has sparked much attention in both the scientific and general community regarding their potential as an alternative to aborted fetal tissue for cell replacement therapies. There is also great interest in developing the ES cell system as a platform for pharmaceutical and toxicological screening. Progress has been made in developing protocols for dopaminergic neuronal specification in ES cell development. Research to define the criteria for the 'right' category of therapeutic dopaminergic cells is underway. However, the promise of human ES cells rests largely on our ability to expand stem cells without genetic and epigenetic compromise, and to direct stem cell differentiation with absolute phenotypic fidelity. The delivery of these goals will require a much better understanding of the control of ES cell self-renewal, proliferation and the commitment of differentiation.     

Neural commitment of embryonic stem cells: molecules, pathways and potential for cell therapy

The study of neuronal differentiation of embryonic stem cells has raised major interest over recent years. It allows a better understanding of fundamental aspects of neurogenesis and, at the same time, the generation of neurons as tools for various applications ranging from drug testing to cell therapy and regenerative medicine. Since the first report of human embryonic stem (ES) cells derivation, many studies have shown the possibility of directing their differentiation towards neurons. However, there are still many challenges ahead, including gaining a better understanding of the mechanisms involved and developing techniques to allow the generation of homogeneous neuronal and glial subtypes. We review the current state of knowledge of embryonic neurogenesis which has been acquired from animal models and discuss its translation into in vitro strategies of neuronal differentiation of ES cells. We also highlight several aspects of current protocols which need to be optimized to generate high-quality embryonic stem cell-derived neuronal precursors suitable for clinical applications. Finally, we discuss the potential of embryonic stem cell-derived neurons for cell replacement therapy in several central nervous system diseases.     

HTS/HCS to Screen Molecules Able to Maintain Embryonic Stem Cell Self-Renewal or to Induce Differentiation: Overview of Protocols

Embryonic stem (ES) cells, combining self-renewal ability with wide range tissue-specific cell differentiation, represent one of the most powerful model systems in basic research, drug discovery and biomedical applications. In the field of drug development, ES cells are instrumental in high-throughput/content screening (HTS/HCS) for the evaluation of large compound libraries to test biological activity and toxic properties. Since it is a high priority to test new compounds in vitro, before starting animal and human treatments, there is an increasing demand for new in vitro models that can be used in HTS/HCS to facilitate drug development. In order to achieve this objective, several methods for ES cell self-renewal or differentiation have been evaluated to assess their compatibility with HTS/HCS. This review describes protocols used to screen molecules able to maintain self-renewal or to induce differentiation in ectodermal, mesodermal, endodermal, and their derivative cell lines.     

Oct4 dependence of chromatin structure within the extended Nanog locus in ES cells

Embryonic stem (ES) cells offer insight into early developmental fate decisions, and their controlled differentiation may yield vast regenerative potential. The molecular determinants supporting ES cell self-renewal are incompletely understood. The homeodomain proteins Nanog and Oct4 are essential for mouse ES cell self-renewal. Using a high-throughput approach, we discovered DNaseI hypersensitive sites and potential regulatory elements along a 160-kb region of the genome that includes GDF3, Dppa3, and Nanog. We analyzed gene expression, chromatin occupancy, and higher-order chromatin structure throughout this gene locus and found that expression of the reprogramming factor Oct4 is required to maintain its integrity.     

Self-renewal of embryonic stem cells through culture on nanopattern polydimethylsiloxane substrate

Embryonic stem (ES) cells can undergo continual proliferation and differentiation into cells of all somatic cell lineages in vitro; they are an unlimited cell source for regenerative medicine. However, techniques for maintaining undifferentiated ES cells are often inefficient and result in heterogeneous cell populations. Here, we determined effects of nanopattern polydimethylsiloxane (PDMS) as a culture substrate in promoting the self-renewal of mouse ES (mES) cells, compared to commercial plastic culture dishes. After many passages, mES cells efficiently maintained their undifferentiated state on nanopattern PDMS, but randomly differentiated on commercial plastic culture dishes, as indicated by partially altered morphologies and decreases in alkaline phosphatase activity and stage-specific expression of embryonic antigen-1. Under nanopattern PDMS conditions, we found increased activities of STAT3 and Akt, important proteins involved in maintaining the self-renewal of mES cells. The substrate-cell interactions also enhanced leukemia inhibitory factor (LIF)-downstream signaling and inhibited spontaneous differentiation, concomitant with reduced focal adhesion kinase (FAK) signaling. This reduction in FAK signaling was shown to be important for promoting mES cell self-renewal. Thus, our data demonstrates that nanopattern PDMS contributes to maintaining the self-renewal of mES cells and may be applicable in the large-scale production of homogeneously undifferentiated mES cells.     

Stable suppression of gene expression in murine embryonic stem cells by RNAi directed from DNA vector-based short hairpin RNA

Murine embryonic stem (ES) cells are an ideal system for the research of directed differentiation in vitro. Long double-stranded RNA, which can induce RNA interference (RNAi) effectively in many organisms, has been shown to suppress target gene expression efficiently and specifically in undifferentiated ES cells. However, it cannot be used in differentiated ES cells due to unspecific inhibition of gene expression resulting from the activation of interferon pathway following differentiation. Using green fluorescent protein (GFP) as a reporter system, we show here that a short hairpin RNA (shRNA) expression vector driven by the murine U6 small nuclear RNA promoter can specifically induce potent gene knockdown effect (i.e., inhibit GFP expression specifically) when transfected transiently into ES cells. Furthermore, when the expression vector is stably integrated into the genome of the cell, it can still show specific RNAi effect, which can be maintained at least for 10 days. These transfected ES cells showed no obvious differences in the morphology or growth rate in culture compared with untransfected cells, suggesting that the activation of shRNA-directed RNAi did not affect the properties of ES cells and that the RNAi effect in ES cells is specific and persistent. Our results prove the feasibility of the U6 promoter-driven shRNA expression technique to be used to study the function of genes expressed in ES cells. These ES cells, after integration of the U6-based RNAi vector into their genome, could be used to generate gene knockdown mice.