Differential gene expression in ES-derived neural stem cells by using RT-PCR.

Embryonic stem (ES) cells hold promise to treat a variety of disease. The major obstacle is to determine the requirements that will drive these cells to a particular lineage. Two approaches to examine lineage commitment are the addition of growth factors or directed differentiation of ES cells. Although many neural genes have been identified, the cascade of gene expression that directs neural differentiation is not well understood. Today, with microarray technology, large data sets of differential gene expression patterns are used to identify genes that may be used as indicators of a particular cell lineage or tissue type. Semiquantitative polymerase chain reaction (PCR) can be carried out to verify the expression of individual genes, followed by quantitative PCR to precisely determine the level of mRNA expression. However, functional analysis of potential neurogenic genes must be done to identify those genes that play a critical role in neural lineage commitment.

Coupled global and targeted proteomics of human embryonic stem cells during induced differentiation.

Elucidating the complex combinations of growth factors and signaling molecules that maintain pluripotency or, alternatively, promote the controlled differentiation of human embryonic stem cells (hESCs) has important implications for the fundamental understanding of human development, devising cell replacement therapies, and cancer cell biology. hESCs are commonly grown on irradiated mouse embryonic fibroblasts (MEFs) or in conditioned medium from MEFs. These culture conditions interfere with many experimental conclusions and limit the ability to perform conclusive proteomics studies. The current investigation avoided the use of MEFs or MEF-conditioned medium for hESC culture, allowing global proteomics analysis without these confounding conditions, and elucidated neural cell-specific signaling pathways involved in noggin-induced hESC differentiation. Based on these analyses, we propose the following early markers of hESC neural differentiation: collapsin response mediator proteins 2 and 4 and the nuclear autoantigenic sperm protein as a marker of pluripotent hESCs. We then developed a directed mass spectrometry assay using multiple reaction monitoring (MRM) to identify and quantify these markers and in addition the epidermal ectoderm marker cytokeratin-8. Analysis of global proteomics, quantitative RT-PCR, and MRM data led to testing the isoform interference hypothesis where redundant peptides dilute quantification measurements of homologous proteins. These results show that targeted MRM analysis on non-redundant peptides provides more exact quantification of homologous proteins. This study describes the facile transition from discovery proteomics to targeted MRM analysis and allowed us to identify and verify several potential biomarkers for hESCs during noggin-induced neural and BMP4-induced epidermal ectoderm differentiation.

Transplacental RNAi: deciphering gene function in the postimplantation-staged embryo.

RNAi offers the opportunity to examine the role in postimplantation development of genes that cause preimplantation lethality and to create allelic series of targeted embryos. We have delivered constituitively expressed short hairpin (sh) RNAs to pregnant mice during the early postimplantation period of development and observed gene knockdown and defects that phenocopy the null embryo. We have silenced genes that have not yet been "knocked out" in the mouse (geminin and Wnt8b), those required during earlier cleavage stages of development (nanog), and genes required at implantation (Bmp4, Bmp7) singly and in combination (Bmp4 + Bmp7), and obtained unique phenotypes. We have also determined a role in postimplantation development of two transcripts identified in a differential display RT-PCR screen of genes induced in ES cells by noggin exposure, Aggf1 and an Est (GenBank AK008955). Systemic delivery of shRNAs provides a valuable approach to gene silencing in the embryo.

Gene silencing using RNA interference in embryonic stem cells.

Pluripotent embryonic stem (ES) cells are an important model system to examine gene expression and lineage segregation during differentiation. One powerful approach to target and inhibit gene expression, RNAi, has been applied to ES cells with the goal of teasing out the cascades of gene expression/repression that shape the early embryo. In this chapter, we describe the current understanding of the mechanisms of gene silencing by small hairpin RNAs, as well as controls and caveats to using this approach in ES cells. A consideration of synthetic vs plasmid-based RNAi vectors, design of targeting constructs, transfection of ES cells, and flow sorting of targeted cells is followed by methods for the analysis of phenotype and behavior of targeted cell populations using immunohistochemistry, reverse transcriptase polymerase chain reaction, Western blotting, and scanning electron microscopy.

RNA inhibition of BMP-4 gene expression in postimplantation mouse embryos.

Short, hairpin RNA (shRNA) directed against bone morphogenetic protein 4 (Bmp-4) was delivered to early postimplantation staged mouse embryos via tail vein injection of pregnant dams. As early as 24 h postinjection, embryos expressed a DsRed marker and later exhibited defects of neural fold elevation and closure and of cardiac morphogenesis. Immunohistochemical analysis of sectioned embryos indicated that Bmp-4 protein was depleted and gene expression analysis indicated there was a reduction in Bmp-4 mRNA and an upregulation of the Bmp-4 antagonists, noggin and chordin, in embryos exposed to the shRNA, but not in control embryos. There was no change in the expression of Gata4, brachyury, or claudin6 in RNAi exposed embryos, indicating that RNA silencing was specific to Bmp-4 rather than producing widespread gene inhibition. Delivery of shRNA to embryos has the potential to specifically knockdown the expression of developmentally essential genes and to rescue gene mutations, significantly decreasing the time required to analyze the function(s) of individual genes in development.

Noggin and chordin have distinct activities in promoting lineage commitment of mouse embryonic stem (ES) cells.

To examine the role of secreted signaling molecules and neurogenic genes in early development, we have developed a culture system for the controlled differentiation of mouse embryonic stem (ES) cells. In the current investigation, two of the earliest identified BMP antagonists/neural-inducing factors, noggin and chordin, were expressed in pluripotent mouse ES cells. Neurons were present as early as 24 h following transfection of ES cells with a pCS2/noggin expression plasmid, with differentiation peaking at 72 h. With neuronal differentiation, stem cell marker genes were down-regulated and neural determination genes expressed. Coculture experiments and exposure to noggin-conditioned medium produced similar neuronal differentiation of control ES cells, while addition of BMP-4 to noggin expressants strikingly inhibited neuronal differentiation. Transfection of ES cells with a pCS2/chordin expression vector or exposure to chordin-conditioned medium produced a more complex pattern of differentiation; ES cells formed neurons, mesenchymal cells as well as N-CAM-positive, nestin-positive neuroepithelial progenitors. These data suggest that, consistent with their different expression fields, noggin and chordin may play distinct roles in patterning the early mouse embryo.