Serial Analysis of Gene Expression (SAGE): a useful tool to analyze the cardiac transcriptome.

Serial analysis of gene expression (SAGE), a functional genomics technique, can be used for global profiling of gene transcripts. It relies on the preparation and sequencing of cDNA concatemers, but it does not require prior knowledge of the genes to be assayed (as with microarrays). Once analyzed, SAGE data provide both a qualitative and quantitative assessment of potentially every transcript present in a particular cell or tissue type. In this chapter, we describe the fundamental principles of SAGE, describe a complete protocol for the generation of SAGE libraries, and show how it has been employed to generate the first SAGE reference data set of the mouse myocardium. Following the protocols described here, investigators should be able to generate unique mouse heart SAGE libraries, which can be directly compared with our reference library. This permits the identification of transcripts that are differentially expressed as a function of time, age, genetic background or transgenic state, among other factors. SAGE is thus a powerful technique that permits a comprehensive analysis of changes in mRNA abundance. The results provide a snapshot of altered patterns of gene expression in response to any genetic or environmental stimulus that can be used to generate new biological hypotheses or test existing paradigms.

Signals from embryonic fibroblasts induce adult intestinal epithelial cells to form nestin-positive cells with proliferation and multilineage differentiation capacity in vitro.

The intestinal epithelium has one of the greatest regenerative capacities in the body; however, neither stem nor progenitor cells have been successfully cultivated from the intestine. In this study, we applied an "artificial niche" of mouse embryonic fibroblasts to derive multipotent cells from the intestinal epithelium. Cocultivation of adult mouse and human intestinal epithelium with fibroblast feeder cells led to the generation of a novel type of nestin-positive cells (intestinal epithelium-derived nestin-positive cells [INPs]). Transcriptome analyses demonstrated that mouse embryonic fibroblasts expressed relatively high levels of Wnt/bone morphogenetic protein (BMP) transcripts, and the formation of INPs was specifically associated with an increase in Lef1, Wnt4, Wnt5a, and Wnt/BMP-responsive factors, but a decrease of BMP4 transcript abundance. In vitro, INPs showed a high but finite proliferative capacity and readily differentiated into cells expressing neural, pancreatic, and hepatic transcripts and proteins; however, these derivatives did not show functional properties. In vivo, INPs failed to form chimeras following injection into mouse blastocysts but integrated into hippocampal brain slice cultures in situ. We conclude that the use of embryonic fibroblasts seems to reprogram adult intestinal epithelial cells by modulation of Wnt/BMP signaling to a cell type with a more primitive embryonic-like stage of development that has a high degree of flexibility and plasticity.

Crucial role of the sarcoplasmic reticulum in the developmental regulation of Ca2+ transients and contraction in cardiomyocytes derived from embryonic stem cells.

In adult myocardium, excitation-contraction coupling is critically regulated by sarcoplasmic reticulum (SR) Ca2+ release via type 2 ryanodine receptor (RyR2), but generally, it is believed that SR-function is rudimentary in the fetal heart and in embryonic stem (ES) cell-derived cardiomyocytes (ESCMs), a possible source for cell replacement therapies. This study used wild-type (RyR2+/+) and RyR2 null (RyR2-/-) ESCMs as an in vitro model of cardiomyogenesis, together with pharmacological approaches and expression profiles of genes relevant for SR function, to elucidate the functional importance of RyR2 and SR on the regulation of Ca2+ transients and contraction during early cardiomyocyte development. During differentiation of RyR2+/+ ESCMs, SR function developed progressively with increased basal cytosolic free Ca2+ concentration ([Ca2+]i), enhanced frequency and amplitude, and decreased duration of Ca2+ transients that were inhibited by ryanodine and thapsigargin. These functional traits correlated with SR Ca2+ load and the expression of RyR2, SERCA2a, and phospholamban. RyR2-/- ESCMs, comparatively, demonstrated a significantly prolonged time-to-peak and reduced frequency of Ca2+ transients and contractions. Beta-adrenergic stimulation of RyR2+/+ ESCMs increased the frequency and amplitude of Ca2+ transients with differentiation but was much weaker in RyR2-/- ESCMs. We conclude that functional SR and control of RyR2-mediated SR Ca2+ release directly contribute to the spontaneous and beta-adrenergic receptor-stimulated contraction of ESCMs, even at very immature stages of development.