A Gene Expression–Based Screening System for Compounds Influencing Differentiation of Mouse Embryonic Stem Cells

Abstract

Low molecular weight compounds have been shown to be useful for controlling stem cells, and various high-throughput screening systems have been developed for identifying compounds that regulate the differentiation of stem cells. However, the effects of such compounds on stem cell differentiation are usually evaluated by assessing a single parameter, which is insufficient for proper monitoring of the cellular status. In this study, to classify a number of compounds, the authors established a gene expression-based screening system using mouse embryonic stem (ES) cells that monitored multiple parameters. ES cells were differentiated into three germ layers by embryoid body formation and then treated with the test compounds. Next, cellular changes were assessed by analyzing the expression of multiple genes with the multiplex quantitative reverse transcriptase polymerase chain reaction. By screening a library of pharmacologically active compounds with this system, the authors were able to classify 52 compounds that influenced the gene expression profile of ES cells. They also found that some compounds identified by screening could enhance osteogenic or adipogenic differentiation of human mesenchymal stem cells. These results indicate that the screening system is effective for identifying compounds involved in regulating the differentiation of both ES cells and adult stem cells.

Definitive Endoderm Formation from Plucked Human Hair-Derived Induced Pluripotent Stem Cells and SK Channel Regulation

“…To test the differentiation potential of our established hiPSC lines into definitive endoderm (DE), previously published protocols were combined in terms of a small molecule-driven approach [43–45]. Small molecule-based assays are less biased by batch-to-batch variations and are usually more cost effective.…”

“…From day 3 on, cells were cultured in RPMI 1640 supplemented with 500 nm IDE1, 5  µ M LY294002, and 50 ng/mL FGF2. The respective figure contains an experimental outline illustrating detailed culture conditions and treatment regimens [40–45] (Figure 2(a)). …”

High-Content Imaging-Based Screening of Microenvironment-Induced Changes to Stem Cells

“…Several tools have emerged to precisely characterize how stem cells react to various stimuli (e.g., growth factors or externally applied forces) and substrate properties. 1,2 Typical screening methods focus on either the detection of lineage-specific markers or gene expression levels of whole stem cell populations, 3–6 which are difficult to scale down quantitatively to single-cell levels. In addition, due to the end-point nature of these assays, stem cells have to be cultured for several weeks before these methods can be applied to assess cellular responses.…”

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

“…Indeed, by quantitative RT-PCR assays the expression of eight genes was examined: Flk1, Actc1, Acta2, Tie2 (mesodermal markers), Nefh, Tubb3 (ectodermal markers), Nanog (pluripotent marker), and Gapdh (an internal control). The analysis revealed that the gene expression profile was altered from treatment with several compound, suggesting that a potential range of effects on differentiation can be detectable with this assay [77]. A drawback of these last two methods is that they require significant manipulations that make them difficult to use to screen for a large number of compounds.…”