Fluorescence microcopy image of human embryonic stem cells stained with green fluorescent protein
From Ref. 1 © 2010 H.-H. Ng and F. Bard
Human embryonic stem cells (hESCs) are an important tool for research and a viable source of potential replacement cells for clinical applications. There are two properties that make hESCs unique: the ability to differentiate into any cell type, a property known as pluripotency; and the ability to go through numerous cycles of cell division without changing their undifferentiated state, a property called self-renewal. To exploit the full potential of hESCs, scientists must have a detailed understanding of the genetic network that governs these properties. For this reason, Huck-Hui Ng, Fred Bard and co-workers from the A*STAR Genome Institute of Singapore, the A*STAR Institute of Molecular and Cell Biology, Nanyang Technological University and the National University of Singapore have carried out a whole-genome RNA interference screen to comb out genes that regulate these functions of self-renewal and pluripotency.
The researchers generated a stable hESC line expressing green fluorescent protein (pictured). As only undifferentiated hESCs of this line exhibit green fluorescence, the researchers could estimate the effect of a particular gene on hESC survival and proliferation by measuring the fluorescent intensity and number of hESC nuclei.
The researchers used the hESC line to screen a library of 21,121 human genes. This process identified 566 genes that had the largest effect on hESC survival and proliferation. Analyses of gene ontology and biological reactions for the 566 genes revealed a wealth of factors and pathways involved in transcription and translation. Among these were components of the transcriptional regulator PRDM14 and the chromatin remodeling complex INO80.
Previous studies have demonstrated the possibility of reprogramming human fibroblasts into pluripotent stem cells (PSCs), known as human induced PSCs, through the introduction of four transcription factors: OCT4, SOX2, KLF4 and c-MYC. The researchers assessed how the introduction of additional transcriptional factors, such as PRDM14 and NFRKB, could affect the pluripotency reprogramming process. They found that the introduction of PRDM14 and NFRKB could accelerate the reprogramming process by factors of 7 and 3.5, respectively. The results not only demonstrate the power of the whole-genome RNA interference screen, but also reveal the roles of many new factors and pathways.
“We are extremely excited by our new findings,” said Ng. “Our data have shown us the intricate network of genes and factors involved in the maintenance of the hESC state.”
Further studies revealed that PRDM14 directly regulates the expression of the pluripotency gene POU5F1, and colocalizes with transcription factors OCT4, NANOG and SOX2. The findings suggest that PRDM14 is a key transcription factor required for the maintenance of hESC pluripotency.
The A*STAR-affiliated researchers contributing to this research are from the Genome Institute of Singapore and the Institute of Molecular and Cell Biology.
