Item 2: hESCs differentiation, hESC self renewal, induced pluripotency. | Characterisation via a GFP-marked hES cell line as primary screen of aspects (my emphasis - HG) of: hESC self renewal; natural differentiation to plutipotency; and reversal of pluripotency to undifferentiated hESCs for reprogramming. |
Of the 21,121 human genes screened for their expressiveness in undifferentiated human embryonic cell lines via the technique of RNA silencing 94 genes were found that down regulated expression of the transcription factor, OCT4, and 54 that down regulated expression of the NANOG transcription factor. OCT4 and NANOG are known to be essential for induced pluripotency. That is, these two transcription factors are known to be important for reinstating an earlier stage of differention in developed somatic cells so that they can be reprogrammed.
Though a number of human embryonic cells lines were screened the experimental results led the researchers to confirm as the primary screen the cell line marked by green flourescent protein incorporated into a reporter construct driven by an upstream regulatory region of the gene POU5F1. POU5F1 is a gene known to be indicative of pluripotency, and it is regulated by the transcription factor PRDM14. So the researchers set out to learn more about PRDM14. They report that their experiments colocalised PRDM14 within the genome with OCT4, NANOG and another transcription factor, SOX2. Thus confirming PRDM14 as critical in the transcriptional regulatory network.
During the screen the researchers also noted that a number of genes not known to be associated with undifferentiated hESCs, such as those coding for the eukaryotic initiation factor complex and the chromatin remodelling complex, in fact are associated with hESCs.
The researchers ran a gain of function experiment to assess in more detail the role of PRDM14. They observed that PRDM14 in combination with other transcription factors enhanced artificial induction of pluripotency.
By depleting the level of PRDM14 the researchers also observed that 358 of 2,645 genes bound by PRDM14 were down regulated whilst 638 were upregulated.
PRDM14 contains domains known to be involved in proteolytic activity.
The experimental cell lines discussed in this paper are at the cusp of the developmental transition from a degree of totipotency to colonies of pluripotency. In vivo they would be around the time of implantation to the uterus.
Published 12.11.10 by Snippets of science with apologies to readers for my delay in preparing and posting this item.
References I thought were interesting when thinking about what the biophysics within the biochemistry might be of the Nature paper on RNAi silencing I selected as one of the two items for this week's issue of Snippets of science are the following two powerful biochemical papers, one a report of experimental observations of PRDM14 from 1998 (1) and one a very helpful biochemical review of protein chemistry as it was understood in 2002 (2), and placing the review in the shifting biomedical arena in which aberrations in protein degradation pathways are seen increasingly as significant to the future direction of medicine.
1. PRDM14 suppresses expression of differentiation marker genes in human embryonic stem cells. by Tsuneyoshi N, Sumi T, Onda H, Nojima H, Nakatsuji N, Suemori H.
Biochem. Biophys. Res. Commun. 2008 Mar 21, 367(4):899-905.
and
The Ubiquitin-Proteasome Proteolytic Pathway: Destruction for the Sake of Construction by Michael H. Glickman and Aaron Ciechanover from the Faculty of Biology and the Institute for Catalysis Science and Technology, and Department of Biochemistry, Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel. Physiological Reviews, Vol. 82, No. 2, April 2002. http://physrev.physiology.org/cgi/content/full/82/2/373#SEC1
My current musing as a science writer and former member of the Medical Journalists' Association, with intent to rejoin when funds allow, are that combined thinking about evolutionary biology and comparative genomics are essential to the future of human medicine.
A genome-wide RNAi screen reveals determinants of human embryonic stem cell identity. DOI:10.1038/nature09531. |
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