Embryonic stem cells or ES cells are cell lines derived from the inner cell mast of blastocyst stage embryos. ES cells have two key properties that make them an ideal model system to study human development. The first, self-renewal, is the ability to replicate almost indefinitely in cell culture. This allows researchers to obtain large and homogenous populations of cells to study. The second property, pluripotency, is the ability to differentiate into all of the of different cell types that make up an adult organism.
The science of regenerative medicine hopes one day to discover how to create healthy tissues to replace those that lose function due to disease, injury, or age. Since ES cells have the developmental potential to differentiate into any tissue are believed to hold great promise for regenerative medicine
In 2006 groundbreaking experiments by Takahashi and Yamanaka demonstrated that pluripotent cells could be created from somatic cells by forced expression of the ES cell transcription factors Oct4, Sox2, Klf4 and cMyc. These induced pluripotent stem cells, or iPS cells, are highly similar to embryonic stem cells.
. The generation of iPS cells was a huge breakthrough in regenerative medicine because they are similar to embryonic stem cells and can be derived in a patient-specific manner, from adult somatic cells, requiring no embryonic tissue.
Since the initial reprogramming experiments in 2006, the pace of research in this field has been very rapid. In chimera experiments iPS were show to be able to give rise to germ line cells. Then in tetraploid complementation assays, it was demonstrated iPS cells can contribute to all of the tissues that make up an adult organism Since their initial generation in mouse, iPS cells have been made in several species including human . They have been made from a large number of different starting cell types and using many different sets of ES cell transcription factors.
One of the original reprogramming transcription factors, Myc, is a known oncogene. It has since been shown that iPS cells can be made without the use of myc. It has also been shown that iPS cells can be made with viral vectors than can later be excised from the genome or with vectors that do not integrate into the genome at all. iPS cells have also been made using small molecules and proteins as reprogramming agents.
Finally, a number of in vitro disease models have been made using iPS cells. For example, iPS cells were made from patients suffering from Parkinson's disease and then differentiated in vitro into neuronal cells. Since these neurononal cells have the genome of a person who developed Parkinson’s disease, they may be an important tool for understanding the disease pathology.
As I said earlier ES cells and iPS cells are highly similar. They express the same markers of pluripotency and have the same cell morphology. They also behave similarly in a broad range of phenotypic assays including - embroid body formation - teratoma formation - germline transmission - and tetraploid complementation Whether ES cells and iPS cell are truly equivalent is a very important question, since if they are equivalent, then the knowledge that has been gained in years of embryonic stem cell research will be applicable to iPS cells as well.
Recently, several studies have been published saying that there are differences in the gene expression programs of ES and iPS cells. If it is true, that ES and iPS cells have different gene expression programs, then this indicates that they are not equivalent cell types. This has huge implications for the entire field of stem cell research. If ES and iPS cells are not equivalent, then iPS cells might not hold the promise for regenerative medicine that we had hoped.
We decided that it would be critical to answer for ourselves whether ES and iPS cells are equivalent cell types. We reasoned that obtaining genome-wide maps of H3K4me3 and H3K27me3 histone modifications in addition to gene expression profiles for a panel of ES and iPS cell lines would allow us to obtain a highly detailed and quantitative assessment of both the current transcriptional state of ES and iPS cells as well as their future developmental potential.
WE decided chose to profile the H3K4me3 and H3K27me3 histone modications because that are two of the most well studied histone modifications. H3K4me3 is generally associated active genes while H3K27me3 is associated with genes that are not being transcribed. In ES cells active genes have the K4 mark at their transcription start sites. There is another class of genes that are occupied by both K4 and K27. These genes are especially interesting because they are include many of the most important genes for controlling differentiated cell types.
Homeobox transcription factors for example are nearly all occupied by both K4 and K27 in ES cells. These genes with both K4 and K27 are not expressed in ES cells, otherwise they would promote differentiation, but they are poised for later expression. In a differentiated cell that expresses one of these genes, the K27 mark is lost while K4 is retained. In a differentiated cell that turns off one of these genes, the K4 mark will be lost and K27 is retained. At some genes, both K4 and K27 are retained which means that this gene is still poised for expression as the cell differentiates further.Together the genome wide locations of the K4 and K27 marks reflect both of the current transcriptional state of a cell as well as it future developmental potential.
We reasoned …
By comparing these data we can accurately asses whether there are truly differences between ES and iPS cells. We assembled a collection of 6 independent ES cell lines each from separate donors, and 6 iPS lines, 4 derived from one fibroblast donor and 2 from another donor. We also examined fibroblast cells as a control.
Together H3K4me3 and H3K27me3 location analysis and microarray based gene expression experiments allow us to get a highly quantitative measure of cell state This reflects both the current transcriptional state of the cell as well as its future developmental potential.
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