Synthetic version of Oct4 robustly supports induced pluripotent stem cell formation


A recent paper by members of the Danish Stem Cell Centre in Copenhagen, the MRC Centre for Regenerative Medicine in Edinburgh, and Memorial Sloan Kettering Cancer Center in New York has used a synthetic version of the Oct4 protein to dissect the precise role of this protein in stem cells, and to more effectively generate induced pluripotent stem cells.
Oct4 is a member of the “class V POU” transcription factors. POU stands for Pit-Oct-Unc, which are the founding members of this group of transcription factors. Transcription factors are proteins that bind to specific sequences of DNA and turn on gene expression. The POU family of transcription factors was originally defined on the basis of a common region of ~150–160 amino acids that was identified in the transcription factors Pit-1, Oct-1, and Oct-2, which were known from mammals, and the nematode factor Unc-86. This common POU protein domain is the DNA binding region that consists of two subdomains joined by a common linker.

Embryonic development in mammals is controlled by regulatory genes, many of which regulate the expression of other genes. These regulators activate or repress patterns of gene expression that mediate the changes characteristic of development. Oct4, like other members of the POU family of transcription factors, activates the expression of their target genes by binding an eight-base sequence motif that usually has some similarity to this sequence: AGTCAAAT. During embryonic development, Oct4 is expressed initially in all the cells of the embryo, but eventually becomes restricted to the Inner Cell Mass (ICM) and Oct4 expression fades in the outer cells (known collectively as the trophectoderm). At maturity, Oct4 expression is confined exclusively to the developing germ cells. Disruption of Oct4 in mice produces embryos without a pluripotent ICM. This suggests that Oct4 is required for maintaining pluripotency.

Given the importance of Oct4 in early development, it is no surprise that it plays an important role in embryonic stem cell maintenance. Oct4 also plays an essential role reprogramming adult cells from their mature state to the embryonic state. In the absence of Oct4, embryonic stem cells differentiate. Oct4 plays a powerful role in regulating stem cell genes. However, while large quantities of Oct4 are needed, too much of it can hamstring the properties of stem cells.

Given these data, does Oct4 maintain pluripotency by activating the expression of particular genes or by repressing those genes necessary for differentiation? These scientists, whose work is published in the journal Cell Reports, made fusions of Oct4 with proteins that are known to activate gene expression or fusions with proteins known to repress gene expression. Then they accessed the ability of these fused versions of Oct4 to support pluripotency in embryonic stem cells or induce pluripotency in adult cells.

The synthetic version of Oct4 fused to known activator of gene expression were much more efficient in turning on genes that instruct cells how to be stem cells.  Cells also did not require as much of this synthetic Oct4; stem cells required less of the synthetic Oct4 to remain stem cells and adult cells required less to become reprogrammed as stem cells.  Those synthetic versions of Oct4 that were fused to known transcriptional repressors caused cells to differentiate, and such synthetic versions of Oct4 could not replace endogenous Oct4 in stem cells.

Further tests with the activating synthetic Oct4 showed that it could support stem cells under conditions that are usually not conducive to their growth.  This provides a way to generate stem cells in the laboratory when growth conditions are less than optimal.  Because the activator version of synthetic Oct4 could replace endogenous Oct4 and not the repressor version of synthetic Oct4, Oct4 must work primarily as an activator of gene expression rather than a repressor of gene expression.

Professor Joshua Brickman, who is affiliated with The Danish Stem Cell Center (DanStem), University of Copenhagen and Medical Research Council Centre for Regenerative Medicine at the University of Edinburgh. said “Our discovery is an important step towards generating and maintaining stem cells much more effectively.  Embryonic stem cells are characterized, among other things, by their ability to perpetuate themselves indefinitely and differentiate into all the cell types in the body – a trait called pluripotency. But to be able to use them medically, we need to be able to maintain them in a pure state, until they’re needed. When we want to turn a stem cell into a specific cell, such as insulin producing beta cell, or a nerve cell in the brain, we’d like this process to occur accurately and efficiently. This will not be possible if we don’t understand how to maintain stem cells as stem cells. As well as maintaining embryonic stem cells in their pure state more effectively, the artificially created Oct4 was also more effective at reprogramming adult cells into so-called induced Pluripotent Stem cells (iPSCs), which have many of the same traits and characteristics as embryonic stem cells but can derived from the patients to both help study degenerative disease and eventually treat them..”

Exploitation of such technology could improve the efficiency of protocols to generate iPSCs in the laboratory and the clinic.  Such cells could be used to produce individualized cells for developing individualized therapies for degenerative diseases such as type 1 diabetes and neuro-degenerative diseases.