The research appears in the November 2007 issue of Experimental Biology and Medicine.

Researchers know very little about how human embryonic stem cells (hESC) self-renew. To fully understand these cells' self renewal capacity and pluripotency, and their regulation, it is necessary to efficiently generate genetically modified cells and analyze the consequences of elevated and reduced expression of genes.

The research team, led by the University of Minnesota's Meri Firpo, Ph.D., included gene therapy researchers at Los Angeles Children's Hospital, and developmental biologists at the University of Michigan.

The researchers used knockdown technology to reduce the expression of oct4, a gene known to be necessary for self renewal of mouse and human embryonic stem cells. As seen in work done with mouse cells by knockdown and other genetic means, they showed that reducing the amount of oct4 in human ES cells induced differentiation. The researchers then used a plasmid vector to transiently increase levels of oct4 in hESC. This also resulted in differentiation as expected, but with differentiation patterns similar to those seen with the knockdown. This was an unexpected result, because when expression of oct4 is up-regulated in mouse ES cells, they differentiate into a different type of cell than if the expression of oct4 is down-regulated.

"This suggests a key difference in the regulation of early development between mouse and human embryos" Firpo said. While animal models are clearly important, this research shows that scientists need human models to truly understand what happens in early human development.

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E-NTPDase2 quickly latches on to ATP converting it to ADP. This meant that where and when the researchers introduced E-NTPDase2 it led to nearby cells experiencing much higher levels of ADP. The Warwick team hypothesized that ATP must be released in a short burst from the location where the eye will develop so that it can be converted to ADP by E-NTPDase2, thereby providing the trigger for eye development. They were able to measure these short bursts of ATP using ATP sensors specially developed by Professor Dale. This is the first time researchers have been able to see and measure bursts of ATP so early in the development of living creatures.

The genes that initiate and direct eye development are well known and are collectively termed the Eye Field Transcription Factors (EFTFs). One of the mysteries of the field is how these genes get turned on in the correct location and at the correct time to initiate eye development. The Warwick research shows that this short burst of ATP followed by accumulation of ADP is a key signal for initiating expression of the EFTFs and hence the development of the eye.

The discovery of this surprising new signal that literally switches on eye development it is not restricted to frogs. Mutations to the E-NTPDase2 gene on the human 9th chromosome is already known to cause severe head and eye defects. This suggests that this newly discovered mechanism for triggering eye development applies across a wide range of species.

This new understanding of how eye development is triggered will greatly assist researchers exploring stem cells connected to eye development and opens up an avenue of a research that could in just a few decades lead to the ability to produce an eye in a dish.

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