Getting stem cells to change to the required target cell and reproduce faultlessly is difficult, whether using adult or embryonic stem cells. An international team of researchers has recently developed a two part system that can change the cells to the targets and then remove the leftovers of that conversion, leaving only the desired DNA behind to duplicate.
This is a major breakthrough that can be used for many types of regenerative stem cell treatments.
Xiaojun Lian, assistant professor of biomechanical engineering, biology and a member of the Huck Institutes of the Life Sciences, Penn State explained that a difficulty with human pluripotent stem cells is that they can’t be used directly, as they have to be functional cell types. To treat damage caused by a heart attack for example, cells that are able to repair the heart wall are required.
Under normal conditions, pluripotent stem cells induced from both embryonic and adult cells are sent a chemical signal that instructs them to change from a stem cell to a specific functional cell. Although pluripotent stem cells are able to change to any cell in the human body, the natural process is part of a complex series of DNA controlled triggers. In the past, researchers have inserted DNA into the pluripotent cells to get them to convert. The problem with this method is however that leftovers of the inserted DNA remain behind.
The research was published in a recent issue of Scientific Reports. The team does not incorporate a piece of DNA that instructs the cells to convert, but DNA that makes the cell glow green when lit up by a blue light. This marker enables the researchers to see when the DNA plasmid has been incorporated into the cell, and also that it completely disappears upon removal. A plasmid is a round piece of DNA that holds the functional DNA fragments that are able to control gene expression in cells.
Lauren N. Randolph, doctoral student in bioengineering, Penn State explained that they were exploring the limits for turning the conversion off and on and also wanted to be able to control the level of expression, as well as the removal of DNA after conversion.
Approaches in the past merged the suitable DNA to switch on the conversions, but was not able to remove all the DNA inserted completely.
The researchers used a Tet-On 3G inducible PiggyBac system. This plasmid, named XLone, was used to accomplish insertion, activation and removal. The PiggyBac part of the system includes the DNA that is used to insert the required DNA into the cell’s DNA. The Tet-On 3G part incorporates the signaling information required. The system also makes the cells more sensitive to doxycycline, the drug employed to initiate the conversion.
Lian added that they use plentiful multiple copies of the plasmid to improve the probability that it gets in to do what it is supposed to do, and to ensure that it follows through with the reproduction of the cells.
If only a single or a few plasmids were to be inserted into the cell, the new DNA could simply be silenced. By inserting multiple plasmids, the team is sure that at least one will work.
Randolph noted that a huge advantage with this system is that it does not have any leakage expression. If the system is not induce with doxycycline, nothing happens.
Another advantage is that once the cells start reproducing to create the required cells, for example nerve cells or heart cells, the plasmid is removed and the cells carry on reproducing without any remnant of the plasmid system.
Although the researchers are currently simply trying to study and understand gene function and directed cell differentiation in human stem cells, they would like to eventually be able to create cell based therapies for a wide range of degenerative diseases.