Removing roadblocks to therapeutic cloning to produce stem cells

Dolly sheep cloning somatic cell nuclear transfer epigenetics
Dolly the sheep, the first mammalian example of successful somatic cell nuclear transfer. (Toni Barros/Wikimedia Commons)

We all remember Dolly the sheep, the first mammal to be born through a cloning technique called somatic cell nuclear transfer (SCNT). As with the thousands of other SCNT-cloned animals ranging from mice to mules, researchers created Dolly by using the nucleus from a grown animal’s cell to replace the nucleus of an egg cell from the same species.

The idea behind SCNT is that the egg’s cellular environment kicks the transferred nucleus’s genome into an embryonic state, giving rise to an animal genetically identical to the nucleus donor. SCNT is also a technique for generating embryonic stem cells for research purposes.

While researchers have accomplished SCNT in many animal species, it could work better than it does now. It took the scientists who cloned Dolly 277 tries before they got it right. To this day, SCNT efficiency—that is, the percent of nuclear transfers it takes generate a living animal—still hovers around 1 to 2 percent for mice, 5 to 20 percent in cows and 1 to 5 percent in other species. By comparison, the success rate in mice of in vitro fertilization (IVF) is around 50 percent.

“The efficiency is very low,” says Yi Zhang, PhD, a stem cell biologist in Boston Children’s Hospital’s Program in Cellular and Molecular Medicine. “This indicates that there are some barriers preventing successful cloning. Thus our first goal was to identify such barriers.”

Unsilencing genes

To help find those barriers, Zhang and researchers Shogo Matoba, PhD, ran a simple and elegant experiment, which they reported in the journal Cell. They generated mouse embryos through either SCNT or IVF and compared their gene expression profiles at the very early stages of development.

That comparison revealed 222 regions of the genome that were turned on in the IVF embryos but silent in the SCNT ones.

“In normal development or in IVF, many genes need to be activated for the embryo to develop.” Zhang explains. “What we found was that these genes are successfully activated in IVF embryos, but fail to be activated in SCNT embryos.”

Probing deeper, they found that the inactive genes in the SCNT-generated embryos were held in check by an epigenetic silencing mark—a methylation tag on histone H3, a protein that packages DNA inside the cell. When Zhang and Matoba removed those tags—either by activating enzymes to cut them off, or turning off enzymes that attach the tags—they were able to increase SCNT efficiency from 1 to 2 percent to 8 to 9 percent.

Mouse embryos cloning somatic cell nuclear transfer epigenetics
Mouse embryos generated by SCNT. The ones on the right have had gene-silencing methylation tags removed, greatly increasing cloning efficiency. (Image courtesy Shogo Matoba)

A better way to create stem cells for research?

Their results suggest that the genomic “rebooting” process triggered by SCNT is incomplete, and that certain epigenetic and genetic programs that govern the developing embryo’s growth and survival are not activated properly. Zhang and Matoba note that 49 of the genes that stay off in SCNT embryos are known to play important roles in turning other genes on, which might also help explain why the embryo gene programs aren’t activated properly.

The study also indicates that removing epigenetic locks could help increase SCNT efficiency. Much more needs to be done to understand the remaining roadblocks, but if those too could be overcome, Zhang sees a bright future for SCNT as a tool for generating embryonic stem (ES) cells for research, augmenting the current technology of reprogramming mature cells to create induced pluripotent stem cells (iPS cells).

“While induced pluripotent stem cells are ES cell–like, they aren’t real ES cells,” Zhang says. “With SCNT you can generate real ES cells, and published results suggest that the quality of ES cells derived from SCNT-mediated reprogramming is better than that of iPS cells. If we can raise the efficiency of SCNT to the level of IVF, it could be very important to regenerative medicine research.”