Another Hurdle Cleared

Roughly a year ago, stem cell biologist Yi Zhang, Harvard Medical School professor of genetics and the Fred S. Rosen Professor of Pediatrics at Boston Children’s Hospital, reported on his efforts to make a cloning technique called somatic cell nuclear transfer (SCNT) more efficient.

With SCNT, researchers take an egg cell and replace its nucleus with that of an adult cell, such as a skin cell, from another individual. The donated nucleus basically reboots an embryonic state, creating a clone of the original cell.

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It’s a hot topic in both agriculture and regenerative medicine. SCNT-generated cells can be used to clone an animal—such as Dolly the sheep—or produce embryonic stem (ES) cell lines for research.

But it’s an inefficient process, producing very few animal clones or ES lines for the effort and material it takes.

Zhang’s team was able to significantly boost SCNT’s efficiency by removing an epigenetic roadblock that kept embryonic genes in the donated nucleus from activating in cloned cells.

Now, in a new paper in Cell Stem Cell, Zhang and his collaborators report that they’ve extended their work to improve SCNT efficiency in human cells.

Making a more stem-y stem cell

Just as with mice, SCNT in human cells is complicated. It requires donated egg cells to receive the transferred nucleus, and human donors are hard to come by. In addition, not all donors, or egg cells from the same donor, are good for SCNT.

So why do this? Last year’s mouse study was about increasing the efficiency of animal cloning for research. With people, it’s about creating ES cell lines for research, particularly patient-derived cells for disease studies. And while, yes, one can use induced pluripotent stem (iPS) cell technology to create stem cells from a person’s own cells, Zhang says iPS cells don’t necessarily cut the mustard.

“There are two major ways to derive stem cells, SCNT and iPS. But iPS generates only ES-like cells,” Zhang explained. “They are pluripotent, but not totipotent like early stage pre-implantation embryos’ cells,” and thus do not achieve a true embryonic genomic state and all of the differentiating power that comes with it. SCNT, he added, results in true embryonic cells.

Same trick, different cells

To see if they could make human SCNT more efficient, Zhang’s team tried the same trick that worked with mice: injecting the cloned cell with RNA for an enzyme that removes the silencing methylation tag.

It wasn’t a sure bet that their mouse findings would be directly transferable to humans, Zhang noted. “Human and mouse ES cells are very different, and so we couldn’t assume that what worked in mice would work in human cells.”

But the results were strikingly similar. Without the RNA injection, none of the team’s SCNT embryos could be sustained to what’s called the expanded blastocyst stage (the stage at which the team could derive embryonic stem cells). With the treatment, however, the team had a 14 percent success rate.

Zhang sees this as a major step for regenerative medicine research.

“For years people have tried to change in vitro culture systems to improve SCNT efficiency,” he said. “We have instead removed a barrier within the cells themselves, not some part of their environment. And because we use RNA, rather than genetically inserting transcription factors as with iPS cells, there’s nothing left over in the cells after the reprogramming process is complete. The RNA is degraded when it is no longer needed.”

“I think this will become a standard procedure for researchers who want to derive ES cell lines,” he said. “It will let researchers generate more ES cell lines with fewer donors, and also generate banks of many, many patient-derived ES cell lines for research.”

He also thinks that SCNT could open the door to new approaches to treating mitochondrial diseases.

“You cannot fix diseases caused by mitochondrial DNA mutations using iPS cells, because in generating these cells only the nuclear genomic DNA is reprogrammed, not the mitochondrial DNA,” he said. “But with SCNT, you use a healthy donor egg, so the cells you generate have the patient’s genomic DNA and the donor’s mitochondrial DNA.”

This study was supported by the Howard Hughes Medical Institute and the Bio & Medical Technology Development Program (2012M3A9C6049723 and 2015M3A9C6028961) of NRF and MSIP of the Republic of Korea.

Adapted from a post on Vector, the clinical and research innovation blog at Boston Children’s.