"The failure rate is so impressive, it's a struggle every day to get anything to go," says Peter Mombaerts, developmental biologist at the Rockefeller University in New York. "We're all working on trying to understand why that is. Is our technique not good enough? Maybe we're not gentle enough with the cells? Or, are there biological explanations—maybe we were using the wrong cell type the whole time."
Now Mombaerts, working with Elaine Fuchs, a mammalian cell biologist at Rockefeller and Howard Hughes Medical Institute, adds another data point for determining cloning's best practices. The researchers' labs announce in this week's issue of the Proceedings of the National Academies of Science the creation of mice cloned from mouse skin cells, several of which survived into adulthood; the eldest of the litter is now nearly two years old.
Nuclear transfer has been used to clone mice using an array of different cell types: Lymphocytes, neurons and connective tissues have all been used. Nuclear transfer involves the injection of the nucleus from a stem (or differentiated) cell into an egg that has had its nucleus removed. The hybrid cells are cultured to create a blastocyst—a precursor of an embryo—and then implanted in a mouse's uterus, where it develops into a fetus. The most common type of nuclear transfer donor cell comes from the cumulus, which surrounds a developing egg or oocyte. (Considering their source, it should be no surprise that cumulus cells can only clone female mice.)
In the current study, Fuchs' team used keratinocyte stem cells, involved in the hair growth cycle and in healing wounds to the skin, from a part of the mouse's hair follicle called the bulge. These adult stem cells are easy to collect, quiescent—so they are not continually dividing—and remain able to develop into many cell types. Cells were taken from the mouse's back, and stem cells were then sorted out (using techniques Fuchs' team developed a few years ago) and delivered to Mombaerts to undergo nuclear transfer. Along with the keratinocyte stem cells, the team also performed nuclear transfer with cumulus cells and further differentiated skin cells.
"What we have done in this case is to use well-defined stem cells to ask: Do they have a better percentage of efficiency than a typical differentiated cell like a cumulus cell?" Mombaerts explains. "The answer is, well, yes. It's not dramatic, but yes."
Surrogate mothers birthed a total of 32 mice—19 of which began as keratinocyte stem cells, six came from cumulus cells and seven from mature skin cells. For females, the keratinocytes barely outperformed cumulus cells in terms of cloning efficiency: 1.6 percent of the former's blastocytes transferred to a uterus developed into a birthed pup versus 1.2 percent for the latter. Male pups that began as keratinocytes, however, had a 5.4 percent cloning efficiency. "We didn't set out to compare males and females," notes Mombaerts.
"The difference in cloning rates of male and female stem cells seems likely to involve epigenetics," adds Fuchs, referring to effects on genetic function that do not require a change in DNA sequence. Epigenetics is important to nuclear transfer, because the chromosomes in the stem cells must be reprogrammed to behave like embryonic chromosomes. In the case of female cells, however, only one of the two X chromosomes in each cell is active making the reassignment more difficult. "I think it's intuitive to immediately think of X chromosome inactivation as an additional hurdle that reprogramming has to solve for female cells," says Mombaerts.
Jerry Yang, director of the Center for Regenerative Biology at the University of Connecticut—whose lab reported last year that fully differentiated bone marrow cells performed better in cloning mice than stem cells from the same source—says the new study "confirms that adult stem cells are as good for nuclear transfer cloning as the cumulus cells they used." He adds that the incongruity observed between male and female cloning efficiency will open doors to more research on why male cells may appear easier to reprogram than female cells.
Fuchs says the finding indicates that nuclear transfer could one day be used to create new embryonic stem cell lines from adult stem cells, allowing researchers to not only study diseases like Alzheimer's and Parkinson's, but also to create a set of stem cells specific to a person from his or her own skin, avoiding complications like immune response rejection.
"In this paper, we don't derive embryonic stem cell lines," Mombaerts makes clear, adding, "Given the preciousness of human oocytes, we have to really make sure we use the best possible conditions and the best possible cell types. We cannot afford wasting oocytes."