Cloning Offers Intriguing Stem Cell-Making Tips

Two recent studies— one human, one mouse— have found cloning creates better pluripotent stem cells than the Nobel Prize-winning induced pluripotent stem cell (iPSC) method.

Cloning involves fusing an old cell (or nucleus) with an egg cell and letting the egg reprogram and rejuvenate it— just as it reprograms and rejuvenates mature sperm during conception. It is a physiologic process. The more artificial iPSC method involves genetically tweaking four genes.

“Based on our recent study, oocyte [egg] cytoplasmic factors induce reprogramming of somatic cells into superior quality embryonic/pluripotent state than conventional iPSC approaches,” the author of one of the studies, Shoukhrat Mitalipov, told Bioscience Technology in an email.

As if choreographed, a perfectly timed, third study came out last week supporting the conclusions. The third study identified, in egg cells, one protein that may help explain the egg’s aforementioned super-powers.

“A very nice study,” Mitalipov said of the third paper. Mitalipov is director of the Center for Embryonic Cell and Gene Therapy at the Oregon Health and Science University. Earlier, he was the first scientist to clone human cells. “It makes sense to understand and mimic oocyte factors for reprogramming so we can produce better iPS cells.”

Cloned stem cells may be epigenetically superior

Mitalipov’s paper, published in Nature, was the first to compare human pluripotent cells (embryonic stem, or ES, cells) from IVF clinic embryos to those made via cloning, and via the iPSC method— “isogenic” cells from the same individual. Noting that ES cells (which form all somatic tissues of the body) from IVF clinic embryos are the “gold standard,” they compared the cells in many ways.

There were no statistically significant de novo CNVs (copy number variations) caused by reprogramming, the paper noted. CNVs are abnormal swaths of DNA sometimes linked to cancer.

But there was a significant, eight-fold greater number of methylation sites in iPSCs— carried over from parent cells— than in stem cells made via cloning. Indeed, the cloned cells resembled “gold standard” ES cells in epigenetic methylations patterns (which determine what genes turn on) more than iPSCs.

Furthermore, the corresponding transcriptional signatures (gene expression patterns) of the cloned stem cells were more similar to “gold standard” IVF ES cells than to iPSCs. That is, turned-on or expressed genes were more alike between ES and cloned cells (in line with the above methylation data).

“Transcription factor-based reprogramming (the iPSC method) is associated with incomplete epigenetic reprogramming,” the paper continued. “In contrast, the same somatic cells reprogrammed by SCNT (somatic cell nuclear transfer, or cloning) displayed epigenetic and transcriptional signatures remarkably similar to those of IVF ES cell controls…An explanation for this more effective reprogramming by SCNT is that the ooplasm provides ‘physiologic’ levels of reprogramming factors…It has been suggested that oocyte factors rapidly demethylate the somatic genome…Clearly elucidation of oocyte-based reprogramming mechanisms will support the development of improved reprogramming methods.”

The study also noted that while CNV numbers — which can influence tumorigenicity— were not statistically different, the team did not study point mutations and other potential cancer influences.

Cloned stem cells may be genetically superior

But a study appearing in an abstract at the June ISSCR meeting— and as a paper in an earlier Stem Cell Reports— did find more (potentially cancer-causing) point mutations in the iPSC method than in cloning. Abe Masumi, director of the National Institute of Radiological Sciences in Chiba, Japan, found there was “an obvious difference in the frequency and mode of point mutations” between iPSCs and cloned cells, with more appearing in iPSCs, he said in an interview with Bioscience Technology at the ISSCR. The study was done with mouse cells from the same embryos. He also found “a large number” of iPSC point mutations were not pre-existing, but de novo, and that there was heterogeneity of point-mutation profiles in identical iPSCs. “The heterogeneity of the point mutation patterns in a single iPSC (identical subline) reflects the history of the emergence of each mutation.”

By email later, Masumi added: “Although a large number of point mutations have been identified in iPSC genomes by several groups thus far, no relation has been found between such point mutations and any functions. In-depth and large-scale analyses should be required for addressing this issue, e.g. relation between genetic aberrations and the differentiation-defective cells described by Dr. Yamanaka.”

Masumi was referring to Shinya Yamanaka, the University of Kyoto scientist who won the Nobel Prize for his creation of iPSCs. At the ISSCR, Yamanaka presented evidence that it may be possible to isolate the iPSC method’s reprogramming-defective cells, leaving behind more healthy iPSCs.

Is the egg already yielding secrets?

In the meantime, in the July 17 Science, another group has found a critical reprogramming protein in the egg. The team of the University of Michigan’s Jose Cibelli, a cloning pioneer, reported that overexpression of ASF1A, a histone remodeling chaperone enriched in the metaphase II human oocyte, is necessary to reprogram cells. “Our report underscores the importance of studying the unfertilized MII oocyte as a means to understand the molecular pathways governing somatic cell reprogramming,” Cibelli’s paper concluded.

A key reason to improve iPSCs with cloning tips, rather than switch to cloning: cloning uses precious human eggs. The iPSC method can theoretically give adults uncontroversial supplies of their own, genetically identical pluripotent stem cells.

Other papers finding cloned stem cells superior to iPSCs include an April 2014 Stem Cell Reports study by University of California stem cell researcher Yang Xu; a January 2014 Cell Stem Cell study by the National Institute of Biological Sciences’ Sharong Gao; and a January 2013 Cell Research study by Jinsong Li.