The efficiency of making “induced pluripotent stem cells” (iPSC) cells has leapt overnight from less than 10% to over 90%, according to Nature.
The Nobel Prize-winning iPSC approach—which lets researchers create potent embryonic-like stem cells from ordinary adult cells—has suffered glitches, key among them inefficiency. Very few cells in any batch go “stem.”
The new approach, devised by the Israeli Weizmann Institute team of Jacob Hanna, has astronomically improved efficiency.
“This is certainly a salient advance,” Stanford University developmental biologist Kyle Loh tells Bioscience. Loh wrote a Nature News & Views commentary on the paper with Harvard Medical School associate professor Bing Lim.
Furthermore, it is a relatively simple and logical advance. The Israeli team simply blocked a gene, Mbd3, that is normally blocked during pre-implantation development.
Last year, Kyoto University stem cell expert Shinya Yamanaka won the Nobel Prize for his work in 2006 and 2007 tinkering with four genes, which let him turn back the clock on old cells, and create out of them embryonic-like stem cells.
The approach has been hailed as key for research. Diseased human cells were hard to acquire for labwork as they can be scarce (humans do not often part with their neurons, for example). And they couldn’t replicate—a trait of most mature adult cells.
Turning adult cells into iPSCs solves these problems. They replicate as easily as embryonic cells.
The iPSC approach has more warily been hailed as a potential clinical advance. IPSCs are identical immunologically to patients—unlike embryonic cells, which come from spare IVF clinic embryos. But now the process is too tumorigenic, needing work. (A possible exception: an upcoming clinical trial in Japan.)
But another iPSC problem has now been solved: efficiency. Only between .1 percent and 10 percent of cells now crank backwards in the dish. Due to Hanna’s team, researchers should soon get plenty of designer iPSCs by suppressing a single gene, Mbd3, which is normally suppressed during embryonic development. This reopens the floodgates to dedifferentiation.
To be precise, by switching off Mbd3—or tinkering with cells engineered to lack the Mbd3 gene—the team reprogrammed blood and skin cells from mice, and skin cells from human adults, with near-perfect efficiency in just seven days. In terms of gene expression, the iPSCs they created were identical to embryonic stem cells.
“It will be of great interest to explore whether direct reprogramming in the absence of Mbd3 repression also improves the quality of reprogrammed cells and reduces the frequency of obtaining aberrantly reprogrammed iPS cells,” the Hanna group wrote.
Loh notes incremental increases in iPSC reprogramming efficiency “ have been reported before, but it remained unclear why it was such an infrequent and sporadic event. The (new) data are fairly compelling that Mbd3 suppression leads to extremely high reprogramming efficiencies from diverse mouse cell types—skin cells, blood cells, and neural progenitors alike—and even more significantly, it yields high-reprogramming efficiencies from human cells notoriously refractory to reprogramming. “
He cautions that no groups have yet tried to reproduce Hanna’s “extraordinary” results, although earlier research, led by epigeneticist Ingrid Grummt of Deutsches Krebsforschungszentrum, did find that partial Mbd3 knockdown modestly improved reprogramming efficiencies.
Loh says that while most of the “hype” surrounding the recent paper centers on the “exceptional” reprogramming coup, “even more significant” is Hanna’s explanation for the molecular mechanisms behind iPSC reprogramming, which until now “have remained obscure…a phenomenological 'black box'.”
Hanna’s paper showed, Loh notes, that reprogramming factors wear many hats. “Though they try to resurrect stem cell pluripotency genes in skin cells, paradoxically they also recruit transcriptional repressors (Mbd3) to inhibit pluripotency gene expression.”
So the four “Yamanaka factors” switch stem cell genes both on and off, which was apparently what led to earlier “extremely inefficient reprogramming. Eliminating Mbd3 enables reprogramming factors to act unilaterally to decisively reactivate stem cell genes. The elucidation of such an elegant molecular mechanism was the most decisive point of the paper.”
A potential roadblock: fully eliminating Mbd3 causes the subsequent iPSCs to be unable to differentiate forward into cells of interest. “However, this impediment only arises when both copies of the Mbd3 gene are deleted. The authors show that if only one Mbd3 copy is removed, reprogramming efficiencies are increased to >90%, yet there is sufficient residual Mbd3 to theoretically permit successful iPSC differentiation into committed cell types.”
But the Hanna group then showed that “temporarily knocking down Mbd3 during iPSC reprogramming, but restoring it upon iPSC formation (using transient siRNA knockdown) permits similar gains in efficiency without altering the differentiation characteristics of the subsequent iPSCs,” he notes.
“So transient Mbd3 inhibition to improve reprogramming will probably become the standard.”
Whether Mbd3 is related to natural tissue regeneration in certain animals such as newts, which dedifferentiate cells naturally, “is an interesting question, although we do not see much of a connection between stem cell reprogramming in vitro and physiological regeneration in vivo. People have speculated as to such a connection before, but empirical evidence is lacking.”
Mbd3 is part of the NuRD complex—a protein complex that causes gene expression to drop by changing the DNA packaging. Active in fertilized eggs, Mbd3 expression drops during the earliest stages of embryonic development. It returns at later stages. This temporary drop in expression may let the stem cells naturally expand, before they naturally differentiate.