The New Stem Cells

Labs globally are electrified by the notion that just “stressing out” ordinary adult cells may transform them into extraordinary cells like the “induced pluripotent stem cells” (iPSCs) of Nobel Prize winner Shinya Yamanaka.

But if it turns out to be true, will it be about co-opting an evolutionarily conserved, natural way of making “young” cells out of old? Or will it be a via dolorosa—a natural (or unnatural) way of hastening old cells’ death?

No one knows for sure yet. But few are blase. For the work--described in two Nature papers by crews at the US’s Harvard University and Japan’s Riken Institute—has led to a jaw-dropping realization: anyone (in the bioscience world) may be able to make pluripotent stem cells.

“Two remarkable papers,” Harvard cell reprogrammer Mathias Stadtfelt tells Bioscience Technology. “When I read them I felt equal amounts of excitement and skepticism. The data seem very solid and the authors did all the key tests for pluripotency. As the basic protocol is straightforward it should only take weeks to confirm. Many are trying. Us included. If this is validated and extended to adult mammalian cells—especially human—it would enormously facilitate the production of pluripotent cell lines and raise important questions. Why does a rather unspecific stress stimulus lead to the highly efficient induction of pluripotency? Why has this mechanism evolved? What is the body using it for, if anything?”

Weizmann Institute iPSC geneticist Joseph Hanna emails that, if the papers are correct, the new STAP (stimulus-triggered acquisition of pluripotency) cells “are not molecularly indistinguishable from embryonic stem cells (ESCs) or IPSCs,“ but “they make adult chimeras and tetraploid embryos so are conclusively pluripotent. A very interesting phenomenon.” He, too, is trying to duplicate results.

The Nature papers find that many external stressors can dedifferentiate mature lymphocytes to a pluripotent state. But a low pH bath of 5.7, less acidic than coffee, is most efficient—and far faster and easier than standard genetic/chemical techniques. The new way takes days; the old, weeks.

Senior author Charles Vacanti, a Harvard professor, and Brigham and Women’s Hospital Chair of Anesthesiology, tells Bioscience that making STAPs can become "easy." Different cells require different external stimuli. (See upcoming Bioscience interview with Vacanti.)

There are cautionary notes. Among other things, the published work was done on adult mouse cells that were neonatal, so immature.  (Although Vacanti, who tells Bioscience he has created STAPs from adult human neonatal fibroblasts, says he is testing STAPs made from "wholly adult" monkey cells. He has not tried wholly adult human cells. Wholly adult mouse cells were difficult.) But if replicated, the published work sets “a new principle,” wrote University of Edenburg stem cell expert Austin Smith in Nature. “A physical stimulus can be sufficient to dismember gene-control cir­cuitry and create a ‘plastic’ state from which a previously unattainable level of potency can rapidly develop.”

Says Jonathan Slack, a University of Minnesota regeneration pioneer: “This paper is creating considerable interest. If it holds up, it is probably of considerable practical value.”

But if it does hold up, a key next question is: is it physiologic, or a culture dish accident?

Dedifferentiation of mature cells to a stem-like state, in response to mechanical stress like amputation, occurs in regeneration of zebra fish, jellyfish, plants, and amphibians (newts not axolotls). And: “It is known that dissociation of amphibian embryo cells can re-set their fate,” offers Cambridge Nobel-Prize winning developmental biologist John Gurdon, via email.

Less common: reports of dedifferentiation via the stress of low pH. But examples do exist. The Nature work cited a classic 1947 Johannes Holtfreter paper where embryonic frog cells, due to form skin, switch to neurons in low pH. But it is not clear if dissociation or low pH is responsible, say Gurdon, and Indiana University Center for Regenerative Biology and Medicine Director David Stocum. (Classic 1941/1944 papers of Lester Barth/Holtfreter show dissociation to be the agent, Stocum says.)

But there may be a good classic example: “In his 1952 book, Regeneration and Wound Healing, A.E. Needham describes experiments that measure pH of the blastema fluid of regenerating salamander limbs. The pH was acidic,” says Stocum. Experiments on limb tissues in low pH could prove that link.

There is recent evidence for a low pH role. A 2013 Cell paper finds “chief” cells in mouse intestines exposed to low pH regularly dedifferentiate into stem cells. Adds that senior author, Hans Clevers of the Hubrecht Institute, via email: “You may think also of Barrett's esophagus,” where human esophagus epithelium dedifferentiates “to a gut (simple) mucous epithelium, presumably by acid reflux from the stomach, leading to cancer.”

Human Schwann cells dedifferentiate after spinal cord injury. (Notes University of London regenerative biologist Jeremy Brockes: “Until recently there were few examples of mammalian dedifferentiation generally accepted. The clearest was Schwann cells.”) And in a surprising article appearing in a 2012 issue of Cell, mouse lineage tracing showed the initial loss of beta cell mass—to the stress of diabetes—is due to dedifferentiation not death.

The Nature paper states that low pH—of seven stressors—yeilded most STAP cells (about eight percent), but may be upstream of key stressors: “A remaining question is whether cellular reprogramming is initiated specifically by the low-pH treatment or also by some other types of sublethal stress such as physical damage, plasma membrane perforation, osmotic pressure shock, growth-factor deprivation, heat shock or high Ca2+ exposure.” 

Stocum thinks it may be about the membrane. In the Nature work, “cells were dissociated, but did not dedifferentiate without another stress stimulus like low pH. So disassociation alone did not do it.” Disrupting membrane, via low pH or other stressors, alters ion permeability, then gene expression, resulting perhaps in dedifferentiation. “The membrane may be the target.”

Michael Levin of Tufts University agrees. “Physiological changes in pH and resting potential are hugely important in regulating cell states.” Levin is a cell behavior expert who feels the “really cool” Nature work is no “culture dish accident; I think such physiological cues are a crucial part of normal cell regulation. I think they are definitely co-opting a natural (endogenous) process.” Barth showed ions in medium can “control the differentiation path of cells from embryos. My lab has shown resting potential changes can reprogram gut cells into vertebrate eyes, regenerate tails and limbs…. My guess is they have found another way to perturb ionic signaling at the membrane, crucial for differentiation” normally, and in cancer.

The reason we may retain this ancient ability? Vacanti says he thinks his team's stressors mimic those of extreme stress/injury, when mature cells may “revert to stemness,” proliferate, and form “repair” cells. Intriguingly, in 2011, Weizmann Institute stem cell researcher Dov Zipori predicted this. (See Bioscience’s next stem cell blog.)

Slack thinks the mechanism is a “general opening of chromatin which creates `mixed up’ cells with many genes active, and many transcription factors accessing sites normally in closed chromatin. A few such cells, by chance, will approximate to a pluripotent state and be selected to grow to colonies by the medium.” In the paper, many cells are only partly reprogrammed. “I would expect a wide range of abnormal phenotypes.”

Says Hanna: “This begs the question whether chromatin "opens" due to cellular stresses, not transcription factor-induced chromatin remodeling. Interplay is going on for sure. Figuring it out is the future.”

All this may explain past “sightings” of pluripotent cells in adults. Says Slack: “Developmental biology theory would not lead one to expect their existence. Careful studies of (pluripotency marker) Oct4 show this does not occur outside the germ line in postnatal animals. But if it is possible to generate pluripotent cells by unspecific stresses, selection for their expansion in culture will do the rest.”

Agrees Stocum: “The first thing I noticed is how unlike ESCs or iPSCs these cells are.” They resemble the “spore cells" in adult tissue described by Vacanti in 2001, and the pluripotent cells in adult tissue described by Mercer University’s Asa Black in 2004, among others. “There is lots of evidence for this cell morphology in literature.” To prove such cells are made with low pH, or other stressors, “is potentially significant, the simplest way yet described to convert somatic to pluripotent cells.”

Indeed, Vacanti tells Bioscience he believes his induced cells are the same cells he described in papers in 2001 and 2011. He believes they are similar to pluripotent cells some other groups have described in adult tissue, including "MUSE" cells. 

Gideon Grafi, a Ben Gurion University plant science expert, is wary. Stress has long been known to induce stem cell properties in adult plant cells, he says. And “differentiated red blood cells can, with appropriate stimuli, dedifferentiate,” as can chicken erythrocyte nuclei in extracts from Xenopus eggs or frog blood cells via electromagnetic field.

He warns: “The major issue is conversion into healthy cells. These cells are pushed down a ‘via dolorosa’ path. They are removed from their natural location, placed in a culturing dish, and transplanted back, subjected to stress cycles that provide an environment for activation of hazardous nuclear reactions (DNA transposition and recombination).” This can lead, in geneticist Barbara McClintock’s words, to genomic restructuring—“not rejuvenation, but aging, of cells.”

One argument against that: karyotyping of the neonatal cells is so far normal.

But all agree on one thing: the field is so excited, the wait for proof will be short.