While many stem cell scientists are basking in the afterglow of induced pluripotency, and working on ways to de-differentiate and re-differentiate cells, a few researchers are already thinking about a shortcut.
Transdifferentiation allows cells to go straight from point A to point B without “passing Go”, that is, without re-entering an embryonic-like, pluripotent state. Discredited a decade ago, transdifferentiation is back, buoyed by the success of induced pluripotent stem (iPS) cells (reviewed in Zhou and Melton, 2008: PMID18940730).
“I think the field is poised for some major discoveries that could be very important for biology and therapeutics,” said Qiao (Joe) Zhou, who devotes his Harvard University lab to the topic.
Research on nuclear transfer reprogramming and iPS cells showed that cell fate is not forever, but whether mature cells could transdifferentiate was “a lingering question in the field,” said Marius Wernig of the Stanford University School of Medicine in Palo Alto, California. A few research papers indicated it was possible to make a small jump, from one lymphocyte type to another, for example (Xie et al., 2004: PMID15163413), or from fibroblast to muscle (Choi et al., 1990: PMID2172969).
Wernig and his colleagues set out to make a big jump. They selected easy-access fibroblasts as their starting point, and neurons as a goal. These cell types do not even come from the same germ layer; fibroblasts are mesodermal and neurons are ectodermal.
The Stanford scientists tried a pool of 19 candidate genes, expressed by lentiviral vectors, to induce a neuronal fate in mouse embryonic and three-day-old tail-tip fibroblasts. Ultimately, they whittled the pool down to three transcription factors that can turn nearly 20 percent of fibroblasts into neurons, complete with the ability to form synapses and transmit action potentials (Vierbuchen et al., 2010: PMID20107439). They christened their creation iN, for induced neuronal, cells. The efficiency of the process was “very surprising to us,” Wernig said, considering how few cells make it to the finish line in iPS protocols. Perhaps, he speculated, iPS methods are not yet optimal, and he was just lucky to land on the right iN program.
The iN cells are not exactly what one would expect from the transcription factors used, notes Arnold Kriegstein of the University of California in San Francisco, who was not involved in the project (Nicholas and Kriegstein, 2010: PMID20182502). He would have predicted an inhibitory neural type, but Wernig and colleagues got excitatory neurons instead. “The real issue is, what is the fidelity of these cells?” Kriegstein said. “Are these true bona fide neurons?”
Another key study in the transdifferentiation revival was conducted by Zhou and colleagues at Harvard (Zhou et al., 2008: PMID18754011). They sought to make a smaller jump, from pancreatic exocrine cells to the islet -cells that secrete insulin, but took the additional step of performing the transition in vivo. That way, the transitioning cells were exposed to the appropriate environmental niche. In addition, the starting point was normal adult tissue, not cells already altered by living in a culture dish, said Robert Blelloch of the University of California in San Francisco, who was not involved in the study (Blelloch, 2008: PMID18833266).
Zhou and colleagues injected the pancreases of mice with an adenovirus pool containing genes for nine different transcription factor candidates. One month later, they looked for extra insulin-producing cells. They narrowed their pool to a three-part cocktail, which converted more than 20 percent of infected cells into insulin producers. The new -cells looked like a -cell, expressed genes like a -cell, and secreted insulin like a -cell. However, they did not join natural islets in the pancreas, or form new islets.
Transdifferentiation bests iPS cells in some arenas, but falls short in others. “The main advantage is that you actually skip a lot of intermediate steps,” Zhou said. And because the cells do not go through a rapidly dividing stage, they should be less likely to form tumors. However, that also means transdifferentiation cannot make more cells than it starts with. And it is harder to be sure what a transdifferentiated cell population really contains. Because they do not proliferate, clonal analysis is impossible.
Transdifferentiated cells may retain some of their original programming, or represent an in-between state. That will matter for some applications, but perhaps not for others. “If you are dying from a heart attack, even a half-functioning cell is going to be better than nothing,” Zhou said.
But getting the new cell type just right could be very important for researchers deriving cell culture models of disease from the fibroblasts of patients (for example, Park et al., 2008: PMID18691744). In these experiments, an intermediate or mixed-up cell type could skew results. But if transdifferentiation can make good mimics, it may be especially useful for cell culture models, Wernig said, because it is easier and faster than iPS procedures.
A decade ago, researchers thought they had evidence for natural transdifferentiation. For example, scientists suggested that stem cells from brain or muscle turn into blood cell types. However, later experiments showed these results were likely due to blood contamination of other tissues, or fusion of two disparate cell types (reviewed in Wagers and Weissman, 2004: PMID15006347). “The wind was lost and the sails deflated, and this whole idea of transdifferentiation developed sort of a bad reputation,” Kriegstein said. Reports of iPS cell reprogramming (Takahashi and Yamanaka, 2006: PMID16904174) provided a fresh breeze, and inspired scientists to try again, carefully. “Obviously, we have to be very cautious in making claims,” Zhou said.
It is not clear how transdifferentiation occurs, and many researchers wonder if cells actually go through a bit of de-differentiation and re-differentiation along the way. “It probably goes through molecular programs that do not exist naturally,” Blelloch suggested.
How will transdifferentiation measure up to the iPS whirlwind? It is too early to say. Harvard scientist Douglas Melton, co-author on the -cell paper, noted that the new techniques add to, but do not detract from, embryonic stem and iPS cell approaches. “We need to attack problems from multiple angles,” he said in a 2008 press release.
“Certainly, I think [the iN paper] has shaken things up, and generated a great deal of laboratory activity,” Kriegstein said. “If there are not new reports of transdifferentiation of a variety of other cell types in the near future, I would assume it is going to be very difficult to do.”
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