Untangling the knot

USC researchers publish results in Cell Stem Cell that could prove the key to effective, reproducible cell reprogramming

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LOS ANGELES—Cellular reprogramming—changing one type of cell into another—holds a great deal of potential for regenerative medicine and the study of disease pathology, but inefficiency and unreliability have plagued current methods. At present, existing methods only work as intended around 1 percent of the time.
To reprogram a cell, its DNA must be uncoiled from the traditional double helix. But as the University of Southern California (USC) team that led a recent study explained, that’s where the problem lies: the more scientists try to untangle DNA, the more knotted up it becomes.
“Think of it as a phone cord, which is coiled to begin with, then gets more coils and knots when something is trying to harm it,” said Justin Ichida, assistant professor in the department of stem cell biology and regenerative medicine at the Keck School of Medicine of USC.
To combat this, the USC team used a chemical and genetic cocktail that activates topoisomerases. These enzymes open DNA molecules, allowing the DNA to lay smoothly and enabling more efficient reprogramming, which in turn increases how many cells can undergo simultaneous transcription and proliferation.
In contrast to current approaches, the USC technique reportedly worked nearly 100 percent of the time in both animal and human cells. The USC team worked with mouse and human fibroblasts for this work, according to Ichida. Skin fibroblasts and muscle cells were used as the starting point, and were then reprogrammed into a variety of different cell types, including excitatory neurons, spinal-motor neurons and inner ear hair cells.
“This is a strategy for greatly improving our ability to perform cellular reprogramming, which could enable the regeneration of lost tissues and the study of diseases that cannot be biopsied from living patients today,” said Ichida.
Another key difference from existing techniques is that the USC approach does not involve stem cells, because the reprogrammed cells are the same age as the parent cell.
“The key is to understand development of disease at a cellular level and how disease affects organs,” Ichida remarked. “This is something you can do with stem cells, but in this case, it skips a stem cell state. That’s important because stem cells reset epigenetics and make new, young cells, but this method allows you to get adult cells of same age to better study diseases in aged individuals, which is important as the elderly suffer more diseases.”
According to Ichida, there are three main ways used at present to reprogram cells: using transcription factors to change patterns of gene expression; using small molecules or chemicals to block certain proteins, which impacts transcription factors in the cell and changes gene expression; or fusing cells together.
“I think the method of the transcription factors is the most wildly used. I’d say the shortcomings of that are that sometimes when you add transcription factors to cells, those are encoded by DNA, so sometimes you get the DNA of those transcription factors basically integrated into the genome of the resulting cells,” he tells DDNews. “That can lead to safety issues—for example, if you’re trying to transplant those cells, you don’t know if constant expression of those transcription factors might cause problems for which cells are transplanted into people.”
“I think the chemical method gets around that, because you don’t actually have to add any DNA to the cells to reprogram them, but it’s much harder to find these chemical cocktails that can effectively reprogram cells, so that’s got more limited utility at the moment,” Ichida continues. “And cell fusion is more of a tool to just study reprogramming; it’s not something that is widely applicable to the clinic just because you end with twice the amount of DNA in the cells, and it’s not a product or anything you’d want to put into people. Even studying disease biology becomes very difficult when you have twice the amount of normal DNA.”
The study of disease biology and progression is one of the key areas Ichida expects to benefit most immediately from this new reprogramming method. With the ability to generate any type of cell, he also sees utility eventually as a potential treatment.
“What I like about this method is that you may be able to use it to regenerate lost tissues in vivo, without having to actually make them outside of the body in the laboratory and then transplant them into the body,” he comments. “For example, you can imagine doing this for neurological diseases, especially the neurodegenerative diseases where you need to regenerate lost neurons, I think this would be a good application there—so things like ALS, or Alzheimer’s disease, Parkinson’s disease or frontotemporal dementia. And then of course hearing loss is even more widespread of a problem, and if we could regenerate hair cells in the ear, that would be very powerful.”
Ichida notes that a key difference between the USC technique and existing options is that their approach is “pretty generalizable,” which should see it translate across multiple cell types.
“People have found ways to make [reprogramming] more efficient for one type of conversion between one particular cell type and one particular target cell type, but nobody had found things that had worked in a general sense, for many different types of cell transitions,” he says. “So we set out in a screening fashion to find either genetic factors or chemical factors that could make reprogramming experiments very efficient, no matter what the starting cell was and no matter what the finishing cell was. And that led us to the problem of DNA tangling; if we untangled the DNA, that was a common problem for all of these transitions, and that made all of these transitions much more efficient.”
The next goal that Ichida and colleagues intend to tackle, beyond the aforementioned usages in disease study and tissue regeneration, is to identify ways to untangle DNA in a way better suited for clinical use, Ichida tells DDNews.

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