LOS ANGELES—University of Southern California researchers have been busy jumping over a hurdle in regenerative medicine that has constrained the ability to use repurposed cells to treat diseases: the scientists have learned how to reprogram cells much more reliably than previous methods have allowed.
The new technique uses enzymes to untangle reprogramming DNA, and it works with near-perfect efficiency in both mice and human cells tested in the laboratories of USC’s Stem Cell Center. These findings are significant because they clear an obstacle to help scientists find treatments for a wide range of diseases, especially neurologic impairments and conditions such as hearing loss.
“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 Justin Ichida, assistant professor in the department of stem cell biology and regenerative medicine at the Keck School of Medicine of USC.
An article on this technique appears in Cell Stem Cell. Ichida, the lead author, was joined by a team of researchers at the Keck School of Medicine.
Cellular reprogramming has great potential as a disease cure; it enables scientists to study cells and molecular processes at each step of disease progression in controlled conditions that have been impossible until now. Reprogramming involves changing a cell into another type of cell, e.g., a blood cell into a muscle or nerve cell. The technique can be used to recreate tissues lost to disease and to study diseases in tissues that cannot be biopsied from living patients.
The technique has been around for decades, but hasn’t yet met its potential. According to the USC team, DNA doesn’t respond well to change when manipulated itself, due to its double helix configuration. Reprogramming DNA requires uncoiling, yet when scientists begin to unravel the molecules, they knot up even more. Nucleotides become much more difficult to work with and cells won’t replicate properly, explained Ichida. Current untangling techniques only work 1 percent of the time.
“In this study, we've identified the roadblock that prevents cells from switching their identity. It turns out to be tangles on the DNA within cells that form during the reprogramming process,” Ichida noted. “By activating enzymes that untangle the DNA, we enable near 100-percent reprogramming efficiency.”
“By examining reprogramming of fibroblasts into motor neurons and multiple other somatic lineages, we find that epigenetic barriers to conversion can be overcome by endowing cells with the ability to mitigate an inherent antagonism between transcription and DNA replication,” the authors explain in their article. “We show that transcription factor overexpression induces unusually high rates of transcription and that sustaining hypertranscription and transgene expression in hyperproliferative cells early in reprogramming is critical for successful lineage conversion. However, hypertranscription impedes DNA replication and cell proliferation, processes that facilitate reprogramming. We identify a chemical and genetic cocktail that dramatically increases the number of cells capable of simultaneous hypertranscription and hyperproliferation by activating topoisomerases.”
Researchers treated cells with a chemical and genetic cocktail, which used the topoisomerase enzymes to open the DNA molecules and release the coiled tension so the molecule lays smoothly. In turn, that leads to more efficient cellular reprogramming. This increases the number of cells capable of simultaneous transcription and proliferation, which is needed to promote tissue growth. This “DNA detangler” relaxes the tension of reprogramming transcription and makes it easier to replicate new cell colonies or tissues.
The technique has multiple advantages over existing current practice. It worked nearly 100 percent of the time, and was proven in human and animal cells. It can be employed now in laboratories to study disease development and drug treatments. This technique has immediate utility for studying schizophrenia, Parkinson’s, ALS and other neurological diseases; new cells can be created to replace lost cells, or to acquire cells that can’t be extracted from people.
“We have identified combined hypertranscription and hyperproliferation as a central driver of reprogramming that is able to overcome molecular barriers to lineage conversion across multiple species and somatic cell states,” continues the article. “Combined hypertranscription and hyperproliferation is rare because transcription and proliferation antagonize each other during reprogramming.”
“Forced expression of the reprogramming transcription factors increases genomic stress in the form of R-loops, DNA torsion, and reduced processivity of DNA replication forks. Consequently, reprogramming remains restricted to rare cells with high transcriptional and proliferative capacity that reprogram at near-deterministic rates. By introducing chemical and genetic perturbations that mitigate antagonism by activating topoisomerases, we expand capacity for high rates of coincident transcription and proliferation and extend conversion to otherwise unreprogrammable cells,” the article adds.
The technique also has the benefit of not involving stem cells. These cells are the same age as the parent cell, which is advantageous for studying age-related disease. The reprogrammed cells may be better at creating age-accurate in-vitro models of human disease, which are useful for studying diverse degenerative diseases and accelerated aging syndromes.
“The key is to understand development of disease at a cellular level and how disease affects organs,” Ichida concluded. “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.”
This advance in regenerative medicine is a complement to other recent technological gains like gene editing, tissue engineering and stem cell development. It has the potential to accelerate targeted medical treatments and precision medicine.
