From connective tissue to neuronal cells with fewer steps

Duke researchers use CRISPR to directly activate genes to generate neuronal cells from mouse connective tissue

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About a decade ago, Japanese researcher Shinya Yamanaka, then a professor at the Institute for Frontier Medical Sciences at Kyoto University, made a breakthrough in working out how to take fibroblasts and revert them back to stem cells with the ability to differentiate into other cells types—what we refer to today as induced pluripotent stem cells. Yamanaka won the Nobel Prize in medicine several years ago thanks to that work.
A multitude of researchers since that discovery in 2006 have worked on various ways to convert cells between different types, typically through the insertion of extra genes, but Duke University reported this month that researchers on its campus have developed a strategy that avoids the need for the extra gene copies. Instead, they use a modification of the CRISPR genetic engineering technique “to directly turn on the natural copies already present in the genome,” as Ken Kingery wrote Aug. 11 in an article for the university’s website. In so doing, the researchers have converted cells isolated from mouse connective tissue directly into neuronal cells. Uses for such cells could include efforts to model neurological disorders and work to discover new therapeutics and therapeutic targets—and, eventually, aid in the development of personalized medicines and implementation of cell therapy.
According to Kingery’s article, early results indicate that the newly converted neuronal cells show a more complete and persistent conversion than methods in which new genes are permanently added to the genome. A paper discussing the research was published Aug. 11 in the journal Cell Stem Cell under the title “Targeted Epigenetic Remodeling of Endogenous Loci by CRISPR/Cas9-Based Transcriptional Activators Directly Converts Fibroblasts to Neuronal Cells.”
As the authors note in the summary of their study, “Overexpression of exogenous fate-specifying transcription factors can directly reprogram differentiated somatic cells to target cell types. Here, we show that similar reprogramming can also be achieved through the direct activation of endogenous genes using engineered CRISPR/Cas9-based transcriptional activators. We use this approach to induce activation of the endogenous Brn2, Ascl1 and Myt1l genes (BAM factors) to convert mouse embryonic fibroblasts to induced neuronal cells. This direct activation of endogenous genes rapidly remodeled the epigenetic state of the target loci and induced sustained endogenous gene expression during reprogramming. Thus, transcriptional activation and epigenetic remodeling of endogenous master transcription factors are sufficient for conversion between cell types. The rapid and sustained activation of endogenous genes in their native chromatin context by this approach may facilitate reprogramming with transient methods that avoid genomic integration and provides a new strategy for overcoming epigenetic barriers to cell fate specification.”
“This technique has many applications for science and medicine. For example, we might have a general idea of how most people’s neurons will respond to a drug, but we don’t know how your particular neurons with your particular genetics will respond,” said Charles Gersbach, the Rooney Family Associate Professor of Biomedical Engineering and director for the Center for Biomolecular and Tissue Engineering at Duke in Kingery’s article for the university. “Taking biopsies of your brain to test your neurons is not an option. But if we could take a skin cell from your arm, turn it into a neuron and then treat it with various drug combinations, we could determine an optimal personalized therapy.”
“The challenge is efficiently generating neurons that are stable and have a genetic programming that looks like your real neurons,” added Joshua Black, the graduate student in Gersbach’s lab who led the work. “That has been a major obstacle in this area.”
In the Duke research, Black, Gersbach and their colleagues used CRISPR to precisely activate the three genes that naturally produce the master transcription factors that control the neuronal gene network, rather than having a virus introduce extra copies of those genes. The CRISPR system was administered to mouse fibroblasts in the laboratory. The tests showed that, once activated by CRISPR, the three neuronal master transcription factor genes robustly activated neuronal genes. This caused the fibroblasts to conduct electrical signals—a hallmark of neuronal cells. And even after the CRISPR activators went away, the cells retained their neuronal properties.
“When blasting cells with master transcription factors made by viruses, it’s possible to make cells that behave like neurons,” said Gersbach. “But if they truly have become autonomously functioning neurons, then they shouldn’t require the continuous presence of that external stimulus ... The method that introduces extra genetic copies with the virus produces a lot of the transcription factors, but very little is being made from the native copies of these genes. In contrast, the CRISPR approach isn’t making as many transcription factors overall, but they’re all being produced from the normal chromosomal position, which is a powerful difference since they are stably activated. We’re flipping the epigenetic switch to convert cell types rather than driving them to do so synthetically.”
Among the next steps are to perfect the strategy for use in human cells and increase efficiency of the technique so that modeling of specific diseases could be carried out using the newly produced cells. And, from there, perhaps therapeutics as well.
“In the future, you can imagine making neurons and implanting them in the brain to treat Parkinson’s disease or other neurodegenerative conditions,” said Gersbach. “But even if we don’t get that far, you can do a lot with these in the lab to help develop better therapies.”
SOURCE: Duke University article “Directly Reprogramming a Cell's Identity with Gene Editing”

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