Designing switches to control cell fate
Researchers at the University of Wisconsin-Madison have developed a strategy to reprogram cells from one type to another more efficiently than other methods.
The ability to convert cells from one type to another holds great promise for engineering cells and tissues for use in therapy. The new University of Wisconsin at Madison study could help speed research and bring the new technology into the clinic faster.
The approach was published Dec. 5, 2016, in the Proceedings of the National Academy of Sciences (PNAS). Scientists searched a library of artificial molecules that bind to DNA — transcription factors — in order to find one that switches on genes and converts cells from one type to another.
Natural transcription factors are molecules in each cell that bind to DNA, turning genes on or off. Turning on specific sets of genes must occur before a cell will commit to become a particular cell type. Artificial transcription factors — made in a lab — can tell researchers which molecules best mimic natural cell fate changes.
According to Asuka Eguchi, member of Aseem Ansari's laboratory in the UW-Madison Department of Biochemistry, and lead author: "Our interest in changing cell fate comes from understanding how cells use information in our genomes to make specific cell types. If a patient needs a certain cell type ...we can reprogram their own cells to what they need, rather than rely on donor cells. This allows us to potentially avoids issues with immune response."
Conventional methods for changing cell fate requires a laborious, slow and failure-prone trial-and-error approach. Scientists need to know which combination — out of thousands — might possibly influence a cell's tightly choreographed timeframe in order to reprogram its fate.
The new method uses "libraries" of millions of artificial transcription factors — designed to bypass natural controls — to turn on genes which might be activated for a given cell type.
By exposing their new library of artificial factors to various cells, they could observe if any cell's fate changed. They then revisited changed cells to see which transcription factors were responsible. Wisconsin researchers chose mice fibroblasts, the most common connective tissue cells, to attempt reprogramming them into pluripotent stem cells.
Given the proper cues, pluripotent stem cells can become any type of cell in an animal's body, including humans. Being able to reprogram a cell to become pluripotent, could mean that artificial transcription factors might trigger the right genes to cause a cell to shift from one type to another.
In the process of testing their new tool, researchers discovered three combinations of artificial factors which reprogram a fibroblast into a stem cell. These factors play a role similar to a natural transcription factor — called Oct4.
"In this unbiased approach, we try to basically cast a wide net on the whole genome and let the cell tell us if there are important genes perturbed," explains Ansari, also affiliated with UW-Madison's Genome Center of Wisconsin. "It's a way to induce cell fate conversions without having to know what genes might be important because we are able to test so many by using an unbiased library of molecules that can search nearly every corner of the genome."
The reprogramming of fibroblasts into stem cells is well studied. It is known this approach requires significant changes to a cell. Now, with their proof of concept, Wisconsin scientists hope more researchers use their method to discover new genes which drive even more difficult cell fate conversions.
"Generating pluripotent stem cells also helps us avoid having to make embryonic stem cells — which can be controversial. We can also start better investigating direct conversions, which are conversions from one cell type to another without the need to go to the pluripotent stage first — because that can cause problems in some contexts. This tool opens up the doors to research these areas more effectively."
Asuka Eguchi PhD, Aseem Ansari laboratory, Department of Cellular and Molecular Biology Training Program, Univeristy of Washington, Madison, Wisconsin, USA, and lead author.
The ability to convert cells into desired cell types enables tissue engineering, disease modeling, and regenerative medicine; however, methods to generate desired cell types remain difficult, uncertain, and laborious. We developed a strategy to screen gene regulatory elements on a genome scale to discover paths that trigger cell fate changes. The proteins used in this study cooperatively bind DNA and activate genes in a synergistic manner. Subsequent identification of transcriptional networks does not depend on prior knowledge of specific regulators important in the biological system being tested. This powerful forward genetic approach enables direct cell state conversions as well as other challenging manipulations of cell fate.
Artificial transcription factors (ATFs) are precision-tailored molecules designed to bind DNA and regulate transcription in a preprogrammed manner. Libraries of ATFs enable the high-throughput screening of gene networks that trigger cell fate decisions or phenotypic changes. We developed a genome-scale library of ATFs that display an engineered interaction domain (ID) to enable cooperative assembly and synergistic gene expression at targeted sites. We used this ATF library to screen for key regulators of the pluripotency network and discovered three combinations of ATFs capable of inducing pluripotency without exogenous expression of Oct4 (POU domain, class 5, TF 1). Cognate site identification, global transcriptional profiling, and identification of ATF binding sites reveal that the ATFs do not directly target Oct4; instead, they target distinct nodes that converge to stimulate the endogenous pluripotency network. This forward genetic approach enables cell type conversions without a priori knowledge of potential key regulators and reveals unanticipated gene network dynamics that drive cell fate choices.
Keywords: artificial transcription factor genome-scale library cell fate reprogramming gene regulatory networks
The W.M. Keck Foundation provided funding for the study, as did the National Institutes of Health's Progenitor Cell Biology Consortium. The NIH consortium brought together leading researchers from different scientific disciplines across campus as well as from the University of Minnesota and across the country.
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