Synthetic Circuits Reveal the Key to Rewinding the Cellular Clock


Most people wonder how their lives would change if they could turn back time and remake past decisions. While a seemingly impossible feat, stem cell biologists Kazutoshi Takahashi and Shinya Yamanaka at Kyoto University first turned back the cellular clock in 2006.1 By overexpressing four transcription factors in fully differentiated fibroblasts, Takahashi and Yamanaka reprogrammed the cells to a pluripotent state and called them induced pluripotent stem cells (iPSC).

Although researchers employ iPSC in the laboratory and the clinic, scientists struggle to efficiently produce large quantities of iPSC.2 “Reprogramming is still very inefficient,” said Thorsten Schlaeger, a stem cell biologist at the Boston Children’s Hospital. “It is still not fully understood why 98 or 99.9 percent of the cells do not end up reprogramming into iPSC.” In a recently published Science Advances paper, Schlaeger and his team developed a system for tracking the fate of cells with different transcription factor expression dynamics during reprogramming.3 These findings will enable researchers in the field to improve iPSC yield.

Scientists suspected that the heterogeneity in reprogramming outcomes results from variations in the levels and durations of transcription factors’ expression. Consequently, several research groups have attempted to correlate the success of reprogramming to the levels of the octamer-binding transcription factor 4 (OCT4), which is one of Takahashi and Yamanaka’s transcription factors that is essential for the reprogramming process. However, these studies used population-based measurements and failed to consider the contribution of endogenous OCT4 to reprogramming.

Compelled to overcome these limitations, Schlaeger’s coauthor Domitilla Del Vecchio, a discipline-straddling mechanical engineer at the Massachusetts Institute of Technology, developed an innovative OCT4 expression system. “The idea really was to try to use a more sophisticated way of overexpressing transcription factors to reprogram stem cells,” Del Vecchio recalled.

Del Vecchio’s team designed a synthetic gene circuit to ectopically overexpress a fluorescently-tagged version of the OCT4 transcription factor, while simultaneously blocking the expression of the endogenous OCT4 through microRNA. This allowed the researchers to control the total OCT4 protein levels within the cell and quantify them by measuring the fluorescence. Additionally, the ectopic OCT4 gene was controlled by an inducible and noisy promoter, which meant that the system generated variability in the expression of the OCT4 conjugate and a broad range of trajectories to assess, such as cells that maintained high OCT4 expression throughout reprogramming or those whose expression decreased over time.

     Photos of the authors of the study: Thorsten Schlaeger (left) and Domitilla Del Vecchio (right).

Thorsten Schlaeger and Domitilla Del Vecchio developed a synthetic gene circuit-based system that allowed them to control and monitor the total OCT4 protein levels within   fibroblasts during reprogramming.

Thorsten Schlaeger and Domitilla Del Vecchio

To determine which OCT4 trajectories successfully reprogrammed human dermal fibroblasts into iPSC, Del Vecchio, Schlaeger, and their team transduced the cells with lentiviral vectors encoding their OCT4 trajectory generator and followed the levels of fluorescently-tagged OCT4 proteins within the cells over time through imaging. The researchers then fixed the resulting cell colonies and immunostained them for two pluripotent stem cell surface markers.

They observed that the colonies fell into three categories: type I colonies were positive for only one of the surface markers; type II colonies showed the exact opposite staining pattern from type I; and type III colonies were positive for both markers. They considered cells within type III colonies as iPSC and categorized the cells within type I and II colonies as incompletely reprogrammed. Despite these differences, cells in all three colony types stably expressed supraphysiological levels of OCT4 during reprogramming, indicating that successfully reprogramming human dermal fibroblasts into iPSC requires consistently high levels of the OCT4 transcription factor. But this parameter alone is not sufficient to promote iPSC generation.

“The paper is innovative in a technical sense. It is consistent with work that has been done showing that elevated levels of OCT4 are important for the reprogramming process,” said Dean Tantin, a geneticist at the University of Utah, who was not involved in the study. 

Although Tantin thought Del Vecchio’s system presented a clever way to directly examine total OCT4 protein levels within live cells, he suggested that examining protein levels alone may not convey the whole story. “The level of a protein based on a fluorescent marker is not the same as the activity of a protein—its ability to bind DNA [or] its ability to regulate transcription once it binds,” he noted. “So, I think where the field needs to go now is [to find out] how OCT4 activity is really dynamically regulated during [reprogramming].” he noted. Building on this idea, Tantin and his team recently determined that OCT4 activity during reprogramming and differentiation is redox-regulated, and he suspects that regulation of other reprogramming components will be of interest in the years to come.4 

Del Vecchio hopes that her work will inspire other researchers to think beyond the standard methods they employ to dissect molecular pathways. “This study is showing how more sophisticated genetic engineering tools can be used in the context of a highly complex biological process and help you get information that will be difficult to get otherwise,” Del Vecchio said. 

Schlaeger wants to leverage the knowledge gained in this study to develop off-the-shelf iPSC-based therapies, such as CAR T cells, and he believes that precision engineering will be the key to safely bringing these products to the clinic. “With the cells, we want to get to this precise control and that can only be done with complex genetic switches and circuits,” Schlaeger said. 

References

  1. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126(4):663-676.
  2. Shi Y, et al. Induced pluripotent stem cell technology: A decade of progress. Nat Rev Drug Discov. 2017;16(2):115-130.
  3. Ilia K, et al. Synthetic genetic circuits to uncover the OCT4 trajectories of successful reprogramming of human fibroblasts. Sci Adv. 2023;9(48):eadg8495.
  4. Shen Z, et al. Oct4 redox sensitivity potentiates reprogramming and differentiation. Preprint. bioRxiv. 2023:2023.02.21.529404.

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