Traditional organ transplants are difficult operations. It can take years to find a match due to extended wait times and examining the immunological similarities of patients and potential donors. Every day, seventeen Americans die while waiting for an organ transplant, according to the Health Resources and Services Administration.

Scientists are investigating alternatives to transplantation, such as the use of human pluripotent stem cells (hPSCs) as therapies. hPSCs are precursor cells that can differentiate into any cell type in the body if given the right environment or culture conditions. In vitro, stem cells from patients can develop tissues and organs that are less likely to be rejected than donor material.  As a result, more output is required to meet demand. Current approaches for increasing hPSC production rely on complex culture conditions that imitate the cellular environment.2 As a result, researchers are looking into different methods to lessen the amount of human input required to manufacture copious amounts of stem cells.

“In the ideal world, we would generate functional cells from stem cells by using synthetic gene circuits to increase [stem cell] yields or cell types to eventually be used in cell and gene therapies,” said Peter Zandstra, a research scientist at the University of British Columbia. Zandstra and his colleagues, including first author Laura Prochazka, intentionally developed a synthetic gene circuit to detect and control the differentiated state of a cell. This research was recently published in the journal Molecular Systems Biology.

A synthetic gene circuit is a gene network that has been created to coordinate a desired output via transcriptional or post-transcriptional regulation. Zandstra’s team used a post-transcriptional regulatory approach via tailored microRNA (miRNA)-based logic circuits to govern hPSC cell-state transitions. The circuits fine-tune gene expression by taking use of miRNAs’ ability to bind to and enhance the degradation of RNA containing miRNA response elements (MREs). The circuits in this work were developed to identify the state of a cell (hPSC vs non-hPSC) or to adjust the expression of user-specified genes to control the differentiation state.

“Controlling the cell has been an important goal,” said Gábor Balázsi, a synthetic biologist at Stony Brook University in New York, who was not involved in this study. “It’s an interesting way to couple these two efforts [detecting cell state and differentiation], basically removing human input.”

The researchers tested their circuits in hPSCs to see if they could influence cellular differentiation as a proof of concept. They used BMP4 expression as the output, which is essential for hPSC differentiation into various cell types. The researchers noticed a dose-dependent response in differentiation when they controlled BMP4 expression using distinct MRE variations known as miRNA silencing-mediated-fine-tuners (misFITs). For example, variant cells expressing low levels of BMP4 remained undifferentiated pluripotent. Cells expressing moderate to high amounts of BMP4, on the other hand, formed gastrulation patterns comparable to cells exposed to exogenous BMP4. This demonstrated that the circuits may coordinate cell-state transitions dose-dependently.

Zandstra is optimistic about the future after a successful proof of concept. “I think we’re really at the very beginning of starting to introduce more complex circuits into pluripotent stem cells, and this paper shows is that it’s possible,” he said. The researchers want to use these complicated circuits in the future to kill undesirable differentiated cells or track cell states to monitor hPSC generation for applications in regenerative and transplantation medicine.