Inhibiting Naturally Occurring Diapause in Pluripotent Stem Cell Cultures

The ultimate goal of my research is to screen inhibitors that will mimic naturally occurring diapause in pluripotent stem cells cultures (PSCs).

Diapause is a naturally occurring state that occurs throughout the mammalian kingdom. Embryonic diapause is when pre-implantation embryos suspend development at the blastocyst stage for an extended duration of time. This phenomenon is observed in over 130 species of mammal including bears, mice and marsupials. The induction of natural diapause can be due to any type of stress in the animal’s environment such as food supply, temperature, photoperiod and lactation. Previous labs have tested if diapause is a mammalian evolutionary conserved state. This was accomplished by transferring an oocyte from a mammal that does not have the ability to enter diapause into a known diapausing mammal. The oocyte paused then was transferred back into the host animal where development reinstated and carried to term without any developmental abnormalities. This opened the field to the possibility of inducing diapause in any mammal.

Current maintenance of PSCs in vitro leads to multiple passages in order to maintain a cell line, allowing only a short window of usable passages. Higher passage numbers can lead to an abnormal karyotype, thus decreasing the quality and value in respect to regenerative

medicine.

The project began by defining molecules regulating developmental potential of metabolically pausing PSCs. The next step was the assessment and titrating of small molecule inhibitors for the reversable pausing of mouse PSCs. These candidates were also tested on a multitude of other species cell lines. Once candidates were discovered the next aim was to develop and optimize conditions for reversable pausing of human PSCs. Optimization occurred through the use of high-throughput live imaging and immunofluorescence to track the initial growth , pause and reversal of the cultures. The reversable cultures were paused up to a week with no developmental inhibition. The imaging was quantified in Fiji with the help of python. We were able to observe a number of inhibitors with the ability to pause as well as restart with no notable problems. We had also begun testing optimal media conditions for the cells to be paused in as well as testing if the cells could be paused and left at RT. Due to the shutdowns following the coronavirus pandemic, I was sadly unable to see the project though.

Though the success of the project will lead to better maintenance of PSCs cultures by minimizing tissue culture manipulation. The quality of the cultures would also increase due to decreasing the amount of passaging needed. Through improving the maintenance and quality of

PSCs which are commonly regarded to as a staple of regenerative medicine we enhance the foundation in which all of the experiments are built.