(H-306) Slowly, Then All at Once: Cellular Dynamics and Potential Limits of Transient Reprogramming in Human Fibroblasts

Introduction:: Aging is a growing public health problem and the single greatest risk factor for most types of chronic and infectious disease. Recent studies have demonstrated that transient reprogramming, which describes temporary expression of the Yamanaka factors Oct4, Sox2, Klf4, and c-Myc (OKSM), can reverse many of the phenotypic and epigenetic hallmarks of aging and potentially improve health in vivo. However, we still do not have a complete understanding of the cellular effects of different degrees of transient reprogramming, nor how long cells can be transiently reprogrammed without a subset of them dedifferentiating fully and forming teratomas. This is crucial information to obtain as therapies based on this method begin to move towards the clinic, and will enable increased efficiency in future transient reprogramming studies as well.


Materials and Methods:: To explore this question, we used a precisely controllable secondary reprogramming system called the hiF-T cell line, which are immortalized human fibroblasts that can be induced to express OKSM through exposure to doxycycline (Cacchiarelli et al., 2015). We modified these cells to express GFP using a lentivirus, then applied doxycycline to them for a series of different durations ranging from 2 to 21 days and tracked the growth of each condition for the full three weeks using live cell imaging on a Sartorius Incucyte. At the end of this period, we fixed the cells and used alkaline phosphatase staining to quantify the number of colonies that had formed in each condition. After collecting our phenotypic data, we used the edgeR and limma packages in R to explore potential mechanistic bases for our findings in a pre-existing RNA-seq data set (Cacchiarelli et al., 2015).


Results, Conclusions, and Discussions:: We found that transient expression of OKSM led to a marked increase in division rate over the first 4 days after its induction. Following this period, the cells began to die at an approximately linear rate for a duration proportional to the duration of OKSM expression. After 10 days of OKSM expression, the cells appeared to pass a tipping point, after which they largely continued to die even following the removal of doxycycline (Fig. 1). Notably, the cells also generally returned to an approximately normal phenotype after doxycycline removal in every condition prior to Day 10, beyond which they never completely recovered. Day 10 is also a significant cutoff for final induced pluripotent stem cell (iPSC) colony formation 21 days following OKSM induction, as this condition showed a sharp increase in colony formation over 7 days of OKSM, while conditions with more than 10 days of OKSM increased their colony formation rate more gradually (Fig. 2). The Day 7 condition did, however, form a small number of colonies, indicating the presence of a subset of primed cells that could still reprogram fully even with very limited doxycycline exposure. Looking at previously described gene expression trajectories in this cell line, we saw that the day 10 turning point we describe appears to align with an upregulation of Nanog, which has been identified as a potential point of no return for reprogramming cells (Samavarchi-Tehrani et al., 2010).

Overall, in this study we have begun to outline some potential limits on transient reprogramming in human fibroblast cells. Our results indicate that 10 days could be a cutoff point for teratoma-free reprogramming of human cells, and that even 7 days may not be entirely safe from teratoma formation. The rapid period of growth we observed following OKSM induction highlights the potential applications of transient reprogramming in regenerative medicine, but the ensuing period of cell death also suggests that even shorter periods of reprogramming may be optimal in contexts where avoiding significant cell death is important. These results suggest more concrete guidelines for subsequent reprogramming experiments, and potentially for reprogramming-based therapies in the future.


Acknowledgements (Optional): : I would like to acknowledge the Raj Lab and Arjun Raj for allowing me to explore my interests entering my PhD, and Naveen Jain for being my primary mentor during this project so far.

References (Optional): : 1. Cacchiarelli, D., Trapnell, C., Ziller, M. J., Soumillon, M., Cesana, M., Karnik, R., Donaghey, J., Smith, Z. D., Ratanasirintrawoot, S., Zhang, X., Ho Sui, S. J., Wu, Z., Akopian, V., Gifford, C. A., Doench, J., Rinn, J. L., Daley, G. Q., Meissner, A., Lander, E. S., & Mikkelsen, T. S. (2015). Integrative Analyses of Human Reprogramming Reveal Dynamic Nature of Induced Pluripotency. Cell, 162(2), 412–424.
2. Samavarchi-Tehrani, P., Golipour, A., David, L., Sung, H.-K., Beyer, T. A., Datti, A., Woltjen, K., Nagy, A., & Wrana, J. L. (2010). Functional genomics reveals a BMP-driven mesenchymal-to-epithelial transition in the initiation of somatic cell reprogramming. Cell Stem Cell, 7(1), 64–77.