My Ph.D. Thesis

Some time passed since it was written, corrected, submitted and approved. I guess enough time... I want it to be here, public, not for you to read it, I know nobody will, but for me to notice the passed time.

It's about stem cells differentiation and reprogramming. About timing, phases of early development, gene expression and DNA methylation. About modeling, partial differential equations and numerical calculation.

And of course about philosophy: "What we cannot speak about we must pass over in silence" (Ludwig Wittgenstein)

my Ph.D. thesis

Abstract

During early development, after fertilization, the totipotent zygote’s progeny forms the trophoblast cells and the inner cell mass cells that mainly give rise to the embryo. During the gastrulation, pluripotent embryonic stem cells which take their origin from inner cell mass cells differentiate to the three germ layers: endoderm, mesoderm and ectoderm, afterwards multipotent progenitor cells continue differentiation into the somatic adult cells. In every differentiation step, the cell nucleus becomes more restricted by chosen cell identity that is regulated by transcription factors and the underlying epigenetic profile. 

Although the somatic cell nucleus loses its developmental potential, adult cells can be reprogrammed to pluripotency via ectopic delivery of the Oct4, Sox2, Klf4 and c-Myc transcription factors. Despite ongoing progress, the process that coordinates exiting from the pluripotent state as well as reprogramming from somatic to pluripotent state remains unclear. 

To better understand the boundaries of pluripotency, we systematically destabilized the pluripotent state through exiting and re-entry to pluripotency. Using this approach we defined three early differentiated phases; exiting pluripotency, the cell progresses through a brief transient phase toward differentiation commitment. During the transient period, cells are still responsive to high efficiency reversion to the pluripotent state by induction of exogenous reprogramming factors. However, once reaching the commitment phase, the reprogramming efficiency of the cells rapidly declines to somatic-like reprogramming kinetics.

Studying the transcriptional and epigenetic dynamics of this process, we found several key molecular parameters that define the pluripotency boundary. We find a distinct subset of Oct4 pluripotent-state enhancers that are protected from immediate silencing during exit from the pluripotent state. During the transient phase, those enhancers stay accessible to reprogramming factors that in their turn activate the pluripotency program of the cell.

We propose an effective model for transcription regulation that describes cells early differentiation. Using this model, we investigate dynamical properties of the system and suggest a few predictions for future study. Moreover, we present a new concept of effective gene network that can be useful for studying complex biological systems.