טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentYakov Altshuler Anna
SubjectIluninating Early Stages of Differentation of Pluripotent
and Limbal Stem Cells
DepartmentDepartment of Medicine
Supervisor Professor Ruby Shalom-Feuerste
Full Thesis textFull thesis text - English Version


Abstract

Stem cells (SCs) hold a huge potential in regenerative medicine, study of embryonic development, disease modeling and drug screening. While pluripotent SCs (PSCs) are found in the early stage of embryogenesis, generating the entire organism, adult SCs (ASCs) possess a more restricted potential of producing cell types of a particular lineage, compensating for tissue cell loss. The mechanisms by which SCs self-renew and differentiate are poorly understood. Consequently, the applications of these SCs in research, drug discovery and cell therapy are limited.

Embryonic SCs (ESCs) are PSCs that are derived from the inner cell mass of blastocyst stage embryo and are considered as the most “naïve” and undifferentiated cells. The blastocyst develops into epiblast which contains slightly differentiated so-called “primed” PSCs. Interestingly, while murine ESCs (mESCs) are stabilized in culture in naïve state, human ESC (hESCs) are stabilized in primed state. Notably, primed state is (i) considered as less potent, (ii) less efficiently amenable for genetic manipulations and (iii) differentiation. The fact that hESCs are primed raised unmet need for clarifying the mechanisms underlying the transition between these states.

The convention to primed state is hallmarked by epithelial to mesenchymal transition, changes in metabolism and epigenetic landscape. Since these changes also hallmark neoplastic cell transformation, in the first part of my thesis, I hypothesized that oncogenic RAS may be involved in this process. Indeed, I found that RAS activity was induced upon early differentiation, or transition of naïve mESCs into the primed state. Forced expression of active RAS induced glycolysis, de novo expression of N-CADHERIN, and expression of repressive epigenetic marks. By contrast, inhibition of RAS significantly attenuated differentiation. Altogether, this study indicates that RAS controls key processes in priming of naive cells.

In the second part of the thesis, I aimed to study the cell state signature that controls the differentiation of ASCs using the cornea as a model. Although limbal epithelial SCs (LSCs) were among the first quiescent SCs (qLSCs) ever discovered, their signature and properties remain largely unknown. I combined single-cell RNA sequencing and quantitative lineage tracing for analysis of murine limbal epithelium. The data revealed the co-existence of two novel LSC populations: (i) the “outer” limbus contains widespread population of qLSCs that uniformly express Krt15/Gpha2/Ifitm3/Cd63 proteins, and play role mainly in boundary formation and (ii) the “inner” limbus contains active LSCs (aLSCs) that express Krt15-GFP/Atf3/Mt1-2/Socs3 and participate in corneal epithelial homeostasis. Quantitative lineage tracing revealed that both populations follow stochastic rules of self-renewal and differentiation, while qLSCs generate aLSCs. Moreover, I discovered that T cells serve as niche cells regulating quiescence and wound response. Taken together, we revealed the co-existence of two novel LSC populations, their hierarchy, cell state signature and function.

Conclusively, this study shed light on key regulatory mechanisms that modulate SC (i.e. PSC and LSC) self-renewal of differentiation. The identification of the genetic signature of SCs and their regulatory network opens new avenues for research and optimal application of SCs in regenerative medicine.