Deciphering a regulatory function of endolysosomes in asymmetric cell division of human neural stem cells via the Notch signalling pathway

Stem cells are defined by their ability to both self-renew as well as to produce cells that differentiate to somatic cells. The switch between symmetric and asymmetric cell divisions is the conserved strategy by which stem cells can accomplish these two tasks. In this thesis, human induced pluripotent stem cell (iPSC)-derived neural stem cells (NSCs) were used to identify intracellular determinants that influence the cell fate of daughter cells during asymmetric cell division. Amongst various analysed vesicular compartments, LAMP1/2+ and CD63+ vesicles were identified as potential asymmetry factors. These represent late stages within the endolysosomal pathway. Withdrawal of growth factors from the culture medium increased the occurrence of asymmetrically segregated LAMP1+ vesicles during cell division, linking asymmetric vesicle distribution to neuronal differentiation of NSCs. Further, Notch1/2 receptors, as well-known stem cell fate determinants, were found to be present in these LAMP1+ vesicles. The intracellular transport of Notch receptors was dependent on Dynamin and coincided with the internalization of recombinant Notch receptor ligands. Inhibition of vesicular acidification led to decreased Notch cleavage and reduced expression of Notch target genes underlining the potential function of endolysosomal vesicles as signalling hubs for the activation of Notch receptors. Establishing a reporter cell line expressing tdTomato under the endogenous HES1 promoter, Notch pathway activity was studied in actively dividing NSCs. The analysis of HES1 expression dynamics uncovered a link between high endolysosome content within a daughter cell during mitosis and a higher HES1 expression after cell division. This endolysosome-mediated bias in Notch signalling activity can be the basis for the decision of neuronal differentiation versus NSC fate maintenance. This is finally supported by the finding obtained in 3D forebrain organoids, that neurogenic cell divisions were associated with an accumulation of LAMP1+ vesicles as well as Notch1 receptors in the remaining NSC. Taken together, a putative, so far unrecognized function of LAMP1+ vesicles as signalling hubs for Notch receptors during asymmetric cell division of human NSCs was discovered. Endocytosis and the increasingly acidic environment thereafter facilitates receptor cleavage and hence activation of the pathway. The active shuttling of endolysosomes to one daughter cell during mitosis leads to a biased Notch signalling activity potentially influencing the cell fate of the respective daughter cells