Stem cells : proliferation, differentiation, migration

Abstract

A stem cell is demanding and requires precise conditions to carry out its unique tasks. This includes proliferation as an undifferentiated stem cell, differentiation to more mature cell types, migration through tissue to its final destination, and functional integration into the cellular network. All these steps are tightly regulated by both intrinsic and extrinsic factors. The aim of this thesis was to write a distinctive chapter in a long story about stem cell proliferation, differentiation and migration.

Stem cells need mitogens and growth factors to proliferate and to remain undifferentiated. These factors are normally secreted from neighboring cells in vivo and are necessary media supplements to stem cells when cultured in vitro. Our experiments identify the growth factor amphiregulin as a mitogen for adult neural stem cells in vitro. These amphiregulin-amplified adult neural stem cells remain multipotent and upon differentiation they generate the cell types characteristic of the brain i.e. neurons, astrocytes and oligodendrocytes. The in vivo role of amphiregulin in the adult brain is not known, but its expression lines in close contact to the adult neural stem cell niche and indicates a possible function for amphiregulin in maintaining stem cell proliferation.

The fate of stem cells can be directed by changing the extracellular milieu or by altering cellintrinsic conditions, for example by expression of ectopic genes. Since gene delivery to adult neural stem cells has proven difficult, we have developed and optimized both viral and nonviral delivery methods for in vitro as well as in vivo applications. Viral methods usually show higher delivery efficiency than non-viral methods. In vivo, the delivery of a reporter gene can be directed to stem or progenitor cells by the use of a specific promoter.

We utilized retroviruses to deliver neurogenin 2 in order to direct the differentiation of adult neural stem and progenitor cells in vitro. The neurogenic activity of neurogenin 2 was almost complete, directing 90% of the transduced cells to a neuronal fate. To identify additional genes promoting neuronal or other specific lineage choices, we developed a rapid highthroughput screen in mouse embryonic stem cells. The potential of this method is vast since genes directing either ectodermal or mesodermal cell fates can be identified, as well as genes maintaining embryonic stem cells in an undifferentiated state. By using a rearrayed cDNA library of 8000 genes in expression vectors we will screen nearly one third of the mouse genome.

New neurons in the adult brain have to migrate from the ventricular zone to the olfactory bulb. Several guidance molecules direct their way, for example, the Robo receptor and the Slit ligand. Little is known about the molecular mechanisms downstream of the Robo2 receptor. We propose a mechanism where the Robo2 receptor is cleaved and the intracellular part translocates to the nucleus whereupon it may influence gene expression. These studies shed light on some of the very complex steps in the transition from stem cell to mature cell.