Isolation and characterization of adult neural stem cells

Abstract

Injury to the central nervous system (CNS) is one of man's most handicapping situations resulting in severe functional impairment and in some cases a vegetative state where life is supported artificially. The brain and spinal cord, constituting the CNS, have been viewed for decades as having limited capacity of regeneration with no neurogenesis in the adult. The failure to regenerate the CNS was postulated to be due to either: (1) weak intrinsic capacity of neurons to re-grow axons to their target, (2) formation of a compact glial scar (3) the presence of axon-growth inhibiting factors or (4) the lack of neurogenesis. Likely it is a combination of factors, rather than one single factor, that explains the limited regenerative capacity. The focus of this study has been to analyze the mechanisms behind scar formation in response to injury and to try to identify and characterize neural stem cells in the adult CNS.

We first focused our studies on the role of the intermediate filaments (IFs) in scar formation in the CNS, using a well-characterized injury model in transgenic animals devoid of different IFs. Glial scar formation appeared normal after spinal cord or brain lesions in GFAP-/- or vimentin-/- mice but was impaired in GFAP-/-vimentin-/- mice. These mice developed less dense scar tissue, frequently accompanied by bleeding. These results show that GFAP and vimentin are required for proper glial scar formation in the injured CNS. Interestingly, we found that nestin, an intermediate filament protein expressed in neuroepithelial stem cells during nervous system development, is re-expressed in the injured CNS. The expression of nestin in adult animals is restricted to endothelial and ependymal cells. After a spinal cord injury, nestin expression is rapidly increased in ependymal cells and the expression then spreads along the midline towards the lesion site. This dynamic nestin expression pattern in response to injury suggested that a latent population of stem cells lining the central canal contribute to scar formation.

Previous work suggested that neural stem cells reside in the subventricular zone (SVZ). However, we found that in the spinal cord injury paradigm, ependymal cells proliferate and their progeny migrate and differentiate to astrocytes. Using an antibody that recognize the extracellular domain of Notch 1, the fluorescent dye (Dil) and cell morphology (ependymal cells are ciliated), ependymal cells were isolated and cultured in vitro and clonally derived cultures differentiated into the three major cell types of the CNS. Longterm BrdU experiments and lineage analysis revealed that forebrain ependymal cells divide slowly and give rise to rapidly dividing SVZ cells. Infection of forebrain ependymal cells with adeno- or retrovirus expressing the reporter gene lacZ, or labeling with Dil, showed that progeny of infected ependymal cells migrate to the olfactory bulb and differentiate to interneurons.

In order to investigate if the adult human brain harbors neural stem cells we obtained hippocampus and lateral ventricle wall tissue from adult patients. We found that both regions harbor cells that are self-renewing and multipotent in vitro, i.e. bona tide neural stem cells.

It was previously thought that the differentiation potential of a stem cell is restricted to the cell types present in the tissue in which it resides. We challenged this view by first co-culturing neural stem cells with embryonic stem (ES) cells. ES cells have the potential to differentiate to all cells in the embryo, and hence we argued that they may be able to instruct the neural stem cells to generate non-neural cells. Neural stem cells from ROSA26 mice were isolated and co-cultured with wild type ES cells. Some of the cells derived from the neural stem cells expressed the muscle specific markers desmin and myosin heavy chain. We next injected neural stem cells into early chick embryos. The injected cells followed the developmental cues in the different germ layers and we found that neural stem cells can differentiate to heart muscle, kidney, liver, epithelial cells and neural tissue. Injection of neural stern cells into mouse blastocysts further supported the notion that neural stem cells can contribute to embryo formation and differentiate to a variety of different cell types.

In conclusion, this thesis demonstrates that IFs are necessary for proper scar formation in response to CNS injury. The spinal cord ependymal cells contribute to scar formation and along with ependymal cells in the brain they have been isolated and characterized as neural stem cells. Moreover, these neural stem cells are not restricted to a neural fate but can differentiate to cells of all three germ layers given the proper permissive environment.