Manipulating neural stem cells

Abstract

The stem cell field is a fairly young but fast growing scientific field. Our understanding of stem cells has changed tremendously over the last couple of decades. This in mainly due to the development of new techniques that has enabled us to study stem cells from a completely new perspective. Several groundbreaking findings were made through technical advances, such as the development of somatic cell nuclear transfer in the 50s and the production of the first cloned mammal, Dolly the sheep in 1996, which demonstrated that nuclei of differentiated cells could be reprogrammed. The development of cultures of adult neural stem cells in the 90s demonstrated the presence of stem cells in vivo that could be propagated in vitro. More recently, the development of artificial embryonic stem cells, the so called induced pluripotent stem cells (iPS cells), were obtained through the delivery of only four exogenous factors, Oct4, klf4, Sox2 and cMyc to cultured fibroblasts in 2006.

The publications presented in this thesis are based on several of these groundbreaking findings. A common theme in the work of the thesis is the use of neural stem cell for the purpose of addressing biological questions in vitro or in vivo. Publication I describes a new miniaturized platform for culturing embryonic stem cells and adult neural stem cells. This platform contains 672 wells, each one holding 500nL. The volume, large enough to culture stem cells over several days is also small enough to drastically reduce reagent costs. The platform is suitable for large-scale screening, where a large number of substances or culture conditions can be studied simultaneously. Publication II presents the development of a rapid and efficient transfection method that allows localized in vivo transfection of plasmid DNA within adult neurogenic niches by electroporation. This strategy allows gain-and loss-of function studies as well as fate mapping experiments. Using this approach, we found that cadherins play an essential role for maintaining the integrity of the lateral ventricle wall. Both publication III and manuscript IV aim to further improve the iPS cell production. Publication III is one of the first publications where reprogramming of neural stem cells without Sox2 is reported. The iPS cells generated displayed pluripotency by teratoma formation and production of chimeric mice upon blastocyst injection. Two of the mice developed tumours displaying residual proviral expression. The use of exogenous DNA presents a potential oncogenic risk. Choice of delivery method for the reprogramming factors in combination with reduction of the number of factors required might bring iPS cells closer to therapeutic use. Manuscript IV attempts to push adult neural stem cell towards a reprogrammed state through an allchemical based strategy, omitting the need for exogenous DNA. Adult neural stem cells endogenously express three of the four reprogramming factors. This allows reprogramming of adult neural stem cell through the addition of only one factor, Oct4. In manuscript IV Oct4 expression is chemically induced. Endogenous expression of the pluripotency factors Nanog and Rex1 were measured to investigate whether Oct4-inducing conditions could lead to a reprogrammed state.