Brain Microenvironment and its Influence on Gene Expression and Differentiation of Murine Embryonic Stem Cells . Implications for Cell Replacement Strategies

ABSTRACT: The discovery of pluripotent embryonic stem (ES) cells more than 20 years ago marked the beginning of a new era in developmental biology and biomedical research. The outstanding endogenous attributes of embryonic stem cells and the availability of in vitro ES cell differentiation protocols emphasise the great potential of these cells for clinical application in CNS disorders. Particularly for the treatment of disease states in which no clinical therapy has proven effective to date, such as traumatic brain injury, stem cell-based cell replacement strategies are advancing. However, in animal models of traumatic brain injury it has been shown that soluble signalling molecules, extracellular matrix-associated activities and the cellular environment at the site of implantation exert great influence on engrafted stem cells and can be decisive for success or failure of functional engraftment. This doctoral thesis aims at elucidating the role of naïve and pathological environmental conditions on plasticity of murine embryonic stem cells. Utilising a sophisticated in vitro approach i) the influence of endothelial and astroglial cells on stem cell self-renewal and differentiation capacity, ii) the impact of traumatic brain injury-associated environment on stem cell fate and iii) the source of motor functional improvements, observed after stem cell transplantation into the traumatically injured brain in vivo were investigated. To achieve this, embryonic stem cells were co-cultured with cerebral endothelial or astroglial cells, both belonging to the neural stem cell niche in vivo, or treated with tissue extract derived from traumatised or healthy rat brain, respectively. Subsequently, embryonic stem cells were analysed for expression of developmental marker genes (RT-PCR), mitotic activity (FACS), production of neurotrophic factors (ELISA) and morphological appearance (microscopy). Results indicate that environmental conditions, both on a cellular and molecular level, distinctively manipulate stem cell fate. It is demonstrated that neuronal lineage commitment in ES cells is mainly mediated via humoral factors present in conditioned medium, whereas the vicinity of living endothelial cells and astrocytes delays differentiation activities and instead promotes oligodendroglial cell fates. Furthermore, it is shown that traumatic brain injury-related parameters support the rapid differentiation of single stem cells to mature neuronal phenotypes. However, many cells are lost due to the deleterious effects of trauma environment. Moreover, cerebral tissue extract is shown to either enhance or restrict the release of trophic factors by embryonic stem cells, and additionally, has the ability to induce growth factor production in fibroblasts. Thus, co-transplanted fibroblasts are proposed to represent an alternative source for functional recovery observed after ES cell transplantation. With its findings, this study tries to bridge a gap between basic biological research and clinical science in a common effort to aid the human patient finally