Neurogenesis from Neural Stem Cells, Ependymal Cells and Fibroblasts

Abstract: Stroke is a major cause of death and disability around the world. Stroke leads to loss of neurons and also other cells in the brain due to lack of blood supply. Currently no therapies are available to treat stroke-related disability. It has been shown that stroke leads to increased neurogenesis, birth of new neurons, within the brain. This increased neurogenesis is not sufficient to restore lost function. There is a need to develop therapies for neuronal replacement by improving neurogenesis within the brain and / or transplanting neurons. Cortical strokes lead to more disability after stroke as compared to those affecting the striatum, and whether cortical neurogenesis occurs after stroke is controversial. Cell transplantation may be the key to cortical repair after stroke. Reports have identified positive but very few negative regulators of neurogenesis after stroke, and suggested that ependymal cells can also contribute to stroke-induced neurogenesis. Transplantation of neurons generated from different sources such as fetal brain, embryonic stem cells and induced pluripotent stem cells are associated with ethical issues and carry the risk of immune rejection and tumorigenicity. Direct conversion of patient’s own skin cells to neurons could overcome these problems and potentially restore function after transplantation in stroke-damaged brain. In this thesis we have used transgenic models, viral vectors, electroporation-mediated gene delivery and overexpression of transcription factors to demonstrate neurogenesis from neural stem cells, ependymal cells in the lateral ventricular wall and fibroblasts. We show that Lnk, a known inhibitor of hematopoietic stem cell self-renewal, is also expressed in the brain. Overexpression or removal of Lnk expression leads to decreased or increased neurogenesis in vitro respectively. When brain is damaged by stroke there is increased proliferation of neural stem cells in animals without Lnk expression. This was not observed in status epilepticus, a severe form of epilepsy. We determined that upregulation of Stat1/3 after stroke leads to increased Lnk expression. Subsequently Lnk inhibits cellular response to increased IGF1 stimulation after stroke, by decreasing Akt phosphorylation. We have identified Lnk signaling as a novel mechanism of influencing neurogenic response to stroke. We next determined if ependymal cells in lateral ventricular wall of adult rat brain contribute to neurogenesis after stroke. We identified FoxJ1 as a marker of ependymal cells in rats similar to mice, and used FoxJ1 promoter in piggyBac system to genetically label these cells with fluorescent reporter proteins GFP or RFP by electroporation. Tracing the lineage of the labeled cells in intact and stroke-damaged brain, we identified that FoxJ1 expressing cells contribute to olfactory bulb neurogenesis while the striatal neurogenic response was not significant. Thus, FoxJ1 expressing cells probably have only a minor role in repair after stroke. We then tested whether human fetal lung fibroblasts could be directly converted to cortical neurons. We overexpressed sets of transcription factors that are known to be involved in cortical neuron development. We found that overexpression of different sets of these factors in fibroblasts converted them to cortical-like neurons. These neurons expressed markers of cortical neurons and were functional by electrophysiology. In summary, these results raise the possibility that inhibition of Lnk, a negative regulator of neurogenesis from the brain’s own neural stem cells, and intracortical transplantation of cortical neurons directly converted from fibroblasts could be developed into novel therapeutic strategies for stroke in the future.