Generation of induced neurons via direct conversion in vivo and in vitro

Sammanfattning: Cellular reprogramming is when one cell is changed into another. This involves structural modifications on the DNA of a cell resulting in a transcriptional change. This occurs naturally during development when early pluripotent cells gradually differentiate into more specialized cells that finally result in a complete organism. This is a finely orchestrated event that includes both extrinsic and intrinsic signaling. Cellular reprogramming can be induced artificially by exposing a somatic cell to a foreign microenvironment or by the forced expression of various transcription factors. Recent studies have shown the possibility to revert a somatic cell back into a pluripotent stem cell, termed induced pluripotent stem cell (iPS) or directly to a different somatic cell using this strategy. In this thesis I focus on the direct reprogramming where one terminally differentiated cell is directly converted into another without passing a pluripotent state. Using lentiviral vectors we could convert embryonic and postnatal human fibroblasts into functional neurons (iN) by the forced expression of Ascl1, Brn2 and Myt1L (ABM). By including the additional factors, Foxa2 and Lmx1a, subtype specific neurons could be obtained that release dopamine, express specific markers and exhibit electrophysiological properties characteristic of dopaminergic neurons. Further we show the possibility to transplant fibroblasts and astrocytes into brains of adult rats and then convert them into neurons in vivo. These cells expressed pan- neuronal markers and converted at similar rates as reported in vitro. Using Cre inducible lentiviral vectors, coding for ABM and inject these into the brains of transgenic mice expressing Cre under the GFAP promoter, we could specifically target astrocytes and convert these into neurons in vivo. Using the same strategy we cloned the three factors, Ascl1, Lmx1a and Nurr1 (ALN) together with GFP, into Cre inducible recombinant adeno associated viral vectors (rAAV) with the aim to convert NG2 glia into dopaminergic neurons. rAAV vectors are interesting tools for clinical applications because of their low pathogenicity and their ability to infect both dividing and non-dividing cells. By including a synapsin promoter for the GFP reporter we could specifically visualize converted cells that expressed the pan neuronal markers NeuN and MAP2 but failed to induce a dopaminergic phenotype. More studies aim to study these cells after a longer maturation time and their functional properties in terms of electrophysiology and synaptic formation. Cellular reprogramming of somatic cells is an interesting option to previously studied sources in cell replacement therapies that often are associated with logistical and ethical concerns. They are readily available cells that can be obtained from the skin of a patient and direct conversion offers further advantages over iPS cells as they are non-proliferating cells eliminating the risk of forming tumors when transplanted. Further, in vivo reprogramming offers an alternative to traditional cell therapy by creating new neurons in the brain removing the need of an exogenous cell source. The brain is of particular interest for cell replacement therapies as its capacity to repair itself after injuries like stroke is limited and treatments for neurological disorders like Parkinson’s disease (PD) progressively decline in effectiveness and are associated with severe side effects. In summary, this thesis shows the possibility to directly convert human, adult fibroblasts into functional dopaminergic neurons by the forced expression of transcription factors important in neural development. We further show the possibility to transplant fibroblasts and astrocytes into the brains of rats and convert them into neurons in situ. We also show the possibility to convert two types of glia cells, astrocytes and NG2 glia residing in the brain into neurons by using transgenic mice and Cre inducible vectors. This could also be done by using a rAAV vector commonly used in clinical trials. Future studies should focus on factors involved in the specificity of the required cell and how well the cell that is formed correspond genetically, functionally and viably to its endogenous counterpart.