| Description |
This thesis addresses the development of an in vitro model system that will allow researchers to study the development and function of neurons from people with neurological disorders. Future studies predicated on the development of this in vitro system will help define how neurons malfunction, thus facilitating therapeutic development and possibly a cure for certain neurological diseases. The first goal of this thesis was to determine if terminally differentiated blood monocytes harvested from human peripheral blood could be reprogrammed into neurons. To accomplish this conversion, human monocytes were de-differentiated to reach a pluripotent state, termed induced pluripotent stem (iPS) cells. These iPS cells have many of the same characteristics as human embryonic stem (hES) cells, including immortality and pluripotency. Previous work has demonstrated that terminally differentiated primary human skin fibroblasts have been de-differentiated into iPS cells. The expression of four transcription factors, Oct-4, Sox-2, Klf-4 and c-Myc, is essential for de-differentiation of primary cells, such as monocytes from humans. Forced expression of these transcription factors can be accomplished by using retroviral vectors. iPS cells are then isolated, expanded, and characterized to ensure immortality and pluripotency. Subsequently these pluripotent cells are directed by different growth factors to differentiate into neurons. The results presented in this thesis demonstrate the differentiation of the H9 line of hES cells into neurons. To confirm that H9 cells were converted into neurons, gene iv and protein expression levels of stem cell and neuron markers were determined by realtime polymerase chain reaction (PCR), microarray and flow cytometry, respectively. Furthermore, iPS cells have been created from mouse fibroblasts using a lentivirus vector thereby demonstrating the efficacy of this system. In conclusion, this thesis will aid in the derivation of iPS cells from blood monocytes and their subsequent differentiation into neurons. Future studies will be directed towards terminally differentiating iPS cells into neurons that could potentially be used to study developmental, genetic and phenotypic abnormalities of cells from patients with neurological disorders. |
