Development of Tools to Study Neural Stem Cell Differentiation in Real Time.

摘要

Insults to the central nervous system (CNS), including multiple sclerosis (MS) and spinal cord injury (SCI), drastically decrease the quality of life by debilitating native function of CNS cells. SCI affects approximately 276,000 people in the U.S with 12,500 new patients each year, while MS affects approximately 400,000 people in the United States and 2.5 million worldwide. Oligodendrocytes play a fundamental role in the CNS by myelinating axons and greatly impact CNS function. Replacement of oligodendrocytes either by neural stem cell transplants or targeting of endogenous stem cells is an attractive potential treatment for CNS tissue repair. However, there is no current consensus on how to efficiently differentiate oligodendrocytes. Developing a standardized protocol is important to better employ cell therapies for axon remyelination and CNS regeneration.;We aim to monitor oligodendrocyte differentiation by tracking reporter gene expression of neural stem cells transduced with a high-throughput array of genetic reporters. In parallel to optimizing a differentiation protocol by comparing previously established methods, we will construct lentiviral reporters for proteins produced at various stages of neural stem cell differentiation to track the stages of live cells. In this system, the same live cells can be tracked over time to achieve single cell data, which is often more representative of the underlying biological processes when investigating heterogeneous stem cell populations. This thesis describes the successful development of a lentivirus reporter for neural stem cell differentiation, and in particular the neuronal lineage verified using bioluminescence and immunostaining. In addition, a direct comparison of the effects of multiple soluble factors reported to mediate early-stage oligodendrocyte differentiation on human neural stem cells was performed. Neural stem cells were differentiated for three and five weeks in various combinations of epidermal growth factor (EGF), platelet derived growth factor (PDGF), retinoic acid (RA) and purmorphamine (PM). Cells cultured in EGF with PM or RA proliferate and express oligodendrocyte specific markers making EGF, PM and RA attractive factors for future studies.;Preliminary data from these studies will be used to set up upcoming experiments for the construction of additional lentivirus reporters and optimizing a protocol for oligodendrocyte differentiation. In the future, this knowledge can be applied to develop effective neural stem-cell based therapies for neuroregeneration.