University of Vermont (P30 GM103498)
"Center for Neuroscience Excellence"

Pilot Project 3: "Determinants of Multipotency and Neurogenesis From Reactive Astrocytes"
Investigator: Jeffrey Spees, Ph.D.

Identifying cell-extrinsic and cell-intrinsic determinants that control self-renewal and differentiation has become a fundamental area of discovery for stem cell biology and regenerative medicine. Cell-extrinsic determinants include: growth factors and cytokines, hormones, extracellular matrix proteins, ions (e.g. Calcium), oxygen tension, mechanical forces (stretch), and neural (electrical) input. Cell-intrinsic determinants include transcription factors, microRNAs, and epigenetic modifiers that control chromatin status and gene promoter activity (e.g. DNA methylation, histone modifications). The field of cellular reprogramming has elaborated many cell-intrinsic regulators of embryonic and adult stem cells, and also intrinsic mechanisms that control the fate and phenotype of differentiated adult cell types. Improved understanding of mechanisms controlling stem cell self-renewal and cell fate decisions may provide powerful tools to manipulate the behavior of stem cells and improve tissue repair after injury. In circumstances where it may prove difficult to transplant cultured stem/progenitor cells or to mobilize endogenous reparative cells to improve healing, growing evidence indicates that we may be able to learn how to alter cellular phenotype in situ. For example, after CNS injuries such as stroke, it may be possible to repair neural tissue by converting proliferating reactive astrocytes of the peri-infarct area into multipotent neural stem/progenitor cells (NSCs/NPCs) that can then be directed to produce neurons. The ability to convert reactive astrocytes into NPCs/NSCs could potentially be very useful because there is no neuroblast migration from the subventricular zone (SVZ) niche toward the infarct core after focal cortical strokes that do not injure the striatum. In addition, larger-size strokes that do damage the striatum still do not elicit a sufficient response from SVZ-derived neuroblasts to replace the vast numbers of neurons destroyed by cerebral ischemia.

 Recently, with lineage-tracing based on expression of glial fibrillary acidic protein (GFAP), we identified proliferating cortical reactive astrocytes of the peri-infarct area as a potential source of multipotent NSCs after stroke. Similar to adult SVZ-NSCs, we found that reactive astrocyte-derived NSCs (Rad-NSCs) grew in culture as neural spheres, self-renewed, and differentiated into neurons, astrocytes, and oligodendrocytes. We found that signals from injured peri-infarct tissue were critical to generate Rad-NSCs, as we could not produce them from uninjured contralateral tissues. Notably, lineage-tracing in vivo for 1 month showed that proliferating reactive astrocytes did not generate neurons in the peri-infarct area after stroke. Therefore, signals may be required from both the peri-infarct area and the culture environment to induce multipotency and neurogenesis from reactive astrocytes of peri-infarct tissues. In studies with conditional knockout mice, we demonstrated that Notch1 signaling through Notch Intracellular Domain (NICD1) was required to produce Rad-NSCs from cortical peri-infarct tissues (see preliminary data). Because hypoxia in peri-infarct tissues increases levels of Hypoxia-Inducible Factor 1 alpha (HIF1 alpha), a transcription factor that stabilizes Notch1 signaling, HIF1 alpha is likely also to control the formation of Rad-NSCs from reactive astrocytes. Furthermore, we were able to generate multipotent Rad-NSCs from cortical peri-infarct tissues with NSC medium that contained only EGF. Therefore, initial Notch1/HIF1 alpha/EGFR signaling from the peri-infarct area combined with high dose EGFR signaling in culture may regulate Rad-NSC formation, self-renewal, and/or differentiation. Improved understanding of the mechanisms controlling Rad-NSCs and their formation from reactive astrocytes may identify ways to promote multipotency and neurogenesis in situ from reactive astrocytes of peri-infarct tissues after stroke.