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"Analyses of Murine Neural Precursor Responses to Leukemia Inhibitory Factor and Hypoxia/Ischemia"

by
Krista D. Buono
Department of Neurology & Neurosciences
B.S. 2001, Loyola University



Thesis Advisor: Steven W. Levison, Ph.D.
Professor
Department of Neurology & Neurosciences

Tuesday, June 21, 2011
12:00 PM, Cancer Center, G Level Seminar Room


Abstract

New discoveries in basic science can generate ideas that advance therapeutic approaches in modern medicine. Stem cells have emerged as a potential means to treat many disorders, diseases and tissue injuries that are presently incurable. The present work focuses on neonatal hypoxia/ischemia (H/I), which is the major cause of permanent neurological disabilities sustained during childhood. Neonatal H/I has an incidence of 2-4 per 1,000 live births and there are presently no treatments to promote recovery. The rational for this dissertation research has been to identify the mechanisms that regulate the expansion of the endogenous stem and progenitor cells that reside within the subventricular zone as a first step in producing new therapeutics to stimulate regeneration in the neonatal brain. There were 4 goals for this dissertation: 1) to developed a flow cytometry protocol to enable the isolation and characterization of growth requirements for primitive, intermediate and lineage-restricted neural precursors (NP); 2) to determine how LIF affects neonatal NP dynamics; 3) to determine which NP population(s) expand after neonatal H/I; and 4) to determine whether LIF is an essential regulator of NP expansion within the SVZ after injury. The overall hypothesis of this dissertation is that LIF is transiently increased in the SVZ after neonatal H/I to stimulate expansion of NPs, particularly NSCs.
To evaluate the outcomes of perturbing the signals that regulate NPs, two multicolor flow cytometry protocols that utilized markers of primitive, intermediate and lineage-restricted neural precursors were developed. Using a 6-color panel, the NSC, at least 3 classes of multipotential progenitors and 4 classes of bipotential restricted precursors were isolated and identified. Using flow cytometry to analyze the composition of the SVZs of LIF null neonates, we discovered that LIF is essential to maintaining NSCs and for the production of immature oligodendrocytes. LIF gain of function studies confirmed that LIF regulates self-renewal of NSCs, while it also maintains 3 LIF-responsive intermediate progenitors and expands immature oligodendrocytes. Furthermore, we found that LIF increases the expression of self-renewing genes Klf4, Fbx15, Nanog, Sox2 and c-Myc. These data demonstrate that LIF is necessary to maintain the normal balance of CNS precursors within the SVZ.
Using a modified Vannucci model for H/I, we discovered that LIF mRNA increases robustly within the murine SVZ by 24 h of recovery and then rapidly returns to baseline. Astrocytes are the primary source of LIF, and EGF stimulated its release. Using multimarker flow cytometry, we found that NSCs diminish after neonatal H/I, whereas multipotential progenitors and 3 unique glial-restricted precursors expand. In LIF haplodeficient mice, NP expansion after H/I was inhibited.
Altogether, we have established that LIF both expands NSCs and sustains their self-renewal. Furthermore, LIF is critical for NP expansion after H/I. However, LIF is not the only cytokine that increases after neonatal brain injury, thus it is likely that other secreted factors collaborate with LIF to generate intermediate progenitors and, as a consequence, diminish the brainís reservoir of stem cells.


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