This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Magdalena Kucia, PI Stem cells provide a novel potential source to replace dead neurons and supporting tissue in brain damaged by chronic ischemia as seen for example in sleep apnea (SA). Some investigations of animals and short-term human bone marrow (BM) transplants have demonstrated that bone marrow cells provide a source of neurons and can repair brain damage (e.g., during stroke). The mechanisms of this functional improvement are currently the focus of intense research, creating a need for new study methodologies to assess the effectiveness of such strategies. Elucidation of stem cell-related mechanisms of regeneration is crucial to developing effective stem cell-based therapies that could extend the lifespan of patients with diseases that are treatable by tissue regeneration. One such disorder is chronic brain damage due to hypoxia resulting from sleep apnea. Based on preliminary data, a novel hypothesis is presented that the pool of CXCR4+ epiblast derived VSEL is deposited in the BM during early development. These cells subsequently serve as a reserve mobile pool of stem cells that may be mobilized into peripheral blood and play an important role in brain regeneration - where they are chemoattracted by an SDF-1 gradient. Furthermore, it is hypothesized that an age-related decrease in the marrow pool of these circulating VSEL may contribute to aging of the central nervous system (CNS), resulting in less effective repair. To investigate these issues, four specific aims are proposed.
Specific Aim 1. Neural differentiation of bone marrow-derived VSEL. We have presented evidence that BM contains a population of VSEL and that these epiblast-derived cells are deposited there early in development during rapid body growth/expansion. We will optimize their isolation from bone marrow and neural differentiation (ability to grow neurospheres). Next we will evaluate the age-related presence of VSEL in bone marrow tissue. Once it is determined whether VSEL circulate under normal steady-state conditions in the peripheral blood (PB) at very low, but detectable, levels, we will study their mobilization in a murine model of sleep apnea.
Specific Aim 2. Optimize mobilization of VSEL into peripheral blood. Since mobilized peripheral blood (mPB) may be a source of VSEL for potential neural regeneration, we will optimize their mobilization into PB. We will test the effect of various mobilizing agents involving selected growth factors (G-CSF, Flt3-ligand, VEGF, HGF) and small-molecule inhibitors (CXCR4-antagonist T140, C3aR antagonist) on the efficacy of their mobilization. We also will investigate mobilization of these cells in response to hypoxia damage and the role of the SDF-1CXCR4 axis in this process. It is hypothesized that CXCR4+ VSEL are mobilized and subsequently chemoattracted into a damaged brain in an SDF-1-dependent manner.
Specific Aim 3. Develop an approach to expand VSEL. Since the number of VSEL that can be isolated from the BM and mPB of older individuals is relatively low, an efficient ex vivo expansion system may be needed to obtain a sufficient number of these cells for neural regeneration. It also is possible that ex vivo culture-derived VSEL will better engraft and regenerate brain. We will employ selected strategies to expand these cells ex vivo involving cocktails of selected growth factors, BM stroma support, and our new strategy based on the expansion of stem cells in the presence of membrane-derived microvesicles isolated from embryonic stem cells (ESMV). We observed that purified VSEL cells are able to form spheres in co-cultures with C2C12 myoblastic cell line feeder layer that resemble embryoid bodies. Cells from these spheres may again (up to 5-7 passages) grow new secondary spheres, or if plated into cultures promoting tissue differentiation, expand into cells from all three germ-cell layers. We will employ this system to expand neural cells from VSEL-derived spheres.
Specific Aim 4. Determine the efficacy of VSEL in brain regeneration in vivo in a murine model of sleep apnea (SA). The contribution of VSEL to functional regeneration of damaged tissues will be tested in an in vivo mouse model of SA. We will compare the regeneration potential of syngeneic VSEL isolated from enhanced green immunofluorescence protein (EGFP+) transgenic mice to rescue brain damaged by hypoxia and the role of the SDF-1CXCR4 axis in this process. We will employ freshly isolated VSEL from BM or mPB as well as VSEL expanded in ex vivo cultures. As a read-out of brain regeneration, we will use selected behavioral tests.
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