Mitochondrial dysfunction is associated with hematopoietic disorders. Overlooked in terms of stem cell function and its regulation are mitochondria, central effectors of cells that generate cellular energy, regulate apoptosis, and other functions. We hypothesize hematopoietic stem (HSC) and progenitor (HPC) cell functions are intimately associated with, and directly linked in cause-effect regulatory mode with mitochondrial activity, mass, morphology, and DNA content, reactive oxygen species (ROS), and growth factor combinations, as well as stromal cells. Our long term goals are to elucidate a mechanistic role for mitochondria in HSC/HPC function, after synergistic stimulation by combinations of cytokines, and in context also of stromal cells, and to use this information to enhance HSC engraftment, and HSC and HPC activities in vivo for therapeutic advantage.
Our Specific Aims are: 1.) Determine role of mitochondrial (mt) respiratory activities, especially electron transport chain (ETC) complex content and functional parameters, bioenergetic status, total mt-mass, mt-morphology, and mtDNA copy number/content, anti-oxidant enzyme expression/content, and glycolytic activities on HSC/HPC functions (survival, self-renewal, proliferation, differentiation, and HSC attrition) under: steady state conditions, varying oxygen tensions (normoxia (~20%), and lowered (5% and 1-3%)), in context of aging and with a special focus on molecular mechanisms and regulation of ROS generation and its effects on HSC/HPC after stimulation with SDF-1/CXCL12, SCF, and other relevant growth factors, especially synergistic combinations, and with regards to the Rapamycin-sensitive mTOR pathway. 1a. Study phenotypically- defined/purified and functionally-assessed populations of mouse bone marrow (BM) HSC and HPC from: normal mice, hematopoietic (H) tissue-specific STAT3 -/- mice and SDF-1 TG mice. 1b. Evaluate purified HSC/HPC from human umbilical cord blood (CB) and adult BM. 1c. At single cell level, determine how mitochondrial segregation into daughter cells and mitochondrial activity relates to the functional activities of HSC/HPC. 2). Use "detoxification" (e.g., N-acetylcysteine (NAC), and hypoxia) strategies in HSC/HPC during harvest and manipulation to reduce HSC/HPC death and differentiation for pre-clinical study. 2a. Evaluate anti-oxidant enzyme expression (i.e. mnSOD) and respiratory ETC efficiency and parameters on ROS levels and generation and link this information mechanistically to HSC/HPC function with a focus on synergistic growth factor combinations, and in relationship to the Rapamycin/mTOR pathway. 2b. Modify ROS generation and its effects on HSC/HPC via treatment with ROS scavengers (i.e. NAC), and pro-oxidant conditions (i.e. rotenone) in context of hypoxia vs. normoxia.
Little is known regarding biology and regulation of hematopoietic stem cells (HSC), and their response to aging and stress. More in-depth knowledge of factors regulating HSC function will make it possible to control/modulate HSC function and fate and develop more efficacious treatments for disease and decline in performance during aging and stress. Understanding mitochondrial behavior could lead to better understanding of how HSC deal with or respond to increased oxidative risk, information of use in studies of stem cells in general.
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