Browsing by Author "O'Leary, Heather"

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    3058 – Sars-Cov-2 Binding in Hematopoietic Stem and Progenitor Cells Under Low Oxygen Conditions
    (Elsevier, 2021) Dausinas, Paige; Hartman, Melissa; Allman, Lauren; O'Leary, Heather; Anatomy, Cell Biology and Physiology, School of Medicine
    The SARS-CoV-2 pandemic highlighted a need for in-depth understanding of interaction/identification of receptors and mechanisms/functional consequences of viral binding/entry. SARS-CoV-2 spike protein (SBP) facilitates viral entry via ACE2 and/or NRP1 binding, with DPP4 as a potential co-receptor. These binding partners are expressed on various cell types including hematopoietic stem and progenitor (HSC/HSPC) cells [1-3]. HSC/HSPCs generate blood cells and reside in the low oxygen (lowO2, 1-4%) bone marrow niches that provide critical signals for maintenance, self-renewal, and differentiation. To investigate aspects of SARS-CoV-2 interactions with HSC/HSPC such as endogenous receptor expression, SPB binding and subsequent functional alterations in native low O2, we performed transcriptional and phenotypic/functional analysis. In lowO2, we identified increased surface expression of ACE2, DPP4 and NRP1, and enhanced binding of SBP to HSC/HSPC populations which amplified proliferation of SBP bound in lowO2. ACE2 and DPP4 surface expression were ∼2-fold higher in HSPCs (p=0.017, p=0.001) and HSCs (p=0.010, p=0.03), and NRP1 was ∼1.5-fold (p=0.002) higher in HSPCs in lowO2 compared to air. Interestingly, in lowO2, overall SBP binding was enhanced in HSPC (2.2-fold, p<.001) and HSC (2.6-fold, p=.018). Although not all cells expressing ACE2/DPP4/NRP1 bind SBP (∼50%), all cells exhibiting SBP binding in HSC/HSPC populations are triple positive for ACE2, NRP1, and DPP4. Additionally, we observed greater than a 2-fold increase in proliferation of SBP bound vs unbound cells in replating assays in lowO2 (p<.001). These data impart compelling evidence that SBP binding/functional outcomes are unique in low O2, providing a foundation that may have potential clinical implications for COVID19 treatment and expanding our baseline understanding of SARS-CoV-2 viral binding implications.
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    Generation and Exploration of a Novel Low Oxygen Landscape for Hematopoietic Stem and Progenitor Cells
    (2022-10) Dausinas, Paige Burke; Elmendorf, Jeffrey; O'Leary, Heather; Bidwell, Joseph; Wan, Jun; Zhang, Ji
    Hematopoietic stem (HSC) and progenitor (HSPC) cells reside in low oxygen (~1- 4%, low O2) bone marrow niches which provide critical signals for maintenance, selfrenewal, and differentiation. Exposure of HSC/HSPCs to air (~21%) for less than 10 minutes irreversibly diminishes numbers of phenotypic and functional stem cells, a phenomenon termed extra physiologic oxygen stress/shock. Yet, most studies harvest and analyze HSC/HSPCs in air and often in fixed cells, leaving endogenous signaling mechanisms unidentified. To better understand the endogenous mechanisms regulating HSCs and HSPCs, we generated the first low O2 landscape of phenotypic/functional/signaling alterations in live, low O2 harvested/sorted HSC/HSPCs utilizing novel technology. HSC (LSKCD150+) and HSC/HSPC (LSK) expression, frequency, and stem cell maintenance retention were enhanced in low O2 relative to historic data and our air data. Transcriptomics uncovered low O2 differential pathway regulation of HSC/HSPCs and HSCs with analysis identifying low O2 enrichment of genes/pathways including Ca2+ ion binding, altered sodium hydrogen (Na+/H+) activity, viral entry, and transmembrane receptor activity in both HSCs and HSPCs. In exploring the low O2 landscape, we investigated differential low O2 regulation of Ca2+ and SARS-CoV-2 related pathways/mechanisms in HSCs and HSPCs. Differential Ca2+ regulation was observed in our transcriptional/proteomic analysis corroborated by phenotypic/functional data demonstrating increases in low O2 of cytosolic and mitochondrial Ca2+ flux, ABC Transporter (ABCG2) and Na+/H+ (NHE1) expression, discovery of a novel low O2 Ca2+ high HSPC population that enhances HSC maintenance compared to Ca2+ low populations and blunting of this population and subsequent enhanced stem cell maintenance upon NHE1 inhibition (Cariporide). Multi-omics analyses also identified enhancements in COVID19-related pathways in low O2 that corresponded with enhanced expression of SARS-CoV-2 receptors/co-receptors, SARS-CoV-2 spike protein (SP) binding, and expansion of SP-bound HSC/HSPCs in low O2 compared to air, as well as enhanced stem cell maintenance of SP-bound, versus unbound, cells in low O2. Together, these data presented show low O2 harvest/retention of HSC/HSPCs enhances stem cell maintenance, which could be utilized to improve HSC expansion, and leads to differential pathway/signaling regulation of various biological pathways in HSC/HSPCs including Ca2+ and SARS-CoV-2/viral infection that results in phenotypic and functional consequences.