- Browse by Subject
Browsing by Subject "hematopoietic stem cell"
Now showing 1 - 7 of 7
Results Per Page
Sort Options
Item 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, JiHematopoietic 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.Item In Vivo Effects of Myeloablative Alkylator Therapy on Survival and Differentiation of MGMTP140K-Transduced Human G-CSF-Mobilized Peripheral Blood Cells(Elsevier, 2006-05-01) Cai, Shanbao; Hartwell, Jennifer R.; Cooper, Ryan J.; Juliar, Beth E.; Kreklau, Emi; Abonour, Rafat; Goebel, W. Scott; Pollok, Karen E.; Pediatrics, School of MedicineHigh-intensity alkylator-based chemotherapy is required to eradicate tumors expressing high levels of O6-methylguanine DNA methyltransferase (MGMT). This treatment, however, can lead to life-threatening myelosuppression. We investigated a gene therapy strategy to protect human granulocyte colony-stimulating factor-mobilized peripheral blood CD34+ cells (MPB) from a high-intensity alkylator-based regimen. We transduced MPB with an oncoretroviral vector that coexpresses MGMTP140K and the enhanced green fluorescent protein (EGFP) (n = 5 donors). At 4 weeks posttransplantation into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice, cohorts were not treated or were treated with low- or high-intensity alkylating chemotherapy. In the high-intensity-treated cohort, it was necessary to infuse NOD/SCID bone marrow (BM) to alleviate hematopoietic toxicity. At 8 weeks posttreatment, human CD45+ cells in the BM of mice treated with either regimen were EGFP+ and contained MGMT-specific DNA repair activity. In cohorts receiving low-intensity therapy, both primitive and mature hematopoietic cells were present in the BM. Although B-lymphoid and myeloid cells were resistant to in vivo drug treatment in cohorts that received high-intensity therapy, no human CD34+ cells or B-cell precursors were detected. These data suggest that improved strategies to optimize repair of DNA damage in primitive human hematopoietic cells are needed when using high-intensity anti-cancer therapy.Item LDB1 Enforces Stability on Direct and Indirect Oncoprotein Partners in Leukemia(American Society for Microbiology., 2020-05) Layer, Justin H.; Christy, Michael; Placek, Lindsey; Unutmaz, Derya; Guo, Yan; Davé, Utpal P.; Medicine, School of MedicineThe LMO2/LDB1 macromolecular complex is critical in hematopoietic stem and progenitor cell specification and in the development of acute leukemia. This complex is comprised of core subunits of LMO2 and LDB1 as well as single-stranded DNA-binding protein (SSBP) cofactors and DNA-binding basic helix-loop-helix (bHLH) and GATA transcription factors. We analyzed the steady-state abundance and kinetic stability of LMO2 and its partners via Halo protein tagging in conjunction with variant proteins deficient in binding their respective direct protein partners. We discovered a hierarchy of protein stabilities (with half-lives in descending order) as follows: LDB1 > SSBP > LMO2 > TAL1. Importantly, LDB1 is a remarkably stable protein that confers enhanced stability upon direct and indirect partners, thereby nucleating the formation of the multisubunit protein complex. The data imply that free subunits are more rapidly degraded than those incorporated within the LMO2/LDB1 complex. Our studies provided significant insights into LMO2/LDB1 macromolecular protein complex assembly and stability, which has implications for understanding its role in blood cell formation and for therapeutically targeting this complex in human leukemias.Item Loss of SIMPL increases TNFα sensitivity during hematopoiesis(2008-10) Benson, Eric Ashley; Harrington, Maureen A.; Goebl, Mark; Clapp, Wade; Skalnik, DavidThe innate and adaptive immune responses are critical for host survival. The TNFα/NF-κB signaling pathway is a major regulator of the immune response. The TNFα/NF-κB signaling pathway has also been proposed to play a role in the regulation of hematopoiesis. In the TNFα signaling pathway, full induction of NF-κB (specifically the p65 subunit) dependent transcription is regulated by a co-activator SIMPL. The biological significance of SIMPL in TNFα dependent responses is poorly understood. To study SIMPL in vitro and in vivo in mammalian cells, a knockdown system utilizing shRNA (short hairpin RNA) was used. Analysis of hematopoietic progenitor cells infected with a retrovirus encoding the SIMPL shRNA was used to study the role of SIMPL in hematopoiesis. The ability of progenitor cells lacking SIMPL to grow and differentiate was not compromised. In contrast in the progenitors cells lacking SIMPL, TNFα mediated inhibition of colony formation was significantly enhanced. These growth inhibitory effects of SIMPL were not due to an increase in apoptosis. The enhanced inhibitory affects were specific for TNFα and not found in other common hematopoietic inhibitors (TGF-β1 and IFNγ). Results of this work reveal that SIMPL is a component of the hematopoiesis that is required for TNFα dependent effects upon myeloid progenitors.Item Pharmacological inhibition of AKT activity in human CD34+ cells enhances their ability to engraft immunodeficient mice(Elsevier, 2017-01) Chen, Sisi; Gao, Rui; Kobayashi, Michihiro; Yu, Hao; Yao, Chonghua; Kapur, Reuben; Yoder, Mervin C.; Liu, Yan; Biochemistry and Molecular Biology, School of MedicineAlthough practiced clinically for more than 40 years, the use of hematopoietic stem cell (HSC) transplantation remains limited by the inability to expand functional HSCs ex vivo. To determine the role of phosphoinositide 3-kinase (PI3K)/AKT signaling in human hematopoietic stem and progenitor cell (HSPC) maintenance, we examined the effect of genetic and pharmacological inhibition of AKT on human umbilical cord blood (UCB) CD34+ cells. We found that knock-down of AKT1 in human UCB CD34+ cells using short interfering RNAs targeting AKT1 enhances their quiescence and colony formation potential in vitro. We treated human UCB CD34+ cells with an AKT-specific inhibitor (AKTi) and performed both in vitro and in vivo stem and progenitor cell assays. We found that ex vivo treatment of human HSPCs maintains CD34 expression and enhances colony formation in serial replating assays. Moreover, pharmacological inhibition of AKT enhances the short-term repopulating potential of human UCB CD34+ cells in immunodeficient mice. Mechanistically, genetic and pharmacological inhibition of AKT activity promotes human HSPC quiescence. These preclinical results suggest a positive role for AKTi during ex vivo culture of human UCB HSPCs.Item Prostaglandin E₂ promotes recovery of hematopoietic stem and progenitor cells after radiation exposure(2014-07-11) Stilger, Kayla N.; Broxmeyer, Hal E.; Kacena, Melissa A.; Srour, Edward F.; Pelus, LouisThe hematopoietic system is highly proliferative, making hematopoietic stem and progenitor cells (HSPC) sensitive to radiation damage. Total body irradiation and chemotherapy, as well as the risk of radiation accident, create a need for countermeasures that promote recovery of hematopoiesis. Substantive damage to the bone marrow from radiation exposure results in the hematopoietic syndrome of the acute radiation syndrome (HS-ARS), which includes life-threatening neutropenia, lymphocytopenia, thrombocytopenia, and possible death due to infection and/or hemorrhage. Given adequate time to recover, expand, and appropriately differentiate, bone marrow HSPC may overcome HS-ARS and restore homeostasis of the hematopoietic system. Prostaglandin E2 (PGE2) is known to have pleiotropic effects on hematopoiesis, inhibiting apoptosis and promoting self-renewal of hematopoietic stem cells (HSC), while inhibiting hematopoietic progenitor cell (HPC) proliferation. We assessed the radiomitigation potential of modulating PGE2 signaling in a mouse model of HS-ARS. Treatment with the PGE2 analog 16,16 dimethyl PGE2 (dmPGE2) at 24 hours post-irradiation resulted in increased survival of irradiated mice compared to vehicle control, with greater recovery in HPC number and colony-forming potential measured at 30 days post-irradiation. In a sublethal mouse model of irradiation, dmPGE2-treatment at 24 hours post-irradiation is associated with enhanced recovery of HSPC populations compared to vehicle-treated mice. Furthermore, dmPGE2-treatment may also act to promote recovery of the HSC niche through enhancement of osteoblast-supporting megakaryocyte (MK) migration to the endosteal surface of bone. A 2-fold increase in MKs within 40 um of the endosteum of cortical bone was seen at 48 hours post-irradiation in mice treated with dmPGE2 compared to mice treated with vehicle control. Treatment with the non-steroidal anti-inflammatory drug (NSAID) meloxicam abrogated this effect, suggesting an important role for PGE2 signaling in MK migration. In vitro assays support this data, showing that treatment with dmPGE2 increases MK expression of the chemokine receptor CXCR4 and enhances migration to its ligand SDF-1, which is produced by osteoblasts. Our results demonstrate the ability of dmPGE2 to act as an effective radiomitigative agent, promoting recovery of HSPC number and enhancing migration of MKs to the endosteum where they play a valuable role in niche restoration.Item S6K1 regulates hematopoietic stem cell self-renewal and leukemia maintenance.(ASCI) Ghosh, Joydeep; Kobayashi, Michihiro; Ramdas, Baskar; Chatterjee, Anindya; Ma, Peilin; Mali, Raghuveer Singh; Carlesso, Nadia; Liu, Yan; Plas, David R.; Chan, Rebecca J.; Kapur, Reuben; Department of Microbiology and Immunology, IU School of MedicineHyperactivation of the mTOR pathway impairs hematopoietic stem cell (HSC) functions and promotes leukemogenesis. mTORC1 and mTORC2 differentially control normal and leukemic stem cell functions. mTORC1 regulates p70 ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E–binding (eIF4E-binding) protein 1 (4E-BP1), and mTORC2 modulates AKT activation. Given the extensive crosstalk that occurs between mTORC1 and mTORC2 signaling pathways, we assessed the role of the mTORC1 substrate S6K1 in the regulation of both normal HSC functions and in leukemogenesis driven by the mixed lineage leukemia (MLL) fusion oncogene MLL-AF9. We demonstrated that S6K1 deficiency impairs self-renewal of murine HSCs by reducing p21 expression. Loss of S6K1 also improved survival in mice transplanted with MLL-AF9–positive leukemic stem cells by modulating AKT and