Browsing by Author "Cooper, Scott"

Now showing 1 - 10 of 17
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    Activation of OCT4 enhances ex vivo expansion of human cord blood hematopoietic stem and progenitor cells by regulating HOXB4 expression
    (SpringerNature, 2016-01) Huang, Xinxin; Lee, Man-Ryul; Cooper, Scott; Hangoc, Giao; Hong, Ki-Sung; Chung, Hyung-Min; Broxmeyer, Hal E.; Department of Microbiology & Immunology, IU School of Medicine
    Although hematopoietic stem cells (HSC) are the best characterized and the most clinically used adult stem cells, efforts are still needed to understand how to best ex vivo expand these cells. Here we present our unexpected finding that OCT4 is involved in the enhancement of cytokine-induced expansion capabilities of human cord blood (CB) HSC. Activation of OCT4 by Oct4-activating compound 1 (OAC1) in CB CD34(+) cells enhanced ex vivo expansion of HSC, as determined by a rigorously defined set of markers for human HSC, and in vivo short-term and long-term repopulating ability in NSG mice. Limiting dilution analysis revealed that OAC1 treatment resulted in 3.5-fold increase in the number of SCID repopulating cells (SRCs) compared with that in day 0 uncultured CD34(+) cells and 6.3-fold increase compared with that in cells treated with control vehicle. Hematopoietic progenitor cells, as assessed by in vitro colony formation, were also enhanced. Furthermore, we showed that OAC1 treatment led to OCT4-mediated upregulation of HOXB4. Consistently, siRNA-mediated knockdown of HOXB4 expression suppressed effects of OAC1 on ex vivo expansion of HSC. Our study has identified the OCT4-HOXB4 axis in ex vivo expansion of human CB HSC.
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    Ames hypopituitary dwarf mice demonstrate imbalanced myelopoiesis between bone marrow and spleen
    (Elsevier, 2015-06) Capitano, Maegan L.; Chitteti, Brahmananda R.; Cooper, Scott; Srour, Edward F.; Bartke, Andrzej; Broxmeyer, Hal E.; Department of Microbiology & Immunology, IU School of Medicine
    Ames hypopituitary dwarf mice are deficient in growth hormone, thyroid-stimulating hormone, and prolactin. The phenotype of these mice demonstrates irregularities in the immune system with skewing of the normal cytokine milieu towards a more anti-inflammatory environment. However, the hematopoietic stem and progenitor cell composition of the bone marrow (BM) and spleen in Ames dwarf mice has not been well characterized. We found that there was a significant decrease in overall cell count when comparing the BM and spleen of 4-5 month old dwarf mice to their littermate controls. Upon adjusting counts to differences in body weight between the dwarf and control mice, the number of granulocyte-macrophage progenitors, confirmed by immunophenotyping and colony-formation assay was increased in the BM. In contrast, the numbers of all myeloid progenitor populations in the spleen were greatly reduced, as confirmed by colony-formation assays. This suggests that there is a shift of myelopoiesis from the spleen to the BM of Ames dwarf mice; however, this shift does not appear to involve erythropoiesis. The reasons for this unusual shift in spleen to marrow hematopoiesis in Ames dwarf mice are yet to be determined but may relate to the decreased hormone levels in these mice.
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    DPP4 Truncated GM-CSF & IL-3 Manifest Distinct Receptor Binding & Regulatory Functions Compared to their Full Length Forms
    (Nature Publishing group, 2017-11) O’Leary, Heather Ann; Capitano, Maegan; Cooper, Scott; Mantel, Charlie; Boswell, H. Scott; Kapur, Reuben; Ramdas, Baskar; Chan, Rebecca; Deng, Lisa; Qu, Cheng-Kui; Broxmeyer, Hal E.; Microbiology and Immunology, School of Medicine
    Dipeptidylpeptidase 4 (DPP4/CD26) enzymatically cleaves select penultimate amino acids of proteins, including colony stimulating factors (CSFs), and has been implicated in cellular regulation. To better understand the role of DPP4 regulation of hematopoiesis, we analyzed the activity of DPP4 on the surface of immature blood cells and then comparatively assessed the interactions and functional effects of full-length (FL) and DPP4 truncated factors [(T)-GM-CSF and- IL-3] on both in vitro and in vivo models of normal and leukemic cells. T-GM-CSF and T-IL-3 had enhanced receptor binding, but decreased CSF activity, compared to their FL forms. Importantly, T-GM-CSF and T-IL-3 significantly, and reciprocally, blunted receptor binding and myeloid progenitor cell proliferation activity of both FL-GM-CSF and FL-IL-3 in vitro and in vivo. Similar effects were apparent in vitro using cluster forming cells from patients with Acute Myeloid Leukemia (AML) regardless of cytogenetic or molecular alterations and in vivo utilizing animal models of leukemia. This suggests that DPP4 T-molecules have modified binding and functions compared to their FL counterparts and may serve regulatory roles in normal and malignant hematopoiesis.
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    Effects of Eupalinilide E and UM171, Alone and in Combination on Cytokine Stimulated Ex-Vivo Expansion of Human Cord Blood Hematopoietic Stem Cells
    (Elsevier, 2020-09) Zhang, Jing; Huang, Xinxin; Guo, Bin; Cooper, Scott; Capitano, Maegan L.; Johnson, Trevor C.; Siegel, Dionicio R.; Broxmeyer, Hal E.; Microbiology and Immunology, School of Medicine
    Eupalinilide E was assessed for ex-vivo expansion activity on hematopoietic stem cells (HSCs) from human cord blood (CB) CD34+ cells in serum-free, SCF, TPO and FL stimulated 7 day cultures. Eupalinilide E ex-vivo enhanced phenotyped (p) HSCs and glycolysis of CD34+ cells isolated 7 days after culture as measured by extracellular acidification rate, but did not alone show enhanced NSG engrafting capability of HSCs as determined by chimerism and numbers of SCID Repopulating cells, a quantitative measure of functional human HSCs. This is another example of pHSCs not necessarily recapitulating functional activity of these cells. Lack of effect on engrafting HSCs may be due to a number of possibilities, including down regulation of CXCR4 or of the homing capacity of these treated cells. However, Eupalinilide did act in an additive to synergistic fashion with UM171 to enhance ex vivo expansion of both pHSCs, and functionally engrafting HSCs. While reasons for the disconnect between pHSC and function of HSCs with Eupalinilide E alone cultured CB CD34+ cells is yet to be determined, the data suggest possible future use of Eupalinilide and UM171 together to enhance ex vivo production of CB HSCs for clinical hematopoietic cell transplantation.
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    Enhanced Collection of Phenotypic and Engrafting Human Cord Blood Hematopoietic Stem Cells at 4°C
    (Oxford University Press, 2020-10) Broxmeyer, Hal E.; Cooper, Scott; Capitano, Maegan L.; Microbiology and Immunology, School of Medicine
    The number of hematopoietic stem cells (HSCs) collected in cord blood (CB) at the birth of a baby is a limiting factor for efficacious use of CB in hematopoietic cell transplantation (HCT). We now demonstrate that collecting and processing of human CB at 4°C within minutes of the baby's birth results in significantly enhanced numbers of rigorously defined phenotypic HSC and self-renewing NSG immune-deficient mouse engrafting and SCID-repopulating cells. This was associated with decreased numbers of hematopoietic progenitor cells (HPC), as noted previously for hypoxia collected/processed cells blocking ambient air induced differentiation of HSC to HPC. We have thus defined a simple, cost-effective, means to collect increased numbers of CB HSC, of potential use for clinical CB HCT.
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    Enhancing Hematopoietic Stem Cell Transplantation Efficacy by Mitigating Oxygen Shock
    (Elsevier, 2015-06-18) Mantel, Charlie R.; O'Leary, Heather A.; Chitteti, Brahmananda R.; Huang, XinXin; Cooper, Scott; Hangoc, Giao; Brustovetsky, Nickolay; Srour, Edward F.; Lee, Man-Ryul; Messina-Graham, Steve; Haas, David M.; Falah, Nadia; Kapur, Reuben; Pelus, Louis M.; Bardeesy, Nabeel; Fitamant, Julien; Ivan, Mircea; Kim, Kye-Seong; Broxmeyer, Hal E.; Department of Microbiology & Immunology, IU School of Medicine
    Hematopoietic stem cells (HSCs) reside in hypoxic niches within bone marrow and cord blood. Yet, essentially all HSC studies have been performed with cells isolated and processed in non-physiologic ambient air. By collecting and manipulating bone marrow and cord blood in native conditions of hypoxia, we demonstrate that brief exposure to ambient oxygen decreases recovery of long-term repopulating HSCs and increases progenitor cells, a phenomenon we term extraphysiologic oxygen shock/stress (EPHOSS). Thus, true numbers of HSCs in the bone marrow and cord blood are routinely underestimated. We linked ROS production and induction of the mitochondrial permeability transition pore (MPTP) via cyclophilin D and p53 as mechanisms of EPHOSS. The MPTP inhibitor cyclosporin A protects mouse bone marrow and human cord blood HSCs from EPHOSS during collection in air, resulting in increased recovery of transplantable HSCs. Mitigating EPHOSS during cell collection and processing by pharmacological means may be clinically advantageous for transplantation.
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    Glucocorticoid hormone-induced chromatin remodeling enhances human hematopoietic stem cell homing and engraftment
    (Nature Publishing Group, 2017-04) Guo, Bin; Huang, Xinxin; Cooper, Scott; Broxmeyer, Hal E.; Microbiology and Immunology, School of Medicine
    Efficient hematopoietic stem cell (HSC) homing is important for hematopoietic cell transplantation (HCT), especially when HSC numbers are limited, as in the use of cord blood (CB). In a screen of small-molecule compounds, we identified glucocorticoid (GC) hormone signaling as an activator of CXCR4 expression in human CB HSCs and hematopoietic progenitor cells (HPCs). Short-term GC pretreatment of human CB HSCs and HPCs promoted SDF-1-CXCR4-axis-mediated chemotaxis, homing, and long-term engraftment when these cells were transplanted into primary- and secondary-recipient NSG mice. Mechanistically, activated glucocorticoid receptor binds directly to a glucocorticoid response element in the CXCR4 promoter and recruits the SRC-1-p300 complex to promote H4K5 and H4K16 histone acetylation, facilitating transcription of CXCR4. These results suggest a new and readily available means to enhance the clinical efficacy of CB HCT.
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    Human Hematopoietic Stem, Progenitor, and Immune Cells Respond Ex Vivo to SARS-CoV-2 Spike Protein
    (Springer, 2021-02) Ropa, James; Cooper, Scott; Capitano, Maegan L.; Van't Hof, Wouter; Broxmeyer, Hal E.; Microbiology and Immunology, School of Medicine
    Despite evidence that SARS-CoV-2 infection is systemic in nature, there is little known about the effects that SARS-CoV-2 infection or exposure has on many host cell types, including primitive and mature hematopoietic cells. The hematopoietic system is responsible for giving rise to the very immune cells that defend against viral infection and is a source of hematopoietic stem cells (HSCs) and progenitor cells (HPCs) which are used for hematopoietic cell transplantation (HCT) to treat hematologic disorders, thus there is a strong need to understand how exposure to the virus may affect hematopoietic cell functions. We examined the expression of ACE2, to which SARS-CoV-2 Spike (S) protein binds to facilitate viral entry, in cord blood derived HSCs/HPCs and in peripheral blood derived immune cell subtypes. ACE2 is expressed in low numbers of immune cells, higher numbers of HPCs, and up to 65% of rigorously defined HSCs. We also examined effects of exposing HSCs/HPCs and immune cells to SARS-CoV-2 S protein ex vivo. HSCs and HPCs expand less effectively and have less functional colony forming capacity when grown with S protein, while peripheral blood monocytes upregulate CD14 expression and show distinct changes in size and granularity. That these effects are induced by recombinant S protein alone and not the infectious viral particle suggests that simple exposure to SARS-CoV-2 may impact HSCs/HPCs and immune cells via S protein interactions with the cells, regardless of whether they can be infected. These data have implications for immune response to SARS-CoV-2 and for HCT.
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    The IL-33 Receptor/ST2 acts as a positive regulator of functional mouse bone marrow hematopoietic stem and progenitor cells
    (Elsevier, 2020-09) Capitano, Maegan L.; Griesenauer, Brad; Guo, Bin; Cooper, Scott; Paczesny, Sophie; Broxmeyer, Hal E.; Microbiology and Immunology, School of Medicine
    There is a paucity of information on a potential role for the IL-33 receptor/ST2 in the regulation of mouse bone marrow (BM) hematopoietic stem (HSC) and progenitor (HPC) cells. Comparing the BM of st2-/- and wild type (WT) control mice using functional assays, it was found that st2-/- BM cells had poorer engrafting capacity than WT BM in a competitive repopulating assay using congenic mice, with no changes in reconstitution of B-, T- and myeloid cells following transplantation. The BM of st2-/- mice also had fewer granulocyte-macrophage, erythroid, and multipotential progenitors than that of WT BM and these st2-/- HPC were in a slow cycling state compared to that of the rapidly cycling HPC of the WT mice. While functional assessment of HSC and HPC demonstrated that ST2 has a positive influence on regulation of HSC, we could not pick up differences in st2-/- compared to WT BM using only phenotypic analysis of HSC and HPC populations prior to transplantation, again demonstrating that phenotypic analysis of HSC and HPC do not always recapitulate the functional assessments of these immature hematopoietic cells.
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    Mild Heat Treatment Primes Human CD34(+) Cord Blood Cells for Migration Toward SDF-1α and Enhances Engraftment in an NSG Mouse Model
    (Wiley Blackwell (John Wiley & Sons), 2015-06) Capitano, Maegan L.; Hangoc, Giao; Cooper, Scott; Broxmeyer, Hal E.; Department of Microbiology and Immunology, IU School of Medicine
    Simple efforts are needed to enhance cord blood (CB) transplantation. We hypothesized that short-term exposure of CD34(+) CB cells to 39.5°C would enhance their response to stromal-derived factor-1 (SDF-1), by increasing lipid raft aggregation and CXCR4 expression, thus leading to enhanced engraftment. Mild hyperthermia (39.5°C) significantly increased the percent of CD34(+) CB that migrated toward SDF-1. This was associated with increased expression of CXCR4 on the cells. Mechanistically, mild heating increased the percent of CD34(+) cells with aggregated lipid rafts and enhanced colocalization of CXCR4 within lipid raft domains. Using methyl-β-cyclodextrin (MβCD), an agent that blocks lipid raft aggregation, it was determined that this enhancement in chemotaxis was dependent upon lipid raft aggregation. Colocalization of Rac1, a GTPase crucial for cell migration and adhesion, with CXCR4 to the lipid raft was essential for the effects of heat on chemotaxis, as determined with an inhibitor of Rac1 activation, NSC23766. Application-wise, mild heat treatment significantly increased the percent chimerism as well as homing and engraftment of CD34(+) CB cells in sublethally irradiated non-obese diabetic severe combined immunodeficiency IL-2 receptor gamma chain d (NSG) mice. Mild heating may be a simple and inexpensive means to enhance engraftment following CB transplantation in patients.