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  1. Article ; Online: Assay optimization for the objective quantification of human multilineage colony-forming units.

    Thompson, Evrett N / Carlino, Maximillian J / Scanlon, Vanessa M / Grimes, H Leighton / Krause, Diane S

    Experimental hematology

    2023  Volume 124, Page(s) 36–44.e3

    Abstract: Colony-forming unit (CFU) assays are a powerful tool in hematopoietic research because they allow researchers to functionally test the lineage potential of individual stem and progenitor cells. Assaying for lineage potential is important for determining ... ...

    Abstract Colony-forming unit (CFU) assays are a powerful tool in hematopoietic research because they allow researchers to functionally test the lineage potential of individual stem and progenitor cells. Assaying for lineage potential is important for determining and validating the identity of progenitor populations isolated by methods such as fluorescence-activated cell sorting (FACS). However, current methods for CFU assays are limited in their ability to robustly assay multipotent progenitors with the ability to differentiate down the myeloid, erythroid, and megakaryocytic lineages because of the lack of specific growth factors necessary for certain lineage outputs. In addition, manual counting of colony types is subjective resulting in user to user variability in assessments of cell types based on colony and cell morphologies. We demonstrate that the addition of granulocyte colony-stimulating factor (G-CSF), macrophage (M)-CSF, and granulocyte-macrophage (GM)-CSF into a collagen-based MegaCult medium containing IL-3, IL-6, SCF, EPO, and TPO allows for the differentiation of common myeloid progenitors into expected proportions of colonies containing granulocytic (G), monocytic (M), erythroid (E), and megakaryocytic (Mk) cells. Additionally, we demonstrate an objective method using in situ immunofluorescence (IF) with anti-CD66b, anti-CD14, anti-CD235a, and anti-CD41 to detect G, M, E, and Mk cells, respectively. IF stained colonies can be analyzed individually at a microscope or using high-throughput microscopy. Thus, our improvements to the culture conditions and method for assay readout increase the accuracy, reproducibility, and throughput of the myeloid CFU assay.
    MeSH term(s) Humans ; Interleukin-3 ; Reproducibility of Results ; Granulocyte-Macrophage Colony-Stimulating Factor ; Hematopoietic Stem Cells ; Colony-Forming Units Assay ; Cells, Cultured
    Chemical Substances Interleukin-3 ; Granulocyte-Macrophage Colony-Stimulating Factor (83869-56-1)
    Language English
    Publishing date 2023-06-02
    Publishing country Netherlands
    Document type Journal Article ; Research Support, N.I.H., Extramural
    ZDB-ID 185107-x
    ISSN 1873-2399 ; 0531-5573 ; 0301-472X
    ISSN (online) 1873-2399
    ISSN 0531-5573 ; 0301-472X
    DOI 10.1016/j.exphem.2023.05.007
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    In stock of ZB MED Cologne/Königswinter

    Zs.A 922: Show issues Location:
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  2. Article: Unravelling human hematopoietic progenitor cell diversity through association with intrinsic regulatory factors.

    Favaro, Patricia / Glass, David R / Borges, Luciene / Baskar, Reema / Reynolds, Warren / Ho, Daniel / Bruce, Trevor / Tebaykin, Dmitry / Scanlon, Vanessa M / Shestopalov, Ilya / Bendall, Sean C

    bioRxiv : the preprint server for biology

    2023  

    Abstract: Hematopoietic stem and progenitor cell (HSPC) transplantation is an essential therapy for hematological conditions, but finer definitions of human HSPC subsets with associated function could enable better tuning of grafts and more routine, lower-risk ... ...

    Abstract Hematopoietic stem and progenitor cell (HSPC) transplantation is an essential therapy for hematological conditions, but finer definitions of human HSPC subsets with associated function could enable better tuning of grafts and more routine, lower-risk application. To deeply phenotype HSPCs, following a screen of 328 antigens, we quantified 41 surface proteins and functional regulators on millions of CD34+ and CD34- cells, spanning four primary human hematopoietic tissues: bone marrow, mobilized peripheral blood, cord blood, and fetal liver. We propose more granular definitions of HSPC subsets and provide new, detailed differentiation trajectories of erythroid and myeloid lineages. These aspects of our revised human hematopoietic model were validated with corresponding epigenetic analysis and
    Language English
    Publishing date 2023-08-30
    Publishing country United States
    Document type Preprint
    DOI 10.1101/2023.08.30.555623
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  3. Article ; Online: Epithelial (E)-Cadherin is a Novel Mediator of Platelet Aggregation and Clot Stability.

    Scanlon, Vanessa M / Teixeira, Alexandra M / Tyagi, Tarun / Zou, Siying / Zhang, Ping-Xia / Booth, Carmen Jane / Kowalska, M Anna / Bao, Jialing / Hwa, John / Hayes, Vincent / Marks, Michael S / Poncz, Mortimer / Krause, Diane S

    Thrombosis and haemostasis

    2019  Volume 119, Issue 5, Page(s) 744–757

    Abstract: Cadherins play a major role in mediating cell-cell adhesion, which shares many parallels with platelet-platelet interactions during aggregate formation and clot stabilization. Platelets express epithelial (E)-cadherin, but its contribution to platelet ... ...

    Abstract Cadherins play a major role in mediating cell-cell adhesion, which shares many parallels with platelet-platelet interactions during aggregate formation and clot stabilization. Platelets express epithelial (E)-cadherin, but its contribution to platelet function and/or platelet production is currently unknown. To assess the role of E-cadherin in platelet production and function in vitro and in vivo, we utilized a megakaryocyte-specific E-cadherin knockout mouse model. Loss of E-cadherin in megakaryocytes does not affect megakaryocyte maturation, platelet number or size. However, platelet dysfunction in the absence of E-cadherin is revealed when conditional knockout mice are challenged with acute antibody-mediated platelet depletion. Unlike wild-type mice that recover fully, knockout mice die within 72 hours post-antibody administration, likely from haemorrhage. Furthermore, conditional knockout mice have prolonged tail bleeding times, unstable clot formation, reduced clot retraction and reduced fibrin deposition in in vivo injury models. Murine platelet aggregation in vitro in response to thrombin and thrombin receptor activating peptide is compromised in E-cadherin null platelets, while aggregation in response to adenosine diphosphate (ADP) is not significantly different. Consistent with this, in vitro aggregation of primary human platelets in response to thrombin is decreased by an inhibitory E-cadherin antibody. Integrin activation and granule secretion in response to ADP and thrombin are not affected in E-cadherin null platelets, but Akt and glycogen synthase kinase 3β (GSK3β) activation are attenuated, suggesting a that E-cadherin contributes to aggregation, clot stabilization and retraction that is mediated by phosphoinositide 3-kinase/Akt/GSK3β signalling. In summary, E-cadherin plays a salient role in platelet aggregation and clot stability.
    MeSH term(s) Animals ; Bleeding Time ; Blood Coagulation ; Blood Platelets/physiology ; Cadherins/genetics ; Cadherins/metabolism ; Cell Adhesion ; Cells, Cultured ; Humans ; Liver/pathology ; Megakaryocytes/physiology ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Mice, Transgenic ; Platelet Aggregation ; Signal Transduction ; Thrombin/metabolism ; Thrombosis/metabolism
    Chemical Substances Cadherins ; Thrombin (EC 3.4.21.5)
    Language English
    Publishing date 2019-03-12
    Publishing country Germany
    Document type Journal Article
    ZDB-ID 518294-3
    ISSN 2567-689X ; 0340-6245
    ISSN (online) 2567-689X
    ISSN 0340-6245
    DOI 10.1055/s-0039-1679908
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    In stock of ZB MED Cologne/Königswinter

    Uh III Zs.192: Show issues Location:
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  4. Article ; Online: Multiparameter analysis of timelapse imaging reveals kinetics of megakaryocytic erythroid progenitor clonal expansion and differentiation.

    Scanlon, Vanessa M / Thompson, Evrett N / Lawton, Betty R / Kochugaeva, Maria / Ta, Kevinminh / Mayday, Madeline Y / Xavier-Ferrucio, Juliana / Kang, Elaine / Eskow, Nicole M / Lu, Yi-Chien / Kwon, Nayoung / Laumas, Anisha / Cenci, Matthew / Lawrence, Kalyani / Barden, Katie / Larsuel, Shannon T / Reed, Fiona E / Peña-Carmona, Gabriela / Ubbelohde, Ashley /
    Lee, June P / Boobalan, Shakthi / Oppong, Yvette / Anderson, Rachel / Maynard, Colby / Sahirul, Kaylie / Lajeune, Callista / Ivathraya, Varsha / Addy, Tiffany / Sanchez, Patricia / Holbrook, Colin / Van Ho, Andrew Tri / Duncan, James S / Blau, Helen M / Levchenko, Andre / Krause, Diane S

    Scientific reports

    2022  Volume 12, Issue 1, Page(s) 16218

    Abstract: Single-cell assays have enriched our understanding of hematopoiesis and, more generally, stem and progenitor cell biology. However, these single-end-point approaches provide only a static snapshot of the state of a cell. To observe and measure dynamic ... ...

    Abstract Single-cell assays have enriched our understanding of hematopoiesis and, more generally, stem and progenitor cell biology. However, these single-end-point approaches provide only a static snapshot of the state of a cell. To observe and measure dynamic changes that may instruct cell fate, we developed an approach for examining hematopoietic progenitor fate specification using long-term (> 7-day) single-cell time-lapse imaging for up to 13 generations with in situ fluorescence staining of primary human hematopoietic progenitors followed by algorithm-assisted lineage tracing. We analyzed progenitor cell dynamics, including the division rate, velocity, viability, and probability of lineage commitment at the single-cell level over time. We applied a Markov probabilistic model to predict progenitor division outcome over each generation in culture. We demonstrated the utility of this methodological pipeline by evaluating the effects of the cytokines thrombopoietin and erythropoietin on the dynamics of self-renewal and lineage specification in primary human bipotent megakaryocytic-erythroid progenitors (MEPs). Our data support the hypothesis that thrombopoietin and erythropoietin support the viability and self-renewal of MEPs, but do not affect fate specification. Thus, single-cell tracking of time-lapse imaged colony-forming unit assays provides a robust method for assessing the dynamics of progenitor self-renewal and lineage commitment.
    MeSH term(s) Cell Differentiation ; Cell Lineage ; Erythropoietin/pharmacology ; Humans ; Megakaryocytes ; Thrombopoietin/pharmacology
    Chemical Substances Erythropoietin (11096-26-7) ; Thrombopoietin (9014-42-0)
    Language English
    Publishing date 2022-09-28
    Publishing country England
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 2615211-3
    ISSN 2045-2322 ; 2045-2322
    ISSN (online) 2045-2322
    ISSN 2045-2322
    DOI 10.1038/s41598-022-19013-x
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  5. Article: Epithelial (E)-Cadherin is a Novel Mediator of Platelet Aggregation and Clot Stability

    Scanlon, Vanessa M. / Teixeira, Alexandra M. / Tyagi, Tarun / Zou, Siying / Zhang, Ping-Xia / Booth, Carmen Jane / Kowalska, M. Anna / Bao, Jialing / Hwa, John / Hayes, Vincent / Marks, Michael S. / Poncz, Mortimer / Krause, Diane S.

    Thrombosis and Haemostasis

    2019  Volume 119, Issue 05, Page(s) 744–757

    Abstract: Cadherins play a major role in mediating cell–cell adhesion, which shares many parallels with platelet–platelet interactions during aggregate formation and clot stabilization. Platelets express epithelial (E)-cadherin, but its contribution to platelet ... ...

    Abstract Cadherins play a major role in mediating cell–cell adhesion, which shares many parallels with platelet–platelet interactions during aggregate formation and clot stabilization. Platelets express epithelial (E)-cadherin, but its contribution to platelet function and/or platelet production is currently unknown. To assess the role of E-cadherin in platelet production and function in vitro and in vivo, we utilized a megakaryocyte-specific E-cadherin knockout mouse model. Loss of E-cadherin in megakaryocytes does not affect megakaryocyte maturation, platelet number or size. However, platelet dysfunction in the absence of E-cadherin is revealed when conditional knockout mice are challenged with acute antibody-mediated platelet depletion. Unlike wild-type mice that recover fully, knockout mice die within 72 hours post-antibody administration, likely from haemorrhage. Furthermore, conditional knockout mice have prolonged tail bleeding times, unstable clot formation, reduced clot retraction and reduced fibrin deposition in in vivo injury models. Murine platelet aggregation in vitro in response to thrombin and thrombin receptor activating peptide is compromised in E-cadherin null platelets, while aggregation in response to adenosine diphosphate (ADP) is not significantly different. Consistent with this, in vitro aggregation of primary human platelets in response to thrombin is decreased by an inhibitory E-cadherin antibody. Integrin activation and granule secretion in response to ADP and thrombin are not affected in E-cadherin null platelets, but Akt and glycogen synthase kinase 3β (GSK3β) activation are attenuated, suggesting a that E-cadherin contributes to aggregation, clot stabilization and retraction that is mediated by phosphoinositide 3-kinase/Akt/GSK3β signalling. In summary, E-cadherin plays a salient role in platelet aggregation and clot stability.
    Keywords cadherins ; clot stability ; haemostasis ; platelet aggregation
    Language English
    Publishing date 2019-03-12
    Publisher Georg Thieme Verlag KG
    Publishing place Stuttgart ; New York
    Document type Article
    ZDB-ID 518294-3
    ISSN 2567-689X ; 0340-6245
    ISSN (online) 2567-689X
    ISSN 0340-6245
    DOI 10.1055/s-0039-1679908
    Database Thieme publisher's database

    Full text online

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    In stock of ZB MED Cologne/Königswinter

    Uh III Zs.192: Show issues Location:
    Je nach Verfügbarkeit (siehe Angabe bei Bestand)
    bis Jg. 2021: Bestellungen von Artikeln über das Online-Bestellformular
    ab Jg. 2022: Lesesaal (EG)
    Zs.MO 433: Show issues

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