Browsing by Author "Beasley, Lindsay"

Now showing 1 - 6 of 6
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    Abstract 26: The Role of Oxygen in Cord Blood Hematopoietic Stem and Progenitor Cell Expansion and Engraftment
    (Oxford University Press, 2023-09-04) Ropa, James; Gutch, Sarah; Beasley, Lindsay; Van't Hof, Wouter; Kaplan, Mark; Capitan, Maegan; Microbiology and Immunology, School of Medicine
    Introduction: Hematopoietic stem (HSC) and progenitor cells (HPCs) are exposed to differing oxygen tensions ranging from <1% to 21% as they reside in/move through different tissues or are harvested for clinical utility. Functional changes in HSCs/HPCs are induced by acute changes in oxygen tension (e.g., a change in percent of cells in cycle). Objectives: We sought to determine if variable oxygen levels affect expansion and/or functional properties of cord blood (CB) HSCs/HPCs ex vivo and in vivo. Methods: Human CB CD34+ cells were grown in expansion culture +/-UM171, an agonist of HSC self-renewal that expands transplantable CB HSCs, in five oxygen tensions: 1%O2, 3%O2, 5%O2, 14%O2, and 21%O2. HSCs/HPCs were enumerated by flow cytometry. Functional HPCs were enumerated by plating in semi solid media for colony forming unit assays (CFU). Cell cycle and reactive oxygen species (ROS) were measured by flow cytometry. Ability of expanded cells to engraft was determined by transplantation in non-lethally irradiated NSG mice. Results: Immunophenotypic HPCs and functional HPC CFUs expanded significantly more after 7 days of growth in higher oxygen tensions (5%O2-21%O2) compared to lower (1%O2-3%O2), while immunophenotypic HSCs expanded best at 5% O2. HSCs/HPCs grown in low oxygen tensions had significantly lower ROS levels, significantly higher percentage of cells in G0, and were slightly but reproducibly smaller/less granular than those grown in high oxygen levels. HSC/HPC numbers were reduced in high oxygen tensions 1-2 days after plating but were better maintained in low, suggesting cells undergo a culture shock/stress after plating that is mitigated by reduced oxygen. In the presence of UM171, HSCs expanded significantly better at higher oxygen levels, but HPCs are better maintained in 5%O2. Ex vivo CD34+ expansions maintained under physiological O2 levels (1-14%O2) demonstrated significantly better/faster neutrophil recovery following transplantation compared to cells expanded at 21%O2 or input. Discussion: HSCs/HPCs proliferate rapidly in high oxygen but have fewer quiescent cells, higher ROS, and are larger and more granular which are all characteristics associated with exhaustion. While high oxygen allows for faster growth, low tensions may mitigate cell stress and allow for prolonged growth (i.e., HSC/HPC expansion) while maintaining functional properties.
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    Abstract 27: Physiologic Oxygen Expansion Enhances Lymphocyte and Neutrophil Recovery Following Transplantation
    (Oxford University Press, 2024-08-21) Gutch, Sarah; Ropa, Jim; Beasley, Lindsay; Whitacre, Grace; Van't Hof, Wouter; Capitano, Maegan; Medical and Molecular Genetics, School of Medicine
    Introduction: Expeditious recovery of lymphocytes after hematopoietic cell transplantation is a major determinant of patient outcome. There are few efficient clinical therapies to enhance lymphocyte recovery, indicating a clear unmet need. Ex vivo expansion of cord blood (CB) units is an approved therapy to increase numbers of hematopoietic stem and progenitor cells, but impact on lymphocyte recovery remains uncertain. Moreover, culture in physioxic (physiological oxygen) conditions results in increased lymphoid-biased RNA levels. Objectives: We hypothesize that ex vivo expansion in physioxic conditions will increase lymphoid-biased cells and increase lymphocyte counts post-transplantation (PT). The objective of this study is to increase lymphocyte numbers following transplantation without sacrificing reconstitution of potent hematopoietic cells. Methods: Three independent transplants were conducted. 1) Murine lineage- bone marrow (BM) was expanded for 7 days then transplanted into lethally irradiated mice with/without additional common lymphoid progenitors (CLPs). 2) Murine lineage- BM was expanded in 1%, 3%, 5%, 14%, and 21% O2 for 7 days and transplanted into lethally irradiated mice. 3) Human CD34+ CB cells were expanded for 7 days in 1%, 3%, 5%, 14%, and 21% O2 and transplanted into NSG mice. Unexpanded BM or CB cells were used as controls. Results: Additional CLPs did not contribute to lymphocyte recovery. Mice transplanted with expanded BM had increased lymphocyte counts compared to transplantations with unexpanded BM at weeks 2 and 5 PT via complete blood count (CBC) and increased B-cell reconstitution in the spleen, BM, and peripheral blood (PB) at weeks 2, 5, and 8 PT. LSK (Lin- SCA1+ cKIT+) and neutrophil frequencies were increased at 3% O2 in the BM and 5% O2 in the PB, respectively. Compared to 21% O2, CB cells expanded at 1%, 3%, 5%, 14% O2 had increased neutrophil and lymphocyte frequencies in the PB at weeks 2 and 10, respectively, and demonstrated greater recovery than unexpanded at week 2. Discussion: Expansion increases lymphocyte counts via CBC and immunophenotyping. Physioxic expansion increases numbers of potent hematopoietic cell subpopulations and frequencies of specific lymphocyte compartments in multiple organs. Thus, expansion under physioxia is a viable strategy to enhance recovery of lymphocytes PT.
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    Abstract 33: Transcriptomic Identification of Functionally Potent Umbilical Cord Blood Units
    (Oxford University Press, 2024-08-21) Ropa, James; Gutch, Sarah; Beasley, Lindsay; Van't Hof, Wouter; Sun, Jessica; Capitano, Maegan; Kaplan, Mark; Medical and Molecular Genetics, School of Medicine
    Introduction: Umbilical cord blood (UCB) is an important donor source for standard of care cellular therapies as well as innovative new treatments. Universal potency criteria for cord blood unit (CBU) selection for different cellular therapy applications are still desired and efficient methods to elucidate these criteria remain elusive. Objectives: Our goal is to find molecular markers that identify potent CBUs for use in cellular therapies. Here, we utilized transcriptomics to reveal genes associated with hematopoietic stem and progenitor cell (HSC/HPC) potency in hematopoietic cell transplantation. Methods: We performed three separate transcriptomic analyses of human UCB used in mouse models of transplantation. This included bulk RNA-sequencing of HSCs/HPCs from CBUs with known engraftment capacities (n=9 CBUs), bulk RNA-sequencing of homed/early engrafted CD34+ cells (n=3 CBUs), and single cell RNA-sequencing of CD34+ cells expanded in varying oxygen tensions, which affects their transplantation potency (n=4 CBUs). Results: HSCs/HPCs enriched for dehydrogenase and cell cycle associated genes yield better repopulating cell frequency. Early homed CD34+ cells have enriched expression of immune activation and cell cycle genes compared to input transplanted cells. Distinct clusters of UCB cells marked by genes such as PRSS2 and AVP are enriched in oxygenation conditions that drive increased potency. Dehydrogenase and stress response genes are enriched in populations predicted to be more functional regardless of HSC/HPC subpopulation. Integration of all three studies reveals genes that may define highly potent CBUs, including DDIT4, a stress response gene. Indeed, DDIT4 independently predicts engraftment outcomes in mouse models of transplantation. Future work will examine a qPCR based gene panel potency assay to predict outcomes in patient transplantations. Discussion: We have identified genes associated with HSC/HPC potency using transcriptomic approaches. These findings have immediate translational implications for CBU selection for transplantation, but also provides a blueprint for finding CBUs best suited for use in developing off-the-shelf immune effector therapies or those that are best for treating non-hematologic central nervous system disorders such as cerebral palsy, among other applications. Importantly, this study highlights the importance for omics technology as a valuable tool to define potency criteria for UCB as a donor source for cellular therapies.
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    BATF sustains homeostasis and functionality of bone marrow Treg cells to preserve homeostatic regulation of hematopoiesis and development of B cells
    (Frontiers Media, 2023-02-22) Tikka, Chiranjeevi; Beasley, Lindsay; Xu, Chengxian; Yang, Jing; Cooper, Scott; Lechner, Joseph; Gutch, Sarah; Kaplan, Mark H.; Capitano, Maegan; Yang, Kai; Pediatrics, School of Medicine
    Bone marrow Treg cells (BM Tregs) orchestrate stem cell niches crucial for hematopoiesis. Yet little is known about the molecular mechanisms governing BM Treg homeostasis and function. Here we report that the transcription factor BATF maintains homeostasis and functionality of BM Tregs to facilitate homeostatic regulation of hematopoiesis and B cell development. Treg-specific ablation of BATF profoundly compromised proportions of BM Tregs associated with reduced expression of Treg effector molecules, including CD44, ICOS, KLRG1, and TIGIT. Moreover, BATF deficiency in Tregs led to increased numbers of hematopoietic stem cells (HSCs), multipotent progenitors (MPPs), and granulocyte-macrophage progenitors (GMPs), while reducing the functionality of myeloid progenitors and the generation of common lymphoid progenitors. Furthermore, Tregs lacking BATF failed to support the development of B cells in the BM. Mechanistically, BATF mediated IL-7 signaling to promote expression of effector molecules on BM Tregs and their homeostasis. Our studies reveal a previously unappreciated role for BATF in sustaining BM Treg homeostasis and function to ensure hematopoiesis.
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    Glucose intolerance as a consequence of hematopoietic stem cell dysfunction in offspring of obese mice
    (Elsevier, 2024) Denizli, Merve; Ropa, James; Beasley, Lindsay; Ghosh, Joydeep; DeVanna, Kelli; Spice, Taylor; Haneline, Laura S.; Capitano, Maegan; Kua, Kok Lim; Pediatrics, School of Medicine
    Objective: Maternal obesity is increasingly common and negatively impacts offspring health. Children of mothers with obesity are at higher risk of developing diseases linked to hematopoietic system abnormalities and metabolism such as type 2 diabetes. Interestingly, disease risks are often dependent on the offspring's sex, suggesting sex-specific reprogramming effect of maternal obesity on offspring hematopoietic stem and progenitor cell (HSPC) function. However, the impact of maternal obesity exposure on offspring HSPC function, and the capability of HSPC to regulate offspring metabolic health is largely understudied. This study aims to test the hypothesis that offspring of obese mice exhibit sex-differences in HSPC function that affect offspring's metabolic health. Methods: We first assessed bone marrow hematopoietic stem and progenitor cell phenotype using postnatal day 21 (P21) and 8-week-old C57BL/6J mice born to control and diet-induced obese dams. We also sorted HSPC (Lineage-, Sca1+, cKit + cells) from P21 mice for competitive primary and secondary transplant, as well as transcriptomic analysis. Body weight, adiposity, insulin tolerance test and glucose tolerance tests were performed in primary and secondary transplant recipient animals. Results: We discovered sex-differences in offspring HSPC function in response to maternal obesity exposure, where male offspring of obese dams (MatOb) showed decreased HSPC numbers and engraftment, while female MatOb offspring remained largely unaffected. RNA-seq revealed immune stimulatory pathways in female MatOb offspring. Finally, only recipients of male MatOb offspring HSPC exhibited glucose intolerance. Conclusions: This study demonstrated the lasting effect of maternal obesity exposure on offspring HSPC function and implicates HSPC in metabolic regulation.
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    Protocol for enrichment and functional analysis of human hematopoietic cells from umbilical cord blood
    (Elsevier, 2024) Gutch, Sarah; Beasley, Lindsay; Cooper, Scott; Kaplan, Mark H.; Capitano, Maegan L.; Ropa, James; Microbiology and Immunology, School of Medicine
    Umbilical cord blood (CB) is a donor source for hematopoietic cell therapies. Understanding what drives hematopoietic stem and progenitor cell function is critical to our understanding of the usage of CB in hematopoietic cell therapies. Here, we describe how to isolate and analyze the function of human hematopoietic cells from umbilical CB. This protocol demonstrates assays that measure phenotypic properties and hematopoietic cell potency. For complete details on the use and execution of this protocol, please refer to Broxmeyer et al.