Browsing by Author "Van't Hof, Wouter"

Now showing 1 - 5 of 5
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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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    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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    Insights into highly engraftable hematopoietic cells from 27-year cryopreserved umbilical cord blood
    (Elsevier, 2023) Broxmeyer, Hal E.; Luchsinger, Larry L.; Singer Weinberg, Rona; Jimenez, Alexandra; Masson Frenet, Emeline; Van't Hof, Wouter; Capitano, Maegan L.; Hillyer, Christopher D.; Kaplan, Mark H.; Cooper, Scott; Ropa, James; Microbiology and Immunology, School of Medicine
    Umbilical cord blood transplantation is a life-saving treatment for malignant and non-malignant hematologic disorders. It remains unclear how long cryopreserved units remain functional, and the length of cryopreservation is often used as a criterion to exclude older units. We demonstrate that long-term cryopreserved cord blood retains similar numbers of hematopoietic stem and progenitor cells compared with fresh and recently cryopreserved cord blood units. Long-term cryopreserved units contain highly functional cells, yielding robust engraftment in mouse transplantation models. We also leverage differences between units to examine gene programs associated with better engraftment. Transcriptomic analyses reveal that gene programs associated with lineage determination and oxidative stress are enriched in high engrafting cord blood, revealing potential molecular markers to be used as potency markers for cord blood unit selection regardless of length of cryopreservation. In summary, cord blood units cryopreserved for extended periods retain engrafting potential and can potentially be used for patient treatment.