Browsing by Author "Capitano, Maegan"
Now showing 1 - 10 of 14
Results Per Page
Sort Options
Item Abstract 16: Insights into Highly Engraftable Hematopoietic Cells from 27-Year Cryopreserved Umbilical Cord Blood(Oxford University Press, 2023-09-04) Broxmeyer, Hal; Luchsinger, Larry; Weinberg, Rona; Jimenez, Alexandra; Masson Frenet, Emeline; van't Hof, Wouter; Capitano, Maegan; Hillyer, Christopher; Kaplan, Mark; Cooper, Scott; Ropa, James; Microbiology and Immunology, School of MedicineIntroduction: Cord blood banking has consistently outpaced the utilization of cord blood units (CBUs). Thus, the average duration of cryopreservation among banked CBUs will likely continue to increase. It remains unclear how long cryopreserved CBUs remain functional, and it is critical to determine whether duration of cryopreservation should be used as an exclusionary criterion during selection for clinical use or if alternative post-thaw metrics can identify potent cryopreserved CBUs regardless of age. Objectives: Our goal was to determine whether long-term (27-year) cryopreserved CBUs retain viable and functional hematopoietic stem (HSCs) and progenitor cells (HPCs). We further sought to leverage differences in HSC/HPC function (measured by in vivo engraftment) to demonstrate the utility of using omics approaches to identify candidate genes for use as molecular potency markers. Methods: We performed comprehensive ex vivo, in vivo, and molecular analyses on the numbers, viability, and function of three 27-year cryopreserved CBUs using 3-year cryopreserved and fresh CBUs for comparison. Assays included viability staining, immunophenotyping by flow cytometry, primary and secondary colony forming unit (CFU) assays, ex vivo expansion of immunophenotypic HSCs/HPCs/CFUs, limiting dilution transplantations into immune-deficient mice, secondary transplantations, and RNA-sequencing of sorted HSCs and multipotent progenitor cells. Results: Compared to fresh and recently cryopreserved CBU controls, long-term cryopreserved CBUs yield statistically similar numbers of viable immunophenotypic HSCs, multipotent HPCs, and committed myeloid and lymphoid HPCs. They retain highly functional cells, demonstrating similar primary and secondary CFU numbers and expansion capacity compared to controls, as well as robust engraftment, SCID repopulating cell frequency, and secondary engraftment capacity in mouse models of transplantation. Transcriptomic modelling revealed 18 genes, including MALT1 and MAP2K1, and several gene programs, including lineage determination programs and oxidative stress responses, that are strongly enriched in high engrafting HSCs/HPCs. Discussion: CBUs cryopreserved for up to 27 years retain highly functional HSCs/HPCs. Thus, duration of cryopreservation alone is not an ideal exclusionary criteria for selection of CBUs. Preserving older CBUs may help to maintain a large and diverse pool of donors for clinical selection. Further, transcriptomics can identify candidate genes associated with engraftment for elucidation of possible CBU potency markers regardless of the duration of cryopreservation.Item 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 MedicineIntroduction: 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.Item 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 MedicineIntroduction: 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.Item Age-related decline in LEPR+ hematopoietic stem cell function(Springer Nature, 2023) Trinh, Thao; Ropa, James; Cooper, Scott; Aljoufi, Arafat; Sinn, Anthony; Capitano, Maegan; Broxmeyer, Hal E.; Kaplan, Mark H.; Microbiology and Immunology, School of MedicineItem 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 MedicineBone 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.Item Chromatin Remodeling Subunit BRM and Valine Regulate Hematopoietic Stem/Progenitor Cell Function and Self-Renewal Via Intrinsic and Extrinsic Effects(Springer Nature, 2022) Naidu, Samisubbu R.; Capitano, Maegan; Ropa, James; Cooper, Scott; Huang, Xinxin; Broxmeyer, Hal E.; Microbiology and Immunology, School of MedicineLittle is known of hematopoietic stem (HSC) and progenitor (HPC) cell self-renewal. The role of Brahma (BRM), a chromatin remodeler, in HSC function is unknown. Bone marrow (BM) from Brm-/- mice manifested increased numbers of long- and short-term HSCs, GMPs, and increased numbers and cycling of functional HPCs. However, increased Brm-/- BM HSC numbers had decreased secondary and tertiary engraftment, suggesting BRM enhances HSC self-renewal. Valine was elevated in lineage negative Brm-/- BM cells, linking intracellular valine with Brm expression. Valine enhanced HPC colony formation, replating of human cord blood (CB) HPC-derived colonies, mouse BM and human CB HPC survival in vitro, and ex vivo expansion of normal mouse BM HSCs and HPCs. Valine increased oxygen consumption rates of WT cells. BRM through CD98 was linked to regulated import of branched chain amino acids, such as valine, in HPCs. Brm-/- LSK cells exhibited upregulated interferon response/cell cycle gene programs. Effects of BRM depletion are less apparent on isolated HSCs compared to HSCs in the presence of HPCs, suggesting cell extrinsic effects on HSCs. Thus, intracellular valine is regulated by BRM expression in HPCs, and the BRM/valine axis regulates HSC and HPC self-renewal, proliferation, and possibly differentiation fate decisions.Item Deficits in Our Understanding of Natural Killer Cell Development in Mouse and Human(Wolters Kluwer, 2023) Schorr, Christopher; Krishnan, Maya Shraddha; Capitano, Maegan; Microbiology and Immunology, School of MedicinePurpose of review: Natural killer (NK) cells are a type of immune cell that play a crucial role in the defense against cancer and viral infections. The development and maturation of NK cells is a complex process, involving the coordination of various signaling pathways, transcription factors, and epigenetic modifications. In recent years, there has been a growing interest in studying the development of NK cells. In this review, we discuss the field's current understanding of the journey a hematopoietic stem cell takes to become a fully mature NK cell and detail the sequential steps and regulation of conventional NK leukopoiesis in both mice and humans. Recent findings: Recent studies have highlighted the significance of defining NK development stages. Several groups report differing schema to identify NK cell development and new findings demonstrate novel ways to classify NK cells. Further investigation of NK cell biology and development is needed, as multiomic analysis reveals a large diversity in NK cell development pathways. Summary: We provide an overview of current knowledge on the development of NK cells, including the various stages of differentiation, the regulation of development, and the maturation of NK cells in both mice and humans. A deeper understanding of NK cell development has the potential to provide insights into new therapeutic strategies for the treatment of diseases such as cancer and viral infections.Item 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 MedicineDipeptidylpeptidase 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.Item 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 MedicineObjective: 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.Item Modulation of Hematopoietic Chemokine Effects In Vitro and In Vivo by DPP-4/CD26(Mary Ann Liebert, 2016-04-15) Broxmeyer, Hal E.; Capitano, Maegan; Campbell, Timothy B.; Hangoc, Giao; Cooper, Scott; Department of Microbiology and Immunology, School of MedicineDipeptidyl peptidase 4 (DPP4)/CD26 truncates certain proteins, and this posttranslational modification can influence their activity. Truncated (T) colony-stimulating factors (CSFs) are decreased in potency for stimulating proliferation of hematopoietic progenitor cells (HPCs). T-CXCL12, a modified chemokine, is inactive as an HPC chemotactic, survival, and enhancing factor for replating or ex-vivo expansion of HPCs. Moreover, T-CSFs and T-CXCL12 specifically downmodulates the positively acting effects of their own full-length molecule. Other chemokines have DPP4 truncation sites. In the present study, we evaluated effects of DPP4 inhibition (by Diprotin A) or gene deletion of HPC on chemokine inhibition of multicytokine-stimulated HPC, and on chemokine-enhancing effects on single CSF-stimulated HPC proliferation, as well as effects of DPP4 treatment of a number of chemokines. Myelosuppressive effects of chemokines with, but not without, a DPP4 truncation site were greatly enhanced in inhibitory potency by pretreating target bone marrow (BM) cells with Diprotin A, or by assaying their activity on dpp4/cd26(-/-) BM cells. DPP4 treatment of myelosuppressive chemokines containing a DPP4 truncation site produced a nonmyelosuppressive molecule, but one which had the capacity to block suppression by that unmodified chemokine both in vitro and in vivo. Additionally, DPP4 treatment ablated the single cytokine-stimulated HPC-enhancing activity of CCL3/MIP-1α and CCL4/MIP-1β, and blocked the enhancing activity of each unmodified molecule, in vitro and in vivo. These results highlight the functional posttranslational modulating effects of DPP4 on chemokine activities, and information offering additional biological insight into chemokine regulation of hematopoiesis.