IU Indianapolis ScholarWorks :: Browsing by Author "Ropa, James"

Browsing by Author "Ropa, James"

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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 Medicine
Introduction: 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.
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.
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.
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 Medicine
An IL-23-STAT4 pathway is required for the proinflammatory function of classical dendritic cells during CNS inflammation
(National Academy of Sciences, 2024) Alakhras, Nada S.; Zhang, Wenwu; Barros, Nicolas; Sharma, Anchal; Ropa, James; Priya, Raj; Yang, X. Frank; Kaplan, Mark H.; Biochemistry and Molecular Biology, School of Medicine
Although many cytokine pathways are important for dendritic cell (DC) development, it is less clear what cytokine signals promote the function of mature dendritic cells. The signal transducer and activator of transcription 4 (STAT4) promotes protective immunity and autoimmunity downstream of proinflammatory cytokines including IL-12 and IL-23. In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), Stat4-/- mice are resistant to the development of inflammation and paralysis. To define whether STAT4 is required for intrinsic signaling in mature DC function, we used conditional mutant mice in the EAE model. Deficiency of STAT4 in CD11c-expressing cells resulted in decreased T cell priming and inflammation in the central nervous system. EAE susceptibility was recovered following adoptive transfer of wild-type bone marrow-derived DCs to mice with STAT4-deficient DCs, but not adoptive transfer of STAT4- or IL-23R-deficient DCs. Single-cell RNA-sequencing (RNA-seq) identified STAT4-dependent genes in DC subsets that paralleled a signature in MS patient DCs. Together, these data define an IL-23-STAT4 pathway in DCs that is key to DC function during inflammatory disease.
CaMKK2 Knockout Bone Marrow Cells Collected/Processed in Low Oxygen (Physioxia) Suggests CaMKK2 as a Hematopoietic Stem to Progenitor Differentiation Fate Determinant
(Springer, 2022) Broxmeyer, Hal E.; Ropa, James; Capitano, Maegan L.; Cooper, Scott; Racioppi, Luigi; Sankar, Uma; Microbiology and Immunology, School of Medicine
Little is known about a regulatory role of CaMKK2 for hematopoietic stem (HSC) and progenitor (HPC) cell function. To assess this, we used Camkk2−/− and wild type (WT) control mouse bone marrow (BM) cells. BM cells were collected/processed and compared under hypoxia (3% oxygen; physioxia) vs. ambient air (~21% oxygen). Subjecting cells collected to ambient air, even for a few minutes, causes a stress that we termed Extra Physiological Shock/Stress (EPHOSS) that causes differentiation of HSCs and HPCs. We consider physioxia collection/processing a more relevant way to assess HSC/HPC numbers and function, as the cells remain in an oxygen tension closer physiologic conditions. Camkk2−/− cells collected/processed at 3% oxygen had positive and negative effects respectively on HSCs (by engraftment using competitive transplantation with congenic donor and competitor cells and lethally irradiated congenic recipient mice), and HPCs (by colony forming assays of CFU-GM, BFU-E, and CFU-GEMM) compared to WT cells processed in ambient air. Thus, with cells collected/processed under physioxia, and therefore never exposed and naïve to ambient air conditions, CaMKK2 not only appears to act as an HSC to HPC differentiation fate determinant, but as we found for other intracellular mediators, the Camkk−/− mouse BM cells were relatively resistant to effects of EPHOSS. This information is of potential use for modulation of WT BM HSCs and HPCs for future clinical advantage.
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 Medicine
Little 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.
Consequences of coronavirus infections for primitive and mature hematopoietic cells: new insights and why it matters
(Wolters Kluwer, 2021) Ropa, James; Trinh, Thao; Aljoufi, Arafat; Broxmeyer, Hal E.; Microbiology and Immunology, School of Medicine
Purpose of review: In recent history there have been three outbreaks of betacoronavirus infections in humans, with the most recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; causing Coronavirus disease 2019 [COVID-19]) outbreak leading to over two million deaths, with a rapidly rising death toll. Much remains unknown about host cells and tissues affected by coronavirus infections, including the hematopoietic system. Here, we discuss the recent findings examining effects that coronavirus infection or exposure has on hematopoietic cells and the clinical implications for these effects. Recent findings: Recent studies have centered on SARS-CoV-2, demonstrating that hematopoietic stem and progenitor cells and mature immune cells may be susceptible to infection and are impacted functionally by exposure to SARS-CoV-2 Spike protein. These findings have important implications regarding hematologic complications arising from COVID-19 and other coronavirus-induced disease, which we discuss here. Summary: Infection with coronaviruses sometimes leads to hematologic complications in patients, and these hematologic complications are associated with poorer prognosis. These hematologic complications may be caused by coronavirus direct infection or impact on primitive hematopoietic cells or mature immune cells, by indirect effects on these cells, or by a combination thereof. It is important to understand how hematologic complications arise in order to seek new treatments to improve patient outcomes.
CXCL15/Lungkine has Suppressive Activity on Proliferation and Expansion of Multi-potential, Erythroid, Granulocyte and Macrophage Progenitors in S-Phase Specific Manner
(Elsevier, 2021) Broxmeyer, Hal E.; Cooper, Scott H.; Ropa, James; Microbiology and Immunology, School of Medicine
Cytokines/chemokines regulate hematopoiesis, most having multiple cell actions. Numerous but not all chemokine family members act as negative regulators of hematopoietic progenitor cell (HPC) proliferation, but very little is known about such effects of the chemokine, CXCL15/Lungkine. We found that CXCL15/Lungkine-/- mice have greatly increased cycling of multi cytokine-stimulated bone marrow and spleen hematopoietic progenitor cells (HPCs: CFU-GM, BFU-E, and CFU-GEMM) and CXCL15 is expressed in many bone marrow progenitor and other cell types. This suggests that CXCL15/Lungkine acts as a negative regulator of the cell cycling of these HPCs in vivo. Recombinant murine CXCL15/Lungkine, decreased numbers of functional HPCs during cytokine-enhanced ex-vivo culture of lineage negative mouse bone marrow cells. Moreover, CXCL15/Lungkine, through S-Phase specific actions, was able to suppress in vitro colony formation of normal wildtype mouse bone marrow CFU-GM, CFU-G, CFU-M, BFU-E, and CFU-GEMM. This clearly identifies the negative regulatory activity of CXCL15/Lungkine on proliferation of multiple types of mouse HPCs.
DEK, a nuclear protein, is chemotactic for hematopoietic stem/progenitor cells acting through CXCR2 and Gαi signaling
(Wiley, 2022) Capitano, Maegan L.; Sammour, Yasser; Ropa, James; Legendre, Maureen; Mor-Vaknin, Nirit; Markovitz, David M.; Microbiology and Immunology, School of Medicine
Few cytokines/growth modulating proteins are known to be chemoattractants for hematopoietic stem (HSC) and progenitor cells (HPC); stromal cell-derived factor 1α (SDF1α/CXCL12) being the most potent known such protein. DEK, a nuclear DNA-binding chromatin protein with hematopoietic cytokine-like activity, is a chemotactic factor attracting mature immune cells. Transwell migration assays were performed to test whether DEK serves as a chemotactic agent for HSC/HPC. DEK induced dose- and time-dependent directed migration of lineage negative (Lin–) Sca-1+ c-Kit+ (LSK) bone marrow (BM) cells, HSCs and HPCs. Checkerboard assays demonstrated that DEK's activity was chemotactic (directed), not chemokinetic (random migration), in nature. DEK and SDF1α compete for HSC/HPC chemotaxis. Blocking CXCR2 with neutralizing antibodies or inhibiting Gαi protein signaling with Pertussis toxin pretreatment inhibited migration of LSK cells toward DEK. Thus, DEK is a novel and rare chemotactic agent for HSC/HPC acting in a direct or indirect CXCR2 and Gαi protein-coupled signaling-dependent manner.

Browsing by Author "Ropa, James"

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