Background/Objectives: Glioblastoma is an aggressive primary brain tumor that develops within a chronically low-oxygen microenvironment, yet most preclinical studies are performed under atmospheric oxygen conditions that poorly reflect in vivo physiology. This study investigated how sustained culture under physiological oxygen tension (physioxia) influences glioblastoma cell behavior, signaling, and therapeutic response. Methods: Multiple patient-derived glioblastoma models were cultured under normoxia (21% O2) or sustained physioxia (5% O2) for at least seven days before experimentation. Cell migration, proliferation, cell cycle distribution, expression of the epithelial-to-mesenchymal transition-associated transcription factor Slug (SNAI2), PDGFRβ-associated signaling, and sensitivity to 5-fluorouracil were evaluated using transwell migration assays, cell counting, flow cytometry, RT-qPCR, immunoblotting, and BrdU incorporation assays. To examine the effects of oxygen history, we established and maintained additional patient-derived cultures grown under physioxia. Results: Sustained physioxia consistently increased migration across all glioblastoma models while reducing proliferation in normoxia-adapted cell lines through increased G0/G1 cell cycle arrest. Physioxia significantly increased Slug expression in all models and enhanced PDGFRβ, AKT, and ERK phosphorylation in a cell line-dependent manner. Therapeutic sensitivity to 5-fluorouracil was altered, with physioxia conferring increased resistance in some glioblastoma models but not universally. Patient-derived cell lines cultured continuously under physioxia retained enhanced migratory capacity and exhibited increased proliferation compared to cells grown in normoxia, suggesting that prior oxygen exposure influences proliferative responses while the pro-migratory phenotype remains conserved. Conclusions: Physiological oxygen tension is a major regulator of glioblastoma cell behavior, influencing migration, proliferation, signaling, and therapeutic response. These findings demonstrate that conventional normoxic culture conditions can obscure biologically relevant phenotypes and support the consideration of physioxia in experimental design. Such technical changes may improve the physiological and translational relevance of preclinical glioblastoma research.

Abstract Background Allogeneic hematopoietic cell transplantation is a potentially curative treatment for malignant and non-malignant hematologic disorders, wherein healthy hematopoietic stem (HSC) and progenitor cells (HSCs) reconstitute a functional blood and immune system following ablation of diseased cells. Functionally potent HSCs/HPCs from bone marrow, mobilized peripheral blood, or umbilical cord blood drive recovery of the hematopoietic system following transplantation. Improving initial characterization of the engraftment potential of donor grafts and recovery time will lead to improved patient outcomes, particularly in the case of umbilical cord blood transplantation, which is limited by low total numbers of cells. However, we still lack a complete understanding of the molecular programs that regulate potency, which is the ability of cells to home to and engraft in a hematopoietic niche, then self-renew and differentiate driving multi-lineage repopulation. Here, we use transcriptomic approaches to elucidate gene programs associated with hematopoietic cell potency in CD34+ cells, which are enriched for HSCs/HPCs. Methods 1) We compared transcriptomic differences between immunophenotypically defined HSCs/HPCs, which have different potentials. 2) We examined the transcriptomic profile of “homing capable” human CD34+ cells from transplantation recipient mouse bone marrow compared to all CD34+ cells used for transplantation. 3) We elucidated the transcriptome of Aldehyde dehydrogenase Bright (AldhBr) CD34+ cells, which are clinical indicators of potent cord blood units, compared to more functionally deficient populations. 4) We used single cell RNA-seq to compare the transcriptome of fresh cord blood HSCs/HPCs with those that are predicted to have lost potency due to cell culture. 5) We analyzed the gene expression profile of potent HSCs from cord blood based on their high long-term engraftment SCID repopulating cell frequencies in an in vivo mouse model compared to those with low engraftment capacity. We validated a novel target using recombinant protein or siRNA mediated knock down in CD34+ expansion assays. Results: Integration of all five data sets identified genes and molecular networks that are highly represented in potent HSCs/HPCs defined by different aspects of potency (homing capacity, cell state, and long-term engraftment capacity). Unsurprisingly, we found that pathways associated with stress, interferon responses, and proliferation were associated with altered potency. We also found a consensus across several or all screens that genes associated with mitochondrial regulation, aldehyde dehydrogenase activity, and hypoxia regulated programs were enriched in potent HSCs/HPCs. By computational integration and manual filtering, we identified a list of genes highly associated with potency that includes PRSS2, AVP, SPP1, MDK, SPINK2, CXCL11, ENO1, BEX2 and PIM1, as well as several mitochondrial genes with expression that negatively correlates with potency such as MT-CO2. Interestingly, many of the potency associated genes code for secreted factors, which may indicate a level of HSC/HPC auto-regulation with regards to potency. We found that treatment with a subset of the identified secreted factors or siRNA-mediated knockdown of genes encoding those proteins potency lead to increased or reduced HPC CFU capacity, respectively, confirming their importance in hematopoietic function ex vivo. Conclusions We found gene programs associated with hematopoietic cell potency, and validated a subset as important for hematopoietic function ex vivo. The identified gene networks could be used to develop a new potency assay based on gene expression that would simplify donor unit selection for cell therapies. For example, following potency assay validation, cord blood units could be selected for transplantation based on expression levels of PRSS2, BEX2, ENO1, and AVP. Next, we identified molecular programs that can be stabilized to improve cell function for use in therapies, or that could be targetable vulnerabilities in diseased hematopoiesis. These findings could also be important for other cell therapies such as gene editing or CAR-T or CAR-NK therapies derived from HSCs/HPCs, which also require the creation or maintenance of potent cells. In all, these transcriptomic screens are the first step toward elucidating the complex network of molecular programs that contribute to hematopoietic cell function and potency.

Barth syndrome (BTHS) is an X-linked recessive disorder characterized by cardiomyopathy, skeletal muscle myopathy and fatigue, growth restriction, and neutropenia. Neutropenia increases the risk of life-threatening bacterial infections, a major cause of death in individuals with BTHS. Currently, there is no curative treatment for BTHS or associated neutropenia. The development of therapeutic strategies to correct BTHS-associated neutropenia has been hindered by a limited understanding of the underlying molecular mechanisms involved. BTHS is caused by a mutation in the Tafazzin gene encoding a transacylase required for the maturation of cardiolipin, an inner mitochondrial membrane phospholipid crucial for mitochondrial structure and function. We introduced a BTHS patient’s point mutation (TAZD75H) into the mouse Tafazzin enzyme’s critical acyltransferase site using CRISPR/Cas9-mediated genome editing, resulting in a patient-tailored point mutant knock-in BTHS model (TazD75H) that expresses a stable mutant TazD75H protein lacking transacylase activity. TazD75H mice were then used to investigate how loss of Tafazzin enzymatic activity impacts hematopoiesis. Male TazD75H mice exhibited impaired granulopoiesis and neutropenia secondary to impaired function of hematopoietic progenitors. Furthermore, they demonstrated age-dependent neutrophil maturation impairment reflecting the variable neutropenia observed in BTHS patients. Additionally, male TazD75H mice exhibit chronic lymphopenia that persists post TazD75H bone marrow transplantation. Mechanistically, the TAZD75H point mutation caused hematopoietic cell mitochondrial dysfunction in patient-derived immortalized TAZD75H lymphoblasts, increasing reactive oxygen species production and mitochondrial membrane depolarization. Likewise, Cyclosporine A treatment rescued these mitochondrial phenotypes in vitro, confirming TAZD75H mitochondrial dysfunction. Overall, our findings demonstrate that mitochondrial dysfunction secondary to TAFAZZIN loss of enzymatic function underlies BTHS-associated neutropenia and lymphopenia.

Glioblastoma (GBM) is an aggressive brain tumor with a median survival 15–20 months, despite maximal treatment including surgery, chemotherapy, radiation, and tumor-treating fields. Recurrence is nearly inevitable, and long-term survival remains low. GBM often contains necrotic regions with decreased perfusion and oxygen delivery. Our goal is to better understand how chronic hypoxia affects GBM signaling and cancer phenotypes. Normoxia(~21% O₂) reflects ambient air; physioxia(~5% O₂) more closely resembles physiological oxygen levels in brain tissue. We hypothesized that chronic physioxia alters GBM phenotypes, metabolism, and treatment responses through modulation of key signaling pathways, including ERK. ERK signaling, a key regulator of tumor survival and therapy resistance, is influenced by oxygen levels. Under normoxia, ERK is activated by receptor tyrosine kinases and the PI3K/AKT pathway. Physioxia modulates ERK activity, potentially enhancing resistance via signaling crosstalk. Most GBM studies use short-term hypoxia(<48 hours), but in situ hypoxia is chronic. Here, GBM cells were maintained under physioxia for two passages(> 10 days) prior to experiments for improved clinical relevance. We investigated patient-derived GBM lines (GB10 and GB43) under normoxia and physioxia. Cell proliferation decreased under physioxia, with significantly reduced doubling times in GB10(p=0.0013) and GB43(p=0.0124). Physioxia also decreased cell migration in GB43(p=0.0123, transwell assay). We examined markers of EMT, with RT-PCR showing increased Slug (p=0.0228 at d3) and decreased IGFBP1(p<0.0001 at d7) transcripts in GB43 cells maintained in physioxia. Physioxia also increased phosphorylation of ERK(GB43), PDGFRβ, and AKT (GB10, Western Blot). Physioxia increased chemoresistance to temozolomide in GB10(p<0.001, BrdU). Thus chronic exposure to physioxia alters cell proliferation, migration, and therapeutic responsiveness with correlation to cell signaling via ERK/AKT signaling pathways. Future work will explore how corticosteroids like dexamethasone affect cancer phenotypes and signaling under both oxygen conditions, contributing to a better understanding of cancer cell phenotypes in clinically relevant conditions in GBM.

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.1

Abstract NF2-related schwannomatosis is a tumor predisposition syndrome caused by mutations in the NF2 gene and associated with spinal ependymomas (SP-EPN). SP-EPNs are suspected to originate from aberrations in the radial glia (RG) cell lineage. These tumors are only effectively treated through high-risk surgical resection, emphasizing the critical need for identification of targets for medical therapy. Yet, the role of NF2-dependent disruption in RG cell development is poorly understood. We hypothesize that this mutation may be halting normal RG cell differentiation, resulting in a RG-like stem cell that is driving SP-EPN formation. An NF2-knockout was generated in neuroepithelial stem (NES) cells using CRISPR/Cas9. Knockouts were validated using Western blot and Sanger sequencing. In vitro differentiation was induced with removal of growth factors. NF2-knockout phenotypes were assessed with real-time polymerase chain reaction and compared with wildtype NES cells. Preliminary data has shown that NF2-knockout cells express similar levels of early pan-neural and neural stem cell genes compared to wildtype after CRISPR editing. The NF2-knockout cells retain this stem cell-like gene expression following attempted differentiation, whereas wildtype cells take on a primarily neural phenotype. The knockout cells also form what appears to be pre-neoplastic spheres when allowed to differentiate. NF2-knockout NES cells fail to differentiate normally compared to wildtype NES cells. They retain early RG-like markers, including SOX2, BLBP, and GFAP. Furthermore, formation of spheres when growth factors were removed hints at NF2 loss being relevant to formation of pre-neoplastic growths. Future work will include investigating downstream effects of NF2 loss in this model.

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.

Hematopoietic cell transplantation (HCT) is a curative treatment for hematologic disorders wherein healthy donor hematopoietic stem (HSCs) and progenitor cells (HPCs) collected from bone marrow (BM), mobilized peripheral blood (PB) or umbilical cord blood (CB) reconstitute an ablated hematopoietic system. There is a strong need to enhance functional competency of HSCs/HPCs for HCT to prevent complications like graft versus host disease (GVHD), relapse, and graft failure. Dipeptidyl peptidase 4 (DPP4) is a protein that cleaves N-terminal dipeptides from hematopoietic factors, impacting their function. Inhibition of DPP4 improves HSC/HPC competency in mouse models of HCT and clinically speeds neutrophil recovery after CB transplantation and reduces incidences of GVHD after PB transplantation. Thus, DPP4 inhibition is a viable component of a multi-pronged strategy to improve engraftment and prevent GVHD. While DPP4 inhibition improved early neutrophil recovery in clinical trials, the effects were modest, required high dose treatment, and effects on platelet recovery were unclear. Examining mechanisms by which DPP4 inhibition drives HSC/HPC functional competency may yield insight into targets that can enhance DPP4 driven improvements to HCT. We harvested BM from mice treated with the DPP4 inhibitor Diprotin A (DPA) and sorted immunophenotypically defined HSCs, multipotent progenitors (MPPs), common myeloid progenitors (CMPs), and granulocyte-macrophage progenitors (GMPs) for transcriptomic analysis. Linear modeling to examine differential gene expression controlling for cell type revealed that DPP4 inhibition gave strong enrichment of gene programs associated with mitochondrial function. Using a mitochondrial stress test and the Seahorse metabolic flux analyzer, we found that lineage depleted mouse BM cells treated ex vivo with DPA had lower basal oxygen consumption rates and spare respiratory capacity. These seemingly paradoxical data are consistent with reports that highly functional HSCs have higher mitochondrial mass but exhibit less mitochondrial activity. Thus, DPP4 inhibition may preserve high levels of functional competency by reducing cellular dependence on oxidative phosphorylation. To examine DPP4 inhibition effects on individual subpopulations of HSCs/HPCs, we modeled each cell type separately for differential gene expression. We found that gene programs associated with neutrophil development were significantly enriched in HSCs and MPPs from mice treated with DPA, including genes Elane, Lcn2, Prtn3 and Ctsg/h, concomitant with downregulation of platelet development programs in HSCs and GMPs from mice treated with DPA, including genes Gp6/9, P2ry12 and Clec1b. This suggests that DPP4 drives improved engraftment in part by priming HSCs/HPCs for neutrophil recovery but does not enhance platelet development. We then sought to target a platelet activating pathway in combination with DPP4 inhibition to improve HCT. Recipient mice were treated on days -2, -1, and 0 with 75 mg/kg DPA and day 0 with 50 µg/kg and days 1, 3, 5, and 7 with 25 µg/kg recombinant human Thrombopoietin (TPO), a platelet activating agonist. On day -1 mice were lethally irradiated and transplanted via tail vein with 2.5x10 5 whole BM donor cells on day 0. Complete blood counts 2 weeks following transplantation showed that mice treated with DPA alone exhibited worse platelet recovery, in line with our transcriptomic data. However, mice treated with the combination of DPA and TPO exhibited significantly better platelet recovery than vehicle controls and DPA alone. Further, neutrophil recovery trended towards improved recovery in the combination treatment compared to the single agent treatments. Flow cytometry of the PB revealed no differences in BM homing or in donor PB chimerism one month following HCT in the combination treatment compared to single agents (though all treatments showed improved homing over vehicle controls), suggesting the primary effects were on early recovery and not on initial or long-term engraftment. These data elucidate the DPP4 dependent transcriptome in the hematopoietic compartment, providing molecular insights into genes that drive enhanced functional competency of HSCs/HPCs. We also demonstrate that the DPP4 dependent transcriptome can reveal gene programs that can be targeted in combination to provide additive improvement on transplantation recovery.

Diffuse midline glioma (DMG) is the most lethal of all childhood cancers. DMGs are driven by histone-tail-mutation-mediated epigenetic dysregulation and partner mutations in genes controlling proliferation and migration. One result of this epigenetic and genetic landscape is the overexpression of LIN28B RNA binding protein. In other systems, LIN28B has been shown to prevent let-7 microRNA biogenesis; however, let-7, when available, faithfully suppresses tumorigenic pathways and induces cellular maturation by preventing the translation of numerous oncogenes. Here, we review the current literature on LIN28A/B and the let-7 family and describe their role in gliomagenesis. Future research is then recommended, with a focus on the mechanisms of LIN28B overexpression and localization in DMG.

Background: Medulloblastoma (MB) is the most common intracranial tumor in children. While molecular classification of MB is well-established, detailing cell origin, biological properties, and biomarkers, little research has been performed concerning the MB tumor microenvironment. Hypoxia is significantly associated with tumor spread, poor prognosis, malignant phenotype, and resistance to radiotherapy and chemotherapy in numerous cancer types. The aim of the present study was to assess a possible role for hypoxia in MB and the potential effect on clinical outcomes. Methods: We, therefore, performed a systematic review examining the role of hypoxia in MB as well as pediatric brain tumors in general. In vitro studies have identified a role for HIF-1α in chemotherapy resistance, while patient samples suggest hypoxia-induced changes in gene expression as well as proteomic, metabolomic, and lipidomic profiles. Based on this literature review, 55 candidate hypoxia-related genes were identified. The PedcBioPortal for Integrated Childhood Cancer Genomics was used to assess expression differences in pediatric patient samples for these genes of interest. Results: RNA expression was analyzed for correlation with survival, molecular group, and Chang stage. Expression of DDAH1, HYOU1, MYC, and RBX1 were significantly associated with survival. ANOVA and Ttest with a Bonferroni correction were used to assess for expression differences between groups and Chang stage. Multiple hypoxia candidate genes (ARNT2, BHLHE40, CYP3A5, DDAH1, DDIT4L, EGLN3, MT3, MYC, MYCN, TGFBR2, TP53, VEGFA) were significantly correlated with molecular group. Expression levels of TGFBR3 and GPR37 were associated with Chang stage. Conclusion and Potential Impact: The results of our systematic review and gene expression analyses support a role for hypoxia in the pathogenesis and potentially the clinical outcomes of children with MB. Future studies comparing gene expression levels at normal oxygen tension (21%) and physiologic oxygen tension (1-3%) will allow us to assess the role of hypoxia in medulloblastoma pathogenesis.

Abstract 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.

Experimental hematopoietic stem cell transplantation (HSCT) is an invaluable tool in determining the function and characteristics of hematopoietic stem cells (HSC) from experimental mouse and human donor groups. These groups could include, but are not limited to, genetically altered populations (gene knockout/knockin models), ex vivo manipulated cell populations, or in vivo modulated cell populations. The basic fundamentals of this process involve taking cells from a mouse/human donor source and putting them into another mouse (recipient) after preconditioning of the recipient with either total body irradiation (TBI) for mouse donor cells or into sublethally irradiated immune-deficient mice for human donor cells. Then, at pre-determined time points post-transplant, sampling a small amount of peripheral blood (PB) and at the termination of the evalaution, bone marrow (BM) to determine donor contribution and function by phenotypic analysis. Exploiting the congenic mouse strains of C57BL/6 (CD45.1- CD45.2+), BoyJ (CD45.1+ CD45.2-), and their F1-crossed hybrid C57BL/6 × BoyJ (CD45.1+ CD45.2+), we are able to quantify donor, competitor, and recipient mouse cell contributions to the engraftment state. Human donor cell engraftment (e.g., from the cord blood [CB], mobilized PB, or BM) is assessed by human cell phenotyping in sublethally irradiated immune-deficient mouse recipients (e.g., NOD scid gamma mice that are deficient in B cells, T cells, and natural killer cells and have defective dendritic cells and macrophages). Engraftment of cells from primary mouse recipients into secondary mice allows for an estimation of the self-renewal capacity of the original donor HSC. This chapter outlines concepts, methods, and techniques for mouse and human cell models of HSCT and for assessment of donor cells collected and processed in hypoxia versus ambient air.

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.

Umbilical cord blood (CB) is a promising source of hematopoietic stem cells (HSCs) for hematopoietic cell transplantation (HCT), a life-saving treatment, due to lower incidence of graft versus host disease and its availability. CB HCT is limited by low numbers of HSCs, thus there is a strong need to increase numbers of engraftable HSCs, for instance by expanding functionally competent HSCs ex vivo. We have shown that extracellular DEK protein, an epigenetic regulator that is secreted in response to stress and inflammation, is a novel hematopoietic regulatory chemokine. Treatment with extracellular DEK in growth stimulating media significantly enhances the expansion of engraftable HSCs in mouse bone marrow (BM) and human CB (Capitano, et al. JCI 2019; 129(6):2555-2570). To examine mechanisms driving enhanced expansion of functional HSCs after DEK treatment, we performed RNA-sequencing on CB HSCs, multipotent progenitor cells (MPPs), common myeloid progenitors (CMPs), and granulocyte macrophage progenitors (GMPs) after overnight DEK treatment in expansion media. All cell populations upregulated gene programs related to antioxidant responses upon DEK treatment. Indeed, 7/10 genes upregulated by DEK in all HSC/HPC populations (NMRAL2P, PIR, NQO1, SELENOW, FTL, RIT1, and QPCT) promote antioxidant activity. Overnight DEK treatment significantly reduced mean fluorescence of the activated reactive oxygen species (ROS) indicator CM-H2DCFDA in HSCs, MPPs, CMPs, and GMPs. Thus, DEK may induce antioxidant activity. We next found that all 10 genes commonly upregulated in HSCs/HPCs are either direct transcriptional targets of NRF2 (NMRAL2P, LUCAT1,PIR, NQO1, FTL, and QPCT) or are directly affected by changes in NRF2 activation (SELENOW, EBI3, TNFRSF9, and RIT1). Further, several genes highly upregulated by DEK treatment in HSCs (i.e., CCL2, KYNU, and CXCL8) are NRF2 targets. To examine if DEK activates NRF2 transcriptional activity, we utilized a transgene reporter assay where expression of red fluorescent protein (RFP) is driven by activation of promoter antioxidant response elements (ARE), to which active NRF2 binds. DEK treatment of hematopoietic derived cell lines (mouse MLL-AF9 AML and human HL-60 APL) expressing the transgene significantly increased RFP signal, indicating that DEK could activate NRF2. We further examined whether DEK treatment affects mitochondrial metabolism, a source of ROS and tightly regulated process in hematopoiesis. Seahorse XFe analysis, which measures metabolic flux, showed DEK treatment of MLL-AF9 cells significantly reduced mitochondrial oxygen consumption rate, suggesting a reduced reliance on oxidative phosphorylation as an energy source in DEK treated cells. These data suggest that DEK treatment during expansion of primitive hematopoietic cells drives activation of NRF2 transcription, upregulation of antioxidant response genes, reduction of intracellular ROS levels, and may reduce the mitochondrial metabolic rate. These effects in a sense "mimic” the in vivo niche of HSCs, where they reside in low oxygen tensions, have low intracellular ROS, and exhibit low levels of oxidative phosphorylation. To test this hypothesis, we examined if DEK treatment has similar effects to maintaining HSCs in physiologic oxygen tensions (3%) compared to HSCs exposed to extraphysiologic ambient air (21%), which drives oxidative stress (Mantel, et al. Cell 2015; 161(7):1553-65). Isolating and maintaining mouse BM in physiologic oxygen tensions or treating mice in vivo with recombinant DEK preserved HSC numbers compared to exposing the BM cells to ambient air or treating with vehicle. However, combining in vivo DEK treatment with physiologic isolation of HSCs/HPCs had no additive or synergistic effect, suggesting that the overall effects of DEK treatment and maintaining low oxygen tensions may be similar. Thus, extracellular DEK treatment leads to expansion of functionally competent HSCs and reduction of oxidative stress. These data have important implications for the regulation of HSCs/HPCs in stress conditions, where DEK is most likely to be secreted. Further, DEK may present a novel way to mimic physiologic oxygen conditions for enhancement of functional HSC expansion, and the elucidated mechanisms may be exploited for more efficient targeting of molecular programs critical for functional HSC competency, potentially providing new ways to enhance HCT.

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.

Hematopoietic stem cells (HSCs) manifest impaired recovery and self-renewal with a concomitant increase in differentiation when exposed to ambient air as opposed to physioxia. Mechanism(s) behind this distinction are poorly understood but have the potential to improve stem cell transplantation. Single-cell RNA sequencing of HSCs in physioxia revealed upregulation of HSC self-renewal genes and downregulation of genes involved in inflammatory pathways and HSC differentiation. HSCs under physioxia also exhibited downregulation of the epigenetic modifier Tet2. Tet2 is α-ketoglutarate, iron- and oxygen-dependent dioxygenase that converts 5-methylcytosine to 5-hydroxymethylcytosine, thereby promoting active transcription. We evaluated whether loss of Tet2 affects the number and function of HSCs and hematopoietic progenitor cells (HPCs) under physioxia and ambient air. In contrast to wild-type HSCs (WT HSCs), a complete nonresponsiveness of Tet2-/- HSCs and HPCs to changes in oxygen tension was observed. Unlike WT HSCs, Tet2-/- HSCs and HPCs exhibited similar numbers and function in either physioxia or ambient air. The lack of response to changes in oxygen tension in Tet2-/- HSCs was associated with similar changes in self-renewal and quiescence genes among WT HSC-physioxia, Tet2-/- HSC-physioxia and Tet2-/- HSC-air. We define a novel molecular program involving Tet2 in regulating HSCs under physioxia.