Normal Mouse Bone Marrow Research Articles

The bone marrow of mouse-rat chimeras contains progenitors of multiple pulmonary cell lineages.

Radiation-induced lung injury (RILI) is a common complication of anti-cancer treatments for thoracic and hematologic malignancies. Bone marrow (BM) transplantation restores hematopoietic cell lineages in cancer patients. However, it is ineffective in improving lung repair after RILI due to the paucity of respiratory progenitors in BM transplants. In the present study, we used blastocyst injection to create mouse-rat chimeras, these are artificial animals in which BM is enriched with mouse-derived progenitor cells. FACS-sorted mouse BM cells from mouse-rat chimeras were transplanted into lethally irradiated syngeneic mice, and the contribution of donor cells to the lung tissue was examined using immunostaining and flow cytometry. Donor BM cells provided long-term contributions to all lung-resident hematopoietic cells which includes alveolar macrophages and dendritic cells. Surprisingly, donor BM cells also contributed up to 8% in pulmonary endothelial cells and stromal cells after RILI. To identify respiratory progenitors in donor BM, we performed single-cell RNA sequencing (scRNAseq). Compared to normal mouse BM, increased numbers of hematopoietic progenitors were found in the BM of mouse-rat chimeras. We also identified unique populations of hemangioblast-like progenitor cells expressing Hes1, Dntt and Ebf1, along with mesenchymal stromal cells expressing Cpox, Blvrb and Ermap that were absent or ultra-rare in the normal mouse BM. In summary, by using rats as "bioreactors", we created a unique mouse BM cell transplant that contributes to multiple respiratory cell types after RILI. Interspecies chimeras have promise for future generations of BM transplants enriched in respiratory progenitor cells.

Murine Bone Marrow Erythroid Cells Have Two Branches of Differentiation Defined by the Presence of CD45 and a Different Immune Transcriptome Than Fetal Liver Erythroid Cells.

Mouse erythropoiesis is a multifaceted process involving the intricate interplay of proliferation, differentiation, and maturation of erythroid cells, leading to significant changes in their transcriptomic and proteomic profiles. While the immunoregulatory role of murine erythroid cells has been recognized historically, modern investigative techniques have been sparingly applied to decipher their functions. To address this gap, our study sought to comprehensively characterize mouse erythroid cells through contemporary transcriptomic and proteomic approaches. By evaluating CD71 and Ter-119 as sorting markers for murine erythroid cells and employing bulk NanoString transcriptomics, we discerned distinctive gene expression profiles between bone marrow and fetal liver-derived erythroid cells. Additionally, leveraging flow cytometry, we assessed the surface expression of CD44, CD45, CD71, and Ter-119 on normal and phenylhydrazine-induced hemolytic anemia mouse bone marrow and splenic erythroid cells. Key findings emerged: firstly, the utilization of CD71 for cell sorting yielded comparatively impure erythroid cell populations compared to Ter-119; secondly, discernible differences in immunoregulatory molecule expression were evident between erythroid cells from mouse bone marrow and fetal liver; thirdly, two discrete branches of mouse erythropoiesis were identified based on CD45 expression: CD45-negative and CD45-positive, which had been altered differently in response to phenylhydrazine. Our deductions underscore (1) Ter-119's superiority over CD71 as a murine erythroid cell sorting marker, (2) the potential of erythroid cells in murine antimicrobial immunity, and (3) the importance of investigating CD45-positive and CD45-negative murine erythroid cells separately and in further detail in future studies.

Reducing genotoxicity of cyclophosphamide by hydro-coholic leaves extract of allium porrum in normal mouse bone marrow stem cells

This paper evaluates percentage of chromosomal aberration (CA )and mitotic index (MI) in mice bone marrow as a method for assessing the ability of hydro-ethanolic leaves extract of leek ( Allium Porrum) to reduce genotoxicity of Cyclophosphamide (CP) . Mice were allocated in to 4 groups, two negative controls ,the first group receive ( 0.2 ml) D.W. and the second group was treated with (0.1ml) DMSO, the positive control group, (CP) 40mg/kg was given single intraperitoneally dose for 24 hr , while the treatment groups were given different doses of extract (75, 150,200and 250 mg/kg ) for 5 consecutive days and the same dose of CP . Experimental data was analyzed using (one –way ANOVA). The results demonstrated the intermediate dose (150 mg/kg) of extract increased in MI significantly and reduced CA (P<0.01) significantly .This study concluded that Allium porrum leaves were able to reduce genotoxicity of CP and protect normal proliferating cells in bone marrow from the effect of CP.

Differentiation and Immunological Function of MDSC-Derived Dendritic Cells.

Dendritic cells (DCs) play a key role in initiating and regulating immune responses, and in addition to their roles in vivo, DCs are used as natural adjuvants for various tumor vaccines. In vitro, monocytes can be used to induce DCs, but in tumor patients, due to insufficient bone marrow hematopoiesis, extramedullary hematopoiesis and tumor-associated myeloid cells expand, and monocytes mainly exist in the form of myeloid-derived suppressor cells (MDSCs). The purpose of this experiment was to explore the differences in the differentiation and immune function of DCs induced by MDSCs in tumor patients. In a mouse model, we used normal mouse bone marrow cell-derived DCs as control cells, and in a tumor-bearing model, we induced MDSCs in the spleen to generate DCs (MDSC-DCs). Through flow cytometry, we found that the production of MDSC-DCs was significantly higher than that of control mice, and the secretion of interferon-γ of MDSC-DCs was significantly reduced. Through OVA antigen presentation experiments, we found that the antigen presentation ability of MDSC-DCs was significantly decreased. Through adoptive treatment of tumor-bearing mice cells, we found that the antitumor immune function of MDSC-DCs was significantly reduced. After that, we explored the mechanism of the decrease of immune function activity of MDSC-DCs. We determined that the surface markers of MDSC-DCs were changed by flow cytometry. Through flow sorting and RNA sequencing, we found that some pathways and key gene expression in MDSC-DCs were changed. In conclusion, this study found that the immune function of MDSC-DCs decreased and explored the mechanism of the decreased immune function activity.

Dual IKZF2 and CK1α Degrader Targets Acute Myeloid Leukemia Cells

Acute myeloid leukemia (AML) is an aggressive hematologic disease for which several epigenetic regulators have been identified as therapeutic targets. We previously found that a transcription factor and Ikaros family member, IKZF2 (HELIOS) is required for myeloid leukemic stem cell activity (Park et al. Cell Stem Cell 2019). Additionally, IKZF2 enhances self-renewal by increasing HOXA9 gene expression and in parallel blocks myeloid differentiation by reducing C/EBP expression. Thus, our studies suggested that targeting IKZF2 could be a new therapeutic strategy in AML. Immunomodulatory drugs (IMiDs; lenalidomide and others) have been used clinically to specifically target and degrade other Ikaros family members. However, these have been unable to target IKZF2. Utilizing a structure-guided approach, we developed a small molecule with low nanomolar degrader activity against IKZF2 (DEG-35). DEG-35 treatment in different AML cell lines induce apoptosis and differentiation with IC50 values of 5 ±0.4 nM in MOLM-13 cells to 27± 2.5 uM in NB4 cells. To understand if DEG35 has activity against other substrates, we performed global quantitative proteomics and found casein kinase 1 alpha (CK1α), a serine/threonine kinase previously identified to be critical for myeloid leukemogenesis and a known IMiD substrate, to be the most depleted protein (Jaras et al. JEM). Also, we performed a PRISM (Profiling Relative Inhibition Simultaneously in Mixtures) screen assay which utilizes 770 barcoded cell lines from 20 lineages and identified multiple correlations with the p53 pathway. Previous studies found that CK1α suppresses the activity of p53 phosphorylation or binding MDM proteins. To further probe the mechanism behind DEG35 activity, we performed RNA sequencing at 24hrs in MOLM-13 cells. Gene set enrichment analysis (GSEA) using the rank list of differentially expressed genes from the MOLM-13 cells treated with DMSO and DEG-35 revealed enrichment for genes upregulated in CK1α KO keratinocytes and p53 targets. Additionally, we found enrichment for the MLL-AF9 IKZF2 KO LSC gene sets, myeloid differentiation, loss of MEIS1-HOXA9 and MYC targets. Immunoblotting validated the reduction of MYC and HOXA9 protein in DEG-35 treated MOLM-13 cells. To determine the contribution of CK1α and IKZF2 to the cellular phenotypes in MOLM13 cells, we developed CK1α and IKZF2 nondegradable mutants. CK1α, G40N was able to significantly reduce the cell killing activity of DEG-35 by 94% compared to vector cells (**p<0.01). Moreover, p53 deletion was also able to block DEG-35 induced apoptosis by 98% and differentiation by 88% compared to control. Additionally, expression of IKZF2 H141Q rescued DEG-35 mediated differentiation by 55% (***p<0.001), but not apoptosis. Thus, we demonstrate that the degradation of IKZF2 and CK1α by DEG-35 blocks cell growth and induces myeloid differentiation in human AML cell lines through CK1α-p53- and IKZF2-dependent pathways. To further probe the efficacy and therapeutic index of targeting both IKZF2 and CK1α. We found that AML cells were more sensitive to DEG-35 compared to normal bone marrow cells (murine MLL-AF9 Crbn I1391V cells has 10 fold and AML PDX cells have 2-5 fold higher sensitivity to normal mouse or human bone marrow cells).Treatment of DEG-35 at 50 mg/kg in AML PDX-transplanted mice led to significant prolonged survival compared to vehicle treated mice (median survival of DMSO mice: 45 days vs DEG-35 mice: 61 days; ***p< 0.001) and reduced disease burden. To improve pharmacological properties, we developed analog DEG-77, which has 10-fold increased solubility than DEG-35 in DMSO. DEG77 exhibited better in vivo efficacy in the MLL-AF9 Crbn I1391V model (Increase in median survival by DEG-35: 2 days *p<0.05 vs DEG-77: 31 days ****p<0.0001). Importantly, the increase of median survival of 30 days (****p<0.0001) was also observed in the secondarily transplanted DEG-77 mice compared to DMSO mice, suggesting that DEG-35 can reduce LSC activity. In summary, we report the development of nanomolar monofunctional cereblon-dependent degraders of IKZF2 and casein kinase 1 alpha (CK1α). DEG-35 or the analog DEG-77 delays leukemia progression in murine and human AML mice models. Thus, we provide a novel strategy for multi-targeted degradation of IKZF2/CK1α to enhance efficacy against AML that may be expanded to additional targets and indications.

In vivo generation of bone marrow from embryonic stem cells in interspecies chimeras.

Generation of bone marrow (BM) from embryonic stem cells (ESCs) promises to accelerate the development of future cell therapies for life-threatening disorders. However, such approach is limited by technical challenges to produce a mixture of functional BM progenitor cells able to replace all hematopoietic cell lineages. Herein, we used blastocyst complementation to simultaneously produce BM cell lineages from mouse ESCs in a rat. Based on fluorescence-activated cell sorting analysis and single-cell RNA sequencing, mouse ESCs differentiated into multiple hematopoietic and stromal cell types that were indistinguishable from normal mouse BM cells based on gene expression signatures and cell surface markers. Receptor-ligand interactions identified Cxcl12-Cxcr4, Lama2-Itga6, App-Itga6, Comp-Cd47, Col1a1-Cd44, and App-Il18rap as major signaling pathways between hematopoietic progenitors and stromal cells. Multiple hematopoietic progenitors, including hematopoietic stem cells (HSCs) in mouse-rat chimeras derived more efficiently from mouse ESCs, whereas chondrocytes predominantly derived from rat cells. In the dorsal aorta and fetal liver of mouse-rat chimeras, mouse HSCs emerged and expanded faster compared to endogenous rat cells. Sequential BM transplantation of ESC-derived cells from mouse-rat chimeras rescued lethally irradiated syngeneic mice and demonstrated long-term reconstitution potential of donor HSCs. Altogether, a fully functional BM was generated from mouse ESCs using rat embryos as 'bioreactors'.

Chromatin remodeling subunit BRM and valine regulate hematopoietic stem/progenitor cell function and self-renewal via intrinsic and extrinsic effects.

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.

FGF7 Supports Hematopoietic Stem and Progenitor Cells and Niche-Dependent Myeloblastoma Cells Via Autocrine Action On Bone Marrow Stromal Cells In Vitro

Some fibroblast growth factor (FGF)/FGF receptor (FGFR) systems, namely FGF1 and FGF2 which are produced mainly by megakaryocytes, and their cognate receptors expressed on either hematopoietic stem or progenitor cells (HSPCs) or bone marrow (BM) stromal cells, are reported to support HSPCs under stressed conditions. In this study, we show that mesenchymal cell-derived FGF7 (also called keratinocyte growth factor or KGF) may be a novel niche factor for HSPC support and niche-dependent leukemia-initiating cell growth. We previously reported that mouse embryonic fibroblasts (MEFs) have characteristics that mimic BM stromal cells and possess an ability of HSPCs support in vitro, and that this ability is profoundly attenuated in MEFs deficient for the MED1 subunit of the Mediator transcriptional master coregulator complex, subcomplex of RNA polymerase II holoenzyme. We found through the microarray analyses that the FGF7 expression (both at mRNA and protein levels) was specifically and profoundly attenuated in Med1-/- MEFs compared with Med1+/+ MEFs. When normal mouse BM cells were cocultured with mitomycin C-treated Med1+/+ MEFs or BM stromal cells (OP-9 or MS-5) in the presence of anti-FGF7 antibody, the DNA synthesis and resultant growth of BM cells, and the number of long-term culture-initiating cells (LTC-ICs), were significantly suppressed. However, the addition of anti-FGF7 antibody did not enhance cell death in BM cells. In contrast, the addition of recombinant FGF7 to the coculture of BM cells with mitomycin C-treated Med1-/- MEFs robustly upregulated the DNA synthesis and growth of BM cells, and the number of LTC-ICs. The Ph+ myeloblastoma cells MB-1, originally isolated from a patient with blast crisis chronic myeloid leukemia, is niche-dependent myeloblastic cells that require mesenchymal stromal cells for survival and growth. During coculture typically with OP-9 BM stromal cells, these MB-1 cells form cobblestone areas, which most probably reconstitute a stochastic model of leukemic stem or initiating cells in vitro. We next analyzed the role of FGF7 in support of MB-1 cells for growth and survival during coculture. Indeed, the addition of anti-FGF7 antibody to the coculture with mitomycin C-treated BM stromal cells (either OP-9 or MS-5) substantially attenuated DNA synthesis and resultant proliferation of stromal cell-dependent myeloblastoma cells MB1. Notably, cobblestone formation (i.e., signature of stem cell phenotype) of MB-1 cells was significantly attenuated when anti-FGF7 antibody was added to the coculture with OP-9 cells. However, the addition of anti-FGF7 antibody did not elicit cell death of MB-1 cells. Thus, FGF7 appears to promote both proliferation and maintenance of leukemia stem or initiating cell feature of MB-1 cells. While neither FGF7 nor its cognate receptor FGFR2IIIb mRNAs were detectable in BM cells, the mesenchymal cells including Med1+/+ MEFs and (OP-9 and MS-5) BM stromal cells expressed them. The expression levels of FGFR2IIIb were comparable between Med1+/+ and Med1-/- MEFs, and BM stromal cells. Therefore, we hypothesized that FGFR2IIIb on these mesenchymal stromal cells might be functional and possibly elicit secretion of niche signal(s) secondarily in response to the downstream target gene(s) of FGFR2IIIb, followed by subsequent support of HSPCs and leukemic cells. To prove that FGF7 is functional in these cells, we examined response of the downstream targets of FGFR2IIIb in these cells, including phosphorylation of FRS2 (the immediate downstream target of FGFR2IIIb tyrosine kinase), phosphorylation of MAP kinases (the intermediate hub of various intracellular signals), and mRNA expression of immediate early response genes including c-Jun, c-Fos and c-Myc. Indeed, exogenously added recombinant FGF7 elicited phosphorylation of both FRS2 and MAP kinases within 10 min, and full induction of these immediate early response genes within 30 min, similarly in both Med1+/+ and Med1-/- MEFs. The OP-9 and MS-5 BM stromal cells also exhibited similar responses to exogenously added FGF7. These results indicate that FGFR2IIIb expressed on mesenchymal stromal cells are functional, and that FGF7 and FGFR2IIIb produced by these cells constitute an autocrine (or paracrine) loop. Taken together, we propose that FGF7 produced by BM stromal cells supports HSPCs and leukemic progenitor cells indirectly via FGFR2IIIb expressed on the BM stromal cells. Disclosures:No relevant conflicts of interest to declare.

CXCL15/Lungkine has suppressive activity on proliferation and expansion of multi-potential, erythroid, granulocyte and macrophage progenitors in S-phase specific manner

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.

Abraxane-induced bone marrow CD11b+ myeloid cell depletion in tumor-bearing mice is visualized by μPET-CT with 64Cu-labeled anti-CD11b and prevented by anti-CSF-1.

To investigate the utility of noninvasive µPET-CT with 64Cu-DOTA-anti-CD11b (64Cu-αCD11b) in assessing bone marrow status after anticancer therapies, and the protective role of anti-CSF-1 (αCSF-1) against bone marrow suppression induced by Abraxane.Methods: MDA-MB-435 tumor-bearing mice were treated with Abraxane, αCSF-1, or αCSF-1 plus Abraxane. µPET-CT and biodistribution of 64Cu-αCD11b were performed after intravenous injection of the radiotracer. Cells from mouse bone marrow and MDA-MB-435 tumor were analyzed by flow cytometry. A humanized αCSF-1 was investigated for its role in protecting bone marrow cells, using a transgenic mouse model that expresses functional human CSF-1.Results: μPET-CT showed that 64Cu-αCD11b had high uptake in the bone marrow and spleen of both normal and tumor-bearing mice. Abraxane significantly reduced 64Cu-αCD11b uptake in the bone marrow and spleen of treated mice compared to untreated mice. Interestingly, 64Cu-αCD11b μPET-CT revealed that αCSF-1 alleviated the depletion of bone marrow cells by Abraxane. These changes in the bone marrow population of CD11b+ myeloid cells were confirmed by flow cytometry. Moreover, αCSF-1 potently enhanced tolerance of bone marrow granulocytic myeloid cells to Abraxane, decreased cell migration, and suppressed recruitment of myeloid cells to the tumor microenvironment. The humanized αCSF-1 also alleviated the effects of Abraxane on bone marrow cells in transgenic mice expressing human CSF-1, suggesting clinical relevance of αCSF-1 in prevention of bone marrow suppression in addition to its role in reducing tumor-infiltrating myeloid cells.Conclusions: Abraxane-induced bone marrow CD11b+ myeloid cell depletion in tumor-bearing mice could be noninvasively assessed by μPET-CT with 64Cu-αCD11b and prevented by αCSF-1.

Mitochondrial Potentiation Ameliorates Age-Related Heterogeneity in Hematopoietic Stem Cell Function.

Aging is associated with reduced fitness and increased myeloid bias of the hematopoietic stem cell (HSC) compartment, causing increased risk of immune compromise, anemia, and malignancy. We show that mitochondrial membrane potential (MMP) can be used to prospectively isolate chronologically old HSCs with transcriptional features and functional attributes characteristic of young HSCs, including a high rate of transcription and balanced lineage-affiliated programs. Strikingly, MMP is a stronger determinant of the quantitative and qualitative transcriptional state of HSCs than chronological age, and transcriptional consequences of manipulation of MMP in HSCs within their native niche suggest a causal relationship. Accordingly, we show that pharmacological enhancement of MMP in old HSCs invivo increases engraftment potential upon transplantation and reverses myeloid-biased peripheral blood output at steady state. Our results demonstrate that MMP is a source of heterogeneity in old HSCs, and its pharmacological manipulation can alter transcriptional programs with beneficial consequences for function.

The role and mechanism of myeloid-derived suppressor cells in mice with nonalcoholic steatohepatitis

Objective To investigate the effects of myeloid-derived suppressor cells (MDSCs) on nonalcoholic steatohepatitis mice. Methods ⑴ Male C57BL/6 mice aged 6-8 weeks were randomly divided into normal diet group, CDAA group(choline deficient amino acid diet) and CSAA group (choline sufficient amino acid diet). ⑵ After the success of the non-alcoholic steatohepatitis model, serum was collected from some mice to detect biochemical indexes; Liver tissue was retained for microscopic observation by hematoxylin-eosin (HE) staining; The changes of T cell subsets in peripheral blood of mice in each group were detected by flow cytometry. In addition, The proportion and subtype of CD11b+ Gr-1+ MDSCs cells in liver, spleen, blood and bone marrow were also detected by flow cytometry. ⑶ The bone marrow-derived Gr-1highLy-6G+ was purified by magnetic bead sorting technique, and the purified Gr-1highLy-6G+ was transferred into non-alcoholic steatohepatitis (NASH) mice by tail vein injection. The role of MDSCs in NASH was analyzed by detection of serological indexes of liver function and pathological dyeing. Results ⑴ There was no significant difference in body weight and liver index between the groups (P>0.05). Serological indicators: alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood glucose, interleukin (IL)-6 and interferon-γ (INF-γ) in the model group were significantly higher than those in the normal group (P 0.05); the MDSCs of liver in CDAA group is lower than that of normal group (P normal group (P<0.01), CD11b+ Gr-1dimLy-6G-(M-MDSC) showed a downward trend, CDAA group < normal group; ⑶ Serum AST and ALT levels of NASH mice who receiving Gr-1highLy-6G+ MDSCs from normal bone marrow were significantly decreased (P<0.001), and histopathological changes were alleviated. Conclusions ⑴ The NASA mouse model can be successfully established on the CDAA diet. ⑵ The CDAA-induced NASH mice may have immune dysfunction, mainly manifesting in the change of bone marrow MDSCs subpopulations and the increase of CD11b+ Gr-1highLy-6G+ (G-MDSC). ⑶ MDSCs derived from normal mouse bone marrow can alleviate the pathological changes of NASH. Key words: Myeloid-derived suppressor cells; Non-alcoholic steatohepatitis; T-lymphocytes; Interleukin-6; Mice

Restraining Lysosomal Activity Preserves Hematopoietic Stem Cell Quiescence and Potency.

Quiescence is a fundamental property that maintains hematopoietic stem cell (HSC) potency throughout life. Quiescent HSCs are thought to rely on glycolysis for their energy, but the overall metabolic properties of HSCs remain elusive. Using combined approaches, including single-cell RNA sequencing (RNA-seq), we show that mitochondrial membrane potential (MMP) distinguishes quiescent from cycling-primed HSCs. We found that primed, but not quiescent, HSCs relied readily on glycolysis. Notably, invivo inhibition of glycolysis enhanced the competitive repopulation ability of primed HSCs. We further show that HSC quiescence is maintained by an abundance of large lysosomes. Repression of lysosomal activation in HSCs led to further enlargement of lysosomes while suppressing glucose uptake. This also induced increased lysosomal sequestration of mitochondria and enhanced the competitive repopulation ability of primed HSCs by over 90-fold invivo. These findings show that restraining lysosomal activity preserves HSC quiescence and potency and may be therapeutically relevant.

Chromatin Remodeling Subunit Brm Regulates Hematopoietic Stem Cell Self-Renewal By Limiting Intracellular Valine Levels

Chromatin remodeling complexes facilitate gene expression and control cell fate decisions. The ATPase subunit of chromatin remodeling complex BRG1 is essential for stem cell function, but the role of its paralog Brm remains essentially unknown. To assess a role(s) for Brm in hematopoietic cell regulation in vivo, we studied hematopoietic stem (HSCs) and progenitor cells (HPCs) in bone marrow (BM) of Brm -/- vs. wildtype (WT) control mice. While BM from Brm -/- mice contain increased numbers of rigorously-defined phenotypic populations of long- and short-term repopulating HSCs and granulocyte macrophage progenitors (GMPs) and increased numbers and cycling status of functional HPC (assessed by CFU-GM, BFU-E, and CFU-GEMM colony assays), they were defective in self-renewal capacity upon in vivo serial transplantation using congenic mice (CD45.2+ donor cells, CD45.1+ competitor cells, and F1 (CD45.2+/CD45.1+) recipient mice). Increased numbers of HSCs from Brm-/- BM failed to show competitive advantage over wild type (WT) control BM cells in primary (1°) transplantation in lethally irradiated mice (based on month 4 donor cell chimerism and phenotypically defined HSC numbers). Moreover, 2° and 3° engraftment at 4 months post transplantation each, a measure of HSC self-renewal capacity, revealed much reduced engraftment of donor Brm -/- BM cell chimerism and HSC numbers compared to the extensive 2° and 3° engraftment of control WT BM. No significant differences in myeloid/lymphoid ratios were noted in 1° or 2° engrafted mice, suggesting no differentiation lineage bias of donor Brm -/- BM cells. This demonstrates a critical role for Brm in controlling in vivo self-renewal of mouse BM HSCs. Valine [(2S)-2 amino-3 methylbutanoic acid (C5H11N02)] is implicated in hematopoietic regulation, since depleting dietary valine permitted non-myeloablative mouse HSC transplantation (Taya et. al. Science 354:1152-1155, 2016). Metabolic analysis of lineage negative (lin-) cells demonstrated that valine, but not leucine, levels were very highly elevated in Brm -/- BM cells, thus linking intracellular valine levels with Brm expression. Exogenously added valine significantly increased basal oxygen consumption rates of both total WT BM and WT lin- cells, but not of total or lin-Brm -/- BM cells in vitro (via Seahorse machine analysis). To study effects of valine on HPCs, we assessed the addition of exogenously added valine on mouse BM and human cord blood (CB) cells cultured in the presence of cytokines with either non-dialyzed or dialyzed fetal bovine serum (FBS). Valine, but not leucine, dose-dependently enhanced HPC (CFU-GM, BFU-E, and CFU-GEMM) colony formation and secondary replating capacity of cytokine stimulated CFU-GM and CFU-GEMM derived colonies of normal mouse BM cells in vitro in presence of non-dialyzed FBS; additional enhanced valine effects were noted when dialyzed FBS (lacking valine and other amino acids) was used. Valine also enhanced mouse BM HPC survival in vitro in context of delayed addition of growth factors, and cytokine stimulated (SCF, FL, TPO) ex-vivo expansion of normal mouse BM HSCs and HPCs. Valine enhancement of the above noted functional mouse BM HPC assays in the presence of dialyzed FBS was also apparent with low density and CD34+ purified CB cells, demonstrating that valine effects are not species specific. Our results suggest that valine is an enhancing factor for HSC maintenance of self-renewal capacity and HPC proliferation, and that Brm gene expression limits intracellular valine levels, thereby controlling HSC self-renewal and HPC proliferation. This information is of potential use for future translation to modulate self-renewal of HSCs and survival and proliferation of HPCs for clinical advantage. Disclosures No relevant conflicts of interest to declare.

Ganoderma spore lipid protects mouse bone marrow mesenchymal stem cells and hematopoiesis from the cytotoxicity of the chemotherapeutic agent.

Cancer chemotherapeutic agents are frequently toxic to bone marrow and impair bone marrow functions. It is unclear whether ganoderma spore lipid (GSL) can protect bone marrow cells from the cytotoxicity of chemotherapy. To investigate the protective effects of GSL on bone marrow mesenchymal stem cells (MSCs) and hematopoiesis, we examined the effects of GSL on MSCs in vitro and hematopoiesis in vivo after treatment with the chemotherapeutic agent cyclophosphamide. MSCs and peripheral blood cells were isolated and counted from the bone marrow of normal mice were pre-treated with GSL before CTX treatment or co-treated with GSL and CTX, followed by examining the changes in phenotype, morphology, proliferation, apoptosis, and differentiation potentials. The results showed that GSL could reduce the CTX-induced changes in the phenotype of MSCs and maintain the elongated fibroblast-like morphology. MTT and annexin V/propidium iodide (PI) analyses found that GSL pre-treatment and co-treatment increased the proliferation and decreased the apoptosis in CTX-treated MSCs. Furthermore, GSL improved the osteogenic and adipogenic differentiation potentials of CTX-treated MSCs. In vivo, GSL treatment increased the number of peripheral blood cells including white blood cells (WBC) and platelets (PLT) in the CTX-treated mice and enhanced the in vitro formation of hematopoietic lineage colonies (erythrocyte colony forming unit, CFU-E; erythroid burst-forming units, BFU-E; and granulocyte macrophage colony-forming units, CFU-GM) from bone marrow cells in these mice. These findings suggest GSL could protect MSCs and hematopoiesis from the cytotoxicity of CTX and might become an effective adjuvant to attenuate side effects of chemotherapy during cancer treatment.

Functional analysis of a biflavone, as a novel inhibitor of TRPV4‐dependent macrophages foam cell formation

Cardiovascular disease is the number one cause of death in United States, and atherosclerosis, a chronic inflammatory disease, is the most dominant underlying pathology. Macrophages, an essential cell type in atherogenesis, are known to directly respond to changes to their surrounding mechanical environment. As TRPV4 (transient receptor potential channel of the vanilloid subfamily) channels can be sensitized by changes in matrix stiffness, we hypothesize that, TRPV4 may upregulate inflammatory/atherogenic macrophage functions. We performed a high‐throughput screening for antagonists of TRPV4 from a library of 2000 compounds using a fluorometric imaging plate reader (FLIPR)‐based Ca2+ influx assay; we identified ginkgetin, a biflavone, as a novel antagonist to TRPV4. Primary and secondary screening was performed with normal human dermal fibroblasts and mouse bone marrow derived macrophages (BMDMs) to assess the ability of ginkgetin and other candidates to inhibit TRPV4‐elicited Ca2+ influx after TRPV4 activation with its selective agonist GSK1016790. In our hand's, ginkgetin demonstrated an IC50 of 0.5 μM for inhibition of TRPV4‐elicited Ca2+ influx. We show that antagonism of TRPV4 channel by ginkgetin blocks oxidized LDL (oxLDL)‐induced macrophage foam cell formation in a concentration dependent manner. Mechanistically, we show that ginkgetin attenuated oxLDL uptake but not its cell surface binding in BMDMs. Altogether, our results show that ginkgetin is a novel inhibitor of TRPV4‐mediated macrophage foam cell formation by modulating uptake of oxLDL.Support or Funding InformationAHA (13SDG17310007), Startup grant from University of Maryland, NIH (1R01EB024556‐01), and NSF (CMMI‐1662776) grants to Shaik O RahamanThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract LB-332: Evidence for cutaneous squamous tumors derived from carcinogen-exposed bone marrow

Abstract Cytokeratins are frequently found in the blood and bone marrow of patients with epithelial cancers and are attributed to metastasis. We wondered whether we could find keratin expression in blood and bone marrow in untreated adult mice. To address this problem, we have used classical immunoreactivity, Krt1-14;mTmG transgenic mice together with fluorescence activated cell sorting, and quantitative reverse transcriptase polymerase chain reaction. We have made several novel findings. First, we discovered, rare but distinctive, keratin-14 and keratin-15 immunoreactive cells the size of small lymphocytes in blood and bone marrow of untreated mice. Second, using Krt1-14;mTmG transgenic mice, we found low (8.6 GFP positive cells per 10^6 cells analyzed), but significant numbers (p&amp;lt0.0005) of GFP positive cells in bone marrow of normal adult mice when compared with negative controls. Third, qRT-PCR demonstrated very low but reproducibly detectable expression of keratin-14 mRNA in blood and bone marrow when compared with epidermal keratinocytes: with blood expressing one thousand times, and bone marrow, one hundred thousand times, less than epidermal keratinocytes. Moreover, FACS analysis of fresh bone marrow disclosed a subpopulation of keratin-14 immunoreactive cells that was negative for hematopoietic lineage markers. Furthermore, and even more curious, recipients of bone marrow transplants from DMBA-treated UBC/GFP mice (“toxic bone marrow”) developed GFP-immunoreactive skin tumors upon TPA tumor promotion: four of fifteen mice developed GFP-immunoreactive skin tumors. Of these tumors, four were squamous papillomas, and one was a squamous cell carcinoma. None of the ten control mice receiving normal whole bone marrow developed any skin tumors upon tumor promotion. We conclude from these observations that keratin-14 protein and mRNA are expressed at low, but detectable levels in the blood and bone marrow of mice, and that a subset of “toxic bone marrow” derived cells can be recruited to form epithelial benign and malignant squamous tumors. Building upon these observations should open new avenues of understanding for cutaneous biology, non-melanoma skin cancer, and other epithelia and their cancers. Citation Format: Sonali Lad, Kelly Johnson, Kelsey Boland, Nyssa Readio, Todd Schuster, Yong Li, Derek Gordon, Rebecca J. Morris. Evidence for cutaneous squamous tumors derived from carcinogen-exposed bone marrow [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-332.

Abstract 126: MYC-driven lymphomas suppress NK surveillance by blocking maturation of early NK cells

Abstract The MYC oncogene is commonly overexpressed in hematopoietic cancers. In a transgenic mouse model of MYC-induced T-cell lymphoma, we found that there was a systemic reduction in NK cell (CD3- NKp46High) numbers and activation in lymphoid organs such as spleen and bone marrow. MYC inactivation in lymphoma-bearing MYCON mice partially restored NK cell numbers and activation. Hence, the MYC oncogene appears to suppress NK cell-mediated immune surveillance of lymphomas. We evaluated whether reduction in mature and activated NK cells (CD3- NKp46High) in MYCON mice was due to increased death of NK cells in these mice when compared to normal and MYCOFF mice. Using flow cytometry, NK cells were measured in age matched spleens and bone marrows of normal (n = 8), MYCON (n = 8), and MYCOFF (n = 8) mice. Surprisingly, we observed a significant reduction in proportions of dead NK cells (NKp46+ PI+) in spleen and bone marrow of MYCON mice, in comparison to normal and MYCOFF cohorts. Hence, the reduction in activated NK cell numbers during MYC-driven lymphomagenesis does not occur because of increased death of NK cells. Next, we investigated whether MYC arrests early NK cell development in the bone marrow of lymphoma mice, leading to systemic NK suppression. NK cell lineage specification begins with CD122 expression, that is maintained throughout NK development starting at the precursor stage. NK precursors (NKP) transition to immature NK (iNK) cells by expressing the cytotoxicity marker NKp46. iNK cells leave the bone marrow to populate peripheral lymphoid organs. We measured NKP (CD122+ NKP46-), and iNK (CD122+ NKp46+) cells in bone marrow of normal (n = 8), MYCON (n = 8), and MYCOFF (n = 8) mice. We observed no significant changes in the percentages of NKP. However, the proportions of iNK cells were significantly reduced in MYCON mice, in comparison to MYCOFF and normal mice. Our results suggest that MYC may block transition from NKP to iNK stage during early NK cell development. The reduction of iNK cells in spleens of MYCON mice was concordant with the reduction of iNK cells in bone marrow from the same mice. We conclude that MYC-induced lymphomagenesis blocks early NK cell development, thereby suppressing NK-mediated immune surveillance. Citation Format: Line D. Heftdal, Srividya Swaminathan, Dean W. Felsher. MYC-driven lymphomas suppress NK surveillance by blocking maturation of early NK cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 126.

Stable lines and clones of long-term proliferating normal, genetically unmodified murine common lymphoid progenitors

AbstractCommon lymphoid progenitors (CLPs) differentiate to T and B lymphocytes, dendritic cells, natural killer cells, and innate lymphoid cells. Here, we describe culture conditions that, for the first time, allow the establishment of lymphoid-restricted, but uncommitted, long-term proliferating CLP cell lines and clones from a small pool of these cells from normal mouse bone marrow, without any genetic manipulation. Cells from more than half of the cultured CLP clones could be induced to differentiate to T, B, natural killer, dendritic, and myeloid cells in vitro. Cultured, transplanted CLPs transiently populate the host and differentiate to all lymphoid subsets, and to myeloid cells in vivo. This simple method to obtain robust numbers of cultured noncommitted CLPs will allow studies of cell-intrinsic and environmentally controlled lymphoid differentiation programs. If this method can be applied to human CLPs, it will provide new opportunities for cell therapy of patients in need of myeloid-lymphoid reconstitution.

Sitagliptin-mediated preservation of endothelial progenitor cell function via augmenting autophagy enhances ischaemic angiogenesis in diabetes.

Recently, the dipeptidyl peptidase‐4 (DPP‐4) inhibitor sitagliptin, a major anti‐hyperglycaemic agent, has received substantial attention as a therapeutic target for cardiovascular diseases via enhancing the number of circulating endothelial progenitor cells (EPCs). However, the direct effects of sitagliptin on EPC function remain elusive. In this study, we evaluated the proangiogenic effects of sitagliptin on a diabetic hind limb ischaemia (HLI) model in vivo and on EPC culture in vitro. Treatment of db/db mice with sitagliptin (Januvia) after HLI surgery efficiently enhanced ischaemic angiogenesis and blood perfusion, which was accompanied by significant increases in circulating EPC numbers. EPCs derived from the bone marrow of normal mice were treated with high glucose to mimic diabetic hyperglycaemia. We found that high glucose treatment induced EPC apoptosis and tube formation impairment, which were significantly prevented by sitagliptin pretreatment. A mechanistic study found that high glucose treatment of EPCs induced dramatic increases in oxidative stress and apoptosis; pretreatment of EPCs with sitagliptin significantly attenuated high glucose‐induced apoptosis, tube formation impairment and oxidative stress. Furthermore, we found that sitagliptin restored the basal autophagy of EPCs that was impaired by high glucose via activating the AMP‐activated protein kinase/unc‐51‐like autophagy activating kinase 1 signalling pathway, although an autophagy inhibitor abolished the protective effects of sitagliptin on EPCs. Altogether, the results indicate that sitagliptin‐induced preservation of EPC angiogenic function results in an improvement of diabetic ischaemia angiogenesis and blood perfusion, which are most likely mediated by sitagliptin‐induced prevention of EPC apoptosis via augmenting autophagy.