Bone Marrow Nuclear Cells Research Articles

Integrated transcriptomic and metabolomic profiles reveal the protective mechanism of modified Danggui Buxue decoction on radiation-induced leukopenia in mice.

Leukopenia caused by radiation hinders the continuous treatment of cancers. Danggui Buxue Decoction (DBD) has been widely used in clinical owing to low toxicity and definite therapeutic effects to increase leukocytes. Meanwhile, icaritin (ICT) has also been proved to have the effect of boosting peripheral blood cells proliferation. However, there is no study to prove the efficacy of MDBD (Modified Danggui Buxue Decoction), a derivative herbal formula composed of DBD and ICT, in the treatment of radiation-induced leukopenia. In this study, we performed a model of 3.5Gy whole-body radiation to induce leukopenia in mice. The results of pharmacodynamic studies demonstrated that MDBD could significantly increase the white blood cells in peripheral blood by improving the activity of bone marrow nuclear cells, reducing bone marrow damage, modulating spleen index, and regulating hematopoietic factors to alleviate leukopenia. We also analyzed the integrated results of metabolomics and transcriptomics and found that MDBD could relieve leukopenia and alleviate bone marrow damage by targeting steroid biosynthesis and IL-17 signaling pathway, in which the key genes are Jun, Cxcl2 and Egr1. Therefore, our study provides a basis for the effectiveness and compatibility in the combination of traditional Chinese medicine formula and small molecule drugs.

Molecular Response Analysis By High Throughput Sequencing in Higher Risk Myelodysplastic Syndrome (HR-MDS) Treated Intensively with CPX-351

Context and objective Intensive chemotherapy (IC) remains an important therapeutic option in HR-MDS, but in-depth evaluation of response to IC remains ill studied. We previously reported results of CPX-351 in 31 untreated higher risk MDS patients (pts) (GFM-CPX-MDS study, NCT04273802, ASH 2021 Abst #243). Responses were associated with a significant decrease of CD34/CD117 progenitors and restoration of normal hematopoiesis patterns in bone marrow (BM) samples in multiparameter flow cytometry. In this ancillary study, the aim was to evaluate molecular responses by high-throughput sequencing (HTS) centrally. This involved description of the pts molecular landscape and an assessment of variant allele frequency (VAF) variations after induction treatment with CPX-351. Methods The mutational landscape was explored in BM nuclear cells by capture HTS of 42 genes (SureSelect XTHS2 Agilent) with unique molecular identifiers (UMI), unique dual index (UDI) and Illumina paired-end sequencing (Miseq and Nextseq). Somatic mutations (SM) were considered significant for VAFs >2%. Molecular response was evaluated by comparing baseline and post-induction. Molecular clearance was defined according to (i) clearance (<2%) of all SM excluding the "pre-leukemic” SM of DNMTA, TET2, ASXL1 (DTA) (ii) clearance of all non-DTA SM < HTS detection threshold (iii) normalized VAF decrease of the major non-DTA baseline SM (nmVAF). Results Median age of the 31 pts included was 62 years (range 31-69). There were 26 MDS-EB2 and 5 CMML-2. IPSS was int-2 in 26 pts and high in 5. Cytogenetic IPSS-R was very good (6.5%), good (71%, including normal in 22 pts), int (9.5%), poor (6.5%) or very poor (6.5%). After induction with CPX-351 (3 days cycle and a possible 2-day second induction cycle for non-responders), 16 pts (52%) reached CR, 4 (13%) CRi, 7 (22%) MLFS while 4 (13%) were in stable disease according to ELN 2017 criteria (which appeared more adequate than MDS IWG 2006, as reported at ASH 2021, Abst #243). Median follow-up was 511 days (179-710), 29/31 patients (94%) received allogeneic hematopoietic stem cell transplantation (Allo-HSCT) after, no, 1, 2, 3 or 4 CPX-351 consolidation cycles (that included only 1 day of CPX-351) in 10, 16, 7, 3 and 1 patients respectively. So far, 4 relapses have occurred, including 3 in transplanted pts. At inclusion, all pts had at least one mutation (median 3, [1-14]) with a mean VAF of 30% (2-89), implicating at least one DTA (71%; 22/31) or one non-DTA genes (93%; 29/31) (fig). Very high or high risk was seen in 19/31 (61%) pts according to the new IPSSm classification. Post induction molecular response was evaluated at a median of 53 days from CPX-351 onset (28-112) in 26 of the 27 who reached at least MLFS, and had a post-induction sample. Molecular clearance was observed (i) at the 2% threshold of non-DTA genes in 17/26 (65%) pts (ii) at the HTS detection threshold (0.1% i.e. minimum considered 3X with median coverage > 3000X) in 11/26 pts (42%) (iii) by nmVAF decrease in 25/26 pts (96%) with a median of -62% (-2 to -100%) (fig) while the remaining pt had an increase of SRSF2 (+67%) and RUNX1 (+46%) SMs but disappearance of three STAG2 SMs and was still in CR after allogeneic SCT. Of note, DTA SMs remained detectable in all pts with a median nmVAF modulation of -86% (+83% to -99.6%, fig) from baseline to post-induction. Of the 4 non-responders to CPX-351, 2 had high risk anomalies (inv(3) and EZH2 / RUNX1 SMs). The 3 relapses observed after alloHSCT, at 7, 8 and 15 months had nmVAFs decrease from baseline to post-induction of -95%, -98% and -50% respectively. Among them, one had a very complex karyotype and a homozygous TP53 mutation, the two others had a high mutational load and EZH2 mutations and one of the latest was transplanted in refractory state. Clonal evolution was observed in 5/26 pts with significant emergence of new mutations involving PPM1D, ASXL1 or JAK2 and in one pt the emergence of EZH2, RUNX1 and TET2 SMs. Four of these pts remained in morphological CR while one progressed to AML. Conclusion In 25/26 CPX-351 responders, a significant decrease of nmVAF was observed, confirming the interest of CPX-351 treatment in higher risk MDS, especially as a bridge to transplant. Follow up is ongoing and few relapses were seen to evaluate the predictive role of molecular analysis. An update will be presented at the meeting. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

High-Dimensional Mass Cytometric Analysis of the Bone Marrow Compartment Identifies a Disease-Specific Immune Cell Network in Aplastic Anemia

Idiopathic acquired aplastic anemia (AA) is considered an immune-mediated syndrome of bone marrow failure since approximately 70% of patients respond to immunosuppressive therapy (IST). However, the immune response that underlies the pathogenesis of AA remains incompletely understood. Previous studies have extensively studied the immune cell composition in peripheral blood in AA, but a comprehensive overview of the immune landscape at the site of hematopoietic stem and progenitor cell (HSPC) destruction, the bone marrow, is lacking. We investigated the immune composition of the bone marrow in AA to interrogate which immune populations may be implicated in the pathogenesis of AA. A mass cytometric analysis of bone marrow aspirates was performed using a 39-marker panel for broad immunophenotyping of all major immune lineages and HSPCs. Cryopreserved bone marrow nuclear cells from adult AA patients (n=7) before start of first-line IST with horse-derived anti-thymocyte globulin (hATG) in combination with ciclosporin and age- and sex-matched healthy donors (HDs, n=7) were studied. 6 of 7 AA patients responded to IST. For 3 IST responders paired bone marrow samples taken at six months after start of IST were also included. A Hierarchical Stochastic Neighborhood Embedding (HSNE) analysis in Cytosplore revealed that the bone marrow in AA pre-IST features a distinct immune cell composition compared to HDs. At the major immune lineage level, HSPC and myeloid cell depletion (0.6% vs 33% of all immune cells, p<0.001), decreased B-cell frequencies (9% vs 16%, p=0.007), increased CD4+ T-cell frequencies (37% vs 28%, p=0.04) and increased CD8+ T-cell frequencies (37% vs 20%, p=0.007) were observed. Detailed analysis of these 4 lineages identified 121 phenotypically distinct cell clusters, 45 of which were differentially present (p<0.05) in AA patients pre-IST compared to HDs. To capture the relationships between these 45 immune clusters a Spearman's rank correlation analysis was performed using the cluster frequencies. Two cell networks were identified (Figure 1A), one of which (network AA) was significantly increased in frequency in AA pre-IST (22% vs 5% of all immune cells, p=0.001; Figure 1B). This network consisted of CD16+ myeloid cells, CCR6++ B-cells, Th17-like CCR6+ memory CD4+ T-cells and KLRG1+ terminally differentiated effector memory (EMRA) CD8+ T-cells, compatible with a state of chronic inflammation. Successful treatment with IST largely normalized the immune cell composition at the immune lineage level and strongly reduced the levels of CD16+ myeloid cells. However, at six months after start of IST the frequencies of CCR6++ B-cells, CCR6+ memory CD4+ T-cells and KLRG1+ EMRA CD8+ T-cells were not normalized, which could indicate that ciclosporin is still needed. This study provides a unique overview of the immune landscape in bone marrow in AA pre- and post-IST at a single-cell level. Previously reported cell subsets could be further defined and possible new players that contribute to the pathogenesis of AA were identified. Our findings show that AA is characterized by high frequencies of immune cells compatible with a state of chronic inflammation and provide a basis for future studies to characterize the function of the identified subsets in the pathogenesis of AA. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Massive expansion of multiple clones in the mouse hematopoietic system long after whole-body X-irradiation

Clonal hematopoiesis (CH) is prevalent in the elderly and associates with hematologic malignancy and cardiovascular disease. Although the risk of developing these diseases increases with radiation doses in atomic-bomb survivors, the causal relationship between radiation exposure and CH is unclear. This study investigated whether radiation exposure induces CH in mice 12–18 months after 3-Gy whole-body irradiation. We found radiation-associated increases in peripheral blood myeloid cells and red blood cell distribution width (RDW). Deep sequencing of bone marrow and non-hematopoietic tissue cells revealed recurrent somatic mutations specifically in the hematopoietic system in 11 of 12 irradiated mice but none in 6 non-irradiated mice. The irradiated mice possessed mutations with variant allele frequencies (VAFs) of > 0.02 on an average of 5.8 per mouse; mutations with VAFs of > 0.1 and/or deletion were prevalent. Examining hematopoietic stem/progenitor cells in two irradiated mice revealed several mutations co-existing in the same clones and multiple independent clones that deliver 60–80% of bone marrow nuclear cells. Our results indicate development of massive CH due to radiation exposure. Moreover, we have characterized mutations in radiation-induced CH.

Efficacy of bioreactor‐activated bone substitute with bone marrow nuclear cells on fusion rate and fusion mass microarchitecture in sheep

Bioreactors have been used for bone graft engineering in pre-clinical investigations over the past 15years. The ability of bioreactor-incubated bone marrow nuclear cells (BMNCs) to enhance bone-forming potential varies significantly, and the three-dimensional (3D) distribution of BMNCs within the scaffold is largely unknown. The aims of this study were (1) to investigate the efficacy of a carbonated hydroxyapatite (CHA) with/without BMNCs on spine fusion rate and fusion mass microarchitecture using a highly challenging two-level posterolateral spine fusion without instrumentation; and (2) to evaluate 3D distribution of BMNCs within scaffolds characterized by immunohistochemistry. Fusion rate and fusion mass were quantified by micro-CT, microarchitectural analysis, and histology. While the homogenous 3D distribution of BMNCs was not observed, BMNCs were found to migrate towards a substitute core. In the autograft group, the healing rate was 83.3%, irrespective of the presence of BMNCs. In the CHA group, also 83.3% was fused in the presence of BMNCs, and 66.7% fused without BMNCs. A significant decrease in the fusion mass porosity (p=.001) of the CHA group suggested the deposition of mineralized bone. The autograft group revealed more bone, thicker trabeculae, and better trabecular orientation but less connection compared to the CHA group. Immunohistochemistry confirmed the ability of bioreactors to incubate a large-sized substitute coated with viable BMNCs with the potential for proliferation and differentiation. These findings suggested that a bioreactor-activated substitute is comparable to autograft on spine fusion and that new functional bone regeneration could be achieved by a combination of BMNCs, biomaterials, and bioreactors.

Epigenetic Plasticity in ATP-Dependent Chromatin Remodeling Complexes in Human Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is the second most common leukemia worldwide with a median age of ~65 years at diagnosis. Myelodysplastic syndromes (MDS) are hematopoietic stem/progenitor cell (HSPC)-initiated clonal disorders of aged individuals that may transform into secondary AML. Cancer genome sequencing studies have identified somatic mutations in genes that encode chromatin regulators. However, mutational profiling alone may not help identify tumor associated transcriptional plasticity, one important hallmark in tumorigenesis. Nucleosome remodeler and deacetylase (NuRD) is an ATP-dependent chromatin remodeling complex, regulating cell-fate commitment and transcriptional architecture. Loss of Mi-2β (CHD4, ATPase subunit of NuRD) causes erythroid leukemia in mice. However, recent report suggests that CHD4 depletion inhibits AML development (Sperlazza J, et al., Blood 2015). Therefore, NuRD involvement in MDS/AML remains incompletely understood.We have analyzed gene expression of NuRD subunits in a cohort of elderly (median age 60 yrs) patients presenting with MDS. Expression of MBD3 , an integral scaffold of NuRD, but not other NuRD subunits, was significantly downregulated (2.5-fold; P = 0.004) in MDS (n=24) bone marrow (BM) CD34+ HSPCs compared to age-matched normal (n=6) BM CD34+ cells. Clonal hematopoiesis emerges as an integral component of hematopoietic aging and myeloid malignancies. We asked whether loss of MBD3 expression was associated with HSPC-aging. MBD3 expression was similar (P = 0.194) in CD34+ HSPCs derived from umbilical cord blood (n=6) and aged normal BM. Next, we extended our RT-qPCR analysis in a cohort of de novo AML (n=82) patients, which also suggested significant loss (3.5-fold; P = 0.003) of MBD3 in AML BM nuclear cells (BMNCs) compared to normal BM HSPCs. In addition, there were loss of expression of MTA1, MTA2, MTA3 , RBBP7 subunits (P < 0.001) in AML. Immunoblot analysis suggested dramatic loss of MBD3 in AML BMNCs, and co immunoprecipitation (Co-IP) analysis using antibody against intact CHD4, demonstrated association of residual NuRD subunits in primary AML cells. Sucrose density gradient centrifugation analysis further confirmed nucleation of residual NuRD (hereafter known as NuRDΔ) in AML BMNCs. Essentially these data identify HSPC-autonomous loss of MBD3, and indicate NuRDΔ nucleation as a general phenomenon in AML.We sought to determine chromatin occupancy of NuRDΔ in primary AML cells using ChIP-sequencing. CHD4 ChIP-seq identified ~ 9,000 target genes on average in AML (n=3) BMNCs. Interestingly, RNA-seq analysis in matched AML cohort compared to normal hematopoietic cells, and functional annotation clustering using Gene Ontology (GO)-terms on the CHD4 ChIP-seq identified gene set suggested an enrichment of transcripts that are involved in regulating small GTPase signaling, cytoskeletal organization and cell trafficking. Small GTPases play pivotal role in regulating myeloid leukemia cell engraftment and survival (Sengupta A, et al., Blood 2010; Thomas EK, Cancelas JA et al., Cancer Cell 2007). Lentivirus-mediated loss of function experiments and biochemical studies suggested NuRDΔ contribution in myeloid leukemia cell-microenvironment interaction. We further tested the hypothesis whether NuRDΔ function involves MBD2/MeCP1-mediated transcriptional dysregulation. Importantly, MBD3 antagonistically regulated H3K27me3, and consistent with this observation a subset of CHD4 ChIP-seq occupied genes in AML significantly overlapped with H3K27me3 binding. Pathway analysis suggested that those genes are involved in RNA transport and metabolism. Pharmacological inhibition of H3K27me3-methyl transferase activity along with small GTPase inhibition attenuated AML cell survival. SWI/SNF, another ATP-dependent chromatin remodeler, represents one of the most commonly mutated (~ 20%) chromatin modulators in human cancer. Co-IP experiments demonstrated interaction between NuRDΔ and leukemic SWI/SNF (having altered stoichiometry) in AML, and ChIP-seq analysis further identified leukemic SWI/SNF and NuRDΔ co-occupancy in AML BMNCs. In summary, we illustrate that in human primary AML cells, there is absence of specific subunits of NuRD complex; NuRDΔ, crosstalks with leukemic SWI/SNF and effects leukemia cell survival and transcriptional dysregulation. DisclosuresNo relevant conflicts of interest to declare.

Mesenchymal stem cell exosome-derived miR-223 alleviates acute graft-versus-host disease via reducing the migration of donor T cells

BackgroundMesenchymal stem cells (MSCs) have been utilized in treating acute graft-versus-host disease (aGvHD) as they show strong immunosuppressive capacity through the release of various mediators, including immunosuppressive molecules, growth factors, chemokines, and exosomes. MicroRNAs (miRNAs) derived from MSC exosomes (MSCs-Exo) play a critical role in the regulation of immune responses. However, the function of miRNAs in treating aGvHD remains unknown. Here, we performed expression profiling of exosome-miRNAs from human umbilical cord MSCs (huc-MSCs) and murine compact bone MSCs (mb-MSCs) to investigate their immunoregulation effects in aGvHD.MethodsHuc-MSCs-Exo and mb-MSCs-Exo were isolated and constructed MSCs-Exo-derived miRNA expression profiling using high-throughput sequencing. High expression of miR-223 was identified in both kinds of MSCs-Exo by bioinformatics analysis and quantitative real-time PCR (qPCR). In vitro cell crawling assay, transmigration assay and adhesion assay were subsequently applied to investigate the regulation of miR-223 on T cells. MiR-223 target gene was analyzed by western blot, luciferase analysis, and qPCR. Moreover, murine aGvHD model was established by infusing splenocytes and bone marrow nuclear cells from C57BL/6j mice (H-2Kb) into BALB/c recipient mice (H-2Kd). For therapeutic effect, MSCs or miR-223 Agomir were injected via tail vein. The general conditions of the mice in each group were monitored. Hematoxylin-eosin (H&E) staining was used to detect pathological changes of mice spleen, liver, and intestine. Mechanistically, immunofluorescence and flow cytometry were used to evaluate donor T cell migration, and enzyme-linked immunosorbent assay (ELISA) was used to detect the expression of serum inflammatory cytokines IFN-γ, TNF-α, and IL-17.ResultsHigh-throughput sequencing revealed high expression of miR-223 in huc-MSCs-Exo and mb-MSCs-Exo. MiR-223 could restrain adhesion and migration of T cells by inhibiting ICAM-1 expression in mouse lymphatic endothelial cells. MiR-223Agomir infusion attenuated aGvHD clinical symptoms, reduced the donor T cell infiltration into the spleen, liver, and intestine, and decreased inflammatory cytokines IFN-γ, TNF-α, and IL-17.ConclusionMSCs-Exo-derived miR-223 could attenuate aGvHD in mice through decreasing donor T cell migration. Our results unveil a new role of MSCs-Exo containing miR-223 in the treatment of aGvHD.

Collagen-hydroxyapatite composite substitute and bone marrow nuclear cells on posterolateral spine fusion in sheep.

The effects of a large collagen-hydroxyapatite (COL-HA) substitute with or without isolated bone marrow nuclear cells (BMNC) on spine fusion were evaluated using a two-level posterolateral spinal f...

B Lymphocyte Chemoattractant (CXCL13) Is an Indicator of Acute Gastrointestinal GVHD in Murine Model.

Gastrointestinal acute graft-vs.-host disease (GI aGVHD) remains a significant obstacle to the success of allogeneic hematopoietic cell transplantation and is a major cause of morbidity and mortality. In addition, GI aGVHD is often clinically indistinguishable from other causes of GI dysfunction such as conditioning regimen toxicity, infections, or medications, which complicates the diagnosis. Thus, specific biomarkers are needed to help improve diagnosis and obtain a deeper understanding of the cytokine changes in GI aGVHD. An MHC-mismatched model of aGVHD was established by transplanting 1×107 bone marrow nuclear cells and 3×107 spleen cells from C57/Bl6 mice or from BALB/c mice into lethally irradiated BALB/c recipients. The mice in the allogeneic transplantation group were intraperitoneally treated with 20mgkg-1day-1 cyclosporin A after aGVHD developed. Five micrograms of lipopolysaccharide were administered intraperitoneally daily to syngeneic recipients at day 11 to imitate infection; the same volume of phosphate-buffered saline was administered to control mice. The mice were killed at the indicated time points. Forty molecules derived from the GI tract were screened cytokine array. The data demonstrated that the expression of B lymphocyte chemoattractant (CXCL13) was increased by ~10-, 12-, and 16-fold upon the occurrence of aGVHD compared with infection, aGVHD after treatment, and the syngeneic control group, respectively. Thus, the elevation of BLC (CXCL13) is an indicator of acute GI GVHD.

Saponins from Sanguisorba officinalis Improve Hematopoiesis by Promoting Survival through FAK and Erk1/2 Activation and Modulating Cytokine Production in Bone Marrow.

Radix Sanguisorbae, the root of Sanguisorba officinalis L. is used as traditional Chinese medicine. In recent decades, it has been reported to be clinically effective against myelosuppression induced by chemotherapy and/ or radiotherapy. However, the underlining mechanism has not been well studied. In this work, we evaluated the hematopoietic effect of total saponins from S. officinalis L. on myelosuppressive mice induced by cyclophosphamide and by60Co-γ-irradiation and confirmed the therapeutic effect. Then, we found total saponins and their characteristic constituents Ziyuglycoside I and Ziyuglycoside II can inhibit apoptosis of TF-1 cells caused by cytokine deprivation, and promote survival of mouse bone marrow nuclear cells through focal adhesion kinase (FAK) and extracellular signal-regulated kinase 1/2 (Erk1/2) activation in vitro. In addition, they can down-regulate macrophage inflammatory protein 2 (MIP-2), platelet factor 4 (PF4) and P-selectin secretion, which are reported to be suppressive to hematopoiesis, both in vitro and in vivo. These results suggest that promotion of survival through FAK and Erk1/2 activation and inhibition of suppressive cytokines in the bone marrow is likely to be the pharmacological mechanism underlying the hematopoietic effect of saponins from S. officinalis L.

Hypermethylation of CpG sites at the promoter region is associated with deregulation of mitochondrial ATPsyn-β and chemoresistance in acute myeloid leukemia.

Aberrant DNA methylation status of some genes has been shown to be involved in chemoresistance of acute myeloid leukemia (AML). We have recently found that down-regulation of the β subunit of mitochondrial ATP synthase (ATPsyn-β) leads to adriamycin resistance in acute and chronic myeloid leukemia cells, and hypermethylation of the ATPsyn-β gene promoter is associated with chemoresistance in chronic myeloid leukemia. To further investigate the relationship between methylation of ATPsyn-β gene, mRNA expression as well as chemoresistance in AML. Quantitative RT-PCR and methylation specific PCR were performed to assess mRNA expression and methylation status of ATPsyn-β gene on primary bone marrow nuclear cells (BMMCs), and cell proliferation assay was used to determine the sensitivity of BMMCs to adriamycin. Hypermethylation status of ATPsyn-β gene promoter existed in those relapsed/refractory AML patients, and this hypermethylation of the gene was associated with a suppressed mRNA expression levels. Four patients at diagnosis and relapse underwent gene methylation status shift from hypermethylation to hypomethylation, which was accompanied by reduced mRNA expression of the gene. 5-azacitidine(5-Aza)- a demethylating agent, could restore ATPsyn-β mRNA expression and increase the adriamycin sensitivity of primary leukemic cells from seven relapsed/ refractory AML patients. Hypermethylation of ATPsyn-β gene promoter is associated with a down-regulated mRNA expression and chemoresistance in AML patients.

Protective effects of WR2721 on early bone marrow hematopoietic function in mice exposed to 6.5 Gy of (60)Co γ-rays

The aim of this study was to investigate the effect of WR2721(amifostine) against bone marrow hematopoietic damage of mice exposed to 6.5 Gy of (60)Co-γ ray. A total of 60 C57/BL6J mice was divided into 3 groups:normal group (mice were injected with physiological salt solution), irradiation group (mice were injected with physiologic salt solution before irradiation) and WR2721 group (mice were injected with WR2721 before irradiation). The WBC, neutrophil (Neut), Plt and RBC levels in peripheral blood of 3 group mice were counted within 60 days after irradiation; the bone marrow nuclear cells (BMNC) were counted at 2 and 24 hours after irradiation; the hematopoietic stem/progenitor cell (LK/LSK) level and colony formation capability were detected by flow cytometry at 2 and 24 hours after irradiation. The results indicated that the counts of WBC and neut at 4 and 18 days, Plt at 7-18 days and RBC at 10-30 day after irradiation in WR2721 group were higher than those in irradiation group (P < 0.05); the BMNC, LSK and LK levels obviously increased at 24 hours after irradiation (P < 0.05), the CFU-GEMM, CFU-GM, CFU-MK BFU-E and CFU-E all significantly increased at 2 and 24 hours after irradiation (P < 0.01), as compared with irradiation group. It is concluded that WR2721 can effectively alleviate early hematopoietic damage and promote the fast recovery of peripheral blood cells in mice exposed to γ-ray, suggesting that the WR2721 has significant radioprotective effect on hematopoietic system.

Infusion of freshly isolated autologous bone marrow derived mononuclear cells prevents endotoxin-induced lung injury in an ex-vivo perfused swine model

IntroductionThe acute respiratory distress syndrome (ARDS), affects up to 150,000 patients per year in the United States. We and other groups have demonstrated that bone marrow derived mesenchymal stromal stem cells prevent ARDS induced by systemic and local administration of endotoxin (lipopolysaccharide (LPS)) in mice.MethodsA study was undertaken to determine the effects of the diverse populations of bone marrow derived cells on the pathophysiology of ARDS, using a unique ex-vivo swine preparation, in which only the ventilated lung and the liver are perfused with autologous blood. Six experimental groups were designated as: 1) endotoxin alone, 2) endotoxin + total fresh whole bone marrow nuclear cells (BMC), 3) endotoxin + non-hematopoietic bone marrow cells (CD45 neg), 4) endotoxin + hematopoietic bone marrow cells (CD45 positive), 5) endotoxin + buffy coat and 6) endotoxin + in vitro expanded swine CD45 negative adherent allogeneic bone marrow cells (cultured CD45neg). We measured at different levels the biological consequences of the infusion of the different subsets of cells. The measured parameters were: pulmonary vascular resistance (PVR), gas exchange (PO2), lung edema (lung wet/dry weight), gene expression and serum concentrations of the pro-inflammatory cytokines IL-1β, TNF-α and IL-6.ResultsInfusion of freshly purified autologous total BMCs, as well as non-hematopoietic CD45(-) bone marrow cells significantly reduced endotoxin-induced pulmonary hypertension and hypoxemia and reduced the lung edema. Also, in the groups that received BMCs and cultured CD45neg we observed a decrease in the levels of IL-1β and TNF-α in plasma. Infusion of hematopoietic CD45(+) bone marrow cells or peripheral blood buffy coat cells did not protect against LPS-induced lung injury.ConclusionsWe conclude that infusion of freshly isolated autologous whole bone marrow cells and the subset of non-hematopoietic cells can suppress the acute humoral and physiologic responses induced by endotoxemia by modulating the inflammatory response, mechanisms that do not involve engraftment or trans-differentiation of the cells. These observations may have important implications for the design of future cell therapies for ARDS.

Dual control of Shuanghuang Shengbai granule on upstream and downstream signal modulators of CyclinD-CDK4/6 signaling pathway of cell cycle in Lewis-bearing mice with cyclophosphamide-induced myelosuppression

BackgroundThis study investigated the dual control mechanism of the Shuanghuang Shengbai granule in modulating the cell cycle in Lewis-bearing mice with cyclophosphamide induced myelosuppression.MethodsThirty Lewis-bearing mice were randomly grouped into an untreated group, control group, and treated group. Both treated and untreated groups were intraperitoneally injected with cyclophosphamide to produce a myelosuppression model. Mice in the treated group were fed with the Shuanghuang Shengbai granule (40 g/day) for 6 consecutive days. Standard blood tests and the count of bone marrow nuclear cells were performed, and the cell reproductive cycles of bone marrow and tumors were measured in these mice. In addition, the western blot approach was used to measure the upstream activating signals of CyclinD-CDK4/6 such as c-Myc and CDC25A, the upstream suppression signals such as p16INK4a, and the expression of downstream activated signals such as Rb, pRB, and E2F. All of the tested results were validated by reverse transcription quantitative real-time polymerase chain reaction.ResultsThe results showed that the Shuanghuang Shengbai granule could elevate the count of leukocyte and bone marrow nuclear cells of Lewis-bearing mice with cyclophosphamide induced myelosuppression. It could also stimulate bone marrow cells to move from G0/G1 phases to S phase, accelerating the progress of the cell reproductive cycle and increasing the cell proliferation index. Simultaneously, the Shuanghuang Shengbai granule could also suppress cancer cells moving from G0/G1 phase to S phase, reducing the proliferation index. The tumor weight of Lewis-bearing mice in the treated group was much less than those of the control group. Expression levels of c-Myc, CDC25A, Rb, pRb, and E2F of bone marrow in Shuanghuang Shengbai granule-treated mice was higher compared to the control group, whereas they were lower in the cancer cells.ConclusionThe experimental results demonstrate that the Shuanghuang Shengbai granule has dual control on the cell reproductive cycles in cancer cells and bone marrow nuclear cells in Lewis-bearing mice.

Treatment of pressure ulcers with autologous bone marrow nuclear cells in patients with spinal cord injury

ContextPressure ulcers are especially difficult to treat in patients with spinal cord injury (SCI) and recurrence rates are high. Prompted by encouraging results obtained using bone marrow stem cells to treat several diseases including chronic wounds, this study examines the use of autologous stem cells from bone marrow to promote the healing of pressure ulcers in patients with SCI.ObjectiveTo obtain preliminary data on the use of bone marrow mononuclear cells (BM-MNCs) to treat pressure ulcers in terms of clinical outcome, procedure safety, and treatment time.ParticipantsTwenty-two patients with SCI (19 men, 3 women; mean age 56.41 years) with single type IV pressure ulcers of more than 4 months duration.InterventionsBy minimally invasive surgery, the ulcers were debrided and treated with BM-MNCs obtained by Ficoll density gradient separation of autologous bone marrow aspirates drawn from the iliac crest.ResultsIn 19 patients (86.36%), the pressure ulcers treated with BM-MNCs had fully healed after a mean time of 21 days. The number of MNCs isolated was patient dependent, although similar clinical outcomes were observed in each case. Compared to conventional surgical treatment, mean intra-hospital stay was reduced from 85.16 to 43.06 days. Following treatment, 5 minutes of daily wound care was required per patient compared to 20 minutes for conventional surgery. During a mean follow-up of 19 months, none of the resolved ulcers recurred.ConclusionsOur data indicate that cell therapy using autologous BM-MNCs could be an option to treat type IV pressure ulcers in patients with SCI, avoiding major surgical intervention.

HLA-Haploidentical Hematopoietic Stem Cell Transplantation In An Infant with Malignant Infantile Osteopetorosis.

AbstractAbstract 4585Malignant infantile osteopetrosis is an autosomal recessive disease characterized by a lack of osteoclastic function with incidence of 1: 200,000 to 1: 300,000. In consequence of disturbed bone building and remodeling, affected patients have osteosclerosis, dense fragile bone, and a marked reduction in the bone marrow cavity. Clinical features, such as anemia, thrombocytopenia, hepatosplenomegaly, bone fractures, bone deformity, and cranial nerve entrapment, appear soon after birth or within the first years of life. In the natural course of the disease, only 30% of children can survive to more than six years old. Because osteoclasts are of hematopoietic origin, allogeneic stem cell transplantation is the only curable therapy. Recently, successful HLA-haploidentical hematopoietic stem cell transplantation (HSCT) has been reported for patients without HLA- matched donors. We also present an infant with malignant infantile osteopetrosis who underwent HLA-haploidentical HSCT. A four-month-old boy was referred to our hospital for splenomegaly and pancytopenia. A physical examination revealed failure to thrive, distention of anterior fontanel, and hepatosplenomegaly. Laboratory findings indicated thrombocytopenia and anemia, and elevated alkaline phosphatase. Osteoclasts were hyperplastic in bone marrow. Radiography showed homogeneous and sclerotic bones with absence of corticomedullary junction, and bone marrow scintigraphy showed no accumulation of bone marrow. Subsequently, TCIRG1 mutations are identified. All these findings are compatible with malignant infantile osteopetrosis and the diagnosis was made. We conducted HSCT to revive his osteoclastic function. However, he didn't have HLA-matched donors not only in relatives but also in the bone marrow bank and the cord blood bank of Japan. We attempted HLA-haploidentical bone marrow transplantation (BMT) from his father with written informed consent. The patient was prepared for BMT at 11 months of age by conditioning with busulfan (6 mg/kg × 4/day × 4 days), cyclophosphamide (360mg/m2 × 2 days) and antithymocyte globulin (2 mg/kg/day × 3 days). Prophylaxis for graft-versus-host disease (GVHD) included tacrolimus and short-term methotrexate. Bone marrow nuclear cells (1×109/kg) were collected from his father and transplanted without any manipulations. The engraftment of neutrophils was confirmed on day 9 after BMT. Acute GVHD was limited to the skin (grade I). At 2 months after BMT, neutrophil counts, platelet counts, and hemoglobin level were all within normal limits. Bone marrow scintigraphy revealed a significant uptake in bone marrow. There have been no chronic GVHD up to six months after BMT.HSCT for patients with osteopetrosis is challenging as it is reported that HLA-haploidentical HSCT has the high rate of acute and chronic GVHD. Fortunately, HLA-haploidentical BMT was successful in our patient. This might be due to our regimen for prophylaxis of rejection and GVHD. We used ATG in conjunction with tacrolimus: this regimen may be prophylactic not only for rejection but also for GVHD as previously reported (Schulz et al. Blood 2002; 99: 3458–3460). In conclusion, HLA-haploidentical family donors should be considered as the alternative hematopoietic stem cell source for patients with malignant infantile osteopetrosis in condition without HLA-matched donors. Disclosures:No relevant conflicts of interest to declare.

Interphase Fluoroscence in Situ Hybridization Improves the Detection Rate of CNS Involvement in Childhood Acute Lymphoblastic Leukemia.

Abstract 4714 BackgroundMorphologic examination of CSF cytospin is the standard method in the detection of CNS involvement of childhood acute lymphoblastic leukemia (ALL). However, the result widely varies depending on the status of cell degeneration caused by cytospin procedure. We performed interphase fluoroscence in situ hybridization (FISH) on CSF cytospin cells to examine its potential usefulness in detecting malignant cell population in the CSF. Materials and MethodsThe study used 19 CSF cytospin specimens of the childhood ALL patients under treatment. All of them had cytogenetic abnormalities in the bone marrow nuclear cells at the initial diagnosis. None of the patients showed malignant cells in morphologic examination of the cytospin. Interphase FISH was performed to detect recurrent genetic abnormalities including rearrangement of BCR/ABL, TEL/AML1, MLL and IgH rearrangement, and p16 deletion using LSI BCR/ABL dual color, dual fusion translocation probe, LSI TEL/AML1 ES dual color translocation probe, LSI MLL dual color, break apart rearrangement probe, LSI IGH dual color, break apart rearrangement probe, and LSI p16/CEP 9 dual-color probe (Abbot Molecular Diagnostics). FISH signals were interpreted using automated slide scanning system (Metafer 4 system) and manually read for confirmation. ResultTwo patients had the same cytogenetic abnormalities in CSF cytospin as observed in the bone marrow mononuclear cells: one patient had 4 copies of AML1 gene, suggesting tetrasomy 21 or multiple fusion partner gene other than chromosome 12, both in bone marrow and in CSF; the other had 3 copies of p16 gene including one with low signal intensity both in bone marrow and in CSF. Ten patients did not have any cytogenetic abnormalities. We could not interpret the result in the other 7 patients due to low cell count and/or low signal intensity. ConclusionOut of 19 who were diagnosed not to have malignant cell in CSF in morphologic examination, 2 patients had cells with the same clonal abnormalities in the CSF as bone marrow. Our result indicates that FISH study would be helpful in evaluating CSF involvement of hematologic malignancies including ALL. In order to improve the performance, it is required to prevent degeneration of the cells by prompt specimen delivery and slide preparation. Disclosures:No relevant conflicts of interest to declare.

Adipocyte-Derived Stromal Cells Facilitate Hematopoiesis in Vitro and in Vivo: Advantages Over Bone Marrow-Derived Stromal Cells

Mesenchymal stromal cells have emerged as a new therapeutic modality that could improve the engraftment in stem cell transplantation since the injection of bone marrow-derived stromal cells (BMSCs) into the bone marrow cavity had been proved to reconstitute the hematopoietic microenvironment. However, BMSCs can be obtained in a limited number and cannot be so easily expandable. Recent studies showed that a large number of stromal cells can be isolated from fat tissue easily and expand rapidly ex vivo, suggesting that adipocyte-derived stromal cells (ADSCs) might be a promising alternative cell source. However, it has been reported that ADSC is not completely identical to BMSC. Thus, it is largely unknown whether ADSCs can be useful for stem cell transplantation so far. These facts prompted us to elucidate extensively the property of ADSCs on hematopoiesis. We isolated ADSCs from the inguinal fat tissue of C57/BL6 mice and compared with BMSCs from various aspects. Proliferation assays showed that the number of mouse primary ADSCs increased 4~5 times faster that of BMSCs in vitro. Similarly, ADSCs, when injected into the bone marrow cavity of NOD/SCID mouse, propagated themselves rapidly. In vitro coculture assays with stromal cells and human CD34+ peripheral blood stem cells (PBSCs) showed that ADSC layer propagated more suspension cells, which were CD45 and CD33 positive, significantly than BMSC layer (p< 0.01). In vitro progenitor assays using methylcellulose media revealed that addition of BMSCs or ADSCs to human CD34+ PBSCs yielded more colony formation units (CFUs) than PBSCs alone. Interestingly, more increased numbers of CFUs were observed in the wells of ADSCs plus PBSCs than in the wells of BMSCs plus PBSCs (p< 0.01). To address whether ADSCs contributes to the development of hematopoiesis in vivo, we analyzed the engraftment after transplantation with or without ADSCs. ADSCs (1 × 105) or BMSCs (1 × 105) were injected into the right tibiae of lethally irradiated C57/BL6 mice (N=10), and then bone marrow nuclear cells (BMNCs) were administered via tail vein into each mouse. Control mice received the same amount of PBS in the right tibia. At 6 days after transplantation, 5 of mice were humanely killed, and the tibiae were excised. Surprisingly, microscopic analysis showed that the right tibiae with ADSCs possessed increased cellularity compared to the BMSC-injected and controls (BMSCs facilitated hematopiesis mildly in comparison with controls). The rest of mice were viable even 3 months after transplantation. We performed secondary transplantation experiments to evaluate the effect of ADSCs on long-term hematopoietic stem cells (LT-HSCs). Bone marrow nuclear cells (BMNCs) obtained from the tibia, where the mixture of ADSCs and BMNCs were injected previously, of primary mice were intravenously transplanted into lethally irradiated secondary recipient mice (C57/BL6, N=5). BMNCs obtained from the tibia, where the mixture of BMSCs and BMNCs were injected, were used as a control. Three months after secondary transplantation, all of mice (N=5) which received BMNCs obtained from the ADSCs-injected tibia survived, and their blood cell counts were almost similar with controls, suggesting that ADSCs did not possess any harmful effect on LT-HSCs. Taken together, we conclude that ADSCs have various advantage and could be useful for stem cell transplantation.

Effects of Transmigration of Bone Marrow Nuclear Cells Through Endothelial Cell and Stromal Cell by Leukotriene B4and Inhibition of Reactive Oxygen Species

Objectives:Leukotriene B4(LTB4) is lipid mediator derived from membrane phospholipids during the process of inflammation, having many roles(ie;inducer of chemotaxis, the production of nitric oxide, transepithelial migration of neutrophil). The major activities of LTB4 include the recruitment and activation of leukocytes, suggesting that it may involve the process for transendothelial migration of nuclear cells in bone marrow environment. Reactive Oxygen Species (ROS) have a cell signaling roles that are involved in signal transduction cascades of numerous growth factor-, cytokine-, and hormone-mediated pathways, and regulate many biological systems. In this present study, we focused on the role of LTB4 and ROS on transmigration of bone marrow nuclear cells across endothelial or stromal cell monolayer. Methods:MS-5, murine stromal cell line cells, or bEnd.3, murine microvascular cell line cells, were grown to confluence on microporous transwell membrane. Murine marrow cells were placed on top of the prepared transwell membrane. The transwells were then seated in wells containing media and LTB4 with or without pretreatment of N-acetylcysteine(NAC), an oxygen free radical scavenger, or diphenylene iodonium(DPI), an inhibitor of NADPH oxidase-like flavoproteins. Cells that migrated through the stromal or endothelial layer into the wells were assayed for transendothelial migration. Results:The numbers of migrated bone marrow nuclear cells through the bEnd.3 were increased by treatment of LTB4(control, 12.5±0.2%;50nM, 22.7±0.9%;100nM, 44.3±1.4%;200 nM, 36.3±0.9%;p<0.05). The numbers of migrated bone marrow nuclear cells through the MS5 were also increased by treatment of LTB4(control, 11.0±0.9%;50nM, 25.7±0.9%;100nM, 35.8±1.8%;200nM, 32.1±0.9%;p<0.05). However, increasing effect of LTB4 to the transmi-

Effects of notoginosides on proliferation and upregulation of GR nuclear transcription factor in hematopoietic cells

To investigate the effects of panax notoginosides (PNS) on the proliferation of human hematopoietic stem/progenitor cells, and to explore the signaling pathway of the nuclear transcription factor of the glucocorticoid receptor (GR-NTF) initiated by PNS related with the proliferation. The human CD34+ cells and bone marrow nuclear cells were exposed to PNS at a concentration of 0, 10, 25, 50, and 100 mg/L, respectively, in semi-solid culture system to observe colony forming unite of all lineages, granulocyte, erythrocyte, and megakaryocyte (CFUGEMM, CFU-GM, CFU-E, and CFU-MK). Three lineages of human hematopoietic cell lines, including granulocytic HL-60, erythrocytic K562, megakaryocytic CHRF- 288, and Meg-01 cells were incubated with PNS at 20 mg/L for 14 d. Meanwhile, dexamethasone (Dex) was used as a positive control. The nuclear protein of the cells was analyzed by Western blotting with monoclonal antibodies against the amino or carboxyl terminus of GR-NTF. Electrophoretic mobility shift assay performed by using the 32P-radiolabeled GR-NTF consensus oligonucleotide. PNS promoted the proliferation of CD34+ cells and significantly raised the colony numbers of CFU-GEMM by 34.7%+/-16.0% over the non-PNS control (P<0.01). PNS also enhanced the proliferation of CFU-GM, CFU-E, and CFU-MK by 39.3%+/- 5.7%, 33.3%+/-7.3%, and 26.2%+/-3.2%, respectively. GR-NTF protein levels of either the amino or carboxyl terminus in K562, CHRF-288, and Meg-01 treated by PNS increased by 2.4-2.8 fold and 1.3- 3.9 fold over the untreated cells. GR-NTF binding activity, initiated by either PNS or Dex, was apparently elevated to form the complex of GR-NTF with DNA as higher density bands in K562 and CHRF-288 cells, and some activity appeared as a band in HL-60 cells induced by PNS. PNS displayed the action of hematopoietic growth factor-like or synergistic efficacy to promote proliferation of human progenitor cells, may play a role in the upregulation of gene expression related to proliferation of hematopoietic cells through increasing the GR-NTF synthesis and its DNA binding activity.