Subpopulations Of Bone Marrow Cells Research Articles

FLAER Revealed Normally Expected Non-PNH FLAER-Dim Immature Myeloid Cells (CD117+/CD34-) In Bone Marrow Aspirates and Could Be Utilized as a Marker of Hierarchical Hematopoiesis.

Fluorescently labeled aerolysin (FLAER) is widely used for the identification of paroxysmal nocturnal hemoglobinuria (PNH) clones in peripheral blood (PB) samples. However, there are only a few reports on the differential fluorescent intensity of FLAER in normal bone marrow (BM) cell subpopulations. The purpose of this study was to evaluate FLAER expression during normal and pathological hematopoiesis, to map the critical existence of non-PNH FLAER-dim cells. A total of 54 BM aspirates were prospectively analyzed with FLAER-based flow cytometric (FC) protocols, during their routine work-up. These were obtained from patients with the following diagnoses: PNH (3), infections/reactive (5), myelodysplastic syndromes (MDS) (7), myelodysplastic/myeloproliferative neoplasms (MDS/MPN) (4), chronic myelogenous leukemia (CML) (3), acute myelogenous leukemia (AML) at diagnosis (20), AML in measurable residual disease (MRD) assessment (7), and B-cell acute lymphoblastic leukemia (B-ALL) in MRD assessment (5). The applied protocols consisted of FLAER, HLA-DR, CD14, CD33, CD34, CD66b, CD38, CD117, CD64, CD45, and FLAER, CD66c, CD14, CD33, CD34, CD66b, CD123, CD16, CD64, and CD45, respectively. FLAER expression was assessed in CD34++/CD38- and CD34+/CD38+ stem cells, CD34-/CD117+/HLA-DR+/CD33+ myeloid precursors, and CD64+/CD14-/HLA-DR+ monocyte precursors but also in mature myeloid cells. All patients revealed an intermediate FLAER intensity in CD34++/CD38- stem cells, with a discrete FLAER-negative subpopulation observed only in maturing CD34+/CD38+ stem cells of patients with PNH. The lowest FLAER intensity was noticed in CD34-/CD117+/HLA-DR+/CD33+ myeloid precursors, not only in patients with PNH but also in PNH-negative BM aspirates. An ascending FLAER intensity was further observed during monocytic and granulocytic maturation, with a discrete FLAER-negative population in CD64+/CD14-/HLA-DR+ monocyte precursors and maturing neutrophils and monocytes of patients with PNH only. The maturation pattern of FLAER expression was further confirmed in a patient with acute promyelocytic leukemia treated with all-trans retinoic acid (ATRA), where FLAER was concurrently upregulated with CD66b in a consecutive series of PB samples tested over a 20-day-period after diagnosis. The application of FLAER in PNH-positive and PNH-negative reactive or malignant BM aspirates identified normally expected non-PNH FLAER-dim CD34-/CD117+/HLA-DR+/CD33+ myeloid precursors in all samples. A specific FLAER-associated maturation pattern was observed, which is proposed for further study within MRD and diagnostic protocols.

Flow cytometry of DNMT1 as a biomarker of hypomethylating therapies.

The 5-azacytidine (AZA) and decitabine (DEC) are noncytotoxic, differentiation-inducing therapies approved for treatment of myelodysplastic syndrome, acute myeloid leukemias (AML), and under evaluation as maintenance therapy for AML postallogeneic hematopoietic stem cell transplant and to treat hemoglobinapathies. Malignant cell cytoreduction is thought to occur by S-phase specific depletion of the key epigenetic regulator, DNA methyltransferase 1 (DNMT1) that, in the case of cancers, thereby releases terminal-differentiation programs. DNMT1-targeting can also elevate expression of immune function genes (HLA-DR, MICA, MICB) to stimulate graft versus leukemia effects. In vivo, there is a large inter-individual variability in DEC and 5-AZA activity because of pharmacogenetic factors, and an assay to quantify the molecular pharmacodynamic effect of DNMT1-depletion is a logical step toward individualized or personalized therapy. We developed and analytically validated a flow cytometric assay for DNMT1 epitope levels in blood and bone marrow cell subpopulations defined by immunophenotype and cell cycle state. Wild type (WT) and DNMT1 knock out (DKO) HC116 cells were used to select and optimize a highly specific DNMT1 monoclonal antibody. Methodologic validation of the assay consisted of cytometry and matching immunoblots of HC116-WT and -DKO cells and peripheral blood mononuclear cells; flow cytometry of H116-WT treated with DEC, and patient samples before and after treatment with 5-AZA. Analysis of patient samples demonstrated assay reproducibility, variation in patient DNMT1 levels prior to treatment, and DNMT1 depletion posttherapy. A flow-cytometry assay has been developed that in the research setting of clinical trials can inform studies of DEC or 5-AZA treatment to achieve targeted molecular pharmacodynamic effects and better understand treatment-resistance/failure.

Low Circulating B Lymphocytes in Newly Diagnosed Follicular and Diffuse Large B Cell Lymphoma: Differences Compared to Normal Subjects and Possible Prognostic Role

Introduction: Reduction of the lymphocyte to monocyte ratio (LMR) has an unfavorable impact on overall survival in Diffuse Large B Cell Lymphoma (DLBCL), and on treatment response in Follicular Lymphoma (FL). Abnormal LMR seems primarily due to a reduction in absolute value of lymphocytes. Based on these observations, we have prospectively investigated peripheral blood (PB) and Bone Marrow (BM) lymphocyte subpopulations in a series of previously untreated DLBCL and FL. Aim of the study was: i. to assess whether LMR alteration is linked to a distinct lymphocyte subset; ii. to correlate PB with BM cell subpopulations; iii. to compared DLBCL and FL with regards to PB and BM lymphocyte subpopulations. Methods: Overall, PB cells from 58 DLBCL and 27 FL at diagnosis were investigated; BM cell populations were analyzed in 77 DLBCL and 54 FL. None of the studied patients had BM involvement nor circulating lymphoma cells detectable by cytometry; in addition, none of the patients had been previously treated with immunosuppressive drugs. PB from 40 healthy subjects and BM samples from 69 subjects without primary BM dysfunctions were used as control. Multicolor flow cytometry (MFC) was used to evaluate percentages and absolute values of the following cell subsets: CD3+ T cell, CD3-CD56+ NK cells, CD19+CD20+ B cells. Among B cells, t he analysis included transitional (CD38+high/CD24+high/IgD+/CD27-), naive (CD38+/CD24+/IgD+/CD27-), memory IgD+ (CD38-/+CD24+/IgD+/CD27+), and memory IgD- (CD38-/CD24+high/IgD-/CD27+) subsets; B cell precursors were investigated as CD19+CD20-CD10+ and mature B cells as CD19+CD20+CD10-. Results: A significant reduction in median absolute lymphocyte count was seen in PB from lymphoma patients (median: 1,526/µL) compared to controls (median: 2,195/ µL). Moreover, absolute lymphocytes were more markedly reduced in DLBCL (median: 1,386 /µL) compared to FL (median: 1883/µl) (p<0.001). This resulted in a significantly lower median PB-LMR in DLBCL (median LMR: 3) compared to FL patients (median LMR: 5) (p=0.001). Among PB cell subpopulations, a marked reduction in circulating B cells was observed in lymphoma patients (median 96/µL) compared to controls (median 224/µL). Again, loss of circulating B-cells was more markedly pronounced in DLBCL (median: 81.5 cell/µL) compared to FL (median: 150 cell/µL) (p=0.013). Differences in circulating B-cells among DLBCL, FL and controls are shown in Figure 1A. At BM analysis, a reduction of B cell precursor (CD19+/CD20-/CD10+) could be documented only in DLBCL. A reduction in circulating T-lymphocytes in lymphoma patients compared to controls was detected as well, although at a lower extent compared to B-cells. No significant abnormalities in the amount of circulating NK-cells was seen in both DLBCL and FL patients. When these data were matched with treatment outcome, the reduction of circulating B cells proved to be associated with an unfavorable outcome in younger patients with DLBCL. Indeed, among DLBCL patients aged < 60 yrs, a poorer progression free survival was seen in those having less than 96 B-cells/µL compared to those with PB B-cells equal or above 96 B-cells/µl (Figure 1B). Conclusions: The study indicates that circulating B lymphocytes are significantly reduced in both DLBCL and FL compared to age-matched healthy controls; however, their loss is much more pronounced in DLBCL compared to FL. Indeed, abnormalities in B-cell progenitors are present in BM from DLCBL but not from FL. In addition, B-cell reduction may have an unfavorable impact in the long-term outcome of DLBCL, at least in younger patients. Lastly, both B and T-cell subsets are decreased in DLBCL and FL whereas circulating NK subset does not seem to be affected. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Development of a method for the identification of receptor activator of nuclear factor-κB+ populations in vivo

ObjectivesOsteoclasts are induced by macrophage colony-stimulating factor-1 (CSF-1) and receptor activator of nuclear factor-κB (RANK) ligand (RANKL). Monocyte/macrophage lineages are thought to be osteoclast precursors; however, such cells have not been fully characterized owing to a lack of tools for their identification. Osteoclast precursors express colony-stimulating factor-1 receptor (CSF-1R) and RANK. However, the capacity of conventional methods using anti-RANK antibodies to detect RANK+ cells by flow cytometry is insufficient. Here, we developed a high-sensitivity method for detecting RANK+ cells using biotinylated recombinant glutathione S-transferase-RANKL (GST-RANKL-biotin). MethodsWe sorted sub-populations of mouse bone marrow (BM) or peripheral blood (PB) cells using GST-RANKL-biotin, anti-CSF1R, and anti-B220 antibodies and induced osteoclastogenesis in vitro. ResultsThe frequency of the RANK+ population in BM detected by GST-RANKL-biotin was significantly higher than that detected by anti-RANK antibodies. Although RANK+ cells were detected in both the B220+ and B220− populations, the macrophage lineage was present only in B220−. Unexpectedly, a significantly higher number of osteoclasts was induced in RANK−CSF-1R+ cells than in RANK+CSF-1R+ cells contained in the B220− population. In contrast, the PB-derived B220−RANK+CSF-1R+ population contained a significantly higher frequency of osteoclast precursors than the B220−RANK−CSF-1R+ population. ConclusionsThese results suggest that GST-RANKL-biotin is useful for the detection of RANK+ cells and that RANK and CSF-1R may be helpful indicators of osteoclast precursors in PB.

Single-cell profiling reveals the trajectories of natural killer cell differentiation in bone marrow and a stress signature induced by acute myeloid leukemia

SummaryNatural killer (NK) cells are innate cytotoxic lymphoid cells (ILCs) involved in the killing of infected and tumor cells. Among human and mouse NK cells from the spleen and blood, we previously identified by single-cell RNA sequencing (scRNAseq) two similar major subsets, NK1 and NK2. Using the same technology, we report here the identification, by single-cell RNA sequencing (scRNAseq), of three NK cell subpopulations in human bone marrow. Pseudotime analysis identified a subset of resident CD56bright NK cells, NK0 cells, as the precursor of both circulating CD56dim NK1-like NK cells and CD56bright NK2-like NK cells in human bone marrow and spleen under physiological conditions. Transcriptomic profiles of bone marrow NK cells from patients with acute myeloid leukemia (AML) exhibited stress-induced repression of NK cell effector functions, highlighting the profound impact of this disease on NK cell heterogeneity. Bone marrow NK cells from AML patients exhibited reduced levels of CD160, but the CD160high group had a significantly higher survival rate.

The effect of ionising radiation on the phenotype of bone marrow-derived extracellular vesicles.

Ionising radiation-induced alterations affecting intercellular communication in the bone marrow (BM) contribute to the development of haematological pathologies. Extracellular vesicles (EVs), which are membrane-coated particles released by cells, have important roles in intercellular signalling in the BM. Our objective was to investigate the effects of ionising radiation on the phenotype of BM-derived EVs of total-body irradiated mice. CBA mice were irradiated with 0.1 Gy or 3 Gy X-rays. BM was isolated from the femur and tibia 24 h after irradiation. EVs were isolated from the BM supernatant. The phenotype of BM cells and EVs was analysed by flow cytometry. The mean size of BM-derived EVs was below 300 nm and was not altered by ionising radiation. Their phenotype was very heterogeneous with EVs carrying either CD29 or CD44 integrins representing the major fraction. High-dose ionising radiation induced a strong rearrangement in the pool of BM-derived EVs which were markedly different from BM cell pool changes. The proportion of CD29 and CD44 integrin-harbouring EVs significantly decreased and the relative proportion of EVs with haematopoietic stem cell or lymphoid progenitor markers increased. Low-dose irradiation had limited effect on EV secretion. Ionising radiation induced selective changes in the secretion of EVs by the different BM cell subpopulations. The novelty of the paper consists of performing a detailed phenotyping of BM-derived EVs after in vivo irradiation of mice.

P-257 Deep polychromatic flow cytometry characterization of circulating endothelial cells in metastatic colorectal cancer patients

Circulating endothelial cells (CEC) and their progenitors (EPC) are restricted subpopulations of peripheral blood (PB), cord blood (CB), and bone marrow (BM) cells, involved in the endothelial remodeling and turnover. Both CEC and EPC have been studied as potential biomarkers in colon cancer, but a lack of specificity has limited their clinical application. In this work, a highly sensitive polychromatic flow cytometry method has been applied for CEC characterization, exploring the role of different circulating endothelial cell subpopulations as potential and predictive biomarkers in colorectal cancer patients.

IMMUNOLOGICAL PARAMETERS OF BONE MARROW IN NON-SMALL CELL LUNG CANCER

Introduction Generation of most immunocompetent cells takes place in bone marrow Bone marrow. As well, bone marrow is a peripheral lymphoid organ where antitumor effector cells and memory cells are present. The aim of the work is to estimate peripheral lymphoid cell subpopulations in bone marrow of lung cancer patients. Materials and methods Study has been done in 68 pts with lung cancer: squamous cell cancer (n = 28), adenocarcinoma (n = 38), other forms (n = 2). In all cases standard diagnostic and staging procedures were performed, as well as morphological (myelogram) and immunological study of bone marrow lymphocyte subpopulations. Multicolor Flow cytomtry was used for study of bone marrow lymphocyte populations. We studied T-cells and its subpopulations, B-cells, NK-cells, perforin-positive cells, and CD27-positive cells. Results Squamous cell lung cancer was characterized by higher content of bone marrow mature T-cells (CD3), and CD8 lymphocytes. More typical for adenocarcinoma was mature B-cell reaction (CD20). Effector (perforin-positive) populations of lymphocytes also were related to histological type of cancer: for adenocarcinoma presence of CD4-positive cytotoxic lymphocytes and CD27-expression on effector cells. Perforin-containing lymphoid cells were in opposite correlation to erythrocaryocytes. Conclusion Subpopulational lymphocyte content of bone marrow is related to histological variant of cancer and erythropoiesis in lung cancer patients.

Aberrant RHAMM Splicing in Multiple Myeloma (MM) and Its Implications for Immunotherapy

Background: Receptor for hyaluronan-mediated motility (RHAMM) or CD168 has been a promising target for MM immunotherapy because it is overexpressed in MM cells. RHAMM has been tested as a target for an anti-RHAMM peptide vaccination approach in MM and other hematological malignancies. Although RHAMM peptide-induced immune response in patients, clinical outcomes were mixed, that can be a result of equal expression levels of RHAMM in different subpopulation of bone marrow (BM) cells in MM patients and healthy donors (HD). To enhance current RHAMM-peptide and future immunotherapeutic approaches, we investigated the cause of altered RHAMM mRNA splicing in MM patients. mRNA splicing has the potential to produce numerous mis-spliced genes, creating novel disease markers; some resulting proteins are likely to contain neoantigens selectively expressed on MM tumor cells. Methods/results: Splicing alterations can be caused by single nucleotide variations (SNVs) that affect splicing regulatory elements (SREs), or by deregulated splicing factors (SFs). We evaluated the incidence of SNVs located in the vicinity of the RHAMM. We identified a total of 57 SNVs: 72% SNVs are in the intronic region, and 28% are in the RHAMM coding region. We used the "HEXplorer" tool and predicted that four SNVs have the potential to contribute to aberrant RHAMM splicing in MM either by altering SF binding to SREs or by impacting splice site selection. Predicted SNVs were evaluated using an in vivo splicing assay to identify SNV-clusters causing aberrant RHAMM splicing. We have observed progressive overexpression of core SF PTBP1/2 (polypyrimidine track-binding protein) in MM patients and associated with disease progression. Since SNVs on the RHAMM modulate canonical SF binding sites, we tested the effects of PTBP1/2 deregulation on RHAMM splicing. We expressed PTBP1/2 in H929 cells, and then evaluated the RHAMM splicing pattern in transfected cells at a single cell (SC) level. SC analyses showed that overexpression of PTBP1/2 increased (2.5-fold) the RHAMM-V3:FL ratio in MM cells. SC analyses also identified overexpression of the RHAMM-V3 splice variant in 18% of H929 SCs expressing PTBP1, and in 37% of cells expressing PTBP2, confirmed at the single cell (SC) level. In BM-infiltrating myeloid cells, analyses showed 50% of myeloid cells express the RHAMM-V3 variant alone, and 79% of plasma cells (PCs) express this variant in combination with RHAMM-FL. Moreover, the RHAMM-V3/FL ratio in PCs is elevated (2.6-fold), further confirming a correlation between the RHAMM variant ratio and the clinical outcome. Next, we determined RHAMM-V3/FL ratios in BM stromal cells from 16 MM patients: MM-BMSC samples exclusively express the RHAMM-V3 in combination with RHAMM-FL and the RHAMM-V3:FL is 1.8 fold. In BMSC samples derived from healthy donors (HD), we detected relatively low-level expression of RHAMM-FL as compared to expression levels of RHAMM-FL in MM patients, while RHAMM-V3 transcripts were undetectable. SC analysis of RHAMM FL and splice variant transcripts in MM BMSC and HD-BMSC agreed with the analyses done on the MM HD-BMSC bulk population. We did not detect any MM BMSC cells expressing RHAMM-V3 alone and the RHAMM V3/FL ratio was 1.6-fold, which is lower than that in MM-PCs. MM-BMSC screening also identified a new splice variant of RHAMM, that was absent in MM PCs or in MM myeloid cells. Conclusions: Our study suggests that aberrant RHAMM splicing in MM can result from SNPs/SNVs affecting SRE due to the upregulation of PTBP1/2. Our study is the first to show that the RHAMM-V3 variant is associated with PTBP2 overexpression. The identification of cell type-specific RHAMM splicing events identifies novel targets for improved immunotherapy in MM. Disclosures Chu: Celgene: Honoraria; AstraZeneca: Honoraria; Gilead: Honoraria; Teva: Consultancy; Amgen Inc.: Honoraria. Anderson:C4 Therapeutics: Other: Scientific founder ; OncoPep: Other: Scientific founder ; Gilead Sciences: Other: Advisory Board; Janssen: Other: Advisory Board; Sanofi-Aventis: Other: Advisory Board.

Changes in the Main Bone Marrow Cell Subpopulations May Potentially Contribute to the Initiation of Acute Myeloid Leukemia

Introduction: In acute myeloid leukemia (AML), differentiation and proliferation of early progenitors is inhibited, leading to blast accumulation in the bone marrow (BM) and in the peripheral blood (PB). Hematopoietic stem cells (HSCs) reside in specialized BM microenvironment (ME), mainly containing osteocytes and adipocytes, derived from BM mesenchymal stem cells (MSCs). Recent studies suggest a possible role of the ME in various hematological malignancies, that either serves as a potential source of an altered milieu or mediates abnormal interaction with HSCs. BM ME contributes to disease pathogenesis, chemotherapy resistance and recurrence in certain types of hematological cancers, but there is limited evidence that BM cells themselves may initiate AML. BM derived MSCs are characterized by a potential to mainly differentiate to adipocytes, osteocytes or chondrocytes. Our experiments have revealed an imbalance between osteocytes and adipocytes in the BM of AML patients that is not observed in the BM of healthy donors. We hypothesize that this imbalance could result in the development of leukemic stem cells/pre-leukemic cells, leading to leukemia initiation. The current study aimed to characterize the composition of AML BM derived adipocytes and osteocytes and evaluate the ability of these subpopulations to support AML cell viability. Methods: Secreted levels of adipogenic and osteogenic biomarkers in the BM plasma were measured using ELISA. The potential of AML MSCs to differentiate to adipocytes and osteocytes was assessed using FACS analysis. Exome sequencing was applied for genetic alteration screening in AML MSCs. Obtained adipocytes and osteocytes were separately co-cultured with AML cells and colony forming unit (CFU) assays were used to evaluate AML cell viability. ProcartaPlex immunoassay was used to evaluate levels of cytokines and other potential mediators secreted to the conditioned media. Results: The study included BM samples of 10 AML patients and 13 healthy donors. Levels of secreted FABP4 and osteocalcin biomarkers were reduced in AML plasma (279.7±164 pg/ml and 0.00057±0.017 pg/ml, respectively) compared to that of healthy controls (1230±1502 pg/ml and 0.0016±0.00052 pg/ml, respectively), with P=0.02 for FABP4 and P=0.017 for osteocalcin. According to adipocyte cell differentiation, patients were divided into two clusters. Cluster-1 demonstrated a higher expression level of FABP4 (mean 65.2±11.4%) compared to healthy controls (mean: 47.5±19.2 %; P=0.05), while cluster-2 exhibited a lower level of FABP4 expression (mean 30.78±6.1%) than healthy controls (mean 47.6±19.2%; P=0.01). A nonsynonymous somatic mutation in the AHNAK2 gene (V3209L) was found in patients' mesenchymal stromal cells. A statistically significant reduction in AML cell proliferation (P&lt;0.01) was revealed following co-culture with adipocyte cells of cluster-1 patients. Of the 10 cytokines tested, only ANG1 levels were significantly elevated in the co-culture of adipocytes from cluster-1 (189.5±17.3pg/ml), cluster-2 (258.08±34.33pg/ml) or osteocytes (392.04±58.3pg/ml) with AML cells of the same patients compared to the levels observed in controls (0.002±0.052pg/ml for adipocytes and 168.663pg/ml for osteocytes; P&lt;0.01). Conclusions: The current study has demonstrated that changes in the secretion of stromal specific biomarkers and in the MSC ability to differentiate to adipocytes and osteocytes indicate impaired functional properties of BM MSCs in AML. Within the adipocyte subpopulation in the AML BM, clusters with either increased or inhibited adipogenesis have been revealed. These variations in adipocyte and osteocyte cells reflect interpatient heterogeneity in AML BM subpopulations. The AHNAK2 gene mutation found in AML MSCs in our study and the previously suggested role of this gene in adipocyte differentiation regulation, may explain in part the phenotype diversity observed in AML patients. Adipocyte cells, derived from an AML patient, were found to inhibit normal hematopoiesis in HSCs of the same patient. This specific crosstalk may be related to the elevated levels of ANG1 mediating abnormal signals from adipocytes to HSCs. Overall, the findings of this study may support the contribution of abnormal composition of the main BM subpopulations to leukemogenesis and could ultimately pave the way to novel therapeutic strategies targeting the BM niche. Disclosures No relevant conflicts of interest to declare.

Evaluation of the Selected Parameters of Bone Marrow Cell Subpopulations by Flow Cytometry and Analysis of Their Diagnostic and Prognostic Value in Patients with Myelodysplastic Syndromes

Evaluation of the Selected Parameters of Bone Marrow Cell Subpopulations by Flow Cytometry and Analysis of Their Diagnostic and Prognostic Value in Patients with Myelodysplastic Syndromes

PS1235 RED BLOOD CELL GENERATION FROM HUMAN INDUCED PLURIPOTENT STEM CELLS

Background: Multiparameter flow cytometry (MFC) is an essential tool for the diagnosis and follow-up of hematological malignancies. Numerous panels have been devised that best allow to achieve these goals. However, analysis of MFC graphs still heavily relies on the expertise of biologists and on specifically well adapted cascade-gating strategies. Because of the development of mass cytometry, which generates data in a multidimensional environment of up to 50 dimensions, new strategies have been developed, that could be applied to classical MFC. Aims: In this study, the capacities of a rapid new tool for multidimensional analysis, FlowSOM (self-organizing maps) have been combined to a classical MFC analysis tool, Kaluza®, for the identification of normal and diseased human bone marrow (BM) cell subpopulations. Methods: Normal BM samples collected during bone surgery were analyzed in MFC with four antibody panels adapted to the exploration of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) respectively. The listmode files obtained were merged and submitted to FlowSOM analysis, using R packages, which yielded trees of 100 nodes defining normal BM cell subsets. FlowSOM parameters were then transferred to Kaluza® for a bidimensional representation of the trees within which selected navigation could be performed with Kaluza® tools. In a second part of the study, the same stainings were performed on myelodysplastic or leukemic (AML or ALL) samples (diagnosis or follow-up), which were submitted to FlowSOM together with the reference merged BM. Results: Node-by-node selection allowed for a precise definition of each of the unsupervised subsets generated by the software. This disclosed a selective segregation of delicate subsets such as non-classical monocytes, hematogones or stem cells. Direct simultaneous comparison of diseased samples and normal BM allowed for a precise definition of abnormal cells (blasts) at diagnosis, and their immediate recognition as minimal residual disease in follow-up samples. Summary/Conclusion: Flow SOM appears as a new and efficient tool for a rapid identification of BM cell subsets, highlighting differences from normal BM in dysplastic of leukemic samples. The node-by-node approach facilitates the definition of abnormal cells and their quantification, supervision intervening only after the generation of subsets delineated by artificial intelligence.

Identification of the xenograft and its ascendant sphere-forming cell line as belonging to EBV-induced lymphoma, and characterization of the status of sphere-forming cells

BackgroundWe have characterized the human cell line arised from the Epstein–Barr virus (EBV) positive multiple myeloma aspirate subjected to the long-term cultivation. This cell line has acquired the ability to form free-floating spheres and to produce a xenograft upon transplantation into NOD/SCID mice.MethodsCells from both in vitro culture and developed xenografts were investigated with a number of analytical approaches, including pathomorphological analysis, FISH analysis, and analysis of the surface antigens and of the VDJ locus rearrangement.ResultsThe obtained results, as well as the confirmed presence of EBV, testify that both biological systems are derived from B-cells, which, in turn, is a progeny of the EBV-transformed B-cellular clone that supplanted the primordial multiple myeloma cells. Next we assessed whether cells that (i) were constantly present in vitro in the investigated cell line, (ii) were among the sphere-forming cells, and (iii) were capable of internalizing a fluorescent TAMRA-labeled DNA probe (TAMRA+ cells) belonged to one of the three types of undifferentiated bone marrow cells of a multiple myeloma patient: CD34+ hematopoietic stem cells, CD90+ mesenchymal stem cells, and clonotypic multiple myeloma cell.ConclusionTAMRA+ cells were shown to constitute the fourth independent subpopulation of undifferentiated bone marrow cells of the multiple myeloma patient. We have demonstrated the formation of ectopic contacts between TAMRA+ cells and cells of other types in culture, in particular with CD90+ mesenchymal stem cells, followed by the transfer of some TAMRA+ cell material into the contacted cell.

Single-Molecule Real-Time (SMRT) Full-Length RNA-Sequencing Reveals Novel and Distinct mRNA Isoforms in Human Bone Marrow Cell Subpopulations.

Hematopoietic cells are continuously replenished from progenitor cells that reside in the bone marrow. To evaluate molecular changes during this process, we analyzed the transcriptomes of freshly harvested human bone marrow progenitor (lineage-negative) and differentiated (lineage-positive) cells by single-molecule real-time (SMRT) full-length RNA-sequencing. This analysis revealed a ~5-fold higher number of transcript isoforms than previously detected and showed a distinct composition of individual transcript isoforms characteristic for bone marrow subpopulations. A detailed analysis of messenger RNA (mRNA) isoforms transcribed from the ANXA1 and EEF1A1 loci confirmed their distinct composition. The expression of proteins predicted from the transcriptome analysis was evaluated by mass spectrometry and validated previously unknown protein isoforms predicted e.g., for EEF1A1. These protein isoforms distinguished the lineage negative cell population from the lineage positive cell population. Finally, transcript isoforms expressed from paralogous gene loci (e.g., CFD, GATA2, HLA-A, B, and C) also distinguished cell subpopulations but were only detectable by full-length RNA sequencing. Thus, qualitatively distinct transcript isoforms from individual genomic loci separate bone marrow cell subpopulations indicating complex transcriptional regulation and protein isoform generation during hematopoiesis.

Silk fibroin/poly(L-lactic acid-co-ε-caprolactone) electrospun nanofibrous scaffolds exert a protective effect following myocardial infarction.

Electrospinning using biocompatible polymer scaffolds, seeded with or without stem cells, is considered a promising technique for producing fibrous scaffolds with therapeutic possibilities for ischemic heart disease. However, no optimal scaffolds for treating ischemic heart disease have been identified thus far. In the present study, it was evaluated whether electrospun silk fibroin (SF)-blended poly(L-lactic acid-co-ε-caprolactone) [P(LLA-CL)] scaffolds that were seeded with cluster of differentiation 117 (c-kit)+ bone marrow (BM) cells may serve a protective role in cardiac remodeling following myocardial infarction (MI). Mechanical characteristics and cytocompatibility were compared between SF/P(LLA-CL) and P(LLA-CL) electrospun nanofibrous scaffolds in vitro. It was observed that MI led to a significant increase of the c-kit+ BM cell subpopulation in mice. Magnetic activated cell sorting was performed to harvest the c-kit+ cell population from the BM of mice following MI. c-kit+ BM cells were seeded on SF/P(LLA-CL) and P(LLA-CL) electrospun nanofibrous scaffolds. Results indicated that SF/P(LLA-CL) electrospun nanofibrous scaffolds were superior to P(LLA-CL) electrospun nanofibrous scaffolds in improving c-kit+ BM cell proliferation. Additionally, compared with pure SF/P(LLA-CL) electrospun nanofibrous scaffolds, SF/P(LLA-CL) scaffolds seeded with c-kit+ BM cells resulted in lower levels of MI markers and reduced infarct size, leading to greater global heart function improvement in vivo. The findings of the present study indicated that SF/P(LLA-CL) electrospun nanofibrous scaffolds seeded with c-kit+ BM cells exert a protective effect against MI and may be a promising approach for cardiac regeneration after ischemic heart disease.

The LSD1 Inhibitor RN-1 Increases γ-Globin Expression in Baboons By Targeting an Early Event Responsible for γ-Globin Repression

The HbF to HbA developmental globin switch is recapitulated during adult erythroid differentiation and involves the acquisition of repressive epigenetic marks at the γ-globin promoter catalyzed by “druggable” enzymes such as histone deacetylases (HDACs), DNA methyltransferase (DNMT1), LSD1 (KDM1A), and G9A (EHMT2) that are functional components of multiprotein co-repressors recruited to the γ-globin gene promoter by the trans-acting repressors TR2/TR4, BCL11A, and ZBTB7A. A logical approach to increase HbF that has been successfully pursued by our laboratory is to intervene with the epigenetic repression mechanism that executes the switch from HbF to HbA using pharmacological inhibitors of these enzymes. Simian primates such as the baboon are widely acknowledged as the best animal models for testing the ability of new drugs to increase γ-globin expression because results in baboons are predictive of effects in man due to conservation of the structure and developmental stage-specific regulation of the β-like globin genes in simian primates.Our laboratory has developed and utilized an in vivo baboon model for over thirty years to investigate globin gene regulation and the ability of pharmacological inhibitors of enzymes that catalyze repressive epigenetic modifications to increase HbF, generating results with DNMT inhibitors that were extended and confirmed in a number of clinical studies in patients with SCD and β-thalassemia. Following studies that identified LSD1 as an additional therapeutic target (Shi et al, Nat Med 19:291, 2013), we showed that the LSD1 inhibitor RN-1 dramatically increased HbF, F cells and F retics in baboons (Rivers et al, Haematol 101:698, 2015) and that these effects were sustained upon long-term treatment (>265d; Ibanez et al, Blood 129:260, 2017). ChIP analysis showed increased levels of Histone H3 di and tri-methyl K4 at the γ-globin gene, consistent with LSD1 inhibition. This current investigation seeks to identify the stage in the erythroid differentiation pathway targeted by LSD1 inhibitors to increase HbF and also to characterize additional effects of these drugs on erythroid differentiation that potentially impact mechanism of action. Subpopulations of bone marrow (BM) cells highly enriched in BFUe (CD105+CD34+CD117+bRBC-), CFUe (CD105+CD34+CD117+bRBC+), proerythroblasts (CD105+CD34-CD117+bRBC+) were purified from RN-1 treated (0.25mg/kg/d; 3d) and untreated baboons by immunomagnetic column separation combined with FACS. RT-PCR analysis of γ-globin expression within purified BM erythroid subpopulations showed that relative levels of γ-globin mRNA (γ/γ+β) in untreated baboons were 4 fold higher in BFUe than in CFUe and 11 fold higher in BFUe than in proerythroblasts consistent with repression of the γ-globin gene during the BFUe to CFUe transition. RN-1 treatment increased relative γ-globin expression (γ/γ+β) 4 fold in BFUe, 25 fold in CFUe, and >100 fold in proerythroblasts compared to untreated controls and thus partially prevented repression of the γ-globin expression at early stages of differentiation. Additional effects of the LSD1 inhibitor RN-1 on erythroid differentiation were measured by flow cytometry in baboon BM aspirates following RN-1 administration. RN-1 treatment (0.25mg/kg/d; 3d) increased the proportion of CD105+CD117+bRBC+BM cells (proerythroblasts) (24.2% pre-treatment; 48.4% post-treatment; p<0.02) and decreased the proportion of CD105+CD117-bRBC+ terminal erythroid BM cells (51.1% pre-treatment; 29.1% post-treatment; p<0.02). RNAseq analysis of CD105+CD34-CD117+bRBC+ FACS-purified BM cells (proerythroblasts) of untreated control (n=2) and RN-1 treated baboons (0.25mg/kg/d; 3d; n=4; 2 treated for 3d; 2 treated >265d) identified a common set of 41 genes whose expression was increased while the expression of 6 genes was decreased. RT-PCR assays confirmed increased expression of γ-globin, GATA-2, GFi-1B, DAP, IFI6, and Lyn in proerythroblasts of RN-1-treated baboons. We conclude that the LSD1 inhibitor RN-1 administered to baboons 1) restrains erythroid differentiation by modulating the expression of genes such as GATA-1 and GFi-1B that regulate erythroid differentiation, and 2) targets an early event responsible for γ-globin repression to increase γ-globin expression. DisclosuresSaunthararajah:Novo Nordisk, A/S: Patents & Royalties; EpiDestiny, LLC: Patents & Royalties. Lavelle:Global Blood therapeutics: Research Funding.

Identification of differential growth differentiation factor 6 signalling responses in subpopulations of human bone marrow and adipose-derived stem cells: implications for intervertebral disc regeneration

Identification of differential growth differentiation factor 6 signalling responses in subpopulations of human bone marrow and adipose-derived stem cells: implications for intervertebral disc regeneration

Abnormalities of the Bone Marrow Immune Microenvironment in Patients with Prolonged Isolated Thrombocytopenia after Allogeneic Hematopoietic Stem Cell Transplantation

Background:Prolonged Isolated Thrombocytopenia (PT), is a serious complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and defined as the engraftment of all peripheral blood cell lines other than a PLT count ≤20×10E+9/L or dependence on PLT transfusions for more than 90 days after allo-HSCT. Nevertheless, the mechanisms underlying PT remain unclear. Recent studies have presumed that the mechanism of PT might be similar, at least in part, to that of Immune Thrombocytopenia (ITP). BM immune microenvironment is considered to be involved in the regulation of hematopoiesis, and also influence the production of platelets. There is growing evidence that activated CD8+ T cells in the bone marrow (BM) of patients with ITP might suppress megakaryocyte apoptosis, leading to impaired platelet production. In our previous study, we also found the deregulated T cells responses in BM were associated with ITP patients. Therefore, we hypothesized aberrant immune microenvironment may also influence the production of platelet after allo-HSCT, contributing to the occurrence of PT, so we conducted a study to analyze the alteration of T cell subpopulations and cytokines in BM micro-environment of allotransplant patients.Aims:To compare the cellular compositions and function of T cells in BM microenvironment between patients with PT and good graft function (GGF) after allo-HSCT.Methods:Using a prospective nested case-control study, the T cell subpopulations in BM were analyzed by flow cytometry in 15 patients with PT, 30 matched patients with GGF after allo-HSCT, and 15 healthy donors (HDs). The fractions of T cells, including Th1, Tc1,Th2, Tc2 ,Th17 and Treg were identified as CD3+CD8-IFN-gama+, CD3+CD8-IFN-gama+, CD3+CD8+IL4+, CD3+CD8+IL-4+, CD3+CD8-IL17A+ and CD3+CD4+CD25+Foxp3+, respectively. The levels of IFN-gama, IL-4 and IL-17A in BM plasma were detected by cytometric beads assay.Results: The demographic and clinical characteristics were similar between allo-HSCT patients with PT and those with GGF. The T cell subset analysis revealed that the proportion of CD8+ T cells in BM was higher in PT patients. The in vitro cytokine stimulated tests demonstrated a significant higher proportion of Th1 in PT patients (29.8% ±13.0% vs. 21.7%±12.2%, P=0.01), and we also found an elevated percentage of Tc1 in PT patients when compared with GGF (39.3% ±19.3% vs. 23.0% ± 14.0%, P=0.01). Meanwhile, the similar percentage of Th2 and Tc2 were found in PT patients. The type-1/ type-2 response ratio was calculated by the percentages of Th1/Th2 and Tc1/Tc2. A significant elevation in the ratio of Tc1/Tc2 (37.3 vs. 22.1 vs. 15.6, P<0.05) was observed in PT when compared with those in GGF and HDs, whereas the ratio of Th1/Th2 did not differ from GGF. Moreover, we also found the significant elevated percentage of Th17 (3.1% ±2.1% vs. 1.1%± 0.7%, P<0.01) and the similar percentage of Treg in PT patients compared with GGF, leading to a higher ratio of Th17/Treg (0.9 vs. 0.6 vs. 0.3, P<0.05). The changes of IFN-gama, IL-4 and IL-17A levels in BM plasma detected by cytometric beads assay were in accordance with the intracellular cytokine results analyzed by flow cytometry.Summary/Conclusion:Our study demonstrated that the abnormal BM immune microenvironment including the higher percentage of Th1, Tc1, and Th17 cells in patients with PT, suggesting that the dysfunction of T cells response in BM immune microenvironment may contribute to the occurrence of PT after allo-HSCT. DisclosuresNo relevant conflicts of interest to declare.

Evaluation of oxidative stress, antioxidant enzyme activities, and genetic damage in sprayers exposed to pesticides

The potential immunotoxic effect of the carbamate pesticide aminocarb on murine bone marrow cell subpopulations was evaluated by flow cytometry, C57B16 mice were exposed by gavage to sublethal doses of the pesticide and lymphocyte precursors from bone marrow population were stained with PNA lectin and a panel of monoclonal antibodies against cell surface antigens. In regard to the microenvironment-dependent maturation of B lymphocytes, the pesticide effect on the lymphoproliferative potential of bone marrow was assessed by marrow transplantation from aminocarb-exposed donor mice to normal, syngeneic, X-irradiated recipient mice. The sublethal exposure of 0.08–5.0 mg/kg body wt aminocarb to donor animals did not affect regenerating bone marrow in the recipient mice. No marked effect on bone marrow cell number was noted in pesticide-exposed animals. However, a marked shift in surface IgM density on marrow B cells was noted at 0.08 and 0.31 mg/kg body wt aminocarb. This was correlated with decreased cell frequency in G0G1 phase and increased frequency of cells in the S phase of the cell cycle. Thus, altered maturation of B lymphocytes, expressed as a shift in the density of surface IgM on mature B cells and not the lymphocyte count, was related to the direct effect of aminocarb and/or to the pesticide-related changes in bone marrow microenvironment. Overall, exposure to the carbamate pesticide aminocarb activated the cell maturation process in bone marrow.

Identification of Bone Marrow Cell Subpopulations Associated With Improved Functional Outcomes in Patients With Chronic Left Ventricular Dysfunction: An Embedded Cohort Evaluation of the FOCUS-CCTRN Trial.

In the current study, we sought to identify bone marrow-derived mononuclear cell (BM-MNC) subpopulations associated with a combined improvement in left ventricular ejection fraction (LVEF), left ventricular end-systolic volume (LVESV), and maximal oxygen consumption (VO2 max) in patients with chronic ischemic cardiomyopathy 6 months after receiving transendocardial injections of autologous BM-MNCs or placebo. For this prospectively planned analysis, we conducted an embedded cohort study comprising 78 patients from the FOCUS-Cardiovascular Cell Therapy Research Network (CCTRN) trial. Baseline BM-MNC immunophenotypes and progenitor cell activity were determined by flow cytometry and colony-forming assays, respectively. Previously stable patients who demonstrated improvement in LVEF, LVESV, and VO2 max during the 6-month course of the FOCUS-CCTRN study (group 1, n = 17) were compared to those who showed no change or worsened in one to three of these endpoints (group 2, n = 61) and to a subset of patients from group 2 who declined in all three functional endpoints (group 2A, n = 11). Group 1 had higher frequencies of B-cell and CXCR4+ BM-MNC subpopulations at study baseline than group 2 or 2A. Furthermore, patients in group 1 had fewer endothelial colony-forming cells and monocytes/macrophages in their bone marrow than those in group 2A. To our knowledge, this is the first study to show that in patients with ischemic cardiomyopathy, certain bone marrow-derived cell subsets are associated with improvement in LVEF, LVESV, and VO2 max at 6 months. These results suggest that the presence of both progenitor and immune cell populations in the bone marrow may influence the natural history of chronic ischemic cardiomyopathy-even in stable patients. Thus, it may be important to consider the bone marrow composition and associated regenerative capacity of patients when assigning them to treatment groups and evaluating the results of cell therapy trials.