Cytometric Analysis Of Bone Marrow Research Articles

Minimal/Measurable Disease Analysis in Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma by Flow Cytometry.

CLL/SLL is the most common leukemia in the western world. The disease is indolent; however, most patients require treatment at some point of the disease course. Outside of allogeneic transplants, the treatment is rarely curative but often controls CLL/SLL manifestations for many years. Several lines of therapy may be used sequentially to prolong clinical remission. Because of the prolonged disease course, CLL/SLL monitoring represents a sizable portion of the workload in a typical flow cytometry laboratory involved in the diagnosis and monitoring of hematopoietic neoplasms. Minimal/measurable disease monitoring of CLL/SLL has emerged as a key component in treatment monitoring and sequencing. In the face of effective therapies, clinical laboratories are tasked with monitoring ever smaller proportions of MRD with high precision and accuracy. With the recent addition of surface antigen-targeting biologics such as antibodies and CAR-T cells, the task has become more complex due to the unavailability of commonly analyzed antigens for flow cytometric analysis. This article details a flow cytometric test developed at Memorial Sloan Kettering Cancer Center that has proven to consistently achieve high sensitivity (<0.01% of nucleated cells) in the bone marrow and peripheral blood, even when CD19 is lost or unavailable for analysis. Moreover, the test helps distinguish between CLL and other CD5-positive B cell neoplasms. The Basic Protocol provides a detailed operational procedure for processing, staining, and cytometric acquisition of samples. The Support Protocol provides typical steps and caveats for MRD data analysis in CLL/SLL and in distinguishing CLL/SLL from other B cell neoplasms and normal CD5-positive B cells. © 2022 Wiley Periodicals LLC. Basic Protocol: Processing, staining, and cytometric analysis of bone marrow or peripheral blood cells for MRD analysis of CLL/SLL Support Protocol: Analysis and interpretation of CLL MRD assay.

Predictive Value of CD229, CD319 and c-Maf Overexpression for Treatment Response in Multiple Myeloma Patients

Background: Ly-9 (CD229) and SLAMF7 (CD319) are more stable markers that could be used to replace the less stable conventional markers CD138 and CD38 in multiple myeloma (MM) patient follow-up. In those patients, the correlation between these markers and the histopathologic marker c-Maf has not been well investigated. Objective: To assess the predictive value of CD229, CD319, and c-MAF overexpression on outcome of treatment in MM patients. Patients and Methods: 61 newly diagnosed multiple myeloma patients were involved in this prospective study. Flow cytometric analysis of bone marrow (BM) samples for CD229 PE, CD319 PE, CD138 PerCP and CD38FITC as well as BM biopsy immunohistochemically were analyzed for c-Maf expression Results: CD229 and CD319 were highly expressed in (≥93.95%) and (≥95.6%) of MM patients respectively. c-Maf was positive in (29.5%) of patients. In MM patients with high expression of CD229 and CD319 as well as those with c-Maf positivity showed high serum Ca++, β2M, CD138 and CD38. CD229 was significantly correlated with CD319, CD38, CD138, β2M and serum Ca (p-values were< 0.001,0.01, 0.01, 0.009, and 0.02 respectively). Patients with overexpression of CD 229, CD319 and c-Maf were more refractory to treatment (p-values were <0.001,<0.001 and <0.001 respectively). Conclusion: We detected that both CD229 and CD319 were significantly overexpressed on MM cells and correlated significantly with CD138 and CD38, suggesting their use as alternative markers for MM diagnosis as well as follow-up. Patients with CD229,CD319, and c-Maf overexpression were associated with a significant poor response to therapy.

Nivolumab (Nivo) with Azacytidine (AZA) in Patients (pts) with Relapsed Acute Myeloid Leukemia (AML) or Frontline Elderly AML

Nivolumab (Nivo) with Azacytidine (AZA) in Patients (pts) with Relapsed Acute Myeloid Leukemia (AML) or Frontline Elderly AML

Highly sensitive assays are mandatory for the differential diagnosis of patients presenting with symptoms of mast cell activation: diagnostic work-up of 38 patients

Mastocytosis is a heterogeneous disease caused by excessive mast cell (MC) proliferation. Diagnosis of systemic mastocytosis (SM) is based on the presence of major and minor criteria defined by the World Health Organization. Symptoms of MC activation can also occur in patients without SM or without allergic or inflammatory disease. These MC activation syndromes (MCAS) can be divided into primary (monoclonal) MCAS (MMAS) vs. secondary and idiopathic MCAS. In this single center study, the diagnostic work-up of 38 patients with a clinical suspicion of SM and/or with elevated basic tryptase levels is presented. Clinical symptoms, biochemical parameters, results of bone marrow investigation, flow cytometric immunophenotyping, and molecular analysis were retrospectively reviewed. Twenty-three patients were found to have a monoclonal MC disorder of which 19 were diagnosed with SM and 4 with MMAS. In 13/19 SM patients, multifocal MC infiltrates in the bone marrow were found (major criterion), while in 6 the diagnosis was based on the presence of ≥3 minor criteria. Flow cytometric analysis of bone marrow showed CD25 expression of MCs in all patients with SM and MMAS (range: 0.002–0.3% of cells). In bone marrow, the KIT D816V mutation was detected in all SM patients but in only 2 patients with MMAS (range: 0.007–9% mutated cells). Basic tryptase elevation was demonstrated in 16/19 patients with SM but also in 9/19 patients without SM. Our study reveals the heterogeneity of primary MC disorders and the importance of sensitive assays in patients suspected of having SM.

Flow cytometric analysis as an additional predictive tool of treatment response in children with chronic-phase chronic myeloid leukemia treated with imatinib.

Bone marrow samples of newly diagnosed children with chronic-phase chronic myeloid leukemia (CML) were obtained at diagnosis and after imatinib initiation and stained with anti-human CD34, CD38, CD123, CD45RA, cMpl, and lineage antibodies. Flow cytometric analysis revealed that granulocyte macrophage progenitor predominance in CML progenitors at diagnosis and elevated cMpl expression in bone marrow progenitors at 3 months may predict poor outcome in children with chronic-phase CML treated with imatinib. We recommend flow cytometric analysis of bone marrow in the early phase of treatment, as it is a convenient tool that may predict treatment response and guide CML management.

Abnormal hematopoiesis and autoimmunity in human subjects with germline IKZF1 mutations

Ikaros, which is encoded by IKZF1, is a transcriptional factor that play a critical role in hematopoiesis. Somatic IKZF1 alterations are known to be involved in the pathogenesis of leukemia in human subjects. Recently, immunodeficiency caused by germline IKZF1 mutation has been described. We sought to describe the clinical and immunologic phenotypes of Japanese patients with heterozygous IKZF1 mutations. We performed whole-exome sequencing in patients from a dysgammaglobulinemia or autoimmune disease cohort and used a candidate gene approach in 4 patients. Functional and laboratory studies, including detailed lymphopoiesis/hematopoiesis analysis in the bone marrow, were performed. Nine patients from 6 unrelated families were identified to have heterozygous germline mutations in IKZF1. Age of onset was 0 to 20years (mean, 7.4years). Eight of 9 patients presented with dysgammaglobulinemia accompanied by B-cell deficiency. Four of 9 patients had autoimmune disease, including immune thrombocytopenic purpura, IgA vasculitis, and systemic lupus erythematosus. Nonautoimmune pancytopenia was observed in 1 patient. All of the mutant Ikaros protein demonstrated impaired DNA binding to the target sequence and abnormal diffuse nuclear localization. Flow cytometric analysis of bone marrow revealed reduced levels of common lymphoid progenitors and normal development of pro-B to pre-B cells. Germline heterozygous IKZF1 mutations cause dysgammaglobulinemia; hematologic abnormalities, including B-cell defect; and autoimmune diseases.

Inv(16) AML Engrafts Human Cytokine Knock-in Mice

Favorable-risk acute myeloid leukemia (AML) constitutes up to 40% of newly diagnosed AML cases. However, only 66% of younger and 33% of older patients are alive 3 years post diagnosis. Studying the biology of these entities in vivo has been inherently difficult: in fact, faithful xeno-engraftment of human AML in immunodeficient mice has been limited to higher-risk AML.To address this limitation we hypothesized that a human myelopoiesis-supportive environment may permit xeno-engraftment of favorable-risk AML: we transplanted primary human AML with isolated NPM1 mutation and AML with inv(16) into knock-in (KI) mice with human versions of genes encoding M-CSF, IL-3, GM-CSF and Thrombopoietin. Newborn MISTRG mice (Rongvaux et al. Nat Biotechnol. 2014) were sub-lethally irradiated, intra-hepatically injected with AML blasts and analyzed 16-24 weeks post transplantation.Flow cytometric analysis of bone marrow (BM) and blood of cytokine KI versus NSG mice revealed profound differences: Average engraftment was 36% in the BM of cytokine KI mice compared to only 10% in NSG mice across all patient samples transplanted. In line with engraftment levels, engraftment efficacy was higher in cytokine KI mice. Importantly, average engraftment in cytokine KI mice approached BM and peripheral blast count observed in human AML patients, while NSG engraftment levels were significantly lower. AML engraftment in cytokine KI mice was found to be independent of expression of the SIRPα transgene in mice expressing all four cytokines. NMP1mut AML showed a trend towards higher BM engraftment in MI(S)TRG versus NSG mice. Expression of the NPM1mutallele was more than 3-fold higher in MI(S)TRG mice compared to NSG mice. Performing targeted next generation sequencing in human and engrafted murine BM, we found comparable allelic frequency ranging from 38% to 51% for NPM1 mutation in a patient’s leukemia and engrafted cytokine KI mice. Of note, even the composition of NPM1 mutation subclones was constant between patient and cytokine KI recipients.In contrast to NPM1mut, a striking difference in quantitative inv(16) AML engraftment was observed between cytokine KI and NSG mice, both by aggregate and split-sample analysis. Serial transplantation further validated engraftment of an inv(16) leukemia-initiating cell population in cytokine KI mice. To dissect the contribution of respective single human KI cytokines to inv(16) AML propagation, we analyzed engraftment in Rag2-/-gc-/- mice carrying, in addition to a human SIRPα transgene, further KI genes for human thrombopoietin (STRG), M-CSF and Thrombopoietin (MSTRG), and M-CSF, IL-3, GM-CSF and Thrombopoietin (MI(S)TRG). STRG mice, as NSG mice, did not support engraftment. In contrast, addition of M-CSF in MSTRG mice led to robust engraftment of inv(16) AML, while addition of IL-3 and GM-CSF in MI(S)TRG mice resulted in only marginal, if any, improvement over MSTRG mice. Of note, knock-in of human M-CSF has previously been shown to be sufficiently cross-reactive to rescue the murine M-CSF knock-out phenotype (Rathinam et al. Blood 2011). We thus reasoned that the engraftment advantage observed in MSTRG mice was likely due to human M-CSF effects on blasts and not due to reduction in mouse macrophages. Together, this establishes a central role for M-CSF in maintenance of inv(16) AML.When profiling cytokine receptor expression for TPO, M-CSF, IL-3 and GM-CSF on inv(16) AML blasts in comparison to NPM1mut AML and an aggregate of non-favorable-risk AML cases, using microarray data from an international cohort study (Wouters et al. Blood 2009), inv(16) AML showed significantly higher expression of each of the 4 receptors compared to NPM1mut AML, and significantly higher expression of M-CSFR and GM-CSFR compared to non-favorable-risk AML; the overall highest difference in expression was observed for M-CSFR. Moreover, gene set enrichment analysis demonstrated enrichment of M-CSF-inducible genes in inv(16) AML compared to NPM1mutAML and non-favorable-risk AML cases, confirming increased signaling through the M-CSF pathway.In sum, we here describe the first faithful and highly efficacious xenotransplantation model of favorable-risk, and specifically, inv(16) AML. We further demonstrate a strong dependency of this subtype on M-CSF, a finding that might provide a basis for therapy optimization studies informed by an improved understanding of favorable-risk AML biology. DisclosuresNo relevant conflicts of interest to declare.

Flow cytometric analysis of bone marrow and spleen cells in adjuvant-treated animals (P4502)

Abstract Despite the poorly understood mechanisms, vaccine adjuvants have been extensively used to enhance the immunogenicity of specific antigens in vaccination. To examine the effect of adjuvants on the differentiation of the hematopoietic system, C57BL/6J mice were immunized with ovalbumin (OVA) with alum, followed by flow cytometric analysis of cells in the bone marrow and spleens after vaccination. Our data showed that treatment of animals with alum upregulated the expression of AA4.1 in the Dump(CD3/CD8/Gr-1/F4/80)(-)CD24(+) population, and a concomitant increase of both B220(-)CD11b(+) and CD24(+)CD43(+) cells in the spleen, indicating the differentiation effect on the hematopoietic cell development. Our results also showed that Dump(-)B220(-)CD24(+) population in the bone marrow were significantly elevated, suggesting the potential induction of myeloid differentiation from proB populations in the adjuvant-treated animals. Similar results were obtained in the complete-Freund's adjuvant (CFA)-treated mice. Treatment of animals with CFA was found to elevate the lineage2 (CD3/CD19/NK1.1/B220/Ter119)(-)CD11b(+) population at the expense of lineage2(+) cells, suggesting the potential differentiation of hematopoietic stem cells towards the myeloid lineage from the lymphoid progenitors. These results raised the potential effect of vaccine adjuvants on modulation of hematopoietic hierarchy, explaining their actions of mechanism on stimulation of the specific immunological responses.

Altered immunophenotypic features of peripheral blood platelets in myelodysplastic syndromes

Multiparameter flow cytometric analysis of bone marrow and peripheral blood cells has proven to be of help in the diagnostic workup of myelodysplastic syndromes. However, the usefulness of flow cytometry for the detection of megakaryocytic and platelet dysplasia has not yet been investigated. The aim of this pilot study was to evaluate by flow cytometry the diagnostic and prognostic value of platelet dysplasia in myelodysplastic syndromes. We investigated the pattern of expression of distinct surface glycoproteins on peripheral blood platelets from a series of 44 myelodysplastic syndrome patients, 20 healthy subjects and 19 patients with platelet alterations associated to disease conditions other than myelodysplastic syndromes. Quantitative expression of CD31, CD34, CD36, CD41a, CD41b, CD42a, CD42b and CD61 glycoproteins together with the PAC-1, CD62-P, fibrinogen and CD63 platelet activation-associated markers and platelet light scatter properties were systematically evaluated. Overall, flow cytometry identified multiple immunophenotypic abnormalities on platelets of myelodysplastic syndrome patients, including altered light scatter characteristics, over-and under expression of specific platelet glycoproteins and asynchronous expression of CD34; decreased expression of CD36 (n = 5), CD42a (n = 1) and CD61 (n = 2), together with reactivity for CD34 (n = 1) were only observed among myelodysplastic syndrome cases, while other alterations were also found in other platelet disorders. Based on the overall platelet alterations detected for each patient, an immunophenotypic score was built which identified a subgroup of myelodysplastic syndrome patients with a high rate of moderate to severe alterations (score>1.5; n = 16) who more frequently showed thrombocytopenia, megakaryocytic dysplasia and high-risk disease, together with a shorter overall survival. Our results show the presence of altered phenotypes by flow cytometry on platelets from around half of the myelodysplastic syndrome patients studied. If confirmed in larger series of patients, these findings may help refine the diagnostic and prognostic assessment of this group of disorders.

Chronic RPS19 Deficiency Leads to Bone Marrow Failure In a Mouse Model for Diamond-Blackfan Anemia

AbstractAbstract 193Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia associated with physical malformations and predisposition to cancer. Of the many different DBA disease genes known, all encode for ribosomal proteins, suggesting that DBA is a disorder relating to ribosomal biogenesis or function. Among these genes, ribosomal protein S19 (RPS19) is the most frequently mutated (25 % of the patients). The generation of animal models for DBA is pivotal in order to understand the disease mechanisms and to evaluate novel therapies. We have generated two mouse models for RPS19-deficient DBA by taking advantage of RNA interference (Jaako et al, 2009 ASH meeting abstract). These models contain RPS19-targeting shRNAs expressed by a doxycycline-responsive promoter downstream of the Collagen A1 locus allowing an inducible and dose-dependent regulation of shRNA. As we have previously reported, the induction of RPS19 deficiency results in a reduction in the number of erythrocytes, platelets and white blood cells, and flow cytometric analysis of bone marrow after a short-term induction reveals increased frequencies of hematopoietic stem and progenitor cells reflecting the onset of stress hematopoiesis. In the current study we have analyzed the long-term effect of RPS19 deficiency in bone marrow. In contrast to a short-term induction, flow cytometric analysis of bone marrow after 51 days revealed decreased frequencies of hematopoietic stem and progenitor cells that correlate with a severe peripheral blood phenotype. In addition, we observed a 3–6 fold increase in apoptosis in RPS19-deficient bone marrow compared to controls based on TUNEL assay. Furthermore, transplantation of whole bone marrow cells from transgenic donors into wild type lethally irradiated recipients confirms that the observed phenotype is autonomous to the blood system. To study whether long-term RPS19 deficiency functionally impairs hematopoietic stem cells, we pre-induced mice for 30 days followed by 15 days without doxycycline to restore the RPS19 expression. Mice were sacrificed and total bone marrow cells were transplanted together with wild-type competitor cells (1:1) into wild type lethally irradiated recipients without doxycycline. This experimental setting allows us to assess the functionality of pre-induced hematopoietic stem cells in absence of ribosomal stress. Flow cytometric analysis of peripheral blood one month after transplantation clearly demonstrates decreased reconstitution from pre-induced donors compared to the wild-type competitor. While this time point reflects mainly the function of transplanted progenitors, long-term analysis of hematopoietic stem cell function in these recipients is ongoing. To study the molecular mechanisms underlying the hematopoietic defect we performed comparative microarray analysis. We chose to analyze preCFU-E/CFU-E erythroid progenitors since we have previously located the erythroid defect at the CFU-E – proerythroblast transition based on flow cytometry and clonogenic proliferation cultures of prospectively isolated erythroid progenitors. Microarray analysis of preCFU-E/CFU-E progenitors reveals deregulation of several genetic pathways, including a robust upregulation of p53 pathway genes, and these targets have been confirmed by real-time PCR. Furthermore, many of p53 target genes are also upregulated in the Lineage− Sca-1+ c-Kit+ (LSK) population that contains immature hematopoietic progenitors and stem cells suggesting that the activation of p53 is not restricted to the erythroid lineage. To ask whether increased activity of p53 can solely explain the hematopoietic phenotype, we have crossed our mouse model into a p53-null background. In summary, our data suggest that RPS19-deficient mice fail to uphold stress hematopoiesis for extended periods of time, with chronic RPS19 deficiency causing bone marrow failure. Disclosures:No relevant conflicts of interest to declare.

Flow Cytometric Score Correlates with Clinical Response to Azacitidine In Intermediate-2 and High Risk Myelodysplastic Syndrome Patients

AbstractAbstract 441New treatment strategies that potentially change the natural course of intermediate (int)-2 and high risk myelodysplastic syndromes (MDS), such as azacitidine, are emerging. Recently, we reported that flow cytometric analysis of bone marrow (BM) in low and int-1 risk MDS is instrumental to identify clinically relevant subgroups. (Westers et al, Blood 2010) Moreover, it was reported that a flow cytometric scoring system (FCSS) is predictive for worse outcome in MDS. (Wells et al, Blood 2003, van de Loosdrecht et al, Blood 2008) The FCSS is a scoring system that allows for a numerical display of immunophenotypic aberrancies in the (im)mature myelo-monocytic lineage. Scores are generated by enumerating abnormalities; e.g. high scores reflect a high number of aberrancies. The current study aimed to investigate the role of this flow cytometry-based scoring system to assess and monitor response to treatment in int-2 and high risk MDS patients treated with azacitidine. Bone marrow aspirates were analyzed by flow cytometry in 18 MDS patients who were treated with azacitidine. Aspirates were drawn before treatment and after every third cycle of azacitidine. Response to treatment was evaluated using IWG-2006 criteria. The median age was 71 (range 50–78). Distribution over WHO 2001 categories was RCMD-RS n=2, RAEB-2 n=7, AML with 20–30% blasts n=6 and MDS/MPD n=3. International prognostic scoring system (IPSS) categories comprised int-2 n=8 and high n=5. In 5 patients the IPSS score could not be assessed due to lack of cytogenetics, these patients were at least int-2 MDS patients. Flow cytometric follow up was available in 12 patients due to short follow up, i.e. in 4 responders, 4 progressive disease (PD) and 4 stable disease (SD) patients; 3 patients stopped due to non-hematologic toxicity, 4 patients died of PD. Median follow up was 7 months (range 3–12). Median pre-treatment Hb was 6.7 mmol/L, platelets 35.5*10e9/L and absolute neutrophil count (ANC) 0.82*10e9/L. Responders had a significant increase in Hb (median 7.8 mmol/L, p=0.04), platelets (291.5 *10e9/L, p=0.03) and ANC (1.4*10e9/L, p=n.s. compared with baseline values). SD and PD patients had a median Hb of 6.5 mmol/L, platelets 69*10e9/L and ANC 0.77*10e9/L. The median pre-treatment flow score was 6 (range 3–8). Interestingly, responders had a significant decrease in flow score from median 5 to median 2 (range 1–3, p=0.005) after 3 months of treatment. No change in flow scores was seen in PD and SD patients after 3 months of treatment (median=6, range 4–8 and pre-treatment FCSS median=6.5, range 3–8). A sustained decrease in flow score was seen in 4 responders after 6 months of treatment (median 2, range 1–3) parallel to a further increase in median Hb (9.0 mmol/L), platelets (278.5 *10e9/L) and ANC (1.7*10e9/L). After 9 cycles of azacitidine, 3 patients were still responsive to treatment (median Hb 9.9 mml/L, platelets 169, ANC 4.07, median flow score 5, range 2–4). The majority of SD and PD MDS patients had aberrant marker expression on myeloid progenitors, such as CD5, CD7 and/or CD56 compared with responsive MDS patients, (63% (5/8) vs 25% (1/4). Moreover, initial loss of aberrant marker expression on myeloid progenitors was detected in one patient who responded to azacitidine treatment. At 9 months, this initially responsive patient and a patient from the stable disease group developed progressive disease. Interestingly, the initially responsive patient showed an increase in the percentage of myeloid progenitors with an aberrant immunophenotype at 6 months; of note, the total percentage of CD34+ cells was less than 3% by flow cytometry. The initial loss of aberrant marker expression on myeloid progenitors might be caused by a relative increase of normal progenitors and decrease of malignant progenitors caused by azacitidine treatment. In conclusion, our data indicate that flow cytometry identifies MDS patients who may benefit from azacitidine treatment by detection of aberrant marker expression on myeloid progenitors. Moreover, the data indicate that the FCSS may be instrumental in selection of SD patients who may benefit from prolonged treatment with azacitidine. Disclosures:Ossenkoppele:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Van de Loosdrecht:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Src-like adaptor protein (SLAP) deficiency leads to decreased autoantibody production (99.37)

Abstract Background: Intrinsic defects in lymphocyte signaling have been implicated in the pathogenesis of autoimmunity. SLAP has been shown to influence BCR levels and signaling in developing B cells. We hypothesize that SLAP deficiency will enhance the negative selection of autoreactive B cells leading to decreased autoantibody production. Methods: To test this hypothesis we immunized SLAP-deficient and wild-type BALB/c mice with a peptide mimitope for dsDNA that leads to the development of dsDNA antibodies and a systemic lupus erythematosus-like autoimmune disease. In addition, we used a second model where we generated SLAP-deficient mice expressing an autoreactive BCR heavy chain 3H9H/E56R (56R) that binds dsDNA to determine how SLAP deficiency impacts BCR repertoire selection and autoantibody production. Results: SLAP-deficiency led to decreased serum levels of dsDNA antibodies in both experimental systems with a 6-fold reduction in the mimitope treated mice and a 3-fold reduction in dsDNA antibodies in the SLAP-deficient 56R mice. Flow cytometric analysis of bone marrow B cell subsets in SLAP-deficient 56R mice revealed a 27% reduction in fraction D, 37% in E and a 2-fold reduction in F, with a 19% increase in lambda usage compared to controls. Conclusions: These data support the hypothesis that SLAP deficiency enhances negative selection of autoreactive B cells leading to decreased autoantibody production.

Hematopoietic Stem Cell Defects in Novel Fanconi Anemia Mouse Models

Fanconi Anemia (FA) is an autosomal recessive disease characterized by congenital defects, crosslinker hypersensitivity, progressive bone marrow failure, and a defect in hematopoietic stem cell (HSC) function. There are thirteen known Fanconi Anemia genes (A, B, C, D1, D2, E, F, G, I, J, L, M, N) and disruption of these genes appears to account for a variable degree of HSC dysfunction. Another gene (Usp1), encoding a deubiquitinating enzyme which is not a FA gene per se, is a critical regulator of the FA pathway. Understanding the mechanism of action of the FA proteins may allow better diagnosis and treatment for Fanconi Anemia and other aplastic anemia patients. Here, we have characterized mouse models for FA, including Fancd2−/− and Usp1 −/− mice. Similar to a previously reported Fancd2 mouse model (Houghtaling S et al, Genes & Development , 17:2021–2035, 2003), the Fancd2−/− mice developed in our laboratory displayed FA phenotypes including cellular hypersensitivity to mitomycin C (MMC), hypogonadism and reduced fertility. Interestingly, Usp1−/− mice developed for the first time in our laboratory exhibited severe FA phenotypes, with increased cellular hypersensitivity to MMC, 80% perinatal lethality, testicular atrophy, and depletion of germ cells. Quantitative assessment of the Fancd2 protein expression in the bone marrow using the AQUA imaging system demonstrated low levels of Fancd2 staining in the Lin+ cells compared to the Lin− cells in wild-type (WT) mice, with an absence of Fancd2 expression in the Fancd2 −/− mice. Analysis of hematopoietic parameters in both the Fancd2 and Usp1 mouse models showed normal white blood cell counts and normal hemoglobin levels in the peripheral blood. However, bone marrow from Fancd2−/− mice as well as Usp1−/− mice exhibited phenotypic and functional differences compared to the bone marrow from WT mice. Multiparameter flow cytometric analysis of bone marrow revealed increased frequencies of MEPs (megakaryocyte-erythroid progenitors) in Usp1−/− mice and decreased frequencies of LSK (Lin-Sca-1+Kit+) population in Fancd2−/− mice in comparison to WT controls. In addition, bone marrow from both Fancd2−/− mice and Usp1−/− mice contained significantly reduced frequencies of late-developing day 28 CAFCs (cobblestone area-forming cells) compared to the bone marrow from WT mice. Furthermore, lethally irradiated recipients transplanted with bone marrow from Fancd2−/− or Usp1−/− mice showed reduced donor-type blood cell chimerism at 16 weeks post-transplant, compared to the recipients transplanted with WT bone marrow. Collectively, our data indicate that mice deficient in Fancd2 or Usp1 have hematopoietic stem cell defects and that these mouse models can be useful for development of therapeutics for FA.

Identification of Chromatin Remodeling Genes Arid4a and Arid4b as Leukemia Suppressor Genes

BackgroundLeukemia evolves through a multistep process from premalignancy to malignancy. Epigenetic alterations, including histone modifications, have been proposed to play an important role in tumorigenesis. The involvement of two chromatin remodeling genes, retinoblastoma-binding protein 1 (Rbbp1/Arid4a) and Rbbp1-like 1 (Rbbp1l1/Arid4b), in leukemogenesis was not characterized.MethodsThe leukemic phenotype of mice deficient for Arid4a with or without haploinsufficiency for Arid4b was investigated by serially monitoring complete blood counts together with microscopic histologic analysis and flow cytometric analysis of bone marrow and spleen from the Arid4a−/− mice or Arid4a−/−Arid4b+/− mice. Regulation in bone marrow cells of downstream genes important for normal hematopoiesis was analyzed by reverse transcription–polymerase chain reaction. Genotypic effects on histone modifications were examined by western blotting and immunofluorescence analysis. All statistical tests were two-sided.ResultsYoung (2–5 months old) Arid4a-deficient mice had ineffective blood cell production in all hematopoietic lineages. Beyond 5 months of age, the Arid4a−/− mice manifested monocytosis, accompanied by severe anemia and thrombocytopenia. These sick Arid4a−/− mice showed bone marrow failure with myelofibrosis associated with splenomegaly and hepatomegaly. Five of 42 Arid4a−/− mice and 10 of 12 Arid4a−/−Arid4b+/− mice progressed to acute myeloid leukemia (AML) and had rapid further increases of leukocyte counts. Expression of Hox genes (Hoxb3, Hoxb5, Hoxb6, and Hoxb8) was decreased in Arid4a-deficient bone marrow cells with or without Arid4b haploinsufficiency, and FoxP3 expression was reduced in Arid4a−/−Arid4b+/− bone marrow. Increases of histone trimethylation of H3K4, H3K9, and H4K20 (fold increases in trimethylation = 32, 95% confidence interval [CI] = 27 to 32; 45, 95% CI = 41 to 49; and 2.2, 95% CI = 1.7 to 2.7, respectively) were observed in the bone marrow of Arid4a-deficient mice.ConclusionsArid4a-deficient mice initially display ineffective hematopoiesis, followed by transition to chronic myelomonocytic leukemia (CMML)–like myelodysplastic/myeloproliferative disorder, and then transformation to AML. The disease processes in the Arid4a-deficient mice are very similar to the course of events in humans with CMML and AML. This mouse model has the potential to furnish additional insights into the role of epigenetic alterations in leukemogenesis, and it may be useful in developing novel pharmacological approaches to treatment of preleukemic and leukemic states.

Improved Analysis of Hematopoietic Engraftment by Non-Invasive In Vivo Bioluminescent Imaging of Transplanted Human Embryonic Stem Cell-Derived Hematopoietic Cells.

Animal transplantation models are essential to characterize the long-term in vivo engraftment capacity of putative hematopoietic stem cells derived from human embryonic stem cells (hESCs). We have previously demonstrated that hESCs can be routinely utilized to derive multiple hematopoietic cell lineages. Here, we use in vivo bioluminescence imaging (BLI) of stable luciferase (luc)-expressing hESCs to noninvasively monitor the dynamics of transplantation, engraftment, and growth of hESC-derived hematopoietic cells within individual animals over an extended time course. Luc expression under control of an EF1α promoter was introduced into the H1 hESC line using a self-inactivating lentiviral vector. Undifferentiated hESC colonies that stably expressed luciferase were established and selected, and the pluripotent capability of luc+ hESCs was first explored by teratoma formation. Undifferentiated luc+ human ES cells were intramuscularly injected into NOD/SCID mice. The dynamics of survival and growth of the hESCs was monitored by BLI using the IVIS Imaging System (Xenogen) at regular time points post-transplantation. There was a decrease of luminescent signal during the first 1–2 weeks. This was followed by a dramatic increase in luminescent signals after about 5 weeks, which correlated with teratoma size. Immunohistochemical analysis confirmed stable luc-expression in multiple differentiated cell types within the teratomas. We next used BLI to examine luc+ H1 hESCs that were induced to undergo hematopoietic differentiation by co-culture with S17 cells, to give rise to H1/S17 cells. Flow cytometric studies confirmed hematopoietic cells (CD34+, CD45+, CD31+, and c-kit+ cells) were derived from these differentiated luc+ hESCs, with 5–10% of H1/S17 cells being CD34+. Hematopoietic progenitors that gave rise to colonies of mature luc+ blood cells in a standard CFU assay were also observed from the H1/S17 cells. Luc-expression of differentiated hESCs was maintained at similar levels to those of the undifferentiated ES cells. To define the in vivo potential of luc+ hESC-derived hematopoietic cells, hESCs were allowed to differentiate on S17 cells for two weeks. SCID-repopulating cell studies were done by intravenous (iv) injection into sublethally irradiated NOD/SCID mice. After iv injection of 2–3 x106 unsorted luc+ H1/S17 cells, BLI showed the brightest signal in the lung at day 0 (within 2 hours), followed by a rapid decline in signal on the next day (day1). On day 8, most luc+ cells were detected in the abdomen and liver. Subsequently, after 6–12 weeks, multiple engraftment loci were identified in hematopoietic tissues. Flow cytometric analysis of bone marrow from these mice confirmed the presence of hESC-derived human CD45+ cells. Engraftment was also demonstrated after direct intra-bone marrow injection with as few as 60,000 CD34+ cells sorted from luc+ H1/S17 cells. Again, stable engraftment can be monitored by BLI for 8+ weeks. These results demonstrate that BLI has several important advantages as an effective non-invasive approach to track and quantitatively monitor in vivo engraftment of hematopoietic or other cell lineages derived from hESCs.

Cre recombinase-controlled expression of the mb-1 allele.

The mb-1 gene is located on chromosome 7 of the mouse genome and it is composed of five exons encoding Ig, which is a B cell-specific protein and an element of the B cell antigen receptor complex (BCR) (Sakaguchi et al., 1998; Hombach et al., 1990). Expression of Igis required for the transport of BCR onto the cell surface. Together with Ig, Igforms a heterodimer that mediates signal transduction from the BCR (Kim et al., 1993). The mb-1 gene is expressed at every stage of B cell development and activation, excluding terminally differentiated plasma cells. The knock-out of mb-1 results in complete block of B cell development at the progenitor stage in mice, as well as in humans (Minegishi et al., 1999; Pelanda et al. unpublished observations). Thus, we created conditional alleles of mb-1 to study the behavior of mature B cells with or without Igexpression. Cre-mediated recombination of inverted loxP sites results in the inversion of the loxP-flanked (floxed) DNA sequence (Nagy 2000). By flanking the mb-1 coding sequence with inverted loxP sites we generated a conditional mb-1 allele that allows switching “on” or “off” (and vice versa) of mb-1 expression under the control of Cre recombinase. In addition, BAP31N-EGFP, which encodes a membrane-associated form of the enhanced green fluorescent protein (mEGFP), was cloned in the opposite transcriptional orientation relative to mb-1 coding sequence into the conditional mb-1 allele. This allows the detection of cells at the “off” state of mb-1 transcription by the expression of mEGFP. Embryonic stem (ES) cells carrying the conditional mb-1 allele were generated by homologous recombination following standard techniques (Torres and Kuhn 1997). By transiently transfecting these latter cells with a Cre-expressing vector, we isolated ES clones carrying the deletion of the neomycin resistance (neo) cassette and the floxed mb-1 targeted allele in either orientation, thus resulting in the generation of mice carrying either mEGFP/mb-1 or mb-1/mEGFP conditional alleles. A schematic representation and Southern analysis of wild-type and targeted mb-1 alleles are shown in Figure 1a–c. Expression of mEGFP (Fig. 2) and of Ig(data not shown) by the targeted mb-1 alleles was confirmed by flow cytometric analysis of bone marrow, spleen, and lymph node cells isolated from heterozygous mEGFP/mb-1 and homozygous mb-1/mEGFP mice, respectively. The mEGFP/mb-1 allele directs expression of mEGFP, but not Ig. Thus we found that homozygous mEGFP/mb-1 mice lack any Igexpression and their B cell development is absolutely blocked at the B cell progenitor stage (data not shown). On the other hand, the mb-1/mEGFP allele encodes for Ig, but not mEGFP (Fig. 2c and data not shown). The expression of Igby the conditional allele was found to be reduced compared to wild-type (data not shown). As expected and observed in previous cell line experiments, the continuous presence of Cre in the nuclei results in the repetitive inversion (flipping) of the floxed DNA sequence that carries inverted loxP sites (data not shown). Removal of nuclear Cre stops further loxP recombination, stabilizing the floxed DNA into one of the two possible orientations with equal probability. Thus, the resulting cell population contains cells with either orientation of the floxed DNA sequence. The conditional mb-1 mice were tested by breeding them to either polyI/C, (MxCre, Kuhn et al., 1995) or hydroxy-tamoxifen, (MerCreMer, Hobeika et al., unpublished) inducible Cre-transgenic mouse strains. Induction of Cre recombinase in mEGFP/mb-1;MxCre heterozygous mice resulted in the elimination of mEGFP expression in approximately 30% of the bone marrow and splenic B cells (Fig. 2a). Southern blot analysis confirmed the presence of cells carrying mb-1/mEGFP alleles, which were the result of Cre-mediated recombination of mEGFP/mb-1 alleles (Fig. 2b). On the other hand, Cre induction in mb-1/mEGFP;MerCreMer bone marrow cells in vitro resulted in the de novo expression of mEGFP in approximately 9% of B cells (Fig. 2c). Thus, our experiments demonstrated that the mb-1 targeted alleles described here are functional with respect to their capacity to be conditionally activated by inducible Cre recombinase. Conditional mb-1 mice will allow study of the kinetics and functions of B cell development by the induction of Igexpression and the role of Igand BCR signaling in mature B cells, by suppression of Igexpression.

Serum tryptase levels combined with flow cytometric analysis of bone marrow aspirate mast cells differentiate systemic mastocytosis from idiopathic syndromes

Serum tryptase levels combined with flow cytometric analysis of bone marrow aspirate mast cells differentiate systemic mastocytosis from idiopathic syndromes

Differential Analysis of Rat Bone Marrow by Flow Cytometry

A procedure for rat bone marrow differential analysis using flow cytometry and commercially available monoclonal antibodies is described. The method uses a combination of the differential expression of leucocyte common antigen (CD45) on different cell lineages and the expression of transferrin receptor (CD71). This is coupled with the side scatter measurement (SSC) for morphological complexity and nucleic acid staining with LDS-751 for the separation of all nucleated cells from mature red blood cells. The relative number of erythroid, myeloid and lymphoid cells as well as the M:E ratio was generated by the flow cytometric method with acceptable reproducibility and a good agreement with microscopic analysis. There were no significant differences in mean values of the three cell populations between males and females or between bone marrow samples collected from left and right femurs. The flow cytometric analysis of bone marrow provides a semi-automated and rapid method that is suitable for the evaluation of rat bone marrow in the preclinical toxicity investigations.

Effects of sublethal radiation on bone marrow cells: induction of apoptosis and inhibition of antibody formation

As part of the study to investigate the mechanism of radiation-induced immunosuppression, the survival and functional ability of bone marrow cells was analyzed by exposing C57Bl/6 mice whole body to 2.0-Gy γ-rays. There was a rapid induction of DNA fragmentation in the total bone marrow cells and the kinetics indicated that apoptosis reached a peak by 4 h and then dropped back to normal control levels within 10 h after irradiation. To determine the functional ability of bone marrow cells which survive the radiation treatment, animals were immunized with antigen trinitrophenyl (TNP)-lipopolysaccharide. There was a significant decrease of anti-TNP plaque-forming cells in the bone marrow of irradiated mice compared to control animals. Flow cytometric analysis of bone marrow revealed a significant depletion of both immature (B220 +, Ig −) as well as mature (B220 +, Ig +) B cells compared to control group. In summary, the present study showed that sublethal whole body irradiation inhibits antibody responses elicited by bone marrow cells. This decreased immune response may have been due to depletion of B lineage subsets as well as generalized apoptosis in the entire bone marrow cells

Bone marrow cellular composition in Listeria monocytogenes infected mice detected using ER-MP12 and ER-MP20 antibodies: a flow cytometric alternative to differential counting

Detailed assessment of bone marrow cellular composition is essential in the evaluation of various experimental in vivo systems, such as expression of transgenes, null mutations and stimulation of host defence in infection. Traditional morphological analysis of mouse bone marrow is laborious, requires specific cytological expertise, and is somewhat subjective. As an alternative, we have examined whether double labelling of bone marrow with the anti-precursor monoclonal antibodies ER-MP12 and ER-MP20 could be used for differential analysis by flow cytometry, as these antibodies define six relatively homogeneous cell populations in mouse bone marrow. Following a sublethal infection of mice with Listeria monocytogenes, we monitored changes in cellular composition of the bone marrow at various time points in three ways: differential morphological count; single-color flow cytometric analysis using markers for the myeloid, erythroid and lymphoid lineages; and double labelling with ER-MP12 and ER-MP20. As expected, the bone marrow composition changed dramatically during infection, leading to an increase of myeloid cells which peaked after 1 week of infection. Data determined by ER-MP12/20 flow cytometric analysis appeared to be in close agreement with both morphology and lineage marker analysis. In addition, ER-MP12/20 analysis provided more detailed information with regards to the presence of early myeloid precursors compared to lineage marker analysis. These data show that flow cytometric analysis of bone marrow using ER-MP12 and ER-MP20 monoclonal antibodies provides a relatively simple, rapid and objective assay when evaluating cellular composition in the bone marrow of the mouse.