Colonies In Semisolid Cultures Research Articles

Combinatorial CSF3R and RUNX1 Mutations Involved in the Leukemic Progression of Severe Congenital Neutropenia Confer a G-CSF-Dependent Premalignant State in Mice

Background: Severe congenital neutropenia (SCN) is a genetically heterogeneous disorder characterized by a lack of peripheral neutrophils, leading to life-threatening bacterial infections. The disease is alleviated by sustained G-CSF treatment, which restores neutrophil production in the majority of patients. While on G-CSF therapy, SCN patients frequently develop hematopoietic clones with acquired CSF3R mutations. The appearance of CSF3R mutant clones is seen in multiple genetic subtypes of SCN and is strongly associated with progression towards MDS or AML. At this stage, CSF3R mutant clones acquire secondary mutations, most often (in ~75% of cases) in RUNX1 . The majority of these mutations affect the Runt homology domain (RHD) of RUNX1. This combination of CSF3R and RUNX1 mutations is exclusively seen in SCN/MDS/AML, suggesting a unique pattern of leukemic progression of SCN. However, the molecular and cellular consequences of combined CSF3R and RUNX1 mutations and the effects of sustained G-CSF therapy on leukemia development are unknown.Aim: To determine the cooperative role of CSF3R and RUNX1 mutations in sequential steps of malignant transformation in vivo and to investigate how sustained G-CSF therapy affects this process.Methods: We used our previously generated Csf3r -d715 mouse strain, which copies the most frequent G-CSFR truncation mutant in SCN patients. Lethally irradiated wild type mice were transplanted with Csf3r-d715 HSPCs transduced with an IRES-GFP backbone lentiviral vector expressing RHD mutant RUNX1-D171N or empty vector control. Mice were treated 3x per week with G-CSF or PBS (solvent control). Biweekly, peripheral blood (PB) samples were analyzed for GFP-marked engraftment, immunophenotypes and cytology. At regular intervals, in vitro progenitor cell colony assays were performed. Bone marrow (BM) and spleen samples were used for phenotypic analyses and secondary transplantations. RNA-seq experiments were carried out on FACS purified LK populations to identify molecular pathways associated with the observed phenotypes.Results: Mice transplanted with Csf3r -d715/ RUNX1 -D171N (RHD mutant) BM cells started to accumulate GFP-marked Lin negative, c-Kit positive (LK) progenitor cells in the PB, 6 weeks after initiation of G-CSF treatment. The percentages of GFP+ LK cells in the PB ranged from 0.7% to 27.8% (Mean=5.9%; n=11) and fluctuated over time in individual mice, indicating that they were maintained but not vastly expansive at this stage. This phenotype was fully dependent on the combined presence of the Csf3r and RUNX1 mutations and was only seen in the G-CSF treated mice. The PB-LK cells had a blast-like morphology and formed myeloid colonies in semi-solid cultures. Transcriptome analysis of in vitro expanded LK cells by RNA-seq and gene set enrichment analysis (GSEA) showed that the combination of Csf3r -d715 and RUNX1 -D171N mutations and G-CSF stimulation selectively enhanced c-Myc , NF-κB and E2F1-driven transcriptional programs. Of note, fully differentiated GFP-positive neutrophils were also formed, both in vitro and in vivo and mice did not succumb to this pre-leukemic condition. Csf3r -d715/ RUNX1 -D171N BM cells isolated after 30 weeks could successfully engraft secondary recipients (n=6), again resulting in sustained G-CSF dependent accumulation of immature blast (LK) cells in the PB. In these mice, the blast cells in the BM exceeded 85% at week 7 after initiation of G-CSF treatment. At this stage, the blasts had become entirely defective in neutrophil differentiation, indicating that they had now undergone full transformation to AML. RNA-seq and genomic analyses of these AML blasts are currently underway to assess the molecular steps involved in the transition from the premalignant to the fully transformed state.Conclusion: The combination of Csf3r -d715 and RUNX1 -D171N (RHD) mutations frequently seen in SCN/MDS/AML perturbs myeloid development in an in vivo transplant model in a G-CSF dependent manner. A sustained pre-leukemic state characterized by the selective accumulation of LK cells is seen in primary recipients, followed by a fully transformed AML phenotype in secondary recipients. This model is attractive for unraveling the molecular steps of malignant transformation driven by CSF3R and RUNX1 mutations and for preclinical testing of new therapeutic options for SCN/AML. DisclosuresNo relevant conflicts of interest to declare.

Characterization, regulation, and targeting of erythroid progenitors in normal and disordered human erythropoiesis.

The erythroid progenitors burst-forming unit-erythroid and colony-forming unit-erythroid have a critical role in erythropoiesis. These cells represent a heterogeneous and poorly characterized population with modifiable self-renewal, proliferation and differentiation capabilities. This review focuses on the current state of erythroid progenitor biology with regard to immunophenotypic identification and regulatory programs. In addition, we will discuss the therapeutic implications of using these erythroid progenitors as pharmacologic targets. Erythroid progenitors are classically characterized by the appearance of morphologically defined colonies in semisolid cultures. However, these prior systems preclude a more thorough understanding of the composite nature of progenitor populations. Recent studies employing novel flow cytometric and cell-based assays have helped to redefine hematopoiesis, and suggest that erythroid progenitors may arise from different levels of the hematopoietic tree. Moreover, the identification of cell surface marker patterns in human burst-forming unit-erythroid and colony-forming unit-erythroid enhance our ability to perform downstream functional and molecular analyses at the population and single cell level. Advances in these techniques have already revealed novel subpopulations with increased self-renewing capacity, roles for erythroid progenitors in globin gene expression, and insights into pharmacologic mechanisms of glucocorticoids and pomalidomide. Immunophenotypic and molecular characterization resolves the diversity of erythroid progenitors, and may ultimately lead to the ability to target these progenitors to ameliorate diseases of dyserythropoiesis.

Loss of the ability to generate large burst-forming unit-like megakaryocytic colonies from thawed cord blood in semisolid cultures after short term suspension culture.

Total colony-forming cells from thawed cord blood units (CBUs) include megakaryocytic colony-forming units (CFU-Mks), which survive the freezing process. The aim of this study was to evaluate whether different megakaryocytic progenitors from unseparated CBUs survive the freezing process and a short-term liquid culture. Thawed samples of CBUs were cultured in liquid medium. During the cultures, serial samples were drawn to assess the growth of different megakaryocytic progenitors in a semisolid collagen medium with identical cytokines as in the liquid medium. Megakaryocytic cells were detected using immunohistochemistry and flow cytometry. In suspension culture, the megakaryocytic progenitors almost completely lost the ability to generate large (burst-forming unit-like, BFU-like) megakaryocytic colonies in semisolid cultures (large colonies, median count per chamber d0: 7.25 vs. d7: 1.5; P < 0.0001), whereas the number of small colonies (median count per chamber d0: 7.25 vs. d7: 16.0; P = 0.0505) peaked at day seven. Further 7-day culture in suspension resulted in the decline of small colonies as well (d7: 16.0 vs. d14: 5.75; P = 0.0088). Total CFU-Mk count declined from 23.3 (range 12.5-34.0) at d0 to 7.25 (range 1.0-13.5) at d14 (P < 0.0001). Immediately post-thaw, CBUs possess an ability to generate large BFU-like megakaryocytic colonies, whereas the colonies were not detectable in most CBUs in semisolid culture after a short suspension culture. Small CFU-Mks were observed throughout the cultures. It may be that the BFU-Mk colonies matured and acquired CFU-Mk behaviour.

Abstract 4301: Transformation by the D816V mutant of c-Kit is dependent on the PI3-kinase isoform p110δ but independent of its lipid kinase activi.

Abstract Gain-of-function mutations of the receptor tyrosine kinase c-Kit are frequently seen in several malignanices with the D816V mutation being the most common. In this study we demonstrate that a mutant of c-Kit/D816V that is deficient in binding of the p85 subunit of PI3-kinase (M724A) has lost its ability to induce ligand-independent cell survival and proliferation while c-Kit/D816V induced PI3-kinases activity was only marginally reduced. Furthermore, inhibitors of PI3-kinase were unable to block c-Kit/D816V-induced cell survival. These data suggest that PI3-kinase plays a role in c-Kit/D816V induced cell transformation which is independent of its lipid kinase activity. By mass spectrometry, we identified a heavily tyrosine-phosphorylated 110 kDa protein associated with p85 to be the p110δ subunit of PI3-kinase and the site of phosphorylation as Tyr 523, and the phosphorylation is not dependent on the lipid kinase activity of PI3-kinase. Knockdown of p110δ in c-Kit/D816V expressing Ba/F3 cells led to reduced cell survival and proliferation while the PI3-kinase activity remained almost intact. The c-Kit/D816V expressing Ba/F3 cells lost their ability to form colonies in semi-solid culture if p110δ was knocked down. Additionally, knockdown of p110δ λεδ τo ρεδυχεδ τυμoρ σιζε, τυμoρ ωειγητ ανδ blood vessel formation ωηεν c-Kit/D816V expressing Ba/F3 cells were subcutaneously injected into nude mice. Taken together, these results suggest a kinase independent role of p110δ in the D816Vmutant of c-Kit mediated cell transformation and suggest that p110δ could potentially be a selective therapeutic target for c-Kit/D816V expressing tumors. Citation Format: Jianmin Sun, Sofie Mohlin, Alicia Lundby, Ulf Hellman, Sven Påhlman, Jesper V. Olsen, Lars Rönnstrand. Transformation by the D816V mutant of c-Kit is dependent on the PI3-kinase isoform p110δ but independent of its lipid kinase activi. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4301. doi:10.1158/1538-7445.AM2013-4301

Reactive oxygen species and aldehyde dehydrogenase activity in Hodgkin lymphoma cells

Tumor cells with tumorigenic potential might be limited to a small population of cells, called cancer-initiating cells (CICs). CICs efficiently form colonies in vitro, yield both CIC and non-CIC populations, maintain reactive oxygen species (ROS) at low levels, show high aldehyde dehydrogenase (ALDH) activity, and are mostly in a quiescent state of the cell cycle. CICs of Hodgkin lymphoma (HL) are small in size, with low levels of ROS. The relationship between ROS level and ALDH activity in CICs was examined in HL cell lines. ROS-low and ALDH-high populations formed colonies in semi-solid cultures more efficiently than ROS-high and ALDH-low populations. ALDH-high populations yielded both ALDH-low and -high populations, whereas ALDH-low populations rarely yielded an ALDH-high population. The number of cells in a quiescent state was significantly greater in ROS-low than in ROS-high cells, whereas that of ALDH-high and ALDH-low cells was comparable to each other. These findings show that ALDH-high and ROS-low cells share CIC-like potential, but they differ in their cell cycle status, suggesting that CICs are comprised of cells with heterogeneous characteristics.

Pharmacological Inhibition or Genetic Knockdown of Kdm1a (Lsd1 or Aof2) Induces Differentiation of MLL Acute Myeloid Leukaemia Stem Cells

Abstract 58Histone demethylases are candidate therapeutic targets in cancer. We analysed a previously published microarray expression dataset of murine acute myeloid leukemias (AML) experimentally initiated with human MLL fusion oncogenes to identify histone demethylases whose expression correlated with the frequency of leukemia stem cells (LSCs). Only expression of Kdm1a correlated positively and significantly (p<0.001) with LSC frequency, a finding confirmed by quantitative PCR of selected leukemias. To investigate whether Kdm1a has a critical role in MLL LSCs, we targeted Kdm1a transcripts for knockdown in murine MLL-AF9 AML cells using lentivirally expressed shRNAs. Knockdown AML cells formed significantly fewer colonies in semi-solid culture compared with AML cells expressing a non-targeting control shRNA (12±7% of control; n=4; p=0.01), and the extent of Kdm1a knockdown correlated significantly (six shRNAs tested) with loss of AML cell colony forming (CFC) potential (p<0.005). Residual Kdm1a knockdown colonies predominantly exhibited “Type 2” morphology (i.e. containing macrophages), a finding confirmed by examination of cytospin preparations. The phenotype was rescued by forced expression of human KDM1A. Kdm1a knockdown MLL-AF9 AML cells were unable to initiate leukemia in secondary transplant assays, in contrast to control cells which readily induced short latency AML. These data demonstrate that Kdm1a is required to prevent differentiation of murine MLL-AF9 AML LSCs.Tranylcypromine (TCP) is a licensed monoamine oxidase inhibitor that inhibits KDM1A, with an IC50 of 5–10uM. Treatment of murine MLL-AF9 AML cells, or human THP1 MLL-AF9 cells, with TCP phenocopied Kdm1a knockdown at a dose close to the KDM1A IC50; both murine and human MLL-AF9 cells exhibited reduced proliferation, fewer colonies and increased macrophage differentiation. Next, we tested the effect of KDM1A knockdown or TCP treatment (25uM) on primary human MLL leukemia cells. KDM1A knockdown reduced proliferation, induced morphological differentiation and induced apoptosis in the two samples of MLL-AF6 leukemia tested (5–20 fold fewer cells remained after a ten day culture versus control cells). Likewise, TCP treatment reduced proliferation 4-fold over five days (MLL-AF6 n=2; MLL-AF10 n=1), blocked CFC formation, reduced the proportion of blasts and increased the proportion of cells with features of monocyte/macrophage differentiation. Of note, addition of 25uM TCP to normal murine KIT+ BM stem and progenitor cells cultured in methylcellulose did not significantly reduce CFC frequency or size or induce macrophage differentiation, suggesting that MLL LSCs are more sensitive than normal BM cells to a given level of KDM1A inhibition.To investigate the mechanism by which KDM1A inhibits a differentiation programme in MLL-AF9 LSCs, we performed exon array analysis of Kdm1a knockdown versus control LSCs 48 hours following lentiviral infection, prior to appearance of morphologically differentiated cells. Down regulated transcriptional regulators included Id2, Hmgb3 and Egr1, but not Hoxa, Meis1 or Myb. Up regulated transcriptional regulators included Irf8. Knockdown of Id2 in murine MLL-AF9 AML cells reduced CFC frequency. In contrast, forced expression of Id2 enhanced CFC frequency both of control MLL-AF9 AML cells and of Kdm1a knockdown LSCs, the latter representing a partial phenotypic rescue of Kdm1a knockdown.These data demonstrate that KDM1A prevents differentiation of MLL LSCs by regulating the relative expression of pro- versus anti-differentiation transcriptional regulators and highlight it as a novel therapeutic target in MLL leukemias. Disclosures:No relevant conflicts of interest to declare.

IFNy Regulates the Balance Between Monocyte and Neutrophil Production During Immune Activation.

AbstractAbstract 1562Recruitment of the appropriate cells from the innate immune system to sites of infection is essential for the defense against pathogens. Depending on the type of invading pathogen, infection induces a rapid release from innate immune cells from the bone marrow, followed by an increased production of those myeloid cells that are most suitable to assist the ensuing immune response in the rapid eradication of the pathogen. However, it is thus far poorly understood what the cellular and molecular mechanism is that underlies this change in hematopoiesis.Using a mouse model with increased numbers of IFNγ-producing T cells in the bone marrow, we have previously found that IFNg plays an important role in the production of myeloid cells and can directly inhibit the formation of eosinophils (De Bruin et al., Blood. 2010 Jun 29). Here we demonstrate that IFNg also skews the balance between monocyte and neutrophil production in the direction of the monocytes. Increased monocyte production was also observed in WT mice infected with LCMV, which was IFNg-dependent, as it was not observed in IFNg-deficient mice. Subsequently, we set out to investigate how IFNγ can modulate the balance between monocyte and neutrophil production. We found that IFNγ reduces the proliferation and differentiation of murine hematopoietic progenitors in vitro in response to G-CSF, while M-CSF responses were not inhibited, but even slightly increased. In addition, IFNγ reduces the capacity of GMPs to form granulocyte colonies in semi-solid cultures and increases their ability to form monocyte/macrophage colonies. On a molecular level, we demonstrate that IFNγ induces a strong upregulation of SOCS3 levels and thereby reduces the phosphorylation of STAT3 in response to G-CSF, thus explaining the reduction in granulocyte formation. The expression levels of M-CSF-R and G-CSF-R by GMPs were not affected by IFNγ, but we found that IFNγ does induce the expression of the monocyte-inducing transcription factors PU.1 and IRF8 in these cells. In conclusion, these data demonstrate that IFNγ can direct myelopoiesis in a lineage-specific manner, thereby modulating the production of the appropriate type of innate immune cells required to combat infection. Disclosures:No relevant conflicts of interest to declare.

Expression and Function of B7.2 and B7-H2 Molecules on Myeloma Cells

(INTRODUCTION) The B7 family molecules, which play an important role in the immune response by costimulating or coinhibiting T cells via antigen–T-cell receptor interactions, are expressed not only on professional antigen-presenting cells but also on some tumor cells. We previously reported that the expression of B7.2 and B7-H2 on leukemic cells was associated with poor prognosis in acute myeloid leukemia. In this study, we investigated whether functional B7.2 and B7-H2 molecules are expressed on myeloma cells and, if so, whether these molecules are associated with pathophysiology in multiple myeloma (MM).(METHODS AND RESULTS)Using flow cytometry (FCM), we examined the expression of B7.2 and B7-H2 molecules on fresh plasma cells in bone marrow samples from 10 normal individuals, 21 monoclonal gammopathy of undetermined significance (MGUS) patients, and 40 MM patients. The percentages of B7.2+ cells in plasma cells were much higher in MM patients than in MGUS patients or in normal individuals (MM vs. MGUS, P = 0.0128; MM vs. normals, P = 0.0053). When MM patients were divided into two groups, those in whom more than 40% of myeloma cells expressed B7.2 (n = 20, called B7.2high+ MM patients in this study) showed significantly lower levels of hemoglobin and platelets and higher levels of M-protein compared with other MM patients (B7.2−/low MM patients in this study, n = 20). Meanwhile, B7-H2 expression on plasma cells was clearly documented only in 4 patients with intractable disease: 1 plasma cell leukemia, 1 progressive disease, and 2 chemotherapy-resistant MM. Notably, the expression of B7.2 and B7-H2 on myeloma cells was augmented at the refractory stage compared with expression at the initial diagnosis in 2 patients examined. All of the above findings support the concept that these molecules are associated with disease progression in MM.Based on the above data, we speculated that B7.2 or B7-H2 expression on myeloma cells was associated with their proliferative potential. When KMS-27 cells (a human myeloma cell line [HMCL]), part of which expressed these B7 family molecules, were analyzed in FCM, B7.2+ and B7-H2+ cells had significantly fewer G0/G1-phase cells and more G2/M-phase cells compared with B7.2− and B7-H2− cells, respectively. Consistent with these results, when four cell populations (B7.2+, B7.2−, B7-H2+, and B7-H2− cells) were isolated and examined, B7.2+ and B7-H2+ KMS-27 cells proliferated more rapidly in liquid cultures and formed more colonies in semisolid cultures compared with B7.2− and B7-H2− KMS- 27 cells, respectively. The same growth advantage of myeloma cells expressing B7.2 and B7-H2 molecules was also documented in all other HMCLs examined. When B7.2 and B7-H2 expression was induced on HMCL RPMI8226 cells by cultivation with tumor necrosis factor-a, the cell cycle was clearly stimulated. Furthermore, when 293T cells were transfected with either B7.2 or B7-H2 gene or Mock, B7.2 or B7-H2 gene induction induced cell cycle activation.Finally, we examined whether B7.2 and B7-H2 molecules on myeloma cells affect T cell immunology. In the mixed lymphocyte-myeloma reaction using KMS-27 cells and CD4+ T cells, an antagonistic monoclonal antibody (mAb) against B7.2 decreased the production of interleukin (IL)-10 as well as that of interferon (IFN)-g and IL-2. Meanwhile, an antagonistic mAb against inducible costimulator (ICOS), which blocks the B7-H2-ICOS pathways, decreased the production of IL-10 and IFN-g but not that of IL-2. The finding that both B7.2 and B7-H2 molecules on myeloma cells enhanced IL-10 production is particularly interesting because IL-10 not only reduces the antitumor immune response in general but also is a growth factor for myeloma cells.(CONCLUSIONS) Our results show that functional B7.2 and B7-H2 molecules are expressed on myeloma cells and that these molecules may be associated with a proliferative advantage of MM cells and therefore contribute to the pathophysiology of MM.

Generation of Blood Cells from Human Ips Cells in Vitro through the Hematopoietic Progenitors Concentrated within the Unique Structures, Ips-Sac.

Human embryonic stem cells (hESCs) are proposed as an alternative source for transfusion therapy or studies of hematopoiesis. We have recently established an in vitro culture system whereby hESCs can be differentiated into hematopoietic progenitors within the ‘unique sac-like structures’ (ES-sacs), that are able to produce megakaryocytes and platelets (Takayama et al., Blood , 111, 5298–306, 2008). However there is a little concern that repetitive transfusion with same human ESC-derived platelets may induce immunological rejection against transfused platelets expressing allogenic HLA. Meanwhile, induced pluripotent stem (iPS) cells established from donor with identical HLA are well known as a potential and given source on platelet transfusion devoid of rejection. To examine if human iPS cells could generate platelets as well as from hESCs, we utilized 3 different human iPS cell lines; two were induced by transduction of 4 genes (Oct3/4, Klf4, Sox2, and c-Myc) in adult dermal fibroblasts, and one was by 3 genes without c-Myc. Sac-like structures (iPS-sac), inducible from 3 iPS cell lines, concentrated hematopoietic progenitors that expressed early hemato-endothelial markers, such as CD34, CD31, CD41a (integrin αIIb) and CD45. These progenitors were able to form hematopoietic colonies in semi-solid culture and differentiate into several blood cells including leukocytes, erythrocytes or platelets. Of these, obtained platelets responded to agonist stimulation, in which the function was as much as human ESC-derived platelets, as evidenced by PAC-1 binding with activated αIIbβ3 integrin or full spreading onto fibrinogen. These results collectively indicated that human dermal fibroblasts could generate functional and mature hematopoietic cells through the reprogramming process and this method may be useful for basic studies of hematopoietic disorders and clinical therapy in the future.

Generation of functional platelets from human embryonic stem cells in vitro via ES-sacs, VEGF-promoted structures that concentrate hematopoietic progenitors

AbstractHuman embryonic stem cells (hESCs) could potentially represent an alternative source for blood transfusion therapies and a promising tool for studying the ontogeny of hematopoiesis. When we cultured hESCs on either C3H10T1/2 or OP-9 cells to facilitate hematopoiesis, we found that exogenous administration of vascular endothelial growth factor promoted the emergence of sac-like structures, which we named embryonic stem cell–derived sacs (ES-sacs). These ES-sacs consisted of multiple cysts demarcated by cellular monolayers that retained some of the properties of endothelial cells. The spherical cells inside ES-sacs expressed primarily CD34, along with VE-cadherin, CD31, CD41a, and CD45, and were able to form hematopoietic colonies in semisolid culture and to differentiate into mature megakaryocytes by day 24 in the presence of thrombopoietin. Apparently, ES-sacs provide a suitable environment for hematopoietic progenitors. Relatively large numbers of mature megakaryocytes could be induced from the hematopoietic progenitors within ES-sacs, which were then able to release platelets that displayed integrin αIIbβ3 activation and spreading in response to ADP or thrombin. This novel protocol thus provides a means of generating platelets from hESCs, which could serve as the basis for efficient production of platelets for clinical transfusion and studies of thrombopoiesis.

Human Embryonic Stem Cell-Derived “NET-Like” Structure Serves as a Hematopoietic Progenitor Niche and Favors Generation of Mature Megakaryocytes and Functional Platelets.

Human embryonic stem cells (hESCs) are proposed as an alternative source in regenerative medicine while being a promising tool to trace earliest ontogeny in vivo. Anucleate platelets can be irradiated to eliminate residual ESCs that potentially cause immunological rejection and teratoma formation in transplantation. Herein, we describe for the first time the generation of functional platelets from hESCs (Kyoto hES1, -2, and -3) by a novel method that is distinct from previously established OP9 co-culture. Exogenous combination of TPO, VEGF and IGF-II allowed hECSs to facilitate the differentiating kinetics of hematopoietic marker emergence when cultured on feeder cells, 10T1/2, whereas independent single administration had little effect on hematopoietic differentiation (P<0.01). Consequently, unique “NET-like” structures expressing VE-cadherin low+, PECAM-1 high+, CD34 high+, CD45 + and VEGF-R2 + were observed around 14–15 days of culture. These structures contained hematopoietic progenitors capable of forming hematopoietic colonies in semisolid culture, or of differentiating into mature megakaryocytes in the presence of TPO, IL-6, and IL-11 (around 45 % of total cells displayed GPIbα +GPIX + by day 23). While totally 24–25 days culture was required for production of hESC-derived integrin αIIb +GPIX + platelets in this way, the platelets exhibited specific fibrinogen-bound to integrin αIIbβ3 in the presence of G-protein coupled receptor agonists in a dose-dependent manner. The efficiency of platelet production appeared to be well correlated with the frequency of “NET-like” structure. Thus, this structure may be a useful hallmark in our newly obtained culture method that may serve as an invaluable tool for not only generation of hESC-derived functional platelets but also studying niche machinery in hematopoiesis.

Inactivation of PU.1 in adult mice leads to the development of myeloid leukemia

Genetically primed adult C57BL mice were deleted of exon 5 of the gene encoding the transcription factor PU.1 by IFN activation of Cre recombinase. After a 13-week delay, conditionally deleted (PU.1(-/-)) mice began dying of myeloid leukemia, and 95% of the mice surviving from early postinduction death developed transplantable myeloid leukemia whose cells were deleted of PU.1 and uniformly Gr-1 positive. The leukemic cells formed autonomous colonies in semisolid culture with varying clonal efficiency, but colony formation was enhanced by IL-3 and sometimes by granulocyte-macrophage colony-stimulating factor. Nine of 13 tumors analyzed had developed a capacity for autocrine IL-3 or granulocyte-macrophage colony-stimulating factor production, and there was evidence of rearrangement of the IL-3 gene. Acquisition of autocrine growth-factor production and autonomous growth appeared to be major events in the transformation of conditionally deleted PU.1(-/-) cells to fully developed myeloid leukemic populations.

Expression and distribution of the Kit receptor in bovine bone marrow cells.

To characterize the expression and distribution of the Kit receptor in bovine bone marrow cells (BMC) and to define the function of its ligand, stem cell factor (SCF). Six 7- to 70-day-old healthy male Holstein-Friesian calves. Expression and distribution of the Kit receptor were assessed by use of flow cytometry with monoclonal antibodies (mAb) against the bovine Kit protein. Using Giemsa-stained centrifuged preparations, the histologic appearance of Kit receptor positive (Kit+) BMC were evaluated. Semisolid cultures supplemented with granulocyte colony-stimulating factor (G-CSF) and SCF were used to measure the colony formation capacity of Kit+ BMC. The Kit receptor was expressed on approximately 18% of total BMC. Most of Kit+ BMC did not coexpress lineage markers, but a small subset of this population did coexpress CD3. The Kit+CD3- BMC were a heterogeneous cell population comprising blast-like cells such as myeloblasts, promyelocytes, rubriblasts, and prorubricytes. Conversely, Kit+CD3+ BMC had a lymphocyte-like appearance. Kit+ BMC formed colonies in semisolid culture with G-CSF, whereas Kit- BMC failed to grow. Addition of SCF to G-CSF resulted in superadditive enhancement in colony numbers and size. The Kit receptor is expressed primarily on immature blood cells in bovine bone marrow, and Kit+ BMC contain hematopoietic progenitor cells that are reactive to G-CSF. In addition, SCF synergizes with G-CSF to stimulate colony formation by these cells. Our results suggest that the Kit receptor and its ligand, SCF, are involved in early stages of granulopoiesis in calves.

Colony-stimulating factor-1 impairs both proliferation and differentiation signals of erythropoietin during the commitment of bipotential NFS-60 cell line to the monocytic lineage

The interleukin-3 (IL-3) dependent cell line NFS-60 contains bipotential progenitors that exhibit both erythroid and myelomonocytic potentials. In order to study their commitment to the monocytic lineage, NFS-60 cells were retrovirally transduced with mouse c-fms cDNA, which encodes the colony-stimulating factor-1 receptor (CSF-1R), resulting in the N-Fms cell line. N-Fms cells proliferated in response to CSF-1 with a growth rate similar to that obtained in response to IL-3 and progressively differentiated from myeloid blasts to monocytic cells within 3 days of culture. When maintained in IL-3, about 3% of N-Fms cells formed large hemoglobinized colonies in semisolid cultures supplemented with erythropoietin (EPO). However, this property was lost after a 24-hour cultivation in the presence of CSF-1 or, interestingly, both CSF-1 and IL-3. This loss of response to EPO was reverted following a brief passage (24 hours) in IL-3, but the rescued colonies did not undergo terminal erythrocytic differentiation. Furthermore, CSF-1 also affected proliferative response to EPO of N-Fms cells constitutively expressing EPO receptors. Our data strongly suggest that CSF-1 can suppress erythroid potential in bipotential N-Fms cells by altering proliferative and differentiation signal of EPO.

Does transmembrane communication through gap junctions enable stem cells to overcome stromal inhibition?

When long-term bone marrow cultures are treated with Amphotericin B (AB) their haemopoietic stem cells (HSC) cease growing. This is not a toxic effect of the drug because once that is removed, HSC resume clonal growth and, given sufficient time, form as many cells as HSC in untreated cultures. Amphotericin B-evoked inhibition of blood formation is probably mediated by transmembrane communication between HSC and stroma for the following reasons: (1) AB does not stop HSC forming colony-forming units in culture (CFU-c) when HSC are separated from stroma by culturing them on Transwell inserts above the stroma. (2) Conditioned media (CM) from AB-containing or normal long-term cultures (LTC) does not inhibit normal marrow cells forming colonies in semi-solid cultures without stromal underlays. (3) AB itself does not stop bone marrow cells forming colonies in semi-solid cultures nor does it stop stromal cells growing or prejudice their long-term maintenance. (4) Furthermore, growing stromal cells with AB does not alter the number of transcripts they form for cytokines and chemokines to any large extent, including TGF-beta1. We have extensive, though circumstantial, evidence that gap junctions are involved in this communication. AB only stopped the growth of HSC when we blocked intercellular communication via gap junctions (GJIC) (tested by micro-injection of lucifer yellow). Lipophilic compounds that do not affect GJIC had no effect on the growth of HSC. Looking at a series of stromal cell lines from foetal liver and neonatal bone marrow we found that extensive GJIC correlated with stromal support of the late-appearing clones formed by primitive HSC (week 3-5 cobblestone-area forming cells, CAFC). We propose that the proliferation of HSC is regulated via transmembrane communication between stromal and HSC. Our findings support the proposal that gap junctions play a part in this stromal-dependent regulation.

In vitro proliferation and differentiation of erythroid progenitors of cord blood.

Stem cell factor (SCF) is known to synergize with erythropoietin (EPO) for erythropoiesis in vitro. Clonogenic assay and suspension culture were used to assess the effect of EPO alone or its combination with SCF on the proliferation and differentiation of erythroid progenitors of cord blood. Colony formation, increase in cell count, and cell cycling status for the proliferation as well as expression of Glycophorin A (Gly A) and hemoglobinization as the marker of differentiation were determined with each stimulation. The cell cycle status of the cells in suspension cultures was determined using FACScan after labeling of cells with propidium iodide. Expression of Gly A and degree of hemoglobinization were determined by FACScan and spectrophotometer on the cells plucked from colonies in semisolid culture. Larger increases in cell counts in suspension culture were observed with EPO + SCF after 12 days of inoculation than with EPO alone. Mean doubling time was 14.2 h with EPO + SCF and 22.7 h with EPO alone. The proportion of cells in S and G2 + M phase in day 14 suspension culture was 48% with EPO + SCF and 43% with EPO alone (no significant difference). Mean colony counts per 10(5) nonadherent mononuclear cells were 76 +/- 14 with EPO + SCF and 51 +/- 15 with EPO at day 14 (p < 0.05). The number of macroscopic colonies with > 0.5 mm diameter was 10.7 +/- 1.2 with EPO + SCF and 0.3 +/- 0.5 with EPO (p < 0.05). Percent of Gly A+ cells was 75% for both EPO + SCF and EPO colonies at day 14. Hemoglobin concentration/10(5) cells at day 14 was 0.70 +/- 0.17 microgram with EPO + SCF, and 1.16 +/- 0.32 micrograms with EPO alone (p < 0.05). In conclusion, SCF in the combination with EPO showed a synergistic effect for erythroid proliferation in colony number as well as colony size derived from cord blood, while SCF with EPO decreased hemoglobin synthesis but not Gly A expression at day 14.

Identification of Human Juvenile Chronic Myelogenous Leukemia Stem Cells Capable of Initiating the Disease in Primary and Secondary SCID Mice

Most juvenile chronic myelogenous leukemia (JCML) cells have limited long-term proliferative capacity, and only a minority of immature cells give rise to colonies in semisolid cultures. Clonogenic JCML progenitors cannot be maintained in culture because they differentiate, and within a few weeks the leukemic clone is lost. This makes it difficult to identify the cell that initiates and maintains the disease in patients. To determine the proliferative capacity of JCML cells in vivo, bone marrow (BM), peripheral blood, or spleen cells from eight patients with JCML either at diagnosis or during treatment were transplanted into sublethally irradiated severe combined immune deficient (SCID) mice. JCML cells from all patients homed to the murine BM and proliferated extensively in response to exogenous stimulation with granulocyte-macrophage colony-stimulating factor. Within a few weeks, highly engrafted mice became ill and cachectic due to infiltration of leukemic cells and secretion of tumor necrosis factor-alpha. Murine BM, spleen, and liver were infiltrated with leukemic blasts, and typical JCML colony-forming progenitors could be recovered. Kinetic experiments demonstrated that only a small minority of transplanted cells homed to the murine BM, and that these cells initiated and maintained the disease in vivo by extensive proliferation and differentiation. To characterize the cell-surface phenotype of the JCML initiating cell (JCML-IC), JCML blood or spleen cells were fractionated on the basis of CD34/CD38 marker expression and transplanted into SCID mice. Only immature CD34+ cells could initiate the disease, while mature CD34- cells did not engraft. Within the CD34+ compartment, there was enrichment for JCML-ICs by immature cells with a CD34+/CD38- stem-cell-like phenotype. Mice transplanted with more mature CD34+/CD38+ populations that also contained clonogenic JCML progenitors were poorly engrafted. These results indicate that the JCML-IC is an earlier stage of development than clonogenic JCML progenitors. Additional evidence that the JCML-IC has stem-cell properties comes from secondary transplant experiments that test the self-renewal capacity. The JCML-IC from all three patients tested could successfully reinitiate the disease in secondary murine recipients. Thus, we have developed a functional in vivo model that replicates many aspects of human JCML, and have used this model to identify and characterize JCML-ICs and their stem-cell properties.

Prevention of acute graft-versus-host disease by treatment with a novel immunosuppressant. Cholera toxin B subunit.

Transplantation of allogeneic bone marrow (BM) is often complicated by the development of acute graft-vs-host disease (GVHD) caused by contaminating T cells in BM inocula because of activation of the host reactive T effector cells. Using a murine model for acute GVHD caused by injection of parental C57Bl/6 splenocytes into unirradiated (C57Bl/6 x DBA/2) F1 hybrids, we demonstrated that pretreatment of the inocula with a novel immunosuppressant, B subunit of cholera toxin (CT-B) impaired the ability of C57Bl/6 T cells to induce acute GVHD in F1 recipients. F1 mice injected with CT-B-treated C57Bl/6 splenocytes did not develop significant splenomegaly, and no antihost CTLs were found in their spleens. Moreover, these mice did not suffer from aplastic anemia, nor from general immunosuppression. Immunofluorescence studies suggest that treatment of the inducing inocula with CT-B selectively prevents accumulation of the host-reactive CD8+ T cells in F1 mice. Furthermore, our experiments demonstrated that CT-B treatment does not impair the ability of BM progenitors to form colonies in semisolid culture or in lethally irradiated hosts. Thus, taken together, our data suggest that ex vivo CT-B treatment can be used in allogeneic BM transplantation to prevent acute GVHD.

Long-Term Generation of Human Mast Cells in Serum-Free Cultures of CD34+ Cord Blood Cells Stimulated With Stem Cell Factor and Interleukin-3

AbstractThe generation of murine mast cells is supported by several cytokines, and mast cell lines are frequently established in long-term cultures of normal murine marrow cells. In contrast, growth of human mast cells was initially dependent on coculture with murine fibroblasts. The growth factor produced by murine fibroblasts and required to observe differentiation of human mast cells is attributable in part to stem cell factor (SCF). However, other factors are likely involved. We have previously shown that the combination of SCF and interleukin-3 (IL-3) efficiently sustains proliferation and differentiation of colony-forming cells (CFCs) from pre-CFC enriched from human umbilical cord blood by CD34+ selection. With periodic medium changes and the addition of fresh growth factors, five consecutive cultures of different cord blood samples gave rise to differentiated cells and CFCs for more than 2 months. Although differentiated cells continued to be generated for more than 5 months, CFCs were no longer detectable by day 50 of culture. The cells have the morphology of immature mast cells, are Toluidine blue positive, are karyotypically normal, are CD33+, CD34–, CD45+, c-kit–, and c-fms–, and die in the absence of either SCF or IL-3. These cells do not form colonies in semisolid culture and are propagated in liquid culture stimulated with SCF and IL-3 at a seeding concentration of no less than 104 cells/mL At refeedings, the cultures contain a high number (>50%) of dead cells and have a doubling time ranging from 5 to 12 days. This suggests that subsets of the cell population die because of a requirement for a growth factor other than SCF or IL-3. These results indicate that the combination of cord blood progenitor and stem cells, plus a cocktail of growth factors including SCF and IL-3, is capable with high efficiency of giving rise in serum-deprived culture to human mast cells that behave like factor-dependent cell lines. These cells may represent a useful tool for studies of human mast cell differentiation and leukemia.

Erythroid Failure in Diamond-Blackfan Anemia Is Characterized by Apoptosis

Programmed cell death, also known as apoptosis, is frequently initiated when cells are deprived of specific trophic factors. To investigate if accelerated apoptosis contributes to the pathogenesis of Diamond-Blackfan anemia (DBA), a rare pure red blood cell aplasia of childhood, we studied the effect of erythropoietin (epo) deprivation on erythroid progenitors and precursors from the bone marrow of DBA patients as compared with hematologically normal controls. Apoptosis in response to epo deprivation was evaluated by enumeration of colony-forming unit-erythroid (CFU-E)- and burst-forming unit-erythroid (BFU-E)-derived colonies in plasma clot semisolid culture and by the identification of typical DNA oligosomes by gel electrophoresis from marrow mononuclear cells in liquid culture. In all DBA patients there was a marked decrease in CFU-E- and BFU-E-derived colony formation compared with normal controls at comparable time points of epo deprivation, with a complete loss of CFU-E-derived colonies in semisolid culture by 9 hours of epo deprivation versus 48 hours in controls. The BFU-E-derived colony response to epo deprivation displayed a similar pattern of decrement. Apoptotic changes assessed by the presence of characteristic DNA fragmentation began in the absence of epo deprivation and were readily detected within 3 hours of epo deprivation in DBA cultures versus 9 hours in controls. We conclude that DBA is characterized by accelerated apoptosis as measured by the loss of erythroid progenitor clonogenicity and increased progenitor and precursor DNA fragmentation leading to the formation of characteristic oligosomes, consistent with an intrinsic erythroid-progenitor defect in which increased sensitivity to epo deprivation results in erythroid failure.