Scoring CFU-GM colonies in vitro by data fusion: a first account.
OBJECTIVE: In vitro models of hematopoiesis used in investigative hematopathology and in safety studies on candidate drugs,involve clonogenic assays on colony-forming unit granulocyte macrophage (CFU-GM). These assays require live and unstained colonies to be counted. Most laboratories still rely on visual scoring,which is time-consuming and error-prone. As a consequence,automated scoring is highly desired. An algorithm that recognizes and scores CFU-GM colonies by data fusion has been developed. Some preliminary results are presented in this article. METHODS: CFU-GM assays were carried out on hematopoietic progenitors (human umbilical cord blood cells) grown in methylcellulose. Colony images were acquired by a digital camera and stored. RESULTS: The classifier was designed to process images of layers sampled from a three-dimensional (3D) domain and forming a stack. Structure and texture information was extracted from each image. Classifier training was based on a 3D colony model applied to the image stack. The number of scored colonies (assigned class) was required to match the count supplied by the human expert (class of belonging). The trained classifier was validated on one more stack and then applied to a stack with overlapping colonies. Scoring in distortion- and caustic-affected border areas was also successfully demonstrated. Because of hardware limitations,compact colonies in some cases were missed. CONCLUSIONS: The industry's scoring methods all rely on structure alone and process 2D data. Instead,the classifier here fuses data from a whole stack and is capable,in principle,of high-throughput screening.
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Berthier R et al. (MAR 1993)
Stem cells (Dayton,Ohio) 11 2 120--9
Serum-free medium allows the optimal growth of human megakaryocyte progenitors compared with human plasma supplemented cultures: role of TGF beta.
The growth of human megakaryocyte progenitors from human bone marrow (BM) cells was compared using a methylcellulose semisolid assay supplemented either by normal human plasma or by a serum-free medium. Far better growth of megakaryocyte colonies from CD34+ BM cells stimulated by interleukin 3 (IL-3) and interleukin 6 (IL-6) was observed in serum-free medium compared with human plasma supplemented cultures. These results were confirmed in liquid cultures using the same serum-free medium composition. The megakaryocytes were identified by using an immunocytochemical procedure after labeling with an anti-GPIIb-IIIa monoclonal antibody. High percentages (15 to 20%) of megakaryocytes were present in serum-free cultures stimulated by IL-3 alone or combined with IL-6. The absolute number of megakaryocytes in serum-free medium exceeds by 3.3 (IL-3 plus IL-6) to 4.4 (IL-3 alone) times the corresponding number of megakaryocytes observed in human plasma supplemented cultures. The optimal concentration of IL-3 alone was 5 ng/ml,and an optimal synergistic effect of IL-6 (5 ng/ml) was obtained when combined with a suboptimal dose of IL-3 (1 ng/ml). The poor growth of megakaryocyte colonies from CD34+ BM cells in human plasma suggested the presence of an inhibitory factor. When a neutralizing monoclonal antibody against transforming growth factor beta (TGF beta) is present in human plasma supplemented cultures of CD34+ BM cells,the number of megakaryocyte colonies is increased to the level observed in corresponding serum-free cultures. The high efficiency of this serum-free medium to promote the growth of human megakaryocytes will be useful to study the effects of regulators and platelet agonists acting on human megakaryocytes,without interference from factors in the serum.
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产品类型：
产品号#：
04850
04900
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04974
产品名：
MegaCult™-C含脂培养基
MegaCult™-C培养基无细胞因子
MegaCult™-C细胞因子培养基
胶原蛋白溶液
MegaCult™-C胶原蛋白和不含细胞因子的培养基
MegaCult™-C胶原蛋白和细胞因子培养基
MegaCult™-C cfu染色试剂盒
双室载玻片试剂盒
MegaCult™-C不含细胞因子完整试剂盒
MegaCult™-C细胞因子完整试剂盒
MegaCult™-C胶原蛋白和脂质培养基
Hisa T et al. (JAN 2004)
The EMBO journal 23 2 450--9
Hematopoietic, angiogenic and eye defects in Meis1 mutant animals.
Meis1 and Hoxa9 expression is upregulated by retroviral integration in murine myeloid leukemias and in human leukemias carrying MLL translocations. Both genes also cooperate to induce leukemia in a mouse leukemia acceleration assay,which can be explained,in part,by their physical interaction with each other as well as the PBX family of homeodomain proteins. Here we show that Meis1-deficient embryos have partially duplicated retinas and smaller lenses than normal. They also fail to produce megakaryocytes,display extensive hemorrhaging,and die by embryonic day 14.5. In addition,Meis1-deficient embryos lack well-formed capillaries,although larger blood vessels are normal. Definitive myeloerythroid lineages are present in the mutant embryos,but the total numbers of colony-forming cells are dramatically reduced. Mutant fetal liver cells also fail to radioprotect lethally irradiated animals and they compete poorly in repopulation assays even though they can repopulate all hematopoietic lineages. These and other studies showing that Meis1 is expressed at high levels in hematopoietic stem cells (HSCs) suggest that Meis1 may also be required for the proliferation/self-renewal of the HSC.
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产品类型：
产品号#：
04960
04902
04900
04961
04901
04963
04962
04970
04971
产品名：
MegaCult™-C胶原蛋白和不含细胞因子的培养基
胶原蛋白溶液
MegaCult™-C培养基无细胞因子
MegaCult™-C胶原蛋白和细胞因子培养基
MegaCult™-C细胞因子培养基
双室载玻片试剂盒
MegaCult™-C cfu染色试剂盒
MegaCult™-C不含细胞因子完整试剂盒
MegaCult™-C细胞因子完整试剂盒
Wernig G et al. (JUN 2006)
Blood 107 11 4274--81
Expression of Jak2V617F causes a polycythemia vera-like disease with associated myelofibrosis in a murine bone marrow transplant model.
An acquired somatic mutation,Jak2V617F,was recently discovered in most patients with polycythemia vera (PV),chronic idiopathic myelofibrosis (CIMF),and essential thrombocythemia (ET). To investigate the role of this mutation in vivo,we transplanted bone marrow (BM) transduced with a retrovirus expressing either Jak2 wild-type (wt) or Jak2V617F into lethally irradiated syngeneic recipient mice. Expression of Jak2V617F,but not Jak2wt,resulted in clinicopathologic features that closely resembled PV in humans. These included striking elevation in hemoglobin level/hematocrit,leukocytosis,megakaryocyte hyperplasia,extramedullary hematopoiesis resulting in splenomegaly,and reticulin fibrosis in the bone marrow. Histopathologic and flow cytometric analyses showed an increase in maturing myeloid lineage progenitors,although megakaryocytes showed decreased polyploidization and staining for acetylcholinesterase. In vitro analysis of primary cells showed constitutive activation of Stat5 and cytokine-independent growth of erythroid colony-forming unit (CFU-E) and erythropoietin hypersensitivity,and Southern blot analysis for retroviral integration indicated that the disease was oligoclonal. Furthermore,we observed strain-specific differences in phenotype,with Balb/c mice demonstrating markedly elevated leukocyte counts,splenomegaly,and reticulin fibrosis compared with C57Bl/6 mice. We conclude that Jak2V617F expression in bone marrow progenitors results in a PV-like syndrome with myelofibrosis and that there are strain-specific modifiers that may in part explain phenotypic pleiotropy of Jak2V617F-associated myeloproliferative disease in humans.
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产品类型：
产品号#：
04960
04902
04900
04961
04901
04963
04962
04970
04971
产品名：
MegaCult™-C胶原蛋白和不含细胞因子的培养基
胶原蛋白溶液
MegaCult™-C培养基无细胞因子
MegaCult™-C胶原蛋白和细胞因子培养基
MegaCult™-C细胞因子培养基
双室载玻片试剂盒
MegaCult™-C cfu染色试剂盒
MegaCult™-C不含细胞因子完整试剂盒
MegaCult™-C细胞因子完整试剂盒
Gekas C et al. (APR 2009)
Blood 113 15 3461--71
Mef2C is a lineage-restricted target of Scl/Tal1 and regulates megakaryopoiesis and B-cell homeostasis.
The basic helix-loop-helix transcription factor stem cell leukemia gene (Scl) is a master regulator for hematopoiesis essential for hematopoietic specification and proper differentiation of the erythroid and megakaryocyte lineages. However,the critical downstream targets of Scl remain undefined. Here,we identified a novel Scl target gene,transcription factor myocyte enhancer factor 2 C (Mef2C) from Scl(fl/fl) fetal liver progenitor cell lines. Analysis of Mef2C(-/-) embryos showed that Mef2C,in contrast to Scl,is not essential for specification into primitive or definitive hematopoietic lineages. However,adult VavCre(+)Mef2C(fl/fl) mice exhibited platelet defects similar to those observed in Scl-deficient mice. The platelet counts were reduced,whereas platelet size was increased and the platelet shape and granularity were altered. Furthermore,megakaryopoiesis was severely impaired in vitro. Chromatin immunoprecipitation microarray hybridization analysis revealed that Mef2C is directly regulated by Scl in megakaryocytic cells,but not in erythroid cells. In addition,an Scl-independent requirement for Mef2C in B-lymphoid homeostasis was observed in Mef2C-deficient mice,characterized as severe age-dependent reduction of specific B-cell progenitor populations reminiscent of premature aging. In summary,this work identifies Mef2C as an integral member of hematopoietic transcription factors with distinct upstream regulatory mechanisms and functional requirements in megakaryocyte and B-lymphoid lineages.
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产品类型：
产品号#：
03234
03434
03444
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04901
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04971
产品名：
MethoCult™M3234
MethoCult™GF M3434
MethoCult™GF M3434
MegaCult™-C胶原蛋白和不含细胞因子的培养基
胶原蛋白溶液
MegaCult™-C培养基无细胞因子
MegaCult™-C胶原蛋白和细胞因子培养基
MegaCult™-C细胞因子培养基
双室载玻片试剂盒
MegaCult™-C cfu染色试剂盒
MegaCult™-C不含细胞因子完整试剂盒
MegaCult™-C细胞因子完整试剂盒
Baran-Marszak F et al. (DEC 2010)
Blood 116 26 5961--71
Expression level and differential JAK2-V617F-binding of the adaptor protein Lnk regulates JAK2-mediated signals in myeloproliferative neoplasms.
Activating mutations in signaling molecules,such as JAK2-V617F,have been associated with myeloproliferative neoplasms (MPNs). Mice lacking the inhibitory adaptor protein Lnk display deregulation of thrombopoietin/thrombopoietin receptor signaling pathways and exhibit similar myeloproliferative characteristics to those found in MPN patients,suggesting a role for Lnk in the molecular pathogenesis of these diseases. Here,we showed that LNK levels are up-regulated and correlate with an increase in the JAK2-V617F mutant allele burden in MPN patients. Using megakaryocytic cells,we demonstrated that Lnk expression is regulated by the TPO-signaling pathway,thus indicating an important negative control loop in these cells. Analysis of platelets derived from MPN patients and megakaryocytic cell lines showed that Lnk can interact with JAK2-WT and V617F through its SH2 domain,but also through an unrevealed JAK2-binding site within its N-terminal region. In addition,the presence of the V617F mutation causes a tighter association with Lnk. Finally,we found that the expression level of the Lnk protein can modulate JAK2-V617F-dependent cell proliferation and that its different domains contribute to the inhibition of multilineage and megakaryocytic progenitor cell growth in vitro. Together,our results indicate that changes in Lnk expression and JAK2-V617F-binding regulate JAK2-mediated signals in MPNs.
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产品类型：
产品号#：
03434
03444
04960
04902
04900
产品名：
MethoCult™GF M3434
MethoCult™GF M3434
MegaCult™-C胶原蛋白和不含细胞因子的培养基
胶原蛋白溶液
MegaCult™-C培养基无细胞因子
Goossens S et al. (MAY 2011)
Blood 117 21 5620--30
The EMT regulator Zeb2/Sip1 is essential for murine embryonic hematopoietic stem/progenitor cell differentiation and mobilization.
Zeb2 (Sip1/Zfhx1b) is a member of the zinc-finger E-box-binding (ZEB) family of transcriptional repressors previously demonstrated to regulate epithelial-to-mesenchymal transition (EMT) processes during embryogenesis and tumor progression. We found high Zeb2 mRNA expression levels in HSCs and hematopoietic progenitor cells (HPCs),and examined Zeb2 function in hematopoiesis through a conditional deletion approach using the Tie2-Cre and Vav-iCre recombination mouse lines. Detailed cellular analysis demonstrated that Zeb2 is dispensable for hematopoietic cluster and HSC formation in the aorta-gonadomesonephros region of the embryo,but is essential for normal HSC/HPC differentiation. In addition,Zeb2-deficient HSCs/HPCs fail to properly colonize the fetal liver and/or bone marrow and show enhanced adhesive properties associated with increased β1 integrin and Cxcr4 expression. Moreover,deletion of Zeb2 resulted in embryonic (Tie2-Cre) and perinatal (Vav-icre) lethality due to severe cephalic hemorrhaging and decreased levels of angiopoietin-1 and,subsequently,improper pericyte coverage of the cephalic vasculature. These results reveal essential roles for Zeb2 in embryonic hematopoiesis and are suggestive of a role for Zeb2 in hematopoietic-related pathologies in the adult.
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产品类型：
产品号#：
03434
03444
04960
04902
04900
产品名：
MethoCult™GF M3434
MethoCult™GF M3434
MegaCult™-C胶原蛋白和不含细胞因子的培养基
胶原蛋白溶液
MegaCult™-C培养基无细胞因子
Dobo I et al. (JAN 2001)
The hematology journal : the official journal of the European Haematology Association / EHA 2 6 396--403
Comparison of four serum-free, cytokine-free media for analysis of endogenous erythroid colony growth in polycythemia vera and essential thrombocythemia.
INTRODUCTION: The assay of endogenous erythroid colony formation (EEC),a characteristic of polycythemia vera and essential thrombocythemia,is not standardized. In this multicentric study,we tested four semisolid,serum-free,cytokine-free media based on either methylcellulose (M1,M2) or collagen (C1,C2) commercialized for the EEC assay. MATERIALS AND METHODS: Bone marrow mononuclear cells (BMMC) from 73 individuals (62 patients with either polycythemia vera (26),essential thrombocythemia (19),secondary polyglobuly (17) or chronic myeloid leukemia (2) and 11 healthy donors) were grown in parallel in the four media without,or with 0.01 U/ml erythropoietin (EPo). RESULTS: In all four media EEC formation was specific,as it was not observed in cultures of patients with secondary polyglobuly or chronic myeloid leukemia,nor of healthy donors. Analysis of fresh or MGG-stained collagen gel cultures allowed detection of EEC formation significantly more frequently than methylcellulose-based media; addition of 0.01 U/ml of EPo had little or no effect on EEC formation. Collagen-based medium C1 gave better results than the other media tested: the 'C1' EEC assay was positive for 68.2% of polycythemia vera cultures with significantly higher median EEC numbers (6.5/10(5) BMMC for patients with one major criteria of polycythemia vera and 19 and 21/10(5) BMMC for patients with two or three major criteria,respectively). Medium C1 was also better for essential thrombocythemia cultures with 47.4% of positive results but with a low median EEC number (6.7/10(5) BMMC). When associated with the ELISA dosage of serum EPo,the 'C1' EEC assay allowed confirmation or elimination of the diagnosis of polycythemia vera for 91% (20/22) of polyglobulic patients. CONCLUSION: We propose that serum-free collagen-based culture systems be considered to standardize the EEC assay,now part of the new criteria of polycythemia vera.
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产品类型：
产品号#：
04850
04900
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04902
04960
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04974
产品名：
MegaCult™-C含脂培养基
MegaCult™-C培养基无细胞因子
MegaCult™-C细胞因子培养基
胶原蛋白溶液
MegaCult™-C胶原蛋白和不含细胞因子的培养基
MegaCult™-C胶原蛋白和细胞因子培养基
MegaCult™-C cfu染色试剂盒
双室载玻片试剂盒
MegaCult™-C不含细胞因子完整试剂盒
MegaCult™-C细胞因子完整试剂盒
MegaCult™-C胶原蛋白和脂质培养基
Houwerzijl EJ et al. (JAN 2004)
Blood 103 2 500--6
Ultrastructural study shows morphologic features of apoptosis and para-apoptosis in megakaryocytes from patients with idiopathic thrombocytopenic purpura.
To investigate whether altered megakaryocyte morphology contributes to reduced platelet production in idiopathic thrombocytopenic purpura (ITP),ultrastructural analysis of megakaryocytes was performed in 11 ITP patients. Ultrastructural abnormalities compatible with (para-)apoptosis were present in 78% +/- 14% of ITP megakaryocytes,which could be reversed by in vivo treatment with prednisone and intravenous immunoglobulin. Immunohistochemistry of bone marrow biopsies of ITP patients with extensive apoptosis showed an increased number of megakaryocytes with activated caspase-3 compared with normal (28% +/- 4% versus 0%). No difference,however,was observed in the number of bone marrow megakaryocyte colony-forming units (ITP,118 +/- 93/105 bone marrow cells; versus controls,128 +/- 101/105 bone marrow cells; P =.7). To demonstrate that circulating antibodies might affect megakaryocytes,suspension cultures of CD34+ cells were performed with ITP or normal plasma. Morphology compatible with (para-)apoptosis could be induced in cultured megakaryocytes with ITP plasma (2 of 10 samples positive for antiplatelet autoantibodies). Finally,the plasma glycocalicin index,a parameter of platelet and megakaryocyte destruction,was increased in ITP (57 +/- 70 versus 0.7 +/- 0.2; P =.009) and correlated with the proportion of megakaryocytes showing (para-) apoptotic ultrastructure (P =.02; r = 0.7). In conclusion,most ITP megakaryocytes show ultrastructural features of (para-) apoptosis,probably due to action of factors present in ITP plasma.
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产品类型：
产品号#：
09600
09650
04960
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04971
产品名：
StemSpan™ SFEM
StemSpan™ SFEM
MegaCult™-C胶原蛋白和不含细胞因子的培养基
胶原蛋白溶液
MegaCult™-C培养基无细胞因子
MegaCult™-C胶原蛋白和细胞因子培养基
MegaCult™-C细胞因子培养基
双室载玻片试剂盒
MegaCult™-C cfu染色试剂盒
MegaCult™-C不含细胞因子完整试剂盒
MegaCult™-C细胞因子完整试剂盒
Pirson L et al. (JUL 2006)
Stem cells (Dayton,Ohio) 24 7 1814--21
Despite inhibition of hematopoietic progenitor cell growth in vitro, the tyrosine kinase inhibitor imatinib does not impair engraftment of human CD133+ cells into NOD/SCIDbeta2mNull mice.
There is potential interest for combining allogeneic hematopoietic cell transplantation (HCT),and particularly allogeneic HCT with a nonmyeloablative regimen,to the tyrosine kinase inhibitor imatinib (Glivec; Novartis,Basel,Switzerland,http://www.novartis.com) in order to maximize anti-leukemic activity against Philadelphia chromosome-positive leukemias. However,because imatinib inhibits c-kit,the stem cell factor receptor,it could interfere with bone marrow engraftment. In this study,we examined the impact of imatinib on normal progenitor cell function. Imatinib decreased the colony-forming capacity of mobilized peripheral blood human CD133(+) cells but not that of long-term culture-initiating cells. Imatinib also decreased the proliferation of cytokine-stimulated CD133(+) cells but did not induce apoptosis of these cells. Expression of very late antigen (VLA)-4,VLA-5,and CXCR4 of CD133(+) cells was not modified by imatinib,but imatinib decreased the ability of CD133(+) cells to migrate. Finally,imatinib did not decrease engraftment of CD133(+) cells into irradiated nonobese diabetic/severe combined immunodeficient/beta2m(null) mice conditioned with 3 or 1 Gy total body irradiation. In summary,our results suggest that,despite inhibition of hematopoietic progenitor cell growth in vitro,imatinib does not interfere with hematopoietic stem cell engraftment.
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产品类型：
产品号#：
05150
04435
04445
04960
04902
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产品名：
MyeloCult™H5100
MethoCult™H4435富集
MethoCult™H4435富集
MegaCult™-C胶原蛋白和不含细胞因子的培养基
胶原蛋白溶液
MegaCult™-C培养基无细胞因子
MegaCult™-C胶原蛋白和细胞因子培养基
MegaCult™-C细胞因子培养基
双室载玻片试剂盒
MegaCult™-C cfu染色试剂盒
MegaCult™-C不含细胞因子完整试剂盒
MegaCult™-C细胞因子完整试剂盒
Tober J et al. (FEB 2007)
Blood 109 4 1433--41
The megakaryocyte lineage originates from hemangioblast precursors and is an integral component both of primitive and of definitive hematopoiesis.
In the adult,platelets are derived from unipotential megakaryocyte colony-forming cells (Meg-CFCs) that arise from bipotential megakaryocyte/erythroid progenitors (MEPs). To better define the developmental origin of the megakaryocyte lineage,several aspects of megakaryopoiesis,including progenitors,maturing megakaryocytes,and circulating platelets,were examined in the murine embryo. We found that a majority of hemangioblast precursors during early gastrulation contains megakaryocyte potential. Combining progenitor assays with immunohistochemical analysis,we identified 2 waves of MEPs in the yolk sac associated with the primitive and definitive erythroid lineages. Primitive MEPs emerge at E7.25 along with megakaryocyte and primitive erythroid progenitors,indicating that primitive hematopoiesis is bilineage in nature. Subsequently,definitive MEPs expand in the yolk sac with Meg-CFCs and definitive erythroid progenitors. The first GP1bbeta-positive cells in the conceptus were identified in the yolk sac at E9.5,while large,highly reticulated platelets were detected in the embryonic bloodstream beginning at E10.5. At this time,the number of megakaryocyte progenitors begins to decline in the yolk sac and expand in the fetal liver. We conclude that the megakaryocyte lineage initially originates from hemangioblast precursors during early gastrulation and is closely associated both with primitive and with definitive erythroid lineages in the yolk sac prior to the transition of hematopoiesis to intraembryonic sites.
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EasySep™小鼠TIL（CD45）正选试剂盒
挂图Human Hematopoietic Stem and Progenitor Cell Phenotyping Overview of subset surface markers, frequencies and assays for analysis
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