Angiopoietin-like 5 and IGFBP2 stimulate ex vivo expansion of human cord blood hematopoietic stem cells as assayed by NOD/SCID transplantation.
Hematopoietic stem cells (HSCs) are the basis of bone marrow transplantation and are attractive target cells for hematopoietic gene therapy,but these important clinical applications have been severely hampered by difficulties in ex vivo expansion of HSCs. In particular,the use of cord blood for adult transplantation is greatly limited by the number of HSCs. Previously we identified angiopoietin-like proteins and IGF-binding protein 2 (IGFBP2) as new hormones that,together with other factors,can expand mouse bone marrow HSCs in culture. Here,we measure the activity of multipotent human severe combined immunodeficient (SCID)-repopulating cells (SRCs) by transplantation into the nonobese diabetic SCID (NOD/SCID) mice; secondary transplantation was performed to evaluate the self-renewal potential of SRCs. A serum-free medium containing SCF,TPO,and FGF-1 or Flt3-L cannot significantly support expansion of the SRCs present in human cord blood CD133+ cells. Addition of either angiopoietin-like 5 or IGF-binding protein 2 to the cultures led to a sizable expansion of HSC numbers,as assayed by NOD/SCID transplantation. A serum-free culture containing SCF,TPO,FGF-1,angiopoietin-like 5,and IGFBP2 supports an approximately 20-fold net expansion of repopulating human cord blood HSCs,a number potentially applicable to several clinical processes including HSC transplantation.
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产品号#：
09600
09650
28600
产品名：
StemSpan™ SFEM
StemSpan™ SFEM
L-Calc™有限稀释软件
Lohmann F and Bieker JJ (JUN 2008)
Development (Cambridge,England) 135 12 2071--82
Activation of Eklf expression during hematopoiesis by Gata2 and Smad5 prior to erythroid commitment.
The hierarchical progression of stem and progenitor cells to their more-committed progeny is mediated through cell-to-cell signaling pathways and intracellular transcription factor activity. However,the mechanisms that govern the genetic networks underlying lineage fate decisions and differentiation programs remain poorly understood. Here we show how integration of Bmp4 signaling and Gata factor activity controls the progression of hematopoiesis,as exemplified by the regulation of Eklf during establishment of the erythroid lineage. Utilizing transgenic reporter assays in differentiating mouse embryonic stem cells as well as in the murine fetal liver,we demonstrate that Eklf expression is initiated prior to erythroid commitment during hematopoiesis. Applying phylogenetic footprinting and in vivo binding studies in combination with newly developed loss-of-function technology in embryoid bodies,we find that Gata2 and Smad5 cooperate to induce Eklf in a progenitor population,followed by a switch to Gata1-controlled regulation of Eklf transcription upon erythroid commitment. This stage- and lineage-dependent control of Eklf expression defines a novel role for Eklf as a regulator of lineage fate decisions during hematopoiesis.
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产品号#：
03234
产品名：
MethoCult™ M3234
Zhou L et al. (OCT 2008)
Blood 112 8 3434--43
Inhibition of the TGF-beta receptor I kinase promotes hematopoiesis in MDS.
MDS is characterized by ineffective hematopoiesis that leads to peripheral cytopenias. Development of effective treatments has been impeded by limited insight into pathogenic pathways governing dysplastic growth of hematopoietic progenitors. We demonstrate that smad2,a downstream mediator of transforming growth factor-beta (TGF-beta) receptor I kinase (TBRI) activation,is constitutively activated in MDS bone marrow (BM) precursors and is overexpressed in gene expression profiles of MDS CD34(+) cells,providing direct evidence of overactivation of TGF-beta pathway in this disease. Suppression of the TGF-beta signaling by lentiviral shRNA-mediated down-regulation of TBRI leads to in vitro enhancement of hematopoiesis in MDS progenitors. Pharmacologic inhibition of TBRI (alk5) kinase by a small molecule inhibitor,SD-208,inhibits smad2 activation in hematopoietic progenitors,suppresses TGF-beta-mediated gene activation in BM stromal cells,and reverses TGF-beta-mediated cell-cycle arrest in BM CD34(+) cells. Furthermore,SD-208 treatment alleviates anemia and stimulates hematopoiesis in vivo in a novel murine model of bone marrow failure generated by constitutive hepatic expression of TGF-beta1. Moreover,in vitro pharmacologic inhibition of TBRI kinase leads to enhancement of hematopoiesis in varied morphologic MDS subtypes. These data directly implicate TGF-beta signaling in the pathobiology of ineffective hematopoiesis and identify TBRI as a potential therapeutic target in low-risk MDS.
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产品号#：
03534
03436
84434
84444
产品名：
MethoCult™ GF M3534
MethoCult™ SF M3436
Lu S-J et al. (SEP 2008)
Regenerative medicine 3 5 693--704
Robust generation of hemangioblastic progenitors from human embryonic stem cells.
BACKGROUND: Human embryonic stem cells (hESCs) are a potentially inexhaustible source of cells for replacement therapy. However,successful preclinical and clinical progress requires efficient and controlled differentiation towards the specific differentiated cell fate. METHODS: We previously developed a strategy to generate blast cells (BCs) from hESCs that were capable of differentiating into vascular structures as well as into all hematopoietic cell lineages. Although the BCs were shown to repair damaged vasculature in multiple animal models,the large-scale generation of cells under these conditions was challenging. Here we report a simpler and more efficient method for robust generation of hemangioblastic progenitors. RESULTS: In addition to eliminating several expensive factors that are unnecessary,we demonstrate that bone morphogenetic protein (BMP)-4 and VEGF are necessary and sufficient to induce hemangioblastic commitment and development from hESCs during early stages of differentiation. BMP-4 and VEGF significantly upregulate T-brachyury,KDR,CD31 and Lmo2 gene expression,while dramatically downregulating Oct-4 expression. The addition of basic FGF during growth and expansion was found to further enhance BC development,consistently generating approximately 1 x 10(8) BCs from one six well plate of hESCs. CONCLUSION: This new method represents a significantly improved system for generating hemangioblasts from hESCs,and although simplified,results in an eightfold increase in cell yield.
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产品号#：
05850
05857
05870
05875
85850
85857
85870
85875
产品名：
mTeSR™1
mTeSR™1
Donahue RE et al. (JAN 2000)
Blood 95 2 445--52
High levels of lymphoid expression of enhanced green fluorescent protein in nonhuman primates transplanted with cytokine-mobilized peripheral blood CD34(+) cells.
We have used a murine retrovirus vector containing an enhanced green fluorescent protein complimentary DNA (EGFP cDNA) to dynamically follow vector-expressing cells in the peripheral blood (PB) of transplanted rhesus macaques. Cytokine mobilized CD34(+) cells were transduced with an amphotropic vector that expressed EGFP and a dihydrofolate reductase cDNA under control of the murine stem cell virus promoter. The transduction protocol used the CH-296 recombinant human fibronectin fragment and relatively high concentrations of the flt-3 ligand and stem cell factor. Following transplantation of the transduced cells,up to 55% EGFP-expressing granulocytes were obtained in the peripheral circulation during the early posttransplant period. This level of myeloid marking,however,decreased to 0.1% or lower within 2 weeks. In contrast,EGFP expression in PB lymphocytes rose from 2%-5% shortly following transplantation to 10% or greater by week 5. After 10 weeks,the level of expression in PB lymphocytes continued to remain at 3%-5% as measured by both flow cytometry and Southern blot analysis,and EGFP expression was observed in CD4(+),CD8(+),CD20(+),and CD16/56(+) lymphocyte subsets. EGFP expression was only transiently detected in red blood cells and platelets soon after transplantation. Such sustained levels of lymphocyte marking may be therapeutic in a number of human gene therapy applications that require targeting of the lymphoid compartment. The transient appearance of EGFP(+) myeloid cells suggests that transduction of a lineage-restricted myeloid progenitor capable of short-term engraftment was obtained with this protocol. (Blood. 2000;95:445-452)
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产品号#：
04436
04064
04100
04230
04236
04431
04434
04444
04464
04531
04535
04545
04536
04564
04035
04330
04034
04044
04435
04445
04534
04544
产品名：
MethoCult™ SF H4436
MethoCult™ H4034 Optimum 入门试剂盒
MethoCult™ H4100
MethoCult™ H4230
MethoCult™ SF H4236
MethoCult™ H4431
MethoCult™ H4434 Classic
MethoCult™ H4434 Classic
MethoCult™ H4434 Classic 套装
MethoCult™ H4531
MethoCult™ H4535 Enriched，不含EPO
MethoCult™ H4535 Enriched，不含EPO
MethoCult™ SF H4536
MethoCult™ H4534 Classic 无 EPO 入门试剂盒
MethoCult™ 不含EPO的H4035 Optimum
MethoCult™ H4330
MethoCult™ H4034 Optimum
MethoCult™ H4034 Optimum
MethoCult™ H4435 Enriched
MethoCult™ H4435 Enriched
MethoCult™ H4534 Classic（不含 EPO）
MethoCult™ H4534 Classic（不含 EPO）
Corti S et al. (OCT 2008)
The Journal of clinical investigation 118 10 3316--30
Neural stem cell transplantation can ameliorate the phenotype of a mouse model of spinal muscular atrophy.
Spinal muscular atrophy (SMA),a motor neuron disease (MND) and one of the most common genetic causes of infant mortality,currently has no cure. Patients with SMA exhibit muscle weakness and hypotonia. Stem cell transplantation is a potential therapeutic strategy for SMA and other MNDs. In this study,we isolated spinal cord neural stem cells (NSCs) from mice expressing green fluorescent protein only in motor neurons and assessed their therapeutic effects on the phenotype of SMA mice. Intrathecally grafted NSCs migrated into the parenchyma and generated a small proportion of motor neurons. Treated SMA mice exhibited improved neuromuscular function,increased life span,and improved motor unit pathology. Global gene expression analysis of laser-capture-microdissected motor neurons from treated mice showed that the major effect of NSC transplantation was modification of the SMA phenotype toward the wild-type pattern,including changes in RNA metabolism proteins,cell cycle proteins,and actin-binding proteins. NSC transplantation positively affected the SMA disease phenotype,indicating that transplantation of NSCs may be a possible treatment for SMA.
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产品号#：
01700
01705
01701
01702
产品名：
ALDEFLUOR™ 试剂盒
ALDEFLUOR™ DEAB试剂, 1.5 mM, 1 mL
ALDEFLUOR™检测缓冲液
Stern P et al. (SEP 2008)
Proceedings of the National Academy of Sciences of the United States of America 105 37 13895--900
A system for Cre-regulated RNA interference in vivo.
We report a system for Cre-regulated expression of RNA interference in vivo. Expression cassettes comprise selectable and FACS-sortable markers in tandem with additional marker genes and shRNAs in the antisense orientation. The cassettes are flanked by tandem LoxP sites arranged so that Cre expression inverts the marker-shRNA construct,allowing its regulated expression (and,at the same time,deletes the original selection/marker genes). The cassettes can be incorporated into retroviral or lentiviral vectors and delivered to cells in culture or used to generate transgenic mice. We describe cassettes incorporating various combinations of reporter genes,miRNA-based RNAi (including two shRNA constructs at once),and oncogenes and demonstrate the delivery of effective RNA interference in cells in culture,efficient transduction into hematopoietic stem cells with cell-type-specific knockdown in their progeny,and rapid generation of regulated shRNA knockdown in transgenic mice. These vector systems allow regulated combinatorial manipulation (both overexpression and loss of function) of gene expression in multiple systems in vitro and in vivo.
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产品号#：
09600
09650
产品名：
StemSpan™ SFEM
StemSpan™ SFEM
Madonna R and De Caterina R (NOV 2008)
American journal of physiology. Cell physiology 295 5 C1271--80
In vitro neovasculogenic potential of resident adipose tissue precursors.
Adipose tissue development is associated with neovascularization,which might be exploited therapeutically. We investigated the neovasculogenesis antigenic profile and kinetics in adipose tissue-derived stromal cells (ADSCs) to understand the potential of ADSCs to generate new vessels. Murine and human visceral adipose tissues were processed with collagenase to obtain ADSCs from the stromal vascular fraction. Freshly isolated murine and human ADSCs featured the expression of early markers of endothelial differentiation [uptake of DiI-labeled acetylated LDL,CD133,CD34,kinase insert domain receptor (KDR)],but not markers for more mature endothelial cells (CD31 and von Willebrand factor). In methylcellulose medium,multilocular cells positive for Oil Red O staining appeared after 6 days. After 10 days,clusters of ADSCs spontaneously formed branched tubelike structures,which were strongly positive for CD34 and CD31,while losing their ability to undergo adipocyte differentiation. In Matrigel,in the presence of endothelial growth factors ADSCs formed branched tubelike structures. By clonal assays in methylcellulose we also determined the frequency of granulocyte-macrophage (CFU-GM) and erythroid (BFU-E) colony-forming units from ADSCs,compared with bone marrow-derived stromal cells (BMSCs) used as a positive control. After 4-14 days,BMSCs formed 8 +/- 3 BFU-E and 40 +/- 10 CFU-GM,while ADSCs never produced colonies of myeloid progenitors. The developing adipose tissue has neovasculogenic potential,based on the recruitment of local rather than circulating progenitors. Adipose tissue might therefore be a viable autonomous source of cells for postnatal neovascularization.
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产品号#：
03534
产品名：
MethoCult™ GF M3534
Pimanda JE et al. (DEC 2008)
Blood 112 12 4512--22
Endoglin expression in blood and endothelium is differentially regulated by modular assembly of the Ets/Gata hemangioblast code.
Endoglin is an accessory receptor for TGF-beta signaling and is required for normal hemangioblast,early hematopoietic,and vascular development. We have previously shown that an upstream enhancer,Eng -8,together with the promoter region,mediates robust endothelial expression yet is inactive in blood. To identify hematopoietic regulatory elements,we used array-based methods to determine chromatin accessibility across the entire locus. Subsequent transgenic analysis of candidate elements showed that an endothelial enhancer at Eng +9 when combined with an element at Eng +7 functions as a strong hemato-endothelial enhancer. Chromatin immunoprecipitation (ChIP)-chip analysis demonstrated specific binding of Ets factors to the promoter as well as to the -8,+7+9 enhancers in both blood and endothelial cells. By contrast Pu.1,an Ets factor specific to the blood lineage,and Gata2 binding was only detected in blood. Gata2 was bound only at +7 and GATA motifs were required for hematopoietic activity. This modular assembly of regulators gives blood and endothelial cells the regulatory freedom to independently fine-tune gene expression and emphasizes the role of regulatory divergence in driving functional divergence.
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产品号#：
03434
03444
产品名：
MethoCult™ GF M3434
MethoCult™ GF M3434
Orelio C et al. (DEC 2008)
Blood 112 13 4895--904
Interleukin-1-mediated hematopoietic cell regulation in the aorta-gonad-mesonephros region of the mouse embryo.
Hematopoiesis during development is a dynamic process,with many factors involved in the emergence and regulation of hematopoietic stem cells (HSCs) and progenitor cells. Whereas previous studies have focused on developmental signaling and transcription factors in embryonic hematopoiesis,the role of well-known adult hematopoietic cytokines in the embryonic hematopoietic system has been largely unexplored. The cytokine interleukin-1 (IL-1),best known for its proinflammatory properties,has radioprotective effects on adult bone marrow HSCs,induces HSC mobilization,and increases HSC proliferation and/or differentiation. Here we examine IL-1 and its possible role in regulating hematopoiesis in the midgestation mouse embryo. We show that IL-1,IL-1 receptors (IL-1Rs),and signaling mediators are expressed in the aorta-gonad-mesonephros (AGM) region during the time when HSCs emerge in this site. IL-1 signaling is functional in the AGM,and the IL-1RI is expressed ventrally in the aortic subregion by some hematopoietic,endothelial,and mesenchymal cells. In vivo analyses of IL-1RI-deficient embryos show an increased myeloid differentiation,concomitant with a slight decrease in AGM HSC activity. Our results suggest that IL-1 is an important homeostatic regulator at the earliest time of HSC development,acting to limit the differentiation of some HSCs along the myeloid lineage.
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产品号#：
03434
03444
产品名：
MethoCult™ GF M3434
MethoCult™ GF M3434
Zhao H et al. (JAN 2009)
Blood 113 3 505--16
The c-myb proto-oncogene and microRNA-15a comprise an active autoregulatory feedback loop in human hematopoietic cells.
The c-myb proto-oncogene encodes an obligate hematopoietic cell transcription factor important for lineage commitment,proliferation,and differentiation. Given its critical functions,c-Myb regulatory factors are of great interest but remain incompletely defined. Herein we show that c-Myb expression is subject to posttranscriptional regulation by microRNA (miRNA)-15a. Using a luciferase reporter assay,we found that miR-15a directly binds the 3'-UTR of c-myb mRNA. By transfecting K562 myeloid leukemia cells with a miR-15a mimic,functionality of binding was shown. The mimic decreased c-Myb expression,and blocked the cells in the G(1) phase of cell cycle. Exogenous expression of c-myb mRNA lacking the 3'-UTR partially rescued the miR-15a induced cell-cycle block. Of interest,the miR-15a promoter contained several potential c-Myb protein binding sites. Occupancy of one canonical c-Myb binding site was demonstrated by chromatin immunoprecipitation analysis and shown to be required for miR-15a expression in K562 cells. Finally,in studies using normal human CD34(+) cells,we showed that c-Myb and miR-15a expression were inversely correlated in cells undergoing erythroid differentiation,and that overexpression of miR-15a blocked both erythroid and myeloid colony formation in vitro. In aggregate,these findings suggest the presence of a c-Myb-miR-15a autoregulatory feedback loop of potential importance in human hematopoiesis.
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产品号#：
09500
产品名：
BIT 9500血清替代物
Singh KP et al. (JAN 2009)
Carcinogenesis 30 1 11--9
Treatment of mice with the Ah receptor agonist and human carcinogen dioxin results in altered numbers and function of hematopoietic stem cells.
The aryl hydrocarbon receptor (AhR) mediates the carcinogenicity of a family of environmental contaminants,the most potent being 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Increased incidence of lymphoma and leukemia in humans is associated with TCDD exposure. Although AhR activation by TCDD has profound effects on the immune system,precise cellular and molecular mechanisms have yet to be determined. These studies tested the hypothesis that alteration of marrow populations following treatment of mice with TCDD is due to an effect on hematopoietic stem cells (HSCs). Treatment with TCDD resulted in an increased number and proliferation of bone marrow (BM) populations enriched for HSCs. There was a time-dependent decrease in B-lineage cells with a concomitant increase in myeloid populations. The decrease in the B-cell lineage colony-forming unit-preB progenitors along with a transient increase in myeloid progenitors were consistent with a skewing of lineage development from lymphoid to myeloid populations. However,HSCs from TCDD-treated mice exhibited diminished capacity to reconstitute and home to marrow of irradiated recipients. AhR messenger RNA was expressed in progenitor subsets but is downregulated during HSC proliferation. This result was consistent with the lack of response following the exposure of 5-fluorouracil-treated mice to TCDD. The direct exposure of cultured BM cells to TCDD inhibited the growth of immature hematopoietic progenitor cells,but not more mature lineage-restricted progenitors. Overall,these data are consistent with the hypothesis that TCDD,through AhR activation,alters the ability of HSCs to respond appropriately to signals within the marrow microenvironment.
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