搜索结果: 'methocult media formulations for mouse hematopoietic cells serum containing'

Biochemical and biophysical stimuli of stem cell niches finely regulate the self-renewal/differentiation equilibrium. Replicating this in vitro is technically challenging,making the control of stem cell functions difficult. Cell derived matrices capture certain aspect of niches that influence fate decisions. Here,aligned fibrous matrices synthesized by MC3T3 cells were produced and the role of matrix orientation and stiffness on the maintenance of stem cell characteristics and adipo- or osteo-genic differentiation of murine mesenchymal stem cells (mMSCs) was investigated. Decellularized matrices promoted mMSC proliferation. Fibrillar alignment and matrix stiffness work in concert in defining cell fate. Soft matrices preserve stemness,whereas stiff ones,in presence of biochemical supplements,promptly induce differentiation. Matrix alignment impacts the homogeneity of the cell population,that is,soft aligned matrices ameliorate the spontaneous adipogenic differentiation,whereas stiff aligned matrices reduce cross-differentiation. We infer that mechanical signaling is a dominant factor in mMSC fate decision and the matrix alignment contributes to produce a more homogeneous environment,which results in a uniform response of cells to biophysical environment. Matrix thus produced can be obtained in vitro in a facile and consistent manner and can be used for homogeneous stem cell amplification or for mechanotransduction-related studies. View Publication

Canine leukocyte adhesion deficiency (CLAD) represents the canine counter-part of the human disease leukocyte adhesion deficiency (LAD). Defects in the leukocyte integrin CD18 adhesion molecule in both CLAD and LAD lead to recurrent,life-threatening bacterial infections. We evaluated ex vivo retroviral-mediated gene therapy in CLAD using 2 nonmyeloablative conditioning regimens--200 cGy total body irradiation (TBI) or 10 mg/kg busulfan--with or without posttransplantation immunosuppression. In 6 of 11 treated CLAD dogs,therapeutic levels of CD18(+) leukocytes were achieved. Conditioning with either TBI or busulfan allowed long-term engraftment,and immunosuppression was not required for efficacy. The percentage of CD18(+) leukocytes in the peripheral blood progressively increased over 6 to 8 months after infusion to levels ranging from 1.26% to 8.37% at 1-year follow-up in the 6 dogs. These levels resulted in reversal or moderation of the severe CLAD phenotype. Linear amplification-mediated polymerase chain reaction assays indicated polyclonality of insertion sites. These results describe ex vivo hematopoietic stem cell gene transfer in a disease-specific,large animal model using 2 clinically applicable conditioning regimens,and they provide support for the use of nonmyeloablative conditioning regimens in preclinical protocols of retroviral-mediated gene transfer for nonmalignant hematopoietic diseases such as LAD. View Publication

Human embryonic stem cells (hESCs) can be differentiated into multiple cell types with great therapeutic potential. However,optimizing the often multi-week cultures to obtain sufficient differentiated cell yields has been in part limited by the high variability of even parallel hESC differentiation cultures. We describe the isolation and features of a subline of CA1 hESCs (CA1S) that display a very high 25% cloning efficiency while retaining many properties of the parental hESCs,including being karyotypically normal and their ability to generate teratomas containing all three germ layers. Although more detailed analysis revealed that CA1S cells have a 3.8 Mb genomic duplication on chromosome 20,they remain highly useful. In particular,CA1S cells are readily expanded at high yields in culture and possess greatly reduced well-to-well variation even when seeded at 100 cells/well. Thus,108 CA1S cells can be generated within one week from 106 cells to seed 106 wells. We determined that CA1S cells have the capacity to follow established in vitro differentiation protocols to pancreatic progenitors and subsequent hormone-positive cell types and used CA1S cells to explore definitive endoderm induction in a high performance screen (Z-factor = 0.97). This system revealed that CA1S cells do not require WNT3A to efficiently form definitive endoderm,a finding that was confirmed with H1 hESCs,although H1 cells did show modest benefits of high WNT3A doses. Proliferative index measurements of CA1S cells were shown to rapidly reflect their differentiation status in a high throughput system. Though results obtained with CA1S cells will need to be confirmed using conventional hESC lines,these cells should ease the development of optimized hESC growth and differentiation protocols. In particular,they should limit the more arduous secondary screens using hESCs to a smaller number of variables and doses. Biotechnol. Bioeng. 2013;110: 2706–2716. textcopyright 2013 Wiley Periodicals,Inc. View Publication

A collaborative study of the second international Reference Preparation of Erythropoietin,Human,Urinary,for Bioassay was carried out in 10 laboratories. Combined potency estimates obtained by comparison with the International Reference Preparation,indicate that ampoules of the second Preparation contain 10.0 IU (weighted mean potency) or,taking the unweighted mean potency,9.8 IU,with fiducial limits (P=0.95) of 8.4-11.5 IU. The second Preparation could be used as a standard in estimating the potency of a preparation of sheep plasma erythropoietin (68/307) although,as with the International Reference Preparation,there was a tendency for the sheep plasma preparation to produce log-dose-log-response regression lines that were steeper than those produced by the second Preparation.In accelerated degradation studies of the second Preparation stored as the dry product in ampoules for up to 1 year,there was no consistent trend to indicate instability of the preparation.Following its establishment in 1971,the Second International Reference Preparation was allocated a potency of 10 IU/ampoule. View Publication

With their unique ability to differentiate into all cell types,embryonic stem (ES) cells hold great therapeutic promise. To improve the efficiency of embryoid body (EB)-mediated ES cell differentiation,we studied murine EBs on the basis of their size and found that EBs with an intermediate size (diameter 100-300 microm) are the most proliferative,hold the greatest differentiation potential,and have the lowest rate of cell death. In an attempt to promote the formation of this subpopulation,we surveyed several biocompatible substrates with different surface chemical parameters and identified a strong correlation between hydrophobicity and EB development. Using self-assembled monolayers of various lengths of alkanethiolates on gold substrates,we directly tested this correlation and found that surfaces that exhibit increasing hydrophobicity enrich for the intermediate-size EBs. When this approach was applied to the human ES cell system,similar phenomena were observed. Our data demonstrate that hydrophobic surfaces serve as a platform to deliver uniform EB populations and may significantly improve the efficiency of ES cell differentiation. View Publication

Reported here is a piggyBac transposon-based expression system for the generation of doxycycline-inducible,stably transfected mammalian cell cultures for large-scale protein production. The system works with commonly used adherent and suspension-adapted mammalian cell lines and requires only a single transfection step. Moreover,the high uniform expression levels observed among clones allow for the use of stable bulk cell cultures,thereby eliminating time-consuming cloning steps. Under continuous doxycycline induction,protein expression levels have been shown to be stable for at least 2 mo in the absence of drug selection. The high efficiency of the system also allows for the generation of stable bulk cell cultures in 96-well format,a capability leading to the possibility of generating stable cell cultures for entire families of membrane or secreted proteins. Finally,we demonstrate the utility of the system through the large-scale production (140-750 mg scale) of an endoplasmic reticulum-resident fucosyltransferase and two potential anticancer protein therapeutic agents. View Publication

Cited2 (cAMP-responsive elementbinding protein [CBP]/p300-interacting transactivators with glutamic acid [E] and aspartic acid [D]-rich tail 2) is a newly identified transcriptional modulator. Knockout of the Cited2 gene results in embryonic lethality with embryos manifesting heart and neural tube defects. Cited2-/- fetal liver displayed significant reduction in the numbers of Lin(-)c-Kit+Sca-1+ cells,Lin(-)c-Kit+ cells,and progenitor cells of different lineages. Fetal liver cells from Cited2-/- embryos gave rise to markedly reduced number of colonies in the colony-forming unit assay. Primary and secondary transplantation studies showed significantly compromised reconstitution of T-lymphoid,B-lymphoid,and myeloid lineages in mice that received a transplant of Cited2-/- fetal liver cells. Competitive reconstitution experiments further showed that fetal liver hematopoietic stem cell (HSC) function is severely impaired due to Cited2 deficiency. Microarray analysis showed decreased expression of Wnt5a and a panel of myeloid molecular markers such as PRTN3,MPO,Neutrophil elastase,Cathepsin G,and Eosinophil peroxidase in Cited2-/- fetal livers. Decreased expression of Bmi-1,Notch1,LEF-1,Mcl-1,and GATA2 was also observed in Cited2-/- Lin(-)c-Kit+ cells. The present study uncovers for the first time a novel role of Cited2 in the maintenance of hematopoietic homeostasis during embryogenesis and thus provides new insights into the molecular regulation of hematopoietic development. View Publication