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

Retroviral vectors constitute the most efficient system to deliver and integrate foreign genes into mammalian cells. We have developed a producer cell line that yields high titers of amphotropic retroviral vectors carrying the enhanced green fluorescent protein (EGFP) gene,a codon humanized,red-shifted variant of the green fluorescent protein (GFP) gene,which can be used as a selectable marker. We have used a hybrid vector that has been shown to efficiently drive gene expression in hematopoietic cells. Virtually all murine and human cell lines and primary human hematopoietic cells tested were transduced with varying efficiency after incubation with vector-containing supernatants. Human CD34(+) cells obtained from cord blood or aphereses products were transduced using a protocol that involves daily addition of vector-containing supernatants for 6 consecutive days. At day 6,up to 16% of the cells expressed EGFP,as assessed by flow cytometry. Sorted EGFP-expressing cells were able to produce fluorescent hematopoietic colonies. EGFP's main advantages are its fast flow cytometry determination and the possibility of cell sorting and simultaneous evaluation of the transduction efficiency along with other phenotypic markers. View Publication

BACKGROUND Human embryonic stem cells (hESCs) have been derived and maintained on mouse embryonic fibroblast feeders to keep their undifferentiated status. To realize their clinical potential,a feeder-free and scalable system for large scale production of hESCs and their differentiated derivatives is required. MATERIALS & METHODS hESCs were cultured and passaged on serum/feeder-free 3D microcarriers for five passages. For embryoid body (EB) formation and hemangioblast differentiation,the medium for 3D microcarriers was directly switched to EB medium. RESULTS hESCs on 3D microcarriers maintained pluripotency and formed EBs,which were ten-times more efficient than hESCs cultured under 2D feeder-free conditions (0.11 ± 0.03 EB cells/hESC input 2D vs 1.19 ± 0.32 EB cells/hESC input 3D). After replating,EB cells from 3D culture readily developed into hemangioblasts with the potential to differentiate into hematopoietic and endothelial cells. Furthermore,this 3D system can also be adapted to human induced pluripotent stem cells,which generate functional hemangioblasts with high efficiency. CONCLUSION This 3D serum- and stromal-free microcarrier system is important for future clinical applications,with the potential of developing to a GMP-compatible scalable system. View Publication

Thpo/Mpl signaling plays an important role in the maintenance of hematopoietic stem cells (HSCs) in addition to its role in megakaryopoiesis. Patients with inactivating mutations in Mpl develop thrombocytopenia and aplastic anemia because of progressive loss of HSCs. Yet,it is unknown whether this loss of HSCs is an irreversible process. In this study,we used the Mpl knockout (Mpl(-/-)) mouse model and expressed Mpl from newly developed lentiviral vectors specifically in the physiologic Mpl target populations,namely,HSCs and megakaryocytes. After validating lineage-specific expression in vivo using lentiviral eGFP reporter vectors,we performed bone marrow transplantation of transduced Mpl(-/-) bone marrow cells into Mpl(-/-) mice. We show that restoration of Mpl expression from transcriptionally targeted vectors prevents lethal adverse reactions of ectopic Mpl expression,replenishes the HSC pool,restores stem cell properties,and corrects platelet production. In some mice,megakaryocyte counts were atypically high,accompanied by bone neo-formation and marrow fibrosis. Gene-corrected Mpl(-/-) cells had increased long-term repopulating potential,with a marked increase in lineage(-)Sca1(+)cKit(+) cells and early progenitor populations in reconstituted mice. Transcriptome analysis of lineage(-)Sca1(+)cKit(+) cells in Mpl-corrected mice showed functional adjustment of genes involved in HSC self-renewal. View Publication