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

Hematopoietic stem cells (HSCs) have the capacity to self-renew and the potential to differentiate into all of the mature blood cell types. The ability to prospectively identify and isolate HSCs has been the subject of extensive investigation since the first transplantation studies implying their existence almost 50 years ago. Despite significant advances in enrichment protocols,the continuous in vitro propagation of human HSCs has not yet been achieved. This chapter describes current procedures used to phenotypically and functionally characterize candidate human HSCs and initial efforts to derive permanent human HSC lines. View Publication

NUP98-HOXA9,the chimeric protein resulting from the t(7;11)(p15;p15) chromosomal translocation,is a prototype of several NUP98 fusions that occur in myelodysplastic syndromes and acute myeloid leukemia. We examined its effect on differentiation,proliferation,and gene expression in primary human CD34+ hematopoietic cells. Colony-forming cell (CFC) assays in semisolid medium combined with morphologic examination and flow cytometric immunophenotyping revealed that NUP98-HOXA9 increased the numbers of erythroid precursors and impaired both myeloid and erythroid differentiation. In continuous liquid culture,cells transduced with NUP98-HOXA9 exhibited a biphasic growth curve with initial growth inhibition followed by enhanced long-term proliferation,suggesting an increase in the numbers of primitive self-renewing cells. This was confirmed by a dramatic increase in the numbers of long-term culture-initiating cells,the most primitive hematopoietic cells detectable in vitro. To understand the molecular mechanisms underlying the effects of NUP98-HOXA9 on hematopoietic cell proliferation and differentiation,oligonucleotide microarray analysis was done at several time points over 16 days,starting at 6 hours posttransduction. The early growth suppression was preceded by up-regulation of IFNbeta1 and accompanied by marked up-regulation of IFN-induced genes,peaking at 3 days posttransduction. In contrast,oncogenes such as homeobox transcription factors,FLT3,KIT,and WT1 peaked at 8 days or beyond,coinciding with increased proliferation. In addition,several putative tumor suppressors and genes associated with hematopoietic differentiation were repressed at later time points. These findings provide a comprehensive picture of the changes in proliferation,differentiation,and global gene expression that underlie the leukemic transformation of human hematopoietic cells by NUP98-HOXA9. View Publication

Relative quiescence is a defining characteristic of hematopoietic stem cells. Reasoning that inhibitory tone dominates control of stem cell cycling,we previously showed that mice engineered to be deficient in the cyclin-dependent kinase inhibitor,p21Cip1/Waf1 (p21),have an increased stem cell pool under homeostatic conditions. Since p21 was necessary to maintain stem cell quiescence and its absence sufficient to permit increased murine stem cell cycling,we tested whether reduction of p21 alone in human adult-derived stem cells could affect stem cell proliferation. We demonstrate here that interrupting p21 expression ex vivo resulted in expanded stem cell number and in vivo stem cell function compared with control,manipulated cells. Further,we demonstrate full multilineage reconstitution capability in cells where p21 expression was knocked down. Therefore,lifting the brake on cell proliferation by altering cell cycle checkpoints provides an alternative paradigm for increasing hematopoietic stem cell numbers. This approach may be useful for relative ex vivo human stem cell expansion. View Publication