技术资料

The adult rat hippocampus contains fibroblast growth factor 2-responsive stem cells that are self-renewing and have the ability to generate both neurons and glia in vitro,but little is known about the molecular events that regulate stem cell differentiation. Hippocampus-derived stem cell clones were used to examine the effects of retinoic acid (RA) on neuronal differentiation. Exposure to RA caused an immediate up-regulation of NeuroD,increased p21 expression,and concurrent exit from cell cycle. These changes were accompanied by a threefold increase in the number of cells differentiating into immature neurons. An accompanying effect of RA was to sustain or up-regulate trkA,trkB,trkC,and p75NGFR expression. Without RA treatment,cells were minimally responsive to neurotrophins (NTs),whereas the sequential application of RA followed by brain-derived neurotrophic factor or NT-3 led to a significant increase in neurons displaying mature y-a-minobutyric acid,acetylcholinesterase,tyrosine hydroxylase,or calbindin phenotypes. Although NTs promoted maturation,they had little effect on the total number of neurons generated,suggesting that RA and neurotrophins acted at distinct stages in neurogenesis. RA first promoted the acquisition of a neuronal fate,and NTs subsequently enhanced maturation by way of RA-dependent expression of the Trk receptors. In combination,these sequential effects were sufficient to stimulate stem cell-derived progenitors to differentiate into neurons displaying a variety of transmitter phenotypes. View Publication

Teratoma formation is a critical obstacle to safe clinical translation of human embryonic stem (ES) cell-based therapies in the future. As current methods of isolation are unable to yield 100% pure population of differentiated cells from a pluripotent donor source,potential development of these tumors is a significant concern. Here we used non-invasive reporter gene imaging to investigate the relationship between human ES cell number and teratoma formation in a xenogenic model of ES cell transplantation. Human ES cells (H9 line) were stably transduced with a double fusion (DF) reporter construct containing firefly luciferase and enhanced green fluorescent protein (Fluc- eGFP) driven by a human ubiquitin promoter. Immunodeficient mice received intramyocardial (n = 35) or skeletal muscle (n = 35) injection of 1 × 102,1 × 103,1 × 104,1 × 105 or 1 × 106 DF positive ES cells suspended in saline for myocardium and Matrigel for skeletal muscle. Cell survival and proliferation were monitored via bioluminescence imaging (BLI) for an 8 week period following transplantation. Mice negative for Fluc signal after 8 weeks were followed out to day 365 to confirm tumor absence. Significantly,in this study,a minimum of 1 × 105 ES cells in the myocardium and 1 × 104 cells in the skeletal muscle was observed to be requisite for teratoma development,suggesting that human ES cell number may be a critical factor in teratoma formation. Engraftment and tumor occurrence were also observed to be highly dependent on ES cell number. We anticipate these results should yield useful insights to the safe and reliable application of human ES cell derivatives in the clinic. Keywords View Publication