Background aims
Dendritic cells (DC) are increasingly being used as cellular vaccines to treat cancer and infectious diseases. While there have been some promising results in early clinical trials using DC-based vaccines, the inability to visualize non-invasively the location, migration and fate of cells once adoptively transferred into patients is often cited as a limiting factor in the advancement of these therapies. A novel perflouropolyether (PFPE) tracer agent was used to label human DC ex vivo for the purpose of tracking the cells in vivo by 19F magnetic resonance imaging (MRI). We provide an assessment of this technology and examine its impact on the health and function of the DC.
Methods
Monocyte-derived DC were labeled with PFPE and then assessed. Cell viability was determined by examining cell membrane integrity and mitochondrial lipid content. Immunostaining and flow cytometry were used to measure surface antigen expression of DC maturation markers. Functional tests included bioassays for interleukin (IL)-12p70 production, T-cell stimulatory function and chemotaxis. MRI efficacy was demonstrated by inoculation of PFPE-labeled human DC into NOD-SCID mice.
Results
DC were effectively labeled with PFPE without significant impact on cell viability, phenotype or function. The PFPE-labeled DC were clearly detected in vivo by 19F MRI, with mature DC being shown to migrate selectively towards draining lymph node regions within 18 h.
Conclusions
This study is the first application of PFPE cell labeling and MRI cell tracking using human immunotherapeutic cells. These techniques may have significant potential for tracking therapeutic cells in future clinical trials.
Excitatory amino acids such as glutamate play important roles in the central nervous system. We previously demonstrated that a neurosteroid, dehydroepiandrosterone (DHEA), has powerful effects on the cell proliferation of human neural progenitor cells (hNPC) derived from the fetal cortex, and this effect is modulated through NMDA receptor signaling. Here, we show that glutamate can significantly increase the proliferation rates of hNPC. The increased proliferation could be blocked by specific NMDA receptor antagonists, but not other glutamate antagonists for kainate-AMPA or metabotropic receptors. The NR1 subunit of the NMDA receptor was detectable in elongated bipolar or unipolar cells with small cell bodies. These NR1-positive cells were colocalized with GFAP immunoreactivity. Detection of the phosphorylation of cAMP response element-binding protein (pCREB) revealed that a subset of NR1-positive hNPC could respond to glutamate. Furthermore, we hypothesized that glutamate treatment may affect mainly the hNPC with a radial morphology and found that glutamate as well as DHEA selectively affected elongated hNPC; these elongated cells may be a type of radial glial cell. Finally we asked whether the glutamate-responsive hNPC had an increased potential for neurogenesis and found that glutamate-treated hNPC produced significantly more neurons following differentiation. Together these data suggest that glutamate stimulates the division of human progenitor cells with neurogenic potential.
Human neural progenitor cells (hNPC) isolated from the fetal cortex can be expanded as aggregates of cells termed neurospheres. Traditional methods have used 20 ng/ml epidermal growth factor (EGF) to drive the proliferation of these cells. Here, we show that 100 ng/ml EGF can significantly increase growth rates of hNPC at later passages. This was through increased survival of dividing cells rather than increased proliferation and associated with prolonged activation of ErbB2 and phosphorylated Akt. High EGF also resulted in a larger proportion of elongated "radial glial"-like cells within the growing neurospheres and increased expression of the radial glial markers. The number of new neurons generated from cultures maintained in 100 ng/ml EGF was significantly higher than from 20 ng/ml EGF. Thus, high concentrations of EGF increase the survival of a highly neurogenic human radial glial cell. STEM CELLS 2008;26:348 -355 Disclosure of potential conflicts of interest is found at the end of this article.
The spatiotemporal birth order of the seven major classes of retinal cells is highly conserved among vertebrates. During retinal development, long projection neurons (ganglion cells) are produced first from resident progenitors, followed by the appearance of retinal interneurons, photoreceptors, and Muller glia. This sequence is maintained through the complex orchestration of cell-intrinsic and cell-extrinsic events and factors, including local influences between neighboring cells. Here we asked whether cultures of human prenatal retinal cells might also yield different ratios of cell types based on gestational age and time spent in vitro, thus recapitulating in vivo development. An established chopping technique was used to passage human prenatal retinal cells as neurospheres, avoiding the use of proteases and preserving cell-cell contacts and native microenvironments present in vivo. Retinal neurospheres cultured in this manner demonstrated specific patterns of growth over a limited time period, possibly reflecting trends in normal retinal development. Upon differentiation, immunocytochemical analysis revealed that retinal neurospheres produce predominantly glial cells with increasing gestational age and time in culture. Conversely, the percentage of betaIII tubulin-positive neurons declined over time. This provides information for optimizing culture systems aimed at the study of human retinal development and the generation of specific retinal cell types for therapeutic use or drug testing.
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