A Microfluidic Method for the Selection of Undifferentiated Human Embryonic Stem Cells and in Situ Analysis.

Conventional cell-sorting methods such as fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS) can suffer from certain shortcomings such as lengthy sample preparation time, cell modification through antibody labeling, and cell damage due to exposure to high shear forces or to attachment of superparamagnetic Microbeads. In light of these drawbacks, we have recently developed a label-free, microfluidic platform that can not only select cells with minimal sample preparation but also enable analysis of cells in situ. We demonstrate the utility of our platform by successfully isolating undifferentiated human embryonic stem cells (hESCs) from a heterogeneous population based on the undifferentiated stem-cell marker SSEA-4. Importantly, we show that, in contrast to MACS or FACS, cells isolated by our method have very high viability (~90%). Overall, our platform technology could likely be applied to other cell types beyond hESCs and to a variety of heterogeneous cell populations in order to select and analyze cells of interest.

Calcineurin and vacuolar-type H+-ATPase modulate macrophage effector functions.

While effector molecules produced by activated macrophages (including nitric oxide, tumor necrosis factor alpha, interleukin 1, etc.) help to eliminate pathogens, high levels of these molecules can be deleterious to the host itself. Despite their importance, the mechanisms modulating macrophage effector functions are poorly understood. This work introduces two key negative regulators that control the levels and duration of macrophage cytokine production. Vacuolar-type H+-ATPase (V-ATPase) and calcineurin (Cn) constitutively act in normal macrophages to suppress expression of inflammatory cytokines in the absence of specific activation and to inhibit macrophage cytokine responses induced by bacterial lipopolysaccharide (V-ATPase), interferon gamma (V-ATPase and Cn), and calcium (Ca2+) flux (Cn). Cn and V-ATPase modulate effector gene expression at the mRNA level by inhibiting transcription factor NF-kappaB. This negative regulation by Cn is opposite to its crucial positive role in T cells, where it activates NFAT transcription factor(s) leading to expression of interleukin 2, tumor necrosis factor alpha, and other cytokine genes. The negative effects of V-ATPase and Cn on NF-kappaB-dependent gene expression are not limited to the macrophage lineage, as similar effects have been seen with a murine fibroblast cell line and with primary astrocytes.

Novel genetic regulation of T helper 1 (Th1)/Th2 cytokine production and encephalitogenicity in inbred mouse strains.

Development of T helper cell (Th)1 or Th2 cytokine responses is essential for effector and regulatory functions of T helper cells. We have compared cytokine profiles of myelin basic protein (MBP) Ac1-16 peptide-specific T helper cells from inbred mouse strains expressing identical k haplotype-derived MHC class II molecules B10.A and B10.BR, B10.BR T cell lines (TCL) produced Th1 cytokines (including high levels of TNF-alpha) and induced experimental autoimmune encephalomyelitis after adoptive transfer. In contrast, B10.A TCL produced Th2 cytokines (including low levels of TNF-alpha) and were poorly encephalitogenic. The contributions of the genetic origin of the T cells and the APC were explored. Serial restimulations of the B10.BR TCL with B10.A or (B10.A x B10.BR) F1 splenic antigen presenting cells (APC) during the establishment of TCL markedly reduced both Th1 cytokine production and encephalitogenicity. In addition, a single restimulation with B10. A splenic APC reduced IFN-gamma and TNF-alpha production by established Th1 MBP-specific Ak-restricted B10.BR TCL and by a Th1 KLH-specific, Ek-restricted B10.BR T cell clone. These studies suggest that B10.A and B10.BR APC differ in their ability to stimulate IFN-gamma and TNF-alpha production by mature Th1 cells and also influence their Th1/Th2 commitment in vivo. The nature of the downregulatory activity of B10.A APC on IFN-gamma and TNF-alpha production was explored. 2-hour supernatants from antigen-activated B10.A APC/TCL cultures or from B10.A APC activated by LPS had the same inhibitory effects on IFN-gamma and TNF-alpha production by B10.BR TCL. The downregulatory effects of B10.A APC are independent of TNF-alpha, IL-4, IL-10, IL-12p40, IFN-gamma, IL-13, TGF-beta, and PGE2. Thus, genetic difference(s) between B10.A and B10.BR APC appear(s) to control the production or activity of a novel soluble cytokine regulatory factor that influences Th1/Th2 commitment and controls production of IFN-gamma and TNF-alpha by mature Th1 cells.

Analysis of regulatory elements of the developmentally controlled chorion s15 promoter in transgenic Drosophila.

The Drosophila s15 chorion gene is expressed only in the follicular epithelium surrounding the developing oocyte, with tight quantitative control and a very narrow temporal specificity. We have used germ-line transformation analysis to conduct an extensive mutational dissection of its promoter between -189 and -39 bp relative to the transcriptional start site. Quantitative control and temporal specificity are disrupted by several of these mutations. The results suggest that this 150-bp DNA region encompasses many positive and negative, at least partially degenerate, cis-regulatory elements, which are involved in specifying the highly precise expression pattern of the s15 gene during development.