The relatively new field of stem cell biology is hampered by a lack of sufficient means to accurately determine the phenotype of cells. Cell-type-specific markers, such as cell surface proteins used for flow cytometry or fluorescence-activated cell sorting, are limited and often recognize multiple members of a stem cell lineage. We sought to develop a complementary approach that would be less dependent on the identification of particular markers for the subpopulations of cells and would instead measure their overall character. We tested whether a microfluidic system using dielectrophoresis (DEP), which induces a frequency-dependent dipole in cells, would be useful for characterizing stem cells and their differentiated progeny. We found that populations of mouse neural stem/precursor cells (NSPCs), differentiated neurons, and differentiated astrocytes had different dielectric properties revealed by DEP. By isolating NSPCs from developmental ages at which they are more likely to generate neurons, or astrocytes, we were able to show that a shift in dielectric property reflecting their fate bias precedes detectable marker expression in these cells and identifies specific progenitor populations. In addition, experimental data and mathematical modeling suggest that DEP curve parameters can indicate cell heterogeneity in mixed cultures. These findings provide evidence for a whole cell property that reflects stem cell fate bias and establish DEP as a tool with unique capabilities for interrogating, characterizing, and sorting stem cells. STEM CELLS 2008;26:656 -665 Disclosure of potential conflicts of interest is found at the end of this article.

BackgroundDistinguishing human neural stem/progenitor cell (huNSPC) populations that will predominantly generate neurons from those that produce glia is currently hampered by a lack of sufficient cell type-specific surface markers predictive of fate potential. This limits investigation of lineage-biased progenitors and their potential use as therapeutic agents. A live-cell biophysical and label-free measure of fate potential would solve this problem by obviating the need for specific cell surface markers.Methodology/Principal FindingsWe used dielectrophoresis (DEP) to analyze the biophysical, specifically electrophysiological, properties of cortical human and mouse NSPCs that vary in differentiation potential. Our data demonstrate that the electrophysiological property membrane capacitance inversely correlates with the neurogenic potential of NSPCs. Furthermore, as huNSPCs are continually passaged they decrease neuron generation and increase membrane capacitance, confirming that this parameter dynamically predicts and negatively correlates with neurogenic potential. In contrast, differences in membrane conductance between NSPCs do not consistently correlate with the ability of the cells to generate neurons. DEP crossover frequency, which is a quantitative measure of cell behavior in DEP, directly correlates with neuron generation of NSPCs, indicating a potential mechanism to separate stem cells biased to particular differentiated cell fates.Conclusions/SignificanceWe show here that whole cell membrane capacitance, but not membrane conductance, reflects and predicts the neurogenic potential of human and mouse NSPCs. Stem cell biophysical characteristics therefore provide a completely novel and quantitative measure of stem cell fate potential and a label-free means to identify neuron- or glial-biased progenitors.

A loss of epidermal cohesion in pemphigus vulgaris (PV) results from autoantibody action on keratinocytes (KCs) activating the signaling kinases and executioner caspases that damage KCs, causing their shrinkage, detachment from neighboring cells, and rounding up (apoptolysis). In this study, we found that PV antibody binding leads to activation of epidermal growth factor receptor kinase, Src, p38 MAPK, and JNK in KCs with time pattern variations from patient to patient. Both extrinsic and intrinsic apoptotic pathways were also activated. Although Fas ligand neutralizing antibody could inhibit the former pathway, the mechanism of activation of the latter remained unknown. PV antibodies increased cytochrome c release, suggesting damage to mitochondria. The immunoblotting experiments revealed penetration of PVIgG into the subcellular mitochondrial fraction. The antimitochondrial antibodies from different PV patients recognized distinct combinations of antigens with apparent molecular sizes of 25, 30, 35, 57, 60, and 100 kDa. Antimitochondrial antibodies were pathogenic because their absorption abolished the ability of PVIgG to cause keratinocyte detachment both in vitro and in vivo. The downstream signaling of antimitochondrial antibodies involved JNK and late p38 MAPK activation, whereas the signaling of anti-desmoglein 3 (Dsg3) antibody involved JNK and biphasic p38 MAPK activation. Using KCs grown from Dsg3؊/؊ mice, we determined that Dsg3 did not serve as a surrogate antigen allowing antimitochondrial antibodies to enter KCs. The PVIgG-induced activation of epidermal growth factor receptor and Src was affected neither in Dsg3KCs nor due to absorption of antimitochondrial antibodies. These results demonstrated that apoptolysis in PV is a complex process initiated by at least three classes of autoantibodies directed against desmosomal, mitochondrial, and other keratinocyte self-antigens. These autoantibodies synergize with the proapoptotic serum and tissue factors to trigger both extrinsic and intrinsic pathways of cell death and break the epidermal cohesion, leading to blisters. Further elucidation of the primary signaling events downstream of PV autoantigens will be crucial for the development of a more successful therapy for PV patients.Pemphigus vulgaris (PV) 3 is an IgG autoantibody-mediated disease of skin and mucosa caused by a loss of epidermal cohesion and manifested by progressive blistering and non-healing erosions. The mechanism of detachment of keratinocytes (KCs) in PV, termed acantholysis, is a subject of intensive research. Elucidation of the pathophysiologic mechanism of acantholysis should facilitate development of novel pharmacologic approaches to prevent and treat blistering without systemic corticosteroids, a mainstay of treatment of PV patients. Acantholysis can be blocked both by inhibitors of signaling kinases, such as p38 MAPK (3), mammalian target of rapamycin (4), Src, and epidermal growth factor receptor (EGFR) kinase (2, 4 -7) and by other tyrosine kinases, phospholipase C, calmodulin...

Pemphigus vulgaris (PV) is a mucocutaneous blistering disease characterized by IgG autoantibodies against the stratified squamous epithelium. Current understanding of PV pathophysiology does not explain the mechanism of acantholysis in patients lacking desmoglein antibodies, which justifies a search for novel targets of pemphigus autoimmunity. We tested 264 pemphigus and 138 normal control sera on the multiplexed protein array platform containing 701 human genes encompassing many known keratinocyte cell-surface molecules and members of protein families targeted by organ-non-specific PV antibodies. The top 10 antigens recognized by the majority of test patients’ sera were proteins encoded by the DSC1, DSC3, ATP2C1, PKP3, CHRM3, COL21A1, ANXA8L1, CD88 and CHRNE genes. The most common combinations of target antigens included at least one of the adhesion molecules DSC1, DSC3 or PKP3 and/or the acetylcholine receptor CHRM3 or CHRNE with or without the MHC class II antigen DRA. To identify the PV antibodies most specific to the disease process, we sorted the data based on the ratio of patient to control frequencies of antigen recognition. The frequency of antigen recognition by patients that exceeded that of control by 10 and more times were the molecules encoded by the CD33, GP1BA, CHRND, SLC36A4, CD1B, CD32, CDH8, CDH9, PMP22 and HLA-E genes as well as mitochondrial proteins encoded by the NDUFS1, CYB5B, SOD2, PDHA1 and FH genes. The highest specificity to PV showed combinations of autoantibodies to the calcium pump encoded by ATP2C1 with C5a receptor plus DSC1 or DSC3 or HLA-DRA. The results identified new targets of pemphigus autoimmunity. Novel autoantibody signatures may help explain individual variations in disease severity and treatment response, and serve as sensitive and specific biomarkers for new diagnostic assays in PV patients.

Acetylcholine (ACh) regulates vital functions of T cells by acting on the nicotinic and muscarinic classes of cholinergic receptors, nAChR and mAChRs, respectively. This study was performed in murine splenic T cells. In freshly isolated CD4 and CD8 T cells, we detected mRNAs encoding a5, a9, a10, b1, b2, b4 nAChR subunits and M 1 , M 3 , M 4 and M 5 mAChR subtypes, whereas a2 was detected only in CD8 T cells. In vitro activation of CD4 T cells through T-cell receptor (TCR)/CD3 cross-linking was associated with the appearance of a4 and a7, upregulation of a5, a10, b4, M 1 and M 5 and downregulation of a9 and b2, whereas in vitro activation of CD8 T cells also featured the appearance of a4 and a7, as well as upregulation of a2, a5, b4, M 1 and M 4 , and downregulation of a10, b1, b2 and M 3 . In vitro polarization toward T helper (Th) 1 lineage was associated with a decrease of b2, b4 and M 3 expression; that toward Th2 cells with downregulation of a9 and M 3 , and upregulation of M 1 and M 5 ; and that toward Th17 phenotype with downregulation of a9, a10, b2 and M 3 mAChR. Polarized T cells also expressed a4, but not a1, a2, a3, a6, b3 or M 2 . To determine the role of cholinergic receptors in mediating the immunoregulatory action of autocrine/paracrine ACh, we analyzed the effects of nicotinic and muscarinic agonists ± antagonists on cytokine production in the CD4 þ CD62L þ T cells co-stimulated via TCR/CD3 cross-linking. The nicotinergic stimulation upregulated interferon-g (IFN-g) and downregulated interleukin (IL)-17 secretion, whereas the muscarinic stimulation enhanced IL-10 and IL-17 and inhibited INF-g secretion. These results demonstrated plasticity of the T-cell cholinergic system.