We have generated an induced pluripotent stem cell (iPSC) line KCLi001-A (iOP118) from a female atopic dermatitis (AD) patient, heterozygous for the loss-of-function mutation c.2282del4 in the filaggrin gene (FLG). Epidermal keratinocytes were reprogrammed using non-integrating Sendai virus vectors. The entire process of derivation and expansion of AD-iPSCs were performed under xeno-free culture conditions. Characterization of KCLi001-A line included molecular karyotyping, mutation screening using restriction enzyme digestion and Sanger sequencing, while pluripotency and differentiation potential were confirmed by expression of associated markers in vitro and by in vivo teratoma assay.

Fluorescent probes have found widespread use in biomedical sciences. Particularly since they can be targeted to cellular compartments and further more can report on the properties of their environment such as calcium concentration. Near infrared ultrafast lasers find increasing use for fluorescence applications since femtosecond pulses with a few milliwatts of average power are sufficient to induce significant two photon fluorescence from the probe when focussed into typical samples. The nonlinear optical excitation process allows sectioned imaging of 3-D samples without use of a confocal pinhole. In this paper we describe two aspects of multiphoton microscopy: the two-photon excitation cross section and the fluorescence lifetime. Of interest is the wavelength characterization of two-photon excitation cross-sections of fluorescence probes. We slowly modulate (∼500Hz) the intensity envelope of the input laser pulse train and analyze the emission signal in terms of the amplitude and phase of the harmonics of this modulation. In effect this is a power study that allows separation of different order effects. An application of ultrafast laser excitation that exploits many of the features outlined above is measurement of pH gradients in the skin. This is essential to skin barrier function and disruption of the gradient is though to be a indicating factor in many skin diseases. A probe for which the fluorescence lifetime varies with pH is used. We thus are able to tackle problems associated with inhomogeneous labeling. We have developed a two-photon laser-scanning lifetime microscope and present pH maps of skin obtained with this instrument. © 2002 SPIE · 0277-786X/02/$15.00.

Restoration of barrier-tissue integrity after injury is dependent on the function of immune cells and stem cells (SCs) residing in the tissue. In response to skin injury, hair-follicle stem cells (HFSCs), normally poised for hair generation, are recruited to the site of injury and differentiate into cells that repair damaged epithelium. We used a SC fate-mapping approach to examine the contribution of regulatory T (Treg) cells to epidermal-barrier repair after injury. Depletion of Treg cells impaired skin-barrier regeneration and was associated with a Th17 inflammatory response and failed HFSC differentiation. In this setting, damaged epithelial cells preferentially expressed the neutrophil chemoattractant CXCL5, and blockade of CXCL5 or neutrophil depletion restored barrier function and SC differentiation after epidermal injury. Thus, Treg-cell regulation of localized inflammation enables HFSC differentiation and, thereby, skin-barrier regeneration, with implications for the maintenance and repair of other barrier tissues.

Restoration of barrier-tissue integrity after injury is dependent on the function of immune cells and stem cells (SCs) residing in the tissue. In response to skin injury, hair-follicle stem cells (HFSCs), normally poised for hair generation, are recruited to the site of injury and differentiate into cells that repair damaged epithelium. We used a SC fate-mapping approach to examine the contribution of regulatory T (Treg) cells to epidermal-barrier repair after injury. Depletion of Treg cells impaired skin-barrier regeneration and was associated with a Th17 inflammatory response and failed HFSC differentiation. In this setting, damaged epithelial cells preferentially expressed the neutrophil chemoattractant CXCL5, and blockade of CXCL5 or neutrophil depletion restored barrier function and SC differentiation after epidermal injury. Thus, Treg-cell regulation of localized inflammation enables HFSC differentiation and, thereby, skin-barrier regeneration, with implications for the maintenance and repair of other barrier tissues.