Viable Epidermal Cells Research Articles

A modified protocol for studying filaggrin degradation using a reconstructed human epidermis model under low and high humidity.

Filaggrin (FLG) is an essential protein that plays a vital role in maintaining skin barrier function and moisture levels, allowing the skin to adapt to dry environments. However, the precise temporal dynamics of FLG metabolism in the human epidermis remain poorly understood, and suitable tools to study these time-dependent effects are currently lacking. To investigate the molecular mechanisms and time course of FLG metabolism and skin barrier function under high- and low-humidity conditions, utilizing a reconstructed epidermis model. EpiSkin specimens cultured under humid or dry conditions for varying durations (2-48 h) were compared by assessing FLG degradation and skin barrier formation using immunofluorescence staining and western blotting. Under conditions of low humidity, the proteolysis of FLG in EpiSkin increased between 4 and 12 h and was accompanied by elevated levels of cysteine-aspartic protease (caspase)-14. The expression of peptidyl arginine deiminase 1 and calpain 1 also increased at 4 h. However, after 24 h, the expression of these three FLG-degrading proteins significantly decreased. Conversely, the levels of pyrrolidone-5-carboxylic acid and urocanic acid initially decreased at 2 h and then increased between 12 and 24 h. Additionally, the expression of skin barrier proteins, such as FLG, transglutaminase 5, loricrin and zonula occludens-1, decreased starting from 12 h. Notably, epidermal cell viability and activity were also inhibited. We propose a reliable and ethical model to study the temporal dynamics of FLG metabolism and its role in skin barrier function. Using a commercially reconstructed epidermis to mimic dry skin formation obviates the need for animal and human testing.

90 Efficacy of Porcine Skin Xenotransplants Indistinguishable from Allograft in First-in-human Clinical Evaluation

IntroductionHuman cadaver allograft (HCA) is the current standard of care for temporary wound closure of large burns, but a critical need for high-quality alternatives exists. Porcine skin shares many similarities with human skin, and hyperacute rejection is prevented via a single genetic modification. Like human skin, non-terminally sterilized porcine skin contains viable dermal and epidermal cells and intact vasculature that enables restoration of barrier function. These characteristics are distinct from those of traditional, terminally sterilized “xenografts” and may offer greater therapeutic capability. We report here key efficacy outcomes specific to severe burn care from a first-in-human clinical trial to assess the capability of genetically engineered porcine skin xenotransplants to provide temporary wound closure for severe and extensive deep-partial and full-thickness burn wounds, compared to HCA.MethodsSplit-thickness skin containing epidermal and dermal layers was harvested from Designated Pathogen Free, GalT-KO, porcine donors, cGMP processed to achieve USP< 71 > sterility and cryopreserved to retain >70% cell viability. These were transplanted side by side with HCA on debrided full-thickness burn wounds in six human subjects. Temporary wound closure, incidence of complete wound closure following autografting, and quality of healing, including scarring, contour, and feel of healed skin, normalization of skin markings or pigmentation, were independently assessed.ResultsAcross all patients and assessment time points, adherence, vascularity, and overall appearance were indistinguishable between porcine skin xenotransplants and HCA control. After surgical removal, wound beds treated with each type of dressing were perfused and otherwise appeared equivalent and clinically suitable for autografting. Long-term outcomes were comparable between wound sites treated with porcine skin or HCA with no discernable differences in scarring or cosmesis.ConclusionsSkin xenotransplants effectively provided temporary wound closure and restoration of barrier function via intact native vasculature, active cells, decreased antigenicity, and high-quality tissue architecture unimpacted by cryopreservation and thawing. These results show clinical promise as an interchangeable alternative to HCA in the treatment of severe burns. Expanded clinical evaluation is ongoing.

Structural and physiological effects of chromium uptake in the seagrass Halophila stipulacea

This work aims to provide insight on the effects of chromium (Cr) uptake in seagrasses at environmentally relevant exposure concentrations; information on this issue can further increase the utility of marine angiosperms as indicators of trace metal contamination. Cr uptake kinetics, effects on physiological and structural cell traits, and the uptake-effects relationship in Halophila stipulacea leaves incubated in 0.01–10 mg L−1 (0.2–200 μM) of Cr (VI) were examined. An initial uptake was followed by a decrease at 0.01–0.5 mg L−1 (0.2–10 μM); at higher exposures, the fit of uptake data to a Michaelis-Menten-type equation was significant (p < 0.001). The mean tissue concentration over the exposure period, and the highest tissue concentration attained both increased as exposure concentration (Cw) increased from 0.05 to 10 mg L−1 (1 to 200 μM); uptake rate generally increased with Cw. Cytotoxic effects occurred even at 0.01 mg L−1 (0.2 μM). Actin filaments (AFs) and endoplasmic reticulum (ER) were impacted in a dose- and time- dependent pattern, while microtubule integrity was not markedly affected even at the highest exposure. AF disruption in differentiating cells initiated at 0.01 mg L−1 (0.2 μM), while ER disorganization did so at 0.1 mg L−1 (2 μM); regression equations described the relationships of Cw vs. time required for effect onset, and of Cw vs. AF disturbance progress. Elevated levels of hydrogen peroxide (H2O2) were detected on the 2d. In addition, a significant reduction in epidermal cell viability appeared at 5–10 or 10 mg L−1 (100–200 or 200 μM), depending on the leaf age. The lowest tissue concentration associated with effect onset was 31.05 μg g−1 dry wt (2d, 5 mg L−1); at lower exposures, higher tissue concentrations did not coincide with impact, implying that the uptake rate rather than the tissue concentration relates to the effect onset. The above suggest that (a) Cr (VI) may pose a high risk to seagrass meadows, (b) H. stipulacea can act as a bioindicator of ambient Cr (VI) bioavailability, mainly at elevated exposure concentrations, (c) AF and ER disturbances in seagrass leaf cells could be considered as sensitive biomarkers of Cr (VI)-induced stress, (d) regression equations describing the relationships Cw vs. time required for effect onset and Cw vs. effect progress increase the utility of AF and ER impairment as biomarkers, and (e) the time frame of the exposure should be considered in the interpretation of tissue residues. Our findings improve the utility of seagrasses as indicators of trace metal contamination and could be utilized in the overall effort for seagrass meadows conservation.

Nickel uptake kinetics and its structural and physiological impacts in the seagrass Halophila stipulacea

The present work aims to provide insight into interactions between trace metals and higher plants, focusing on nickel uptake and its effects in seagrasses at environmentally relevant concentrations. Total and intracellular nickel accumulation kinetics, nickel effects on structural cell components, oxidative stress marker and cellular viability, and the accumulation kinetics-toxic effects relationship were investigated in leaves of Halophila stipulacea plants incubated in seawater under laboratory conditions containing nickel ions at 0.01–10 mg L−1 for 14 days. Nickel accumulation kinetics in H. stipulacea young and older apical leaves followed a Michaelis-Menten-type equation, allowing the calculation of uptake parameters; uptake rate (Vc) and equilibrium concentration (Ceq) tended to increase with the increase of nickel concentration in the medium. A dose- and uptake parameter-dependent actin filament (AF) and endoplasmic reticulum (ER) impairment was observed, whereas no effects occurred on microtubules and cell ultrastructure. AF disturbance and ER aggregation were firstly observed in differentiated cells at the lowest concentration on the 12th and 14th day, respectively, while AF disruption in meristematic cells firstly occurred at 0.05 mg L−1; the effects appeared earlier and were more acute at higher concentrations. Increased H2O2 levels were detected, while, at the highest exposures, a significant reduction in epidermal cell viability in older leaves occurred. The lowest total nickel concentrations in young leaves associated with AF disturbance onset at nickel exposure concentrations of 0.01–1 mg L−1 varied between 18.98 and 63.93 μg g−1 dry wt; importantly, they were comparable to nickel concentrations detected in seagrass leaves from various locations. The relationships between exposure concentration, uptake kinetic parameters and toxic effect onset were satisfactorily described by regression models. Our findings suggest that (a) nickel may pose a threat to seagrass meadows, (b) H. stipulacea can be regarded as an efficient biomonitor of nickel, (c) AF and ER impairment in seagrass leaves can be considered as early biomarkers of nickel-induced stress, and (d) the regression models obtained can be used as a tool to evaluate ambient nickel levels and to detect ecotoxicologically significant nickel contamination. The data presented can be utilized in the management and conservation of the coastal environment.

Lipid depletion enables permeation of Staphylococcus aureus bacteria through human stratum corneum

ABSTRACT Atopic dermatitis (AD) is a chronic inflammatory disease that affects approximately 2-5% of adults worldwide. The pathogenesis of AD continues to be a well-debated point of conjecture, with numerous hypotheses having been proposed. AD conditions are associated with increased populations of Staphylococcus aureus and reduced skin lipids. In this study, we evaluate the ability of S. aureus to permeate across human stratum corneum (SC) exhibiting both normal and depleted lipid conditions consistent with AD. This permeation would enable bacteria to interact with underlying viable epidermal cells, which could serve as a trigger for inflammation and disease onset. Our results indicate that permeation of S. aureus through SC exhibiting normal lipid conditions is not statistically significant. However, bacteria can readily permeate through lipid depleted tissue over a 9-d period. These findings suggest that S. aureus may potentially act as the mechanistic cause, rather than merely the result of AD. Abbreviations AD: Atopic dermatitis; SC: Stratum Corneum; AMP: Antimicrobial peptide; DIW: Deionized water; PDMS: Polydimethylsiloxane; GFP: Green fluorescent protein; BHI: Brain heart infusion medium

INF2 regulates oxidative stress-induced apoptosis in epidermal HaCaT cells by modulating the HIF1 signaling pathway

Promoting epidermal cell survival in an oxidative stress microenvironment is vital for skin regeneration after burns and/or wounds. However, few studies have explored the mediators related to epidermal cell apoptosis in an oxidative stress microenvironment. Cellular viability was determined using the MTT assay, TUNEL staining, western blot analysis and LDH release assay. Two independent siRNAs were transfected into HaCaT cell to repress INF2 and/or HIF1 in the presence of H2O2. Mitochondrial function was determined using JC-1 staining, mitochondrial ROS staining, immunofluorescence staining and western blotting. In the present study, our data demonstrated that the expression of inverted formin-2 (INF2) increased rapidly when the cells were exposed to H2O2. Interestingly, INF2 knockdown promoted HaCaT cell survival via reducing H2O2-mediated cell apoptosis. Molecular investigations demonstrated that INF2 deletion attenuated mitochondrial ROS overloading, restored the cellular redox balance, sustained the mitochondrial membrane potential, improved mitochondrial respiratory function and corrected the mitochondrial dynamics disorder in an H2O2-mimicking oxidative stress microenvironment. In addition, INF2 deletion upregulated the expression of HIF1. Interestingly, the inhibition of HIF1 increased cell death and caused mitochondrial stress despite the deletion of INF2, suggesting that the HIF1 signaling pathway is required for INF2 deletion-mediated HaCaT cell survival and mitochondrial protection. Altogether, our results identified INF2 as a novel apoptotic mediator for oxidative stress-mediated HaCaT cell death via modulating mitochondrial stress and repressing the HIF1 signaling pathway. This finding provides evidence to support the critical role played by the INF2-HIF1 axis in regulating mitochondrial stress and epidermal cell viability in an oxidative stress microenvironment.

Abstract 4567: Demonstration of pharmacodynamic effects of Notch and PI3Kinase inhibitors using robust immunohistochemical assays on human skin explant models

Abstract Assessment of pharmacodynamic (PD) effect of novel therapeutic compounds in human tissues is used to demonstrate target engagement and to rationalize dosing strategy decisions for emerging targeted therapeutics in clinical trials. Often, human skin biopsies, a readily accessible surrogate tissue, are used for these purposes. Aberrant signaling in the Notch cell surface receptor and the PI3K signal transduction pathways are increasingly implicated in human cancers. In order to test the utility of immunohistochemical (IHC) PD biomarker assays developed in our laboratory for Notch and PI3K inhibitor development programs, we developed ex vivo PD models using respective inhibitors on human skin biopsy explants. Fresh human skin samples were obtained in the Operating-Room from reduction mammoplasty specimens, serially sliced (1-2 mm) and incubated at 37°C in Williams E. media containing 1 µM Notch (gamma-secretase) inhibitor (15 h); and 50 µM PI3K inhibitor (LY294002, 4 h). Specimens were fixed in 10% neutral buffered formalin for 24 hours, and processed into FFPE blocks. Sections were stained for Notch1 intracellular domain (N1ICD, rabbit monoclonal, D3B8) using DAB and for phospho-S6 (S240/S244, rabbit polyclonal) using an alkaline phosphatase-based detection system. N1ICD and phospho-S6 immunoreactivity in cells of the epidermis was analyzed using positive pixel counting. Viability of epidermal cells throughout ex vivo incubation was verified by Ki67 and histomorphologic assessments of the epidermis. Nuclear N1ICD immunoreactivity, a marker of Notch pathway activation, was decreased by 76% when treated with gamma-secretase inhibitor. Similarly, treatment with PI3K inhibitor decreased cytoplasmic phospho-S6 immunoreactivity by 98%. These ex vivo models of human skin provide scientific evidence to support: 1) The N1ICD and phospho-S6 IHC assays developed in our laboratory can demonstrate ex vivo inhibition of these two pathways in human skin; and 2) Feasibility to translate human skin explant findings to patient skin samples from clinical trials. Our explant model systems are closer to real life PD assessments on patient skin samples from clinical trials and can further substantiate the PD data generated in animal models. In some cases, the ex vivo model could replace animal models. Citation Format: Timothy R. Holzer, Leslie A. O'Neill, Angie D. Fulford, Janet M. Grondin, Bradley L. Ackermann, Robert J. Konrad, Kelly M. Credille, Aejaz Nasir. Demonstration of pharmacodynamic effects of Notch and PI3Kinase inhibitors using robust immunohistochemical assays on human skin explant models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4567. doi:10.1158/1538-7445.AM2014-4567

Marine‐derived nutrient improves epidermal and dermal structure and prolongs the life span of reconstructed human skin equivalents

Imedeen™ is a cosmeceutical that provides nutrients to the skin. One of its active ingredients is the Marine Complex™ (MC). The aim of this study was to evaluate whether MC affects skin morphogenesis differently in female and male human skin equivalents (HSEs). Human skin equivalents were established with cells obtained from female or male donors between 30 and 45 years of age and cultured for seven or 11 weeks in the presence or absence of MC. Using immunohistochemistry, we examined early differentiation by keratin 10 expression, (hyper)proliferation by keratin 17 and Ki67, and basement membrane composition by laminin 332 and collagen type VII. In addition, the expression of collagen type I and the secretion of pro-collagen I were measured. Marine Complex strongly increased the number of Ki67-positive epidermal cells in female HSEs. In the dermis, MC significantly stimulated the amount of secreted pro-collagen I and increased the deposition of laminin 332 and collagen type VII. Furthermore, MC prolonged the viable phase of HSEs by slowing down its natural degradation. After 11 weeks of culturing, the MC-treated HSEs showed higher numbers of viable epidermal cell layers and a thicker dermal extracellular matrix compared with controls. In contrast, these effects were less pronounced in male HSEs. The MC nutrient positively stimulated overall HSE tissue formation and prolonged the longevity of both female and male HSEs. The ability of MC to stimulate the deposition of basement membrane and dermal components can be used to combat 2 human skin aging in vivo.

Differential Effects of Corticosteroids and Pimecrolimus on the Developing Skin Immune System in Humans and Mice

Atopic dermatitis arises primarily in early infancy. In these patients, corticosteroids are used especially with great caution because of their side effects. Calcineurin inhibitors such as pimecrolimus (PIM) could be useful, but safety concerns have been raised in particular because of the lack of knowledge about their effects on the developing skin immune system. This study was designed to investigate the impact of PIM and corticosteroids on epidermal cells (EC) in infants and newborn mice. We found that the percentage of unfractionated viable infant ECs was significantly decreased in the presence of beta-methasone-17-valerate (BMV) but not PIM. Exposure of unfractionated infant ECs to BMV but not to PIM and vehicle control caused a significant inhibition of the upregulation of CD86 molecules on Langerhans cells (LC). The release of cytokines by LCs and ECs, cultured in the presence of BMV and PIM, was not significantly reduced compared with controls. Topical corticosteroid but not PIM application onto newborn mice induced apoptosis in some LC precursors. Our data suggest that similar to the situation in adult skin, corticosteroids may impair LC maturation as well as viability of ECs in infants, effects not seen with PIM.

The mechanical properties of the skin epidermis in relation to targeted gene and drug delivery

A challenge in combating many major diseases is breaching the skin's tough outer layer (the stratum corneum (SC)) and delivering drugs and genes into the underlying abundant immunologically sensitive viable epidermal cells with safe, practical physical technologies. To achieve this effectively and accurately, design information is needed on key skin mechanical properties when pushing into and through epidermal skin cells. We measure these important mechanical properties by penetrating through the intact SC and viable epidermis (VE) of freshly excised murine skin with a NANO-indenter, using custom tungsten probes fabricated with nominally 5 and 2 μm diameters (with nanoscale tips). We show the skin Young's modulus, storage modulus and stress all dramatically decreased through the SC. Also, for a given penetration depth, decreasing the probe size significantly increases the storage modulus. Biological variation in penetrating the skin was shown. These collective findings advance the rational design of physical approaches for delivering genes and drugs within key cells of the VE.

Effects of different preservation solutions on skin graft epidermal cell viability and graft performance in a rat model

This study sought to evaluate the viable epidermal cell count of skin stored at 4 °C in different media, and to determine the longest time that grafts could be stored and still be used for clinical application of skin grafts. Harvested rat skin grafts were divided into four groups: saline (group 1), Roswell Park Memorial Institute-1640 solution (RPMI) (group 2), University of Wisconsin solution (UW) (group 3), and Histidine–tryptophan–ketoglutarate solution (HTK) (group 4). After the designated storage time (7, 14, 21, 28, or 35 days), grafts were divided into two parts. Skin grafts (3 cm × 3 cm) were then autotransplanted onto full-thickness circular wound beds. Percentages of viable keratinocytes (PVK) declined significantly for skin grafts stored in UW, HTK, and saline solutions (Kruskal–Wallis, P < 0.05), while there was an insignificant decline in the PVK of skin grafts stored in RPMI until the 28th day of storage (Kruskal–Wallis, P > 0.05). Compared with UW, HTK, and saline, grafts stored in RPMI had significantly higher percentages of PCNA at the 14th and 21st days of storage (Mann–Whitney U-test, P < 0.05). Grafts stored in RPMI had significantly lower apoptosis rates than did grafts stored in UW or HTK ( P < 0.05). Based on these results, we conclude that RPMI-1640 provides a better environment for skin grafts by increased quality and survival time of skin grafts, as assessed by both microscopic and macroscopic investigations.

Assessment of epidermal cell viability by near infrared multi-photon microscopy following ballistic delivery of gold micro-particles

The use of gene guns in ballistically delivering DNA vaccine coated gold micro-particles to skin can potentially damage targeted cells, therefore influencing transfection efficiencies. In this paper, we assess cell death in the viable epidermis by non-invasive near infrared two-photon microscopy following micro-particle bombardment of murine skin. We show that the ballistic delivery of micro-particles to the viable epidermis can result in localised cell death. Furthermore, experimental results show the degree of cell death is dependant on the number of micro-particles delivered per unit of tissue surface area. Micro-particles densities of 0.16 ± 0.27 (mean ± S.D.), 1.35 ± 0.285 and 2.72 ± 0.47 per 1000 μm 2 resulted in percent deaths of 3.96 ± 5.22, 45.91 ± 10.89, 90.52 ± 12.28, respectively. These results suggest that optimization of transfection by genes administered with gene guns is − among other effects − a compromise of micro-particle payload and cell death.

Sunscreen Penetration of Human Skin and Related Keratinocyte Toxicity after Topical Application

Sunscreen skin penetration and safety assessment should be considered together in order to ensure that in vitro cytotoxicity studies examine relevant doses of these organic chemical UV filters to which viable epidermal cells are realistically exposed. In this study, we sought to determine whether sufficient topically applied sunscreens penetrated into human viable epidermis to put the local keratinocyte cell populations at risk of toxicity. The penetration and retention of five commonly used sunscreen agents (avobenzone, octinoxate, octocrylene, oxybenzone and padimate O) in human skin was evaluated after application in mineral oil to isolated human epidermal membranes. Sunscreen concentration–human keratinocyte culture response curves were then defined using changes in cell morphology and proliferation (DNA synthesis using radiolabelled thymidine uptake studies) as evidence of sunscreens causing toxicity. Following 24 h of human epidermal exposure to sunscreens, detectable amounts of all sunscreens were present in the stratum corneum and viable epidermis, with epidermal penetration most evident with oxybenzone. The concentrations of each sunscreen found in human viable epidermis after topical application, adjusting for skin partitioning and binding effects, were at least 5-fold lower, based on levels detected in viable epidermal cells, than those appearing to cause toxicity in cultured human keratinocytes. It is concluded that the human viable epidermal levels of sunscreens are too low to cause any significant toxicity to the underlying human keratinocytes.

Characterization of powdered epidermal vaccine delivery with multiphoton microscopy

Multiphoton laser scanning microscopy (MPLSM) has been adapted to non-invasively characterize hand-held powdered epidermal vaccine delivery technology. A near infrared femtosecond pulsed laser, wavelength at approximately 920 nm, was used to evoke autofluorescence of endogenous fluorophores within ex vivo porcine and human skin. Consequently, sub cellular resolution three-dimensional images of stratum corneum and viable epidermal cells were acquired and utilized to observe the morphological deformation of these cells as a result of micro-particle penetration. Furthermore, the distributional pattern of micro-particles within the specific skin target volume was quantified by measuring the penetration depth as revealed by serial optical sections in the axial plane obtained with MPLSM. Additionally, endogenous fluorescence contrast images acquired at the supra-basal layer reveal cellular structures that may pertain to dendritic Langerhans cells of the epidermis. These results show that MPLSM has advantages over conventional histological approaches, since three-dimensional functional images with sub-cellular spatial resolution to depths beyond the epidermis can be acquired non-invasively. Accordingly, we propose that MPLSM is ideal for investigations of powdered epidermal vaccine delivery.

Epidermal Remodeling in Psoriasis (II): A Quantitative Analysis of the Epidermal Architecture

Hyperproliferative psoriatic epidermis was quantitatively analyzed using a geometric model of viable epidermis. Our model was based on hexagonally arranged cylindrical papillae, which allowed the determination of the total volume of the viable epidermis and the total area of the interface with the proliferative compartment based on several parameters, such as papillary height, papillary width, and distance between neighboring papillae. The analysis assumed that the total number of viable epidermal cells paralleled the proliferative compartment in a steady state of cell flow, so a quantitative relation could be made between both volume and interface of the viable epidermis. Multiple parameters of the psoriatic epidermal architecture were measured, and variations within psoriasis were predicted by the model. The results predicted were remarkably close to the observed values. The geometric model also indicated that psoriatic epidermis could be subdivided into two distinct types, with and without a granular layer; the latter having a shorter turnover time. This is consistent with the notion that the typical psoriatic epidermis (without the granular layer) represents the expanding hyperproliferative phase, whereas the psoriatic epidermis with a granular layer represents stationary or resolving states. The model of hexagonally arranged cylindrical papillae suggested that the architecture of the psoriatic epidermis is constructed by a simple mechanism, whereby the psoriatic angulated rete-papilla pattern was produced by a two-dimensional increase in the proliferative compartment and a three-dimensional increase in the total volume of the viable epidermis.

On quantification of CD1a, HLA-DR, and HLA class I expression on viable human langerhans cells and keratinocytes

In order to determine precisely the cellular density of surface molecules that are critical for antigen presentation in human epidermis, we utilized a quantitative immunofluorescence indirect assay and performed flow cytometric analysis of human epidermal cell (EC) suspensions. We first demonstrated that Tricolor-labeled streptavidin coupled to Cy-5 (SA-TC) was a reliable marker for non viable EC and that SA-TC+ EC accounted for the frequent nonspecific background of fluorescence due to isotype controls binding, although Langerhans cells (LC) and Keratinocytes (Kc) express Fc receptors for IgG on their surfaces. These results indicate that quantification of cell surface antigens on human EC requires the concomitant use of a marker of viability. Multicolor flow cytometric analysis allowed us to quantify CD1 molecules and major histocompatibility complex (MHC) antigens on viable human LC and Kc. Our results demonstrated a weak expression of MHC class I molecules on viable LC (163 ± 19 × 103 molecules/cell) compared to viable Kc (785 ± 110 × 103 molecules/cell). Mean antigen density of HLA-DR and CD1a molecules on viable LC were 579 ± 82 × 103 molecules/cell and 1600 ± 133 × 103 molecules/cell, respectively. Quantitative flow cytometry of viable EC may be proposed to evaluate the number of membrane antigens whose level of expression is related to cellular maturation or activation that occurs in skin diseases. © 1996 Wiley-Liss, Inc.

Aerosol vehicle for Delivery of Epidermal Cells – An in Vitro Study

This in vitro study investigated the possibility of delivering epidermal cells to cell culture plates using a simple and inexpensive aerosolization apparatus. Full-thickness skin biopsies were incubated in dispase to separate the epidermis from the dermis, and the epidermis was treated with trypsin to separate the epidermal cells from one another. The cells were suspended in aerosolization medium (1x106 cells/mL) and sprayed onto culture plates using a sterilized pump-action aerosol nozzle. The plates were incubated and microscopically examined on a daily basis. The aerosolization process was successful in consistently delivering a uniform distribution of suspended epidermal cells. By day 4 there was evidence of cell proliferation, and by days 7 to 9 a confluent layer of cells was achieved on the plates. The monolayer consisted primarily of keratinocytes interspersed with a few fibroblasts. The aerosol method was shown to be effective at delivering a suspension of viable epidermal cells to a culture plate.

Quantification of CD1a, HLA‐DR, and HLA class I expression on viable human langerhans cells and keratinocytes

In order to determine precisely the cellular density of surface molecules that are critical for antigen presentation in human epidermis, we utilized a quantitative immunofluorescence indirect assay and performed flow cytometric analysis of human epidermal cell (EC) suspensions. We first demonstrated that Tricolor-labeled streptavidin coupled to Cy-5 (SA-TC) was a reliable marker for non viable EC and that SA-TC+ EC accounted for the frequent nonspecific background of fluorescence due to isotype controls binding, although Langerhans cells (LC) and Keratinocytes (Kc) express Fc receptors for IgG on their surfaces. These results indicate that quantification of cell surface antigens on human EC requires the concomitant use of a marker of viability. Multicolor flow cytometric analysis allowed us to quantify CD1 molecules and major histocompatibility complex (MHC) antigens on viable human LC and Kc. Our results demonstrated a weak expression of MHC class I molecules on viable LC (163 ± 19 × 103 molecules/cell) compared to viable Kc (785 ± 110 × 103 molecules/cell). Mean antigen density of HLA-DR and CD1a molecules on viable LC were 579 ± 82 × 103 molecules/cell and 1600 ± 133 × 103 molecules/cell, respectively. Quantitative flow cytometry of viable EC may be proposed to evaluate the number of membrane antigens whose level of expression is related to cellular maturation or activation that occurs in skin diseases. © 1996 Wiley-Liss, Inc.

Quantification of CD1a, HLA-DR, and HLA class I expression on viable human Langerhans cells and keratinocytes.

In order to determine precisely the cellular density of surface molecules that are critical for antigen presentation in human epidermis, we utilized a quantitative immunofluorescence indirect assay and performed flow cytometric analysis of human epidermal cell (EC) suspensions. We first demonstrated that Tricolor-labeled streptavidin coupled to Cy-5 (SA-TC) was a reliable marker for non viable EC and that SA-TC+ EC accounted for the frequent nonspecific background of fluorescence due to isotype controls binding, although Langerhans cells (LC) and Keratinocytes (Kc) express Fc receptors for IgG on their surfaces. These results indicate that quantification of cell surface antigens on human EC requires the concomitant use of a marker of viability. Multicolor flow cytometric analysis allowed us to quantify CD1 molecules and major histocompatibility complex (MHC) antigens on viable human LC and Kc. Our results demonstrated a weak expression of MHC class I molecules on viable LC (163 +/- 19 x 10(3) molecules/cell) compared to viable Kc (785 +/- 110 x 10(3) molecules/cell). Mean antigen density of HLA-DR and CD1a molecules on viable LC were 579 +/- 82 x 10(3) molecules/cell and 1600 +/- 133 x 10(3) molecules/cell, respectively. Quantitative flow cytometry of viable EC may be proposed to evaluate the number of membrane antigens whose level of expression is related to cellular maturation or activation that occurs in skin diseases.

Isolation and characterization of human epidermal stem cells.

1. The keratinocytes in human epidermis are constantly turned over and replaced by a population of stem cells located in the basal epidermal layer. Until recently there were no markers allowing the isolation of viable epidermal stem cells. However, it has now been shown that epidermal stem cells can be isolated both in vitro and direct from the epidermis as they express high levels of functional beta 1 integrin family receptors for extracellular matrix proteins. 2. The evidence for integrins as stem cell markers and the insights that have been gained into stem cell behaviour are reviewed.