Neuroinflammatory CSF biomarkers MIF, sTREM1, and sTREM2 show dynamic expression profiles in Alzheimer's disease.

BACKGROUND: There is a need for novel fluid biomarkers tracking neuroinflammatory responses in Alzheimer's disease (AD). Our recent cerebrospinal fluid (CSF) proteomics study revealed that migration inhibitory factor (MIF) and soluble triggering receptor expressed on myeloid cells 1 (sTREM1) increased along the AD continuum. We aimed to assess the potential use of these proteins, in addition to sTREM2, as CSF biomarkers to monitor inflammatory processes in AD. METHODS: We included cognitively unimpaired controls (n = 67, 63 ± 9 years, 24% females, all amyloid negative), patients with mild cognitive impairment (MCI; n = 92, 65 ± 7 years, 47% females, 65% amyloid positive), AD (n = 38, 67 ± 6 years, 8% females, all amyloid positive), and DLB (n = 50, 67 ± 6 years, 5% females, 54% amyloid positive). MIF, sTREM1, and sTREM2 levels were measured by validated immunoassays. Differences in protein levels between groups were tested with analysis of covariance (corrected for age and sex). Spearman correlation analysis was performed to evaluate the association between these neuroinflammatory markers with AD-CSF biomarkers (Aß42, tTau, pTau) and mini-mental state examination (MMSE) scores. RESULTS: MIF levels were increased in MCI (p < 0.01), AD (p < 0.05), and DLB (p > 0.05) compared to controls. Levels of sTREM1 were specifically increased in AD compared to controls (p < 0.01), MCI (p < 0.05), and DLB patients (p > 0.05), while sTREM2 levels were increased specifically in MCI compared to all other groups (all p < 0.001). Neuroinflammatory proteins were highly correlated with CSF pTau levels (MIF: all groups; sTREM1: MCI, AD and DLB; sTREM2: controls, MCI and DLB). Correlations with MMSE scores were observed in specific clinical groups (MIF in controls, sTREM1 in AD, and sTREM2 in DLB). CONCLUSION: Inflammatory-related proteins show diverse expression profiles along different AD stages, with increased protein levels in the MCI stage (MIF and sTREM2) and AD stage (MIF and sTREM1). The associations of these inflammatory markers primarily with CSF pTau levels indicate an intertwined relationship between tau pathology and inflammation. These neuroinflammatory markers might be useful in clinical trials to capture dynamics in inflammatory responses or monitor drug-target engagement of inflammatory modulators.

C1q is increased in cerebrospinal fluid-derived extracellular vesicles in Alzheimer's disease: A multi-cohort proteomics and immuno-assay validation study.

INTRODUCTION: Extracellular vesicles (EVs) may propagate and modulate Alzheimer's disease (AD) pathology. We aimed to comprehensively characterize the proteome of cerebrospinal fluid (CSF) EVs to identify proteins and pathways altered in AD. METHODS: CSF EVs were isolated by ultracentrifugation (Cohort 1) or Vn96 peptide (Cohort 2) from non-neurodegenerative controls (n = 15, 16) and AD patients (n = 22, 20, respectively). EVs were subjected to untargeted quantitative mass spectrometry-based proteomics. Results were validated by enzyme-linked immunosorbent assay (ELISA) in Cohorts 3 and 4, consisting of controls (n = 16, n = 43, (Cohort3, Cohort4)), and patients with AD (n = 24, n = 100). RESULTS: We found > 30 differentially expressed proteins in AD CSF EVs involved in immune-regulation. Increase of C1q levels in AD compared to non-demented controls was validated by ELISA (∼ 1.5 fold, p (Cohort 3) = 0.03, p (Cohort 4) = 0.005). DISCUSSION: EVs may be utilized as a potential biomarker and may play a so far unprecedented role in immune-regulation in AD.

The effect of PPAR isoform (de)activation on the lipid composition in full-thickness skin models.

Human skin equivalents (HSEs) are 3D-cultured human skin models that mimic many aspects of native human skin (NHS). Although HSEs resemble NHS very closely, the barrier located in the stratum corneum (SC) is impaired. This is caused by an altered lipid composition in the SC of HSEs compared with NHS. One of the most pronounced changes in this lipid composition is a high level of monounsaturation. One key enzyme in this change is stearoyl-CoA desaturase-1 (SCD1), which catalyses the monounsaturation of lipids. In order to normalize the lipid composition, we aimed to target a group of nuclear receptors that are important regulators in the lipid synthesis. This group of receptors are known as the peroxisome proliferating activating receptors (PPARs). By (de)activating each isoform (PPAR-α, PPAR-δ and PPAR-γ), the PPAR isoforms may have normalizing effects on the lipid composition. In addition, another PPAR-α agonist Wy14643 was included as this supplement demonstrated normalizing effects in the lipid composition in a more recent study. After PPAR (ant)agonists supplementation, the mRNA of downstream targets, lipid synthesis genes and lipid composition were investigated. The PPAR downstream targets were activated, indicating that the supplements reached the keratinocytes to trigger their effect. However, minimal impact was observed on the lipid composition after PPAR isoform (de) activation. Only the highest concentration Wy14643 resulted in strong, but negative effects on CER composition. Although the novel tested modifications did not result in an improvement, more insight is gained on the nuclear receptors PPARs and their effects on the lipid barrier in full-thickness skin models.

Correction: Boiten et al. Pathologically Decreased CSF Levels of Synaptic Marker NPTX2 in DLB Are Correlated with Levels of Alpha-Synuclein and VGF. Cells 2021, 10, 38.

The authors want to notify that they have made the following changes to the author list of the paper [...].

Multitargeted Approach for the Optimization of Morphogenesis and Barrier Formation in Human Skin Equivalents.

In vitro skin tissue engineering is challenging due to the manifold differences between the in vivo and in vitro conditions. Yet, three-dimensional (3D) human skin equivalents (HSEs) are able to mimic native human skin in many fundamental aspects. However, the epidermal lipid barrier formation, which is essential for the functionality of the skin barrier, remains compromised. Recently, HSEs with an improved lipid barrier formation were generated by (i) incorporating chitosan in the dermal collagen matrix, (ii) reducing the external oxygen level to 3%, and (iii) inhibiting the liver X receptor (LXR). In this study, we aimed to determine the synergic effects in full-thickness models (FTMs) with combinations of these factors as single-, double-, and triple-targeted optimization approaches. The collagen-chitosan FTM supplemented with the LXR inhibitor showed improved epidermal morphogenesis, an enhanced lipid composition, and a better lipid organization. Importantly, barrier functionality was improved in the corresponding approach. In conclusion, our leading optimization approach substantially improved the epidermal morphogenesis, barrier formation, and functionality in the FTM, which therefore better resembled native human skin.

Improved organotypic skin model with reduced quantity of monounsaturated ceramides by inhibiting stearoyl-CoA desaturase-1.

Full thickness models (FTM) are 3D in vitro skin cultures that resemble the native human skin (NHS) to a great extent. However, the barrier function of these skin models is reduced. The skin barrier is located in the stratum corneum (SC) and consists of corneocytes embedded in a lipid matrix. In this matrix, deviations in the composition of the FTMs lipid matrix may contribute to the impaired skin barrier when compared to NHS. One of the most abundant changes in lipid composition is an increase in monounsaturated lipids for which stearoyl-CoA desaturase-1 (SCD-1) is responsible. To improve the SC lipid composition, we reduced SCD-1 activity during the generation of the FTMs. These FTMs were subsequently assessed on all major aspects, including epidermal homeostasis, lipid composition, lipid organization, and barrier functionality. We demonstrate that SCD-1 inhibition was successful and resulted in FTMs that better mimic the lipid composition of FTMs to NHS by a significant reduction in monounsaturated lipids. In conclusion, this study demonstrates an effective approach to normalize SC monounsaturated lipid concentration and may be a valuable tool in further optimizing the FTMs in future studies.

Pathologically Decreased CSF Levels of Synaptic Marker NPTX2 in DLB Are Correlated with Levels of Alpha-Synuclein and VGF.

Background: Dementia with Lewy bodies (DLB) is a neurodegenerative disease where synaptic loss and reduced synaptic integrity are important neuropathological substrates. Neuronal Pentraxin 2(NPTX2) is a synaptic protein that drives the GABAergic inhibitory circuit. Our aim was to examine if NPTX2 cerebral spinal fluid (CSF) levels in DLB patients were altered and how these levels related to other synaptic protein levels and to cognitive function and decline. Methods: NPTX2, VGF, and α-synuclein levels were determined in CSF of cognitive healthy (n = 27), DLB (n = 48), and AD (n = 20) subjects. Multiple cognitive domains were tested, and data were compared using linear models. Results: Decreased NPTX2 levels were observed in DLB (median = 474) and AD (median = 453) compared to cognitive healthy subjects (median = 773). Strong correlations between NPTX2, VGF, and α-synuclein were observed dependent on diagnosis. Combined, these markers had a high differentiating power between DLB and cognitive healthy subjects (AUC = 0.944). Clinically, NPTX2 levels related to global cognitive function and cognitive decline in the visual spatial domain. Conclusion: NPTX2 CSF levels were reduced in DLB and closely correlated to decreased VGF and α-synuclein CSF levels. CSF NPTX2 levels in DLB related to decreased functioning in the visual spatial domain.

The effects of LXR agonist T0901317 and LXR antagonist GSK2033 on morphogenesis and lipid properties in full thickness skin models.

Full thickness models (FTMs) are 3D-cultured human skin models that mimic many aspects of native human skin (NHS). However, their stratum corneum (SC) lipid composition differs from NHS causing a reduced skin barrier. The most pronounced differences in lipid composition are a reduction in lipid chain length and increased monounsaturated lipids. The liver-X-receptor (LXR) activates the monounsaturated lipid synthesis via stearoyl-CoA desaturase-1 (SCD-1). Therefore, the aim was to improve the SC lipid synthesis of FTMs by LXR deactivation. This was achieved by supplementing culture medium with LXR antagonist GSK2033. LXR agonist T0901317 was added for comparison. Subsequently, epidermal morphogenesis, lipid composition, lipid organization and the barrier functionality of these FTMs were assessed. We demonstrate that LXR deactivation resulted in a lipid composition with increased overall chain lengths and reduced levels of monounsaturation, whereas LXR activation increased the amount of monounsaturated lipids and led to a reduction in the overall chain length. However, these changes did not affect the barrier functionality. In conclusion, LXR deactivation led to the development of FTMs with improved lipid properties, which mimic the lipid composition of NHS more closely. These novel findings may contribute to design interventions to normalize SC lipid composition of atopic dermatitis patients.

Contribution of Palmitic Acid to Epidermal Morphogenesis and Lipid Barrier Formation in Human Skin Equivalents.

The outermost barrier layer of the skin is the stratum corneum (SC), which consists of corneocytes embedded in a lipid matrix. Biosynthesis of barrier lipids occurs de novo in the epidermis or is performed with externally derived lipids. Hence, in vitro developed human skin equivalents (HSEs) are developed with culture medium that is supplemented with free fatty acids (FFAs). Nevertheless, the lipid barrier formation in HSEs remains altered compared to native human skin (NHS). The aim of this study is to decipher the role of medium supplemented saturated FFA palmitic acid (PA) on morphogenesis and lipid barrier formation in HSEs. Therefore, HSEs were developed with 100% (25 µM), 10%, or 1% PA. In HSEs supplemented with reduced PA level, the early differentiation was delayed and epidermal activation was increased. Nevertheless, a similar SC lipid composition in all HSEs was detected. Additionally, the lipid organization was comparable for lamellar and lateral organization, irrespective of PA concentration. As compared to NHS, the level of monounsaturated lipids was increased and the FFA to ceramide ratio was drastically reduced in HSEs. This study describes the crucial role of PA in epidermal morphogenesis and elucidates the role of PA in lipid barrier formation of HSEs.

Unravelling effects of relative humidity on lipid barrier formation in human skin equivalents.

Relative humidity (RH) levels vary continuously in vivo, although during in vitro generation of three-dimensional human skin equivalents (HSEs) these remain high (90-95%) to prevent evaporation of the cell-culture medium. However, skin functionality is directly influenced by environmental RH. As the barrier formation in HSEs is different, there is a need to better understand the role of cell-culture conditions during the generation of HSEs. In this study, we aim to investigate the effects of RH on epidermal morphogenesis and lipid barrier formation in HSEs. Therefore, two types of HSEs were developed at 90% or at 60% RH. Assessments were performed to determine epidermal morphogenesis by immunohistochemical analyses, ceramide composition by lipidomic analysis, and lipid organization by Fourier transform infrared spectroscopy and small-angle X-ray diffraction. We show that reduction of RH mainly affected the uppermost viable epidermal layers in the HSEs, including an enlargement of the granular cells and induction of epidermal cell activation. Neither the composition nor the organization of the lipids in the intercorneocyte space were substantially altered at reduced RH. In addition, lipid processing from glucosylceramides to ceramides was not affected by reduced RH in HSEs as shown by enzyme expression, enzyme activity, and substrate-to-product ratio. Our results demonstrate that RH directly influences epidermal morphogenesis, albeit the in vitro lipid barrier formation is comparable at 90% and 60% RH.

Selectivity in cornified envelop binding of ceramides in human skin and the role of LXR inactivation on ceramide binding.

The cornified lipid envelope (CLE) is a lipid monolayer covalently bound to the outside of corneocytes and is part of the stratum corneum (SC). The CLE is suggested to act as a scaffold for the unbound SC lipids. By profiling the bound CLE ceramides, a new subclass was discovered and identified as an omega-hydroxylated dihydrosphingosine (OdS) ceramide. Bound glucosylceramides were observed in superficial SC layers of healthy human skin. To investigate the relation between bound and unbound SC ceramides, the composition of both fractions was analyzed and compared. Selectivity in ceramide binding towards unsaturated ceramides and ceramides with a shorter chain length was observed. The selectivity in ceramide species bound to the cornified envelope is thought to have a physiological function in corneocyte flexibility. Next, it was examined if skin models exhibit an altered bound ceramide composition and if the composition was dependent on liver X-receptor (LXR) activation. The effects of an LXR agonist and antagonist on the bound ceramides composition of a full thickness model (FTM) were analyzed. In FTMs, a decreased amount of bound ceramides was observed compared to native human skin. Furthermore, FTMs had a bound ceramide fraction which consisted mostly of unsaturated and shorter ceramides. The LXR antagonist had a normalizing effect on the FTM bound ceramide composition. The agonist exhibited minimal effects. We show that ceramide binding is a selective process, yet, still is contingent on lipid synthesized.

Human skin equivalents cultured under hypoxia display enhanced epidermal morphogenesis and lipid barrier formation.

Human skin equivalents (HSEs) are three-dimensional cell models mimicking characteristics of native human skin (NHS) in many aspects. However, a limitation of HSEs is the altered in vitro morphogenesis and barrier formation. Differences between in vitro and in vivo skin could have been induced by suboptimal cell culture conditions, of which the level of oxygen in vitro (20%) is much higher than in vivo (0.5-8%). Our aim is to study how external oxygen levels affect epidermal morphogenesis and barrier formation in HSEs. In the present study, fibroblast and keratinocyte monocultures, and HSEs were generated under 20% (normoxia) and 3% (hypoxia) oxygen level. In all cultures under hypoxia, expression of hypoxia-inducible factor target genes was increased. Characterization of HSEs generated under hypoxia using immunohistochemical analyses of morphogenesis biomarkers revealed a reduction in epidermal thickness, reduced proliferation, similar early differentiation, and an attenuated terminal differentiation program compared to normoxia, better mimicking NHS. The stratum corneum ceramide composition was studied with liquid chromatography coupled to mass spectrometry. Under hypoxia, HSEs exhibited a ceramide composition that more closely resembles that of NHS. Consequently, the lipid organization was improved. In conclusion, epidermal morphogenesis and barrier formation in HSEs reconstructed under hypoxia better mimics that of NHS.

Characterization of human skin equivalents developed at body's core and surface temperatures.

Human skin equivalents (HSEs) are in vitro developed three-dimensional models resembling native human skin (NHS) to a high extent. However, the epidermal lipid biosynthesis, barrier lipid composition, and organization are altered, leading to an elevated diffusion rate of therapeutic molecules. The altered lipid barrier formation in HSEs may be induced by standardized culture conditions, including a culture temperature of 37°C, which is dissimilar to skin surface temperature. Therefore, we aim to determine the influence of culture temperature during the generation of full thickness models (FTMs) on epidermal morphogenesis and lipid barrier formation. For this purpose, FTMs were developed at conventional core temperature (37°C) or lower temperatures (35°C and 33°C) and evaluated over a time period of 4 weeks. The stratum corneum (SC) lipid composition was analysed using advanced liquid chromatography coupled to mass spectrometry analysis. Our results show that SC layers accumulated at a similar rate irrespective of culture temperature. At reduced culture temperature, an increased epidermal thickness, a disorganization of the lower epidermal cell layers, a delayed early differentiation, and an enlargement of granular cells were detected. Interestingly, melanogenesis was reduced at lower temperature. The ceramide subclass profile, chain length distribution, and level of unsaturated ceramides were similar in FTMs generated at 37°C and 35°C but changed when generated at 33°C, reducing the resemblance to NHS. Herein, we report that culture temperature affects epidermal morphogenesis substantially and to a lesser extent the lipid barrier formation, highlighting the importance of optimized external parameters during reconstruction of skin.

Applying a vernix caseosa based formulation accelerates skin barrier repair by modulating lipid biosynthesis.

Restoring the lipid homeostasis of the stratum corneum (SC) is a common strategy to enhance skin barrier function. Here, we used a ceramide containing vernix caseosa (VC)-based formulation and were able to accelerate barrier recovery in healthy volunteers. The recovery was examined over 16 days by monitoring trans-epidermal water loss (TEWL) after barrier disruption by tape-stripping. Four skin sites were used to examine the effects of both treatment and barrier recovery. After 16 days, samples were harvested at these sites to examine the SC ceramide composition and lipid organization. Changes in ceramide profiles were identified using principal component analysis. After barrier recovery, the untreated sites showed increased levels of ceramide subclass AS and ceramides with a 34 total carbon-atom chain length, while the mean ceramide chain length was reduced. These changes were diminished by treatment with the studied formulation, which concurrently increased the formulated ceramides. Correlations were observed between SC lipid composition, lipid organization, and TEWL, and changes in the ceramide subclass composition suggest changes in the ceramide biosynthesis. These results suggest that VC-based formulations enhance skin barrier recovery and are attractive candidates to treat skin disorders with impaired barrier properties.

Altered expression of epidermal lipid bio-synthesis enzymes in atopic dermatitis skin is accompanied by changes in stratum corneum lipid composition.

BACKGROUND: The barrier dysfunction in atopic dermatitis (AD) skin correlates with stratum corneum (SC) lipid abnormalities including reduction of global lipid content, shorter ceramide (CER) as well as free fatty acid (FFA) chain length and altered CER subclass levels. However, the underlying cause of these changes in lipid composition has not been fully investigated. AIM: We investigated whether the expression of CER and FFA biosynthesis enzymes are altered in AD skin compared with control skin and determine whether changes in enzyme expression can be related with changes in lipid composition. METHODS: In AD patients and controls the expression of enzymes involved in the biosynthesis of FFAs and CERs was analyzed in relation to the SC lipid composition. These enzymes include stearoyl CoA desaturase (SCD), elongase 1 (ELOVL1) and ELOVL6 involved in FFA synthesis and ß-glucocerebrosidase (GBA), acid-sphingomyelinase (aSmase), ceramide synthase 3 (CerS3) involved in CER synthesis. In TH2 treated human skin equivalents (AD HSEs) mimicking lesional AD skin, the mRNA expression of these enzymes was investigated. RESULTS: The results reveal an altered expression of SCD and ELOVL1 in AD lesional skin. This was accompanied by functional changes displayed by increased unsaturated FFAs (SCD) and reduced FFA C22-C28 (ELOVL1) in AD lesional skin. The expression of GBA, aSmase and CerS3 were also altered in lesional skin. The CER composition in AD lesional skin showed corresponding changes such as increased CER AS and NS (aSmase) and decreased esterified ω-hydroxy CERs (CerS3). In support of the results from AD skin, the AD HSEs showed reduced mRNA ELOVL1, GBA and a Smase levels. CONCLUSION: This study shows that alterations in the expression of key enzymes involved in SC lipid synthesis contribute to changes in the lipid composition in AD skin and inflammation may influence expression of these enzymes.

Quantitative analysis of ceramides using a novel lipidomics approach with three dimensional response modelling.

In the outermost layer of the skin, the stratum corneum (SC), ceramides form a diverse and essential pool of lipids. Due to their diversity and the limited availability of synthetic standards it is challenging to quantitatively analyse all SC ceramides independently. We aim to perform a detailed analysis of ceramides on SC harvested from in vivo and ex vivo skin, therefore, a LC/MS method was developed in which all steps from sample acquisition until data analysis were examined and optimized. Improving extraction efficiency of ceramides resulted in an increase in efficiency from 71.5% to 99.3%. It was shown that sample harvesting by tape-stripping in vivo was accurate and precise. A full scan MS method was developed, compatible with all sample types, enabling simultaneously qualitative and quantitative data analysis. A novel three dimensional response model was constructed to quantify all detected ceramides from full scan data using a limited amount of synthetic ceramides. The application is demonstrated on various SC sample types. When ex vivo SC was regenerated during human skin culture, increases are observed in the amount of the ceramide sphingosine subclasses, in mono unsaturated ceramides (which have an cis-double bond in the acyl chain), and ceramides with a short C34 carbon chain (ceramides with a total carbon chain of 34 carbon atoms), compared with native human skin. These changes in ceramide levels are also often encountered in diseased skin.

Modulation of stratum corneum lipid composition and organization of human skin equivalents by specific medium supplements.

Our in-house human skin equivalents contain all stratum corneum (SC) barrier lipid classes, but have a reduced level of free fatty acids (FAs), of which a part is mono-unsaturated. These differences lead to an altered SC lipid organization and thereby a reduced barrier function compared to human skin. In this study, we aimed to improve the SC FA composition and, consequently, the SC lipid organization of the Leiden epidermal model (LEM) by specific medium supplements. The standard FA mixture (consisting of palmitic, linoleic and arachidonic acids) supplemented to the medium was modified, by replacing protonated palmitic acid with deuterated palmitic acid or by the addition of deuterated arachidic acid to the mixture, to determine whether FAs are taken up from the medium and are incorporated into SC of LEM. Furthermore, supplementation of the total FA mixture or that of palmitic acid alone was increased four times to examine whether this improves the SC FA composition and lipid organization of LEM. The results demonstrate that the deuterated FAs are taken up into LEMs and are subsequently elongated and incorporated in their SC. However, a fourfold increase in palmitic acid supplementation does not change the SC FA composition or lipid organization of LEM. Increasing the concentration of the total FA mixture in the medium resulted in a decreased level of very long chain FAs and an increased level of mono-unsaturated FAs, which lead to deteriorated SC lipid properties. These results indicate that SC lipid properties can be modulated by specific medium supplements.

Intercellular skin barrier lipid composition and organization in Netherton syndrome patients.

Netherton syndrome (NTS) is a rare genetic skin disease caused by mutations in the serine protease inhibitor Kazal-type 5 gene, which encodes the lympho-epithelial Kazal-type-related inhibitor. NTS patients have profoundly impaired skin barrier function. As stratum corneum (SC) lipids have a crucial role in the skin barrier function, we investigated the SC lipid composition and organization in NTS patients. We studied the SC lipid composition by means of mass spectrometry, and the lipid organization was examined by infrared spectroscopy and X-ray diffraction. Decreased free fatty acid (FFA) chain length and increased levels of monounsaturated FFAs were observed in the SC of NTS patients compared with controls. Furthermore, the level of short-chain ceramides (CERs) was enhanced in NTS patients and a strong reduction in long-chain CER levels was seen in several patients. The changes in lipid composition modified the lipid organization leading to an increased disordering of the lipids compared with the controls. In addition, in a subgroup of patients the organization of the lipid layers changed dramatically. The altered FFA and CER profiles in NTS patients corresponded to changes in the expression of enzymes involved in SC lipid processing. The observed changes in lipid composition, lipid organization, and enzyme expression are likely to contribute to the barrier dysfunction in NTS.

Combined LC/MS-platform for analysis of all major stratum corneum lipids, and the profiling of skin substitutes.

Ceramides (CERs), cholesterol, and free fatty acids (FFAs) are the main lipid classes in human stratum corneum (SC, outermost skin layer), but no studies report on the detailed analysis of these classes in a single platform. The primary aims of this study were to 1) develop an LC/MS method for (semi-)quantitative analysis of all main lipid classes present in human SC; and 2) use this method to study in detail the lipid profiles of human skin substitutes and compare them to human SC lipids. By applying two injections of 10µl, the developed method detects all major SC lipids using RPLC and negative ion mode APCI-MS for detection of FFAs, and NPLC using positive ion mode APCI-MS to analyze CERs and cholesterol. Validation showed this lipid platform to be robust, reproducible, sensitive, and fast. The method was successfully applied on ex vivo human SC, human SC obtained from tape strips and human skin substitutes (porcine SC and human skin equivalents). In conjunction with FFA profiles, clear differences in CER profiles were observed between these different SC sources. Human skin equivalents more closely mimic the lipid composition of human stratum corneum than porcine skin does, although noticeable differences are still present. These differences gave biologically relevant information on some of the enzymes that are probably involved in SC lipid processing. For future research, this provides an excellent method for (semi-)quantitative, 'high-throughput' profiling of SC lipids and can be used to advance the understanding of skin lipids and the biological processes involved.