Addition of either candidate glucosylceramide significantly decreased colony size (Fig. Immunofluorescence staining for involucrin, an early marker of differentiation in cultured keratinocytes (9, 30), was used as a readout of differentiation (Dataset S2). In vertebrate development, melanocytes originate from the neural crest and undergo a complex process of fate specification, proliferation, survival and differentiation, before finally residing in the epidermis … Lipid molecules enriched both upon ELOVL1 or SLC27A1 knockdown and upon suspension-induced differentiation of primary human keratinocytes. Indeed, several studies suggest tight regulation and diverse functions for specific lipid molecular structures in a variety of cellular processes, ranging from cell division (23) to the innate immune response (24). from the Wellcome Trust (Grant 110060/Z/15/Z), and from a grant to F.M.W. Keratinocytes were differentiated in suspension as described previously (8, 9). The contribution of lipid species to components 2 and 3 could therefore identify which discriminating molecules were enriched in the committed and differentiated samples, where a total of 145 lipids were found to accumulate (Fig. S2B), ruling out possible contributions of a sample’s position within a plate. During development, pilosebaceous formation is initiated from an early epidermal structure (the placode) that develops into a fully formed pilosebaceous unit (PSU) through a series of steps involving complex interactions with existing dermal cells. Nevertheless, the results we report corroborate a number of recent studies (23, 24) in underlining the importance and largely unexplored regulatory potential of individual lipid subspecies in cellular function and lay the foundation for further exploration of the role of lipids in epidermal differentiation. Proliferation in the cutaneous epithelium begins with the stem cells. Analysis of lipid changes at the class level showed significant accumulation of ceramides and hexosylceramides as keratinocytes underwent differentiation in suspension, mirroring the increase in these lipid classes that occurs during epidermal differentiation in vivo (14). A class-level analysis of the lipidomics data did not show any obvious divergence in accumulation/depletion trends between the control and knocked-down samples (SI Appendix, Fig. The suspended cells were subsequently incubated at 37 °C. Fatty acid 2-hydroxylase, encoded by FA2H, accounts for differentiation-associated increase in 2-OH ceramides during keratinocyte differentiation. For the knockdown time course lipidomics, n = 3 replicates (independent transfections). Slc27a1−/− mice do not exhibit skin phenotypes (39), and a loss-of-function mutation in humans linked to Melkersson–Rosenthal syndrome does not produce an epidermal phenotype (40). 4C). 1) with the ones that accumulated in ELOVL1 or SLC27A1 knocked-down keratinocytes to see if there were any common species. These cells migrate from the basal layer, home of the stem cell compartment, upward toward the surface of the skin through the spinous and granular layers to finally reach the cornified layer. The roles of ABCA12 in epidermal lipid barrier formation and keratinocyte differentiation. (F) Contribution of the 50 most discriminant lipid species to the separation along the second (Upper panel) and third (Lower panel) components of sPLS. In conclusion, we have systematically shown that the lipid composition of human keratinocytes critically impacts their differentiation, down to the level of individual lipid subspecies. Epidermal stem cells (EPSC) are a type of autologous adult stem cells that are easily obtainable from the skin. Mammalian epidermis, the outermost layer of the skin, is maintained by stem cells that self-renew and produce progeny that undergo terminal differentiation to generate the interfollicular … As a negative control, we employed one ceramide and one sphingomyelin species that did not appear in either of our enriched lipid sets. (E) Separation along three components by sPLS after fitting the samples to the model shown in A. Since it is not technically feasible to specifically target the basal layer of the epidermis for lipid delivery, either in in vitro reconstituted skin or in vivo, the physiological relevance of our findings remains to be explored fully. RNA was extracted from all samples using the RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions and subsequently reverse transcribed using the QuantiTect Reverse Transcription kit (Qiagen) according to the manufacturer’s instructions. Secondary stains were incubated for 2 h at room temperature protected from light, and plates were subsequently washed three times with PBS before being imaged using the Perkin-Elmer Operetta High-Content Imaging System. (A) Heat map representation of the Z-scores of involucrin levels in 258 lipid-modifying enzyme knockdowns (y axis) after 2D clustering of samples (x axis) (Euclidean distance and complete-linkage clustering). ( A ) Heat…, Specific lipid molecules can induce keratinocyte differentiation in culture. This would be consistent with SLC27A1 knockdown affecting differentiation without strongly perturbating the overall lipid composition of keratinocytes. 2A). Keratinocyte lipid composition changes during suspension-induced differentiation. It also remains technically challenging to dissect the exact mechanisms through which the lipid subspecies identified are operating. Unsupervised two-way clustering of the screen results grouped together replicates of the same treatment but not related enzymes (Fig. We therefore introduced perturbations in the lipid composition of adherent cultures of primary human keratinocytes by transfecting them with a panel of 258 siRNAs against lipid-modifying enzymes (23). Lipids in cell biology: How can we understand them better? In the adult mammalian epidermis, it is unclear how molecularly heterogenous stem/progenitor cell populations fit into the complete trajectory of epidermal differentiation. Copyright © 2020 the Author(s). We thank all members of the F.M.W. The cells undergo a differentiation commitment phase after 4 h and increase expression of differentiation markers between 8 and 24 h (8, 9). Epidermal … While the lipids of the outer layers of mammalian epidermis and their contribution to barrier formation have been extensively described, the role of individual lipid species in the onset of keratinocyte differentiation remains unknown. Potential regulators of epidermal stem cell self-renewal and differentiation include epigenetic factors. These results show that during suspension-induced differentiation keratinocytes extensively change their lipid composition and accumulate a number of specific lipid species. For lipid-induced involucrin expression, n ≥ 6 independent treatments. laboratory for helpful discussions. Error bars indicate SDs; P values are calculated using Dunnet’s multiple comparison test (*P < 0.05, **P < 0.01). To maximize separation between the samples and identify discriminating lipid species, we classified the samples according to the model detailed in Fig. We subsequently performed colony formation assays to further validate these effects, and in two cases (siELOVL1 and siSLC27A1) we were able to confirm a significant induction of keratinocyte differentiation on the basis of a reduction in colony formation, a surrogate of stem cell activity (9)(Fig. Our results reveal the potential of lipid subspecies to regulate exit from the epidermal stem cell compartment. (E and F) Contribution of the 50 most discriminant lipid species to the separation along the first and second components of sPLS for the 48 h (E) and 72 h (F) samples. 9661) diluted to 1 µg/mL or according to manufacturer’s instructions in blocking buffer. Plates were then washed three times with PBS, stained with the secondary antibodies Alexa Fluor 555 donkey anti-mouse (Thermo Fisher Scientific) and/or Alexa Fluor 488 donkey anti-mouse (Thermo Fisher Scientific) at 1 µg/mL, the nuclear dye DRAQ5 (abcam) at 10 µM, and Alexa Fluor 647 Phalloidin (Thermo Fisher Scientific) at 12.6 nM in blocking buffer. Science 346, 937–940 (2014). A lipidomic analysis of primary human keratinocytes revealed accumulation of numerous lipid species during suspension-induced differentiation. See figure legends for details of the statistical tests used. Approximately 8,000 keratinocytes were then seeded in every well in 125 µL complete KSFM medium. Consistent with this, principal component analysis (PCA) separated the adherent cell sample from all of the others along the first component and the differentiated cells along the second, while the committed and inhibited cells could not be clearly separated (Fig. Epidermal stem cells divide in a … Epigenetic factors are proteins that modify either DNA or histone. Principal component analysis was consistent with this observation, in that the three different time points, irrespective of treatment, separated neatly along the first component (Fig. Keratinocytes can be induced to undergo terminal differentiation in a near-synchronous manner when maintained as a single-cell suspension in medium containing methylcellulose (7, 8… (B) Sample variation along the first two principal components. The predominant lipid species change across the different epidermal layers, with the basal and spinous layers enriched in more polar lipid classes such as phospholipids and sphingomyelins, the granular layers exhibiting higher levels of glycosylceramides and cholesterol sulfates, and the cornified layer mostly composed of cholesterol, fatty acids, and ceramides, particularly ω-acylceramides that are only found in the outermost epidermal layer (14). The transfected cells were incubated under conditions (feeder-free keratinocyte serum-free medium [KSFM]) that would enrich for undifferentiated cells or were treated with medium supplemented with fetal bovine serum, which is known to stimulate accumulation of differentiated cells (28, 29). Nuclei were initially defined using the DRAQ5 channel; small (<2,000 µm2) and highly irregular (roundness < 0.6) nuclei were excluded from the analysis. S2E). is the head of research and development at Lipotype GmbH. In the principal component analysis of the lipidomic data the majority of the variance (component 1) separated cells that had been disaggregated and immediately lysed (time 0) from cells that had been maintained in suspension (Fig. 1,2 Stem cells in this regard lack many characteristics of terminal differentiation, and have an intrinsically high proliferative … Five hundred keratinocytes were plated on a 3T3 feeder layer per well of a six-well dish. Skin stem cells distributed in the basal layer of the epidermis and hair follicles are important cell sources for skin development, metabolism, and injury repair. This latter concept is consistent with the idea that lipids operate by affecting the functionality of whole assemblies of molecular partners within their localized environment, which makes their effects challenging to evaluate using the more linear approaches that are commonly employed when studying proteins (27). : posttransfection. Researchers are still trying to understand what causes this strong correlation between neural and social networks. Human epidermal stem cell differentiation is modulated by specific lipid subspecies. Nontargeting and no-siRNA wells were used as negative controls, while involucrin-targeting wells were used as positive controls for the estimation of transfection efficiency and validation of antibody specificity. Green). Reverse transfection of siRNAs was performed in accordance with the transfection reagent manufacturer’s instructions. Error bars indicate SDs. Madison KC, Swartzendruber DC, Wertz PW, Downing DT. Enrichment was determined by comparing the median of each sample class. Cell differentiation Epidermal stem cells reside in the lower part of the epidermis (stratum basale) and are attached to the basement membrane through hemidesmosomes. 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