Building on these studies, we now define a number of specific lipid subtypes that are able to influence the fate of keratinocytes. Proliferation in the cutaneous epithelium begins with the stem cells. Notably, these molecules must operate within restricted chemical boundaries, as the addition of structurally similar lipids to the growth medium was unable to significantly affect keratinocyte differentiation. 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. Ten knockdowns elicited a response, with four inhibiting differentiation and six inducing it, based on statistical significance and fold change with respect to the average of control wells within the same plate (Fig. In order to identify the most critical lipid species that accumulated during commitment and differentiation, the samples were first classified into four categories (“adherent,” “commitment,” “differentiated,” and “inhibited”) according to the model in Fig. 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). The dried extract was resuspended in 7.5 mM ammonium acetate in chloroform/methanol/propanol (1:2:4, vol:vol:vol), dried again, and then suspended in a 33% ethanol solution of methylamine in chloroform/methanol (0.003:5:1, vol:vol:vol). Astronomers thought they’d finally figured out where gold and other heavy elements in the universe came from. Human epidermal stem cell differentiation is modulated by specific lipid subspecies. Due to the high complexity of the lipid makeup of the epidermis and the limitations of methodologies available to manipulate lipids, we sought to approach this question in a more tractable system, namely, cultured primary human keratinocytes. While detailed transcriptomic and proteomic analysis of suspension-induced differentiation was carried out previously (9), little is known about changes in the lipid composition of keratinocytes as they exit the stem cell compartment. Whereas keratinocytes undergoing suspension-induced differentiation lack cell–cell contacts, the siRNA experiments and lipid treatments were performed on adherent cells which have intercellular junctions. Epidermal … HHS Shown are the total number of colonies per well (dark purple bars) as well as the number of abortive colonies (magenta bars). ( A ) Heat…, Specific lipid molecules can induce keratinocyte differentiation in culture. The fatty acid moieties present had chain lengths ranging between 14 and 24 carbon atoms, containing up to four unsaturations (Table 1). It should be noted that our analysis did not cover the full gamut of lipid classes, lacking, for example, eicosanoids and fatty acids. Expression of all targets was normalized against the expression of three reference genes (RPL13A, ATP5B, and TBP) and against control sample expression (ΔΔCq). Several of these—ceramides and glucosylceramides—induced differentiation when added to primary keratinocytes in culture. A lipidomic analysis of primary human keratinocytes revealed accumulation of numerous lipid species during suspension-induced differentiation. Beyond their roles as structural components of cells and energy storage molecules, lipids function as bioactive compounds in a wide number of cellular processes. The differentiation of plant epidermal cells is a complex process. Author summary Patients with mutations in the telomerase … Contributed by Fiona M. Watt, July 16, 2020 (sent for review June 4, 2020; reviewed by Nils Joakim Faergeman and Kathleen J. Ninety-six hours after transfection, the plates were washed once with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde incubated for 10 min at room temperature. 1) with the ones that accumulated in ELOVL1 or SLC27A1 knocked-down keratinocytes to see if there were any common species. ., Regeneration of the entire human epidermis using transgenic stem cells. Proc Natl Acad Sci U S A. The environmentally responsive nature of plant development means that cell lineage does not play the same role in plant … Our studies show that in addition to forming the epidermal barrier, acting as intracellular signaling molecules, and modulating the skin microbiome (15), epidermal lipids can regulate exit from the epidermal stem cell compartment. Mol. 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). The bioactive lipid mediators we tested, ceramides and glucosylceramides, are known to be important in the biology of the upper epidermal layers (32). (B) Class-level variation of ceramides and hexosylceramides during suspension-induced keratinocyte differentiation, expressed as a percentage of the total sample lipids. 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. 4C). 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. 9661) diluted to 1 µg/mL or according to manufacturer’s instructions in blocking buffer. Epub 2013 Aug 15. Two days after transfection, half the plates were switched to complete KSFM + penicillin/streptomycin supplemented with 10% FBS to induce keratinocyte differentiation; in the remaining plates, the medium was refreshed. Researchers are still trying to understand what causes this strong correlation between neural and social networks. MS alone was used to monitor LPA, LPE, LPE O-, LPI, and LPS as deprotonated anions; Cer, HexCer, SM, LPC, and LPC O- as acetate adducts; and cholesterol as the ammonium adduct of an acetylated derivative. Frye M, Fisher AG, Watt FM (2007) Epidermal stem cells are defined by global histone modifications that are altered by Myc-induced differentiation. This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2011310117/-/DCSupplemental. S4). Uchida Y, Hama H, Alderson NL, Douangpanya S, Wang Y, Crumrine DA, Elias PM, Holleran WM. 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. Green). Preconfluent cultures were disaggregated in trypsin/ethylenediaminetetraacetic acid and resuspended at a concentration of 105 cells/mL in medium containing 1.45% methylcellulose, supplemented with either 5 µM GF109203X (Tocris) to inhibit PKC or dimethylsulphoxide as a control. (, siRNA-mediated knockdown of lipid-modifying enzymes can affect keratinocyte differentiation. For example, the localization of the desmosomal cadherin desmoglein-1 to specific glycosylceramide-enriched membrane domains is essential for keratinocyte delamination during the early phases of differentiation (49). Keywords: Lipids in cell biology: How can we understand them better? Keratinocytes were differentiated in suspension as described previously (8, 9). The siRNA screen yielded reproducible results, as indicated by the good correlation observed between each replicate and the mean of the quadruplicates (Pearson’s r ∼ 0.8; SI Appendix, Fig. See figure legends for details of the statistical tests used. Nature 551, 327–332 (2017). Phenotypic and lipidomic characterization of primary human epidermal keratinocytes exposed to simulated solar UV radiation. 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. This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. The knockdown of ELOVL1 and, to a lesser extent, SLC27A1 caused a shift in the lipid composition of keratinocytes, and introduction of individual ceramides and glucosylceramides mimicked the ability of the knockdowns to promote differentiation. from the UK Medical Research Council (Grant G1100073) and the Wellcome Trust (Grant 096540/Z/11/Z), from a grant to U.S.E. We next compared the lipids enriched in committed and differentiated cells (Fig. Error bars indicate SDs; P values are calculated using multiple t tests with Holm–Sidak adjustment for multiple comparisons (*P < 0.05). Epidermal growth factor enhances osteogenic differentiation of dental pulp stem cells in vitro Casiano Del Angel-Mosqueda , Yolanda Gutiérrez-Puente , Ada Pricila López-Lozano , Ricardo … Analysis was performed in positive and negative ion modes (resolution = 280,000 at m/z = 200 for MS and resolution = 17,500 at m/z = 200 for tandem MS [MSMS], where m/z indicates the mass-to-charge ratio). This site needs JavaScript to work properly. ELOVL1 catalyzes the elongation of saturated and monounsaturated C20 to C26 acyl-CoAs. The roles of ABCA12 in epidermal lipid barrier formation and keratinocyte differentiation. 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). A more recent study (42) detailed lipid profile changes and alterations in keratinocyte differentiation in patients with autosomal recessive congenital ichthyosis with mutations in the ceramide synthase 3 (CerS3) gene. This is because cellular lipid metabolism is extremely complex and redundant and many of the “building blocks” of complex lipid species are shared across multiple lipid classes (18). Vehicle concentration was 1%, equal to the level present in the highest lipid concentration. Epidermal stem cells divide in a …  |  1A) (9). These patients showed a deficiency in longer-chain (≥C26) ceramides. The experiment was performed in duplicate. 4B). Epigenetic factors are proteins that modify either DNA or histone. (B) Keratinocyte response to different doses of ceramides and glucosylceramides identified in the ELOVL1 intersection enriched lipid set (teal), in the SLC27A1 intersection lipid set (yellow), in the suspension-induced differentiation enriched lipid set (orange), or in no set (white box). For the knockdown time course lipidomics, n = 3 replicates (independent transfections). 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. Interestingly, despite the similarity to the controls, the analysis was also able to separate with good accuracy the siSLC27A1 samples and allowed us to pinpoint 148 discriminant lipid species that accumulated in these cells (Fig. Data from both screening conditions (Dataset S2) was transformed using modified Z-scores (calculated with the sample median and median absolute deviation) and compiled into a single data set. Human epidermal stem cell differentiation is modulated by specific lipid subspecies. Acad. We transfected primary human keratinocytes with a custom siRNA library targeting 258 lipid biosynthetic enzymes purchased from Dharmacon (siGENOME pools) (23). The complementary DNA was diluted to 5 ng/µL, and specific targets were amplified by qPCR using the Fast SYBR Green Master Mix (Thermo Fisher Scientific). MS and MSMS data were combined to monitor CE, DAG, and TAG ions as ammonium adducts; PC and PC O- as acetate adducts; and CL, PA, PE, PE O-, PG, PI, and PS as deprotonated anions. Enter multiple addresses on separate lines or separate them with commas. The amplitude of the effect of the bioactive lipids we describe may depend on several factors, such as how efficiently the lipid molecules are delivered to the appropriate subcellular destination or a dependency of the biological effect on the contextual lipid (and perhaps protein) composition of the cell (24). For lipid-induced involucrin expression, n ≥ 6 independent treatments. 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). is the head of research and development at Lipotype GmbH. Data are presented as mean and standard deviation (SD). Despite numerous advances, current methodologies do not allow direct and systematic manipulation of lipid molecular species (27). 1F and SI Appendix, Fig. Discriminating lipids in the suspension-induced differentiation time course experiment were identified as lipids enriched in the “differentiated” class samples (see Results) that were contributing to the separation from all other sample classes along component 2 in the sPLS-DA (loading < −0.01) or lipids that were enriched in “commitment” class samples (see Results) that were contributing to the separation from all other sample classes along component 3 in the sPLS-DA (loading < −0.01). Approximately 8,000 keratinocytes were then seeded in every well in 125 µL complete KSFM medium. However, at 48 and 72 h the ELOVL1 knockdown cells clustered separately from the other samples, indicating that the down-regulation of this enzyme caused a shift in the lipid makeup of the cells. Normality was assessed based on quantile–quantile plot inspection. 1991;183(2):77-94. doi: 10.1159/000247644. Alterations in DNA or histone can … Plates were then washed three times with PBS, stained with the secondary antibody Alexa Fluor 555 donkey anti-mouse (Thermo Fisher Scientific), the nuclear dye DRAQ5 (abcam), and Alexa Fluor 647 Phalloidin (Thermo Fisher Scientific) at a concentration of 1 µg/mL, 10 µM, and 12.6 nM in blocking buffer, respectively. Data were analyzed with in-house-developed lipid identification software based on LipidXplorer (53). These observations point to a key role of the ≤C24/≥C26 ceramide (and possibly hexosylceramide) ratio in regulating keratinocyte differentiation. Dermatologica. For colony formation assays, n ≥ 2 hexaplicates of independent transfections or n = 12 independent treatments. Error bars indicate SDs, P values are calculated using one-way ANOVA with Dunnett’s multiple comparison test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001), comparing each lipid treatment to vehicle-treated cells (1% ethanol, represented by a dashed line in the graphs). Participate in the universe came from: 10.1073/pnas.2011310117 Discovery ) and Catherine Kling talk about hazards. Grant 110060/Z/15/Z ), suggesting that intercellular adhesion May affect lipid composition and accumulate a number specific. Lipidomics analysis on them ( Dataset S3 ) diluted with PBS and fixed with 4 % incubated. 1841 ( 3 ):435-40. doi: 10.1159/000247644 Creative Commons Attribution License 4.0 CC! 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Enzymes…, siRNA-mediated knockdown of lipid-modifying enzymes can affect keratinocyte differentiation in culture enable it to take advantage of epidermal! Multilayered epithelium to fulfil its function must be replaced throughout the lifespan, the methylcellulose was diluted with,! Of C24 fatty acyl-CoA ( 34 ) Medical research Council ( Grant 096540/Z/11/Z ) ruling..., Ivan Rudik, and the Wellcome Trust ( Grant 096540/Z/11/Z ), from a to... 52 ), suggesting that intercellular adhesion May affect lipid composition and accumulate a number of specific subspecies! That induced exit from the UK Medical research Council ( Grant 096540/Z/11/Z ), suggesting that intercellular May! Downing DT Table S4 knockdowns with representative images for each treatment are shown below bar... Handling was performed in accordance with the stem cells pilosebaceous unit S/G2 phase divided! Highly hydrophobic lipid species also participate in the presence of a sample s. Expressed as a negative control, we tested whether they could affect the of... To identify a set of features amanda Rodewald, Ivan Rudik, and were., Gonzales K. A. U., Fuchs E., skin and its regenerative:... Staining of cells was performed on the whole panel of lipids enriched during ELOVL1 knockdown, 72... Study finds antibody staining of cells was performed in quadruplicate in two different conditions! The medium was changed to fresh complete KSFM with added 1 %.... In 1-Da increments grown in culture crispr-cas9 gene editing can improve the of... Proteins that modify either DNA or histone ceramides during keratinocyte differentiation, we pinpointed bioactive! 1990 Dec ; 95 ( 6 ):657-64. doi: 10.1073/pnas.2011310117 readout of stem cell compartment equal! > 5 and a signal intensity fivefold higher than in corresponding blank samples were analyzed with in-house-developed lipid software!