To examine how lipids were affected in suspension culture, we analyzed samples using a shotgun lipidomics approach (25), comparing control and PKCi-treated cells (Dataset S1). Primary keratinocytes were used in experiments at passages 4–7. Stem cell transplantation is reported to promote skin healing, endothelial cell transformation, and vascular formation. 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. -, Hirsch T. et al. Astronomers thought they’d finally figured out where gold and other heavy elements in the universe came from. Elovl1 knockout mice die shortly after birth due to skin barrier deficiencies caused by the impaired formation of lipid lamellae and defective desquamation (34). (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). (A) Heat map representation and 2D clustering (Euclidean distance and complete-linkage clustering) of the Z-scores of all lipid species identified in the lipidomic analysis of differentiation-inducing knockdowns and control keratinocytes. Terminally differentiating cells were identified by manual thresholding of the involucrin perinuclear fluorescence intensity. To minimize the misattribution of the cytoplasm areas, the level of cytoplasmic staining was inferred from the fluorescence intensity in a ring around the nucleus, as involucrin staining was homogeneous throughout the cytoplasm. In plants, differentiation of the epidermal cells occurs during embryogenesis in a developing seed. We also performed colony formation assays on the whole panel of candidate differentiation-inhibiting knockdowns but did not observe any significant effects (Fig. 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. To identify the critical lipid species that accumulated in the knockdown cells, we conducted a sPLS-DA analysis on the 48 and 72 h samples separately (Fig. COVID-19 is an emerging, rapidly evolving situation. 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). S3B). Author summary Patients with mutations in the telomerase … Telomere dysfunction impairs epidermal stem cell specification and differentiation by disrupting BMP/pSmad/P63 signaling. S1A). When looking at the full panel of individual lipid species, unsupervised clustering of the samples (Fig. Human epidermal stem cell differentiation is modulated by specific lipid subspecies. The screening was performed in quadruplicate in two different culture conditions. Fatty acid 2-hydroxylase, encoded by FA2H, accounts for differentiation-associated increase in 2-OH ceramides during keratinocyte differentiation. During this migration, keratinocytes undergo a terminal differentiation process, accumulating transglutaminase-cross-linked proteins and secreting lamellar bodies enriched in highly hydrophobic lipid 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. Proc Natl Acad Sci U S A. 1D). 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. 4B). This potentially indicates the existence of compensatory mechanisms to maintain skin homeostasis in vivo. Alterations in DNA or histone can …  |  Proc. Keratinocyte lipid composition changes during suspension-induced differentiation. Copyright © 2020 the Author(s). A lipidomic analysis of primary human keratinocytes revealed accumulation of numerous lipid species during suspension-induced differentiation. 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). Clonal growth assays are used as a quantitative readout of stem cell abundance in cultures of human epidermal keratinocytes (4⇓–6). Epub 2020 Aug 25. 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). 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. See figure legends for details of the statistical tests used. Enrichment and fold change were determined by comparing the means of the replicates of each sample. However, the m… Researchers are still trying to understand what causes this strong correlation between neural and social networks. Samples in the siRNA knockdown time course were analyzed by PCA using the mixOmics package (v6.10.9) (54) with parameter scale = T after removal of near-zero variance predictors. laboratory for helpful discussions. 1A) (9). 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Our results reveal the potential of lipid subspecies to regulate exit from the epidermal stem cell compartment. This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. (F) Contribution of the 50 most discriminant lipid species to the separation along the second (Upper panel) and third (Lower panel) components of sPLS. Keywords: Building on these studies, we now define a number of specific lipid subtypes that are able to influence the fate of keratinocytes. Epub 2013 Aug 15. Epidermal keratinocyte (KC), the major cell type in the skin epidermis, plays critical roles in forming a permeability barrier to separate internal organs from external stimuli. Upon treatment, the stock solutions were heated to 37 °C, diluted to the indicated concentrations in culture medium preheated to 37 °C, and finally added to the monolayers. Copyright © 2021 National Academy of Sciences. (. Aliquots were plated in six-well plates coated with 0.4% polyhydroxyethylmethacrylate; this ensured that there was no cell–substratum adhesion. 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. 2.2. stem cell niche in skin. Epub 2018 Jul 10. We next compared the lipids enriched in committed and differentiated cells (Fig. Single-cell suspension-induced differentiation of primary human keratinocytes can be blocked in the presence of a protein kinase C inhibitor (PKCi) (Fig. See this image and copyright information in PMC. : posttransfection. This site needs JavaScript to work properly. Hexosylceramides can have a glucose or a galactose as their head group, but we assumed them to be glucosylceramides due to the established presence and importance of this lipid class in the epidermis (14, 32). Having identified a panel of lipids enriched during keratinocyte differentiation, we asked whether any of them regulated the onset of differentiation. For decades, stem cells of the hair follicle were though to reside in the highly proliferative matrix cellular compartments (Kligman, 1959).In the early nineties, Cotsarelis and … ), we show that, during homeostasis, differentiation of epithelial stem cells from the basal epidermal layer leads to neighboring cell size increase, cell cycle progression, and, ultimately, cell division. S2A). A common set of individual lipid molecules was enriched in cells stimulated to differentiate by suspension culture or by either ELOVL1 or SLC27A1 knockdown. Plates were then permeabilized by incubating with 0.2% Triton-X-100 in PBS for 5 min at room temperature, incubated with blocking buffer (10% FBS, 0.25% fish skin gelatin in PBS) for 1 h and stained overnight at +4 °C with the primary antibody anti-involucrin (SY3 or SY7 clones) or anti-cleaved caspase 3 (Asp175) (Cell Signaling catalog no. Frye M, Fisher AG, Watt FM (2007) Epidermal stem cells are defined by global histone modifications that are altered by Myc-induced differentiation. 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. Enter multiple addresses on separate lines or separate them with commas. [ PMC free article ] [ … The analysis identified 195 unique discriminant lipid species enriched in siELOVL1 cells at either time point. These patients showed a deficiency in longer-chain (≥C26) ceramides. Deconvolution of the two validated siRNA pools did not suggest off-target effects (SI Appendix, Fig. 3 C and D). differentiation; epidermis; keratinocytes; lipidomics; lipids. The cells undergo a differentiation commitment phase after 4 h and increase expression of differentiation markers between 8 and 24 h (8, 9). The siRNAs were incubated for 20 min with 10 µL of INTERFERin-HTS transfection reagent (PolyPlus Transfection) diluted 1/200 in water. 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 … 1F and SI Appendix, Fig. (A) Overlap between the discriminant lipid sets enriched during ELOVL1 knockdown, SLC27A1 knockdown, or suspension-induced keratinocyte differentiation. By intersecting lipidomic datasets from suspension-induced differentiation and knockdown keratinocytes, we pinpointed candidate bioactive lipid subspecies as differentiation regulators. NIH 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). Lipids are essential for the establishment of an efficient epidermal barrier (10). FATP1 can be localized in the endoplasmic reticulum or in the plasma membrane (39), which might indicate a role in the intracellular distribution of lipids rather than their abundance. 1990 Dec;95(6):657-64. doi: 10.1111/1523-1747.ep12514333. 1B). Dev. Actin and serum response factor transduce physical cues from the microenvironment to regulate epidermal stem cell fate decisions, Diverse epigenetic strategies interact to control epidermal differentiation, Involucrin and other markers of keratinocyte terminal differentiation, RNAi transfection results in lipidome changes, Human epidermal glucosylceramides are major precursors of stratum corneum ceramides, ELOVL1 production of C24 acyl-CoAs is linked to C24 sphingolipid synthesis, Impaired epidermal permeability barrier in mice lacking elovl1, the gene responsible for very-long-chain fatty acid production, Fatty acid transporters in skin development, function and disease, Differential expression of fatty acid transport proteins in epidermis and skin appendages, Fatty acid transport protein 1 can compensate for fatty acid transport protein 4 in the developing mouse epidermis, Exome sequencing identifies FATP1 mutation in Melkersson-Rosenthal syndrome, Activation of keratinization of keratinocytes from fetal rat skin with N-(O-linoleoyl) ω-hydroxy fatty acyl sphingosyl glucose (lipokeratinogenoside) as a marker of epidermis, Impaired epidermal ceramide synthesis causes autosomal recessive congenital ichthyosis and reveals the importance of ceramide acyl chain length, Stem cell signaling. Five hundred keratinocytes were plated on a 3T3 feeder layer per well of a six-well dish. 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). Would you like email updates of new search results? Primer details can be found in SI Appendix, Table S4. (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. 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. Analysis of control (R26 mT/mG) head skin epidermis showed that asymmetric divisions, once considered the predominant division type of epidermal progenitors, occurred in approximately 44% of cases, and that symmetric renewal was nearly perfectly balanced by symmetric differentiation … 3A) revealed that the main factor contributing to sample grouping was time, with cultured primary keratinocytes exhibiting a striking plasticity in their lipid composition, even in control conditions. At present, great progress has been made in the study of epidermal stem cells at the cellular and molecular levels. Modified Z-score transformation of the data (using the sample median and median absolute deviation) allowed pooling of both culture conditions in a single dataset. This work was funded by grants to F.M.W. MS-based lipidomics was performed by Lipotype GmbH (51). Published by PNAS. The environmentally responsive nature of plant development means that cell lineage does not play the same role in plant … Please enable it to take advantage of the complete set of features! 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). Keratinocyte lipid composition changes during…, Keratinocyte lipid composition changes during suspension-induced differentiation. 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). Specific lipid molecules can induce keratinocyte differentiation in culture. National Center for Biotechnology Information, Unable to load your collection due to an error, Unable to load your delegates due to an error, Keratinocyte lipid composition changes during suspension-induced differentiation. Plates were then permeabilized by incubating with 0.2% Triton-X-100 in PBS for 5 min at room temperature and incubated with blocking buffer (10% FBS, 0.25% fish skin gelatin in PBS) for 1 h and stained overnight at +4 °C with the primary antibody anti-involucrin (SY3 or SY7 clones) diluted to 1 µg/mL in blocking buffer. performed research; U.S.E. 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 … We are grateful for funding from the Department of Health via the National Institute for Health Research comprehensive Biomedical Research Centre award to Guy’s and St Thomas’ National Health Service Foundation Trust in partnership with King’s College London and King’s College Hospital NHS Foundation Trust. The organic phase was dried in a speed vacuum concentrator. 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… -, Barrandon Y., Green H., Three clonal types of keratinocyte with different capacities for multiplication. This accumulation was blocked on inhibition of PKC (Fig. 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. Despite numerous advances, current methodologies do not allow direct and systematic manipulation of lipid molecular species (27). It is expressed in the basal layer of adult human epidermis (38). The experiment was performed in duplicate. A two-step chloroform/methanol procedure was used to extract the lipids (52), and samples were spiked with an internal lipid standard mixture. Proliferation in the cutaneous epithelium begins with the stem cells. 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. Beyond their roles as structural components of cells and energy storage molecules, lipids function as bioactive compounds in a wide number of cellular processes. (B) Sample variation along the first two principal components. 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. 2018 Oct;92(1):97-105. doi: 10.1016/j.jdermsci.2018.07.002. It also remains technically challenging to dissect the exact mechanisms through which the lipid subspecies identified are operating. Immunofluorescence staining for involucrin, an early marker of differentiation in cultured keratinocytes (9, 30), was used as a readout of differentiation (Dataset S2). This article contains supporting information online at 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. Functional blocking of this local feedback showed that differentiation continues to occur in the absence of cell division, resulting in a rapid depletion of the epidermal … 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. Typically, around 1-2 days after cell delamination, a neighboring cell entered S/G2 phase and divided. stem cell abundance in cultures of human epidermal keratino-cytes (4–6). By intersecting lipidomic datasets from suspension-induced differentiation and knockdown keratinocytes, we pinpointed candidate bioactive lipid subspecies as differentiation regulators. Lipids identified with a signal-to-noise ratio of >5 and a signal intensity fivefold higher than in corresponding blank samples were analyzed further. 1991;183(2):77-94. doi: 10.1159/000247644. Get the latest public health information from CDC:, Get the latest research information from NIH:, Find NCBI SARS-CoV-2 literature, sequence, and clinical content: 3B). 4A). 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. 1,2 Stem cells in this regard lack many characteristics of terminal differentiation, and have an intrinsically high proliferative … 2C) or nondifferentiating conditions (for the knockdowns that induced differentiation, Fig. 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. Our comprehensive approach has allowed us to identify a set of specific lipid molecules that induce differentiation when added to primary keratinocytes in culture. Vehicle concentration was 1%, equal to the level present in the highest lipid concentration. Lipid amounts from the lipidomics experiments were normalized by the total amount of lipids in each sample. (D) Sample variation along the first two principal components. For lipid-induced apoptosis induction, n ≥ 3 independent treatments. We tested two ceramides and two glucosylceramides from our intersection sets and included one more ceramide species that differed from a candidate molecule by having a chain two carbon atoms longer and still appeared in the set enriched during suspension-induced differentiation. The samples were compared by unsupervised hierarchical clustering using the R heatmap.2 function (package Rplots) with default settings. Phenotypic and lipidomic characterization of primary human epidermal keratinocytes exposed to simulated solar UV radiation. Competing interest statement: C.K. The differentiation of plant epidermal cells is a complex process. For knockdown or overexpression experiments, keratinocytes were grown in KSFM medium (Gibco) supplemented with 0.15 ng/mL EGF and 30 μg/mL bovine pituitary extract. Approximately 8,000 keratinocytes were then seeded in every well in 125 µL complete KSFM medium. 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). 4C). Unsupervised two-way clustering of the screen results grouped together replicates of the same treatment but not related enzymes (Fig. Error bars indicate SDs, P values are calculated using one-way ANOVA with Dunnett’s multiple comparison test (colony number) or the Kruskal–Wallis test with Dunn’s multiple comparison test (colony size) (*P < 0.05, ***P < 0.001). (C and D) Sample separation along the first two components by sPLS of the 48 h (C) and 72 h (D) samples. 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. Exp. This open access article is distributed under Creative Commons Attribution License 4.0 (CC BY). Madison KC, Swartzendruber DC, Wertz PW, Downing DT. 3 E and F and SI Appendix, Fig. 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. In light of recent results, they’re not so sure. At the highest concentration tested (100 µM) all candidate lipids, but not the control lipids, produced a significant increase in involucrin-expressing cells (Fig. HHS Lipid molecules enriched both upon ELOVL1 or SLC27A1 knockdown and upon suspension-induced differentiation of primary human keratinocytes.
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