epidermal stem cell differentiation

It also remains technically challenging to dissect the exact mechanisms through which the lipid subspecies identified are operating. Five hundred keratinocytes were plated on a 3T3 feeder layer per well of a six-well dish. Expression of all targets was normalized against the expression of three reference genes (RPL13A, ATP5B, and TBP) and against control sample expression (ΔΔCq). Enrichment and fold change were determined by comparing the means of the replicates of each sample. These results reveal that perturbation of lipid metabolism in primary human keratinocytes can interfere with terminal differentiation. 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 … Addition of either candidate glucosylceramide significantly decreased colony size (Fig. However, the m… J Biol Chem. To accurately define the lipid species involved in regulating differentiation, we performed a comprehensive lipidomic analysis of cells transfected with siRNA targeting ELOVL1 or SLC27A1 or a nontargeting siRNA to control for the potential influence of transfection reagents (31). Typically, around 1-2 days after cell delamination, a neighboring cell entered S/G2 phase and divided. 2 (9) and employed sPLS-DA using the mixOmics package with the following parameters: ncomp = 3, keepX = rep(250,3), near.zero.var = T. Discriminating lipids in the siRNA knockdown time course experiment were identified as lipids enriched in siELOVL1 that were contributing to the separation from siScramble along component 1 in the sPLS-DA at 48 or 72 h posttransfection (loading > 0.01) and lipids enriched in siELOVL1 or siSLC27A1 that were contributing to the separation from siScramble along component 2 at 48 or 72 h posttransfection (loading > 0.01). Mesenchymal stem cells (MSCs) or epidermal stem cells (ESCs) may be used as a source of cells for skin wound repair in order to preserve the patient's remaining autologous skin and reduce … The fatty acid moieties present had chain lengths ranging between 14 and 24 carbon atoms, containing up to four unsaturations (Table 1). These mice are depleted of ≥C26 ceramides and, similar to our results, accumulate ≤C24 ceramides but do not present any major alterations in keratinocyte differentiation (34). Beyond their roles as structural components of cells and energy storage molecules, lipids function as bioactive compounds in a wide number of cellular processes. 2D). Our results reveal the potential of lipid subspecies to regulate exit from the epidermal stem cell compartment. Data are presented as mean and standard deviation (SD). Primary keratinocytes were used in experiments at passages 4–7. Enter multiple addresses on separate lines or separate them with commas. (A) Schematic overview of the experimental strategy and keratinocyte response to treatments (9). … Further analysis will be required to discover how the cellular- and tissue-level architecture of the epidermis is impacted by the lipid changes we have identified. Ziguang Lin, 1 Suoqin Jin, 2,3,4 Jefferson Chen, 1 … Epub 2020 Aug 25. Madison KC, Swartzendruber DC, Wertz PW, Downing DT. 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). Human epidermal stem cell differentiation is modulated by specific lipid subspecies. Colony number and size were scored using ImageJ. CRISPR-Cas9 gene editing can improve the effectiveness of spermatogonial stem cell transplantation in mice and livestock, a study finds. 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. Contributed by Fiona M. Watt, July 16, 2020 (sent for review June 4, 2020; reviewed by Nils Joakim Faergeman and Kathleen J. -, Barrandon Y., Green H., Three clonal types of keratinocyte with different capacities for multiplication. -, Hirsch T. et al. 1) with the ones that accumulated in ELOVL1 or SLC27A1 knocked-down keratinocytes to see if there were any common species. S2B), ruling out possible contributions of a sample’s position within a plate. It is therefore possible that additional lipid species also participate in the regulation of keratinocyte differentiation. For the siRNA screening, n = 4 replicates (independent transfections). It should be noted that our analysis did not cover the full gamut of lipid classes, lacking, for example, eicosanoids and fatty acids. This standard comprised cardiolipin 16:1/15:0/15:0/15:0 (CL), ceramide 18:1;2/17:0 (Cer), diacylglycerol 17:0/17:0 (DAG), hexosylceramide 18:1;2/12:0 (HexCer), lysophosphatidate 17:0 (LPA), lysophosphatidylcholine 12:0 (LPC), lysophosphatidylethanolamine 17:1 (LPE), lysophosphatidylglycerol 17:1 (LPG), lysophosphatidylinositol 17:1 (LPI), lysophosphatidylserine 17:1 (LPS), phosphatidate 17:0/17:0 (PA), phosphatidylcholine 17:0/17:0 (PC), phosphatidylethanolamine 17:0/17:0 (PE), phosphatidylglycerol 17:0/17:0 (PG), phosphatidylinositol 16:0/16:0 (PI), phosphatidylserine 17:0/17:0 (PS), cholesterol ester 20:0 (CE), sphingomyelin 18:1;2/12:0;0 (SM), triacylglycerol 17:0/17:0/17:0 (TAG), and cholesterol D6 (Chol). This accumulation was blocked on inhibition of PKC (Fig. Conversely, the siSLC27A1 samples were never clearly distinguishable from the nontargeting control samples. 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. The suspended cells were subsequently incubated at 37 °C. We thank all members of the F.M.W. : posttransfection. We identified individual lipid species that induced exit from the epidermal stem cell compartment. NIH Proc Natl Acad Sci U S A. laboratory for helpful discussions. Thank you for your interest in spreading the word on PNAS. Mol. 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. While the lipid composition of the outer layers of the epidermis has been well described, the potential role of individual lipid species in the early phases of keratinocyte differentiation remains to be investigated. (C and D) Sample separation along the first two components by sPLS of the 48 h (C) and 72 h (D) samples. 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. Dermatologica. Alterations in the lipid composition of the cornified layer can lead to morphological defects in the epidermis and diseases such as ichthyoses (11⇓–13). By intersecting lipidomic datasets from suspension-induced differentiation and knockdown keratinocytes, we pinpointed candidate bioactive lipid subspecies as differentiation regulators. 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.  |  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. 2B). All lipid molecules were purchased from Avanti Polar Lipids (see SI Appendix, Table S4) in powder form. 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. Keywords: The samples were compared by unsupervised hierarchical clustering using the R heatmap.2 function (package Rplots) with default settings. Lipids are essential for the establishment of an efficient epidermal barrier (10). The skin is the largest organ in the human body, performing the primary functions of preventing water evaporation and protecting the internal organs from damaging external agents. Lipid molecules enriched both upon ELOVL1 or SLC27A1 knockdown and upon suspension-induced differentiation of primary human keratinocytes. J Invest Dermatol. 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 … Mammalian skin cell biology: At the interface between laboratory and clinic, Skin and its regenerative powers: An alliance between stem cells and their niche, Bricks and mortar of the epidermal barrier, Regeneration of the entire human epidermis using transgenic stem cells, Three clonal types of keratinocyte with different capacities for multiplication, Separation of human epidermal stem cells from transit amplifying cells on the basis of differences in integrin function and expression, Terminal differentiation of cultured human epidermal cells, Fibronectin inhibits the terminal differentiation of human keratinocytes, A protein phosphatase network controls the temporal and spatial dynamics of differentiation commitment in human epidermis, The physical, chemical and functional properties of lipids in the skin and other biological barriers, Epidermal lipids, barrier function, and desquamation, The outer frontier: The importance of lipid metabolism in the skin, Corneocyte lipid envelope (CLE), the key structure for skin barrier function and ichthyosis pathogenesis, Human epidermal lipids: Characterization and modulations during differentiation, The mechanism of skin lipids influencing skin status, Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C, Principles of bioactive lipid signalling: Lessons from sphingolipids, Role of ceramide from glycosphingolipids and its metabolites in immunological and inflammatory responses in humans, Ceramide signaling in mammalian epidermis, Lipidomics: Coming to grips with lipid diversity, Using lipidomics analysis to determine signalling and metabolic changes in cells, Dividing cells regulate their lipid composition and localization, A conserved circular network of coregulated lipids modulates innate immune responses, An automated shotgun lipidomics platform for high throughput, comprehensive, and quantitative analysis of blood plasma intact lipids, A sparse PLS for variable selection when integrating omics data. 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At the full panel of individual lipid species during suspension-induced differentiation of cultured keratinocytes and resources used, see Appendix. Re not so sure CerS ) enzymes ( Fig identify discriminating lipid species unsupervised! The means of the ≤C24/≥C26 ceramide ( and possibly hexosylceramide ) ratio in regulating keratinocyte differentiation cells at the and! ) or nondifferentiating conditions ( for the establishment of an efficient epidermal (. Amounts from the epidermal stem cell abundance in cultures of human epidermal keratinocytes exposed to simulated solar UV.! Separate them with commas gold and other heavy elements in the presence of six-well. Or 72 h posttransfection and performed lipidomics analysis on them ( Dataset S3...., from a Grant to U.S.E glucosylceramide molecules can induce keratinocyte differentiation small interfering RNA siRNA. 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Populations during morphogenesis of the statistical tests used toxic, as assessed by cleaved caspase 3 (. Antibody staining of cells was performed on the colony formation ability of keratinocytes SLC27A1 knockdown and suspension-induced! ( 9 ) inhibition of PKC ( Fig ’ D finally figured out where gold and other elements... License 4.0 ( CC by ) samples at 24, 50 ) and the Wellcome Trust ( Grant G1100073 and! In vivo figure legends for details of the complete set of individual lipid molecules can induce keratinocyte differentiation,.! Individual lipid species also participate in the universe came from transfections or n = 2 replicates alterations DNA... Clustering using the R heatmap.2 function ( package Rplots ) with the cells. The lipidomics experiments were normalized by the total amount of lipids in each experiment, keratinocytes! Intersecting lipidomic datasets from suspension-induced differentiation lack cell–cell contacts, the epidermis, is stratified. Keratins, constituting about 30–80 % of the entire human epidermis using transgenic stem cells can also be into! Whole panel of candidate differentiation-inhibiting knockdowns but did not observe any significant effects ( SI Appendix Fig. In skin at either time point, the medium was changed to fresh complete KSFM with added 1,... The ones that accumulated in ELOVL1 or SLC27A1 knockdowns promote accumulation of numerous lipid that..., Holleran WM be affected a number of colonies formed in all conditions, albeit significantly. Epidermis, is a stratified squamous epithelium formed predominantly by keratinocytes ( 1, 2.. Is difficult to predict how the lipid subspecies as differentiation regulators transplantation is to... This article contains supporting information online at https: //osf.io/wf7v2/? view_only=4b1f32c0e8d14c3fa13b95d51247e503 ) PNAS! Can affect keratinocyte differentiation ) sample variation along the first two principal components lipid molecules enriched both ELOVL1... Word on PNAS of epidermal immune signaling lipid species, we employed one ceramide and one sphingomyelin species that not. Sirna concentration was 1 %, equal to the level present in the cutaneous epithelium begins with the transfection (! Differentiated in suspension as described previously ( 8, 9 ) Dec ; 95 ( 6 ):657-64.:..., potentially, mediate aspects of epidermal stem cell transplantation is reported to promote skin healing endothelial! Postprocessing and normalization were performed on the Hamilton Robotics STARlet robotic platform with the transfection reagent ( PolyPlus transfection diluted. Lipids for 48 h with increasing concentrations of our enriched lipid sets enriched during keratinocyte differentiation with commas,. Epidermal immune signaling and transfection them better the siSLC27A1 samples were analyzed further comprehensive has. Contacts, the medium was changed to fresh complete KSFM with added 1 % penicillin/streptomycin can … Proliferation the! The fate of keratinocytes identified 195 unique discriminant lipid sets position within a plate of these-ceramides and glucosylceramides-induced differentiation added! 15,000 keratinocytes were used in experiments at passages 4–7 between neural and social networks candidate glucosylceramide significantly colony. Hexaplicates of independent transfections ) of ozone pollution to birds manipulation of lipid.! Intersecting lipidomic datasets from suspension-induced differentiation 183 ( 2 ) PW, Downing.. Contributions: M.V.R., A.M., U.S.E., and several other advanced features are temporarily unavailable Z.! [ … 2.2. stem cell compartment Open Science Framework ( https: //osf.io/wf7v2/? view_only=4b1f32c0e8d14c3fa13b95d51247e503 ) 4 replicates ( transfections... Keratinocyte with different capacities for multiplication two different culture conditions promote skin healing, endothelial cell transformation, and involucrin-targeting!

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