epidermal stem cell differentiation

Sample analysis (53) was performed on a Q Exactive mass spectrometer (Thermo Scientific) with a TriVersa NanoMate ion source (Advion Biosciences). To investigate whether lipids can regulate cell fate decisions, we carried out a systematic lipidomic analysis and perturbation of lipid metabolism in cultured human epidermal keratinocytes, determining associations with the onset of differentiation. 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). 2.2. stem cell niche in skin. When looking at the full panel of individual lipid species, unsupervised clustering of the samples (Fig. Single-cell suspension-induced differentiation of primary human keratinocytes can be blocked in the presence of a protein kinase C inhibitor (PKCi) (Fig. Author contributions: M.V.R., A.M., U.S.E., and F.M.W. As a negative control, we employed one ceramide and one sphingomyelin species that did not appear in either of our enriched lipid sets. Expression of all targets was normalized against the expression of three reference genes (RPL13A, ATP5B, and TBP) and against control sample expression (ΔΔCq). Building on these studies, we now define a number of specific lipid subtypes that are able to influence the fate of keratinocytes. Beyond their roles as structural components of cells and energy storage molecules, lipids function as bioactive compounds in a wide number of cellular processes. Local stem cells can also be transformed into keratinocytes, sebaceous gland, and other skin-associated tissues. 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). Human epidermis can be reconstituted in culture, forming stratified sheets in which the stem cell compartment and key elements of the terminal differentiation process are preserved (1, 4). In this respect, while the regulatory potential of individual lipid subspecies on cellular function is largely unexplored, there are a number of interesting possibilities. For validation experiments, n ≥ 2. 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). See figure legends for details of the statistical tests used. ), 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. 1991;183(2):77-94. doi: 10.1159/000247644. The analysis identified 195 unique discriminant lipid species enriched in siELOVL1 cells at either time point. Error bars indicate SDs; P values are calculated using Dunnet’s multiple comparison test (*P < 0.05, **P < 0.01). Ziguang Lin, 1 Suoqin Jin, 2,3,4 Jefferson Chen, 1 … At the highest concentration tested (100 µM) all candidate lipids, but not the control lipids, produced a significant increase in involucrin-expressing cells (Fig. 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. When subculturing or seeding cells for an experiment, we filtered the disaggregated keratinocytes through a 40 µm nylon strainer to remove cell clumps and large differentiated keratinocytes. Emergence of distinct stem cell populations during morphogenesis of the pilosebaceous unit. A lipidomic analysis of primary human keratinocytes revealed accumulation of numerous lipid species during suspension-induced differentiation. The lipids were dissolved in ethanol at a stock concentration of 10 mM and stored at −20 °C. For colony formation assays, n ≥ 2 hexaplicates of independent transfections or n = 12 independent treatments. Researchers are still trying to understand what causes this strong correlation between neural and social networks. Phenotypic and lipidomic characterization of primary human epidermal keratinocytes exposed to simulated solar UV radiation. The roles of ABCA12 in epidermal lipid barrier formation and keratinocyte differentiation. -, Barrandon Y., Green H., Three clonal types of keratinocyte with different capacities for multiplication. The amplification program used was as follows: 3 min incubation at 95 °C followed by 40 cycles of denaturation for 3 s at 95 °C and annealing/extension for 25 s at 60 °C; melting curve analysis was also performed to ensure amplifications were specific. The Wnt pathway is a key regulator of stem cells in many, if not all, tissues, and Wnts require lipid modifications for activity (43). Primer details can be found in SI Appendix, Table S4. We incubated cultured primary human keratinocytes for 48 h with increasing concentrations of our candidate bioactive lipids or the control lipids. Validation of differentiation-inhibiting (C) and differentiation-inducing (D) knockdowns by qPCR of differentiation markers (Upper) and immunofluorescence staining of involucrin (Lower); p.t. Lipids are essential for the establishment of an efficient epidermal barrier (10). is the head of research and development at Lipotype GmbH. We sought next to verify whether any of the species identified could stimulate keratinocytes to undergo differentiation. 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. For validation experiments, a similar protocol was performed, but using the ONTARGET plus siRNA series (Horizon Discovery). Get the latest public health information from CDC: https://www.coronavirus.gov, Get the latest research information from NIH: https://www.nih.gov/coronavirus, Find NCBI SARS-CoV-2 literature, sequence, and clinical content: https://www.ncbi.nlm.nih.gov/sars-cov-2/. 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 environmentally responsive nature of plant development means that cell lineage does not play the same role in plant … J Biol Chem. Primary male human keratinocytes (strain Km) isolated from neonatal foreskin were cultured at 37 °C on mitotically inactivated 3T3-J2 cells in complete F12/adenine/Dulbecco’s modified eagle medium (FAD) medium, containing one part Ham’s F-12, three parts Dulbecco’s modified eagle medium, 100 µM adenine, 10% (vol/vol) fetal bovine serum (FBS), 0.5 μg/mL hydrocortisone, 5 μg/mL insulin, 0.1 nM cholera toxin, and 10 ng/mL epidermal growth factor (EGF), as described previously (9). This potentially indicates the existence of compensatory mechanisms to maintain skin homeostasis in vivo. 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. Final volume was 175 µL per well, and the final siRNA concentration was 30 nM. Our comprehensive approach has allowed us to identify a set of specific lipid molecules that induce differentiation when added to primary keratinocytes in culture. S3B). 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. See this image and copyright information in PMC. A two-step chloroform/methanol procedure was used to extract the lipids (52), and samples were spiked with an internal lipid standard mixture. (, Specific lipid molecules can induce keratinocyte differentiation in culture. It is therefore possible that additional lipid species also participate in the regulation of keratinocyte differentiation. 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. A small interfering RNA screen of 258 lipid-modifying enzymes identified two genes that on knockdown induced epidermal differentiation: ELOVL1, encoding elongation of very long-chain fatty acids protein 1, and SLC27A1, encoding fatty acid transport protein 1. S1A). 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. MS-based lipidomics was performed by Lipotype GmbH (51). S3 C and D and Tables S2 and S3). differentiation; epidermis; keratinocytes; lipidomics; lipids. We show that differentiation, from commitment to exit from the stem cell layer, is a multi-day process wherein cells transit through a continuum of transcriptional changes. 3B). from the Wellcome Trust (Grant 110060/Z/15/Z), and from a grant to F.M.W. Primary keratinocytes were used in experiments at passages 4–7. (C and D) Sample separation along the first two components by sPLS of the 48 h (C) and 72 h (D) samples. 2E). (C) Heat map representation and two-dimensional (2D) clustering of samples (Euclidean distance and complete-linkage clustering) based on the Z-scores of all lipid species (y axis) identified in the lipidomic analysis of suspension-induced keratinocyte differentiation; different time points/conditions are shown along the x axis; dendrogram branches are color-coded according to the schematic in A. Aliquots were plated in six-well plates coated with 0.4% polyhydroxyethylmethacrylate; this ensured that there was no cell–substratum adhesion. The suspended cells were subsequently incubated at 37 °C. S1C and Table S1). Cell differentiation Epidermal stem cells reside in the lower part of the epidermis (stratum basale) and are attached to the basement membrane through hemidesmosomes. S4). Several of these-ceramides and glucosylceramides-induced differentiation when added to primary keratinocytes in culture. Enter multiple addresses on separate lines or separate them with commas. Lipids in cell biology: How can we understand them better? 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. Mol. We thank all members of the F.M.W. We collected samples at 24, 48, or 72 h posttransfection and performed lipidomics analysis on them (Dataset S3). 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. 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. 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. COVID-19 is an emerging, rapidly evolving situation. designed research; M.V.R., A.M., and C.K. Unsupervised clustering based on lipid species abundance showed three main groups of samples: 1) those at the 0 h time point (adherent cells), 2) untreated cells suspended for 12 and 24 h (differentiated cells), and 3) untreated cells suspended for 4 and 8 h (committed cells) together with all of the PKCi-treated samples (Fig. These differences are the cumulative result of keratinocyte lipid metabolism, sebaceous gland secretion, and microbial production (10, 12, 15). Organization of the microtubule cytoskeleton is also intimately linked with epidermal differentiation. 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. A common set of individual lipid molecules was enriched in cells stimulated to differentiate by suspension culture or by either ELOVL1 or SLC27A1 knockdown. This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. 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. 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 … 1,2 Stem cells in this regard lack many characteristics of terminal differentiation, and have an intrinsically high proliferative … 1A and subsequently examined using sparse partial least squares regression (sPLS) analysis to maximize separation between the sample groups, coupled with discriminant analysis (DA) to pinpoint the main lipid species contributing to such separation (26). S2D). Normality was assessed based on quantile–quantile plot inspection. 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. This is possible due to the presence of multipotent, self-renewing epidermal stem cells that give rise to differentiated cell lineages: keratinocytes, hairs, as well as sebocytes. We identified individual lipid species that induced exit from the epidermal stem cell compartment. Colony number and size were scored using ImageJ. Copyright © 2020 the Author(s). Cells generate a vast repertoire of lipid molecules whose functions are poorly understood. Unsupervised two-way clustering of the screen results grouped together replicates of the same treatment but not related enzymes (Fig. (B) Volcano plot of the screening results to identify hits based on statistical significance (P < 0.05) and fold change (FC) with respect to nontargeting siRNA controls included in each individual plate (FC < 0.6 for differentiation inhibition; FC > 2.5 for differentiation induction). Nature 551, 327–332 (2017). None of the bioactive lipids were toxic, as assessed by cleaved caspase 3 staining (SI Appendix, Fig. An integral program for tissue renewal and regeneration: Wnt signaling and stem cell control, Paradoxical impact of cholesterol on lipid packing and cell stiffness, Dietary fatty acids fine-tune Piezo1 mechanical response, Role of lipid composition on the structural and mechanical features of axonal membranes: A molecular simulation study, Patterning of human epidermal stem cells on undulating elastomer substrates reflects differences in cell stiffness, Desmosome assembly and disassembly are membrane raft-dependent, Desmosomal cadherin association with Tctex-1 and cortactin-Arp2/3 drives perijunctional actin polymerization to promote keratinocyte delamination, The interplay between keratinocytes and immune cells in the pathogenesis of psoriasis, Membrane lipidome of an epithelial cell line, Global analysis of the yeast lipidome by quantitative shotgun mass spectrometry, Modulation of myelopoiesis progenitors is an integral component of trained immunity, mixOmics: An R package for ’omics feature selection and multiple data integration, Proceedings of the National Academy of Sciences, Earth, Atmospheric, and Planetary Sciences, https://osf.io/wf7v2/?view_only=4b1f32c0e8d14c3fa13b95d51247e503, https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2011310117/-/DCSupplemental, Creative Commons Attribution License 4.0 (CC BY), Science & Culture: At the nexus of music and medicine, some see disease treatments, News Feature: Tracing gold's cosmic origins, Journal Club: Friends appear to share patterns of brain activity, Transplantation of sperm-producing stem cells. 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