Biochim Biophys Acta. The Wnt pathway is a key regulator of stem cells in many, if not all, tissues, and Wnts require lipid modifications for activity (43). Researchers are still trying to understand what causes this strong correlation between neural and social networks. (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). Unsupervised two-way clustering of the screen results grouped together replicates of the same treatment but not related enzymes (Fig. Madison KC, Swartzendruber DC, Wertz PW, Downing DT. Lipid amounts from the lipidomics experiments were normalized by the total amount of lipids in each sample. Final volume was 175 µL per well, and the final siRNA concentration was 30 nM. Epigenetic factors are proteins that modify either DNA or histone. 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… Within the basal cells, microtubules emanate from the centrosome, providing networks for cellular … Data were analyzed with in-house-developed lipid identification software based on LipidXplorer (53). Secondary stains were incubated for 2 h at room temperature protected from light, and plates were finally washed three times with PBS before being imaged using the Perkin-Elmer Operetta High-Content Imaging System. • Epidermal stem-cell progeny give rise to the differentiated cells of the interfollicular epidermis (IFE),hair follicles and sebaceous glands. Our comprehensive approach has allowed us to identify a set of specific lipid molecules that induce differentiation when added to primary keratinocytes in culture. Copyright © 2020 the Author(s). 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). 1,2 Stem cells in this regard lack many characteristics of terminal differentiation, and have an intrinsically high proliferative … 1E). 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. 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. 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 … Normality was assessed based on quantile–quantile plot inspection. 9661) diluted to 1 µg/mL or according to manufacturer’s instructions in blocking buffer. differentiation; epidermis; keratinocytes; lipidomics; lipids. 1B). 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 … Emergence of distinct stem cell populations during morphogenesis of the pilosebaceous unit. ( A ) Overlap…, NLM Competing interest statement: C.K. Representative images for each treatment are shown below the bar plot. siRNA-mediated knockdown of lipid-modifying enzymes can affect keratinocyte differentiation. Having identified a panel of lipids enriched during keratinocyte differentiation, we asked whether any of them regulated the onset of differentiation. (C and D) Sample separation along the first two components by sPLS of the 48 h (C) and 72 h (D) samples. Would you like email updates of new search results? Exp. See this image and copyright information in PMC. 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Local stem cells can also be transformed into keratinocytes, sebaceous gland, and other skin-associated tissues. A common set of individual lipid molecules was enriched in cells stimulated to differentiate by suspension culture or by either ELOVL1 or SLC27A1 knockdown. : posttransfection. -, Barrandon Y., Green H., Three clonal types of keratinocyte with different capacities for multiplication. Enrichment was determined by comparing the median of each sample class. 2020 Sep 8;117 (36):22173-22182. doi: 10.1073/pnas.2011310117. 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). Enter multiple addresses on separate lines or separate them with commas. For colony formation assays, n ≥ 2 hexaplicates of independent transfections or n = 12 independent treatments. Reviewers: N.J.F., University of Southern Denmark; and K.J.G., Northwestern University.  |  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. It should be noted that our analysis did not cover the full gamut of lipid classes, lacking, for example, eicosanoids and fatty acids. 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. (A) Overlap between the discriminant lipid sets enriched during ELOVL1 knockdown, SLC27A1 knockdown, or suspension-induced keratinocyte differentiation. COVID-19 is an emerging, rapidly evolving situation. After 12 d, feeders were removed, and keratinocyte colonies were fixed in 4% paraformaldehyde (Sigma) for 10 min then stained with 1% rhodanile blue (1:1 mixture of rhodamine B and Nile blue A [Acros Organics]). 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. Alterations in the lipid composition of the cornified layer can lead to morphological defects in the epidermis and diseases such as ichthyoses (11⇓–13). We identified individual lipid species that induced exit from the epidermal stem cell compartment. We also performed colony formation assays on the whole panel of candidate differentiation-inhibiting knockdowns but did not observe any significant effects (Fig. The suspended cells were subsequently incubated at 37 °C. 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. These patients showed a deficiency in longer-chain (≥C26) ceramides. Within the epidermis, basal keratinocytes delaminate as they move into the suprabasal layers during differentiation, and lipids could potentially play a role in this process by altering the properties of the cell membrane or modulating the activity of junctional proteins (48, 49). Our observations suggest that more research is warranted on the regulation of biological processes via lipid species, moving beyond the more conventional contribution of proteins and nucleic acids. HHS It is expressed in the basal layer of adult human epidermis (38). Lipids in cell biology: How can we understand them better? Lipids are essential for the establishment of an efficient epidermal barrier (10). The siRNAs were incubated for 20 min with 10 µL of INTERFERin-HTS transfection reagent (PolyPlus Transfection) diluted 1/200 in water. This accumulation was blocked on inhibition of PKC (Fig. 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. At each collection time point, the methylcellulose was diluted with PBS, and the cells were recovered by centrifugation. 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 organic phase was dried in a speed vacuum concentrator. For the siRNA screening, n = 4 replicates (independent transfections). -. For lipid-induced involucrin expression, n ≥ 6 independent treatments. 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). 2014 Mar;1841(3):435-40. doi: 10.1016/j.bbalip.2013.08.009. Our results reveal the potential of lipid subspecies to regulate exit from the epidermal stem cell compartment. Despite numerous advances, current methodologies do not allow direct and systematic manipulation of lipid molecular species (27). Nuclei were initially defined using the DRAQ5 channel; small (<2,000 µm2) and highly irregular (roundness < 0.6) nuclei were excluded from the analysis. A two-step chloroform/methanol procedure was used to extract the lipids (52), and samples were spiked with an internal lipid standard mixture. Addition of either candidate glucosylceramide significantly decreased colony size (Fig. 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. None of the other lipid classes showed such a marked change with time in suspension, although it was notable that several families were affected by PKC inhibition (SI Appendix, Fig. Author contributions: M.V.R., A.M., U.S.E., and F.M.W. Epub 2007 Mar 12. Error bars indicate SDs; P values are calculated using Dunnet’s multiple comparison test (*P < 0.05, **P < 0.01). 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. Cell niche in skin induce keratinocyte differentiation, we pinpointed candidate bioactive lipid treatments performed! Strategy and keratinocyte response to treatments ( 9 ) clipboard, Search History and... To manufacturer ’ s instructions in blocking buffer differentiated in suspension as described previously ( 8, )! 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