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Human Epidermal Stem Cell Differentiation Is Modulated by Specific Lipid Subspecies

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Human Epidermal Stem Cell Differentiation Is Modulated by Specific Lipid Subspecies Human epidermal stem cell differentiation is modulated by specific lipid subspecies Matteo Vietri Rudana, Ajay Mishraa,b, Christian Klosec, Ulrike S. Eggertd, and Fiona M. Watta,1 aCentre for Stem Cells and Regenerative Medicine, King’s College London, SE1 9RT London, United Kingdom; bEuropean Bioinformatics Institute, CB10 1SD Hinxton, United Kingdom; cLipotype GmbH, 01307 Dresden, Germany; and dRandall Centre for Cell and Molecular Biophysics, King’s College London, SE1 1UL London, United Kingdom Contributed by Fiona M. Watt, July 16, 2020 (sent for review June 4, 2020; reviewed by Nils Joakim Faergeman and Kathleen J. Green) While the lipids of the outer layers of mammalian epidermis and such as phospholipids and sphingomyelins, the granular layers their contribution to barrier formation have been extensively exhibiting higher levels of glycosylceramides and cholesterol described, the role of individual lipid species in the onset of sulfates, and the cornified layer mostly composed of cholesterol, keratinocyte differentiation remains unknown. A lipidomic analy- fatty acids, and ceramides, particularly ω-acylceramides that are sis of primary human keratinocytes revealed accumulation of only found in the outermost epidermal layer (14). These differ- numerous lipid species during suspension-induced differentiation. ences are the cumulative result of keratinocyte lipid metabolism, A small interfering RNA screen of 258 lipid-modifying enzymes sebaceous gland secretion, and microbial production (10, 12, 15). identified two genes that on knockdown induced epidermal dif- Beyond their roles as structural components of cells and en- ferentiation: ELOVL1, encoding elongation of very long-chain fatty ergy storage molecules, lipids function as bioactive compounds in acids protein 1, and SLC27A1, encoding fatty acid transport protein a wide number of cellular processes. Posttranslational addition 1. By intersecting lipidomic datasets from suspension-induced dif- of lipid moieties to certain proteins is necessary for their function ferentiation and knockdown keratinocytes, we pinpointed candidate (16), and multiple classes of lipids are involved in endocrine bioactive lipid subspecies as differentiation regulators. Several of (e.g., steroid hormones), paracrine (e.g., eicosanoids), or intra- these—ceramides and glucosylceramides—induced differentiation cellular signaling (e.g., the phosphatidylinositol bisphosphate/ when added to primary keratinocytes in culture. Our results reveal diacylglycerol second-messenger system) (17). However, several the potential of lipid subspecies to regulate exit from the epidermal other lipid classes, such as the sphingolipids ceramide and stem cell compartment. sphingosine-1-phosphate (18, 19), have signaling properties CELL BIOLOGY across multiple tissues, including the epidermis (20). Within each lipids | epidermis | differentiation | keratinocytes | lipidomics lipid class a cell produces a plethora of individual lipid species (i.e., carbon chain length, desaturation, and hydroxylation vari- he skin is the largest organ in the human body, performing ants), whose importance is only beginning to be investigated (21, Tthe primary functions of preventing water evaporation and 22). Indeed, several studies suggest tight regulation and diverse protecting the internal organs from damaging external agents. functions for specific lipid molecular structures in a variety of The outermost part of the skin, the epidermis, is a stratified cellular processes, ranging from cell division (23) to the innate squamous epithelium formed predominantly by keratinocytes (1, 2). immune response (24). These cells migrate from the basal layer, home of the stem cell While the lipid composition of the outer layers of the epi- compartment, upward toward the surface of the skin through the dermis has been well described, the potential role of individual spinous and granular layers to finally reach the cornified layer. lipid species in the early phases of keratinocyte differentiation During this migration, keratinocytes undergo a terminal differenti- remains to be investigated. Due to the high complexity of the ation process, accumulating transglutaminase-cross-linked proteins and secreting lamellar bodies enriched in highly hydrophobic lipid Significance species. Ultimately, keratinocytes lose their nuclei and become corneocytes, held together by cross-linked sheets of apolar lipids in Cells generate a vast repertoire of lipid molecules whose a structure that has been likened to bricks and mortar (1, 3). functions are poorly understood. To investigate whether lipids Human epidermis can be reconstituted in culture, forming can regulate cell fate decisions, we carried out a systematic stratified sheets in which the stem cell compartment and key lipidomic analysis and perturbation of lipid metabolism in cul- elements of the terminal differentiation process are preserved tured human epidermal keratinocytes, determining associa- (1, 4). Clonal growth assays are used as a quantitative readout of tions with the onset of differentiation. We identified individual stem cell abundance in cultures of human epidermal keratino- lipid species that induced exit from the epidermal stem cell cytes (4–6). Keratinocytes can be induced to undergo terminal compartment. Our observations suggest that more research is differentiation in a near-synchronous manner when maintained warranted on the regulation of biological processes via lipid as a single-cell suspension in medium containing methylcellulose species, moving beyond the more conventional contribution of (7, 8). The cells undergo a differentiation commitment phase proteins and nucleic acids. after 4 h and increase expression of differentiation markers be- tween 8 and 24 h (8, 9). While detailed transcriptomic and Author contributions: M.V.R., A.M., U.S.E., and F.M.W. designed research; M.V.R., A.M., and C.K. performed research; U.S.E. contributed new reagents/analytic tools; and M.V.R., proteomic analysis of suspension-induced differentiation was U.S.E., and F.M.W. wrote the paper. carried out previously (9), little is known about changes in the Reviewers: N.J.F., University of Southern Denmark; and K.J.G., Northwestern University. lipid composition of keratinocytes as they exit the stem cell Competing interest statement: C.K. is the head of research and development at compartment. Lipotype GmbH. Lipids are essential for the establishment of an efficient epi- This open access article is distributed under Creative Commons Attribution License 4.0 dermal barrier (10). Alterations in the lipid composition of the (CC BY). cornified layer can lead to morphological defects in the epider- 1To whom correspondence may be addressed. Email: [email protected]. mis and diseases such as ichthyoses (11–13). The predominant This article contains supporting information online at https://www.pnas.org/lookup/suppl/ lipid species change across the different epidermal layers, with doi:10.1073/pnas.2011310117/-/DCSupplemental. the basal and spinous layers enriched in more polar lipid classes www.pnas.org/cgi/doi/10.1073/pnas.2011310117 PNAS Latest Articles | 1of10 Downloaded by guest on September 29, 2021 lipid makeup of the epidermis and the limitations of methodol- inhibition of PKC (Fig. 1B). None of the other lipid classes showed ogies available to manipulate lipids, we sought to approach this such a marked change with time in suspension, although it was question in a more tractable system, namely, cultured primary notable that several families were affected by PKC inhibition (SI human keratinocytes. We have used a combination of small in- Appendix,Fig.S1A). Unsupervised clustering based on lipid species terfering RNA (siRNA) screening and lipidomic analysis of abundance showed three main groups of samples: 1) those at the primary human keratinocytes grown in culture. We find changes 0 h time point (adherent cells), 2) untreated cells suspended for 12 in sphingolipids and phospholipids associated with differentia- and 24 h (differentiated cells), and 3) untreated cells suspended tion and show that several ceramide and glucosylceramide mol- for 4 and 8 h (committed cells) together with all of the PKCi- ecules can induce differentiation of cultured keratinocytes. treated samples (Fig. 1C). Consistent with this, principal component analysis (PCA) separated the adherent cell sample from all of the Results others along the first component and the differentiated cells along the The Lipid Composition of Keratinocytes Changes during Suspension-Induced second, while the committed and inhibited cells could not be clearly Differentiation. Single-cell suspension-induced differentiation of pri- separated (Fig. 1D). mary human keratinocytes can be blocked in the presence of a protein In order to identify the most critical lipid species that accu- kinase C inhibitor (PKCi) (Fig. 1A) (9). To examine how lipids were mulated during commitment and differentiation, the samples affected in suspension culture, we analyzed samples using a shotgun were first classified into four categories (“adherent,”“commit- lipidomics approach (25), comparing control and PKCi-treated cells ment,”“differentiated,” and “inhibited”) according to the model (Dataset S1). Analysis of lipid changes at the class level showed sig- in Fig. 1A and subsequently examined using sparse partial least nificant accumulation of ceramides and hexosylceramides as squares regression (sPLS) analysis to maximize separation be- keratinocytes underwent differentiation in
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