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• W2280356735 abstract "The use of primary human cells for research is hampered by the inability to grow and maintain the substantial amount of cells required for experimental assays. Primary sebocytes (P-Sebs) are especially difficult to grow in culture because of their innate programming that causes the cells to accumulate lipids and undergo holocrine rupture within days of their isolation (Xia et al., 1989Xia L.Q. Zouboulis C. Detmar M. Mayer-da-Silva A. Stadler R. Orfanos C.E. Isolation of human sebaceous glands and cultivation of sebaceous gland-derived cells as an in vitro model.J Invest Dermatol. 1989; 93: 315-321Abstract Full Text PDF PubMed Google Scholar). As such, the use of sebocyte cell lines immortalized by SV40 large T antigen or human papilloma virus E6/E7 oncogenes are used to enhance the proliferative capacity of these cells, but immortalization prevents complete differentiation (Lo Celso et al., 2008Lo Celso C. Berta M.A. Braun K.M. Frye M. Lyle S. Zouboulis C.C. et al.Characterization of bipotential epidermal progenitors derived from human sebaceous gland: contrasting roles of c-Myc and beta-catenin.Stem Cells. 2008; 26: 1241-1252Crossref PubMed Scopus (107) Google Scholar, Thiboutot et al., 2003Thiboutot D. Jabara S. McAllister J. Sivarajah A. Gilliland K. Cong Z. et al.Human skin is a steroidogenic tissue: steroidogenic enzymes and cofactors are expressed in epidermis, normal sebocytes, and an immortalized sebocyte cell line (SEB-1).J Invest Dermatol. 2003; 120: 905-914Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, Zouboulis et al., 1999Zouboulis C. Seltmann H. Neitzel H. Orfanos C. Establishment and characterization of an immortalized human sebaceous gland cell line.J Invest Dermatol. 1999; 113: 1011-1020Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). Y-27632 is a small molecule that, when used at concentrations of less than 10 μM, selectively inhibits both isoforms of Rho-associated protein kinase (ROCK I and II), resulting in blockage of integrin-mediated cell contact inhibition. When used at higher concentrations, Y-27632 will also inhibit protein kinase C, protein kinase A, and myosin light-chain kinases (Narumiya et al., 2000Narumiya S. Ishizaki T. Uehata M. Use and properties of ROCK-specific inhibitor Y-27632.Methods Enzymol. 2000; 325: 273-284Crossref PubMed Google Scholar). Y-27632 has been successfully used to expand and maintain the survival of other primary human cells including keratinocytes, fibroblasts, endothelial cells, and adipose-derived stem cells (Chapman et al., 2010Chapman S. Liu X. Meyers C. Schlegel R. McBride A.A. Human keratinocytes are efficiently immortalized by a Rho kinase inhibitor.J Clin Invest. 2010; 120: 2619-2626Crossref PubMed Scopus (231) Google Scholar, Peh et al., 2015Peh G.S. Adnan K. George B.L. Ang H.P. Seah X.Y. Tan D.T. et al.The effects of Rho-associated kinase inhibitor Y-27632 on primary human corneal endothelial cells propagated using a dual media approach.Sci Rep. 2015; 5: 9167Crossref PubMed Scopus (71) Google Scholar, Piltti et al., 2015Piltti J. Varjosalo M. Qu C. Hayrinen J. Lammi M.J. Rho-kinase inhibitor Y-27632 increases cellular proliferation and migration in human foreskin fibroblast cells.Proteomics. 2015; 15: 2953-2965Crossref PubMed Scopus (15) Google Scholar, Qu et al., 2014Qu C.Q. Li D.W. Shen N. Jiang-Yang J.H. Ji Y.T. Effect of glutathione and Y27632 on the viability of cryopreserved porcine adipose-derived stem cells.Cryo Letters. 2014; 35: 308-311PubMed Google Scholar). As previously shown in keratinocytes (Chapman et al., 2014Chapman S. McDermott D.H. Shen K. Jang M.K. McBride A.A. The effect of Rho kinase inhibition on long-term keratinocyte proliferation is rapid and conditional.Stem Cell Res Ther. 2014; 5: 60Crossref PubMed Scopus (65) Google Scholar), we describe a method of reversibly immortalizing P-Sebs, allowing for their expansion in culture using 3T3 fibroblast feeder layers and Y-27632 (5 μmol/L). Patient consent was not required for use of discarded human skin obtained from facial surgeries under a protocol approved by the institutional review board of the Pennsylvania State University College of Medicine. Whole sebaceous glands were dissected from normal human skin, and P-Seb cultures from multiple sebaceous glands were established (see Supplemental Materials and Methods online). With skin from three different subjects, seven independent P-Seb cultures were established. Each culture was passaged multiple times, with one line (P-Seb A: male, 65-year-old scalp A) continually passaged for over 200 days in culture, before intentionally stopping (Figure 1a and b). P-Seb A exhibited steady cell proliferation with an average of one population doubling every 1.03 days. Periodically, some cultures were deprived of Y-27632, 3T3 fibroblasts, or both. These sebocytes remained viable for an additional 2–5 passages before growth arrest. Those sebocytes with only 3T3 support cells fared better than those with Y-27632 alone (Figure 1b). The growth arrest observed in cultures without Y-27632 confirmed that P-Seb A did not spontaneously immortalize in the presence of Y-27632. Like freshly isolated sebocytes, P-Seb A proliferated as outgrowths from small, round colonies that are three-dimensional and multilayered. Yellow lipid droplets are visible in the cytoplasm (Figure 1c). Genomic stability after long-term exposure to Y-27632 was assessed by karyotype analysis. Passage (p) 13 P-Seb A sebocytes, with approximately 100 days of continuous exposure to Y-27632, were cultured for 5 days without Y-27632 immediately before analysis. These sebocytes are normal with a karyotype of 46,XY (Figure 1d). Spontaneous immortalization with an abnormal karyotype was found in one subculture (p33) (see Supplemental Figure S1 online). We noted variability in the lipogenic properties of P-Sebs from four samples and further characterized the P-Seb A sebocytes (see Supplemental Figures S2 and S3 online). P-Seb A sebocytes were grown in the presence of Y-27632 and 3T3 fibroblasts for several days to achieve sufficient cell numbers, followed by a 5-day washout period with no Y-27632. We examined overall lipid content of the P-Seb A (p15) using Oil Red O staining and observed a slight increase in lipid droplet accumulation after Y-27632 removal (Figure 2a). We then compared total lipogenesis using our standard lipogenesis assay (Smith et al., 2008Smith T.M. Gilliland K. Clawson G.A. Thiboutot D. IGF-1 induces SREBP-1 expression and lipogenesis in SEB-1 sebocytes via activation of the phosphoinositide 3-kinase/Akt pathway.J Invest Dermatol. 2008; 128: 1286-1293Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). After removal of Y-27632 for 5 days, P-Seb A (p27) significantly increased total lipogenesis by 38% (p = .001) (Figure 2b). We further characterized the lipid profile of these P-Seb A sebocytes (p40–42) using the 14C acetate incorporation assay in the presence of Y-27632 or 5 days after its removal (Figure 2c). All categories of lipids were significantly increased: cholesterol by 90%, fatty alcohol by 75%, fatty acid by 80%, triglycerides by 80%, wax esters by 80%, cholesterol oleate by 84%, and squalene by 84% (p < .00001). P-Seb A sebocytes (p19) have significantly increased lipogenic enzyme expression after removal of Y-27632 compared with Y-27632 remaining in the culture medium (Figure 2d). These data show that after removal of Y-27632 from the culture medium, P-Seb A sebocytes were able to differentiate and produce more lipids, especially the sebaceous gland-specific lipids: wax esters and squalene. We have shown that transient immortalization of P-Sebs is possible when using ROCK inhibitor, Y-27632, and fibroblast feeder cells. This model system for sebocyte research allows for expansion of P-Sebs and overcomes the need for oncogene-driven immortalization. We experienced, however, intra- and interindividual variation in the characteristics of the P-Seb cultures. For an unknown reason, perhaps skin site, multiple primary lines were generated from male 65-year-old scalp tissue, whereas only one was possible from each of our other skin samples. We were unable to determine which sebocytes from the parent sebaceous glands responded to Y-27632. We hypothesize that Y-27632 acts on basal sebocytes, because these are the proliferative cells at the edge of the gland and are readily exposed to Y-27632 in culture medium. Our data highlight the fact that careful characterization of each P-Seb culture is necessary before its use in downstream experimental assays. Although each culture was expanded using Y-27632 and fibroblast feeder cells, each culture varied in proliferation rates and lipogenesis. For example, total lipogenesis was increased more in P-Seb E versus P-Seb A after withdrawal of Y-27632. ROCK inhibition also altered the expression of lipogenic transcription factors, which should be taken into consideration when using this model system for the evaluation of sebocyte lipogenesis. We show that P-Sebs can be expanded in culture using ROCK inhibition. These differences in primary cultures may be overcome by pooling cells from multiple glands and skin samples, similar to commercially available human epidermal keratinocytes. This method represents a valuable system for research in human sebocyte biology that overcomes the limitations of freshly isolated P-Sebs and the need for oncogene-driven immortalization. The authors state no conflict of interest. This work is supported by the Department of Dermatology Research Endowment Fund and the Jake Gittlen Laboratories for Cancer Research at Penn State Hershey College of Medicine. Download .pdf (.5 MB) Help with pdf files Supplementary Data" @default.
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• W2280356735 title "Selective Rho Kinase Inhibitor Allows for Expansion of Human Primary Sebocytes In Vitro" @default.
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