Hindawi Publishing Corporation Stem Cells International Volume 2016, Article ID 2809192, 16 pages http://dx.doi.org/10.1155/2016/2809192 Research Article Differential Proteomic Analysis of Human Placenta-Derived Mesenchymal Stem Cells Cultured on Normal Tissue Culture Surface and Hyaluronan-Coated Surface Tzyy Yue Wong,1 Ying-Hui Chen,1 Szu-Heng Liu,1 Mairim Alexandra Solis,1 Chen-Hsiang Yu,2 Chiung-Hsin Chang,2 and Lynn L. H. Huang1,3,4,5,6 1 Institute of Biotechnology, College of Bioscience and Biotechnology, National Cheng Kung University, Tainan 701, Taiwan 2Department of Obstetrics and Gynecology, National Cheng Kung University, Tainan 701, Taiwan 3Department of Biotechnology and Bioindustry Sciences, National Cheng Kung University, Tainan 701, Taiwan 4Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan 5Research Center of Excellence in Regenerative Medicine, National Cheng Kung University, Tainan 701, Taiwan 6Advanced Optoelectronic Technology Center, National Cheng Kung University, Tainan 701, Taiwan Correspondence should be addressed to Lynn L. H. Huang; [email protected] Received 16 May 2015; Revised 7 September 2015; Accepted 7 October 2015 Academic Editor: Andrzej Lange Copyright © 2016 Tzyy Yue Wong et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Our previous results showed that hyaluronan (HA) preserved human placenta-derived mesenchymal stem cells (PDMSC) in a slow cell cycling mode similar to quiescence, the pristine state of stem cells in vivo, and HA was found to prevent murine adipose- derived mesenchymal stem cells from senescence. Here, stable isotope labeling by amino acid in cell culture (SILAC) proteomic profiling was used to evaluate the effects of HA on aging phenomenoninstemcells,comparing(1)oldandyoungpassagePDMSC cultured on normal tissue culture surface (TCS); (2) old passage on HA-coated surface (CHA) compared to TCS; (3) old and young passage on CHA. The results indicated that senescence-associated protein transgelin (TAGLN) was upregulated in old TCS. Protein CYR61, reportedly senescence-related, was downregulated in old CHA compared to old TCS. The SIRT1-interacting Nicotinamide phosphoribosyltransferase (NAMPT) increased by 2.23-fold in old CHA compared to old TCS, and is 0.48-fold lower in old TCS compared to young TCS. Results also indicated that components of endoplasmic reticulum associated degradation (ERAD) pathway were upregulated in old CHA compared to old TCS cells, potentially for overcoming stress to maintain cell function and suppress senescence. Our data points to pathways that may be targeted by HA to maintain stem cells youth. 1. Introduction towards aging. The trigger of cellular aging includes many factors, such as oxidative stress [5], genomic instability [6], Senescence is when cells reach an irreversible growth arrest altered niche microenvironment [6], altered mitochondrial [1]andisknowntoplayrolesinvariousbiologicalpro- function [7, 8], altered epigenetic regulations [9], and stem cesses such as development, apoptosis, and aging [2]. It was cells exhaustion [9]. Previous study showed that genome reviewed that mitotic cells, cells undergoing proliferation, stability such as maintained telomere length extended cell are prone to senescence [3]. Mitotic cells include stem cells, population doubling and reduced cellular aging in normal epithelial, vascular, and fibroblastic cells. The model for retinal pigment epithelial cells and foreskin fibroblasts [10]. cellular senescence first originated from Hayflick and Moor- Thus, the link between limited replicative senescence and head, who reported in 1961 that normal human diploid cells actual aging phenomenon had been established. had limited replicative lifespan [4]. Since then, senescence The known aging mechanisms include telomere shorten- is believed to play a role in two potential circumstances, ing [11], reactive oxygen species (ROS) accumulation, accu- namely, (1) to prevent tumor growth and (2) as a normal way mulated DNA damage-induced cell cycle regulation factors 2 Stem Cells International p53, p16, and p21 [3, 6], and altered cytoplasmic metabolism cultured on polystyrene tissue-culture surface were termed [12], all potentially leading to aging. The aging of stem cells TCS. leading to stem cell population decline had been suggested tooccurinthehumandiseaseknownasHutchinsonGil- 2.2. Differentiation Potential Analysis. For adipogenic dif- ford Progeria Syndrome [13]. The mesenchymal stem cells ferentiation, induction medium contained final concentra- (MSC) from aged Sprague-Dawley rats were found to have tion of 1 M dimethyl sulfoxide (DMSO; Sigma, St. Louis, compromised MSC function compared to that from young MO, USA), 0.2 mM indomethacin (Sigma, St. Louis, MO, Sprague-Dawley rats [14]. The MSC are maintained in vivo as USA), 0.5 mM 3-Isobutyl-1-methylxanthine (IBMX; Sigma, multipotent cells, capable of adipogenesis, osteogenesis, and St. Louis, MO, USA), 10 M insulin, and 10% fetal bovine chondrogenesis differentiation. Although it is well-known serum (FBS; GibcoBRL, Grand Island, NY, USA) in Dul- that stem cells are preserved in a pristine or youthful state becco’s modified Eagle’s medium high glucose medium compared to somatic cells in vivo,themechanisminvolved (DMEM; Gibco BRL, Grand Island, NY, USA). Cells were 4 2 is not fully understood. As a result, MSC are presented as a seeded at density 1×10 per cm until 100% confluence was good model for studying the aging phenomenon. reached, and medium was changed to induction medium for The goal was to study aging phenomenon using stable 4 weeks. Induction medium was changed every 72 hours. Oil isotope labeling by amino acid in cell culture (SILAC), a non- Red O (Sigma, St. Louis, MO, USA) was used to stain oil radioactive labeling method to label human placenta-derived droplets. In brief, cells were fixed in 4% paraformaldehyde, MSC (PDMSC). This study aimed to confirm the previous washed with 1x phosphate buffer saline (PBS), rinsed in results observed with hyaluronan (HA) presence. HA is a 60% isopropanol for three minutes, and stained in Oil Red major component of extracellular matrix (ECM) that belongs Ofor1hour.Afterthat,cellswerewashedusing60% to the glycosaminoglycan (GAG) family. It was observed isopropanol once, then rinsed in double-distilled water, and that HA played a role in maintaining PDMSC in slow- rinsed in 0.05% (w/v) sodium carbonate solution (Riedel-de cell cycling mode similar to stem cell quiescence [15]. HA Haen,¨ Sigma, St. Louis, MO, USA) for two minutes. Finally, induced multidrug resistance in PDMSC via CD44-PI3K- cells were counterstained with hematoxylin for one minute, Akt pathway [16]. HA was found to prevent murine adipose- washed with water, and analyzed under microscope. For derived stromal cells from senescence [17]. PDMSC of older chondrogenic differentiation, induction medium contained passage was compared to younger passage. It was found that final concentration of 6.25 g/mL insulin, 50 nM ascorbic PDMSC underwent replicative senescence after long-term in acid (JT Baker), and 10 ng/mL tumor growth factor-beta1 vitro expansion, in particular, the upregulation of proaging (TGF-1; CellGS, St. Louis, MO, USA) in DMEM high proteins and structural proteins. Molecules pertaining to glucose medium without FBS. Cells were seeded at density 1× 4 2 normal cell function maintenance such as the endoplasmic 10 per cm until 100% confluence was reached, and medium reticulum-associated degradation (ERAD) pathway during was changed to induction medium for 4 weeks. Induction stress and the COP9 signalosome-specific phosphorylation medium was changed every 72 hours. Accumulation of for p53 degradation were upregulated under HA presence glycosaminoglycan was analyzed by staining with Alcian blue compared to without HA, suggesting that the PDMSC under (Sigma, St. Louis, MO, USA). Briefly, cells were fixed in HA culture condition intended to suppress the stress of aging. 4% paraformaldehyde, washed with PBS, and incubated in 1 N hydrogen chloride (HCl; Sigma, St. Louis, MO, USA) 2. Materials and Methods solution for five minutes. Next, cells were stained with 3% Alcian blue solution in 0.1 N HCl for 30 minutes. Finally, 2.1. Cell Isolation. Full-term human placentas were obtained cellswerewashedwithwaterandanalyzedundermicroscope. from mothers at the National Cheng Kung University Osteogenesis differentiation assay was performed using the Hospital with informed consent. Procedure of the human induction medium supplemented with 10 MDMSO,10nM placenta handling and cell isolation was approved by the ascorbic acid, 10 mM 2-glycerophosphate (Sigma, St. Louis, Institutional Review Board. PDMSC were isolated based MO, USA), and 10% FBS in DMEM high glucose medium. on previous method [15]. In brief, the chorionic villi layer 1×104 2 was harvested and rinsed in Hanks’ Balanced Salt Solution Seeding cell density was per cm , and experiment (Sigma, St. Louis, MO, USA). The tissues were cut into was performed upon reaching 100% confluence. Calcification tiny pieces and digested by 347 U/mL Collagenase Type 2 was visualized after staining with Alizarin Red S (Sigma, St. ∘ (Worthington Biochemical Corporation) at 37 Cfor40min. Louis,MO,USA).Cellswerefixedin4%paraformaldehyde, The digested tissues went through filters from 500 and washed