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Transcriptome Profile of Human Fibroblasts in an Ex Vivo Culture Int. J. Med. Sci. 2020, Vol. 17 125 Ivyspring International Publisher International Journal of Medical Sciences 2020; 17(1): 125-136. doi: 10.7150/ijms.35693 Research Paper Transcriptome Profile of Human Fibroblasts in an Ex Vivo Culture Bogusław Machaliński1, Dorota Rogińska1, Kamila Szumilas1, Alicja Zawiślak1, Aleksandra Wilk2, Iwona Stecewicz1, Andrzej Brodkiewicz1, Barbara Wiszniewska2 1. Department of General Pathology, Pomeranian Medical University, Powstańców Wlkp. 72, 70-111 Szczecin, Poland 2. Department of Histology and Embryology, Pomeranian Medical University, Powstańców Wlkp. 72, 70-111 Szczecin, Poland Corresponding author: Bogusław Machaliński, Department of General Pathology, Pomeranian Medical University in Szczecin, Ul. Powstancow Wlkp. 72, 70-111 Szczecin, Poland. Tel: +48914661546; Fax: +48914661548; email: [email protected] © The author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). See http://ivyspring.com/terms for full terms and conditions. Received: 2019.04.12; Accepted: 2019.08.18; Published: 2020.01.01 Abstract Implantation of autologous fibroblasts is a method used to correct age-related changes in facial skin. The aim of this study was to establish the optimal population of cultured human fibroblasts according to the organization of the extracellular matrix in the dermis. Transcriptome profile analysis of cells derived from three consecutive passages indicated that fibroblasts after the second passage were the population with the greatest number of upregulated genes encoding the critical biological processes responsible for skin regeneration, such as extracellular matrix organization, collagen fibril organization, and cell adhesion. Furthermore, genes encoding proteinases responsible for the degradation of dermal extracellular matrix proteins were noticeably downregulated at this stage of culture. Autologous fibroblasts seem to be an optimal and safe biological filler for the renewal of all skin structures. Key words: autologous fibroblasts culture, transcriptome profile, gene expression Introduction Skin aging in male and female subjects is a part disrupting the balance between synthesis and of natural human aging of different organs, tissues degradation processes [7, 8]. The decrease in collagen and cells [1]. Aging is a complex, inevitable process type I disturbs the collagen type III to collagen type I influenced by a combination of endogenous and ratio [7, 9]. Human skin is rich in elastic fibers. exogenous factors. Both types of factors induce However, the relative volume and quantity of the cumulative morphological and physiological fibers varies with age, and numerous age-related alterations and progressive changes in each layer of changes are observed [7, 10-12]. A large amount of skin as well as influence the appearance of skin [1-3]. disintegrated elastic fibers have been reported in However, the most pronounced changes occur in individuals older than 70 years [10, 13]. The elements of the dermis [2, 4], which predominantly remodeling and degradation of elastic fibers are consists of connective tissue. During the aging mostly regulated by MMPs [14, 15], and abnormal process, human skin undergoes structural and localization of elastin in the upper dermis is observed, molecular alterations, especially at the level of type I especially in photo-damaged skin [16]. In addition to collagen, which is the principle structural component collagen and elastin, glycosaminoglycans (GAGs) are of the dermis [5]. These modifications can include abundant structural components of the extracellular rearrangements and reorientation of collagen fibers matrix of human skin. Glycosaminoglycans have and increases in the density of collagen bundles. As a various structural and physiological regulatory consequence, the dermis becomes fragmented, functions in skin, including tissue water maintenance, disorganized, and less soluble [5-7]. Matrix given their high water-holding capacity. The levels metalloproteinases (MMPs) are upregulated and and localization of GAGs change during collagen synthesis is reduced during skin aging, endogenous-induced aging to some extent, especially http://www.medsci.org Int. J. Med. Sci. 2020, Vol. 17 126 during photoaging [17]. The differences in the were taken from the postauricular area, which is one location of dermal GAGs in photoaged skin as well as of the lower exposed to ultraviolet radiation areas of alterations in the size, structure and type of GAGs the head. The skin biopsies were devoted to isolation present in sun-damaged skin may affect their water and culture of dermal fibroblasts and to prepare binding properties and their ability to interact with histological slides. other components of the dermal extracellular matrix The skin specimens were transported to the [18]. Although the total content of GAGs is increased laboratory in ice-cold Ca2+/ Mg2+-free PBS containing in aged skin, they are unable to bind water. Therefore, 1:100 penicillin/streptomycin solution and 1 µg/mL poor hydration and a low turgor capacity contributes Fungizone (both from Thermo Fisher Scientific, to the dry (xerotic) appearance of skin [7, 19]. Waltham, MA, USA) and then processed During aging, fibroblasts also play a key role in immediately. The tissue sample was washed twice maintaining the homeostasis of the extracellular with cold Ca2+/ Mg2+-free PBS, cut into smaller pieces matrix and are subjected to various changes, such as and incubated in 0.6 U/mL Dispase II (Thermo Fisher reduced metabolic activity, e.g., loss of type I collagen Scientific, Waltham, MA, USA) for 1-2 h at 37˚C. The expression [20, 21]. Human fibroblasts located within epidermis was manually removed from each tissue the age-associated dermal extracellular matrix sample, and the dermis was cut into 1-mm3 pieces microenvironment express increased levels of following enzymatic disaggregation with 0.62 collagen-degrading matrix metalloproteinases Wünsch U/mL Liberase DH (Roche Applied Science, (MMPs) and decreased levels of tissue inhibitors of Penzberg, Germany) for 30-40 min at 37˚C. MMPs [14, 15, 22]. Subsequently, tissue pieces were dissociated by Due to structural and functional changes, the vortexing and then passed through a 70-µm cell skin shows obvious signs of the passing time [1]. The strainer Becton Dickinson, Franklin Lakes, NJ, USA). most important and prominent sign of aging is skin The dissociated cells were centrifuged at 1500 rpm for wrinkling [23]. Ongoing efforts for preserving a 5 minutes. The supernatant was discarded, and the youthful appearance demonstrate that youthfulness is pellet was suspended in Medium 199 (Thermo Fisher considered a prerequisite for beauty [4]. Therefore, Scientific, Waltham, MA, USA) containing 10% the maintenance and improvement of facial skin human serum isolated from the patient themselves quality have gained particular attention, and novel and 0.5% penicillin and streptomycin solution. To technologies aimed at preventing skin aging in obtain human autologous serum, 10 mL of whole women and, more recently, in men are being blood from patient was collected into plastic tubes extensively investigated in the field of aesthetic containing a serum separator gel with clot activator dermatology. One of the innovative methods used to (Becton Dickinson, Franklin Lakes, NJ, USA). Serum correct age-related skin changes, e.g., unwanted facial separation was completed after centrifugation at 2,000 wrinkles, involves the use of cultured fibroblasts rpm for 10 minutes. The cells were cultured in a T75 derived from the patient`s own skin [24]. In our tissue culture flask (Becton Dickinson, Franklin Lakes, previous studies [24], we observed a significant NJ, USA) at 37°C in 5% CO2 in a humidified increase in the diameter of collagen fiber bundles and atmosphere. The medium was changed 48-h after an improvement in the density of reticular fibers, plating and every 3-4 days thereafter. When the fibrillin-1-rich microfibrils, and elastic fibers after cultures reached 80% confluency, the cells were reinjection of autologous fibroblasts into the dermis. detached with Accutase (GE Healthcare, Chicago, IL, The aim of this study was to compare global USA), washed with PBS and divided in two equal gene expression profiles in fibroblasts from three parts. Some of the cells were used to isolate total RNA consecutive passages to estimate the optimal for microarray gene expression analysis and the population quality of the tested cells according to remaining part was reseeded in complete medium. potential secretory activity critical for skin The cell cultures were maintained until the 3rd rejuvenation. passage. The samples of skin were fixed in freshly Materials and Methods prepared 4% paraformaldehyde and embedded in paraffin. For the morphological analysis, serial slices Patients and cell culture (3-5 μm in thickness) of skin were mounted onto glass Human skin samples were obtained from slides to histochemical and immunohistochemical volunteer male donor at the age of 46, in accordance studies. with the Declaration of Helsinki and with the approval of Local Ethics Committee of Pomeranian RNA isolation Medical University. Skin biopsies (0.8 cm x 0.6 cm) Total RNA from fibroblast cell cultures was http://www.medsci.org Int. J. Med. Sci. 2020, Vol. 17 127 isolated using the MirVana Kit (Thermo Fisher fixed in methanol. For the morphological analysis of Scientific, Waltham, MA, USA), according
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