Establishment of an Immortalized Skin Keratinocyte Cell Line Derived from the Animal Model Mastomys coucha

RESEARCH ARTICLE Establishment of an Immortalized Skin Keratinocyte Cell Line Derived from the Animal Model Mastomys coucha Daniel Hasche1, Sonja Stephan1, Larissa Savelyeva2, Frank Westermann2, Frank Rösl1*, Sabrina E. Vinzón1¤* 1 Division of Viral Transformation Mechanisms (F030), German Cancer Research Center, Heidelberg, Germany, 2 Division of Neuroblastoma Genomics (B087), German Cancer Research Center, Heidelberg, Germany a11111 ¤ Current address: Laboratory of Molecular and Cellular Therapy, Fundación Instituto Leloir, IIBBACONICET, Buenos Aires, Argentina * ; (SEV); (FR) Abstract OPEN ACCESS Citation: Hasche D, Stephan S, Savelyeva L, Westermann F, Rösl F, Vinzón SE (2016) Establishment of an Immortalized Skin Keratinocyte Cell Line Derived from the Animal Model Mastomys coucha. PLoS ONE 11(8): e0161283. doi:10.1371/ journal.pone.0161283 Editor: Andrzej T Slominski, University of Alabama at Birmingham, UNITED STATES Received: June 16, 2016 Accepted: August 2, 2016 In the present report we describe the establishment of a spontaneous immortalized skin keratinocyte cell line derived from the skin of the multimammate rodent Mastomys coucha. These animals are used in preclinical studies for a variety of human diseases such as infections with nematodes, bacteria and papillomaviruses, especially regarding cutaneous manifestations such as non-melanoma skin cancer. Here we characterize the cells in terms of their origin and cytogenetic features. Searching for genomic signatures, a spontaneous mutation in the splicing donor sequence of Trp53 (G to A transition at the first position of intron 7) could be detected. This point mutation leads to alternative splicing and to a premature stop codon, resulting in a truncated and, in turn, undetectable form of p53, probably contributing to the process of immortalization. Mastomys coucha-derived skin keratinocytes can be used as an in vitro system to investigate molecular and immunological aspects of infectious agent interactions with their host cells. Published: August 17, 2016 Copyright: © 2016 Hasche et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This study was supported by the “Eurostars Project” E! 6872 TOSCA (01QE1203C). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Introduction The multimammate rodent Mastomys [1] serves as a suitable model for diseases caused by numerous infectious agents such as Brugia malayi [2], Trypanosoma [3], Helicobacter pylori [4], Lassa fever virus [5] and papillomaviruses [6, 7]. In vivo models allow the dissection of infection routes, to study cancer development and to test the efficacy of vaccination against the respective infectious agent [8–10]. In our previous studies, we have used Mastomys coucha as a model to study the role of cutaneous papillomaviruses and their function in the context of non-melanoma skin cancer [11, 12]. The animals housed at the German Cancer Research Center (DKFZ) are persistently infected with the Mastomys natalensis papillomavirus (MnPV) and Mastomys coucha papillomavirus 2 (McPV2) [7] and spontaneously develop epithelial lesions like warts, keratoacanthomas and PLOS ONE | DOI:10.1371/journal.pone.0161283 August 17, 2016 1 / 15 Establishment of Kera5 Cell Line squamous cell carcinomas linked to MnPV [11]. We previously showed that the development of skin tumors in these animals can be efficiently prevented by prophylactic vaccination based on virus-like particles (VLP) even under immunosuppressive conditions [11]. Moreover, we recently reported the complete MnPV transcription map derived from productive lesions in animals and found homologous transcripts known from HPVs as well as novel splicing isoforms for proteins of unknown function [13]. Although animal models are essential to mimic a clinical scenario seen in patients, it is also necessary to design reductionist molecular approaches under in vitro conditions, using a homogeneous population of cells to study the bidirectional cross-talk between virus and host, thereby making Mastomys coucha-derived keratinocytes extremely desirable, especially considering the strict species-specificity in the case of papillomavirus infection. Here, we describe the establishment and characterization of such cells (referred as Kera5) obtained from the skin of virus-free animals. Methods Animals Mastomys coucha from the DKFZ breeding colony were maintained under standard conditions in compliance with German and European statutes [11] and all experiments were undertaken with the approval of the responsible Animal Ethics Committee (Regional Council of Karlsruhe, Germany; G26/12, DKFZ 276). Virus-free animals were obtained by hysterectomies of pregnant Mastomys under sterile conditions [11]. The offspring were nursed by foster specified pathogen-free (SPF) mice (Mus musculus), kept in a specific pathogen free isolator unit at the DKFZ. Isolation of skin keratinocytes and fibroblasts Mastomys keratinocytes were isolated as described [14, 15]. Briefly, newborn animals were sacrificed by decapitation and carcasses were disinfected by submersion in iodine solution (5 min) and 70% ethanol (5 min) prior to removal of extremities under aseptic conditions. A longitudinal incision was made from neck to tail and the skin was peeled off. Skins were allowed to float two times (10 min) in gentamycin (0.25 mg/ml in PBS) and were spread out in a petri dish and incubated overnight at 4°C with 5 mg/ml Dispase II (Roche) in dKSFM (Thermo Fisher Scientific) to separate epidermis and dermis. The epidermis was peeled off the dermis and incubated with 1.25% trypsin (Sigma-Aldrich) in PBS (20 min at room temperature) to separate the keratinocytes. To favor the process, the epidermis was ripped with forceps. Trypsinization was stopped by addition of defined Keratinocyte-SFM (dKSFM, Thermo Fisher Scientific) supplemented with 10% FCS (Thermo Fisher Scientific). The suspension was filtered through a 70 μm cell strainer (Falcon) and centrifuged for 5 min at 400xg. The pellet was resuspended in conditioned dKSFM obtained from Mastomys-derived fibroblasts, supplemented with penicillin/streptomycin (Thermo Fisher Scientific) and 1.15 x 105 cells were seeded per 6 cm dish. Medium was changed every two days. In order to establish Mastomys-derived fibroblasts for the production of conditioned keratinocyte medium, the dermis was cut into pieces, spread in a cell culture dish and air dried for 15 min in the cell culture hood prior to the addition of DMEM supplemented with 4.5 g/l glucose (...truncated)