Contents lists available at ScienceDirect Tissue and Cell journal homepage: www.elsevier.com/locate/tice Canine amniotic membrane mesenchymal stromal/stem cells: Isolation, characterization and differentiation Jéssica Borghesi a, ⁎ ,1 , Mariana Ferreira Lima b,1 , Lara Carolina Mario a , Adriana Raquel de Almeida da Anunciação a , Ana Carolina Silveira Rabelo a , Marcella Giancoli Kato Cano da Silva b , Fausto Assunpção Fernandes a , Maria Angélica Miglino a , Ana Claudia Oliveira Carreira a,c, ⁎ , Phelipe Oliveira Favaron a a Department of Surgery, School of Veterinary Medicine and Animal Science, University of Sao Paulo (FMVZ-USP), Sao Paulo, Brazil b University Paulista (UNIP), Sao Paulo, Brazil c NUCEL (Cell and Molecular Therapy Center), School of Medicine, Internal Medicine Department, University, Sao Paulo, Sao Paulo, Brazil ARTICLE INFO Keywords: Cell culture Dog Placenta Tumorigenic potential ABSTRACT The amniotic membrane can be considered as one of the sources of isolation of these cells, since it is found in the fetal maternal interface and has low immunogenicity. Mesenchymal stromal/stem cells (MSCs) have not been identified in canine amniotic membrane (AMC). Therefore, our objective was to isolate, culture, characterize and differentiate cells derived from canine amniotic membrane (AMC) and to verify its immunological and tu- morigenic potential. For this, 12 dogs fetuses of each gestational age 32, 43 and 55 days were used, and the isolation and culture of the AMC were performed. We observed that the cells presented fibroblastoid morphology and high confluence even after freezing. We also observed that, when induced, they were able to differentiate into osteogenic, adipogenic, and chondrogenic cells, as well as being CD34- and CD105+. Regarding the im- munological markers, we found that IL-1, IL-2, IL-6, IL-10 and MHC II were not expressed, whereas MHC I was expressed. After application of AMC cells in nude mice we can verify that there was no tumor formation. Based on this, we conclude that canine amniotic membrane is a good and accessible source for obtaining MSCs of low immunogenic and tumorigenic potential for veterinary therapeutic applications. 1. Introduction Knowledge about mesenchymal/stromal cells has increased in re- cent years, but there is still much to be discovered about the char- acteristics of these cells, not only their morphology, but also about related to their functionality, as well as their real potential for appli- cation in regenerative medicine (Pountos and Giannoudis, 2008; Bianco et al., 2008; Nombela-Arrieta et al., 2011). Besides that, these cells have an immunophenotype characteristic (Dominici et al., 2006), related on repair and reconstruction processes of injured tissues, thus being able to recover local functions at different levels or to replace cells lost (Filip et al., 2004). The stem cells can be classified according to their origin as fetal, embryonic or adult stem cells (Hübner et al., 2003). The benefits of fetal stem cells in relation to adults and embryos ones are due to their origin from extraembryonic tissues. Fetal stem cells can be easily isolated from extraembryonic tissues due to the large volume of this tissue, which is easily accessible during physical manipulation, allowing an increasing in the number of cells that can be isolated. In addition, extraembryonic tissues are usually discarded after delivery, so, there are low ethical problems in relation to their use (Brunstein and Wagner, 2006; Goldstein et al., 2006). Moreover, due the fact that they are at the maternal-fetal interface, they become convenient for transplants since they show (Li et al., 2005; Mihu et al., 2009), very important characteristics, such as low im- munogenicity and immunomodulatory properties. So, when the cells are implanted, they present immune defense mechanisms, reducing their rejection by the organism (Li et al., 2005). In Parolini and Caruso, 2011 described the potential for the clinical application of fetal stem cells from the human amniotic membrane. These cells can be used for treatment of many of diseases, especially those associated with degenerative processes induced by inflammatory https://doi.org/10.1016/j.tice.2019.04.007 Received 4 February 2019; Received in revised form 3 April 2019; Accepted 18 April 2019 ⁎ Corresponding authors at: Orlando Marques de Paiva, 87, Cidade Universitária, 05508-270, Butantã, Cidade Universitária, São Paulo, Brazil. E-mail addresses: jehborghesi@hotmail.com (J. Borghesi), ancoc@iq.usp.br (A.C. Oliveira Carreira). 1 Equal contribution. Tissue and Cell 58 (2019) 99–106 Available online 04 May 2019 0040-8166/ © 2019 Published by Elsevier Ltd. T