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Alessandra de Oliveira Pinheiro,1 Valéria M Lara,1 Aline F Souza,1 Juliana B Casals,2 Fabiana F Bressan,1 Paulo Fantinato Neto,1 Vanessa C Oliveira,1 Daniele S Martins,1 Carlos E Ambrosio1 1Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of São Paulo, Pirassununga, São Paulo, Brazil; 2Private Veterinary Practice, Pirassununga, São Paulo, BrazilCorrespondence: Carlos E AmbrosioDepartment of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of São Paulo, FZEA- Av. Duque de Caxias Norte, 225, ZMV, Pirassununga 13635-900, São Paulo, BrazilTel +55 19 3565-4113 Email ceambrosio@usp.brPurpose: Amniotic membrane stem cells have a high capacity of proliferation, cell expansion, and plasticity, as well as immunomodulatory properties that contribute to maternal-fetal tolerance. Owing to the lack of research on human amniotic membrane at different gestational stages, the canine model is considered ideal because of its genetic and physiological similarities. We aimed to characterize the canine amniotic membrane (CAM) cell lineage in different gestational stages and evaluate the expression of immunomodulatory genes.Materials and Methods: Twenty CAMs from early (20– 30 days) (n=7), mid- (31– 45 days) (n=7), and late gestation (46– 63 days) (n=6) stages were studied. The cell features were assessed by cell viability tests, growth curve, colony-forming units, in vitro differentiation, cell labeling for different immunophenotypes, and pluripotent potential markers. The cells were subjected to RT-PCR and qPCR analysis to determine the expression of IDO, HGF, EGF, PGE2, and IL-10 genes.Results: CAM cells exhibited a fibroblastoid morphology and adherence to plastic with an average cell viability of 78.5%. The growth curve indicated a growth peak in the second passage and we obtained an average of 138.2 colonies. Osteogenic, chondrogenic, and adipogenic lineages were confirmed by in vitro differentiation assays. Cellular immunophenotyping experiments confirmed the presence of positive mesenchymal markers (CD90 and CD105) and the low or negative expression of hematopoietic markers (CD45 and CD34). Qualitative analysis of the immunomodulatory functions indicated the expression of the IDO, HGF, EGF5, and PGE2 genes. When stimulated by interferon-gamma, CAM cells exhibited higher IDO levels throughout gestation.Conclusion: The CAMs from different gestational stages presented features consistent with mesenchymal stem cell lineage; better results were observed during the late gestation stage. Therefore, the gestational stage is a key factor that may influence the functionality of therapies when using fetal membrane tissues from different periods of pregnancy.Keywords: canine stem cells, immunomodulation, fetal annexes

Helena Debiazi Zomer,1,2 Kelly CS Roballo,2 Thais Borges Lessa,2 Fabiana Fernandes Bressan,2 Natália Nardeli Gonçalves,2 Flávio Vieira Meirelles,2,3 Andrea Gonçalves Trentin,1 Carlos Eduardo Ambrósio2,3 1Department of Cell Biology, Embryology and Genetic, Faculty of Biological Sciences, Santa Catarina Federal University (UFSC), Florianópolis, Brazil; 2Department of Surgery, Sector Anatomy, Faculty of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil; 3Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of São Paulo, São Paulo, Brazil Introduction: Owing to their similarity with humans, rabbits are useful for multiple applications in biotechnology and translational research from basic to preclinical studies. In this sense, mesenchymal stem cells (MSCs) are known for their therapeutic potential and promising future in regenerative medicine. As many studies have been using rabbit adipose-derived MSCs (ASCs) as a model of human ASCs (hASCs), it is fundamental to compare their characteristics and understand how distinct features could affect the translation to human medicine.Objective: The aim of this study was to comparatively characterize rabbit ASCs (rASCs) and hASCs to further uses in biotechnology and translational studies.Materials and methods: rASCs and hASCs were isolated and characterized by their immunophenotype, differentiation potential, proliferative profile, and nuclear stability in vitro.Results and discussion: Both ASCs presented differentiation potential to osteocytes, chondrocytes, and adipocytes and shared similar immunophenotype expression to CD105+, CD34–, and CD45–, but rabbit cells expressed significantly lower CD73 and CD90 than human cells. In addition, rASCs presented greater clonogenic potential and proliferation rate than hASCs but no difference in nuclear alterations.Conclusion: The distinct features of rASCs and hASCs can positively or negatively affect their use for different applications in biotechnology (such as cell reprogramming) and translational studies (such as cell transplantation, tissue engineering, and pharmacokinetics). Nevertheless, the particularities between rabbit and human MSCs should not prevent rabbit use in preclinical models, but care should be taken to interpret results and properly translate animal findings to medicine.Keywords: characterization, comparison, MSC, iPS, immunophenotype, proliferation

Atanasio S Vidane,1 Aline F Souza,1 Rafael V Sampaio,1 Fabiana F Bressan,2 Naira C Pieri,1 Daniele S Martins,2 Flavio V Meirelles,2 Maria A Miglino,1 Carlos E Ambrósio2 1Department of Surgery, Faculty of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil; 2Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engineering, University of São Paulo, São Paulo, Brazil Abstract: Amnion-derived mesenchymal stem cells (AMSCs) are multipotent cells with an enhanced ability to differentiate into multiple lineages. AMSCs can be acquired through noninvasive methods, and therefore are exempt from the typical ethical issues surrounding stem cell use. The objective of this study was to isolate and characterize AMSCs from a cat amniotic membrane for future application in regenerative medicine. The cat AMSCs were harvested after mechanical and enzymatic digestion of amnion. In culture medium, the cat AMSCs adhered to a plastic culture dish and displayed a fibroblast-like morphology. Immunophenotyping assays were positive for the mesenchymal stem cell-specific markers CD73 and CD90 but not the hematopoietic markers CD34, CD45, and CD79. Under appropriate conditions, the cat AMSCs differentiated into osteogenic, chondrogenic, and adipogenic cell lineages. One advantage of cat AMSCs was nonteratogenicity, assessed 4 weeks post injection of undifferentiated AMSCs into immunodeficient mice. These findings suggest that cat amniotic membranes may be an important and useful source of mesenchymal stem cells for clinical applications, especially for cell or tissue replacement in chronic and degenerative diseases. Keywords: amnion, cats, cell differentiation, fetal membranes, mesenchymal cells