Characterization and Immunomodulation of Canine Amniotic Membrane Stem Cells
Received 7 November 2019
Accepted for publication 24 March 2020
Published 7 May 2020 Volume 2020:13 Pages 43—55
Checked for plagiarism Yes
Review by Single anonymous peer review
Peer reviewer comments 2
Editor who approved publication: Dr Bernard Binetruy
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, Brazil
Correspondence: Carlos E Ambrosio
Department 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, Brazil
Tel +55 19 3565-4113 Email firstname.lastname@example.org
Purpose: 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
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