Abstract
PurposeAmniotic 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 MethodsTwenty 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.ResultsCAM 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.ConclusionThe 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.
Highlights
De Oliveira Pinheiro et al Immunomodulatory activities occur through direct contact between the Mesenchymal stem cells (MSCs) and tissues or through paracrine interaction mediated by interferon-gamma (IFN-γ), produced by the body’s immune cells, which act on natural killer cells, monocytes, neutrophils, and macrophages
Based on the significant immunomodulatory properties of MSCs and the limited number of studies on canine amniotic membrane (CAM) stem cells from different gestational stages, we aimed to characterize the CAM stem cells derived from different gestational stages and to determine the in vitro immunomodulatory potential and to establish a cell line that can be employed in the treatment of several diseases that affect domestic animals
The diversity of results on the immunomodulation of MSCs may be directly related to the tissue being studied or the mechanisms inherent to each species,[46] such as in dogs.[47]. This is the first study that compared the cellular characterization and gene expression patterns linked to immunomodulation in CAMs from different gestational stages
Summary
Some of the T-helper lymphocytes, cytotoxic T lymphocytes, and B lymphocytes secret soluble factors such as TGF-β, interleukin-10 (IL-10), interleukin-6 (IL-6), indoleamine2,3-dioxygenase (IDO), prostaglandin E2 (PGE2), and soluble human leukocyte antigen-G5 (sHLA-G5), or interact by cell-cell reactions,[21] in addition to preventing the expression of proinflammatory cytokines such as IFN-γ and tumor necrosis factor-α (TNF-α).[22,23]
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