Immunomodulatory effects of mesenchymal stem cells derived from equine amniotic membrane: proteomic and cytokine profile analysis of conditioned medium and its interaction with the immune system.

Abstract

The mesenchymal stem cells and multipotent stromal cells (MSCs) have emerged in recent years as an excellent alternative to embryonic stem cells for their high plastic capacity and its immunomodulatory effects without inducing tumor formation and exhibiting lower ethical barriers in their therapeutic use. Bone marrow is the most used source of MSCs, however their invasive way of harvest and their age-dependent capacity of proliferation and differentiation motivated the search for new sources. Thus, Because they are sources routinely discarded after birth, with non-invasive way of harvest, high potential of differentiation and immunomodulatory effect, MSCs derived from fetal adnnexes have gained attention in recent decades. These characteristics, combined with its low immunogenicity has made the MSC derived from amniotic membrane (AM-MSCs) a great candidate for the formation of stem cell banks for future use in allogeneic therapies for several species. However, research about equine AM-MSCs are still rare. The aim of this study is to evaluate the immunomodulatory effects of AM-MSCs equine seeking the formation of a cell bank. Membrane samples will be collected at delivery and transferred in a cooled transport system to the laboratory. Cells will be obtained by enzymatic digestion and then cultured under special conditions. The conditioned medium will be harvested during in vitro expansion and cryopreserved until use. The kinetic profile of cell proliferation will be assessed through the tests of colony forming unit fibroblast-like cells, population doubling and doubling time. The samples will be identified immunophenotypically for expression of CD29, CD44, CD105, CD34, MHCI and MHCII markers by RT-PCR. Evaluation of the expression of Oct-4, TRA1-60, and SSEA-4 pluripotency markers will be performed by flow cytometry and, if found, the potential of differentiating into lineages will be evaluated by neurogenic in vitro differentiation. The proteomic assessment of conditioned medium will be performed by bi-dimensional electrophoresis and mass spectrometry. The cytokine profile will be evaluated by ELISA. The study of the interaction of AM-MSCs with immune system cells will be performed by the lymphocyte proliferation test. Finally, two different protocols of cryopreservation, for the best moment of freezing cells to form the cell bank, will be compared. With the expected results, we wish to demonstrate the characteristics of MA-equine MSCs as advantageous for creation of an equine stem cells bank and their use in allogeneic therapies.