Title: Investigation of conditioned medium properties obtained from mesenchymal stem cells preconditioned with dimethyloxalylglycine

Abstract:

Background: Mesenchymal stem cells (MSCs) are multipotent progenitor cells with fibroblastic morphology, high proliferation capacities, and trilineage differentiation abilities¹. They can be isolated from many tissues, such as bone marrow, umbilical cord, adipose tissue, and amniotic membrane, and reproduced on a large scale in vitro. Since the umbilical cord is a primitive tissue, cells isolated from it stand out for regenerative treatments with their high proliferation capacity, strong immunomodulation, and immunosuppression abilities. Recent research has shown that the therapeutic value of MSCs is related to secretomes rather than their ability to differentiate^2,3. In this case, a better understanding and enhancement of the paracrine properties of MSCs will increase the therapeutic potential of these cells and the possibility of developing targeted therapy options. Regulation of the secretome profiles of MSCs involves cultivating cells following a specific topography, preconditioning them with hypoxia and various chemical agents⁴. Preconditioning with hypoxia improves the robustness and survival of MSCs and their therapeutic potential by increasing their angiogenic and immune modulatory capacity⁵.

Aim: This study aimed to compare the secretome profiles and exosome contents of two types of conditioned media (CM) obtained from human umbilical cord-derived MSCs preconditioned and non-preconditioned with dimethyloxalylglycine (DMOG), a hypoxia mimetic agent, and to evaluate the ultrastructural changes in both MSC groups.

Methods: First, MSCs were isolated from the human umbilical cord, and the cells were characterized by confirming their immunophenotypic and differentiation properties. The expression of Hif1a in MSCs was analyzed immunocytochemically to decide the appropriate dose and duration of use DMOG. Then, two different CMs were prepared by preconditioning MSCs with/without DMOG. CM contents were analyzed for total protein, IL-4, IL-10, IL-17, IFN-λ, VEGF, NGF, BDNF, and GDNF. Moreover, exosomes were isolated from both conditioned media using a commercial kit, and the isolated exosomes were shown by Western blot and transmission electron microscopy. To observe and compare the effects of conditioned media on proliferation and migration, an in vitro wound healing test was performed. Finally, MSCs in both groups were evaluated ultrastructurally.

Results: After isolation and characterization of MSCs, considering the Hif1a expression results, 1000μM DMOG was applied to MSCs for 24 hours to prepare the conditioned medium. VEGF, NGF, and IL-4 levels were found to be increased in CM from DMOG-preconditioned MSCs compared to CM from normal MSC. BDNF, GDNF, IL-10, IL-17, and IFN-γ levels were not detectable in both groups. Western blot and negative staining in TEM demonstrated the presence of exosomes isolated from CMs of both groups. According to the wound healing test results, both CM increased the migration and proliferation abilities of 3T3 cells. Finally, examination of MSCs by TEM showed that these cells were active, and it was observed that autophagosome, autolysosome, myelin figure, and microvesicular body structures increased in MSCs preconditioned with DMOG.

Conclusion: These findings suggested that preconditioning MSCs with DMOG could alter their secretion profile, modify their ultrastructural morphology accordingly, and make their conditioned medium a more potent therapeutic tool

Biography:

Basak Isıldar completed her bachelor's degree in the fields of Molecular Biology and Genetics at Istanbul University University. She received her master's and doctorate degrees from Istanbul University-Cerrahpasa, Cerrahpasa Faculty of Medicine, Department of Histology and Embryology. In her master's thesis, she investigated the characteristics of mesenchymal stem cells isolated after freezing the umbilical cord in different cryopreservation solutions, and in her doctoral thesis, the effects of conditioned media obtained from MSCs cultured in 2D and 3D environments on experimental autoimmune type 1 diabetes (T1D). Her studies on extracellular vesicles and conditioned medium derived from MSCs and their effects on T1D are still ongoing. She also gives Histology and Embryology lectures at Balikesir University, Faculty of Medicine.