Poster H12 - Effect of Amniotic Fluid Stem Cell Derived Extracellular Vesicles on Regenerative and Fibrotic Macrophage Phenotype

Thursday, October 24, 2024

2:45 PM – 3:45 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Tarren Stephens

Student Assistant IV
University of Colorado Denver Anschutz Medical Campus
Littleton, Colorado, United States

Primary Investigator(s)

Jeffrey G. Jacot, PhD (he/him/his)

Associate professor
University of Colorado Anschutz Medical Campus
Aurora, Colorado, United States

Co-Author(s)

EB

Emily Burtch

Process Engineer
FUJIFILM Cellular Dynamics, Inc, United States

Introduction: Mesenchymal stem cells (MSCs) are being studied for regenerative therapies targeting a variety of conditions. In animal models of spina bifida myelomeningocele, intravenous injection of MSCs from bone marrow, amniotic fluid, or placental tissue improved motor outcomes. However, MSCs are not immune privileged and are rapidly eliminated, sometimes resulting in severe tissue ischemia resulting from embolisms. A functional unit behind some of MSCs’ regenerative properties are extracellular vesicles (EVs) excreted by the MSCs. EVs from MSCs derived from amniotic fluid (AFSCs) become an attractive candidate to study for stem cell based regenerative therapies, since they pose an autologous treatment option using very small particles that do not differentiate or proliferate. In a variety of in-vitro experiments, exposure to AFSC EVs improved proliferation and neurite branching of neuroblastoma cell lines, encouraged regenerative cell migration patterns in human umbilical vein endothelial cells, and proteomic profiling reveals several proteins and enzymes involved in many key metabolic, immunological, and cell proliferation pathways. We can see some supporting evidence from past work that AFSC EVs have broad spectrum regenerative properties that can be applied to different congenital defects. Our interest is in the regenerative properties and potential of AFSC EVs for improving outcomes in congenital heart defect cases. Resident cardiac macrophages play key roles in congenital heart defect disease progression by virtue of their involvement in maintaining cardiac tissues and modulating inflammation. As a result, our focus centers on the effects of AFSC EVs on human monocyte derived macrophage preference for fibrotic or regenerative phenotypes.

Materials and

Methods: AFSCs were separated from amniotic fluid collected during twin-twin transfusion syndrome surgeries and cultured. AFSCs were then stimulated to produce EVs, which were subsequently collected and purified. Cultures of THP-1 Monocytes were also expanded, then differentiated into macrophages under testing conditions of only EVs, EVs and lipopolysaccharide, or no treatment. The resulting macrophages were then fixed, stained, and analyzed for prevalence of phenotypic surface markers CD206 (M2 macrophages, predominantly regenerative) and CD11c (M1 macrophages, predominantly inflammatory). The amount of each marker compared to one another will be analyzed and used to get a preliminary read on the general impact of EVs on macrophage phenotype.

Results, Conclusions, and Discussions: Currently, there are not complete results. We are in the process of expanding lines of THP-1 monocytes for differentiation. However, if EVs have a significant effect on macrophage behavior, EVs should be considered an immune modulator. If the macrophages trend towards a more regenerative phenotype, that would make EVs a stronger candidate for further studies in regenerative stem cell based therapies. If there are no observable effects on macrophage phenotype preference, ongoing research is still a desirable option. Here, we only evaluated two surface markers and broad phenotypes on inflammation and regeneration. Resident cardiac macrophages have several more specific and nuanced roles in cardiac maturation and maintenance. In this study, we did not specifically test resident cardiac macrophages or the effect of EVs on specific roles of cardiac maintenance.

Here, we attempt to better characterize the regenerative properties of AFSC EVs by evaluating their effect on macrophage phenotype preference in order to determine the candidacy of AFSC EVs for further research into regenerative stem cell based therapies for congenital heart defects. Positive results of AFSC EV effects on macrophage phenotype preference offers some preliminary data that supports AFSC EVs’ role in immune modulation. Follow up studies further investigating the more specific effects of AFSC EVs on resident cardiac macrophages should be performed, since macrophages are complex immune cells that perform many specialized tasks beyond modulating inflammation depending on stage of development, organ system, and existing pathology. Here, we only evaluated one aspect of macrophage response to AFSC EV exposure. Additionally, macrophage responses to AFSC EVs may indicate a wider network of immune cells that react similarly or uniquely to AFSC EVs. Follow up studies aiming to further describe the effects of AFSC EVs should evaluate effects on multiple immune cells isolated and within a system. Here, we provide preliminary experimentation supporting the existence of AFSC EV immune modulatory effects.

Acknowledgements (Optional): Dr. Coughlan, director for the Exosome Core for providing guidance, protocols surrounding AFSC EV isolation, and finding our experimental EV size distributions and statistics.

NIH Grant 1R25EB031572-01A1