Available on-demand - F.SM07.01.05
The Plasticity of Primary Human Macrophages When Interacting with Tissue-Engineered Blood Vessels
Beatriz Hernaez Estrada1,2,Edorta Santos Vizcaino2,3,Rosa Hernandez2,3,Kara Spiller1
Drexel University1,University of the Basque Country2,Biomedical Research Networking3
Introduction: Deficient vascularization of engineered tissues is one of the major causes of inadequate tissue integration and function in vivo. A potential solution to this problem is the implantation of pre-vascularized biomaterials, such as those prepared from human adipose microvascular endothelial cells (HAMECS) and adipose-derived mesenchymal stromal cells (MSC) [1-2]. However, these pre-vascularized constructs will encounter an inflammatory environment upon implantation, and it is not known how this will affect the engineered blood vessels. Macrophages, the primary cells of the inflammatory response, are key regulators of tissue vascularization and integration of implanted biomaterials. Even though macrophages can exist as different phenotypes, each with distinct effects on blood vessels, they are also very plastic cells that are able to switch phenotypes depending on microenvironmental stimuli . In the first phase of wound healing, macrophages are primarily pro-inflammatory (M1), whereas the second stage is characterized by a pro-wound resolution phenotype (M2). M2 macrophages can be derived from non-activated (M0) macrophages or they may switch from M1 (resulting in M1-M2). The diversity of these populations and their crosstalk with blood vessels are not known. Therefore, the purpose of this study was to assess the phenotypic subsets of primary human macrophages polarized to pro-inflammatory (M1) and pro-wound resolution (M2 and M1-M2) phenotypes, and how they change when cultured with 3D tissue-engineered human blood vessels in vitro.
Methods: Tissue-engineered blood vessels were formed by co-culturing HAMECs and MSCs within porous gelatin scaffolds (Surgifoam®). M0, M1, M2 and M1-M2 primary macrophages were seeded alone on the scaffolds or in co-culture with the engineered blood vessels as previously described . Then, the scaffolds were digested to generate a single cell suspension and stained with a 10-marker flow cytometry panel: macrophage general marker (CD45), M1 macrophage markers (CCR7, HLA-DR, CD83, CD38 and PD-L1), and M2 macrophage markers (CD206, CD209, CD163 and CXCR4). Data were acquired on BD Fortessa™ flow cytometer. Live, single and CD45 positive cells from all the different conditions were down sampled and merged into a single expression matrix prior to dimensionality reduction analysis in FlowJo®. Cluster analysis was done using manual gating or different plugins such as FlowSom.
Results: Using this approach, we were able to differentiate the effect of the biomaterial itself from the effects of interactions with blood vessels on macrophage phenotype. While macrophages polarized to the different phenotypes were relatively homogenous in 2D culture, after 24 hours in 3D culture, even without tissue-engineered blood vessels, they became a heterogeneous population comprising 3-4 main clusters, each comprising cells that expressed varying levels of each marker. Following crosstalk with tissue-engineered blood vessels, the prior polarization states of the macrophages affected their response. In particular, M1 and M1-M2 macrophages became enriched in clusters characterized by high levels of M1 markers (such as CD38, CD80 and CD38), while M2 and M0 macrophages became enriched in clusters that were predominant in M2 markers (such as CD206, CD163 and CXCR4).
Conclusions: These findings suggest that macrophage phenotype is strongly affected by co-culture with tissue-engineered blood vessels in a way that depends on the prior polarization state of the macrophages.
Acknowledgements: This project was supported by NHLBI HL130037 to KLS. The NanoBioCel group acknowledges SAF2017-82292-R (MINECO/AEI/FEDER, UE). B.Hernaez thanks the Basque Government for the PhD grant. Flow cytometry was conducted in Sidney Kimmel Cancer Center Flow Cytometry Facility (Philadelphia)
References: Freiman et al. Stem Cell Research & Therapy (2016) Ben-Shaul et al. PNAS (2019) Graney, P.L. et al. Science Advances (2020)