Single-cell, high-throughput analysis of cell docking to vessel wall

Abstract

Therapeutic potential of mesenchymal stem cells (MSCs) has been reported consistently in animal models of stroke, with mechanism mainly through immunomodulation and paracrine activity. Intravenous injection has been a prevailing route for MSCs administration, but cell quantities needed when scaling-up from mouse to human are extremely high putting into question feasibility of that approach. Intra-arterial delivery directly routes the cells to the brain thus lowering the required dose. Cell engineering may additionally improve cell homing, further potentiating the value of intra-arterial route. Therefore, our goal was to create microfluidic platform for screening and fast selection of molecules that enhance the docking of stem cells to vessel wall. We hypothesized that our software will be capable of detecting distinct docking properties of naïve and ITGA4-engineered MSCs. Indeed, the cell flow tracker analysis revealed positive effect of cell engineering on docking frequency of MSCs (42% vs. 9%, engineered vs. control cells, p < 0.001). These observations were then confirmed in an animal model of focal brain injury where cell engineering resulted in improved homing to the brain. To conclude, we developed a platform to study the docking of cells to the vessel wall which is highly relevant for intraarterial cell targeting or studies on neuroinflammation.

Keywords: ITGA4; Mesenchymal stem cells; docking; mRNA; microfluidic assay; stroke.

Figure 1.

Experimental study design. Initially, the hBM-MSCs were engineered with mRNA-ITGA4 and the functionality of the ITGA4 protein was analysed. We established a microfluidic assay and generated the necessary data to develop a software program for single-cell, high-throughput, real-time analysis of cell docking in a model of activated blood vessel, with subsequent validation against the manual analysis. In the next step, we compared the interaction of engineered and naïve hBM-MSCs during the flow in channel microfluidic chamber coated with VCAM-1, and, finally, we compared the docking of engineered vs. naïve cells in an animal model of focal brain injury.

Figure 2.

Flow cytometry of control and engineered hBM-MSCs at various time points after mRNA-ITGA4 transfection. The visual illustration of flow cytometry results (a), the graphical presentation of the percentage of ITGA (+) hBM-MSCs (b) and the changes in the fluorescence signal intensity generated by an antibody attached to the ITGA4 antigen on the surface of the cells (c).

Figure 3.

The correlation analysis of the distance traveled by single cells, assessed manually in the ImageJ program and automatically by the cell flow tracker software (a). The speed pattern curves of three random cells measured by the cell flow tracker (red line) and measured manually (blue line) (b).

Figure 4.

The example of movement patterns typical for hBM-MSCs during their interactions with microfluidic channels decorated with the VCAM-1 protein which resembles steps of the leukocyte transmigration process. The flow of unstopped cells (a), rolling (b), arrest (c), and crawling (d).

Figure 5.

The functional effects of mRNA-ITGA4 engineering of hBM-MSCs in vitro. The percentage of mRNA-ITGA4-engineered cells or control hBM-MSCs, which docked in the model of the activated vessel wall (a). The relative ratio of mRNA-ITGA4 engineered to control hBM-MSCs across ranges of the arrest lengths (b). The correlation between the percentage of docked cells and the length of their arrest (c).

Figure 6.

Analysis of the movement dynamics of hBM-MSCs flowing through microfluidic chamber. The rolling of hBM-MSCs prior to their attachment compared to cell flow (a). The crawling of hBM-MSCs prior to their attachment compared to cell flow (b). The comparison of the average speed of mRNA-ITGA4-engineered cells to control hBM-MSCs (c). The comparison of cell size between mRNA-ITGA4-engineered cells and control hBM-MSCs (d).

Figure 7.

The comparison of in vivo docking efficacy between mRNA-ITGA4-engineered cells and control hBM-MSCs in an animal model of focal brain injury under MRI guidance. Examples of MR images (a). The quantification of docking efficacy in both mRNA-ITGA4-engineered cells and control hBM-MSCs (b), with a distribution of difference across brain cross-sections to illustrate the multilevel model used for statistical calculations (c) (control cell group n = 6, mRNA ITGA4 hBM-MSCs group n = 6).