Session: 803. Emerging Tools, Techniques and Artificial Intelligence in Hematology: Poster III
Hematology Disease Topics & Pathways:
emerging technologies, Technology and Procedures, imaging
The standard practice to generate CHIP models relies on transplanting syngeneic or allogeneic cells into a sub-lethally irradiated recipient. However, concerns arise as irradiation compromises the microenvironment10. Successful engraftment in non-conditioned mice can be achieved by injecting a high number of cells (1.5 x 107 cells) 11,12, but modeling early clonal expansion events from single cells remain challenging, as a crowd of cells is often observed in proximity. Here, we established a low dose (0.5-1.5 Gy), whole body or local irradiation regimen that preserves the integrity of the bone marrow microenvironment. We performed high-resolution in-vivo imaging to capture dynamics of single cells and cells undergoing early expansion. Specifically, as little as 0.5Gy irradiation enabled survival of the transplanted cells (2x106 GFP+ healthy whole bone marrow) in non-irradiated side, and allowed direct visualization of early clonal expansion in vivo through 16 weeks. Engraftment was negligible in non-irradiated mice. Through in-vivo, video-rate tracking of Rhodamine dextran dye (70kDa) leakage from vessels into the extravascular space showed no increase in permeability13 or signs of vessel dilation, suggesting minimal inflammation after the radiation insult (N= 3 mice, data points are individual vessel segments. Mann-Whitney U test). Preliminary findings with in-vitro cultures of mesenchymal stromal cells (MSCs) have also suggested that the MSCs retain their ability for tri-lineage differentiation after the low-dose irradiation. With this imaging protocol, we showed that hot spots of cell expansion exist in the Tet2+/- murine model.
To capture the highly localized niche factors responsible for these hot spots, we implemented image-guided live-cell labeling adapted from the previously published Image-seq protocols9. As illustrated in Figure 1, a small channel is etched in the bone with plasma-mediated laser ablation. The micropipette was then inserted to the region of interest for delivery of fluorescent antibodies under image guidance. By administering FITC-conjugated anti-CD45 antibodies into the marrow cavities, we can label and recover ~10,000 live cells with 98% viability from two regions of interest, suitable for next generation single cell sequencing. The technique can be expanded to employ antibody cocktails to visualize and enrich particular cell lineages of interest during cell isolation, while preserving spatial information. Its compatibility with standard cell isolation protocols for FACS may help collect cellular targets that are geometrically inaccessible for cell aspiration.
In conclusion, we established a working model to visualize expansion of healthy and Tet2+/- hematopoietic cells in vivo. This can be followed by image-assisted live-cell tagging to isolate local microenvironment cells and study cell-niche coordination at high spatial precision. The imaging protocol may also be broadly applied to study microenvironment regulations in non-malignant clonal disorders.
Disclosures: No relevant conflicts of interest to declare.