In Vivo flow Cytometry Combined with Intravital Microscopy to Monitor Synchronous Kinetics of Transplanted BM-MNCs in Peripheral Blood and Bone Marrow
Authors: Wang Fen , Wei Dan , Suo Yuanzhen , Zhu Xi , Yuan Yan , Jiang Hua , Wei Xunbin , Chen Tong 
Published: October 2019

Abstract
Limited sources and finite numbers of hematopoietic stem cells (HSCs) have always been difficult problems to overcome in clinical HSC transplantation (HSCT). Improving the bone marrow (BM) homing efficiency of HSC has become an intensely studied topic. HSC homing involves the kinetics of transplanted cells in the peripheral blood (PB), the cell distribution in various organs and tissues, and the targeted homing of HSC to the BM. However, it is difficult to monitor the dynamic homing in living mice. The objective of this study is to establish a platform for monitoring the kinetics of transplanted BM-MNCs in the PB and the synchronous homing of BM-MNCs in the BM in living mice by a combination of in vivo flow cytometry (IVFC) and calvarium intravital microscopy.
IVFC is a new in vivo technology that can quantitatively and continuously monitor fluorescence-labeled cells circulating in an anesthetized mouse. When a circulating fluorescent cell passes through the laser slit across the selected ear artery, fluorescence can be excited and recorded. Therefore, this method can provide continuous information on cellular behavior over time in the same living mouse without requiring extracting a blood sample at every time point. Calvarium intravital microscopy is another in vivo technology that allows a direct view of the dynamic homing of transplanted cells in the BM of a living mouse. Although the skull and femur are different bone types, the homing behavior and function of HSCs in the cross-road intersection region of coronal and sagittal sutures are comparable to those in the long bones in terms of immunophenotype and propagation. With calvarium intravital microscopy combined with in vivo flow cytometry, we here continually monitor the synchronous dynamic homing of transplanted BM-MNCs in the skull marrow in living mouse.
By detecting the fluorescent cells in circulation, we obtained the kinetics of transplanted BM-MNCs in the PB over 24 h. Interestingly, the number of BM-MNCs decreased rapidly about 90% in the PB within the first 4 h after transplantation, further decreasing from 4 h to 6 h, and remaining at a low level from 6 h to 24 h. We further applied calvarium intravital microscopy to directly visualize the localization and the synchronous homing kinetics of BM-MNCs in the mouse skull marrow after transplantation. Interestingly, the BM-MNCs were mainly distributed over the large vessels in the cross-road intersection of coronal and sagittal sutures at 24 h, especially within the anterior half of this region (90-95% of total fluorescent cells). We then mainly measured the synchronous homing kinetics of BM-MNCs in the anterior half region at 2, 6, and 24 h in the mice, and found out that the homing of BM-MNCs in the skull marrow indeed increased gradually in the same mouse with the same pattern in different transplanted mice. Both the data obtained by IVFC and the calvarium intravital microscopy had a good linear correlation with their respective conventional in vitro flow cytometry data. By combining the conventional in vitro data, we also found a similar synchronous kinetics of BM-MNCs in the PB and the BM. All these data suggest that the BM-MNCs cleared from the PB might home to the BM over time after transplantation.
In summary, as an important source of HSCs, BM-MNCs have been successfully applied to clinical HSCT. Understanding the early homing mechanism of transplanted BM-MNCs can enhance the marrow homing efficiency and engraftment of HSCs. Some other in vivo methods have been developed to study the homing of transplanted BM-MNCs to BM, including magnetic resonance imaging (MRI), single-photon emission computed tomography (SPECT), endoscopic probing, and femur drilling to a laser-detectable depth. Compared to these methods, our method by calvarium intravital microscopy is mildly invasive, while the homing kinetics of BM-MNCs to the BM can be directly viewed and measured at the single-cell level.

Disclosures
No relevant conflicts of interest to declare.