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In vivo flow cytometry combined with intravital microscopy to monitor kinetics of transplanted bone marrow mononuclear cells in peripheral blood and bone marrow

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Abstract

Bone marrow mononuclear cells (BM-MNCs) transplantation has evolved as a promising experimental treatment in various regenerative therapy fields, especially in clinical hematopoietic stem cells transplantation (HSCT). In vitro methods have mainly been used to study the pre-clinical kinetics of BM-MNCs in mice after transplantation. And it is difficult to monitor the dynamic homing of BM-MNCs in living mice. The present study obtained the kinetics of transplanted BM-MNCs in the peripheral blood (PB) and the dynamic homing of BM-MNCs in the BM in living mice by a combination of in vivo flow cytometry (IVFC) and calvarium intravital microscopy. We found out that BM-MNCs were cleared rapidly from the PB and mainly localized to various hematopoietic tissues after transplantation. The number of BM-MNCs in the PB decreased over time accompanied by an increase in the BM indeed after transplantation. In addition, a lower number of BM-MNCs were found home to calvaria than long bone, probably indicating long bone marrow might also be an important hematopoietic organ. Clinical studies will benefit from non-invasive measurements to monitor the dynamic homing of transplanted cells. Our pre-clinical kinetics of BM-MNCs in living mice will have important clinical guiding significance in HSCT and other regenerative therapy fields.

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Acknowledgements

This study was supported by National Natural Science Foundation of China (Grant Nos. 81561138002, 91542109), Program of Shanghai Subject Chief Scientist (Grant No. 16XD1400600) to Tong Chen, and National Science Fund for Distinguished Young Scholars (Grant No. 61425006) to Xunbin Wei.

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  1. Fen Wang and Dan Wei have contributed equally to this work.

Authors and Affiliations

  1. Department of Hematology, Huashan Hospital, Fudan University, Shanghai, 200040, China

    Fen Wang, Yan Yuan & Tong Chen

  2. Med-X Research Institute, School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, 200030, China

    Dan Wei, Yuanzhen Suo, Xi Zhu & Xunbin Wei

  3. Obstetrics & Gynecology Hospital, Fudan University, Shanghai, 200011, China

    Hua Jiang

  4. Beijing Advanced Innovation Center for Biomedical Engineering, Beihang University, Beijing, 100083, China

    Xunbin Wei

  5. Department of Pharmacy, Binzhou Medical University, Yantai, 264003, Shandong, China

    Wenyuan Gao

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  1. Fen Wang
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Correspondence to Hua Jiang, Xunbin Wei or Tong Chen.

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Ethical approval

All experimental procedures involving animals were approved by Ethical Committee of Animal Experiments of the School of Pharmacy, Fudan University (2014-09-HSYY-CT-01). All mice were housed and bred at the Laboratory Animal Center of School of Pharmacy affiliated with Fudan University (No. 826, Zhangheng Road, Pudong District, Shanghai).

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Supplementary Figure 1

. Schematic of in vivo flow cytometry (IVFC). In the IVFC, a suitable ear artery was chosen for subsequent signal detection. A He-Ne laser was emitted continuously and focused onto a slit. The recipient mouse was positioned and fixed to ensure the laser slit traverse the width of the selected artery, covering the whole interface. When a circulating DiD-labeled cell passed through the slit, fluorescence was excited and collected by the same microscope objective (40X, NA=0.6). Thus, we could obtain continuous cytometric information on the kinetics of circulating cells in vivo in the same mouse. BS1-2: dichroic beam splitter. M1: mirror. BPF1-2: bandpass filter. AL1-3: achromatic lenses. CL: cylindrical lens. NDF: neutral-density filter. A/D: Analog-to-digital converter. CCD: charge-coupled device. PMT: photo multiplier tube. (TIF 683 KB)

Supplementary Figure 2

. Schematic of calvarium intravital microscopy. Before scanning, the vessels and transplanted cells within the skull marrow were labeled with different fluorescence markers (green for vessels by intravenously injected FITC-dextran; red for transplanted cells by DiD). After deep anesthetization, the mouse scalp was incised to fully expose the cross-road intersection region of coronal and sagittal sutures. Then, the mouse was positioned on the microscope stage comfortably with a heating pad. After the laser from the objective of the microscope scanned the mouse skull marrow, the fluorescence images were acquired. The images were further analyzed by Imaris Software. (TIF 562 KB)

Supplementary Figure 3

. The definition of scanning depth in intravital imaging of calvarial marrow. Red is DiD labeled BM-MNCs. Green is FITC-Dextran labeled vessels. (TIF 2963 KB)

Supplementary Figure 4

. The homing kinetics of BM-MNCs in BM by intravital microscopy in different mice. The statistical difference of the homing cell number to the skull marrow in different mice at various time points (n=5 per time point). The statistical analysis of each panel was presented in Supplementary material (Statistic table S2-2). The data were presented as mean ± SEM. (TIF 1325 KB)

Supplementary Figure 5

. The immunofluorescence histological analysis of tibia at various time points. Blue is DAPI for labeling cell nucleus. Green is GFP labeled BM-MNCs. (TIF 6355 KB)

Supplementary material Tables (DOCX 17 KB)

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Wang, F., Wei, D., Suo, Y. et al. In vivo flow cytometry combined with intravital microscopy to monitor kinetics of transplanted bone marrow mononuclear cells in peripheral blood and bone marrow. Mol Biol Rep 47, 1–10 (2020). https://doi.org/10.1007/s11033-019-04608-x

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