Bone Research: Dr. Bo Shen's Laboratory develops deep imaging technology for the visualization and investigation of bone marrow microenvironment

On January 13, 2025, Dr. Bo Shen’s Laboratory at the National Institute of Biological Sciences, Beijing (NIBS)/Tsinghua Institute of Multidisciplinary Biomedical Research (TIMBR) published a research article titled “Deep imaging of LepR⁺ stromal cells in optically cleared murine bone hemisections” in Bone Research. This study was conducted in collaboration with Dr. Shentong Fang’s Laboratory at China Pharmaceutical University and Dr. Hu Zhao’s Laboratory at the Chinese Institute for Brain Research, Beijing (CIBR). The research introduced an efficient and cost-effective technique for skeletal tissue clearing and three-dimensional imaging, achieving an unbiased complete deep imaging of the bone marrow niches.

Adult bone marrow maintains adult hematopoietic stem cells (HSCs) and skeletal stem cells (SSCs). In this bone marrow microenvironment (also called niche), leptin receptor-expressing stromal cells (LepR⁺ cells) act as a central regulator: they not only secrete key growth factors to maintain HSCs (such as Stem Cell Factor), but themselves also contain most SSCs which would differentiate into osteoblasts and adipocytes. However, traditional imaging techniques have difficulty in clearly capturing the distribution and interactions within the niches.

In this work, authors optimized the tissue clearing process (Fig. 1), and improved the fixation, decalcification, defatting, and clearing steps. The entire sample preparation took only about 11-12 days, thus streamlined the process with enhanced imaging quality. This technique has been effectively applied to various murine skeletal tissues (such as the femur, humerus, calvaria, and tibia). This method also allows multicolor fluorescent labeling, and can be extended to deep imaging of many soft tissues (such as the spleen and lung illustrated in the article).

Figure 1: Flowchart of bone hemisection tissue clearing and deep imaging

To facilitate future researches in adult bone marrow niche and LepR⁺ cells, authors collaborated with Abcam to develop a novel monoclonal antibody (ab318272) targeting Leptin receptor. This antibody works well both in fluorescent imaging and flow cytometry, and labels human LepR⁺ cells.

To further investigate the spatial distribution of LepR⁺ cells, authors further generated a Lepr^mTagBFP2 reporter allele, which enabled precise visualization of LepR⁺ cells in parallel with the monoclonal antibody. Using this deep imaging method, authors observed that Lepr-mTagBFP⁺ cells exclusively located around blood vessels (mostly perisinusoidal, but also around peri-arteriolar locations) within adult bone marrow, while no LepR⁺ cells were found on the endosteum (Fig. 2).

Figure 2: 3D spatial image of Lepr^mTagBFP2 mouse femur hemisection (a)

and 2D projection images at different depths (b)

In summary, this deep imaging method achieved an imaging depth exceeding 500 µm in skeletal and non-skeletal tissues. The development of a LepR monoclonal antibody and Lepr^mTagBFP2 reporter allele provided tools for the future investigations of LepR⁺ cells within the bone marrow and in other tissues as well.

Yuehan Ni (2^nd year Ph.D. candidate from Beijing Normal University), Jiamiao Wu (3^rd year Ph.D. candidate from the Tsinghua University) and Fengqi Liu (3^rd year Ph.D. candidate from China Pharmaceutical University) are the co-first authors of this article. The correspondence authors are Dr. Bo Shen, Dr. Hu Zhao and Dr. Shentong Fang. Other authors of this paper include Xiangjiao Meng, Xiang Gao, Luyi Xiao, Weiwei Zhou, Peng Chu, Ge Shen, and Min Yang from Shen Laboratory, as well as other collaborators. This research was funded by the Natural Science Foundation of China, the Ministry of Science and Technology of China, the Beijing Municipal Science and Technology Commission, and the National Institute of Biological Sciences, Beijing (NIBS).