Multipotent mesenchymal stem cells (MSCs) can be easily derived from bone marrow and other tissues and are of high interest for clinical use. MSCs are characterized by plastic adherence, immunophenotype, and their differentiation potential in vitro. However, the current understanding of their localization and function in vivo remain elusive, because of a lack of specific MSC markers. We therefore aimed to generate a transgenic mouse model in which MSCs are labeled by a live reporter gene.

CD73, an established membrane surface molecule on MSCs, appeared to be a suitable marker with only moderate distribution in other cell types. We confirmed high expression of CD73 in various MSC populations by FACS, immunostainings and PCR. A reporter vector expressing EGFP under control of the CD73 promoter was generated from bacterial artificial chromosomes (BACs). Transfection of the vector into established MSC lines gave rise to EGFP positive cells with the ability to differentiate into the osteogenic and adipogenic lineage, proving expression of the construct in multipotent MSCs. Next, we generated transgenic embryonic stem cell (ESC) lines using the CD73-BAC-EGFP reporter vector. EGFP positive cells with fibroblastic morphology could be detected starting from day six of ESC differentiation. Then, transgenic mice were generated from ESC clones by diploid aggregation. EGFP expressing cells were visible in different tissues of embryonic and adult CD73-BAC-EGFP mice, such as in developing bones at the sites of endochondral ossification, demonstrating osteogenic precursor cells, or in the bone marrow, labeling sinusoidal adventitial cells. Furthermore, primary cultures from epiphysis, bone marrow, white fat, uterus and kidney gave rise to adherent growing EGFP positive cells that were able to differentiate into mesenchymal lineages, underlining the residence of MSCs in various organs.

We are currently further analyzing the transgenic mouse model to characterize the EGFP positive cells and to investigate their in vivo distribution. Long term goal is the identification of the MSC niche in order to better understand the biology of these cells and their physiological and pathophysiological roles.