Research Paper Volume 12, Issue 14 pp 15050—15057

Establishing a density-based method to separate proliferating and senescent cells from bone marrow stromal cells

Fei Xu1,3, , Qiang Zhang2,3, , Haitao Wang3, ,

  • 1 Department of Orthopedics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
  • 2 Department of Orthopaedics, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, China
  • 3 Department of Physiology Biomedical Engineering, Division of Geriatric Medicine and Gerontology, and the Robert and Arlene Kogod Center on Aging, Mayo Clinic, Rochester, MN 55905, USA

Received: February 21, 2020       Accepted: May 27, 2020       Published: July 25, 2020
How to Cite

Copyright © 2020 Xu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


To assist in the study of cellular aging, we established a new method of enriching physiologically aged bone marrow stromal cells (BMSCs) in animals of any age using a Percoll density gradient centrifugation technique. BMSCs from mice 2 months of age were isolated, and their cellular age determined (over 80% Scal-1+ CD29+ CD11b- CD45- CD105- and able to differentiate into osteoblasts, adipocytes, and chondrocytes). As proof –of principle, cells were aged in vitro and confirmed by low bromodeoxyuridine (BrdU) incorporation and senescence-associated β-galactosidase (SA-β-gal) staining. Proliferating cells were enriched in high-density gradient layers, and senescent cells were enriched in low-density gradient layers. We confirmed that over 80% of cells from the low-density gradient layer were senescent with SA-β-gal staining and telomere dysfunction-induced foci (TIF) assay. This density-based method, which can separate proliferating and senescent BMSCs, could be used to study mechanisms of physiologic cell aging and may have implications for the use of BMSCs in clinical transplant applications.


BMSCs: bone marrow stromal cells; CD105: endoglin; CD11b: integrin subunit alpha M; CD29: integrin beta-1; CD45: protein tyrosine phosphatase, receptor type, C; SA-β-gal: senescence-associated β-galactosidase; TIFs: telomere dysfunction-induced foci; Scal-1: smoke- and cancer-associated long noncoding RNA-1.