Related Experiment Video
Updated: Jul 20, 2025
SA-β-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs
Published on: June 28, 2019
Multiparametric senescent cell phenotyping reveals targets of senolytic therapy in the aged murine skeleton
Madison L Doolittle1,2, Dominik Saul1,2,3, Japneet Kaur1,2
1Division of Endocrinology, Diabetes and Metabolism, Mayo Clinic, Rochester, MN, 55905, USA.
Abstract:
Senescence drives organismal aging, yet the deep characterization of senescent cells in vivo remains incomplete. Here, we apply mass cytometry by time-of-flight using carefully validated antibodies to analyze senescent cells at single-cell resolution. We use multiple criteria to identify senescent mesenchymal cells that are growth-arrested and resistant to apoptosis. These p16 + Ki67-BCL-2+ cells are highly enriched for senescence-associated secretory phenotype and DNA damage markers, are strongly associated with age, and their percentages are increased in late osteoblasts/osteocytes and CD24high osteolineage cells. Moreover, both late osteoblasts/osteocytes and CD24high osteolineage cells are robustly cleared by genetic and pharmacologic senolytic therapies in aged mice. Following isolation, CD24+ skeletal cells exhibit growth arrest, senescence-associated β-galactosidase positivity, and impaired osteogenesis in vitro. These studies thus provide an approach using multiplexed protein profiling to define senescent mesenchymal cells in vivo and identify specific skeletal cell populations cleared by senolytics.
Insights
Cellular senescence drives aging. This study identifies specific senescent mesenchymal cells in vivo using mass cytometry and finds that senolytic therapies clear these cells, particularly in bone tissue, offering new aging insights.
Area of Science:
- Cellular Biology
- Gerontology
- Biochemistry
Background:
- Cellular senescence is a key driver of organismal aging.
- Comprehensive characterization of senescent cells in vivo is limited.
- Senescent cells contribute to age-related pathologies.
Purpose of the Study:
- To deeply characterize senescent mesenchymal cells in vivo at single-cell resolution.
- To identify specific senescent cell populations within skeletal tissues.
- To evaluate the efficacy of senolytic therapies against these cells.
Main Methods:
- Mass cytometry by time-of-flight with validated antibodies for high-resolution analysis.
- Multi-parameter criteria to identify senescent mesenchymal cells (p16+, Ki67-, BCL-2+).
- In vivo and in vitro assays to assess cell characteristics and response to senolytics.
Main Results:
- Identified senescent mesenchymal cells exhibiting growth arrest, apoptosis resistance, senescence-associated secretory phenotype, and DNA damage markers.
- Senescent cell percentages increase with age, particularly in late osteoblasts/osteocytes and CD24high osteolineage cells.
- Genetic and pharmacologic senolytic therapies effectively cleared these senescent skeletal cells in aged mice.
- Isolated CD24+ skeletal cells showed growth arrest, senescence markers, and impaired osteogenesis in vitro.
Conclusions:
- Multiplexed protein profiling provides a robust approach to define senescent mesenchymal cells in vivo.
- Specific skeletal cell populations are identified as targets for senolytic intervention.
- Senolytic therapies show promise for clearing senescent cells in aging bone, potentially mitigating age-related bone dysfunction.
