Establishment of In Vitro Models by Stress-Induced Premature Senescence for Characterizing the Stromal Vascular Niche in Human Adipose Tissue

Marlene Wahlmueller; Marie-Sophie Narzt; Karin Missfeldt; Verena Arminger; Anna Krasensky; Ingo Lämmermann; Barbara Schaedl; Mario Mairhofer; Susanne Suessner; Susanne Wolbank; Eleni Priglinger

doi:10.3390/life12101459

Establishment of In Vitro Models by Stress-Induced Premature Senescence for Characterizing the Stromal Vascular Niche in Human Adipose Tissue

Life (Basel). 2022 Sep 20;12(10):1459. doi: 10.3390/life12101459.

Authors

Marlene Wahlmueller^{1

2

3}, Marie-Sophie Narzt^{1

2

3}, Karin Missfeldt^{1

2}, Verena Arminger^{1

2}, Anna Krasensky^{1

2}, Ingo Lämmermann^{4

5}, Barbara Schaedl^{1

2

6}, Mario Mairhofer⁷, Susanne Suessner⁸, Susanne Wolbank^{1

2}, Eleni Priglinger^{1

2

3}

Affiliations

¹ Ludwig Boltzmann Institute for Traumatology in Cooperation with the AUVA, 1200 Vienna, Austria.
² Austrian Cluster for Tissue Regeneration, 1200 Vienna, Austria.
³ MorphoMed GmbH, 1030 Vienna, Austria.
⁴ Christian Doppler Laboratory for the Biotechnology of Skin Aging, Department of Biotechnology, Institute of Molecular Biotechnology, University of Natural Resources and Life Sciences, 1190 Vienna, Austria.
⁵ Rockfish Bio AG, 1010 Vienna, Austria.
⁶ University Clinic of Dentistry, Medical University of Vienna, 1090 Vienna, Austria.
⁷ Department of Hematology and Internal Oncology, Johannes Kepler University, 4020 Linz, Austria.
⁸ Austrian Red Cross Blood Transfusion Service for Upper Austria, 4020 Linz, Austria.

Abstract

Acting as the largest energy reservoir in the body, adipose tissue is involved in longevity and progression of age-related metabolic dysfunction. Here, cellular senescence plays a central role in the generation of a pro-inflammatory environment and in the evolution of chronic diseases. Within the complexity of a tissue, identification and targeting of senescent cells is hampered by their heterogeneity. In this study, we generated stress-induced premature senescence 2D and 3D in vitro models for the stromal vascular niche of human adipose tissue. We established treatment conditions for senescence induction using Doxorubicin (Dox), starting from adipose-derived stromal/stem cells (ASCs), which we adapted to freshly isolated microtissue-stromal vascular fraction (MT-SVF), where cells are embedded within their native extracellular matrix. Senescence hallmarks for the established in vitro models were verified on different cellular levels, including morphology, cell cycle arrest, senescence-associated β-galactosidase activity (SA-βgal) and gene expression. Two subsequent exposures with 200 nM Dox for six days were suitable to induce senescence in our in vitro models. We demonstrated induction of senescence in the 2D in vitro models through SA-βgal activity, at the mRNA level (LMNB1, CDK1, p21) and additionally by G2/M phase cell cycle arrest in ASCs. Significant differences in Lamin B1 and p21 protein expression confirmed senescence in our MT-SVF 3D model. MT-SVF 3D cultures were composed of multiple cell types, including CD31, CD34 and CD68 positive cells, while cell death remained unaltered upon senescence induction. As heterogeneity and complexity of adipose tissue senescence is given by multiple cell types, our established senescence models that represent the perivascular niche embedded within its native extracellular matrix are highly relevant for future clinical studies.

Keywords: adipose tissue; adipose-derived stromal/stem cells (ASCs); doxorubicin; microtissue; senescence; stress-induced premature senescence (SIPS); stromal vascular fraction (SVF).

Grants and funding

40027790/Austrian Research Promotion Agency