Impact of Aging on the Regenerative Properties of Bone Marrow-, Muscle-, and Adipose-Derived Mesenchymal Stem/Stromal Cells

December Impact of Aging on the Regenerative Properties of Bone Marrow-, Muscle-, and Adipose-Derived Mesenchymal Stem/ Stromal Cells Olivia S. Beane 0 5 6 7 Vera C. Fonseca 1 5 6 7 Leroy L. Cooper 2 5 6 7 Gideon Koren 2 5 6 7 Eric M. Darling * 0 1 3 4 5 6 7 0 Center for Biomedical Engineering, Brown University , Providence, Rhode Island , United States of America, 1 Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University , Providence, Rhode Island , United States of America, 2 Cardiovascular Research Center, Rhode Island Hospital, Alpert Medical School of Brown University , Providence, Rhode Island , United States of America, 3 Department of Orthopaedics, Brown University , Providence, Rhode Island , United States of America, 4 School of Engineering, Brown University , Providence, Rhode Island , United States of America 5 Funding: This work was supported by awards from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (R01 AR063642 for EMD), National Institute of General Medical Sciences (P20 GM104937 for EMD), and National Science Foundation (CAREER Award, CBET1253189 for EMD). The content of this article is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or National Science Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript 6 Editor: Xing-Ming Shi, Georgia Regents University , United States of America 7 Impact of Aging on MSC Regenerative Properties Mesenchymal stem/stromal cells (MSCs) are promising cell sources for regenerative therapies due to their multipotency and ready availability, but their application can be complicated by patient-specific factors like age or illness. MSCs have been investigated for the treatment of many musculoskeletal disorders, including osteoarthritis and osteoporosis. Due to the prevalence of these diseases in older populations, researchers have studied how aging affects MSC properties and have found that proliferation and differentiation potential are impaired. However, these effects have never been compared among MSCs isolated from multiple tissue sources in the same, healthy donor. Revealing differences in how MSCs are affected by age could help identify an optimal cell source for musculoskeletal therapies targeting older patients. MSCs were isolated from young and old rabbit bone marrow, muscle, and adipose tissue. Cell yield and viability were quantified after isolation procedures, and expansion properties were assessed using assays for proliferation, senescence, and colony formation. Multipotency was also examined using lineage-specific stains and spectrophotometry of metabolites. Results were compared between age groups and among MSC sources. Results showed that MSCs are differentially influenced by aging, with bone marrow-derived stem cells having impaired proliferation, senescence, and chondrogenic response, whereas muscle-derived stem cells and adipose-derived stem cells exhibited no negative effects. While age reduced overall cell yield and adipogenic potential of all MSC populations, osteogenesis and - clonogenicity remained unchanged. These findings indicate the importance of age as a factor when designing cell-based therapies for older patients. Mesenchymal stem/stromal cells (MSCs) hold promise in regenerative therapies due to their multipotency and availability. MSCs are being considered for the treatment of a wide range of pathologies, and researchers are especially interested in their potential to treat musculoskeletal disorders such as osteoarthritis, osteoporosis, and osteonecrosis [13]. Bone marrow is the most commonly investigated source tissue for these applications, although cells from other tissues like muscle and fat have also been used effectively [47]. Recent reports have determined that bone marrow-derived mesenchymal stem cells (BMSCs) can slow the degradation of articular cartilage or even regenerate it in osteoarthritic animal models [8, 9]. Similarly, muscle-derived stem cells (MDSCs) and adipose-derived stem cells (ASCs) have been used successfully in treating bone defects in vivo [10, 11]. The prevalence of the aforementioned musculoskeletal diseases in older populations has motivated researchers to investigate the impact of aging on MSC properties to evaluate their functionality for autologous treatments. Though conflicting results exist, past findings have indicated that the regenerative potential of MSCs deteriorates with age, which suggests a possible limitation in their use. In individual studies, old BMSCs and ASCs have diminished osteogenesis compared with their younger counterparts [12, 13]. Furthermore, aged MDSC proliferation is slowed compared with young MDSC rates [14]. Similar adverse aging effects have been reported for periosteal progenitor cells and even hematopoietic stem cells [15, 16]. While these past studies are beneficial for understanding how aging might affect a single MSC type, their experimental designs do not allow for a direct, comparative analysis of MSC types in a donor from multiple source tissues. Since MSC properties are variable among donors [17] and experimental approaches are often inconsistent, it is difficult to relate trends across literature to make a reliable and accurate conclusion. The effects of aging have not been compared among MSCs derived from different tissue sources of healthy donors, an important consideration since cell origin has also been shown to influence properties such as differentiation and proliferation [18]. Exploring whether tissue source disparately influences MSC susceptibility to adverse aging effects could identify a cell type that is minimally impaired, making this cell source an optimal candidate for use in regenerative therapies for older patients. This study aimed to investigate the effects of aging on BMSCs, MDSCs, and ASCs derived in matched groups from young and old donor animals. We hypothesized that not all cell sources would see similar impairment of therapeutic characteristics with age. MSCs were isolated from rabbit donors, and their initial viabilities and yields were quantified to evaluate cell susceptibility to isolation procedures and determine the availability of cells in different tissue sources and age groups. Proliferation rates, cell senescence, and clonogenicity were quantified and compared among populations to examine the impact of aging on MSC expansion properties. Lastly, multilineage differentiation potential was investigated by quantifying the production of lineage-specific metabolites for adipogenesis, osteogenesis, and chondrogenesis. Materials and Methods MSC Isolation and Culture BMSCs, MDSCs, and ASCs were isolated from young (46 months) and old (45 years), female, New Zealand white rabbits (RSI Farms, Mocksville, NC, USA) as previously described (N55 for each age group) [5, 19 (...truncated)