Improved Post-Thaw Function and Epigenetic Changes in Mesenchymal Stromal Cells Cryopreserved Using Multicomponent Osmolyte Solutions

Abstract

Current methods for freezing mesenchymal stromal cells (MSCs) result in poor post-thaw function, which limits the clinical utility of these cells. This investigation develops a novel approach to preserve MSCs using combinations of sugars, sugar alcohols, and small-molecule additives. MSCs frozen using these solutions exhibit improved post-thaw attachment and a more normal alignment of the actin cytoskeleton compared to cells exposed to dimethylsulfoxide (DMSO). Osteogenic and chondrogenic differentiation assays show that cells retain their mesenchymal lineage properties. Genomic analysis indicates that the different freezing media evaluated have different effects on the levels of DNA hydroxymethylation, which are a principal epigenetic mark and a key step in the demethylation of CpG doublets. RNA sequencing and quantitative real time-polymerase chain reaction validation demonstrate that transcripts for distinct classes of cytoprotective genes, as well as genes related to extracellular matrix structure and growth factor/receptor signaling are upregulated in experimental freezing solutions compared to DMSO. For example, the osmotic regulator galanin, the antiapoptotic marker B cell lymphoma 2, as well as the cell surface adhesion molecules CD106 (vascular cell adhesion molecule 1) and CD54 (intracellular adhesion molecule 1) are all elevated in DMSO-free solutions. These studies validate the concept that DMSO-free solutions improve post-thaw biological functions and are viable alternatives for freezing MSCs. These novel solutions promote expression of cytoprotective genes, modulate the CpG epigenome, and retain the differentiation ability of MSCs, suggesting that osmolyte-based freezing solutions may provide a new paradigm for therapeutic cell preservation.

Osteogenic Stimulation of Human Adipose-Derived Mesenchymal Stem Cells Using a Fungal Metabolite That Suppresses the Polycomb Group Protein EZH2

“…The therapeutic importance of mesenchymal stem/stromal cells (MSCs) is evident from their use in over 300 ongoing clinical trials that use either allogeneic or autologous MSCs (see http://www.clinicaltrials.gov) for a broad range of clinical conditions, including osteoarthritis, myocardial infarction, diabetes mellitus, as well as pulmonary, neurodegenerative, and immunological disorders . Based on recent concerns regarding variability and inconsistency in tissue healing responses, there is considerable focus on improved characterization and optimization of these cells prior to their application for efficacious tissue regeneration . Strategies using MSCs for treating bone defects (e.g., to support fracture repair in age‐related osteoporosis) require that their quality is validated and optimized.…”

Laboratory Training Guidelines for Clinicians Undertaking Stem Cell Therapy

“…The freeze-thaw process is a critical procedure that, if not done correctly, can result in apoptosis due to plasma membrane, mitochondrial and lysosomal damage [33]. There is also evidence to suggest that the freezethaw process may induce alterations in the cells' epigenetic landscape [34]. However, there is still much controversy over whether the freezethaw process reduces the immunomodulatory response, phenotype or metabolic activity of MSCs [35][36][37][38].…”

Effect of Lidocaine on Viability and Gene Expression of Human Adipose–derived Mesenchymal Stem Cells: An in vitro Study

“…Because human adipose tissue is a readily accessible source of such multipotent adult stem cells, human adipose‐deriveds MSCs have been well characterized and are being considered for stem cell treatments of musculoskeletal tissues disorders . Our research group has extensively studied a number of joint and cartilage disorders that could benefit from MSC therapy as well as examined environmental factors that may impact the effective therapeutic dose of stem cells …”

“…20,21 Our research group has extensively studied a number of joint and cartilage disorders that could benefit from MSC therapy [22][23][24][25][26][27] as well as examined environmental factors that may impact the effective therapeutic dose of stem cells. [28][29][30][31][32][33][34] Injected local anesthetics are commonly applied for both diagnostic and therapeutic purposes in perioperative and ambulatory settings in orthopedic practices 35 and for the treatment of musculoskeletal pain. 36 They are commonly administered periprocedurally along with MSCs in regions surrounding damaged ligament or tendon.…”