Improved Post-Thaw Function and Epigenetic Changes in Mesenchymal Stromal Cells Cryopreserved Using Multicomponent Osmolyte Solutions.
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.
1 Department of Biomedical Engineering, University of Minnesota , Minneapolis, Minnesota.
Kathryn
K
Pollock
2 Department of Orthopedic Surgery, Mayo Clinic , Rochester, Minnesota.
Rebekah M
RM
Samsonraj
2 Department of Orthopedic Surgery, Mayo Clinic , Rochester, Minnesota.
Amel
A
Dudakovic
2 Department of Orthopedic Surgery, Mayo Clinic , Rochester, Minnesota.
Roman
R
Thaler
3 Stem Cell Institute, University of Minnesota , Minneapolis, Minnesota.
Aron
A
Stumbras
4 Department of Laboratory Medicine and Pathology, University of Minnesota , Minneapolis, Minnesota.
David H
DH
McKenna
5 Institute for Therapeutics Discovery and Development, University of Minnesota , Minneapolis, Minnesota.
Peter I
PI
Dosa
2 Department of Orthopedic Surgery, Mayo Clinic , Rochester, Minnesota.
Andre J
AJ
van Wijnen
6 Department of Mechanical Engineering, University of Minnesota , Minneapolis, Minnesota.
Allison
A
Hubel
D008841:Actin Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D016922:Cellular Senescence / Q000187:drug effects; D015925:Cryopreservation; D003451:Cryoprotective Agents / Q000494:pharmacology*; D019175:DNA Methylation / Q000187:drug effects; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D044127:Epigenesis, Genetic / Q000187:drug effects*; D005615:Freezing; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000187:drug effects / Q000378:metabolism*; D009995:Osmosis / Q000187:drug effects*; D010012:Osteogenesis / Q000187:drug effects; D012996:Solutions
D015415:Biomarkers; D003451:Cryoprotective Agents; D012996:Solutions; D004121:Dimethyl Sulfoxide
amino acids; cryopreservation; epigenetic; mesenchymal stromal cells; saccharides