Microfluidic Separation of Canine Adipose-Derived Mesenchymal Stromal Cells
Publication: Tissue Engineering Part C: Methods
Volume 27, Issue Number 8
Abstract
Mesenchymal stromal cells (MSCs) are potential treatments for a variety of veterinary medical conditions. However, clinical trials have often fallen short of expectations, due in part to heterogeneity and lack of characterization of the MSCs. Identification and characterization of subpopulations within MSC cultures may improve those outcomes. Therefore, the functional heterogeneity of different-sized subpopulations of MSCs was evaluated. A high-throughput, biophysical, label-free microfluidic sorting approach was used to separate subpopulations of canine adipose-derived MSCs (Ad-MSCs) based on size for subsequent characterization, as well as to evaluate the impact of culture conditions on their functional heterogeneity. We found that culture-expanded canine Ad-MSCs comprise distinct subpopulations: larger MSCs (mean diameter of 18.6 ± 0.2 μm), smaller MSCs (mean diameter of 15.3 ± 0.2 μm), and intermediate MSCs (mean diameter of 16.9 ± 0.1 μm). In addition, proliferation characteristics, senescence, and differentiation potential of canine Ad-MSCs are also dependent on cell size. We observed that larger MSCs proliferate more slowly, senesce at earlier passages, and are inclined to differentiate into adipocytes compared with smaller MSCs. Most importantly, these size-dependent functions are also affected by the presence of serum in the culture medium, as well as time in culture. Cell surface staining for MSC-specific CD44 and CD90 antigens showed that all subpopulations of MSCs are indistinguishable, suggesting that this criterion is not relevant to define subpopulations of MSCs. Finally, transcriptome analysis showed differential gene expression between larger and smaller subpopulations of MSCs. Larger MSCs expressed genes involved in cellular senescence such as cyclin-dependent kinase inhibitor 1A and smaller MSCs expressed genes that promote cell growth [mechanistic target of rapamycin 1 (mTORC1) pathway] and cell proliferation [myelocytomatosis (myc), e2f targets]. These results suggest that different subpopulations of MSCs have specific properties.
Impact statement
Clinical trials of mesenchymal stromal cells (MSCs) from veterinary species have often fallen short of expectations, due in part to heterogeneity and lack of characterization of the MSCs. A high-throughput, biophysical, label-free microfluidic sorting approach was used to separate subpopulations of canine adipose-derived MSCs (Ad-MSCs) based on size for subsequent characterization. Proliferation characteristics, senescence, and differentiation potential of canine Ad-MSCs are also dependent on cell size. Cell surface staining for MSC-specific cell surface markers showed that all subpopulations of MSCs are indistinguishable, suggesting that this criterion is not relevant to define subpopulations of MSCs.
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Disclaimer
The views expressed in this article are those of the authors and do not necessarily reflect the official policy of the Department of Health and Human Services, the U.S. Food and Drug Administration, or the U.S. Government.
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Information & Authors
Information
Published In
Tissue Engineering Part C: Methods
Volume 27 • Issue Number 8 • August 2021
Pages: 445 - 461
PubMed: 34155926
Copyright
Copyright 2021, Mary Ann Liebert, Inc., publishers.
History
Published online: 18 August 2021
Published in print: August 2021
Published ahead of production: 22 June 2021
Accepted: 17 June 2021
Received: 7 April 2021
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Authors
Authors' Contributions
J.H. and D.Y. developed the microfluidic device. L.R.D. and Z.L. designed experiments. Z.L., L.R.D., and R.S. performed experiments and analyzed the data. L.R.D., M.J.M., and L.B. wrote the article.
Disclosure Statement
No competing financial interests exist.
Funding Information
This work was supported in part by a Challenge Grant from the office of the Chief Scientist and research funds from the Division of Applied Veterinary Research, Center for Veterinary Medicine, FDA.
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