Synergistic Adhesiveness of Fibronectin with PHSRN Peptide in Gelatin Mixture Promotes the Therapeutic Potential of Human ES-Derived MSCs
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Mesenchymal stem cells (MSCs) are promising candidates for cell therapy owing to their therapeutic effect in various diseases. In general, MSCs grow efficiently in serum-containing culture media, indicating an essential role of adhesion in their mesenchymal lineage-specific propagation. Nevertheless, the use of non-human supplements in culture (xeno-free issue) in addition to the lack of control over unknown factors in the serum hampers the clinical transition of MSCs.
In this study, embryonic stem cell derived mesenchymal stem cells (ES-MSCs) were used owing to their scalable production, and they expressed a series of MSC markers same as adipose-derived MSCs. The affinity of the culture matrix was increased by combining fibronectin coating with its adjuvant peptide, gelatin, or both (FNGP) on tissue culture polystyrene to compare the regenerative, therapeutic activities of ES-MSCs with a cell binding plate as a commercial control.
The FNGP culture plate promoted pivotal therapeutic functions of ES-MSCs as evidenced by their increased stemness as well as anti-inflammatory and proangiogenic effects in vitro. Indeed, after culturing on the FNGP plates, ES-MSCs efficiently rescued the necrotic damages in mouse ischemic hindlimb model.
This study suggests a potential solution by promoting the surface affinity of culture plates using a mixture of human fibronectin and its adjuvant PHSRN peptide in gelatin. The FNGP plate is expected to serve as an effective alternative for serum-free MSC expansion for bench to clinical transition.
KeywordsES-MSC Serum-free expansion PHSRN Fibronectin Gelatin
Mesenchymal stem cells
Embryonic stem cell
Embryonic stem cell-derived mesenchymal stem cells
Fibronectin coating with gelatin
Fibronectin coating with adjuvant peptide PHSRN
Fibronectin coating with gelatin and adjuvant peptide PHSRN
Tissue culture plate
Tissue culture plate polystyrene
Adipose derived mesenchymal stem cell
Residual reactive oxygen species
Kruppel-like factor 4
Homeobox protein nanog
Sex determining region Y-box 2
Apurinic/apyrimidinic endonuclease 1
Superoxide dismutase 2
Human umbilical vein endothelial cells
Enzyme-linked immunosorbent assay
Dulbecco’s modified Eagle’s medium
Fetal bovine serum
Cell counting kit-8
Real time quantitative PCR
Glyceraldehyde 3-phosphate dehydrogenase
Red fluorescent protein
H. S. Kim and S. H. Choi contributed equally to this work and are thus listed as the equal first authors. We acknowledge Dr. Dae-Hyun Kim for guiding mouse experiments.
H-J.S. and K.N.K conceived and initiated the entire study. H–S.K. and S.H.C conducted most experiments. M-L. K designed the experiments and analyzed the results. H–S.K., S.H.C., M-L. K., and K-W. L wrote the paper. H-J.S. and K.N.K supervised all aspects of the study.
This work was financially supported by DAEWOONG Pharmaceutical and the National Research Foundation of Korea (NRF) (2016M3A9E9941743 and 2019R1A2C2010802).
The data used to support the findings of this study are included within the article.
Conflict of interest
Hye-Seon Kim, Sung Hyun Choi, Mi-Lan Kang, Ki-Won Lee, Ki Nam Kim and Hak-Joon Sung have no known conflicts of interest or significant financial support associated with this publication that could have influenced its outcome.
All animal studies were carried out in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publication No. 85–23 revised 1985) and approved by IACUC. No human studies were carried out by the authors for this publication.
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