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Synergistic Adhesiveness of Fibronectin with PHSRN Peptide in Gelatin Mixture Promotes the Therapeutic Potential of Human ES-Derived MSCs

  • Hye-Seon Kim
  • Sung Hyun Choi
  • Mi-Lan Kang
  • Ki-Won Lee
  • Ki Nam KimEmail author
  • Hak-Joon SungEmail author
Original Article
  • 19 Downloads

Abstract

Introduction

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.

Methods

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.

Results

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.

Conclusions

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.

Keywords

ES-MSC Serum-free expansion PHSRN Fibronectin Gelatin 

Abbreviations

MSC

Mesenchymal stem cells

ES

Embryonic stem cell

ES-MSCs

Embryonic stem cell-derived mesenchymal stem cells

FN

Fibronectin

FNG

Fibronectin coating with gelatin

FNP

Fibronectin coating with adjuvant peptide PHSRN

FNGP

Fibronectin coating with gelatin and adjuvant peptide PHSRN

TCP

Tissue culture plate

TCPS

Tissue culture plate polystyrene

CB

CellBind®

PHSRN

Pro-His-Ser-Arg-Asn

ADSC

Adipose derived mesenchymal stem cell

ROS

Residual reactive oxygen species

KLF4

Kruppel-like factor 4

NANOG

Homeobox protein nanog

SOX2

Sex determining region Y-box 2

APEX1

Apurinic/apyrimidinic endonuclease 1

SENS1

Scalp-ear-nipple syndrome

SOD2

Superoxide dismutase 2

TXN

Thioredoxin

HUVEC

Human umbilical vein endothelial cells

IL-10

Interleukin 10

IL-6

Interleukin 6

ELISA

Enzyme-linked immunosorbent assay

DMEM

Dulbecco’s modified Eagle’s medium

FBS

Fetal bovine serum

PS

Penicillin streptomycin

CCK-8

Cell counting kit-8

qPCR

Real time quantitative PCR

GAPDH

Glyceraldehyde 3-phosphate dehydrogenase

RGD

Arg-Gly-Asp

RFP

Red fluorescent protein

EB

Embryonic body

Notes

Acknowledgments

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.

Author contributions

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.

Funding

This work was financially supported by DAEWOONG Pharmaceutical and the National Research Foundation of Korea (NRF) (2016M3A9E9941743 and 2019R1A2C2010802).

Data availability

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.

Ethical approval

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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Copyright information

© Biomedical Engineering Society 2019

Authors and Affiliations

  1. 1.Department of Medical EngineeringYonsei University College of MedicineSeoulRepublic of Korea
  2. 2.Cellular Therapeutics TeamDaewoong PharmaceuticalYonginRepublic of Korea

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