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

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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.

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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

References

  1. Bianco, P., X. Cao, P. S. Frenette, J. J. Mao, P. G. Robey, P. J. Simmons, and C. Y. Wang. The meaning, the sense and the significance: translating the science of mesenchymal stem cells into medicine. Nat. Med. 19(1):35–42, 2013.

    Article  Google Scholar 

  2. Bronckaers, A., P. Hilkens, W. Martens, P. Gervois, J. Ratajczak, T. Struys, and I. Lambrichts. Mesenchymal stem/stromal cells as a pharmacological and therapeutic approach to accelerate angiogenesis. Pharmacol. Ther. 143(2):181–196, 2014.

    Article  Google Scholar 

  3. Dankers, P. Y., M. C. Harmsen, L. A. Brouwer, M. J. van Luyn, and E. W. Meijer. A modular and supramolecular approach to bioactive scaffolds for tissue engineering. Nat. Mater. 4(7):568–574, 2005.

    Article  Google Scholar 

  4. De Francesco, F., G. Ricci, F. D’Andrea, G. F. Nicoletti, and G. A. Ferraro. Human adipose stem cells: from bench to bedside. Tissue Eng. Part B Rev. 21(6):572–584, 2015.

    Article  Google Scholar 

  5. Feng, Y., and M. Mrksich. The synergy peptide PHSRN and the adhesion peptide RGD mediate cell adhesion through a common mechanism. Biochemistry 43(50):15811–15821, 2004.

    Article  Google Scholar 

  6. Giam, L. R., M. D. Massich, L. Hao, L. Shin Wong, C. C. Mader, and C. A. Mirkin. Scanning probe-enabled nanocombinatorics define the relationship between fibronectin feature size and stem cell fate. Proc. Natl. Acad. Sci. USA 109(12):4377–4382, 2012.

    Article  Google Scholar 

  7. Hamidian Jahromi, S., C. Estrada, Y. Li, E. Cheng, and J. E. Davies. Human umbilical cord perivascular cells and human bone marrow mesenchymal stromal cells transplanted intramuscularly respond to a distant source of inflammation. Stem Cells Dev. 27(6):415–429, 2018.

    Article  Google Scholar 

  8. Hawkins, K. E., M. Corcelli, K. Dowding, A. M. Ranzoni, F. Vlahova, K. L. Hau, A. Hunjan, D. Peebles, P. Gressens, H. Hagberg, P. de Coppi, M. Hristova, and P. V. Guillot. Embryonic stem cell-derived mesenchymal stem cells (MSCs) have a superior neuroprotective capacity over fetal MSCs in the hypoxic-ischemic mouse brain. Stem Cells Transl. Med. 7(5):439–449, 2018.

    Article  Google Scholar 

  9. Hematti, P. Human embryonic stem cell-derived mesenchymal progenitors: an overview. Methods Mol. Biol. 690:163–174, 2011.

    Article  Google Scholar 

  10. Huang, G. S., L. G. Dai, B. L. Yen, and S. H. Hsu. Spheroid formation of mesenchymal stem cells on chitosan and chitosan-hyaluronan membranes. Biomaterials 32(29):6929–6945, 2011.

    Article  Google Scholar 

  11. Ji, A. R., S. Y. Ku, M. S. Cho, Y. Y. Kim, Y. J. Kim, S. K. Oh, S. H. Kim, S. Y. Moon, and Y. M. Choi. Reactive oxygen species enhance differentiation of human embryonic stem cells into mesendodermal lineage. Exp. Mol. Med. 42(3):175–186, 2010.

    Article  Google Scholar 

  12. Jin, H. J., J. H. Kwon, M. Kim, Y. K. Bae, S. J. Choi, W. Oh, Y. S. Yang, and H. B. Jeon. Downregulation of melanoma cell adhesion molecule (MCAM/CD146) accelerates cellular senescence in human umbilical cord blood-derived mesenchymal stem cells. Stem Cells Transl. Med. 5(4):427–439, 2016.

    Article  Google Scholar 

  13. Kandoi, S., L. Praveen-Kumar, B. Patra, P. Vidyasekar, D. Sivanesan, S. Vijayalakshmi, K. Rajagopal, and R. S. Verma. Evaluation of platelet lysate as a substitute for FBS in explant and enzymatic isolation methods of human umbilical cord MSCs. Sci. Rep. 8(1):12439, 2018.

    Article  Google Scholar 

  14. Kang, K. T., R. Z. Lin, D. Kuppermann, J. M. Melero-Martin, and J. Bischoff. Endothelial colony forming cells and mesenchymal progenitor cells form blood vessels and increase blood flow in ischemic muscle. Sci. Rep. 7(1):770, 2017.

    Article  Google Scholar 

  15. Kao, W. J., and D. Lee. In vivo modulation of host response and macrophage behavior by polymer networks grafted with fibronectin-derived biomimetic oligopeptides: the role of RGD and PHSRN domains. Biomaterials 22(21):2901–2909, 2001.

    Article  Google Scholar 

  16. Kao, W. J., D. Lee, J. C. Schense, and J. A. Hubbell. Fibronectin modulates macrophage adhesion and FBGC formation: the role of RGD, PHSRN, and PRRARV domains. J. Biomed. Mater. Res. 55(1):79–88, 2001.

    Article  Google Scholar 

  17. Kimura, K., A. Hattori, Y. Usui, K. Kitazawa, M. Naganuma, K. Kawamoto, S. Teranishi, M. Nomizu, and T. Nishida. Stimulation of corneal epithelial migration by a synthetic peptide (PHSRN) corresponding to the second cell-binding site of fibronectin. Invest. Ophthalmol. Vis. Sci. 48(3):1110–1118, 2007.

    Article  Google Scholar 

  18. Lee, S. H., Y. Lee, Y. W. Chun, S. W. Crowder, P. P. Young, K. D. Park, and H. J. Sung. In situ crosslinkable gelatin hydrogels for vasculogenic induction and delivery of mesenchymal stem cells. Adv. Funct. Mater. 24(43):6771–6781, 2014.

    Article  Google Scholar 

  19. Mackensen, A., R. Drager, M. Schlesier, R. Mertelsmann, and A. Lindemann. Presence of IgE antibodies to bovine serum albumin in a patient developing anaphylaxis after vaccination with human peptide-pulsed dendritic cells. Cancer Immunol. Immunother. 49(3):152–156, 2000.

    Article  Google Scholar 

  20. Maraldi, T., C. Angeloni, E. Giannoni, and C. Sell. Reactive oxygen species in stem cells. Oxid. Med. Cell Longev. 2015:159080, 2015.

    Google Scholar 

  21. Martin, M. J., A. Muotri, F. Gage, and A. Varki. Human embryonic stem cells express an immunogenic nonhuman sialic acid. Nat. Med. 11(2):228–232, 2005.

    Article  Google Scholar 

  22. Mendicino, M., A. M. Bailey, K. Wonnacott, R. K. Puri, and S. R. Bauer. MSC-based product characterization for clinical trials: an FDA perspective. Cell Stem Cell 14(2):141–145, 2014.

    Article  Google Scholar 

  23. Park, Y. H., J. I. Yun, N. R. Han, H. J. Park, J. Y. Ahn, C. Kim, J. H. Choi, E. Lee, J. M. Lim, and S. T. Lee. Mass production of early-stage bone-marrow-derived mesenchymal stem cells of rat using gelatin-coated matrix. Biomed. Res. Int. 2013:347618, 2013.

    Google Scholar 

  24. Rustad, K. C., V. W. Wong, M. Sorkin, J. P. Glotzbach, M. R. Major, J. Rajadas, M. T. Longaker, and G. C. Gurtner. Enhancement of mesenchymal stem cell angiogenic capacity and stemness by a biomimetic hydrogel scaffold. Biomaterials 33(1):80–90, 2012.

    Article  Google Scholar 

  25. Smirnov, S. V., R. Harbacheuski, A. Lewis-Antes, H. Zhu, P. Rameshwar, and S. V. Kotenko. Bone-marrow-derived mesenchymal stem cells as a target for cytomegalovirus infection: implications for hematopoiesis, self-renewal and differentiation potential. Virology 360(1):6–16, 2007.

    Article  Google Scholar 

  26. Swamynathan, P., P. Venugopal, S. Kannan, C. Thej, U. Kolkundar, S. Bhagwat, M. Ta, A. S. Majumdar, and S. Balasubramanian. Are serum-free and xeno-free culture conditions ideal for large scale clinical grade expansion of Wharton’s jelly derived mesenchymal stem cells? A comparative study. Stem Cell Res. Ther. 5(4):88, 2014.

    Article  Google Scholar 

  27. Tao, H., Z. Han, Z. C. Han, and Z. Li. Proangiogenic features of mesenchymal stem cells and their therapeutic applications. Stem Cells Int. 2016:1314709, 2016.

    Article  Google Scholar 

  28. Tebebi, P. A., S. J. Kim, R. A. Williams, B. Milo, V. Frenkel, S. R. Burks, and J. A. Frank. Improving the therapeutic efficacy of mesenchymal stromal cells to restore perfusion in critical limb ischemia through pulsed focused ultrasound. Sci. Rep. 7:41550, 2017.

    Article  Google Scholar 

  29. Teixeira, F. G., M. M. Carvalho, N. Sousa, and A. J. Salgado. Mesenchymal stem cells secretome: a new paradigm for central nervous system regeneration? Cell. Mol. Life Sci. 70(20):3871–3882, 2013.

    Article  Google Scholar 

  30. Togel, F., Z. Hu, K. Weiss, J. Isaac, C. Lange, and C. Westenfelder. Administered mesenchymal stem cells protect against ischemic acute renal failure through differentiation-independent mechanisms. Am. J. Physiol. Renal Physiol. 289(1):F31–42, 2005.

    Article  Google Scholar 

  31. Tuschong, L., S. L. Soenen, R. M. Blaese, F. Candotti, and L. M. Muul. Immune response to fetal calf serum by two adenosine deaminase-deficient patients after T cell gene therapy. Hum. Gene Ther. 13(13):1605–1610, 2002.

    Article  Google Scholar 

  32. Wang, J., J. Hao, D. Bai, Q. Gu, W. Han, L. Wang, Y. Tan, X. Li, K. Xue, P. Han, Z. Liu, Y. Jia, J. Wu, L. Liu, L. Wang, W. Li, Z. Liu, and Q. Zhou. Generation of clinical-grade human induced pluripotent stem cells in Xeno-free conditions. Stem Cell Res. Ther. 6:223, 2015.

    Article  Google Scholar 

  33. White, S. Reflections upon caring for dying people. Inforum 12:27, 1991.

    Google Scholar 

  34. Wong, S. P., J. E. Rowley, A. N. Redpath, J. D. Tilman, T. G. Fellous, and J. R. Johnson. Pericytes, mesenchymal stem cells and their contributions to tissue repair. Pharmacol. Ther. 151:107–120, 2015.

    Article  Google Scholar 

  35. Zhang, S., and W. Cui. Sox2, a key factor in the regulation of pluripotency and neural differentiation. World J. Stem Cells 6(3):305–311, 2014.

    Article  MathSciNet  Google Scholar 

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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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Author notes

  1. Hye-Seon Kim, Sung Hyun Choi and Ki Nam Kim have contributed equally to this work.

Authors and Affiliations

  1. Department of Medical Engineering, Yonsei University College of Medicine, Seoul, 03722, Republic of Korea

    Hye-Seon Kim, Mi-Lan Kang, Ki-Won Lee & Hak-Joon Sung

  2. Cellular Therapeutics Team, Daewoong Pharmaceutical, Yongin, 17028, Gyeonggi-do, Republic of Korea

    Sung Hyun Choi & Ki Nam Kim

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  1. Hye-Seon Kim
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Correspondence to Ki Nam Kim or Hak-Joon Sung.

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Kim, HS., Choi, S.H., Kang, ML. et al. Synergistic Adhesiveness of Fibronectin with PHSRN Peptide in Gelatin Mixture Promotes the Therapeutic Potential of Human ES-Derived MSCs. Cel. Mol. Bioeng. 13, 73–86 (2020). https://doi.org/10.1007/s12195-019-00604-0

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