Monitoring wound healing of burn in rat model using human Wharton’s jelly mesenchymal stem cells containing cGFP integrated by lentiviral vectors

Document Type: Original Article

Authors

1 Burn and Wound Healing Research Center, Shiraz University of Medical Science, Shiraz, Iran

2 Nour Danesh Institute of Higher Education, Isfahan, Iran

3 Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran

Abstract

Objective(s): Human Wharton’s Jelly mesenchymal stem cells (hWMSCs) are undifferentiated cells commonly used in regenerative medicine. The aim of this study was to develop a reliable tool for tracking hWMSCs when utilized as therapeutics in burnt disorders and also to optimize the cell-based treatment procedure.
Materials and Methods: The hWMSCs were first isolated from fresh umbilical cord Wharton’s jelly and cultured. The 293LTV cell line was transfected by cGFP containing lentiviral vector and the helper plasmids for production of the viral particle. The viral particles were collected to transduce the hWMSCs. The transduced cells were finally selected based on resistance to puromycin. The burned rats (n=24) were treated with cGFP expressing hWMSCs using the cell spray method, with the cells being tracked 7, 14 and 21 days later. The rats were sacrificed 7, 14 and 21 days following treatment and paraffin embedded sections prepared from the burned area for downstream pathological analyses.
Results: The lentiviral particles carrying the cGFP gene were generated and the hWMSCs were transduced. The cGFP-expressing hWMSCs were detected in the burned tissue and the burned injuries were improved dramatically as compared to control.
Conclusion: Because of the establishment of stably transduced cGFP expressing cells and the ability to detect cGFP for a relatively long-time interval, the method was found to be quite efficient for the purpose of cell tracking. The combination of hWMSC-based cell therapy and sterile Gauze Vaseline (GV) as covering was proven much more efficient than the traditional methods based on GV alone.

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Main Subjects


1. Leclerc T, Thepenier C, Jault P, Bey E, Peltzer J, Trouillas M, et al. Cell therapy of burns. Cell Prolif 2011; 44: 48–54.

2. Kagan RJ, Peck MD, Ahrenholz DH, Hickerson WL, Holmes J 4th, Korentager R, et al. Surgical management of the burn wound and use of skin substitutes: an expert panel white paper. J Burn Care Res 2013; 34: e60–79.

3. Puri V, Khare NA, Chandramouli MV, Shende N, Bharadwaj S. Comparative analysis of early excision and grafting vs delayed grafting in burn patients in a developing country. J Burn Care Res 2016; 37:278-282.

4. Hermans, M. H. Porcine xenografts vs. (cryopreserved) allografts in the management of partial thickness burns: is there a clinical difference? Burns 2014; 40: 408-415.

5. Troyer DL, Weiss ML. Concise review: Wharton’s jelly-derived cells are a primitive stromal cell population. Stem Cells 2008; 26: 591-599.

6. Fong CY, Richards M, Manasi N, Biswas A, Bongso A. Comparative growth behaviour and characterization of stem cells from human Wharton’s jelly. Reprod Biomed Online 2007; 15: 708–718.

7. Baksh D, Yao R, Tuan RS. Comparison of Proliferative and Multilineage Differentiation Potential of Human Mesenchymal Stem Cells Derived from Umbilical Cord and Bone Marrow. STEM CELLS 2007; 25: 1384 –1392.

8. Xue L, Xu YB, Xie JL, Tang JM, Shu B, Chen L, et al. Effects of human bone marrow mesenchymal stem cells on burn injury healing in a mouse model. Int J Clin Exp Pathol 2013; 6: 1327-1336.

9. Liu L, Yu Y, Hou Y, Chai J, Duan H, Chu W, et al. Human Umbilical Cord Mesenchymal Stem Cells Transplantation Promotes Cutaneous Wound Healing of Severe Burned Rats. PLoS ONE 2014; 9: 1-7.

10. Burd A, Ahmed K, Lam S, Ayyappan T, Huang L, Stem cell strategies in burns care. Burns 2007; 33: 282-291.

11. Liu P, Deng Z, Han S, Liu T, Wen N, Lu W, et al. Tissue-engineered skin containing mesenchymal stem cells improves burn wounds. Artif Organs 2008; 32: 925-931.

12. Wu Y, Wang J, Scott PG, Tredget EE. Bone marrow-derived stem cells in wound healing: a review. Wound Repair Regen 2007; 15: 18-26.

13. Salehinejad P, Alitheen NB, Ali AM, Omar AR, Mohit M, Janzamin E, et al. Comparison of different methods for the isolation of mesenchymal stem cells from human umbilical cord Wharton's jelly. In Vitro Cell Dev Biol 2012;48:75-83

14. Klages N, Zufferey R, Trono D. A stable system for the high-titer production of multiply attenuated lentiviral vectors. Mol Ther 2000; 2:170-176.

15. Wong D, Makowska IJ, Weary DM. Rat aversion to isoflurane versus carbon dioxide. Biology letters. 2012; 9: 1-4.

16. MEYER, Tufi Neder; SILVA, Alcino Lázaro da. A standard burn model using rats. Acta Cir. Bras 1999;14.

17. Navarro FA, Stoner ML, Park CS, Huertas JC, Lee HB, Wood FM, et al. Sprayed keratinocyte suspensions accelerate epidermal coverage in a porcine micro-wound model. J Burn Care Rehabil 2000; 21: 513-518.

18. Fischer A H, Jacobson K A, Rose J, & Zeller R. Hematoxylin and eosin staining of tissue and cell sections. Cold Spring Harbor Protocols 2008; 3.

19. Wei X, Yang X, Han ZP, Qu FF, Shao L, Shi YF. Mesenchymal stem cells: a new trend for cell therapy. Acta Pharmacologica Sinica 2013; 34: 747–754.

20. Korbling M, Estrove Z. Adult stem cells for tissue repair-a new therapeutic concept? N Engl J Med 2003;349: 570-582.

21. De Girolamo L, Lucarelli E, Alessandri G, et al. Mesenchymal Stem/Stromal Cells: A New "Cells as Drugs" Paradigm. Efficacy and Critical Aspects in Cell Therapy. Current Pharmaceutical Design 2013;19: 2459-2473.

22. Marguerie E, Michael P. Scherrer, Frank D. Ferrari, Mikhail V. Matz. Very Bright Green Fluorescent Proteins from the Ponellid Copepod Pontella mimocerami. PLoS ONE 2010; 5: e11517.

23. Yannas, Ioannis V. Tissue and organ regeneration in adults. Springer Publishing Co; 2015.

24. Ankrum J, Jeffrey M K. Mesenchymal stem cell therapy: Two steps forward, one step back. Trends in Molecular Medicine 2010; 5: 203-209.

25. Hocking AM, Gibran NS. Mesenchymal stem cells: paracrine signaling and differentiation during cutaneous wound repair. Exp Cell Res 2010; 2213–2219.

26. Ko IK, Lee SJ, Atala A, Yoo JJ. In situ tissue regeneration through host stem cell recruitment. Experimental & Molecular Medicine 2013; 45: e57.