Document Type : Original Article

Authors

1 Department of Clinical Biochemistry, Faculty of Medical Science, Tarbiat Modares University, Tehran, Iran

2 Protein Research Center, Shahid Beheshti University, Tehran, Iran

Abstract

Introduction and Aim: Using 3D culture to increase cell density and creating a suitable bedrock for the growth and proliferation and differentiation of stem cells is one of the important methods for their application in tissue engineering. In this study, mesenchymal stem cells of adipose tissue because of ease of access and abundance have been used to differentiate into hepatocyte-like cells to investigate the effect of 3D cultures on gelatin/laminin 3D scaffolds.
Methods: Mesenchymal stem cells were extracted from subcutaneous fat of the abdominal region of C57 mice by enzymatic digestion using collagenase enzyme. The extracted stem cells were confirmed by flow cytometry analysis and observation of the expression of specific stem cells markers such as CD105, CD44, and the absence of specific leukocyte (CD34) and hematopoietic (CD45). The non-toxicity of the scaffold was also investigated by the MTT method. Stem cells were cultured on a polystyrene surface and laminin and gelatin/laminin scaffold and treated with growth factors for 21 days in two stages. Cell differentiation was investigated by biochemical methods including examination of secretion indices such as urea and glycogen storage.
Results: The expression of specific stem cells markers such as CD105, CD44, and the absence of CD34 and CD45 were proved. The ability to differentiate these cells into adipocytes and osteocytes has been proven.
Conclusion: The results show the presence of a three-dimensional gelatin/laminin scaffold increases the adhesion, proliferation, and differentiation of fatty mesenchymal cells to hepatocyte-like cells.

Keywords

Main Subjects

Aldana, A.A. and Abraham, G.A. 2017. Current advances in electrospun gelatin-based scaffolds for tissue engineering applications. International journal of pharmaceutics, 523(2): 441-453.
Alhadlaq, A. and Mao, J.J. 2004. Mesenchymal stem cells: Isolation and therapeutics. Stem cells and development, 13(4): 436-448.
Alison, M.R. 2009. Stem cells in pathobiology and regenerative medicine. The Journal of Pathology: A Journal of the Pathological Society of Great Britain and Ireland, 217(2): 141-143.
Caplan, A.I. 2007. Adult mesenchymal stem cells for tissue engineering versus regenerative medicine. Journal of cellular physiology, 213(2): 341-347.
Chamberlain, G., Fox, J., Ashton, B. and Middleton, J. 2007. Concise review: Mesenchymal stem cells: Their phenotype, differentiation capacity, immunological features, and potential for homing. Stem cells, 25(11): 2739-2749.
Durbeej, M. 2010. Laminins. Cell and tissue research, 339(1): 259-268.
Ferrin, I., Beloqui, I., Zabaleta, L., Salcedo, J.M., Trigueros, C. and Martin, A.G. 2017. Isolation, culture, and expansion of mesenchymal stem cells. In: Stem cell banking. Springer: pp: 177-190.
Fukuchi, Y., Nakajima, H., Sugiyama, D., Hirose, I., Kitamura, T. and Tsuji, K. 2004. Human placenta‐derived cells have mesenchymal stem/progenitor cell potential. Stem cells, 22(5): 649-658.
Hanna, L., Crosby, T. and Macbeth, F. 2015. Practical clinical oncology. Cambridge University Press.
Hussain, S.A., Carafoli, F. and Hohenester, E. 2011. Determinants of laminin polymerization revealed by the structure of the α5 chain amino‐terminal region. EMBO reports, 12(3): 276-282.
Ito, Y., Kajihara, M. and Imanishi, Y. 1991. Materials for enhancing cell adhesion by immobilization of cell‐adhesive peptide. Journal of biomedical materials research, 25(11): 1325-1337.
Jurga, M., Dainiak, M.B., Sarnowska, A., Jablonska, A., Tripathi, A., Plieva, F.M., Savina, I.N., Strojek, L., Jungvid, H. and Kumar, A. 2011. The performance of laminin-containing cryogel scaffolds in neural tissue regeneration. Biomaterials, 32(13): 3423-3434.
Kuijpers, A.J., Engbers, G.H., Krijgsveld, J., Zaat, S.A., Dankert, J. and Feijen, J. 2000. Cross-linking and characterisation of gelatin matrices for biomedical applications. Journal of Biomaterials Science, Polymer Edition, 11(3): 225-243.
Lien, S.-M., Ko, L.-Y. and Huang, T.-J. 2009. Effect of pore size on ecm secretion and cell growth in gelatin scaffold for articular cartilage tissue engineering. Acta biomaterialia, 5(2): 670-679.
Markarian, C.F., Frey, G.Z., Silveira, M.D., Milani, A.R., Ely, P.B., Horn, A.P., Nardi, N.B. and Camassola, M. 2014. Isolation of adipose-derived stem cells: A comparison among different methods. Biotechnology letters, 36(4): 693-702.
Mizuguchi, H., Kawabata, K., Inamura, M. and Furue, M. 2012. Method of differentiation from stem cells to hepatocytes. Google Patents.
Morrison, S.J. and Spradling, A.C. 2008. Stem cells and niches: Mechanisms that promote stem cell maintenance throughout life. Cell, 132(4): 598-611.
Nardi, N.B. and da Silva Meirelles, L. 2008. Mesenchymal stem cells: Isolation, in vitro expansion and characterization. Stem cells: 249-282.
Nussler, A., Konig, S., Ott, M., Sokal, E., Christ, B., Thasler, W., Brulport, M., Gabelein, G., Schormann, W. and Schulze, M. 2006. Present status and perspectives of cell-based therapies for liver diseases. Journal of hepatology, 45(1): 144-159.
Romanov, Y.A., Darevskaya, A., Merzlikina, N. and Buravkova, L. 2005. Mesenchymal stem cells from human bone marrow and adipose tissue: Isolation, characterization, and differentiation potentialities. Bulletin of experimental biology and medicine, 140(1): 138-143.
Sánchez-Elsner, T., Botella, L.M., Velasco, B., Langa, C. and Bernabéu, C. 2002. Endoglin expression is regulated by transcriptional cooperation between the hypoxia and transforming growth factor-β pathways. Journal of Biological Chemistry, 277(46): 43799-43808.
Selden, C. and Hodgson, H. 2004. Cellular therapies for liver replacement. Transplant immunology, 12(3-4): 273-288.
Snykers, S., Vanhaecke, T., Papeleu, P., Luttun, A., Jiang, Y., Vander Heyden, Y., Verfaillie, C. and Rogiers, V. 2006. Sequential exposure to cytokines reflecting embryogenesis: The key for in vitro differentiation of adult bone marrow stem cells into functional hepatocyte-like cells. Toxicological Sciences, 94(2): 330-341.
Sutherland, D.R. and Keating, A. 1992. The cd34 antigen: Structure, biology, and potential clinical applications. Journal of hematotherapy, 1(2): 115-129.
Zhang, Y., Venugopal, J., Huang, Z.-M., Lim, C.T. and Ramakrishna, S. 2006. Crosslinking of the electrospun gelatin nanofibers. Polymer, 47(8): 2911-2917.
Zong, Y. and Friedman, J.R. 2014. Liver development. Liver Disease in Children: 1-813.