Human Platelet lysate as a Xeno Free Alternative of Fetal bovine serum for the In Vitro Expansion of Human Mesenchymal Stromal Cells
Background: Mesenchymal stromal cells (MSCs) are employed in various different clinical settings in order to modulate immune response. Human autologous and allogeneic supplements including platelet derivatives such as platelet lysate (PL), platelet-released factors (PRF) and serum are assessed in clinical studies to replace fetal bovine serum (FBS). The immunosuppressive activity and multi-potential characteristic of MSCs appear to be maintained when the cells are expanded in platelet derivatives.
Materials and Methods: Platelet-rich plasma was collected from umbrical cord blood (UCB). Platelet-derived growth factors obtained by freeze and thaw methods. CD62P expression was determined by flow cytometry. The concentration of PDGF-BB and PDGF-AB was detemined by ELISA. We tested the ability of a different concentration of PL-supplemented medium to support the ex vivo expansion of Wharton's jelly derived MSCs. We also investigated the biological/functional properties of expanded MSCs in presence of different concentration of PL. The conventional karyotyping was performed in order to study the chromosomal stability. The gene expression of Collagen I and II aggrecan and SOX-9 in the presence of different concentrations of PL was evaluated by Real-time PCR.
Results: We observed 5% and 10% PL, causing greater effects on proliferation of MSCs .These cells exhibited typical morphology, immunophenotype and differentiation capacity. The genetic stability of these derivative cells from Wharton's jelly was demonstrated by a normal karyotype. Furthermore, the results of Real-time PCR analysis showed that the expression of chondrocyte specific genes was higher in MSCs in the presence of 5% and 10% PL, compared with FBS supplement.
Conclusions: We demonstrated that PL could be used as an alternative safe source of growth factors for expansion of MSCs and also maintained similar growing potential and phenotype without any effect on chromosomal stability.
Friedenstein AJ, Gorskaja JF, Kulagina NN. Fibroblast precursors in normal and irradiated mouse hematopoietic organs. Exp Hematol. 1976; 4(5):267-74.
Phinney DG, Prockop DJ. Concise review: Mesenchymal stem/multipotent stromal cells: The state of transdifferentiation and modes of tissue repair. current views. Stem Cells. 2007; 25(11):2896-902.
Lian QZ, Lye E, Yeo KS, et al. Derivation of clinically compliant MSCs from CD105+, CD24-differentiated human ESCs. Stem Cells. 2007; 25(2):425-36.
Fibbe WE, Nauta AJ, Roelofs H. Modulation of immune responses by mesenchymal stem cells. Ann N Y Acad Sci. 2007; 1106:272-8.
Aggarwal S, Pittenger MF. Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood. 2005; 105(4):1815-22.
Le Blanc K, Ringden O. Mesenchymal stem cells: properties and role in clinical bone marrow transplantation. Curr Opin Immunol. 2006; 18(5):586-91.
Bieback K, Kinzebach S, Karagianni M. Translating Research into Clinical Scale Manufacturing of Mesenchymal Stromal Cells. Stem Cells Int. 2010; 2010, 193519.
Bernardo ME, Pagliara D, Locatelli F. Mesenchymal stromal cell therapy: a revolution in Regenerative Medicine? Bone Marrow Transpl. 2012;47(2):164-71.
Sundin M, Ringden O, Sundberg B, et al. No alloantibodies against mesenchymal stromal cells, but presence of anti-fetal calf serum antibodies, after transplantation in allogeneic hematopoietic stem cell recipients. Haematologica. 2007; 92(9):1208-15.
Doucet C, Ernou I, Zhang YZ, et al. Platelet lysates promote mesenchymal stem cell expansion: A safety substitute for animal serum in cell-based therapy applications. J Cell Physiol. 2005; 205(2):228-36.
Bernardo ME, Avanzini MA, Perotti C, et al. Optimization of in vitro expansion of human multipotent mesenchymal stromal cells for cell-therapy approaches: Further insights in the search for a fetal calf serum substitute. J Cell Physiol. 2007; 211(1):121-30.
Muller I, Kordowich S, Holzwarth C, et al. Animal serum-free culture conditions for isolation and expansion of multipotent mesenchymal stromal cells from human BM. Cytotherapy. 2006; 8(5):437-44.
Bieback K, Hecker A, Kocaomer A, et al. Human Alternatives to Fetal Bovine Serum for the Expansion of Mesenchymal Stromal Cells from Bone Marrow. Stem Cells. 2009; 27(9):2331-41.
Castegnaro S, Chieregato K, Maddalena M, et al. Effect of Platelet Lysate on the Functional and Molecular Characteristics of Mesenchymal Stem Cells Isolated from Adipose Tissue. Curr Stem Cell Res Ther. 2011; 6(2):105-14.
Abdelrazik H, Spaggiari GM, Chiossone L, et al. Mesenchymal stem cells expanded in human platelet lysate display a decreased inhibitory capacity on T- and NK-cell proliferation and function. Eur J Immunol. 2011; 41(11):3281-90.
Grant MB, Khaw PT, Schultz GS, et al. Effects of Epidermal Growth-Factor, Fibroblast Growth-Factor, and Transforming Growth-Factor-Beta on Corneal Cell Chemotaxis. Invest Ophthalmol Vis Sci. 1992; 33(12):3292-301.
Hecquet C, Morisset S, Lorans G, et al. Effects of Acidic and Basic Fibroblast Growth-Factors on the Proliferation of Rabbit Corneal Cells. Curr Eye Res. 1990; 9(5):429-33.
Li Y, Yu X, Lin S, et al. Insulin-like growth factor 1 enhances the migratory capacity of mesenchymal stem cells. Biochem Biophys Res Commun. 2007; 356(3):780-4.
Kofidis T, de Bruin JL, Yamane T, et al. Insulin-like growth factor promotes engraftment, differentiation, and functional improvement after transfer of embryonic stem cells for myocardial restoration. Stem Cells. 2004; 22(7):1239-45.
Elcin YM, Dixit V, Gitnick T. Extensive in vivo angiogenesis following controlled release of human vascular endothelial cell growth factor: Implications for tissue engineering and wound healing. Artif Organs. 2001; 25(7):558-65.
Hammacher A, Hellman U, Johnsson A, et al. A Major Part of Platelet-Derived Growth-Factor Purified from Human-Platelets Is a Heterodimer of One a-Chain and One B-Chain. J Biol Chem. 1988; 263(31):16493-8.
Hart CE, Bailey M, Curtis DA, et al. Purification of Pdgf-Ab and Pdgf-Bb from Human-Platelet Extracts and Identification of All 3 Pdgf Dimers in Human-Platelets. Biochemistry-Us. 1990; 29(1):166-72.
Carrancio S, Lopez-Holgado N, Sanchez-Guijo FM, et al. Optimization of mesenchymal stem cell expansion procedures by cell separation and culture conditions modification. Exp Hematol. 2008; 36(8):1014-21.
Murphy MB, Blashki D, Buchanan RM, et al. Adult and umbilical cord blood-derived platelet-rich plasma for mesenchymal stem cell proliferation, chemotaxis, and cryo-preservation. Biomaterials. 2012; 33(21):5308-16.
Avanzini MA, Bernardo ME, Cometa AM, et al. Generation of mesenchymal stromal cells in the presence of platelet lysate: a phenotypic and functional comparison of umbilical cord blood- and bone marrow-derived progenitors. Haematologica. 2009; 94(12):1649-60.
Evangelista V, Manarini S, Rotondo S, et al. Platelet/polymorphonuclear leukocyte interaction in dynamic conditions: Evidence of adhesion cascade and cross talk between P-selectin and the beta 2 integrin CD11b/CD18. Blood. 1996; 88(11):4183-94.
Michelson AD, Barnard MR, Krueger LA, et al. Evaluation of platelet function by flow cytometry. Methods. 2000; 21(3):259-70.
Xia WJ, Li H, Wang Z, et al. Human platelet lysate supports ex vivo expansion and enhances osteogenic differentiation of human bone marrow-derived mesenchymal stem cells. Cell Biol Int. 2011; 35(6):639-43.
Ploderl K, Strasser C, Hennerbichler S, et al. Development and Validation of a Production Process of Platelet Lysate for Autologous Use. Platelets. 2011; 22(3):204-9.
Su CY, Kuo YP, Lin YC, et al. A virally inactivated functional growth factor preparation from human platelet concentrates. Vox Sang. 2009; 97(2):119-28.
Nedeau AE, Bauer RJ, Gallagher K, et al. A CXCL5-and bFGF-dependent effect of PDGF-B-activated fibroblasts in promoting trafficking and differentiation of bone marrow-derived mesenchymal stem cells. Exp Cell Res. 2008; 314(11-12):2176-86.
Ding W, Knox TR, Tschumper RC, et al. Platelet-derived growth factor (PDGF)-PDGF receptor interaction activates bone marrow-derived mesenchymal stromal cells derived from chronic lymphocytic leukemia: implications for an angiogenic switch. Blood. 2010; 116(16):2984-93.
Jenhani F, Durand V, Ben Azouna N, et al. Human Cytokine Expression Profile in Various Conditioned Media for In Vitro Expansion Bone Marrow and Umbilical Cord Blood Immunophenotyped Mesenchymal Stem Cells. Transpl P. 2011; 43(2):639-43.
Tokunaga A, Oya T, Ishii Y, et al. PDGF receptor beta is a potent regulator of mesenchymal stromal cell function. J Bone Miner Res. 2008; 23(9):1519-28.
Ponte AL, Marais E, Gallay N, et al. The in vitro migration capacity of human bone marrow mesenchymal stem cells: Comparison of chemokine and growth factor chemotactic activities. Stem Cells. 2007; 25(7):1737-45.
Cheng P, Gao ZQ, Liu YH, et al. Platelet-derived growth factor BB promotes the migration of bone marrow-derived mesenchymal stem cells towards C6 glioma and up-regulates the expression of intracellular adhesion molecule-1. Neurosci Lett. 2009; 451(1):52-6.
Ng F, Boucher S, Koh S, et al. PDGF, TGF-beta, and FGF signaling is important for differentiation and growth of mesenchymal stem cells (MSCs): transcriptional profiling can identify markers and signaling pathways important in differentiation of MSCs into adipogenic, chondrogenic, and osteogenic lineages. Blood. 2008; 112(2):295-307.
Lange C, Cakiroglu F, Spiess AN, et al. Accelerated and safe expansion of human mesenchymal stromal cells in animal serum-free medium for transniantation and regenerative medicine. J Cell Physiol. 2007; 213(1):18-26.
Kocaoemer A, Kern S, Kluter H, et al. Human AB serum and thrombin-activated platelet-rich plasma are suitable alternatives to fetal calf serum for the expansion of mesenchymal stem cells from adipose tissue. Stem Cells. 2007; 25(5):1270-8.
Flemming A, Schallmoser K, Strunk D, et al. Immunomodulative Efficacy of Bone Marrow-Derived Mesenchymal Stem Cells Cultured in Human Platelet Lysate. J Clin Immunol. 2011; 31(6):1143-56.
Tao Y, Zhou X, Liu D, et al. Proportion of collagen type II in the extracellular matrix promotes the differentiation of human adipose-derived mesenchymal stem cells into nucleus pulposus cells. Biofactors. 2016; 42(2):212-23.
Chen WH, Lo WC, Lee JJ, et al. Tissue-engineered intervertebral disc and chondrogenesis using human nucleus pulposus regulated through TGF-beta1 in platelet-rich plasma. J Cell Physiol. 2006; 209(3):744-54.
Park YB, Seo S, Kim JA, et al. Effect of chondrocyte-derived early extracellular matrix on chondrogenesis of placenta-derived mesenchymal stem cells. Biomedical materials. 2015;10(3):035014.
Diederichs S, Gabler J, Autenrieth J, et al. Differential Regulation of SOX9 Protein During Chondrogenesis of Induced Pluripotent Stem Cells Versus Mesenchymal Stromal Cells: A Shortcoming for Cartilage Formation. Stem Cells Dev. 2016; 25(8):598-609.
Binato R, Fernandez TD, Lazzarotto-Silva C, et al. Stability of human mesenchymal stem cells during in vitro culture: considerations for cell therapy. Cell Prolif. 2013; 46(1):10-22.
Borgonovo T, Vaz IM, Senegaglia AC, et al. Genetic evaluation of mesenchymal stem cells by G-banded karyotyping in a Cell Technology Center. Rev Bras Hematol Hemoter. 2014; 36(3):202-7.
Gisselsson D, Palsson E, Yu C, et al. Mitotic instability associated with late genomic changes in bone and soft tissue tumours. Cancer Lett. 2004; 206(1):69-76.
|Issue||Vol 10, No 3 (2016)|
|Mesenchymal stromal cells Umbilical cord blood Platelet lysate Immunomodulatory properties Cell therapy|
|Rights and permissions|
|This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.|