Ex vivo expansion of cord blood mononuclear cells by umbilical cord blood platelet lysate

Abstract

Background and Objective: In spite of promising results in treatment of blood diseases by umbilical cord blood cell transplantation, low number of derived stem cells has been a matter of concern. Since platelets extracts has been introduced as a potent mesenchymal stem cell culture supplement, the purpose of current study was to investigate the probable effects of umbilical cord blood platelet lysate (UCB-PL) in proliferation, survival and differentiation of cord blood mononuclear cells.


Materials and Methods: The umbilical cord blood platelet lysate (UCB-PL) was added to cord blood mononuclear cells culture (1×10 6 /mL) in presence of FLT3L, TPO and SCF. The effective dose of UCB-PL, cell viability and differentiation of mononuclear cells were determined by trypan blue staining and colony assay method, respectively. The number of mononuclear cells was measured by flowcytometric analysis of CD133+ CD34+CD38- cells. Quantitative expression of CD 34 was performed by QRT-PCR method.


Results: Combination of 5% of UCB-PL with mentioned cytokines yielded a 12-fold cell proliferation in comparison with control group. A higher concentration of UCB-PL showed cytotoxic effects. CD 34 also increased significantly in group of 5% UCB-PL treated cells. In this particular group, a significant increase of colony forming units granulocytes-erythroid-macrophages-megakaryocyte (CFU-GMMM) and granulocyte-macrophage (CFU-GM) were detected.


Conclusion: Our results indicate that 5% UCB -PL can be used as a supplement in MNC cultured cells and in cord blood hematopoietic stem cells for cell therapy purposes.

Keywords


1. Haspel RL, Miller KB. Hematopoietic stem cells: source matters. Current Stem Cell Research & Therapy 2008;3:229-36 2. Barker JN, Wagner JE. Umbilical cord blood transplantation: Current Practice and Future Innovations. Critical Reviews in Oncology/Hematology 2003;48:35-43 3. Nishino T, Osawa M, Iwama A. New approaches to expand hematopoietic stem and progenitor cells. Expert Opinion on Biological Therapy 2012;12:743-56 4. Stanevsky A, Shimoni A, Yerushalmi R, Nagler A. Cord blood stem cells for hematopoietic transplantation. Stem Cell Reviews 2011;7:425-33 5. Akala OO, Clarke MF. Hematopoietic stem cell self-renewal. Current Opinion in Genetics & Development 2006;16:496-501 6. Norkin M, Lazarus HM, Wingard JR. Umbilical cord blood graft enhancement strategies: has the time come to move these into the clinic? Bone Marrow Transplantation 2013;48:884-9 7. Lima MD. Mesenchymal Stem Cell (MSC) Based Cord Blood (CB) Expansion (Exp) leads to rapid engraftment of platelets and neutrophils. Blood 2010;116 8. Jaroscak J,Goltry K, Smith A, Waters-Pick B, Martin PL, Driscoll TA, et al. Augmentation of umbilical cord blood (UCB) transplantation with ex vivo-expanded UCB cells: results of a phase 1 trial using the AastromReplicell System. Blood 2003;101: 5061-7 9. Rick G, Smith BS, C.C.P, Craig J, Glassmann C.C.P, and Mark S. Campbell, B.A , C.C.P. Platelet-rich Plasma: Properties and Clinical Applications. The Journal of Lancaster General Hospital 2007; 2 10. Jonsdottir-Buch SM, Lieder R, Sigurjonsson OE. Platelet lysates produced from expired platelet concentrates support growth and osteogenic differentiation of mesenchymal stem cells. PlOS One 2013;8:e68984 11. Heldin CH, Westermark B. Mechanism of action and in vivo role of platelet-derived growth factor. Physiological Reviews 1999;79 :1283-316 12. Bolta PRmaZ. Use of platelet growth factors in treating wounds and soft-tissue injuries. Acta Dermatoven APA 2007;16:10 13. Kita K, Lee JO, Finnerty CC, Herndon DN. Cord blood-derived hematopoietic stem/progenitor cells: current challenges in engraftment, infection, and ex vivo expansion. Stem Cells International 2011;2011:276193 14. Cutler C, Ballen KK. Improving outcomes in umbilical cord blood transplantation: state of the art. Blood Reviews 2012;26 :241-6 15. Ranzato E, Patrone M, Mazzucco L, Burlando B. Platelet lysate stimulates wound repair of HaCaT keratinocytes. The British Journal of Dermatology 2008;159:537-45 16. Ranzato E, Mazzucco L, Patrone M, Burlando B. Platelet lysate promotes in vitro wound scratch closure of human dermal fibroblasts: different roles of cell calcium, P38, ERK and PI3K/AKT. Journal of Cellular and Molecular Medicine 2009;13:2030-8 17. Ranzato E, Boccafoschi F, Mazzucco L, Patrone M, Burlando B. Role of ERK1/2 in platelet lysate-driven endothelial cell repair. Journal of Cellular Biochemistry 2010;110: 783-93 18. Wu JY, Liao C, Chen JS, Xu ZP, Li Y, Sun X, et al. [Analysis of post-thaw infused cell dose for predicting engraftment after unrelated cord blood transplantation]. Journal of Experimental Hematology / Chinese Association of Pathophysiology 2011;19:754-8 19. Kawai-Kowase K, Sato H, Oyama Y, Kanai H, Sato M, Doi H, et al. Basic fibroblast growth factor antagonizes transforming growth factor-beta1-induced smooth muscle gene expression through extracellular signal-regulated kinase 1/2 signaling pathway activation. Arteriosclerosis, Thrombosis, and Vascular Biology 2004;24 :1384-90 20. Coutu DL, Galipeau J. Roles of FGF signaling in stem cell self-renewal, senescence and aging. Aging 2011;3:920-33 21. Zhou D, Deoliveira D, Kang Y, Choi SS, Li Z, Chao NJ, et al. Insulin-like growth factor 1 mitigates hematopoietic toxicity after lethal total body irradiation. International Journal of Radiation Oncology, Biology, Physics 2013;85:1141-8 22. Sattler FR. Growth hormone in the aging male. Best Practice & Research Clinical Endocrinology & Metabolism 2013;27:541-55 23. Barzilai N, Bartke A. Biological approaches to mechanistically understand the healthy life span extension achieved by calorie restriction and modulation of hormones. The journals of gerontology Series A, Biological Sciences and Medical Sciences 2009;64:187-91 24. Faloon P, Arentson E, Kazarov A, Deng CX, Porcher C, Orkin S, et al. Basic fibroblast growth factor positively regulates hematopoietic development. Development 2000;127:1931-41 25. Su RJ, Zhang XB, Li K, Yang M, Li CK, Fok TF, et al. Platelet-derived growth factor promotes ex vivo expansion of CD34+ cells from human cord blood and enhances long-term culture-initiating cells, non-obese diabetic/severe combined immunodeficient repopulating cells and formation of adherent cells. British Journal of Haematology 2002;117:735-46 26. Drygalski A, Xu G, Constantinescu D, Kashiwakura I, Farley T, Dobrila L, et al. The frequency and proliferative potential of megakaryocytic colony-forming cells (Meg-CFC) in cord blood, cytokine-mobilized peripheral blood and bone marrow, and their correlation with total CFC numbers: implications for the quantitation of Meg-CFC to predict platelet engraftment following cord blood transplantation. Bone Marrow Transplantation 2000;25 :1029-34