Comparison of the Ex Vivo Expansion of UCB-Derived CD34+ in 3D DBM/MBA Scaffolds with USSC as a Feeder Layer

Document Type : Original Article


1 Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran

2 Department of Hematology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran



Ex vivo expansion of hematopoitic stem cells is an alternative way to increase umbilical cord blood (UCB)-CD34+ cells for bone marrow transplantation. For this purpose demineralized bone matrix (DBM) and mineralized bone allograft (MBA) as two scaffolds based on bone matrix and stem cell niche, were simultaneously used to enhance the effect of human mesenchymal progenitor cells (MPCs) - unrestricted somatic stem cells (USSCs) - as a feeder layer.
Materials and Methods:
USSCs were isolated and characterized by morphological and immunological analysis then seeded on both scaffolds as a feeder layer. UCB-CD34+ were isolated by MACS method and were co-culture expanded by USSC in 3D and 2D environments. After 3 weeks expansion, cells were counted and were assessed by karyotype, flow cytometry, clonogenic activity, and long-term culture-initiating cells (LTC-IC).
Co-culture expansion in DBM and MBA was 29.22-fold and 27.77-fold, no significant differences in colony and LTC-IC were obtained. Maximum number of colonies belonged to the day 14 with the 73% CFU-GM (Colony Forming Unit- Granulocyte/Macrophage) in contrast to the day 0 which was BFU-E/CFU-E (Burst/Colony Forming Unit-Erythroid). Flow cytometry indicated that the percentage of CD34+ marker was decreased in USSC co-culture and the highest percentage was observed in simple 2D culture.
Because of acid extraction in the DBM production process, mineral materials were removed and the protein background that was more flexible was presented. Therefore these results suggest that USSC-DBM can be a suitable ex vivo mimicry niche by intensifying of surface/volume ratio and supporting the stem cell differentiation and expansion.


1. Bordignon C. Stem-cell therapies for blood diseases. Nature 2006; 441:1100-1112.
2. Siena S, Schiavo R, Pedrazzoli P, Carlo-Stella C. Therapeutic relevance of CD34 cell dose in blood cell transplantation for cancer therapy. J Clin Oncol 2000; 18:1360.
3. Seghatoleslam M, Jalali M, Nikravesh MR, Hosseini M, Alamdari DH, Fazel A. Therapeutic benefit of intravenous administration of human umbilical cord blood-mononuclear cells following intracerebral hemorrhage in rat. Iran J Basic Med Sci 2012; 15:860.
4. Emerson SG. Ex vivo expansion of hematopoietic precursors, progenitors, and stem cells: the next generation of cellular therapeutics. Blood 1996; 87:3082.
5. To L, Haylock D, Simmons P, Juttner C. The biology and clinical uses of blood stem cells. Blood 1997; 89:2233.
6. Bakhtiary M, Marzban M, Mehdizadeh M, Joghataei MT, Khoei S, Tondar M, et al. Combination of stem cell mobilized by granulocyte-colony stimulating factor and human umbilical cord matrix stem cell: therapy of traumatic brain injury in rats. Iran J Basic Med Sci 2011; 14:327-329.
7. Gao L, Chen X, Zhang X, Liu Y, Kong P, Peng X, et al. Human umbilical cord blood-derived stromal cell, a new resource of feeder layer to expand human umbilical cord blood CD34+ cells in vitro. Blood Cells Mol Dis 2006; 36:322-328.
8. Delalat B, Pourfathollah AA, Soleimani M, Mozdarani H, Ghaemi SR, Movassaghpour AA, et al. Isolation and ex vivo expansion of human umbilical cord blood-derived CD34+ stem cells and their cotransplantation with or without mesenchymal stem cells. Hematology 2009; 14:125-132.
9. Hai Jiang W, Xin Na D, Hui Jun D. Expansion of hematopoietic stem/progenitor cells. Am J Hematol 2008; 83:922-926.
Hashemi et al Ex vivo Expansion of UCB-Derived CD34+
Iran J Basic Med Sci, Vol. 16, No. 10, Oct 2013
10. Golipour Z, Ragerdi Kashani I, Akbari Mohammad HGR, Malek F, Mahmoudi R. Differentiation of adipose-derived stem cells into schwann cell phenotype in comparison with bone marrow stem cells. Iran J Basic Med Sci 2010; 13:76-84.
11. Baghban Eslaminezhad M, Nazarian H, Falahi Fahimeh TL, Daneshzadeh M. Ex vivo expansion and differentiation of mesenchymal stem cells from goat bone marrow. Iran J Basic Med Sci 2009; 12:70-79.
12. Song Y, Song G. Stem cell niche and its roles in proliferation and differentiation of stem cells. J Biomed Engin 2009; 26:195.
13. Sachlos E, Czernuszka J. Making tissue engineering scaffolds work. Review: the application of solid freeform fabrication technology to the production of tissue engineering scaffolds. Eur Cell Mater 2003; 5:29-39.
14. Chung HJ, Park TG. Surface engineered and drug releasing pre-fabricated scaffolds for tissue engineering. Adv Drug Deliv Rev 2007; 59:249-262.
15. Kasten P, Luginbühl R, Van Griensven M, Barkhausen T, Krettek C, Bohner M, et al. Comparison of human bone marrow stromal cells seeded on calcium-deficient hydroxyapatite,[beta]-tricalcium phosphate and demineralized bone matrix. Biomaterials 2003; 24:2593-2603.
16. Behnia H, Khojasteh A, Soleimani M, Tehranchi A, Khoshzaban A, Keshel S, et al. Secondary repair of alveolar clefts using human mesenchymal stem cells. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009; 108:e1.
17. Mauney JR, Jaquiéry C, Volloch V, Heberer M, Martin I, Kaplan DL. In vitro and in vivo evaluation of differentially demineralized cancellous bone scaffolds combined with human bone marrow stromal cells for tissue engineering. Biomaterials 2005; 26:3173-3185.
18. Halabian R, Mohammadi MH, Salimi M, Amani M, Roushande AM, Aghaipoor M, et al. Genetically engineered mesenchymal stem cells stably expressing. Iran J Basic Med Sci; 13:24-30.
19. Thibault RA, Scott Baggett L, Mikos AG, Kasper FK. Osteogenic differentiation of mesenchymal stem cells on pregenerated extracellular matrix scaffolds in the absence of osteogenic cell culture supplements. Tissue Engin Part A 2009; 16:431-440.
20. Langenbach F, Naujoks C, Kersten-Thiele PV, Berr K, Depprich RA, Kübler NR, et al. Osteogenic differentiation influences stem cell migration out of scaffold-free microspheres. Tissue Engin Part A 2009; 16:759-766.
21. Hashemi S, Soleimani M, Zargarian S, Haddadi-Asl V, Ahmadbeigi N, Soudi S, et al. In vitro differentiation of human Ccrd blood-derived unrestricted somatic stem cells into hepatocyte-like cells on poly (Caprolactone) nanofiber scaffolds. Cells Tissues Organs 2008; 190:135-149.
22. Miller CL, Eaves CJ. Expansion in vitro of adult murine hematopoietic stem cells with transplantable lympho-myeloid reconstituting ability. Proc Natl Acad Sci 1997; 94:13648-1353.
23. Ueda T, Tsuji K, Yoshino H, Ebihara Y, Yagasaki H, Hisakawa H, et al. Expansion of human NOD/SCID-repopulating cells by stem cell factor, Flk2/Flt3 ligand, thrombopoietin, IL-6, and soluble IL-6 receptor. J Clin Invest 2000; 105:1013-1021.
24. Bhatia M, Bonnet D, Kapp U, Wang JC, Murdoch B, Dick JE. Quantitative analysis reveals expansion of human hematopoietic repopulating cells after short-term ex vivo culture. J Expe Med 1997; 186:619-624.
25. Bagley J, Rosenzweig M, Marks DF, Pykett MJ. Extended culture of multipotent hematopoietic progenitors without cytokine augmentation in a novel three-dimensional device. Exp Hematol 1999; 27:496-504.
26. Franke K, Pompe T, Bornhäuser M, Werner C. Engineered matrix coatings to modulate the adhesion of CD133< sup>+</sup> human hematopoietic progenitor cells. Biomaterials 2007; 28:836-843.
27. Sagar BMM, Rentala S, Gopal P, Sharma S, Mukhopadhyay A. Fibronectin and laminin enhance engraftibility of cultured hematopoietic stem cells. Biochem Biophys Res Commun 2006; 350:1000-1005.
28. Jiang X-S, Chai C, Zhang Y, Zhuo R-X, Mao H-Q, Leong KW. Surface-immobilization of adhesion peptides on substrate for ex vivo expansion of cryopreserved umbilical cord blood CD34< sup>+</sup> cells. Biomaterials 2006; 27:2723-2732.
29. Chua K-N, Chai C, Lee P-C, Tang Y-N, Ramakrishna S, Leong KW, et al. Surface-aminated electrospun nanofibers enhance adhesion and expansion of human umbilical cord blood hematopoietic stem/progenitor cells. Biomaterials 2006; 27:6043-6051.
30. Feng Q, Chai C, Jiang XS, Leong KW, Mao HQ. Expansion of engrafting human hematopoietic stem/progenitor cells in three‐dimensional scaffolds with surface‐immobilized fibronectin. J Biomed Mater Res Part A 2006; 78:781-791.
31. Das H, Abdulhameed N, Joseph M, Sakthivel R, Mao H-Q, Pompili VJ. Ex vivo nanofiber expansion and genetic modification of human cord blood-derived progenitor/stem cells enhances vasculogenesis. Cell Transplant 2009; 18:305.
32. Seong JM, Kim B-C, Park J-H, Kwon IK, Mantalaris A, Hwang Y-S. Stem cells in bone tissue engineering. Biomed Mater 2010; 5:062001.
33. Yildirim S, Boehmler A, Kanz L, Möhle R. Expansion of cord blood CD34&plus; hematopoietic progenitor cells in coculture with autologous umbilical vein endothelial cells (HUVEC) is superior to cytokine-supplemented liquid culture. Bone Marrow Transplant 2005; 36:71-79.
34. Robinson S, Ng J, Niu T, Yang H, McMannis J, Karandish S, et al. Superior ex vivo cord blood expansion following co-culture with bone marrow-derived mesenchymal stem cells. Bone Marrow Transplant 2006; 37:359-366.
35. Nakamura Y, Hiroyama T, Miharada K, Kurita R. Red blood cell production from immortalized progenitor cell line. Int J Hematol 2011; 93:5-9.
36. Schoemans H, Theunissen K, Maertens J, Boogaerts M, Verfaillie C, Wagner J. Adult umbilical cord blood transplantation: a comprehensive review. Bone Marrow Transplant 2006; 38:83-93.