Abstract
The simultaneous expansion and harvest of hematopoietic stem cells and mesenchymal stem cells derived from umbilical cord blood were carried out using bioreactors. The co-culture of umbilical cord blood (UCB)-derived hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) was performed within spinner flasks and a rotating wall vessel (RWV) bioreactor using glass-coated styrene copolymer (GCSC) microcarriers. The medium used was composed of serum-free IMDM containing a cocktail of SCF 15 ng·mL−1, FL 5 ng·mL−1, TPO 6 ng·mL−1, IL-3 15 ng·mL−1, G-CSF 1 ng·mL−1 and GM-CSF 5 ng·mL−1. Accessory stromal cells derived from normal allogeneic adipose tissue were encapsulated in alginate-chitosan (AC) beads and used as feeding cells. The quality of the harvested UCB-HSCs and MSCs was assessed by immunophenotype analysis, methylcellulose colony and multi-lineage differentiation assays. After 12 days of culture, the fold-expansion of total cell numbers, colony-forming units (CFU-C), CD34+/CD45+/CD105− (HSCs) cells and CD34−/CD45−/CD105+ (MSCs) cells using the RWV bioreactor were (3.7 ± 0.3)- , (5.1 ± 1.2)- , (5.2 ± 0.4)- , and (13.9 ± 1.2)-fold respectively, significantly better than those obtained using spinner flasks. Moreover, UCB-HSCs and UCB-MSCs could be easily separated by gravity sedimentation after the co-culture period as only UCB-MSCs adhered on to the microcarriers. Simultaneously, we found that the fibroblast-like cells growing on the surface of the GCSC microcarriers could be induced and differentiated towards the osteoblastic, chondrocytic and adipocytic lineages. Phenotypically, these cells were very similarly to the MSCs derived from bone marrow positively expressing the MSCs-related markers CD13, CD44, CD73 and CD105, while negatively expressing the HSCs-related markers CD34, CD45 and HLA-DR. It was thus demonstrated that the simultaneous expansion and harvest of UCB-HSCs and UCB-MSCs is possible to be accomplished using a feasible bioreactor culture system such as the RWV bioreactor with the support of GCSC microcarriers.
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References
Bellantuono I. Haemopoietic stem cells. Int J Biochem Cell Biol. 2004;36:607.
Masson S, Harrison DJ, Plevris JN, et al. Potential of hematopoietic stem cell therapy in hepatology: aritical review. Stem Cells. 2004;22:897.
Jang YK, Jung DH, Jung MH, et al. Mesenchymal stem cells feeder layer from human umbilical cord blood for exvivo expanded growth and proliferation of hematopoietic progenitor cells. Ann Hematol. 2006;85:212.
Majumdar MK, Thiede MA, Haynesworth SE, et al. Human marrow-derived mesenchymal stem cells (MSCs) express hematopoietic cytokines and support long-term hematopoiesis when differentiated toward stromal and osteogenic lineages. J Hematother Stem Cell Res. 2000;9:841.
Laughlin MJ, Barker J, Bambach B, et al. Hematopoietic engraftment and survival in adult recipients of umbilicalcord blood from unrelated donors. N Engl J Med. 2001;344:1815.
Bensidhoum M, Chapel A, Francois S, et al. Homing of in vitro expanded Stro-1− or Stro-1+ human mesenchymal stem cells into the NOD/SCID mouse and their role in supporting human CD34 cell engraftment. Blood. 2004;103:3313.
Koc ON, Gerson SL, Cooper BW, et al. Rapid hematopoietic recovery after coinfusion of autologousblood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy. J Clin Oncol. 2000;18:307.
Deans RJ, Moseley AB. Mesenchymal stem cells; biology and potential clinical uses. Exp Hematol. 2000;28:875.
Chen X, Xu H, Wan C, et al. Bioreactor expansion of human adult bone marrow-derived mesenchymal stem cells. Stem Cells. 2006;24:2052.
Thalmeier K, Meissner P, Reisbach G, et al. Establishment of two permanent human bone marrow stromal cell lines with long-term post irradiation feeder capacity. Blood. 1994;83:1799.
Fujimoto N, Fujita S, Tsuji T, et al. Microencapsulated feeder cells as a source of soluble factors for expansion of CD34(+) hematopoietic stem cells. Biomaterials. 2007;28:4795.
Song K, Zhao G, Liu T, et al. Effective expansion of umbilical cord blood hematopoietic stem/progenitor cells by regulation of microencapsulated osteoblasts under hypoxic condition. Biotechnol Lett. 2009;31:923.
Liu Y, Liu T, Ma X, et al. Effects of encapsulated rabbit mesenchymal stem cells on ex vivo expansion of human umbilical cord blood hematopoietic stem/progenitor cells. J Microencapsul. 2009;26:130.
Song KD, Liu TQ, Li XQ, et al. Three-dimensional expansion: in suspension culture of SD rat’s osteoblasts in a rotating wall vessel bioreactor. Biomed Environ Sci. 2007;20:91.
Siti-Ismail N, Bishop AE, Polak JM, et al. The benefit of human embryonic stem cell encapsulation for prolonged feeder-free maintenance. Biomaterials. 2008;29:3946.
Kim Tae-Jin, Kim Su-Jin, Jung Hyo-Il. Physical stimulation of mammalian cells using micro-bead impact within a microfluidic environment to enhance growth rate. Microfluid Nanofluid. 2009;6:131.
Fok EY, Zandstra PW. Shear-controlled single-step mouse embryonic stem cell expansion and embryoid body-based differentiation. Stem Cells. 2005;23:1333.
Placzek MR, Chung IM, Macedo HM, et al. Stem cell bioprocessing: fundamentals and principles. J R Soc Interface. 2009;6:209.
Zhu YX, Liu TQ, Song KD, et al. Adipose tissue-derived stem cell: a better stem cell than BMSC. Cell Biochem Funct. 2008;26:664.
Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science. 1999;284:143.
Zhao ZG, Tang XQ, You Y, et al. Assessment of bone marrow mesenchymal stem cell biological characteristics and support hemotopoiesis function in patients with chronic myeloid leukemia. Leukemia Research. 2006;30:993.
Nakahara M, Takagi M, Hattori T, et al. Effect of sub-cultivation of human bone marrow mesenchymal stem cells on their capacities for chondrogenesis, supporting hematopoiesis and telomea length. Cytotechnology. 2005;47(1–3):19–27.
Ishige I, Nagamura-Inoue T, Honda MJ, et al. Comparison of mesenchymal stem cells derived from arterial, venous, and Wharton’s jelly explants of human umbilical cord. Int J Hematol. 2009;90(2):261–9.
Wagner W, Wein F, Seckinger A, et al. Comparative characteristics of mesenchymal stem cells from human bone marrow, adipose tissue, and umbilical cord blood. Exp Hematol. 2005;33(11):1402–16.
Inzunza J, Gertow K, Strömberg MA, et al. Derivation of human embryonic stem cell lines in serum replacement medium using postnatal human fibroblasts as feeder cells. Stem Cells. 2005;23:544.
Liu Y, Liu TQ, Fan XB, et al. Ex vivo expansion of hematopoietic stem cells derived from umbilical cord blood in rotating wall vesse. J Biotechnol. 2006;124:592.
Goncalves R, da Silva CL, Cabral JMS, et al. A Stro-1(+) human universal stromal feeder layer to expand/maintain human bone marrow hematopoietic stem/progenitor cells in a serum-free culture system. Exp Hematol. 2006;34:1353.
Yildirim S, Boehmler AM, Kanz L, et al. Expansion of cord blood CD34+ 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.
Chen TW, Yao CL, Chu IM, et al. Large generation of megakaryocytes from serum-free expanded human CD34+ cells. Biochem Biophys Res Commun. 2009;378:112.
Acknowledgment
This work was supported by the National Science Foundation of China (30670525, 30700181) and the new teacher foundation of Ministry of Education (20070141055). Mr. Hugo Macedo is also grateful to the Portuguese Fundação para a Ciência e Tecnologia for his PhD grant number SFRH/BD/28138/2006.
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Kedong, S., Xiubo, F., Tianqing, L. et al. Simultaneous expansion and harvest of hematopoietic stem cells and mesenchymal stem cells derived from umbilical cord blood. J Mater Sci: Mater Med 21, 3183–3193 (2010). https://doi.org/10.1007/s10856-010-4167-5
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DOI: https://doi.org/10.1007/s10856-010-4167-5