Abstract
Umbilical cord (UC) is a rich source of rapidly proliferating mesenchymal stem cells (MSCs) that are easily cultured on a large-scale. Clinical applications of UC–MSCs include graft-versus-host disease, and diabetes mellitus types 1 and 2. UC–MSCs should be isolated and proliferated according to good manufacturing practice (GMP) with animal component-free medium, quality assurance, and quality control for their use in clinical applications. This study developed a GMP standard protocol for UC-MSC isolation and culture. UC blood and UC were collected from the same donors. Blood vasculature was removed from UC. UC blood was used as a source of activated platelet rich plasma (aPRP). Small fragments (1–2 mm2) of UC membrane and Wharton’s jelly were cut and cultured in DMEM/F12 medium containing 1 % antibiotic–antimycotic, aPRP (2.5, 5, 7.5 and 10 %) at 37 °C in 5 % CO2. The MSC properties of UC–MSCs at passage 5 such as osteoblast, chondroblast and adipocyte differentiation, and markers including CD13, CD14, CD29, CD34, CD44, CD45, CD73, CD90, CD105, and HLA-DR were confirmed. UC–MSCs also were analyzed for karyotype, expression of tumorigenesis related genes, cell cycle, doubling time as well as in vivo tumor formation in NOD/SCID mice. Control cells consisted of UC–MSCs cultured in DMEM/F12 plus 1 % antibiotic–antimycotic, and 10 % fetal bovine serum (FBS). All UC-MSC (n = 30) samples were successfully cultured in medium containing 7.5 and 10 % aPRP, 92 % of samples grew in 5.0 % aPRP, 86 % of samples in 2.5 % aPRP, and 72 % grew in 10 % FBS. UC–MSCs in these four groups exhibited similar marker profiles. Moreover, the proliferation rates in medium with PRP, especially 7.5 and 10 %, were significantly quicker compared with 2.5 and 5 % aPRP or 10 % FBS. These cells maintained a normal karyotype for 15 sub-cultures, and differentiated into osteoblasts, chondroblasts, and adipocytes. The analysis of pluripotent cell markers showed UC–MSCs maintained the expression of the oncogenes Nanog and Oct4 after long term culture but failed to transfer tumors in NOD/SCID mice. Replacing FBS with aPRP in the culture medium for UC tissues allowed the successful isolation of UC–MSCs that satisfy the minimum standards for clinical applications.
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References
Baba K, Yamazaki Y, Ishiguro M, Kumazawa K, Aoyagi K, Ikemoto S, Takeda A, Uchinuma E (2013) Osteogenic potential of human umbilical cord-derived mesenchymal stromal cells cultured with umbilical cord blood-derived fibrin: a preliminary study. J Craniomaxillofac Surg 41:775–782
Badraiq H, Devito L, Ilic D (2015) Isolation and expansion of mesenchymal stromal/stem cells from umbilical cord under chemically defined conditions. Methods Mol Biol 1283:65–71
Bieback K, Hecker A, Kocaomer A, Lannert H, Schallmoser K, Strunk D, Kluter H (2009) Human alternatives to fetal bovine serum for the expansion of mesenchymal stromal cells from bone marrow. Stem Cells 27:2331–2341
Blande IS, Bassaneze V, Lavini-Ramos C, Fae KC, Kalil J, Miyakawa AA, Schettert IT, Krieger JE (2009) Adipose tissue mesenchymal stem cell expansion in animal serum-free medium supplemented with autologous human platelet lysate. Transfusion 49:2680–2685
Buyl K, Vanhaecke T, Desmae T, Lagneaux L, Rogiers V, Najar M, De Kock J (2014) Evaluation of a new standardized enzymatic isolation protocol for human umbilical cord-derived stem cells. Toxicol In Vitro 29:1254–1262
Chen GH, Yang T, Tian H, Qiao M, Liu HW, Fu CC, Miao M, Jin ZM, Tang XW, Han Y, He GS, Zhang XH, Ma X, Chen F, Hu XH, Xue SL, Wang Y, Qiu HY, Sun AN, Chen ZZ, Wu DP (2012) Clinical study of umbilical cord-derived mesenchymal stem cells for treatment of nineteen patients with steroid-resistant severe acute graft-versus-host disease. Zhonghua Xue Ye Xue Za Zhi 33:303–306
Cheng H, Liu X, Hua R, Dai G, Wang X, Gao J, An Y (2014) Clinical observation of umbilical cord mesenchymal stem cell transplantation in treatment for sequelae of thoracolumbar spinal cord injury. J Transl Med 12:253
Ding Y, Yang H, Feng JB, Qiu Y, Li DS, Zeng Y (2013) Human umbilical cord-derived MSC culture: the replacement of animal sera with human cord blood plasma. In Vitro Cell Dev Biol Anim 49:771–777
Ding DC, Chou HL, Chang YH, Hung WT, Liu HW, Chu TY (2015) Characterization of HLA-G and related immunosuppressive effects in human umbilical cord stroma derived stem cells. Cell Transplant. doi:10.3727/096368915X688182
Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop D, Horwitz E (2006) Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 8:315–317
Dongmei H, Jing L, Mei X, Ling Z, Hongmin Y, Zhidong W, Li D, Zikuan G, Hengxiang W (2011) Clinical analysis of the treatment of spinocerebellar ataxia and multiple system atrophy-cerebellar type with umbilical cord mesenchymal stromal cells. Cytotherapy 13:913–917
Escobedo-Lucea C, Bellver C, Gandia C, Sanz-Garcia A, Esteban FJ, Mirabet V, Forte G, Moreno I, Lezameta M, Ayuso-Sacido A, Garcia-Verdugo JM (2013) A xenogeneic-free protocol for isolation and expansion of human adipose stem cells for clinical uses. PLoS One 8:e67870
Fekete N, Rojewski MT, Furst D, Kreja L, Ignatius A, Dausend J, Schrezenmeier H (2012) GMP-compliant isolation and large-scale expansion of bone marrow-derived MSC. PLoS One 7:e43255
Gu F, Wang D, Zhang H, Feng X, Gilkeson GS, Shi S, Sun L (2014) Allogeneic mesenchymal stem cell transplantation for lupus nephritis patients refractory to conventional therapy. Clin Rheumatol 33:1611–1619
Iftimia-Mander A, Hourd P, Dainty R, Thomas RJ (2013) Mesenchymal stem cell isolation from human umbilical cord tissue: understanding and minimizing variability in cell yield for process optimization. Biopreserv Biobank 11:291–298
Jiang Y, Zhu W, Zhu J, Wu L, Xu G, Liu X (2013) Feasibility of delivering mesenchymal stem cells via catheter to the proximal end of the lesion artery in patients with stroke in the territory of the middle cerebral artery. Cell Transplant 22:2291–2298
Jin JL, Liu Z, Lu ZJ, Guan DN, Wang C, Chen ZB, Zhang J, Zhang WY, Wu JY, Xu Y (2013) Safety and efficacy of umbilical cord mesenchymal stem cell therapy in hereditary spinocerebellar ataxia. Curr Neurovasc Res 10:11–20
Jonsdottir-Buch SM, Lieder R, Sigurjonsson OE (2013) Platelet lysates produced from expired platelet concentrates support growth and osteogenic differentiation of mesenchymal stem cells. PLoS One 8:e68984
Kim JW, Kim SY, Park SY, Kim YM, Kim JM, Lee MH, Ryu HM (2004) Mesenchymal progenitor cells in the human umbilical cord. Ann Hematol 83:733–738
Kocaoemer A, Kern S, Kluter H, Bieback K (2007) 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 25:1270–1278
Kong D, Zhuang X, Wang D, Qu H, Jiang Y, Li X, Wu W, Xiao J, Liu X, Liu J, Li A, Wang J, Dou A, Wang Y, Sun J, Lv H, Zhang G, Zhang X, Chen S, Ni Y, Zheng C (2014) Umbilical cord mesenchymal stem cell transfusion ameliorated hyperglycemia in patients with type 2 diabetes mellitus. Clin Lab 60:1969–1976
Lee OK, Kuo TK, Chen WM, Lee KD, Hsieh SL, Chen TH (2004) Isolation of multipotent mesenchymal stem cells from umbilical cord blood. Blood 103:1669–1675
Lee JY, Nam H, Park YJ, Lee SJ, Chung CP, Han SB, Lee G (2011) The effects of platelet-rich plasma derived from human umbilical cord blood on the osteogenic differentiation of human dental stem cells. In Vitro Cell Dev Biol Anim 47:157–164
Liu J, Han D, Wang Z, Xue M, Zhu L, Yan H, Zheng X, Guo Z, Wang H (2013) Clinical analysis of the treatment of spinal cord injury with umbilical cord mesenchymal stem cells. Cytotherapy 15:185–191
Lv YT, Zhang Y, Liu M, Qiuwaxi JN, Ashwood P, Cho SC, Huan Y, Ge RC, Chen XW, Wang ZJ, Kim BJ, Hu X (2013) Transplantation of human cord blood mononuclear cells and umbilical cord-derived mesenchymal stem cells in autism. J Transl Med 11:196
Ma HY, Yao L, Yu YQ, Li L, Ma L, Wei WJ, Lu XM, Du LL, Jin YN (2012) An effective and safe supplement for stem cells expansion ex vivo: cord blood serum. Cell Transplant 21:857–869
Mareschi K, Biasin E, Piacibello W, Aglietta M, Madon E, Fagioli F (2001) Isolation of human mesenchymal stem cells: bone marrow versus umbilical cord blood. Haematologica 86:1099–1100
Marx RE (2004) Platelet-rich plasma: evidence to support its use. J Oral Maxillofac Surg 62:489–496
Mori Y, Ohshimo J, Shimazu T, He H, Takahashi A, Yamamoto Y, Tsunoda H, Tojo A, Nagamura-Inoue T (2015) Improved explant method to isolate umbilical cord-derived mesenchymal stem cells and their immunosuppressive properties. Tissue Eng Part C Methods 21:367–372
Murphy MB, Blashki D, Buchanan RM, Yazdi IK, Ferrari M, Simmons PJ, Tasciotti E (2012) Adult and umbilical cord blood-derived platelet-rich plasma for mesenchymal stem cell proliferation, chemotaxis, and cryo-preservation. Biomaterials 33:5308–5316
Otte A, Bucan V, Reimers K, Hass R (2013) Mesenchymal stem cells maintain long-term in vitro stemness during explant culture. Tissue Eng Part C Methods 19:937–948
Peng W, Sun J, Sheng C, Wang Z, Wang Y, Zhang C, Fan R (2015) Systematic review and meta-analysis of efficacy of mesenchymal stem cells on locomotor recovery in animal models of traumatic brain injury. Stem Cell Res Ther 6:47
Pham PV, Vu NB, Pham VM, Truong NH, Pham TL, Dang LT, Nguyen TT, Bui AN, Phan NK (2014) Good manufacturing practice-compliant isolation and culture of human umbilical cord blood-derived mesenchymal stem cells. J Transl Med 12:56
Phuc PV, Ngoc VB, Lam DH, Tam NT, Viet PQ, Ngoc PK (2012) Isolation of three important types of stem cells from the same samples of banked umbilical cord blood. Cell Tissue Bank 13:341–351
Pineault N, Abu-Khader A (2015) Advances in umbilical cord blood stem cell expansion and clinical translation. Exp Hematol 43:498–513
Rauch C, Feifel E, Amann EM, Spotl HP, Schennach H, Pfaller W, Gstraunthaler G (2011) Alternatives to the use of fetal bovine serum: human platelet lysates as a serum substitute in cell culture media. ALTEX 28:305–316
Romanov YA, Svintsitskaya VA, Smirnov VN (2003) Searching for alternative sources of postnatal human mesenchymal stem cells: candidate MSC-like cells from umbilical cord. Stem Cells 21:105–110
Santos Nascimento D, Mosqueira D, Sousa LM, Teixeira M, Filipe M, Resende TP, Araujo AF, Valente M, Almeida J, Martins JP, Santos JM, Barcia RN, Cruz P, Cruz H, Pinto-do OP (2014) Human umbilical cord tissue-derived mesenchymal stromal cells attenuate remodeling after myocardial infarction by proangiogenic, antiapoptotic, and endogenous cell-activation mechanisms. Stem Cell Res Ther 5:5
Shetty P, Bharucha K, Tanavde V (2007) Human umbilical cord blood serum can replace fetal bovine serum in the culture of mesenchymal stem cells. Cell Biol Int 31:293–298
Shi D, Wang D, Li X, Zhang H, Che N, Lu Z, Sun L (2012a) Allogeneic transplantation of umbilical cord-derived mesenchymal stem cells for diffuse alveolar hemorrhage in systemic lupus erythematosus. Clin Rheumatol 31:841–846
Shi M, Zhang Z, Xu R, Lin H, Fu J, Zou Z, Zhang A, Shi J, Chen L, Lv S, He W, Geng H, Jin L, Liu Z, Wang FS (2012b) Human mesenchymal stem cell transfusion is safe and improves liver function in acute-on-chronic liver failure patients. Stem Cells Transl Med 1:725–731
Sun L, Wang D, Liang J, Zhang H, Feng X, Wang H, Hua B, Liu B, Ye S, Hu X, Xu W, Zeng X, Hou Y, Gilkeson GS, Silver RM, Lu L, Shi S (2010) Umbilical cord mesenchymal stem cell transplantation in severe and refractory systemic lupus erythematosus. Arthritis Rheum 62:2467–2475
Van Pham P, Phan NK (2015) Production of good manufacturing practice-grade human umbilical cord blood-derived mesenchymal stem cells for therapeutic use. Methods Mol Biol 1283:73–85
Wang D, Zhang H, Liang J, Li X, Feng X, Wang H, Hua B, Liu B, Lu L, Gilkeson GS, Silver RM, Chen W, Shi S, Sun L (2013a) Allogeneic mesenchymal stem cell transplantation in severe and refractory systemic lupus erythematosus: 4 years of experience. Cell Transplant 22:2267–2277
Wang L, Li J, Liu H, Li Y, Fu J, Sun Y, Xu R, Lin H, Wang S, Lv S, Chen L, Zou Z, Li B, Shi M, Zhang Z, Wang FS (2013b) Pilot study of umbilical cord-derived mesenchymal stem cell transfusion in patients with primary biliary cirrhosis. J Gastroenterol Hepatol 28(Suppl 1):85–92
Wang S, Cheng H, Dai G, Wang X, Hua R, Liu X, Wang P, Chen G, Yue W, An Y (2013c) Umbilical cord mesenchymal stem cell transplantation significantly improves neurological function in patients with sequelae of traumatic brain injury. Brain Res 1532:76–84
Wang D, Li J, Zhang Y, Zhang M, Chen J, Li X, Hu X, Jiang S, Shi S, Sun L (2014) Umbilical cord mesenchymal stem cell transplantation in active and refractory systemic lupus erythematosus: a multicenter clinical study. Arthritis Res Ther 16:R79
Woodworth TG, Furst DE (2014) Safety and feasibility of umbilical cord mesenchymal stem cells in treatment-refractory systemic lupus erythematosus nephritis: time for a double-blind placebo-controlled trial to determine efficacy. Arthritis Res Ther 16:113
Wu KH, Tsai C, Wu HP, Sieber M, Peng CT, Chao YH (2013a) Human application of ex vivo expanded umbilical cord-derived mesenchymal stem cells: enhance hematopoiesis after cord blood transplantation. Cell Transplant 22:2041–2051
Wu KH, Sheu JN, Wu HP, Tsai C, Sieber M, Peng CT, Chao YH (2013b) Cotransplantation of umbilical cord-derived mesenchymal stem cells promote hematopoietic engraftment in cord blood transplantation: a pilot study. Transplantation 95:773–777
Zhang Z, Lin H, Shi M, Xu R, Fu J, Lv J, Chen L, Lv S, Li Y, Yu S, Geng H, Jin L, Lau GK, Wang FS (2012a) Human umbilical cord mesenchymal stem cells improve liver function and ascites in decompensated liver cirrhosis patients. J Gastroenterol Hepatol 27(Suppl 2):112–120
Zhang H, Zhang B, Tao Y, Cheng M, Hu J, Xu M, Chen H (2012b) Isolation and characterization of mesenchymal stem cells from whole human umbilical cord applying a single enzyme approach. Cell Biochem Funct 30:643–649
Acknowledgments
This research was funded by Ministry of Science and Technology via project Grant No. DTDL.2012-G/23.
Authors’ contributions
PVP conceived the study, performed PRP preparation, evaluated the effects of PRP on mesenchymal stem cell proliferation. NBV primarily cultured mesenchymal stem cells from mononuclear cells; TDXT, PTBL collected umbilical cord blood, isolated mononuclear cells from umbilical cord blood; KHTB carried out the differentiation assays; NTC, NKP evaluated the, karyotype, and tumorigenecity of MSCs in mice model. All authors read and approved the final manuscript.
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Van Pham, P., Truong, N.C., Le, P.TB. et al. Isolation and proliferation of umbilical cord tissue derived mesenchymal stem cells for clinical applications. Cell Tissue Bank 17, 289–302 (2016). https://doi.org/10.1007/s10561-015-9541-6
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DOI: https://doi.org/10.1007/s10561-015-9541-6