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Cell and Tissue Banking

, Volume 17, Issue 2, pp 289–302 | Cite as

Isolation and proliferation of umbilical cord tissue derived mesenchymal stem cells for clinical applications

  • Phuc Van PhamEmail author
  • Nhat Chau Truong
  • Phuong Thi-Bich Le
  • Tung Dang-Xuan Tran
  • Ngoc Bich Vu
  • Khanh Hong-Thien Bui
  • Ngoc Kim Phan
Original Paper

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.

Keywords

Activated platelet rich plasma Clinical application of mesenchymal stem cells Umbilical cord Umbilical cord derived mesenchymal stem cells Good manufacturing practice UC–MSCs 

Notes

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.

Compliance with ethical standards

Conflict of interests

The authors declare that they have no competing interests.

References

  1. 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 41:775–782CrossRefPubMedGoogle Scholar
  2. 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 1283:65–71CrossRefPubMedGoogle Scholar
  3. 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 27:2331–2341CrossRefPubMedGoogle Scholar
  4. 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 49:2680–2685CrossRefPubMedGoogle Scholar
  5. 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 29:1254–1262Google Scholar
  6. 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 33:303–306PubMedGoogle Scholar
  7. 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 12:253CrossRefPubMedPubMedCentralGoogle Scholar
  8. 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 49:771–777CrossRefPubMedGoogle Scholar
  9. 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
  10. 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 8:315–317CrossRefPubMedGoogle Scholar
  11. 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 13:913–917CrossRefPubMedGoogle Scholar
  12. 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:e67870CrossRefPubMedPubMedCentralGoogle Scholar
  13. 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:e43255CrossRefPubMedPubMedCentralGoogle Scholar
  14. 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 33:1611–1619CrossRefPubMedGoogle Scholar
  15. 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 11:291–298CrossRefPubMedGoogle Scholar
  16. 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 22:2291–2298CrossRefPubMedGoogle Scholar
  17. 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 10:11–20CrossRefPubMedGoogle Scholar
  18. 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:e68984CrossRefPubMedPubMedCentralGoogle Scholar
  19. 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 83:733–738CrossRefPubMedGoogle Scholar
  20. 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 25:1270–1278CrossRefPubMedGoogle Scholar
  21. 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 60:1969–1976PubMedGoogle Scholar
  22. Lee OK, Kuo TK, Chen WM, Lee KD, Hsieh SL, Chen TH (2004) Isolation of multipotent mesenchymal stem cells from umbilical cord blood. Blood 103:1669–1675CrossRefPubMedGoogle Scholar
  23. 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 47:157–164CrossRefPubMedGoogle Scholar
  24. 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 15:185–191CrossRefPubMedGoogle Scholar
  25. 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 11:196CrossRefPubMedPubMedCentralGoogle Scholar
  26. 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 21:857–869CrossRefPubMedGoogle Scholar
  27. 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 86:1099–1100PubMedGoogle Scholar
  28. Marx RE (2004) Platelet-rich plasma: evidence to support its use. J Oral Maxillofac Surg 62:489–496CrossRefPubMedGoogle Scholar
  29. 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 21:367–372CrossRefPubMedGoogle Scholar
  30. 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 33:5308–5316CrossRefPubMedGoogle Scholar
  31. 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 19:937–948CrossRefPubMedGoogle Scholar
  32. 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 6:47CrossRefPubMedPubMedCentralGoogle Scholar
  33. 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 12:56CrossRefPubMedPubMedCentralGoogle Scholar
  34. 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 13:341–351CrossRefPubMedGoogle Scholar
  35. Pineault N, Abu-Khader A (2015) Advances in umbilical cord blood stem cell expansion and clinical translation. Exp Hematol 43:498–513Google Scholar
  36. 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 28:305–316CrossRefPubMedGoogle Scholar
  37. 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 21:105–110CrossRefPubMedGoogle Scholar
  38. 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 5:5CrossRefPubMedGoogle Scholar
  39. 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 31:293–298CrossRefPubMedGoogle Scholar
  40. 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 31:841–846CrossRefPubMedGoogle Scholar
  41. 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 1:725–731CrossRefPubMedPubMedCentralGoogle Scholar
  42. 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 62:2467–2475CrossRefPubMedGoogle Scholar
  43. 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 1283:73–85CrossRefPubMedGoogle Scholar
  44. 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 22:2267–2277CrossRefPubMedGoogle Scholar
  45. 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):85–92CrossRefPubMedGoogle Scholar
  46. 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 1532:76–84CrossRefPubMedGoogle Scholar
  47. 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:R79CrossRefPubMedPubMedCentralGoogle Scholar
  48. 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 16:113CrossRefPubMedPubMedCentralGoogle Scholar
  49. 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 22:2041–2051CrossRefPubMedGoogle Scholar
  50. 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 95:773–777CrossRefPubMedGoogle Scholar
  51. 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):112–120CrossRefPubMedGoogle Scholar
  52. 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 30:643–649CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Phuc Van Pham
    • 1
    Email author
  • Nhat Chau Truong
    • 1
  • Phuong Thi-Bich Le
    • 2
  • Tung Dang-Xuan Tran
    • 2
  • Ngoc Bich Vu
    • 1
  • Khanh Hong-Thien Bui
    • 3
  • Ngoc Kim Phan
    • 1
  1. 1.Laboratory of Stem Cell Research and ApplicationUniversity of Science, Vietnam National UniversityHo Chi Minh CityVietnam
  2. 2.Van Hanh Stem Cell UnitVan Hanh HospitalHo Chi Minh CityVietnam
  3. 3.University Medical Center, University of Medicine and PharmacyHo Chi Minh CityVietnam

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